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Sample records for cell surface antigens

  1. Analysis of cell surface antigens by Surface Plasmon Resonance imaging

    NARCIS (Netherlands)

    Stojanovic, I.; Schasfoort, R.B.M.; Terstappen, L.W.M.M.

    2013-01-01

    Surface Plasmon Resonance (SPR) is most commonly used to measure bio-molecular interactions. SPR is used significantly less frequent for measuring whole cell interactions. Here we introduce a method to measure whole cells label free using the specific binding of cell surface antigens expressed on th

  2. Stable isotope labeling of oligosaccharide cell surface antigens

    Energy Technology Data Exchange (ETDEWEB)

    Unkefer, C.J.; Silks, L.A. III; Martinez, R.A. [and others

    1998-12-31

    The overall goal of this Laboratory Directed Research and Development (LDRD) project was to develop new methods for synthesis of {sup 13}C-labeled oligosaccharides that are required for nuclear magnetic resonance (NMR) studies of their solution conformation. Oligosaccharides are components of the cell`s outer surface and are involved in important processes such as cell-cell recognition and adhesion. Recently, Danishefsky and coworkers at Slone-Kettering Cancer Center developed a method for the solid-phase chemical synthesis of oligosaccharides. The specific goal of this LDRD project was to prepare uniform {sup 13}C-labeled aldohexose precursors required for the solid-phase synthesis of the Lewis blood-group antigenic determinants. We report the synthesis of {sup 13}C-labeled D-glucal, D-galactal and Fucosyl precursors. We have been collaborating with the Danishefsky group on the synthesis of the Lewis oligosaccharides and the NMR analysis of their solution conformation.

  3. Pooled protein immunization for identification of cell surface antigens in Streptococcus sanguinis.

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    Xiuchun Ge

    Full Text Available BACKGROUND: Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species. METHODS AND FINDINGS: We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity. CONCLUSIONS: The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.

  4. Surface proteome analysis and characterization of surface cell antigen (Sca or autotransporter family of Rickettsia typhi.

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    Khandra T Sears

    Full Text Available Surface proteins of the obligate intracellular bacterium Rickettsia typhi, the agent of murine or endemic typhus fever, comprise an important interface for host-pathogen interactions including adherence, invasion and survival in the host cytoplasm. In this report, we present analyses of the surface exposed proteins of R. typhi based on a suite of predictive algorithms complemented by experimental surface-labeling with thiol-cleavable sulfo-NHS-SS-biotin and identification of labeled peptides by LC MS/MS. Further, we focus on proteins belonging to the surface cell antigen (Sca autotransporter (AT family which are known to be involved in rickettsial infection of mammalian cells. Each species of Rickettsia has a different complement of sca genes in various states; R. typhi, has genes sca1 thru sca5. In silico analyses indicate divergence of the Sca paralogs across the four Rickettsia groups and concur with previous evidence of positive selection. Transcripts for each sca were detected during infection of L929 cells and four of the five Sca proteins were detected in the surface proteome analysis. We observed that each R. typhi Sca protein is expressed during in vitro infections and selected Sca proteins were expressed during in vivo infections. Using biotin-affinity pull down assays, negative staining electron microscopy, and flow cytometry, we demonstrate that the Sca proteins in R. typhi are localized to the surface of the bacteria. All Scas were detected during infection of L929 cells by immunogold electron microscopy. Immunofluorescence assays demonstrate that Scas 1-3 and 5 are expressed in the spleens of infected Sprague-Dawley rats and Scas 3, 4 and 5 are expressed in cat fleas (Ctenocephalides felis. Sca proteins may be crucial in the recognition and invasion of different host cell types. In short, continuous expression of all Scas may ensure that rickettsiae are primed i to infect mammalian cells should the flea bite a host, ii to remain

  5. Detection of cells captured with antigens on shear horizontal surface-acoustic-wave sensors.

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    Hao, Hsu-Chao; Chang, Hwan-You; Wang, Tsung-Pao; Yao, Da-Jeng

    2013-02-01

    Techniques to separate cells are widely applied in immunology. The technique to separate a specific antigen on a microfluidic platform involves the use of a shear horizontal surface-acoustic-wave (SH-SAW) sensor. With specific antibodies conjugated onto the surface of the SH-SAW sensors, this technique can serve to identify specific cells in bodily fluids. Jurkat cells, used as a target in this work, provide a model of cells in small abundance (1:1000) for isolation and purification with the ultimate goal of targeting even more dilute cells. T cells were separated from a mixed-cell medium on a chip (Jurkat cells/K562 cells, 1/1000). A novel microchamber was developed to capture cells during the purification, which required a large biosample. Cell detection was demonstrated through the performance of genetic identification on the chip.

  6. Longitudinal microarray analysis of cell surface antigens on peripheral blood mononuclear cells from HIV+ individuals on highly active antiretroviral therapy

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    Wang Bin

    2008-03-01

    Full Text Available Abstract Background The efficacy of highly active antiretroviral therapy (HAART determined by simultaneous monitoring over 100 cell-surface antigens overtime has not been attempted. We used an antibody microarray to analyze changes in the expression of 135 different cell-surface antigens overtime on PBMC from HIV+ patients on HAART. Two groups were chosen, one (n = 6 achieved sustainable response by maintaining below detectable plasma viremia and the other (n = 6 responded intermittently. Blood samples were collected over an average of 3 years and 5–8 time points were selected for microarray assay and statistical analysis. Results Significant trends over time were observed for the expression of 7 cell surface antigens (CD2, CD3epsilon, CD5, CD95, CD36, CD27 and CD28 for combined patient groups. Between groups, expression levels of 10 cell surface antigens (CD11a, CD29, CD38, CD45RO, CD52, CD56, CD57, CD62E, CD64 and CD33 were found to be differential. Expression levels of CD9, CD11a, CD27, CD28 and CD52, CD44, CD49d, CD49e, CD11c strongly correlated with CD4+ and CD8+ T cell counts, respectively. Conclusion Our findings not only detected markers that may have potential prognostic/diagnostic values in evaluating HAART efficacy, but also showed how density of cell surface antigens could be efficiently exploited in an array-like manner in relation to HAART and HIV-infection. The antigens identified in this study should be further investigated by other methods such as flow cytometry for confirmation as biological analysis of these antigens may help further clarify their role during HAART and HIV infection.

  7. T cell responses to hepatitis B surface antigen are detectable in non-vaccinated individuals

    Institute of Scientific and Technical Information of China (English)

    Martin R Weihrauch; Michael von Bergwelt-Baildon; Milos Kandic; Martin Weskott; Winfried Klamp; Joachim R(o)sier; Joachim L Schultze

    2008-01-01

    AIM: To evaluate, whether humoral hepatitis-B-vaccine non-responders also fail to mount a T cell response and to compare these results to normal vaccinees.METHODS: Fourty-seven health care employees were enrolled in this study including all available nonresponders (n = 13) with an anti-HBsAg titer 1000 kU/L as controls.PBMC from all subjects were analyzed by IFN-γ and IL-4 ELISPOT assays for the presence of hepatitis B surface antigen (HBsAg) reactive T cells.RESULTS: Non-responders and low-responders had no or only very limited T cell responses, respectively.Individuals responding to vaccination with the induction of a high anti-HBsAg titer showed a strong T cell response after the third vaccination.Surprisingly, these individuals showed response even before the first vaccination.T cell response to control antigens and mitogens was similar in all groups.CONCLUSION: Our data suggest that there is no general immune deficiency in non-/low-responders.Thus,we hypothesize that the induction of anti-HBsAg responses by vaccination is significantly dependent on the pre-existing T cell repertoire against the specific antigen rather than the presence of a general T cell defect.

  8. Identification of Bacterial Surface Antigens by Screening Peptide Phage Libraries Using Whole Bacteria Cell-Purified Antisera

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    Hu, Yun-Fei; Zhao, Dun; Yu, Xing-Long; Hu, Yu-Li; Li, Run-Cheng; Ge, Meng; Xu, Tian-Qi; Liu, Xiao-Bo; Liao, Hua-Yuan

    2017-01-01

    Bacterial surface proteins can be good vaccine candidates. In the present study, we used polyclonal antibodies purified with intact Erysipelothrix rhusiopthiae to screen phage-displayed random dodecapeptide and loop-constrained heptapeptide libraries, which led to the identification of mimotopes. Homology search of the mimotope sequences against E. rhusiopthiae-encoded ORF sequences revealed 14 new antigens that may localize on the surface of E. rhusiopthiae. When these putative surface proteins were used to immunize mice, 9/11 antigens induced protective immunity. Thus, we have demonstrated that a combination of using the whole bacterial cells to purify antibodies and using the phage-displayed peptide libraries to determine the antigen specificities of the antibodies can lead to the discovery of novel bacterial surface antigens. This can be a general approach for identifying surface antigens for other bacterial species. PMID:28184219

  9. Quantifying cell binding kinetics mediated by surface-bound blood type B antigen to immobilized antibodies

    Institute of Scientific and Technical Information of China (English)

    LI BaoXia; CHEN Juan; LONG Mian

    2008-01-01

    Cell adhesion is crucial to many biological processes, such as inflammatory responses, tumor metastasis and thrombosis formation. Recently a commercial surface plasmon resonance (SPR)-based BIAcore biosensor has been extended to determine cell binding mediated by surface-bound biomolecular interactions. How such cell binding is quantitatively governed by kinetic rates and regulating factors, however, has been poorly understood. Here we developed a novel assay to determine the binding kinetics of surface-bound biomolecular interactions using a commercial BIAcore 3000 biosensor. Human red blood cells (RBCs) presenting blood group B antigen and CM5 chip bearing immobilized anti-B monoclonal antibody (mAb) were used to obtain the time courses of response unit, or sensorgrams, when flowing RBCs over the chip surface. A cellular kinetic model was proposed to correlate the sensorgrams with kinetic rates. Impacts of regulating factors, such as cell concentration,flow duration and rate, antibody-presenting level, as well as Ph value and osmotic pressure of suspending medium were tested systematically, which imparted the confidence that the approach can be applied to kinetic measurements of cell adhesion mediated by surface-bound biomolecular interactions.These results provided a new insight into quantifying cell binding using a commercial SPR-based BIAcore biosensor.

  10. Impaired cell surface expression of HLA-B antigens on mesenchymal stem cells and muscle cell progenitors

    DEFF Research Database (Denmark)

    Isa, Adiba; Nehlin, Jan; Sabir, Hardee Jawad;

    2010-01-01

    HLA class-I expression is weak in embryonic stem cells but increases rapidly during lineage progression. It is unknown whether all three classical HLA class-I antigens follow the same developmental program. In the present study, we investigated allele-specific expression of HLA-A, -B, and -C...... at the mRNA and protein levels on human mesenchymal stem cells from bone marrow and adipose tissue as well as striated muscle satellite cells and lymphocytes. Using multicolour flow cytometry, we found high cell surface expression of HLA-A on all stem cells and PBMC examined. Surprisingly, HLA-B was either...... undetectable or very weakly expressed on all stem cells protecting them from complement-dependent cytotoxicity (CDC) using relevant human anti-B and anti-Cw sera. IFNgamma stimulation for 48-72 h was required to induce full HLA-B protein expression. Quantitative real-time RT-PCR showed that IFNgamma induced...

  11. Antigen 43-mediated autotransporter display, a versatile bacterial cell surface presentation system

    DEFF Research Database (Denmark)

    Kjærgaard, Kristian; Hasman, Henrik; Schembri, Mark;

    2002-01-01

    Antigen 43 (Ag43), a self-recognizing outer membrane protein of Escherichia coli, has been converted into an efficient and versatile tool for surface display of foreign protein segments. Ag43 is an autotransporter protein characterized by the feature that all information required for transport...... to the outer membrane and secretion through the cell envelope is contained within the protein itself. Ag43 consists of two subunits (alpha and beta), where the beta-subunit forms an integral outer membrane translocator to which the alpha-subunit is noncovalently attached. The simplicity of the Ag43 system...

  12. [The surface glycolipid antigen specific for the internal cell mass of the mouse blastocyst and of the stem cells of murine teratocarcinoma F9].

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    Anfimova, M L; Bannikov, G A; Troianovskiĭ, S M

    1989-01-01

    A new monoclonal antibody that recognizes a new antigen on the surface of mouse teratocarcinoma F9 stem cells has been described. This antigen is a glycolipid as demonstrated by inhibition of immunofluorescence by different monosaccharides, glycoproteins and glycolipid fraction of F9 cells as well as by chemical analysis. Immunofluorescent staining of in vitro cultivated preimplantation mouse embryos has demonstrated that this antigen is specific only of internal cell mass cells of late blastocyst.

  13. Adhesion molecule expression stimulated by Bacteroides thetaiotaomicron cell-surface antigens.

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    Rokosz, A; Meisel-Mikołajczyk, F; Malchar, C; Nowaczyk, M; Górski, A

    1999-01-01

    Bacteroides thetaiotaomicron, a Gram-negative anaerobic rod belonging to the Bacteroides fragilis group (BFG), is involved in many systemic and local, most frequently suppurative infections in man. The cell envelope of these rods is composed of two carbohydrate-containing antigens: lipopolysaccharide (LPS) and capsular polysaccharide (CPS). Adhesion molecules ICAM-1, VCAM-1 and E-selectin (ELAM-1) are induced on the endothelial cells by mediators of inflammation. The aim of this study was to assay the ability of B. thetaiotaomicron surface antigens to induce adhesion molecule expression on the endothelial cells. The influence of LPS and CPS on the expression of adhesion molecules on HMEC-1 cell line was examined in an ELISA test. ELISA was performed with monoclonal mouse anti-human: ICAM-1, VCAM-1 and E-selectin antibodies of the IgG class. B. thetaiotaomicron lipopolysaccharides revealed the ability to induce ICAM-1, VCAM-1 and E-selectin expression on the endothelial cells. Their activities were similar, but lower than the activity of Eschericha coli LPS. ICAM-1 was the most stimulated adhesion molecule. The strongest activation by LPS was achieved at the concentrations of 10.0 and 1.0 micrograms/ml. The ability of capsular polysaccharide to induce the expression of adhesion molecules was considerably weaker.

  14. Enhanced cell disruption strategy in the release of recombinant hepatitis B surface antigen from Pichia pastoris using response surface methodology

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    Tam Yew

    2012-10-01

    Full Text Available Abstract Background Cell disruption strategies by high pressure homogenizer for the release of recombinant Hepatitis B surface antigen (HBsAg from Pichia pastoris expression cells were optimized using response surface methodology (RSM based on the central composite design (CCD. The factors studied include number of passes, biomass concentration and pulse pressure. Polynomial models were used to correlate the above mentioned factors to project the cell disruption capability and specific protein release of HBsAg from P. pastoris cells. Results The proposed cell disruption strategy consisted of a number of passes set at 20 times, biomass concentration of 7.70 g/L of dry cell weight (DCW and pulse pressure at 1,029 bar. The optimized cell disruption strategy was shown to increase cell disruption efficiency by 2-fold and 4-fold for specific protein release of HBsAg when compared to glass bead method yielding 75.68% cell disruption rate (CDR and HBsAg concentration of 29.20 mg/L respectively. Conclusions The model equation generated from RSM on cell disruption of P. pastoris was found adequate to determine the significant factors and its interactions among the process variables and the optimum conditions in releasing HBsAg when validated against a glass bead cell disruption method. The findings from the study can open up a promising strategy for better recovery of HBsAg recombinant protein during downstream processing.

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

    DEFF Research Database (Denmark)

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

    2006-01-01

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

  16. Proteasomal targeting and minigene repetition improve cell-surface presentation of a transfected, modified melanoma tumour antigen

    DEFF Research Database (Denmark)

    Rasmussen, A B; Zocca, M-B; Bonefeld, C M;

    2004-01-01

    Melanoma antigen recognized by T cell 1 (MART-1) is regarded as a candidate peptide for vaccination against malignant melanoma, and it is of importance to develop strategies to improve the vaccine-elicited T-cell activation towards MART-1. T-cell activation is, among other determinants, dependent......-surface presentation of the epitope and propose both these approaches as potential strategies in DNA vaccines to increase MART-1-specific T-cell activation.......Melanoma antigen recognized by T cell 1 (MART-1) is regarded as a candidate peptide for vaccination against malignant melanoma, and it is of importance to develop strategies to improve the vaccine-elicited T-cell activation towards MART-1. T-cell activation is, among other determinants, dependent...... on the density of specific major histocompatibility complex-peptide complexes on the surface of the antigen-presenting cell. In this study, we explored the cell-surface presentation of a substituted MART-1 peptide encoded by transfected minigenes. We investigated the potential of proteasomal targeting compared...

  17. Flow-induced detachment of red blood cells adhering to surfaces by specific antigen-antibody bonds.

    OpenAIRE

    Xia, Z; Goldsmith, H L; van de Ven, T G

    1994-01-01

    Fixed spherical swollen human red blood cells of blood type B adhering on a glass surface through antigen-antibody bonds to monoclonal mouse antihuman IgM, adsorbed or covalently linked on the surface, were detached by known hydrodynamic forces created in an impinging jet. The dynamic process of detachment of the specifically bound cells was recorded and analyzed. The fraction of adherent cells remaining on the surface decreased with increasing hydrodynamic force. For an IgM coverage of 0.26%...

  18. Functional Specialty of CD40 and Dendritic Cell Surface Lectins for Exogenous Antigen Presentation to CD8(+) and CD4(+) T Cells.

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    Yin, Wenjie; Gorvel, Laurent; Zurawski, Sandra; Li, Dapeng; Ni, Ling; Duluc, Dorothée; Upchurch, Katherine; Kim, JongRok; Gu, Chao; Ouedraogo, Richard; Wang, Zhiqing; Xue, Yaming; Joo, HyeMee; Gorvel, Jean-Pierre; Zurawski, Gerard; Oh, SangKon

    2016-03-01

    Dendritic cells (DCs) are major antigen-presenting cells that can efficiently prime and cross-prime antigen-specific T cells. Delivering antigen to DCs via surface receptors is thus an appealing strategy to evoke cellular immunity. Nonetheless, which DC surface receptor to target to yield the optimal CD8(+) and CD4(+) T cell responses remains elusive. Herein, we report the superiority of CD40 over 9 different lectins and scavenger receptors at evoking antigen-specific CD8(+) T cell responses. However, lectins (e.g., LOX-1 and Dectin-1) were more efficient than CD40 at eliciting CD4(+) T cell responses. Common and distinct patterns of subcellular and intracellular localization of receptor-bound αCD40, αLOX-1 and αDectin-1 further support their functional specialization at enhancing antigen presentation to either CD8(+) or CD4(+) T cells. Lastly, we demonstrate that antigen targeting to CD40 can evoke potent antigen-specific CD8(+) T cell responses in human CD40 transgenic mice. This study provides fundamental information for the rational design of vaccines against cancers and viral infections.

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

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

    2015-05-01

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

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

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    Aneel Paulus

    Full Text Available Waldenströms macroglobulinemia (WM is a subtype of Non-Hodgkin's lymphoma in which the tumor cell population is markedly heterogeneous, consisting of immunoglobulin-M secreting B-lymphocytes, plasmacytoid lymphocytes and plasma cells. Due to rarity of disease and scarcity of reliable preclinical models, many facets of WM molecular and phenotypic architecture remain incompletely understood. Currently, there are 3 human WM cell lines that are routinely used in experimental studies, namely, BCWM.1, MWCL-1 and RPCI-WM1. During establishment of RPCI-WM1, we observed loss of the CD19 and CD20 antigens, which are typically present on WM cells. Intrigued by this observation and in an effort to better define the immunophenotypic makeup of this cell line, we conducted a more comprehensive analysis for the presence or absence of other cell surface antigens that are present on the RPCI-WM1 model, as well as those on the two other WM cell lines, BCWM.1 and MWCL-1. We examined expression of 65 extracellular and 4 intracellular antigens, comprising B-cell, plasma cell, T-cell, NK-cell, myeloid and hematopoietic stem cell surface markers by flow cytometry analysis. RPCI-WM1 cells demonstrated decreased expression of CD19, CD20, and CD23 with enhanced expression of CD28, CD38 and CD184, antigens that were differentially expressed on BCWM.1 and MWCL-1 cells. Due to increased expression of CD184/CXCR4 and CD38, RPCI-WM1 represents a valuable model in which to study the effects anti-CXCR4 or anti-CD38 targeted therapies that are actively being developed for treatment of hematologic cancers. Overall, differences in surface antigen expression across the 3 cell lines may reflect the tumor clone population predominant in the index patients, from whom the cell lines were developed. Our analysis defines the utility of the most commonly employed WM cell lines as based on their immunophenotype profiles, highlighting unique differences that can be further studied for

  1. Epitope distance to the target cell membrane and antigen size determine the potency of T cell-mediated lysis by BiTE antibodies specific for a large melanoma surface antigen.

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    Bluemel, Claudia; Hausmann, Susanne; Fluhr, Petra; Sriskandarajah, Mirnalini; Stallcup, William B; Baeuerle, Patrick A; Kufer, Peter

    2010-08-01

    Melanoma chondroitin sulfate proteoglycan (MCSP; also called CSPG4, NG2, HMW-MAA, MSK16, MCSPG, MEL-CSPG, or gp240) is a surface antigen frequently expressed on human melanoma cells, which is involved in cell adhesion, invasion and spreading, angiogenesis, complement inhibition, and signaling. MCSP has therefore been frequently selected as target antigen for development of antibody- and vaccine-based therapeutic approaches. We have here used a large panel of monoclonal antibodies against human MCSP for generation of single-chain MCSP/CD3-bispecific antibodies of the BiTE (for bispecific T cell engager) class. Despite similar binding affinity to MCSP, respective BiTE antibodies greatly differed in their potency of redirected lysis of CHO cells stably transfected with full-length human MCSP, or with various MCSP deletion mutants and fusion proteins. BiTE antibodies binding to the membrane proximal domain D3 of MCSP were more potent than those binding to more distal domains. This epitope distance effect was corroborated with EpCAM/CD3-bispecific BiTE antibody MT110 by testing various fusion proteins between MCSP and EpCAM as surface antigens. CHO cells expressing small surface target antigens were generally better lysed than those expressing larger target antigens, indicating that antigen size was also an important determinant for the potency of BiTE antibody. The present study for the first time relates the positioning of binding domains and size of surface antigens to the potency of target cell lysis by BiTE-redirected cytotoxic T cells. In case of the MCSP antigen, this provides the basis for selection of a maximally potent BiTE antibody candidate for development of a novel melanoma therapy.

  2. Antigen processing of glycoconjugate vaccines; the polysaccharide portion of the pneumococcal CRM(197) conjugate vaccine co-localizes with MHC II on the antigen processing cell surface.

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    Lai, Zengzu; Schreiber, John R

    2009-05-21

    Pneumococcal (Pn) polysaccharides (PS) are T-independent (TI) antigens and do not induce immunological memory or antibodies in infants. Conjugation of PnPS to the carrier protein CRM(197) induces PS-specific antibody in infants, and memory similar to T-dependent (Td) antigens. Conjugates have improved immunogenicity via antigen processing and presentation of carrier protein with MHC II and recruitment of T cell help, but the fate of the PS attached to the carrier is unknown. To determine the location of the PS component of PnPS-CRM(197) in the APC, we separately labeled PS and protein and tracked their location. The PS of types 14-CRM(197) and 19F-CRM(197) was specifically labeled by Alexa Fluor 594 hydrazide (red). The CRM(197) was separately labeled red in a reaction that did not label PS. Labeled antigens were incubated with APC which were fixed, permeabilized and incubated with anti-MHC II antibody labeled green by Alexa Fluor 488, followed by confocal microscopy. Labeled CRM(197) was presented on APC surface and co-localized with MHC II (yellow). Labeled unconjugated 14 or 19F PS did not go to the APC surface, but PS labeled 14-CRM(197) and 19F-CRM(197) was internalized and co-localized with MHC II. Monoclonal antibody to type 14 PS bound to intracellular type 14 PS and PS-CRM(197). Brefeldin A and chloroquine blocked both CRM(197) and PS labeled 14-CRM(197) and 19F-CRM(197) from co-localizing with MHC II. These data suggest that the PS component of the CRM(197) glycoconjugate enters the endosome, travels with CRM(197) peptides to the APC surface and co-localizes with MHC II.

  3. Structural evidence for evolution of shark Ig new antigen receptor variable domain antibodies from a cell-surface receptor.

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    Streltsov, V A; Varghese, J N; Carmichael, J A; Irving, R A; Hudson, P J; Nuttall, S D

    2004-08-24

    The Ig new antigen receptors (IgNARs) are single-domain antibodies found in the serum of sharks. Here, we report 2.2- and 2.8-A structures of the type 2 IgNAR variable domains 12Y-1 and 12Y-2. Structural features include, first, an Ig superfamily topology transitional between cell adhesion molecules, antibodies, and T cell receptors; and, second, a vestigial complementarity-determining region 2 at the "bottom" of the molecule, apparently discontinuous from the antigen-binding paratope and similar to that observed in cell adhesion molecules. Thus, we suggest that IgNARs originated as cell-surface adhesion molecules coopted to the immune repertoire and represent an evolutionary lineage independent of variable heavy chain/variable light chain type antibodies. Additionally, both 12Y-1 and 12Y-2 form unique crystallographic dimers, predominantly mediated by main-chain framework interactions, which represent a possible model for primordial cell-based interactions. Unusually, the 12Y-2 complementarity-determining region 3 also adopts an extended beta-hairpin structure, suggesting a distinct selective advantage in accessing cryptic antigenic epitopes.

  4. Immunity to intracellular Salmonella depends on surface-associated antigens.

    Directory of Open Access Journals (Sweden)

    Somedutta Barat

    Full Text Available Invasive Salmonella infection is an important health problem that is worsening because of rising antimicrobial resistance and changing Salmonella serovar spectrum. Novel vaccines with broad serovar coverage are needed, but suitable protective antigens remain largely unknown. Here, we tested 37 broadly conserved Salmonella antigens in a mouse typhoid fever model, and identified antigen candidates that conferred partial protection against lethal disease. Antigen properties such as high in vivo abundance or immunodominance in convalescent individuals were not required for protectivity, but all promising antigen candidates were associated with the Salmonella surface. Surprisingly, this was not due to superior immunogenicity of surface antigens compared to internal antigens as had been suggested by previous studies and novel findings for CD4 T cell responses to model antigens. Confocal microscopy of infected tissues revealed that many live Salmonella resided alone in infected host macrophages with no damaged Salmonella releasing internal antigens in their vicinity. In the absence of accessible internal antigens, detection of these infected cells might require CD4 T cell recognition of Salmonella surface-associated antigens that could be processed and presented even from intact Salmonella. In conclusion, our findings might pave the way for development of an efficacious Salmonella vaccine with broad serovar coverage, and suggest a similar crucial role of surface antigens for immunity to both extracellular and intracellular pathogens.

  5. Combined cell surface carbonic anhydrase 9 and CD147 antigens enable high-efficiency capture of circulating tumor cells in clear cell renal cell carcinoma patients.

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    Liu, Shijie; Tian, Zuhong; Zhang, Lei; Hou, Shuang; Hu, Sijun; Wu, Junshen; Jing, Yuming; Sun, Huimin; Yu, Fei; Zhao, Libo; Wang, Ruoxiang; Tseng, Hsian-Rong; Zhau, Haiyen E; Chung, Leland W K; Wu, Kaichun; Wang, Hao; Wu, Jason Boyang; Nie, Yongzhan; Shao, Chen

    2016-09-13

    Circulating tumor cells (CTCs) have emerged as promising tools for noninvasive cancer detection and prognosis. Most conventional approaches for capturing CTCs use an EpCAM-based enrichment strategy, which does not work well in cancers that show low or no expression of EpCAM, such as renal cell carcinoma (RCC). In this study, we developed a new set of cell surface markers including CA9 and CD147 as alternative CTC-capture antigens specifically designed for RCC patients. We showed that the expression of both CA9 and CD147 was prevalent in a RCC patient cohort (n=70) by immunohistochemical analysis, with both molecules in combination covering 97.1% of cases. The NanoVelcro platform combined with CA9-/CD147-capture antibodies demonstrated significantly higher efficiency for capturing both CTC-mimicking renal cancer cells and RCC CTCs in peripheral blood, compared to the conventional EpCAM-based method. Using immunofluorescence cytological validation at the single-cell level, we were able to identify bona fide CTCs in RCC patient blood following the well-accepted criteria in our CTC-capture system. We further demonstrated a significant association of CTC numbers as well as the CTC expression status of Vimentin, a mesenchymal marker, with disease progression, including pathologic features and clinical staging. These results provide new insights into developing novel, effective targets/approaches for capturing CTCs, making CTCs a valuable tool for improved cancer detection, prognosis and treatment in RCC.

  6. Ionizing radiation modulates the surface expression of human leukocyte antigen-G in a human melanoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Michelin, S.; Gallegos, C.E.; Dubner, D. [Radiopathology Laboratory, Nuclear Regulatory Authority, Buenos Aires (Argentina); Favier, B.; Carosella, E.D. [CEA, I2BM, Hopital Saint-Louis, IUH, Service de Recherches en Hemato-Immunologie, Paris (France)

    2009-07-01

    Human leukocyte antigen G (HLA-G) is a nonclassical HLA class I molecule involved in fetus protection from the maternal immune system, transplant tolerance, and viral and tumoral immune escape. Tumor-specific HLA-G expression has been described for a wide variety of malignancies, including melanomas. The aim of this study was to evaluate whether ionizing radiation (IR) could modulate the surface expression of HLA-G1 in a human melanoma cell line that expresses endogenously membrane-bound HLA-G1. For this purpose, cells were exposed to increasing doses of {gamma}-irradiation (0-20 Gy) and HLA-G1 levels at the plasma membrane were analyzed at different times postirradiation by flow cytometry. HLA-G total expression and the presence of the soluble form of HLA-G1 (sHLA-G1) in the culture medium of irradiated cells were also evaluated. IR was capable of down regulating cell surface and total HLA-G levels, with a concomitant increase of sHLA-G1 in the medium. These results could indicate that {gamma}-irradiation decreases HLA-G1 surface levels by enhancing the proteolytic cleavage of this molecule. (authors)

  7. Quantum dots affect expression of CD133 surface antigen in melanoma cells

    Directory of Open Access Journals (Sweden)

    Steponkiene S

    2011-10-01

    Full Text Available Simona Steponkiene1-3, Simona Kavaliauskiene1, Rasa Purviniene4, Ricardas Rotomskis3,5, Petras Juzenas11Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, Radiumhospital, Oslo, Norway; 2Faculty of Natural Sciences, Vilnius University, Vilnius, Lithuania; 3Biomedical Physics Laboratory of Oncology Institute, Vilnius University, Vilnius, Lithuania; 4Immunology Laboratory of Oncology Institute, Vilnius University, Vilnius, Lithuania; 5Biophotonics Laboratory, Laser Research Center, Vilnius University, Vilnius, LithuaniaBackground: In novel treatment approaches, therapeutics should be designed to target cancer stem cells (CSCs. Quantum dots (QDs are a promising new tool in fighting against cancer. However, little is known about accumulation and cytotoxicity of QDs in CSCs.Methods: Accumulation and cytotoxicity of CdTe-MPA (mercaptopropionic acid QDs in CSCs were assessed using flow cytometry and fluorescence-activated cell sorting techniques as well as a colorimetric cell viability assay.Results: We investigated the expression of two cell surface-associated glycoproteins, CD44 and CD133, in four different cancer cell lines (glioblastoma, melanoma, pancreatic, and prostate adenocarcinoma. Only the melanoma cells were positive to both markers of CD44 and CD133, whereas the other cells were only CD44-positive. The QDs accumulated to a similar extent in all subpopulations of the melanoma cells. The phenotypical response after QD treatment was compared with the response after ionizing radiation treatment. The percentage of the CD44high-CD133high subpopulation decreased from 72% to 55%–58% for both treatments. The stem-like subpopulation CD44highCD133low/- increased from 26%–28% in the untreated melanoma cells to 36%–40% for both treatments.Conclusion: Treatment of melanoma cells with QDs results in an increase of stem-like cell subpopulations. The changes in phenotype distribution of the melanoma cells after

  8. Identification of ATP synthase beta subunit (ATPB on the cell surface as a non-small cell lung cancer (NSCLC associated antigen

    Directory of Open Access Journals (Sweden)

    Qian Zhi

    2009-01-01

    Full Text Available Abstract Background Antibody-based immuneotherapy has achieved some success for cancer. But the main problem is that only a few tumor-associated antigens or therapeutic targets have been known to us so far. It is essential to identify more immunogenic antigens (especially cellular membrane markers for tumor diagnosis and therapy. Methods The membrane proteins of lung adenocarcinoma cell line A549 were used to immunize the BALB/c mice. A monoclonal antibody 4E7 (McAb4E7 was produced with hybridoma technique. MTT cell proliferation assay was carried out to evaluate the inhibitory effect of McAb4E7 on A549 cells. Flow cytometric assay, immunohistochemistry, western blot and proteomic technologies based on 2-DE and mass spectrometry were employed to detect and identify the corresponding antigen of McAb4E7. Results The monoclonal antibody 4E7 (McAb4E7 specific against A549 cells was produced, which exhibited inhibitory effect on the proliferation of A549 cells. By the proteomic technologies, we identified that ATP synthase beta subunit (ATPB was the corresponding antigen of McAb4E7. Then, flow cytometric analysis demonstrated the localization of the targeting antigen of McAb4E7 was on the A549 cells surface. Furthermore, immunohistochemstry showed that the antigen of McAb4E7 mainly aberrantly expressed in tumor cellular membrane in non-small cell lung cancer (NSCLC, but not in small cell lung cancer (SCLC. The rate of ectopic expressed ATPB in the cellular membrane in lung adenocarcinoma, squamous carcinoma and their adjacent nontumourous lung tissues was 71.88%, 66.67% and 25.81% respectively. Conclusion In the present study, we identified that the ectopic ATPB in tumor cellular membrane was the non-small cell lung cancer (NSCLC associated antigen. ATPB may be a potential biomarker and therapeutic target for the immunotherapy of NSCLC.

  9. Interferon-alpha-induced changes in surface antigens in a hairy-cell leukemia (JOK-1), and a Burkitt's lymphoma cell line (Daudi) during in vitro culture

    DEFF Research Database (Denmark)

    Nielsen, B; Madsen, P S; Jensen, A W

    1992-01-01

    In further studying the mechanism of action of IFN-alpha in HCL, we cultured the HCL cell line JOK-1 and the IFN-sensitive Burkitt cell line Daudi with and without IFN-alpha and investigated the changes in density of a number of surface antigens by use of mAb and flow cytometry analyses. During...... culture with IFN-alpha, reproducible changes were induced in both cell lines, which were qualitatively similar but differed quantitatively with small and transient changes in JOK-1. Significant decreases in surface antigen expression were observed for CD 19, 23, 37, and for IgM on both cell lines....... Moreover, decreases were seen for CD 10, 22, 45, and MHC class II on Daudi, and for CD 20, 21, 27, and 40 on JOK-1. By contrast, only a few antigens increased in density, including CD 39, A96/G8 and SC9, on both cell lines, CD 22 on JOK-1, and CD 21 on Daudi. The increase in CD 39, A96/G8 and SC9...

  10. Generation of a phage-display library of single-domain camelid VH H antibodies directed against Chlamydomonas reinhardtii antigens, and characterization of VH Hs binding cell-surface antigens.

    Science.gov (United States)

    Jiang, Wenzhi; Rosenberg, Julian N; Wauchope, Akelia D; Tremblay, Jacqueline M; Shoemaker, Charles B; Weeks, Donald P; Oyler, George A

    2013-11-01

    Single-domain antibodies (sdAbs) are powerful tools for the detection, quantification, purification and subcellular localization of proteins of interest in biological research. We have generated camelid (Lama pacos) heavy chain-only variable VH domain (VH H) libraries against antigens in total cell lysates from Chlamydomonas reinhardtii. The sdAbs in the sera from immunized animals and VH H antibody domains isolated from the library show specificity to C. reinhardtii and lack of reactivity to antigens from four other algae: Chlorella variabilis, Coccomyxa subellipsoidea, Nannochloropsis oceanica and Thalassiosira pseudonana. Antibodies were produced against a diverse representation of antigens as evidenced by sera ELISA and protein-blot analyses. A phage-display library consisting of the VH H region contained at least 10(6) individual transformants, and thus should represent a wide range of C. reinhardtii antigens. The utility of the phage library was demonstrated by using live C. reinhardtii cells to pan for VH H clones with specific recognition of cell-surface epitopes. The lead candidate VH H clones (designated B11 and H10) bound to C. reinhardtii with EC50 values ≤ 0.5 nm. Treatment of cells with VH H B11 fused to the mCherry or green fluorescent proteins allowed brilliant and specific staining of the C. reinhardtii cell wall and analysis of cell-wall genesis during cell division. Such high-complexity VH H antibody libraries for algae will be valuable tools for algal researchers and biotechnologists.

  11. Fusion to green fluorescent protein improves expression levels of Theileria parva sporozoite surface antigen p67 in insect cells

    NARCIS (Netherlands)

    Kaba, S.A.; Nene, V.; Musoke, A.J.; Vlak, J.M.; Oers, van M.M.

    2002-01-01

    East Coast fever (ECF) is a fatal disease of cattle caused by the protozoan parasite Theileria parva. The development of a subunit vaccine, based on the sporozoite-specific surface antigen p67, has been hampered by difficulties in achieving high-level expression of recombinant p67 in a near-authenti

  12. Surface antigens of metacyclic trypomastigotes of Trypanosoma cruzi.

    OpenAIRE

    1983-01-01

    The surface antigen makeup of metacyclic trypomastigote forms of strain G of Trypanosoma cruzi, which produce a subpatent infection in mice, differed from those of the virulent strains Y and CL. A 100,000-molecular-weight protein, barely detectable on the Y or CL cell surface, appeared as the main surface antigen of the G metacyclic trypomastigotes. In addition, the G metacyclic forms differed from those of the virulent strains in their susceptibility to complement-mediated immunolysis.

  13. The crystal structure of Haloferax volcanii proliferating cell nuclear antigen reveals unique surface charge characteristics due to halophilic adaptation

    Directory of Open Access Journals (Sweden)

    Morroll Shaun

    2009-08-01

    Full Text Available Abstract Background The high intracellular salt concentration required to maintain a halophilic lifestyle poses challenges to haloarchaeal proteins that must stay soluble, stable and functional in this extreme environment. Proliferating cell nuclear antigen (PCNA is a fundamental protein involved in maintaining genome integrity, with roles in both DNA replication and repair. To investigate the halophilic adaptation of such a key protein we have crystallised and solved the structure of Haloferax volcanii PCNA (HvPCNA to a resolution of 2.0 Å. Results The overall architecture of HvPCNA is very similar to other known PCNAs, which are highly structurally conserved. Three commonly observed adaptations in halophilic proteins are higher surface acidity, bound ions and increased numbers of intermolecular ion pairs (in oligomeric proteins. HvPCNA possesses the former two adaptations but not the latter, despite functioning as a homotrimer. Strikingly, the positive surface charge considered key to PCNA's role as a sliding clamp is dramatically reduced in the halophilic protein. Instead, bound cations within the solvation shell of HvPCNA may permit sliding along negatively charged DNA by reducing electrostatic repulsion effects. Conclusion The extent to which individual proteins adapt to halophilic conditions varies, presumably due to their diverse characteristics and roles within the cell. The number of ion pairs observed in the HvPCNA monomer-monomer interface was unexpectedly low. This may reflect the fact that the trimer is intrinsically stable over a wide range of salt concentrations and therefore additional modifications for trimer maintenance in high salt conditions are not required. Halophilic proteins frequently bind anions and cations and in HvPCNA cation binding may compensate for the remarkable reduction in positive charge in the pore region, to facilitate functional interactions with DNA. In this way, HvPCNA may harness its environment as

  14. The herpes virus Fc receptor gE-gI mediates antibody bipolar bridging to clear viral antigens from the cell surface.

    Directory of Open Access Journals (Sweden)

    Blaise Ndjamen

    2014-03-01

    Full Text Available The Herpes Simplex Virus 1 (HSV-1 glycoprotein gE-gI is a transmembrane Fc receptor found on the surface of infected cells and virions that binds human immunoglobulin G (hIgG. gE-gI can also participate in antibody bipolar bridging (ABB, a process by which the antigen-binding fragments (Fabs of the IgG bind a viral antigen while the Fc binds to gE-gI. IgG Fc binds gE-gI at basic, but not acidic, pH, suggesting that IgG bound at extracellular pH by cell surface gE-gI would dissociate and be degraded in acidic endosomes/lysosomes if endocytosed. The fate of viral antigens associated with gE-gI-bound IgG had been unknown: they could remain at the cell surface or be endocytosed with IgG. Here, we developed an in vitro model system for ABB and investigated the trafficking of ABB complexes using 4-D confocal fluorescence imaging of ABB complexes with transferrin or epidermal growth factor, well-characterized intracellular trafficking markers. Our data showed that cells expressing gE-gI and the viral antigen HSV-1 gD endocytosed anti-gD IgG and gD in a gE-gI-dependent process, resulting in lysosomal localization. These results suggest that gE-gI can mediate clearance of infected cell surfaces of anti-viral host IgG and viral antigens to evade IgG-mediated responses, representing a general mechanism for viral Fc receptors in immune evasion and viral pathogenesis.

  15. The neural cell adhesion molecule L1 is distinct from the N-CAM related group of surface antigens BSP-2 and D2

    DEFF Research Database (Denmark)

    Faissner, A; Kruse, J; Goridis, C

    1984-01-01

    The neural cell adhesion molecule L1 and the group of N-CAM related molecules, BSP-2 and D2 antigen, are immunochemically distinct molecular species. The two groups of surface molecules are also functionally distinct entities, since inhibition of Ca2+-independent adhesion among early post-natal m......-natal mouse cerebellar cells by Fab fragments of both antibodies are at least additive, when compared with equal concentrations of the individual antibodies....

  16. Stem Cell Physics. Laser Manipulation of Blood Types: Laser-Stripping-Away of Red Blood Cell Surface Antigens

    Science.gov (United States)

    Stefan, V. Alexander

    2014-03-01

    A novel mechanism of importance for the transfusion medicine[2] is proposed. The interaction of ultrashort wavelength multilaser beams with the flowing blood thin films can lead to a conversion of blood types A, B, and AB into O type.[3] The stripping away of antigens is done by the scanning-multiple-lasers of a high repetition rate in the blue-purple frequency domain. The guiding-lasers are in the red-green frequency domain. The laser force, (parametric interaction with the antigen eigen-oscillation),[4] upon the antigen protein molecule must exceed its weight. Supported by Nikola Tesla Labs, La Jolla, CA.

  17. Amplification of Surface Antigen P43 Gene and Its Application in Detection of Toxoplasma Gondii in Allogeneic Hematopoietic Stem Cell Transplantation

    Institute of Scientific and Technical Information of China (English)

    ZHOUYongan; YUXinbing; 等

    2002-01-01

    Objective:To establish a rapid,specific and sensitive diagnostic technique for the human Toxoplasma gondii infection in the recipi-ents with allogeneic hematopoietic stem cell transplantation and discuss its clinical significance.Methods:30 patients undergoing allogeneic hematopoietic stem cell transplantation were detected by using ELISA and PCR.Results:Among 30 recipients undergiong allogeneic hematopoietic stem cell transplantation,3 were positive for Toxoplasma gondiii antigen and 5 for surface antigen p43 gene with the positive rate being 13.3% and 16.67% respectively.20 healthy people(negative for anti-Tox antibody)were also tested by using ELISA and PCR.Conclusion:PCR is an accurate,relatively rapid,sensitive and specific method for detecting P43 gene of Toxoplasma gondii.Be-canuse PCR can be applied to a variety of different clinical samples,it can be considered as a valuable additional tool for identification of Toxoplasma gondii infections.

  18. Phenotypic modulation of chronic lymphocytic leukemia cells by phorbol ester: induction of IgM secretion and changes in the expression of B cell-associated surface antigens.

    Science.gov (United States)

    Gordon, J; Mellstedt, H; Aman, P; Biberfeld, P; Klein, G

    1984-01-01

    Freshly explanted neoplastic populations from 22 cases of phenotypically well-characterized chronic type B lymphocytic leukemia were studied for their capacity to respond to the phorbol ester TPA in vitro. In all but four cases the secretion of IgM was either induced or increased, often to a high level. In contrast, the export of free immunoglobulin (Ig) light chains, an almost consistent feature of the B lymphocytic leukemias, remained relatively constant after TPA treatment. Parallel changes in leukemic cell surface phenotype were probed with both "conventional" and monoclonal antibodies, revealing some modulation of markers in every case investigated. A diminution in the level of surface Ig (preferentially IgD) and the accumulation of cytoplasmic Ig observed after phorbol ester treatment were accompanied by a corresponding reduction or loss of the B1 antigen and usually of B2 when present. The most consistent change induced by TPA was the appearance of BB-1, a marker of activated B lymphocytes, which was rarely expressed on fresh leukemic cells. Another marker of activated lymphocytes, LB-1, was also often induced or increased in its expression after exposure of the cells to TPA. The magnitude of the TPA response appeared to relate to the stage of maturation arrest of the individual leukemic clones rather than to any clinical parameter explored. The significance of the findings to normal B cell differentiation and their potential clinical utility are discussed.

  19. Mapping antigenic motifs in the trypomastigote small surface antigen from Trypanosoma cruzi.

    Science.gov (United States)

    Balouz, Virginia; Cámara, María de Los Milagros; Cánepa, Gaspar E; Carmona, Santiago J; Volcovich, Romina; Gonzalez, Nicolás; Altcheh, Jaime; Agüero, Fernán; Buscaglia, Carlos A

    2015-03-01

    The trypomastigote small surface antigen (TSSA) is a mucin-like molecule from Trypanosoma cruzi, the etiological agent of Chagas disease, which displays amino acid polymorphisms in parasite isolates. TSSA expression is restricted to the surface of infective cell-derived trypomastigotes, where it functions as an adhesin and engages surface receptors on the host cell as a prerequisite for parasite internalization. Previous results have established TSSA-CL, the isoform encoded by the CL Brener clone, as an appealing candidate for use in serology-based diagnostics for Chagas disease. Here, we used a combination of peptide- and recombinant protein-based tools to map the antigenic structure of TSSA-CL at maximal resolution. Our results indicate the presence of different partially overlapping B-cell epitopes clustering in the central portion of TSSA-CL, which contains most of the polymorphisms found in parasite isolates. Based on these results, we assessed the serodiagnostic performance of a 21-amino-acid-long peptide that spans TSSA-CL major antigenic determinants, which was similar to the performance of the previously validated glutathione S-transferase (GST)-TSSA-CL fusion molecule. Furthermore, the tools developed for the antigenic characterization of the TSSA antigen were also used to explore other potential diagnostic applications of the anti-TSSA humoral response in Chagasic patients. Overall, our present results provide additional insights into the antigenic structure of TSSA-CL and support this molecule as an excellent target for molecular intervention in Chagas disease.

  20. Amino acid sequences recognized by T cells: studies on a merozoite surface antigen from the FCQ-27/PNG isolate of Plasmodium falciparum.

    Science.gov (United States)

    Rzepczyk, C M; Csurhes, P A; Baxter, E P; Doran, T J; Irving, D O; Kere, N

    1990-08-01

    Twenty-six overlapping peptides, spanning the entire FCQ-27/PNG sequence of the Plasmodium falciparum antigen known as merozoite surface antigen 2 were screened for their ability to induce the proliferation of peripheral blood lymphocytes (PBL) obtained from 12 donors living in Honiara, Solomon Islands where P. falciparum is endemic. A recombinant (r) form of MSA2, known as Ag 1609 was also screened in these assays and tetanus toxoid (TT) antigen was included as a control. The location of the predicted T cell determinants within MSA2 was examined using the algorithm, AMPHI and by scanning MSA2 for amino acid sequences showing the Rothbard motif. There were 13 predicted amphipathic helical sites and five examples of Rothbard sequences in the antigen. The location of these with regard to the peptides tested is shown. Nine of the 12 individuals responded to TT with high stimulation indices (greater than 4) being obtained in the majority of donors. Only three individuals responded to r-MSA2 with the stimulation indices (SI) in the range of 2.4-4.1. Peptides from both the constant and variable regions of MSA2 were recognized in the proliferative assays. However, the majority of the positive proliferative responses were to peptides which spanned the central variable region which included the two copies of the 32-amino-acid repeat occurring in the antigen. High SI comparable to those obtained to TT were seen in some individuals with some peptides. There was considerable variation between donors in number and nature of the peptides recognised and two donors did not respond to any of the antigens tested. The significance of these findings to vaccine development is discussed.

  1. Curcumin induced nanoscale CD44 molecular redistribution and antigen-antibody interaction on HepG2 cell surface

    Energy Technology Data Exchange (ETDEWEB)

    Wang Mu [Department of Chemistry, Jinan University, 601 Huangpu Road West, Tianhe District, Guangzhou 510632 (China); Ruan Yuxia [Department of Ophthalmology, The First Affiliated Hospital, Jinan University, 601 Huangpu Road West, Tianhe District, Guangzhou 510632 (China); Xing Xiaobo; Chen Qian; Peng, Yuan [Department of Chemistry, Jinan University, 601 Huangpu Road West, Tianhe District, Guangzhou 510632 (China); Cai Jiye, E-mail: tjycai@jnu.edu.cn [Department of Chemistry, Jinan University, 601 Huangpu Road West, Tianhe District, Guangzhou 510632 (China)

    2011-07-04

    Graphical abstract: Highlights: > In this study, we investigate the changes of CD44 expression and distribution on HepG2 cells after curcumin treatment. > We find curcumin is able to change the morphology and ultrastructure of HepG2 cells. > Curcumin can reduce the expression of CD44 molecules and induce the nanoscale molecular redistribution on cell surface. > The binding force between CD44-modified AFM tip and the HepG2 cell surface decreases after curcumin-treatment. - Abstract: The cell surface glycoprotein CD44 was implicated in the progression, metastasis and apoptosis of certain human tumors. In this study, we used atomic force microscope (AFM) to monitor the effect of curcumin on human hepatocellular carcinoma (HepG2) cell surface nanoscale structure. High-resolution imaging revealed that cell morphology and ultrastructure changed a lot after being treated with curcumin. The membrane average roughness increased (10.88 {+-} 4.62 nm to 129.70 {+-} 43.72 nm) and the expression of CD44 decreased (99.79 {+-} 0.16% to 75.14 {+-} 8.37%). Laser scanning confocal microscope (LSCM) imaging showed that CD44 molecules were located on the cell membrane. The florescence intensity in control group was weaker than that in curcumin treated cells. Most of the binding forces between CD44 antibodies and untreated HepG2 cell membrane were around 120-220 pN. After being incubated with curcumin, the major forces focused on 70-150 pN (10 {mu}M curcumin-treated) and 50-120 pN (20 {mu}M curcumin-treated). These results suggested that, as result of nanoscale molecular redistribution, changes of the cell surface were in response to external treatment of curcumin. The combination of AFM and LSCM could be a powerful method to detect the distribution of cell surface molecules and interactions between molecules and their ligands.

  2. An immunodominant HLA-A*1101-restricted CD8+ T-cell response targeting hepatitis B surface antigen in chronic hepatitis B patients.

    Science.gov (United States)

    Chen, Xiaoling; Wang, Wenbo; Wang, Shufeng; Meng, Gang; Zhang, Mengjun; Ni, Bing; Wu, Yuzhang; Wang, Li

    2013-12-01

    Hepatitis B virus (HBV) infection is a worldwide public health problem. HBV-specific CD8(+) CTLs are vital for viral clearance. Identification of immunodominant CTL epitopes from HBV-associated antigens is necessary for therapeutic vaccine development. We showed that the HLA-A*1101 allele is one of the most common alleles in both healthy individuals and chronic hepatitis B (CHB) patients in the Chongqing area, China. However, less than 10% of epitopes of HBV-associated antigens have been identified in an HLA-A*1101 context. Here, we describe an immunodominant CD8(+) T-cell response targeting a hepatitis B surface antigen determinant (HBs(295-304)) restricted by HLA-A*1101 in both healthy individuals and CHB patients. Moreover, HBs(295-304) is more immunogenic for CTL induction than a known naturally HLA-A*1101-processed epitope from hepatitis B core antigen (HBc(88-96)). Therefore, the newly identified epitope, HBs(295-304), will benefit the development of immunotherapeutic approaches for HBV infection.

  3. Enrichment and purging of human embryonic stem cells by detection of cell surface antigens using the monoclonal antibodies TG30 and GCTM-2.

    Science.gov (United States)

    Polanco, Juan Carlos; Wang, Bei; Zhou, Qi; Chy, Hun; O'Brien, Carmel; Laslett, Andrew L

    2013-12-06

    Human embryonic stem cells (hESC) can self-renew indefinitely in vitro, and with the appropriate cues can be induced to differentiate into potentially all somatic cell lineages. Differentiated hESC derivatives can potentially be used in transplantation therapies to treat a variety of cell-degenerative diseases. However, hESC differentiation protocols usually yield a mixture of differentiated target and off-target cell types as well as residual undifferentiated cells. For the translation of differentiated hESC-derivatives from the laboratory to the clinic, it is important to be able to discriminate between undifferentiated (pluripotent) and differentiated cells, and generate methods to separate these populations. Safe application of hESC-derived somatic cell types can only be accomplished with pluripotent stem cell-free populations, as residual hESCs could induce tumors known as teratomas following transplantation. Towards this end, here we describe a methodology to detect pluripotency associated cell surface antigens with the monoclonal antibodies TG30 (CD9) and GCTM-2 via fluorescence activated cell sorting (FACS) for the identification of pluripotent TG30(Hi)-GCTM-2(Hi) hESCs using positive selection. Using negative selection with our TG30/GCTM-2 FACS methodology, we were able to detect and purge undifferentiated hESCs in populations undergoing very early-stage differentiation (TG30(Neg)-GCTM-2(Neg)). In a further study, pluripotent stem cell-free samples of differentiated TG30(Neg)-GCTM-2(Neg) cells selected using our TG30/GCTM-2 FACS protocol did not form teratomas once transplanted into immune-compromised mice, supporting the robustness of our protocol. On the other hand, TG30/GCTM-2 FACS-mediated consecutive passaging of enriched pluripotent TG30(Hi)-GCTM-2(Hi) hESCs did not affect their ability to self-renew in vitro or their intrinsic pluripotency. Therefore, the characteristics of our TG30/GCTM-2 FACS methodology provide a sensitive assay to obtain highly

  4. The Leishmania promastigote surface antigen-2 (PSA-2) is specifically recognised by Th1 cells in humans with naturally acquired immunity to L. major

    DEFF Research Database (Denmark)

    Kemp, M; Handman, E; Kemp, K

    1998-01-01

    The promastigote surface antigen-2 (PSA-2) is a Leishmania parasite antigen, which can induce Th1-mediated protection against murine leishmaniasis when used as a vaccine. To evaluate PSA-2 as a human vaccine candidate the specific T-cell response to PSA-2 was characterised in individuals immune...... to cutaneous leishmaniasis. Peripheral blood mononuclear cells from Sudanese individuals with a past history of self-healing cutaneous leishmaniasis proliferated vigorously in response to PSA-2 isolated from Leishmania major, whereas the antigen did not activate cells from presumably unexposed Danes......-specific Th1-like cells, PSA-2 might be considered a vaccine candidate for human leishmaniasis....

  5. HD-03/ES: A Herbal Medicine Inhibits Hepatitis B Surface Antigen Secretion in Transfected Human Hepatocarcinoma PLC/PRF/5 Cells.

    Science.gov (United States)

    Varma, Sandeep R; Sundaram, R; Gopumadhavan, S; Vidyashankar, Satyakumar; Patki, Pralhad S

    2013-01-01

    HD-03/ES is a herbal formulation used for the treatment of hepatitis B. However, the molecular mechanism involved in the antihepatitis B (HBV) activity of this drug has not been studied using in vitro models. The effect of HD-03/ES on hepatitis B surface antigen (HBsAg) secretion and its gene expression was studied in transfected human hepatocarcinoma PLC/PRF/5 cells. The anti-HBV activity was tested based on the inhibition of HBsAg secretion into the culture media, as detected by HBsAg-specific antibody-mediated enzyme assay (ELISA) at concentrations ranging from 125 to 1000  μ g/mL. The effect of HD-03/ES on HBsAg gene expression was analyzed using semiquantitative multiplex RT-PCR by employing specific primers. The results showed that HD-03/ES suppressed HBsAg production with an IC50 of 380  μ g/mL in PLC/PRF/5 cells for a period of 24 h. HD-03/ES downregulated HBsAg gene expression in PLC/PRF/5 cells. In conclusion, HD-03/ES exhibits strong anti-HBV properties by inhibiting the secretion of hepatitis B surface antigen in PLC/PRF/5 cells, and this action is targeted at the transcription level. Thus, HD-03/ES could be beneficial in the treatment of acute and chronic hepatitis B infections.

  6. Evidence that platelet-derived microvesicles may transfer platelet-specific immunoreactive antigens to the surface of endothelial cells and CD34+ hematopoietic stem/ progenitor cells--implication for the pathogenesis of immune thrombocytopenias.

    Directory of Open Access Journals (Sweden)

    Mariusz Z Ratajczak

    2007-03-01

    Full Text Available The pathogenesis and tissue damage that accompanies destruction of platelets in immune thrombocytopenias (IT is still not understood very well and in addition to platelets, other cells (e.g. endothelial cells, CD34+ hematopoietic stem/progenitors may also become affected. Based on our previous work that platelet antigens (e.g., CD41 may be transferred by platelet-derived microvesicles (PMV to the surface of other cells, we asked if platelet derived-antigens, especially those that are involved in the formation of anti-platelet antibodies in IT (e.g., against antigen HPA 1 a could be also transferred by similar mechanism. To address this issue normal human CD34+ cells, human umbilical vein-endothelial cells (HUVEC and monocytic cell line THP-1 were incubated with PMV derived from HPA1a+ donors. We noticed that the HPA1a antigen is highly expressed on PMV-derived from the HPAla positive platelets and is transferred in PMV-dependent manner to the surface of CD34+ cells, HUVEC and monocytic THP-1 cells. These cells covered with HPA1a positive PMV but not by PMV derived from HPAla negative platelets reacted with anti-HPA1a antibodies derived from the alloimmunized pregnant women. More importantly, human hematopoietic cells that were preincubated with HPA1a+ PMV and subsequently exposed to anti-HPA 1 a serum and human NK cells, become subject to elimination by antibody dependent cell cytotoxicity ADCC. Thus, we postulate that PMV-dependent transfer of antigens may playing an important role in "expanding" the population of target cells that may be affected by anti-platelet antibodies and explain several pathologies that accompany IT (e.g. damage of endothelium, cytopenias.

  7. Ultraviolet radiation (UVR) induces cell-surface Ro/SSA antigen expression by human keratinocytes in vitro: a possible mechanism for the UVR induction of cutaneous lupus lesions

    Energy Technology Data Exchange (ETDEWEB)

    Jones, S.K. (Bristol Royal Infirmary (United Kingdom))

    1992-06-01

    Antinuclear antibodies are useful markers of connective tissue disease. In this study, UVB but not UVA induced the expression of Ro/SSA antigen on keratinocyte surfaces in vitro. This expression was also found with the extractable nuclear antigens RnP and Sm, but not with single or double-stranded DNA. The expression was prevented by blocking protein synthesis, suggesting that it was an active process. The results suggest that UVB exposure may result in the expression of Ro/SSA antigen on the surfaces of basal keratinocytes in vivo. This antigen could then bind circulating antibody leading to the cutaneous lesions in neonatal and subacute cutaneous lupus erythematosus. (Author).

  8. Thrombin Increases Expression of Fibronectin Antigen on the Platelet Surface

    Science.gov (United States)

    Ginsberg, Mark H.; Painter, Richard G.; Forsyth, Jane; Birdwell, Charles; Plow, Edward F.

    1980-02-01

    Fibronectins (fn) are adhesive glycoproteins which bind to collagen and to fibrin and appear to be important in cellular adhesion to other cells or surfaces. Fn-related antigen is present in human platelets, suggesting a possible role for fn in the adhesive properties of platelets. We have studied the localization of fn in resting and thrombin-stimulated platelets by immunofluorescence and quantitative binding of radiolabeled antibody. In resting fixed platelets, variable light surface staining for fn was observed. When these cells were made permeable to antibody with detergent, staining for fn was markedly enhanced and was present in a punctate distribution, suggesting intracellular localization. Stimulation with thrombin, which is associated with increased platelet adhesiveness, resulted in increased staining for fn antigen on intact platelets. These stimulated cells did not leak 51Cr nor did they stain for F-actin, thus documenting that the increased fn staining was not due to loss of plasma membrane integrity. The thrombin-induced increase in accessible platelet fn antigen was confirmed by quantitative antibody binding studies in which thrombin-stimulated platelets specifically bound 15 times as much radiolabeled F(ab')2 anti-fn as did resting cells. Thus, thrombin stimulation results in increased expression of fn antigen on the platelet surface. Here it may participate in interactions with fibrin, connective tissue, or other cells.

  9. A comparison of the antigens present on the surface of virus released artificially from chick cells infected with vaccinia virus, and cowpox virus and its white pock mutant

    Science.gov (United States)

    Baxby, Derrick

    1972-01-01

    Antisera prepared against vaccinia and cowpox viruses were absorbed with purified suspensions of vaccinia virus, red cowpox and white cowpox viruses. They were then tested for their ability to neutralize the viruses, and to precipitate the virus soluble antigens. The results showed that some virus specific antigens were not virus surface components and that some components were present on the surface of all three viruses. However, certain components were detected on the surface of vaccinia virus but not on the surface of cowpox virus, and vice versa. Some evidence for the existence of a vaccinia-specific surface component was also obtained. Comparisons between results of cross-neutralization tests and immunodiffusion tests on the absorbed sera indicated that antibody to a number of antigens, including the classical LS, and the cowpox-specific d antigen play no part in the process of poxvirus neutralization. ImagesFig. AFig. BFig. CFig. DFig. EFig. FFig. G PMID:4624399

  10. Antigen-specific memory B cell development.

    Science.gov (United States)

    McHeyzer-Williams, Louise J; McHeyzer-Williams, Michael G

    2005-01-01

    Helper T (Th) cell-regulated B cell immunity progresses in an ordered cascade of cellular development that culminates in the production of antigen-specific memory B cells. The recognition of peptide MHC class II complexes on activated antigen-presenting cells is critical for effective Th cell selection, clonal expansion, and effector Th cell function development (Phase I). Cognate effector Th cell-B cell interactions then promote the development of either short-lived plasma cells (PCs) or germinal centers (GCs) (Phase II). These GCs expand, diversify, and select high-affinity variants of antigen-specific B cells for entry into the long-lived memory B cell compartment (Phase III). Upon antigen rechallenge, memory B cells rapidly expand and differentiate into PCs under the cognate control of memory Th cells (Phase IV). We review the cellular and molecular regulators of this dynamic process with emphasis on the multiple memory B cell fates that develop in vivo.

  11. Cell-surface expression of neuron-glial antigen 2 (NG2) and melanoma cell adhesion molecule (CD146) in heterogeneous cultures of marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Russell, Katie C; Tucker, H Alan; Bunnell, Bruce A; Andreeff, Michael; Schober, Wendy; Gaynor, Andrew S; Strickler, Karen L; Lin, Shuwen; Lacey, Michelle R; O'Connor, Kim C

    2013-10-01

    Cellular heterogeneity of mesenchymal stem cells (MSCs) impedes their use in regenerative medicine. The objective of this research is to identify potential biomarkers for the enrichment of progenitors from heterogeneous MSC cultures. To this end, the present study examines variation in expression of neuron-glial antigen 2 (NG2) and melanoma cell adhesion molecule (CD146) on the surface of MSCs derived from human bone marrow in response to culture conditions and among cell populations. Multipotent cells isolated from heterogeneous MSC cultures exhibit a greater than three-fold increase in surface expression for NG2 and greater than two-fold increase for CD146 as compared with parental and lineage-committed MSCs. For both antigens, surface expression is downregulated by greater than or equal to six-fold when MSCs become confluent. During serial passage, maximum surface expression of NG2 and CD146 is associated with minimum doubling time. Upregulation of NG2 and CD146 during loss of adipogenic potential at early passage suggests some limits to their utility as potency markers. A potential relationship between proliferation and antigen expression was explored by sorting heterogeneous MSCs into rapidly and slowly dividing groups. Fluorescence-activated cell sorting revealed that rapidly dividing MSCs display lower scatter and 50% higher NG2 surface expression than slowly dividing cells, but CD146 expression is comparable in both groups. Heterogeneous MSCs were sorted based on scatter properties and surface expression of NG2 and CD146 into high (HI) and low (LO) groups. Sc(LO)NG2(HI) and Sc(LO)NG2(HI)CD146(HI) MSCs have the highest proliferative potential of the sorted groups, with colony-forming efficiencies that are 1.5-2.2 times the value for the parental controls. The Sc(LO) gate enriches for rapidly dividing cells. Addition of the NG2(HI) gate increases cell survival to 1.5 times the parental control. Further addition of the CD146(HI) gate does not significantly

  12. Bacterial surface antigen-specific monoclonal antibodies used to detect beer spoilage pediococci.

    Science.gov (United States)

    Whiting, M S; Ingledew, W M; Lee, S Y; Ziola, B

    1999-08-01

    Fourteen monoclonal antibodies (Mabs) were isolated that react with surface antigens of Pediococcus beer spoilage organisms, including P. damnosus, P. pentosaceous, P. acidilactici, and unspeciated isolates. Immunoblotting, enzyme immunoassays (EIAs) of protease- and neuraminidase-treated surface antigen extracts, carbohydrate competition EIAs, and cardiolipin EIAs were used to characterize the bacterial antigens involved in Mab binding. Antigen stability in situ was tested by protease treatment or surface antigen extraction of washed bacteria. In most cases, the Mabs bind to Pediococcus surface antigens that appear to be covalently bound cell wall polymers resistant to alteration or removal from the bacterial surface. These bacterial surface antigen reactive Mabs show good potential for rapid, sensitive, and specific immunoassay detection of Pediococcus beer spoilage organisms.

  13. 21 CFR 660.40 - Hepatitis B Surface Antigen.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Hepatitis B Surface Antigen. 660.40 Section 660.40...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.40 Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this...

  14. Correlation of urine cytology with ABO(H) antigenicity in transitional cell carcinoma of the bladder.

    OpenAIRE

    1988-01-01

    Cell surface ABO(H) antigenicity of superficial bladder tumours was assessed by the indirect immunoperoxidase test in 49 patients. Good correlation was obtained between surface antigenicity of tumours and the results of urine cytology. Malignant cells were detected cytologically in 22(56%) of cases with ABO(H) antigen negative tumours which are known to behave more aggressively than ABO(H) antigen positive ones. In contrast, malignant cells were found in the urine cytology of only one (10%) o...

  15. Monoclonal Antibody Production against Human Spermatozoal Surface Antigens

    Directory of Open Access Journals (Sweden)

    M Jedi-Tehrani

    2005-10-01

    Full Text Available Introduction: As monoclonal antibodies are potential tools for characterization of soluble or cellular surface antigens, use of these proteins has always been considered in infertility and reproduction research. Therefore, in this study, monoclonal antibodies against human sperm surface antigens were produced. Material and Methods: To produce specific clones against human sperm surface antigens, proteins were extracted using solubilization methods. Balb/c mice were immunized intraperitoneally with the proteins using complete Freund’s adjuvant in the first injection and incomplete Adjuvant in the following booster injections. Hybridoma cells producing ASA were cloned by limiting dilution. Results: Five stable ASA producing hybridoma clones were achieved and their antibody isotypes were determined by ELISA. All the isotypes were of IgG class. Their cross reactivity with rat and mice spermatozoa was examined but they did not have any cross reactivity. Conclusion: The produced antibodies can be used in further studies to characterize and evaluate each of the antigens present on human sperm surface and determining their role in fertilization.

  16. Fine-mapping of the B-cell epitope domain at the N-terminus of the preS2 region of the hepatitis B surface antigen.

    Science.gov (United States)

    Sominskaya, Irina; Paulij, Wilma; Jansons, Juris; Sobotta, Dirk; Dreilina, Dzidra; Sunnen, Cecile; Meisel, Helga; Gerlich, Wolfram H; Pumpens, Paul

    2002-02-01

    In this study, we report the exact localization and substitutional characterization of a B-cell epitope domain at the N-terminus of the preS2 region of the hepatitis B surface antigen. A set of deletion variants containing preS2 sequences of different length was generated on the basis of frCP as a carrier. It was found after Western blot analysis that three monoclonal antibodies (MAbs) (2-11B1, 3-11C2, HB.OT10) recognized the linear preS2 sequence within the amino acid (aa) stretch 3-WNSTTFHQTLQDP-13. The importance of each aa residue of the epitope was proved by comparison of antibody binding to alanine-substituted peptides in both free-peptide and Pepscan variants.

  17. Cell surface expression level variation between two common Human Leukocyte Antigen alleles, HLA-A2 and HLA-B8, is dependent on the structure of the C terminal part of the alpha 2 and the alpha 3 domains

    DEFF Research Database (Denmark)

    Dellgren, Christoffer; Nehlin, Jan O; Barington, Torben

    2015-01-01

    Constitutive cell surface expression of Human Leukocyte Antigen (HLA) class I antigens vary extremely from tissue to tissue and individual antigens may differ widely in expression levels. Down-regulation of class I expression is a known immune evasive mechanism used by cancer cells and viruses....... Moreover, recent observations suggest that even minor differences in expression levels may influence the course of viral infections and the frequency of complications to stem cell transplantation. We have shown that some human multipotent stem cells have high expression of HLA-A while HLA-B is only weakly...... expressed, and demonstrate here that this is also the case for the human embryonic kidney cell line HEK293T. Using quantitative flow cytometry and quantitative polymerase chain reaction we found expression levels of endogenous HLA-A3 (median 71,204 molecules per cell) 9.2-fold higher than the expression of...

  18. Encephalitis associated with antibodies against neuronal cell surface antigens.%细胞表面抗原抗体相关脑炎

    Institute of Scientific and Technical Information of China (English)

    王佳伟; 宋兆慧

    2012-01-01

    Since encephalitis associated with antibodies against neuronal cell surface antigens hasn't been known enough by people,it has always been delayed diagnosis and treatment,which results in an adverse effect on its prognosis. The target antigens of encephalitis associated with antibodies against neuronal cell surface antigens play an important role in synaptic transmission,plasticity and nerve excitability,including NMDAR,AMPAR,GABABR and other receptors such as LG11 and Caspr2. This kind of disease mainly occurs in children and young people and has relatively severe disease degree and longer disease course,with or without tumor,which has a relatively good prognosis after treatment but has a trend of recurrence. In this study,the introduction of clinical characteristics and the update of diagnosis and treatment were highlighted so as to cause the concerns of clinicians.%由于人们对细胞表面抗原抗体相关脑炎的认识不足,延误诊治,对其预后造成不良影响.细胞表面抗原抗体相关脑炎的靶抗原,在突触传递、重塑和神经兴奋方面起着重要作用,包括N-甲基-D天冬氨酸(NMDA)受体、α-氨基-3-羟基-5-甲基-4-异恶唑基丙酸(AMPA)受体、γ-氨基丁酸B( GABAB)受体和其他自身抗原,如LGI1和Caspr2等.该类疾病主要涉及儿童和年轻人,病情较重,病程较长,可伴或不伴发肿瘤,早期治疗后预后较好,但存在复发趋势.文章旨在阐述该类疾病临床特征,诊断和治疗的新进展,以引起临床医生的关注.

  19. Presentation of antigen by B cells subsets. Pt. 2. The role of CD5 B cells in the presentation of antigen to antigen-specific T cells

    Energy Technology Data Exchange (ETDEWEB)

    Zimecki, Michal [Polish Academy of Sciences, Wroclaw (Poland). Institute of Immunology and Experimental Therapy; Kapp, Judith A. [Emory Univ., Atlanta, GA (United States). School of Medicine

    1994-12-31

    We demonstrate that peritoneal B cells have a much higher ability to present antigen to antigen-specific T cell lines splenic B cells. Presentation of antigen by B cells is abrogated or drastically reduced after removal of Lyb-5{sup +} cells from the population of splenic or peritoneal B cells. Peritoneal B cells, precultured for 7 days prior to the antigen presentation assay, retain their antigen presenting cell (APC) function. Enrichment for CD5{sup +} cells in the peritoneal B cell population results in a more effective antigen presentation. Lastly, stimulation of B cells via CD5 antigen, by treatment of cells with anti-CD5 antibodies or cross-linking of CD5 receptors, enhances APC function of these cells. The results indicate, both indirectly and directly, that CD5{sup +} B cells play a predominant role in the presentation of conventional antigens to antigen-specific T cells. (author). 30 refs, 6 tabs.

  20. Three-dimensional structure and biophysical characterization of Staphylococcus aureus cell surface antigen-manganese transporter MntC.

    Science.gov (United States)

    Gribenko, Alexey; Mosyak, Lidia; Ghosh, Sharmistha; Parris, Kevin; Svenson, Kristine; Moran, Justin; Chu, Ling; Li, Sheng; Liu, Tong; Woods, Virgil L; Jansen, Kathrin U; Green, Bruce A; Anderson, Annaliesa S; Matsuka, Yury V

    2013-09-23

    MntC is a metal-binding protein component of the Mn²⁺-specific mntABC transporter from the pathogen Staphylococcus aureus. The protein is expressed during the early stages of infection and was proven to be effective at reducing both S. aureus and Staphylococcus epidermidis infections in a murine animal model when used as a vaccine antigen. MntC is currently being tested in human clinical trials as a component of a multiantigen vaccine for the prevention of S. aureus infections. To better understand the biological function of MntC, we are providing structural and biophysical characterization of the protein in this work. The three-dimensional structure of the protein was solved by X-ray crystallography at 2.2Å resolution and suggests two potential metal binding modes, which may lead to reversible as well as irreversible metal binding. Precise Mn²⁺-binding affinity of the protein was determined from the isothermal titration calorimetry experiments using a competition approach. Differential scanning calorimetry experiments confirmed that divalent metals can indeed bind to MntC reversibly as well as irreversibly. Finally, Mn²⁺-induced structural and dynamics changes have been characterized using spectroscopic methods and deuterium-hydrogen exchange mass spectroscopy. Results of the experiments show that these changes are minimal and are largely restricted to the structural elements involved in metal coordination. Therefore, it is unlikely that antibody binding to this antigen will be affected by the occupancy of the metal-binding site by Mn²⁺.

  1. Three-Dimensional Structure and Biophysical Characterization of Staphylococcus aureus Cell Surface Antigen-Manganese Transporter MntC

    Energy Technology Data Exchange (ETDEWEB)

    Gribenko, Alexey; Mosyak, Lidia; Ghosh, Sharmistha; Parris, Kevin; Svenson, Kristine; Moran, Justin; Chu, Ling; Li, Sheng; Liu, Tong; Woods, Jr., Virgil L.; Jansen, Kathrin U.; Green, Bruce A.; Anderson, Annaliesa S.; Matsuka, Yury V. [Pfizer; (UCSD)

    2013-08-23

    MntC is a metal-binding protein component of the Mn2 +-specific mntABC transporter from the pathogen Staphylococcus aureus. The protein is expressed during the early stages of infection and was proven to be effective at reducing both S. aureus and Staphylococcus epidermidis infections in a murine animal model when used as a vaccine antigen. MntC is currently being tested in human clinical trials as a component of a multiantigen vaccine for the prevention of S. aureus infections. To better understand the biological function of MntC, we are providing structural and biophysical characterization of the protein in this work. The three-dimensional structure of the protein was solved by X-ray crystallography at 2.2 Å resolution and suggests two potential metal binding modes, which may lead to reversible as well as irreversible metal binding. Precise Mn2 +-binding affinity of the protein was determined from the isothermal titration calorimetry experiments using a competition approach. Differential scanning calorimetry experiments confirmed that divalent metals can indeed bind to MntC reversibly as well as irreversibly. Finally, Mn2 +-induced structural and dynamics changes have been characterized using spectroscopic methods and deuterium–hydrogen exchange mass spectroscopy. Results of the experiments show that these changes are minimal and are largely restricted to the structural elements involved in metal coordination. Therefore, it is unlikely that antibody binding to this antigen will be affected by the occupancy of the metal-binding site by Mn2 +.

  2. Antigen Export Reduces Antigen Presentation and Limits T Cell Control of M. tuberculosis.

    Science.gov (United States)

    Srivastava, Smita; Grace, Patricia S; Ernst, Joel D

    2016-01-13

    Persistence of Mycobacterium tuberculosis results from bacterial strategies that manipulate host adaptive immune responses. Infected dendritic cells (DCs) transport M. tuberculosis to local lymph nodes but activate CD4 T cells poorly, suggesting bacterial manipulation of antigen presentation. However, M. tuberculosis antigens are also exported from infected DCs and taken up and presented by uninfected DCs, possibly overcoming this blockade of antigen presentation by infected cells. Here we show that the first stage of this antigen transfer, antigen export, benefits M. tuberculosis by diverting bacterial proteins from the antigen presentation pathway. Kinesin-2 is required for antigen export and depletion of this microtubule-based motor increases activation of antigen-specific CD4 T cells by infected cells and improves control of intracellular infection. Thus, although antigen transfer enables presentation by bystander cells, it does not compensate for reduced antigen presentation by infected cells and represents a bacterial strategy for CD4 T cell evasion.

  3. A novel multilocus sequence typing scheme for the opportunistic pathogen Propionibacterium acnes and characterization of type I cell surface-associated antigens.

    Science.gov (United States)

    McDowell, Andrew; Gao, Anna; Barnard, Emma; Fink, Colin; Murray, Philip I; Dowson, Chris G; Nagy, Istvan; Lambert, Peter A; Patrick, Sheila

    2011-07-01

    We have developed a novel multilocus sequence typing (MLST) scheme and database (http://pubmlst.org/pacnes/) for Propionibacterium acnes based on the analysis of seven core housekeeping genes. The scheme, which was validated against previously described antibody, single locus and random amplification of polymorphic DNA typing methods, displayed excellent resolution and differentiated 123 isolates into 37 sequence types (STs). An overall clonal population structure was detected with six eBURST groups representing the major clades I, II and III, along with two singletons. Two highly successful and global clonal lineages, ST6 (type IA) and ST10 (type IB(1)), representing 64 % of this current MLST isolate collection were identified. The ST6 clone and closely related single locus variants, which comprise a large clonal complex CC6, dominated isolates from patients with acne, and were also significantly associated with ophthalmic infections. Our data therefore support an association between acne and P. acnes strains from the type IA cluster and highlight the role of a widely disseminated clonal genotype in this condition. Characterization of type I cell surface-associated antigens that are not detected in ST10 or strains of type II and III identified two dermatan-sulphate-binding proteins with putative phase/antigenic variation signatures. We propose that the expression of these proteins by type IA organisms contributes to their role in the pathophysiology of acne and helps explain the recurrent nature of the disease. The MLST scheme and database described in this study should provide a valuable platform for future epidemiological and evolutionary studies of P. acnes.

  4. Antigen presentation by MHC-dressed cells

    Directory of Open Access Journals (Sweden)

    Masafumi eNakayama

    2015-01-01

    Full Text Available Professional antigen presenting cells (APCs such as conventional dendritic cells (DCs process protein antigens to MHC-bound peptides and then present the peptide-MHC complexes to T cells. In addition to this canonical antigen presentation pathway, recent studies have revealed that DCs and non-APCs can acquire MHC class I (MHCI and/or MHC class II (MHCII from neighboring cells through a process of cell-cell contact-dependent membrane transfer called trogocytosis. These MHC-dressed cells subsequently activate or regulate T cells via the preformed antigen peptide-MHC complexes without requiring any further processing. In addition to trogocytosis, intercellular transfer of MHCI and MHCII can be mediated by secretion of membrane vesicles such as exosomes from APCs, generating MHC-dressed cells. This review focuses on the physiological role of antigen presentation by MHCI- or MHCII-dressed cells, and also discusses differences and similarities between trogocytosis and exosome-mediated transfer of MHC.

  5. Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Holers, V.M.; Kotzin, B.L.

    1985-09-01

    The authors used monoclonal anti-nuclear autoantibodies and indirect immunofluorescence to examine normal human peripheral blood mononuclear leukocytes for the presence of cell surface nuclear antigens. Only one monoclonal anti-histone antibody (MH-2) was found to bind to freshly isolated PBL, staining approximately 10% of large cells. However, after cells were placed into culture for 16-24 h, a high percentage (up to 60%) of large-sized cells were recognized by an anti-DNA (BWD-1) and several different antihistone monoclonal antibodies (BWH-1, MH-1, and MH-2). These antibodies recognize separate antigenic determinants on chromatin and histones extracted from chromatin. The histone antigen-positive cells were viable, and the monoclonal antibodies could be shown to be binding to the cell surface and not to the nucleus. Using monoclonal antibodies specific for monocytes and T cells, and complement-mediated cytotoxicity, the cells bearing histone antigens were shown to be primarily monocytes. The appearance of histone and DNA antigen-positive cells was nearly completely inhibited by the addition of low concentrations of cycloheximide at initiation of the cultures. In contrast, little effect on the percentage of positive cells was detected if cells were exposed to high doses of gamma irradiation before culture. These data further support the existence of cell surface nuclear antigens on selected cell subsets, which may provide insight into the immunopathogenesis of systemic lupus erythematosus and related autoimmune diseases.

  6. Harnessing Dendritic Cells for Tumor Antigen Presentation

    Energy Technology Data Exchange (ETDEWEB)

    Nierkens, Stefan [Department of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Geert Grooteplein 28, Nijmegen 6525 GA (Netherlands); Janssen, Edith M., E-mail: edith.janssen@cchmc.org [Division of Molecular Immunology, Cincinnati Children' s Hospital Research Foundation, University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229 (United States)

    2011-04-26

    Dendritic cells (DC) are professional antigen presenting cells that are crucial for the induction of anti-tumor T cell responses. As a consequence, research has focused on the harnessing of DCs for therapeutic interventions. Although current strategies employing ex vivo-generated and tumor-antigen loaded DCs have been proven feasible, there are still many obstacles to overcome in order to improve clinical trial successes and offset the cost and complexity of customized cell therapy. This review focuses on one of these obstacles and a pivotal step for the priming of tumor-specific CD8{sup +} and CD4{sup +} T cells; the in vitro loading of DCs with tumor antigens.

  7. Photoaffinity antigens for human γδ T cells1

    Science.gov (United States)

    Sarikonda, Ghanashyam; Wang, Hong; Puan, Kia-Joo; Liu, Xiao-hui; Lee, Hoi K.; Song, Yongcheng; Distefano, Mark D.; Oldfield, Eric; Prestwich, Glenn D.; Morita, Craig T.

    2009-01-01

    Vγ2Vδ2 T cells comprise the major subset of peripheral blood γ δ T cells in humans and expand during infections by recognizing small, nonpeptide prenyl pyrophosphates. These molecules include (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMBPP), a microbial isoprenoid intermediate, and isopentenyl pyrophosphate (IPP), an endogenous isoprenoid intermediate. Recognition of these nonpeptide antigens is mediated by the Vγ2Vδ2 T cell antigen receptor (TCR). Several findings suggest that prenyl pyrophosphates are presented by an antigen presenting molecule: contact between T cells and APCs is required; the antigens do not bind the Vγ2Vδ2 TCR directly; and antigen recognition is abrogated by TCR mutations in CDRs distant from the putative antigen recognition site. Identification of the putative antigen presenting molecule, however, has been hindered by the inability to achieve stable association of nonpeptide prenyl pyrophosphate antigens with the presenting molecule. In this study, we show that photoaffinity analogs of HMBPP, meta/para-benzophenone-(methylene)-prenyl pyrophosphates (m/p-BZ-(C)-C5-OPP), can cross-link to the surface of tumor cell lines and be presented as antigens to γ δ T cells. Mutant tumor cell lines lacking MHC class I, MHC class II, β2-microglobulin, and CD1, as well as tumor cell lines from a variety of tissues and individuals, will all crosslink to and present m-BZ-C5-OPP. Finally, pulsing of BZ-(C)-C5-OPP is inhibited by IPP and an inactive analog, suggesting that they bind to the same molecule. Taken together, these results suggest that nonpeptide antigens are presented by a novel antigen presenting molecule that is widely distributed, non-polymorphic, but not classical MHC class I, MHC class II, or CD1. This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript

  8. [Association of CD34 cell surface antigen expression with cytomorphological characteristics of acute promyelocytic leukemia blasts and clinical characteristics of patients: one center experience].

    Science.gov (United States)

    Ostojić, Alen; Pazur, Marina; Siftar, Zoran; Paro, Mirjana Mariana Kardum; Jelić-Puskarić, Biljana; Gredelj-Simec, Njetocka; Radić-Kristo, Delfa; Kardum-Skelin, Ika; Vrhovac, Radovan; Jaksić, Branimir

    2011-09-01

    The aims of the study were to investigate the association between cytomorphology and immunophenotypic expression of CD34 cell surface antigen of blasts and their relationship with clinical and laboratory characteristics of patients with acute promyelocytic leukemia (APL). Sixteen consecutive patients (male 69% and female 31%) diagnosed with APL at Department of Hematology, Merkur University Hospital between August 1998 and December 2010 were included in the study. The mean age of patients was 43.9 (range: 18-78, SD 14.9). The patients' clinical and laboratory features, cytomorphological characteristics of APL-blasts and their immunophenotype determined by flow cytometry were analyzed. Patients were divided into two groups, CD34- and CD34+, and were then compared according to clinical and laboratory characteristics. There was no difference according to age, sex or white blood cell count between two groups. The mean value of hypogranular/agranular APL-blasts was markedly higher in CD34+ group than CD34- group (34%, range 9-60, SD 24.4 vs. 11.5%, range 0-38, SD 13.7), with borderline statistical significance (P=0.055). CD34- patients had significantly better overall survival than CD34+ ones (P=0.02). Patients without Auer rods detected in APL-blasts had higher CD34 expression (69.4% +/- 33.8) compared to patients with detected Auer rods (7.3% +/- 24.8), but statistical significance was not reached (p=0.053). Our results are consistent with the results of other published studies and point to the fact that higher CD34 expression and lower cytoplasmic granularity of APL-blasts are factors that seem to define a specific subgroup of APL patients. Together with other diagnostic tools currently available, they could be of value in planning treatment of APL patients.

  9. A Molecular-Level Account of the Antigenic Hantaviral Surface

    Directory of Open Access Journals (Sweden)

    Sai Li

    2016-05-01

    Full Text Available Hantaviruses, a geographically diverse group of zoonotic pathogens, initiate cell infection through the concerted action of Gn and Gc viral surface glycoproteins. Here, we describe the high-resolution crystal structure of the antigenic ectodomain of Gn from Puumala hantavirus (PUUV, a causative agent of hemorrhagic fever with renal syndrome. Fitting of PUUV Gn into an electron cryomicroscopy reconstruction of intact Gn-Gc spike complexes from the closely related but non-pathogenic Tula hantavirus localized Gn tetramers to the membrane-distal surface of the virion. The accuracy of the fitting was corroborated by epitope mapping and genetic analysis of available PUUV sequences. Interestingly, Gn exhibits greater non-synonymous sequence diversity than the less accessible Gc, supporting a role of the host humoral immune response in exerting selective pressure on the virus surface. The fold of PUUV Gn is likely to be widely conserved across hantaviruses.

  10. Inhibition of Viability, Proliferation, Cytokines Secretion, Surface Antigen Expression, and Adipogenic and Osteogenic Differentiation of Adipose-Derived Stem Cells by Seven-Day Exposure to 0.5 T Static Magnetic Fields

    Directory of Open Access Journals (Sweden)

    Jian Wang

    2016-01-01

    Full Text Available After seven-day exposure to 0.5-Tesla Static Magnetic Field (SMF, Adipose-derived Stem Cells (ASCs and those labeled by superparamagnetic iron oxide (SPIO nanoparticles were examined for viability by methyl thiazol tetrazolium (MTT assay, proliferation by cell counting and bromodeoxyuridine (BrdU incorporation, DNA integrity by single cell gel electrophoresis, surface antigen by flow cytometry analysis, and the expression of cytokines and genetic markers by reverse transcription-PCR and underwent adipogenic and osteogenic differentiation assessed by quantifying related specific genes expression. The SMF slightly reduced cell viability and proliferation and inhibited the expression of CD49d, CD54, and CD73 but did not damage DNA integrity. The SMF slightly downregulated the expression of cytokines including Vascular Endothelial Growth Factor (VEGF, Insulin-like Growth Factor-1 (IGF-1, Transforming Growth Factor Beta 1 (TGF-β1, genetic markers comprising Stem Cell Antigen-1 (Sca1, Octamer-4 (Oct-4, ATP-binding Cassette Subfamily B Member 1 (ABCB1, adipogenic marker genes containing Lipoprotein Lipase (LPL, Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ, and osteogenic marker genes including Secreted Phosphor-protein 1 (SPP1 and Osterix (OSX. Exposure to 0.5 T SMF for seven days inhibited viability, proliferation, surface antigen expression, cytokine secretion, stem cell genetic marker expression, and adipogenic and osteogenic differentiation but did not affect the DNA integrity in ASCs with or without SPIO labeling.

  11. Characterization of a surface membrane molecule expressed by natural killer cells in most inbred mouse strains: monoclonal antibody C9.1 identifies an allelic form of the 2B4 antigen

    Science.gov (United States)

    Kubota, K; Katoh, H; Muguruma, K; Koyama, K

    1999-01-01

    A newly generated monoclonal antibody (mAb C9.1) described in this study identifies a surface membrane molecule that is involved in the lytic programme of activated natural killer (NK) cells. This conclusion is based on the facts that, first, this antigen was expressed on the vast majority of surface immunoglobulin (sIg)− CD3− CD4− CD8− spleen lymphocytes, albeit it was also present on minor subsets of sIg+ B (≈7%) and CD3+ T (≈2%) lymphocytes; second, that all splenic NK activity was contained within the C9.1+ cell population, and was almost totally abolished by treatment of spleen cells with mAb C9.1 and complement; third, that mAb C9.1 was capable of increasing interleukin-2-cultured and in vivo polyinosinic:polycytidylic acid-activated, NK cell-mediated, antibody-redirected lysis, but not freshly isolated NK cell-mediated killing. Furthermore, the strain distribution of the C9.1 antigen was shown to be antithetical to that of the 2B4 antigen already described as a molecule associated with major histocompatibility complex-unrestricted killing mediated by activated NK cells. The gene encoding C9.1 antigen was linked to the Akp1 isozyme locus on chromosome 1 close to the 2B4 gene. Although C9.1 and 2B4 were monomeric glycoproteins of 78 000 MW and 66 000 MW, respectively, removal of N-linked sugars from both antigens by endoglycosidase F yielded identical protein backbones of 38 000 MW. Thus, all of these results suggest that mAb C9.1 recognizes an allelic form of the 2B4 antigen. However, the detection of mAb C9.1-reactive antigen on a minor subset of B cells may suggest a possible reactivity of mAb C9.1 with some product of other members of the 2B4 family genes. PMID:10233732

  12. Mapping of immunodominant B-cell epitopes and the human serum albumin-binding site in natural hepatitis B virus surface antigen of defined genosubtype.

    Science.gov (United States)

    Sobotta, D; Sominskaya, I; Jansons, J; Meisel, H; Schmitt, S; Heermann, K H; Kaluza, G; Pumpens, P; Gerlich, W H

    2000-02-01

    Twelve MAbs were generated by immunization of BALB/c mice with plasma-derived hepatitis B virus surface spherical antigen particles subtype ayw2 (HBsAg/ayw2 genotype D). Their epitopes were mapped by analysis of reactivity with plasma-derived HBsAg/ayw2 and HBsAg/adw2 (genotype A) in enzyme immunoassays and blots. Mapping was supported by nested sets of truncated preS2 proteins and preS2 peptides. Five antibodies were S domain-specific, seven were preS2-specific and 11 had a preference for genotype D. According to our data, group I of the three known epitope groups of preS2 has to be divided into IA and IB. Three preS2-specific MAbs forming the new group IA reacted with genotype D residues 3-15 which have not yet been described as an epitope region. IA antibodies strongly inhibited the binding of polymerized human serum albumin. Two antibodies (group II) reacted with the glycosylated N-terminal region of preS2 in plasma-derived HBsAg, but not with a preparation from transfected murine cells. One group III antibody was subtype-specific and reacted with the highly variable preS2 sequence 38-48. Only one antibody (group IB) mapped to the region (old group I) which was believed to be immunodominant and genotype-independent. Geno(sub)type-specific epitopes of preS2 are obviously the immunodominant components of natural HBsAg in BALB/c mice, but these epitopes may be masked by serum albumins in humans. The data may explain why it is difficult to detect anti-preS2 antibodies in human recipients of preS2-containing vaccines, in spite of the preS2 immunodominance in mice.

  13. Regulator T cells: specific for antigen and/or antigen receptors?

    Science.gov (United States)

    Rubin, B; de Durana, Y Diaz; Li, N; Sercarz, E E

    2003-05-01

    Adaptive immune responses are regulated by many different molecular and cellular effectors. Regulator T cells are coming to their rights again, and these T cells seem to have ordinary alpha/beta T-cell receptors (TCRs) and to develop in the thymus. Autoimmune responses are tightly regulated by such regulatory T cells, a phenomenon which is beneficial to the host in autoimmune situations. However, the regulation of autoimmune responses to tumour cells is harmful to the host, as this regulation delays the defence against the outgrowth of neoplastic cells. In the present review, we discuss whether regulatory T cells are specific for antigen and/or for antigen receptors. Our interest in these phenomena comes from the findings that T cells produce many more TCR-alpha and TCR-beta chains than are necessary for surface membrane expression of TCR-alphabeta heterodimers with CD3 complexes. Excess TCR chains are degraded by the proteasomes, and TCR peptides thus become available to the assembly pathway of major histocompatibility complex class I molecules. Consequently, do T cells express two different identification markers on the cell membrane, the TCR-alphabeta clonotype for recognition by B-cell receptors and clonotypic TCR-alphabeta peptides for recognition by T cells?

  14. The Antigen Presenting Cells Instruct Plasma Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Wei eXu

    2014-01-01

    Full Text Available The professional antigen presenting cells (APCs, including many subsets of dendritic cells and macrophages, not only mediate prompt but nonspecific response against microbes, but also bridge the antigen-specific adaptive immune response through antigen presentation. In the latter, typically activated B cells acquire cognate signals from T helper cells in the germinal center of lymphoid follicles to differentiate into plasma cells, which generate protective antibodies. Recent advances have revealed that many APC subsets provide not only signal 1 (the antigen, but also signal 2 to directly instruct the differentiation process of plasma cells in a T cell-independent manner. Herein, the different signals provided by these APC subsets to direct B cell proliferation, survival, class switching and terminal differentiation are discussed. We furthermore propose that the next generation of vaccines for boosting antibody response could be designed by targeting APCs.

  15. EXPRESSION OF HUMAN α-GALACTOSIDASE AND α1,2-FUCOSYL-TRANSFERASE GENES MODIFIES THE CELL SURFACE GALα1,3GAL ANTIGEN AND CONFERS RESISTANCE TO HUMAN SERUM-MEDIATED CYTOLYSIS

    Institute of Scientific and Technical Information of China (English)

    贾延军; 任会明; 高新; 季守平; 杨军; 刘泽鹏; 李素波; 章扬培

    2004-01-01

    Objective To explore the strategies which reduce the amount of xenoantigen Galα1, 3 Gal. Methods Human α-galactosidase gene and α 1,2-fucosyltransferase gene were transferred into cultured porcine vascular endothelial cells PEDSV.15 and human α-galactosidase transgenic mice were produced. The Galα 1,3Gal on the cell surface and susceptibility of cells to human antibody-mediated lysis were analyzed. Results Human α-galactosidase gene alone reduced 78% of Galα1,3Gal on PEDSV.15 cell surface while human α-galactosidase combined with α 1,2-fucosyltransferase genes removed Galα 1,3Gal completely. Decrease of Galα1,3Gal could reduce susceptibility of cells to human antibody-mediated lysis, especially during co-expression of α-galactosidase gene and α1,2-fucosyltransferase gene. RT-PCR indicated positive human α-galactosidase gene expression in all organs of positive human α-galactosidase transgenic F1 mice including heart, liver, kidney, lung, and spleen, the amount of Galα1,3Gal antigens on which was reduced largely. 58% of spleen cells from F1 mice were destroyed by complement-mediated lysis compared with 24% of those from normal mice. Conclusions Human α-galactosidase gene and α1,2-fucosyltransferase gene effectively reduce the expression of Galα1,3Gal antigens on endothelial cell surface and confers resistance to human serummediated cytolysis. The expression of human α-galactosidase in mice can also eliminate the Galα1,3Gal antigens in most tissues and decrease the susceptibility of spleen cells to human serum-mediated cytolysis.

  16. Effect of Vi antigen on assembly of colonization factor antigens from enterotoxigenic Escherichia coli on the cell surface of Salmonella typhi%Vi抗原影响产肠毒素大肠杆菌菌毛 在人伤寒沙门菌表面的装配

    Institute of Scientific and Technical Information of China (English)

    韩照中; 张兆山; 李淑琴; 苏国富; 黄翠芬

    2001-01-01

    Objective To study the effects of Vi antigen on assembly ofcolonization factor antigens from enterotoxigenic Escherichia coli on the cell surface of Salmonella typhi. Methods A Salmonella typhi mutant RH108 was constructed by homological recombination in vivo, in which VipR gene was partially deleted and so with low expression level of Vi antigen as compared with the corresponding wild type, RS406. The two bacteria strains were transformed by recombinant plasmids expressing coli surface antigen CFA-I and CS3 from enterotoxigenic Escherichia coli. The concentrations of CFA-I and CS3 expressed and assembled on the surface of RS406 and RH108 were titered by ELISA method. Results CFA-I and CS3 were expressed and assembled better on the cell surface of RH108. Immuno-gold labeled electroscope analysis gave same results. Conclusion Moderation of Vi antigen may boost expression and assembly of coli surface antigens from enterotoxigenic Escherichia coli on the surface of Salmonella typhi. This result may be useful for construction of effective vaccine against enterotoxigenic Escherichia coli with Salmonella typhi as antigens carrier.%目的 观察人伤寒沙门菌Vi抗原对大肠杆菌菌毛抗原装配的影响。方法 利用体内、外同源重组系统,构建了VipR基因缺失突变的人伤寒沙门菌菌株,导致其Vi抗原的表达较相应野生菌株偏低。用包含产肠毒素大肠杆菌菌毛抗原基因的表达质粒分别转化Vi表达弱化菌株和相应野生菌株,对两者表达的菌毛抗原进行含量分析。结果 产肠毒素大肠杆菌CS3、CFA-Ⅰ在VipR突变体菌株表面的含量,均比在相应野生菌株表面的含量高。结论 Vi抗原的表达弱化可能有利于菌毛抗原在人伤寒沙门菌表面的装配。本研究结果对于产肠毒素大肠杆菌基因工程疫苗的构建有指导意义。

  17. Liposome-coupled antigens are internalized by antigen-presenting cells via pinocytosis and cross-presented to CD8 T cells.

    Directory of Open Access Journals (Sweden)

    Yuriko Tanaka

    Full Text Available We have previously demonstrated that antigens chemically coupled to the surface of liposomes consisting of unsaturated fatty acids were cross-presented by antigen-presenting cells (APCs to CD8+ T cells, and that this process resulted in the induction of antigen-specific cytotoxic T lymphocytes. In the present study, the mechanism by which the liposome-coupled antigens were cross-presented to CD8+ T cells by APCs was investigated. Confocal laser scanning microscopic analysis demonstrated that antigens coupled to the surface of unsaturated-fatty-acid-based liposomes received processing at both MHC class I and class II compartments, while most of the antigens coupled to the surface of saturated-fatty-acid-based liposomes received processing at the class II compartment. In addition, flow cytometric analysis demonstrated that antigens coupled to the surface of unsaturated-fatty-acid-liposomes were taken up by APCs even in a 4°C environment; this was not true of saturated-fatty-acid-liposomes. When two kinds of inhibitors, dimethylamiloride (DMA and cytochalasin B, which inhibit pinocytosis and phagocytosis by APCs, respectively, were added to the culture of APCs prior to the antigen pulse, DMA but not cytochalasin B significantly reduced uptake of liposome-coupled antigens. Further analysis of intracellular trafficking of liposomal antigens using confocal laser scanning microscopy revealed that a portion of liposome-coupled antigens taken up by APCs were delivered to the lysosome compartment. In agreement with the reduction of antigen uptake by APCs, antigen presentation by APCs was significantly inhibited by DMA, and resulted in the reduction of IFN-γ production by antigen-specific CD8+ T cells. These results suggest that antigens coupled to the surface of liposomes consisting of unsaturated fatty acids might be pinocytosed by APCs, loaded onto the class I MHC processing pathway, and presented to CD8+ T cells. Thus, these liposome-coupled antigens

  18. Optimization of a flow cytometric method for the simultaneous measurement of cell surface antigen, DNA content, and in vitro BrdUrd incorporation into normal and malignant hematopoietic cells.

    Science.gov (United States)

    Holm, M; Thomsen, M; Høyer, M; Hokland, P

    1998-05-01

    We have designed an assay for the simultaneous measurement of cell surface phenotype, S-phase fraction, and DNA content by single laser instrumentation for the purpose of determining the labeling index (LI), duration of S-phase (Ts), and the potential doubling time (Tpot) of leukocyte subpopulations. The procedure was optimized with regard to: mode of bromodeoxyuridine (BrdUrd) incorporation, selection of suitable leukocyte differentiation antigens (LDAs) as well as PE-conjugated monoclonal antibodies (MoAbs) against myeloid cells, overnight permeabilization and fixation (paraformaldehyde 1% and 0.05% Nonidet P40), DNase I treatment (250 Kunitz units), concentration of FITC-conjugated anti-BrdUrd MoAb (dilution 1:5), and DNA staining with 7-amino-actinomycin (7-AAD) (10 microg/ml). We validated this assay by measuring LI, Ts, and Tpot repeatedly in four leukemic cell lines and found these to be stable (coefficients of variation (CV): 0.06, 0.13, and 0.08, respectively). Finally, we employed the assay on different leukocyte preparations from normal donors (including purified CD34 + cells) and patients with malignant myeloid disorders, and we concluded that it will yield valuable data regarding the cell cycle kinetics of subsets of leukocytes in heterogeneous mixtures of hematopoietic cells.

  19. Expression of the T1 (CD5, p67) surface antigen in B-CLL and B-NHL and its correlation with other B-cell differentiation markers.

    Science.gov (United States)

    Delia, D; Bonati, A; Giardini, R; Villa, S; De Braud, F; Cattoretti, G; Rilke, F

    1986-01-01

    The T1 surface antigen (CD5,p67) expression on blood lymphocytes (PBL) and lymphoid cells from lymph node biopsies (LN) from 31 patients with B-cell chronic lymphocytic leukemia (B-CLL) and 79 with B non-Hodgkin lymphoma (B-NHL), was detected in 25 B-CLL (80 per cent) and in 11 B-NHL (13 per cent) belonging to the following histologic subtypes: lymphocytic of CLL type (DLWD) one case, lymphoplasmacytoid (DLWD) four cases, centrocytic (DLPD) five cases, immunoblastic (DH) one case. All B-CLL and the T1 + B-NHL were also tested with monoclonal antibodies against the Common Acute Lymphoblastic Leukemia Antigen, B cells (FMC7, FMC8, BA1, Y29-55), T cells (OKT11a), HLA-DR and HLA-DQ monomorphic determinants. All the B-CLL and the T1+ B-NHL were CALLA-, BA1+, Y29.55+. FMC7+ cells were detected in large numbers six B-CLL (three T1+ and three T1-) and in four centrocytic lymphomas. FMC8 reacted with 70 per cent of leukemias (where it stained 30 per cent of neoplastic cells) and with 8/9 T+ B-NHL. HLA-DR and HLA-DQ molecules were detected in 100 per cent and 90 per cent of cases respectively. In vitro treatment of HLA-DQ- or T1- B-CLL with phorbol ester TPA led to the expression of these antigens as well as of the receptors for Interleukin 2 and MLR3 activation antigen. Surface membrane Ig (SIg) was detected in 79 per cent of cases, its density measured by FACS analysis varied, even markedly, from case to case. Among the B-CLL, cells with high SIg content were either T1+ or T1- and more likely FMC7+. The SIg- cases were seven B-CLL (five T1+ and two T1-) and two B-NHL, in which, however, cytoplasmic IgM was detected. This study reveals the existence of four major B-CLL subgroups: T1- SIg-, T1+ SIg+, T1+ SIg+, T1- SIg+. It also indicates that the T1 antigen may be transitionally present during B-cell differentiation and that its expression may precede that of SIg as supported by the in vitro studies. In addition, the finding that some B-NHL are T1+ suggests that they derive

  20. Studies on the surface coat of Paramecium aurelia. II. Relationship to the immobilization antigen.

    Science.gov (United States)

    Wyroba, E

    1977-07-11

    Correlations between the presence of surface coat and immobilization antigen of Paramecium tetraurelia were studied. Supravital, partial removal of the surface coat resulted in accelerated response of monobacterially and axenically grown cells to the homologous antiserum. Ciliates pretreated with trypsin or pronase (0.5 mg/ml for 45 min at 0-4 degrees C) were immobilized approximately twice as fast as untreated control cells. The probable localization of at least part, of the immobilization antigen within the surface coat of P. tetraurelia is discussed.

  1. Improved cell mediated immune responses after successful re-vaccination of non-responders to the hepatitis B virus surface antigen (HBsAg) vaccine using the combined hepatitis A and B vaccine.

    Science.gov (United States)

    Nyström, Jessica; Cardell, Kristina; Björnsdottir, Thora Björg; Fryden, Aril; Hultgren, Catharina; Sällberg, Matti

    2008-11-01

    We successfully re-vaccinated hepatitis B virus (HBV) vaccine non-responders using a double dose of the combined hepatitis A virus (HAV) and HBV vaccine. The hope was to improve priming of hepatitis B surface antigen (HBsAg)-specific cell mediated immune response (CMI) by an increased antigen dose and a theoretical adjuvant-effect from the local presence of a HAV-specific CMI. A few non-responders had a detectable HBsAg-specific CMI before re-vaccination. An in vitro detectable HBsAg-specific CMI was primed equally effective in non-responders (58%) as in first time vaccine recipients (68%). After the third dose a weak, albeit significant, association was observed between the magnitude of HBsAg-specific proliferation and anti-HBs levels. This regimen improves the priming of HBsAg-specific CMIs and antibodies.

  2. 21 CFR 660.1 - Antibody to Hepatitis B Surface Antigen.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Antibody to Hepatitis B Surface Antigen. 660.1... Hepatitis B Surface Antigen § 660.1 Antibody to Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product shall be Antibody to Hepatitis B Surface Antigen. The product...

  3. Expression of maturation-specific nuclear antigens in differentiating human myeloid leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Murao, S.; Epstein, A.L.; Clevenger, C.V.; Huberman, E.

    1985-02-01

    The expression of three myeloid-specific nuclear antigens was studied by indirect immunofluorescence with murine monoclonal antibodies in human myeloid (HL-60, ML-2, KG-1, and B-II) leukemia cells treated with chemical inducers of cell differentiation. Treatment of the promyelocytic HL-60 cells with dimethyl sulfoxide or 1,25-dihydroxyvitamin DT induced the cells to acquire a phenotype that resembled that of granulocytes and monocytesmacrophages, respectively. These phenotypes were characterized by changes in cell growth, cell morphology, expression of specific cell surface antigens, and activities of lysozyme and nonspecific esterase enzymes. Induction of these differentiation markers in the HL-60 cells was associated with induction of the myeloid-specific nuclear antigens. The ML-2 cells, which are arrested at the myeloblast-promyelocyte stage, were also susceptible to the induction of cell differentiation and to changes in the expression of the nuclear antigens, but the degree of susceptibility was less than in the HL-60 cells. The less-differentiated KG-1 and B-II myeloid cells were either not responsive or responded only in a limited degree to the induction of cell differentiation or to changes in the expression of the nuclear antigens. The authors suggest that the reactivity of cells with monoclonal antibodies to specific nuclear antigens can be used as a maturational marker in cell differentiation studies. Furthermore, nuclear antigens expressed early in cellular differentiation may provide information about changes in regulatory elements in normal and malignant cells. 40 references, 2 figures, 1 table.

  4. Conformational dynamics and antigenicity in the disordered malaria antigen merozoite surface protein 2.

    Directory of Open Access Journals (Sweden)

    Christopher A MacRaild

    Full Text Available Merozoite surface protein 2 (MSP2 of Plasmodium falciparum is an abundant, intrinsically disordered protein that is GPI-anchored to the surface of the invasive blood stage of the malaria parasite. Recombinant MSP2 has been trialled as a component of a malaria vaccine, and is one of several disordered proteins that are candidates for inclusion in vaccines for malaria and other diseases. Nonetheless, little is known about the implications of protein disorder for the development of an effective antibody response. We have therefore undertaken a detailed analysis of the conformational dynamics of the two allelic forms of MSP2 (3D7 and FC27 using NMR spectroscopy. Chemical shifts and NMR relaxation data indicate that conformational and dynamic properties of the N- and C-terminal conserved regions in the two forms of MSP2 are essentially identical, but significant variation exists between and within the central variable regions. We observe a strong relationship between the conformational dynamics and the antigenicity of MSP2, as assessed with antisera to recombinant MSP2. Regions of increased conformational order in MSP2, including those in the conserved regions, are more strongly antigenic, while the most flexible regions are minimally antigenic. This suggests that modifications that increase conformational order may offer a means to tune the antigenicity of MSP2 and other disordered antigens, with implications for vaccine design.

  5. Conformational Dynamics and Antigenicity in the Disordered Malaria Antigen Merozoite Surface Protein 2

    Science.gov (United States)

    Andrew, Dean; Krishnarjuna, Bankala; Nováček, Jiří; Žídek, Lukáš; Sklenář, Vladimír; Richards, Jack S.; Beeson, James G.; Anders, Robin F.; Norton, Raymond S.

    2015-01-01

    Merozoite surface protein 2 (MSP2) of Plasmodium falciparum is an abundant, intrinsically disordered protein that is GPI-anchored to the surface of the invasive blood stage of the malaria parasite. Recombinant MSP2 has been trialled as a component of a malaria vaccine, and is one of several disordered proteins that are candidates for inclusion in vaccines for malaria and other diseases. Nonetheless, little is known about the implications of protein disorder for the development of an effective antibody response. We have therefore undertaken a detailed analysis of the conformational dynamics of the two allelic forms of MSP2 (3D7 and FC27) using NMR spectroscopy. Chemical shifts and NMR relaxation data indicate that conformational and dynamic properties of the N- and C-terminal conserved regions in the two forms of MSP2 are essentially identical, but significant variation exists between and within the central variable regions. We observe a strong relationship between the conformational dynamics and the antigenicity of MSP2, as assessed with antisera to recombinant MSP2. Regions of increased conformational order in MSP2, including those in the conserved regions, are more strongly antigenic, while the most flexible regions are minimally antigenic. This suggests that modifications that increase conformational order may offer a means to tune the antigenicity of MSP2 and other disordered antigens, with implications for vaccine design. PMID:25742002

  6. Persistence of Zinc-Binding Bacterial Superantigens at the Surface of Antigen-Presenting Cells Contributes to the Extreme Potency of These Superantigens as T-Cell Activators

    Science.gov (United States)

    2005-09-01

    Contributes to the Extreme Potency of These Superantigens as T-Cell Activators Dorothy D. Pless,† Gordon Ruthel, Emily K. Reinke, Robert G. Ulrich, and Sina...immunoglobulin G, and the cells were analyzed with a FACSort flow cytometer (Becton Dickinson , Mountain View, CA). To measure off rates, 1 or 5 g of SE or

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1984-08-01

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

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

    DEFF Research Database (Denmark)

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

    1999-01-01

    Antibodies against variable antigens expressed on the surface of Plasmodium falciparum-infected erythrocytes are believed to be important for protection against malaria. A target for these antibodies is the P. falciparum erythrocyte membrane protein 1, PfEMP1, which is encoded by around 50 var...... genes and undergoes clonal variation. Using agglutination and mixed agglutination tests and flow cytometry to analyse the recognition of variant antigens on parasitized erythrocytes by plasma antibodies from individuals living in Daraweesh in eastern Sudan, an area of seasonal and unstable malaria...

  9. Regulation of protein synthesis and autophagy in activated dendritic cells: implications for antigen processing and presentation.

    Science.gov (United States)

    Argüello, Rafael J; Reverendo, Marisa; Gatti, Evelina; Pierre, Philippe

    2016-07-01

    Antigenic peptides presented in the context of major histocompatibility complex (MHC) molecules originate from the degradation of both self and non-self proteins. T cells can therefore recognize at the surface of surveyed cells, the self-peptidome produced by the cell itself (mostly inducing tolerance) or immunogenic peptides derived from exogenous origins. The initiation of adaptive immune responses by dendritic cells (DCs), through the antigenic priming of naïve T cells, is associated to microbial pattern recognition receptors engagement. Activation of DCs by microbial product or inflammatory cytokines initiates multiple processes that maximize DC capacity to present exogenous antigens and stimulate T cells by affecting major metabolic and membrane traffic pathways. These include the modulation of protein synthesis, the regulation of MHC and co-stimulatory molecules transport, as well as the regulation of autophagy, that, all together promote exogenous antigen presentation while limiting the display of self-antigens by MHC molecules.

  10. Antigen presentation for priming T cells in central system.

    Science.gov (United States)

    Dasgupta, Shaoni; Dasgupta, Subhajit

    2017-01-01

    Generation of myelin antigen-specific T cells is a major event in neuroimmune responses that causes demyelination. The antigen-priming of T cells and its location is important in chronic and acute inflammation. In autoimmune multiple sclerosis, the effector T cells are considered to generate in periphery. However, the reasons for chronic relapsing-remitting events are obscure. Considering mechanisms, a feasible aim of research is to investigate the role of antigen-primed T cells in lupus cerebritis. Last thirty years of investigations emphasize the relevance of microglia and infiltrated dendritic cells/macrophages as antigen presenting cells in the central nervous system. The recent approach towards circulating B-lymphocytes is an important area in the context. Here, we analyze the existing findings on antigen presentation in the central nervous system. The aim is to visualize signaling events of myelin antigen presentation to T cells and lead to the strategy of future goals on immunotherapy research.

  11. Conservation of a protective surface antigen of Tritrichomonas foetus.

    Science.gov (United States)

    Ikeda, J S; BonDurant, R H; Campero, C M; Corbeil, L B

    1993-12-01

    Bovine trichomoniasis is a sexually transmitted disease caused by the flagellated protozoan Tritrichomonas foetus. A protective surface antigen was previously identified and immunoaffinity purified from T. foetus isolate D1 with cross-reactive monoclonal antibodies (MAbs) TF1.15 and TF1.17 (BonDurant, R. H., R. R. Corbeil, and L. B. Corbeil, Infect. Immun. 61:1385-1394, 1993). This antigen elicited antibody responses in the serum and cervicovaginal mucus of heifers. Thus, it may be useful as an immunodiagnostic reagent as well as a subunit vaccine. Conservation of the antigen in all strains would be crucial for either application. We investigated the conservation of this antigen among 36 isolates of T. foetus from Argentina, Costa Rica, and the United States using MAbs TF1.15 and TF1.17 in an enzyme-linked immunosorbent assay. MAb TF1.17 reacted with 32 of the 36 isolates, whereas MAb TF1.15 reacted with all of the isolates tested. One of the isolates which did not react with MAb TF1.17 (i.e., D1#3) was investigated further by Western blotting (immunoblotting) to determine the reason for the lack of reactivity with one of the two cross-reactive MAbs. The antigenic band that was reactive with MAb TF1.15 had a molecular mass slightly lower than that of the corresponding band from isolate D1, which reacted with both MAbs TF1.15 and TF1.17. Thus, at least a major portion of the antigen appeared to be conserved. This was confirmed in a study of heifers infected with isolate D1#3. The vaginal immunoglobulin A antibodies of these infected heifers reacted with the antigen of isolate D1 that was immunoaffinity purified with MAb TF1.17. Therefore, even though the epitope recognized by MAb TF1.17 was missing in the challenge isolate (D1#3), the heifers developed an immune response to the rest of the molecule. These results indicate that the major portion of the previously described protective antigen is conserved in different isolates of T. foetus. This portion contains the

  12. Cell wall anchoring of the Campylobacter antigens to Lactococcus lactis

    Directory of Open Access Journals (Sweden)

    Patrycja Anna Kobierecka

    2016-02-01

    Full Text Available Campylobacter jejuni is the most frequent cause of human food-borne gastroenteritis and chicken meat is the main source of infection. Recent studies showed that broiler chicken immunization against Campylobacter should be the most efficient way to lower the number of human infections by this pathogen. Induction of the mucosal immune system after oral antigen administration should provide protective immunity to chickens. In this work we tested the usefulness of Lactococcus lactis, the most extensively studied lactic acid bacterium, as a delivery vector for Campylobacter antigens. First we constructed hybrid protein – CjaA antigen presenting CjaD peptide epitopes on its surface. We showed that specific rabbit anti-rCjaAD serum reacted strongly with both CjaA and CjaD produced by a wild type Campylobacter jejuni strain. Next, rCjaAD and CjaA were fused to the C-terminus of the L. lactis YndF containing the LPTXG motif. The genes expressing these proteins were transcribed under control of the L. lactis Usp45 promoter and their products contain the Usp45 signal sequences. This strategy ensures a cell surface location of both analysed proteins, which was confirmed by immunofluorescence assay. In order to evaluate the impact of antigen location on vaccine prototype efficacy, a L. lactis strain producing cytoplasm-located rCjaAD was also generated. Animal experiments showed a decrease of Campylobacter cecal load in vaccinated birds as compared with the control group and showed that the L. lactis harboring the surface-exposed rCjaAD antigen afforded greater protection than the L. lactis producing cytoplasm-located rCjaAD. To the best of our knowledge, this is the first attempt to employ LAB (Lactic Acid Bacteria strains as a mucosal delivery vehicle for chicken immunization. Although the observed reduction of chicken colonization by Campylobacter resulting from vaccination was rather moderate, the experiments showed that LAB strains can be considered

  13. Glycosylation of the major human Pneumocystis carinii surface antigen

    DEFF Research Database (Denmark)

    Lundgren, Bettina; Koch, C; Mathiesen, Lars Reinhardt;

    1993-01-01

    It has recently been shown that the major rat P. carinii surface antigen is important for initial host-organism attachment, possibly through binding to fibronectin, mannose-binding protein, or surfactant protein A. Since a carbohydrate/lectin interaction may be involved in adhesion, we undertook...

  14. CD40-induced aggregation of MHC class II and CD80 on the cell surface leads to an early enhancement in antigen presentation.

    Science.gov (United States)

    Clatza, Abigail; Bonifaz, Laura C; Vignali, Dario A A; Moreno, José

    2003-12-15

    Ligation of CD40 on B cells increases their ability to present Ag and to activate MHC class II (MHC-II)-restricted T cells. How this occurs is not entirely clear. In this study we demonstrate that CD40 ligation on Ag-presenting B cells (APC) for a short period between 30 min and 3 h has a rapid, augmenting effect on the ability of a B cell line and normal B cells to activate T cells. This is not due to alterations in Ag processing or to an increase in surface expression of CD80, CD86, ICAM-1, or MHC-II. This effect is particularly evident with naive, resting T lymphocytes and appears to be more pronounced under limiting Ag concentrations. Shortly after CD40 ligation on a B cell line, MHC-II and CD80 progressively accumulated in cholesterol-enriched microdomains on the cell surface, which correlated with an initial enhancement in their Ag presentation ability. Moreover, CD40 ligation induced a second, late, more sustained enhancement of Ag presentation, which correlates with a significant increase in CD80 expression by APC. Thus, CD40 signaling enhances the efficiency with which APC activate T cells by at least two related, but distinct, mechanisms: an early stage characterized by aggregation of MHC-II and CD80 clusters, and a late stage in which a significant increase in CD80 expression is observed. These results raise the possibility that one important role of CD40 is to contribute to the formation of the immunological synapse on the APC side.

  15. Genetic diversity and antigenicity variation of Babesia bovis merozoite surface antigen-1 (MSA-1) in Thailand.

    Science.gov (United States)

    Tattiyapong, Muncharee; Sivakumar, Thillaiampalam; Takemae, Hitoshi; Simking, Pacharathon; Jittapalapong, Sathaporn; Igarashi, Ikuo; Yokoyama, Naoaki

    2016-07-01

    Babesia bovis, an intraerythrocytic protozoan parasite, causes severe clinical disease in cattle worldwide. The genetic diversity of parasite antigens often results in different immune profiles in infected animals, hindering efforts to develop immune control methodologies against the B. bovis infection. In this study, we analyzed the genetic diversity of the merozoite surface antigen-1 (msa-1) gene using 162 B. bovis-positive blood DNA samples sourced from cattle populations reared in different geographical regions of Thailand. The identity scores shared among 93 msa-1 gene sequences isolated by PCR amplification were 43.5-100%, and the similarity values among the translated amino acid sequences were 42.8-100%. Of 23 total clades detected in our phylogenetic analysis, Thai msa-1 gene sequences occurred in 18 clades; seven among them were composed of sequences exclusively from Thailand. To investigate differential antigenicity of isolated MSA-1 proteins, we expressed and purified eight recombinant MSA-1 (rMSA-1) proteins, including an rMSA-1 from B. bovis Texas (T2Bo) strain and seven rMSA-1 proteins based on the Thai msa-1 sequences. When these antigens were analyzed in a western blot assay, anti-T2Bo cattle serum strongly reacted with the rMSA-1 from T2Bo, as well as with three other rMSA-1 proteins that shared 54.9-68.4% sequence similarity with T2Bo MSA-1. In contrast, no or weak reactivity was observed for the remaining rMSA-1 proteins, which shared low sequence similarity (35.0-39.7%) with T2Bo MSA-1. While demonstrating the high genetic diversity of the B. bovis msa-1 gene in Thailand, the present findings suggest that the genetic diversity results in antigenicity variations among the MSA-1 antigens of B. bovis in Thailand.

  16. Detection of Rare Antigen Presenting Cells through T cell-intrinsic meandering motility, mediated by Myo1g

    OpenAIRE

    Gérard, Audrey; Patino-Lopez, Genaro; Beemiller, Peter; Nambiar, Rajalakshmi; Ben-Aissa, Khadija; Liu, Yin; Totah, Fadi J.; Tyska, Matthew J.; Shaw, Stephen; Krummel, Matthew F.

    2014-01-01

    To mount an immune response, T lymphocytes must successfully search for foreign material bound to the surface of antigen-presenting cells. How T cells optimize their chances of encountering and responding to these antigens is unknown. T cell motility in tissues resembles a random or Levy walk and is regulated in part by external factors including chemokines and lymph node topology, but motility parameters such as speed and propensity to turn may also be cell-intrinsic. Here we found that the ...

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

    DEFF Research Database (Denmark)

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

    2002-01-01

    In areas of intense Plasmodium falciparum transmission, protective immunity is acquired during childhood in parallel with acquisition of agglutinating antibodies to parasite-encoded variant surface antigens (VSA) expressed on parasitized red blood cells. In a semi-immune child in such an area, cl...

  18. Infectious vaccinia virus recombinants that express hepatitis B virus surface antigen

    Science.gov (United States)

    Smith, Geoffrey L.; Mackett, Michael; Moss, Bernard

    1983-04-01

    Potential live vaccines against hepatitis B virus have been produced. The coding sequence for hepatitis B virus surface antigen (HBsAg) has been inserted into the vaccinia virus genome under control of vaccinia virus early promoters. Cells infected with these vaccinia virus recombinants synthesize and excrete HBsAg and vaccinated rabbits rapidly produce antibodies to HBsAg.

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

    NARCIS (Netherlands)

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

    1996-01-01

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

  20. Application of surface-linked liposomal antigens to the development of vaccines that induce both humoral and cellular immunity.

    Science.gov (United States)

    Uchida, Tetsuya; Taneichi, Maiko

    2014-01-01

    The first characteristic identified in surface-linked liposomal antigens was the ability to induce antigen-specific, IgE-selective unresponsiveness. These results remained consistent even when different coupling procedures were employed for antigens with liposomes or for liposomes with different lipid components. The potential usefulness of surface-linked liposomal antigens for application to vaccine development was further investigated. During this investigation, a significant difference was observed in the recognition of liposomal antigens by antigen-presenting cells between liposomes with different lipid components, and this difference correlated closely with the adjuvant activity of liposomes. In addition to this "quantitative" difference between liposomes with differential lipid components, a "qualitative" difference (i.e., a differential ability to induce cross-presentation) was observed between liposomes with different lipid components. Therefore, by utilizing the ability to induce cross-presentation, surface-linked liposomal antigens might be used to develop virus vaccines that would induce cytotoxic T lymphocyte (CTL) responses. We have successfully developed a liposome vaccine that is capable of inducing CTL responses against internal antigens of influenza viruses and thus removing virus-infected cells in the host. This CTL-based liposomal vaccine might be applicable to the development of vaccines against influenza and other viruses that frequently undergo changes in their surface antigenic molecules.

  1. Potent antigen-specific immune response induced by infusion of spleen cells coupled with succinimidyl-4-(N-maleimidomethyl cyclohexane)-1-carboxylate (SMCC) conjugated antigens.

    Science.gov (United States)

    Guo, Yixian; Werbel, Tyler; Wan, Suigui; Wu, Haitao; Li, Yaohua; Clare-Salzler, Michael; Xia, Chang-Qing

    2016-02-01

    In the present study, we report our recently developed new approach to inducing antigen-specific immune response. We use two nucleophilic substitution "click" chemistry processes to successfully couple protein antigens or peptides to mouse spleen cells or T cells by a heterobifunctional crosslinker, succinimidyl-4-(N-maleimidomethyl cyclohexane)-1-carboxylate (SMCC) or sulfo-SMCC. SMCC and its water-soluble analog sulfo-SMCC contain N-hydroxysuccinimide (NHS) ester and maleimide groups, which allow stable covalent conjugation of amine- and sulfhydryl-containing molecules in trans. Protein coupling to cells relies on the free sulfhydryls (thiols) on cell surfaces and the free amines on protein antigens. Although the amount of protein coupled to cells is limited due to the limited number of cell surface thiols, the injection of spleen cells coupled with antigenic proteins, such as keyhole limpet hemocyanin (KLH) or ovalbumin (OVA), induces a potent antigen-specific immune response in vivo, which is even stronger than that induced by the injection of a large dose of protein plus adjuvants. In addition, short peptides coupled to purified splenic T cells also potently elicit peptide-specific T cell proliferation in vivo after injection. Further studies show that antigen-coupled spleen cell treatment leads to augmented IFN-γ-producing T cells. Our study provides a unique antigen delivery method that efficiently distributes antigen to the entire immune system, subsequently eliciting a potent antigen-specific immune response with enhanced IFN-γ production. The findings in the present study suggest that this antigen-cell coupling strategy could be employed in immunotherapy for cancers, infectious diseases as well as immune-mediated disorders.

  2. Physical and functional association between thymic shared antigen-1/stem cell antigen-2 and the T cell receptor complex.

    Science.gov (United States)

    Kosugi, A; Saitoh, S; Noda, S; Miyake, K; Yamashita, Y; Kimoto, M; Ogata, M; Hamaoka, T

    1998-05-15

    Thymic shared antigen-1 (TSA-1)/stem cell Ag-2 (Sca-2) is a glycosylphosphatidylinositol (GPI)-anchored antigen expressed on lymphocytes. We have previously demonstrated that a signal via TSA-1/Sca-2 inhibits T cell receptor (TCR)-mediated T cell activation and apoptosis. To elucidate a molecular mechanism for TSA-1-mediated modulation of the TCR-signaling pathway, we examined whether TSA-1 is physically coupled to the TCR in the present study. TSA-1 was clearly associated with CD3zeta chains in T cell hybridomas, activated T cells, and COS-7 cells transfected with TSA-1 and CD3zeta cDNA. The physical association was confirmed on the surface of T cells in immunoprecipitation and confocal microscopy. The analysis using stable and transient transfectants expressing a transmembrane form of TSA-1 revealed that the association of CD3zeta did not require the GPI anchor of TSA-1. Finally, tyrosine phosphorylation of CD3zeta chains was induced after stimulation with anti-TSA-1, suggesting that a functional association between these two molecules also exists. These results imply that the physical association to CD3zeta underlies a regulatory role of TSA-1/Sca-2 in the TCR-signaling pathway.

  3. Optimization of the expression of surface antigen SAG1/2 of Toxoplasma gondii in the yeast Pichia pastoris.

    Science.gov (United States)

    Thiruvengadam, G; Init, I; Fong, M Y; Lau, Y L

    2011-12-01

    Surface antigens are the most abundant proteins found on the surface of the parasite Toxoplasma gondii. Surface antigen 1 (SAG1) and Surface antigen 2 (SAG2) remain the most important and extensively studied surface proteins. These antigens have been identified to play a role in host cell invasion, immune modulation, virulence attenuation. Recombinant SAG1/2 was cloned and expressed in yeast Pichia pastoris. We describe here optimization of critical parameters involved in high yield expression of the recombinant SAG1/2. Our results suggest that recombinant SAG1/2 were best expressed at 30ºC, pH 6 and 1% methanol as the carbon source by X33 Pichia cells. Additional optimizations included the downstream process such as ammonium sulphate precipitation and dialysis. The fusion protein was purified using Ni-NTA purification system with 80% recovery. The purified protein was 100% specific and sensitive in detection of toxoplasmosis.

  4. Development of an in-house enzyme-linked immunosorbent assay based on surface whole cell antigen for diagnosis of Helicobacter pylori infection in patients with gastroduodenal ulcer disease.

    Science.gov (United States)

    Aziz, Faisal; Sherwani, Sikander Khan; Akhtar, Syed Shakeel; Kazmi, Shahana Urooj

    2014-01-01

    Helicobacter pylori (H. pylori) is a causative agent of gastritis, gastroduodenal ulcers and gastric adenocarcinoma. More than 50% world population is colonized by H. pylori, which is closely related to the chronic gastritis and gastric ulcer infection. In this study, a total of 214 gastritis patient's serum samples were screened for anti-H. pylori IgG antibody. A 96-well plate coated with 20 μg/ml antigen and hundred-fold diluted patient's serum was allowed to react. After extensive washing with buffer, 1:2,500 diluted conjugated secondary antibody was added. Later substrate was added to observe positivity by measuring the intensity of color. Statistical analyses were performed, and p value of <0.01 was taken as significant; 84% male patients and 89% female patients, respectively, tested positive for H. pylori, while agewise distribution was 35-45 years males (40%) and 35-55 years females (52%) were found highest number of H. pylori infected patients. In-house ELISA based on surface whole cell antigen (wELISA) showed a sensitivity of 93%, specificity of 100%, accuracy 94% and κ value 0.86 with significant correlation R-0.77020; p < 0.0001. We conclude that H. pylori local isolates surface antigen was satisfactory for diagnosis as different parameters were adjusted according to the local H. pylori isolates. Fluctuations in serum antibody titer predict the variation in an individual's response of the host against H. pylori. In-house wELISA could provide a reliable and a clinically useful method for the diagnosis of H. pylori infection in patients of Karachi, Pakistan.

  5. A fibroblast-associated antigen: Characterization in fibroblasts and immunoreactivity in smooth muscle differentiated stromal cells

    DEFF Research Database (Denmark)

    Rønnov-Jessen, Lone; Celis, Julio E.; van Deurs, Bo

    1992-01-01

    Fibroblasts with smooth muscle differentiation are frequently derived from human breast tissue. Immunofluorescence cytochemistry of a fibroblast-associated antigen recognized by a monoclonal antibody (MAb), 1B10, was analyzed with a view to discriminating smooth muscle differentiated fibroblasts...... from vascular smooth muscle cells. The antigen was detected on the cell surface and in cathepsin D-positive and acridine orange-accumulating vesicular compartments of fibroblasts. Ultrastructurally, the antigen was revealed in coated pits and in endosomal and lysosomal structures. 1B10 recognized three...... immunoreactivity was specific to fibroblasts and smooth muscle differentiated fibroblasts within the context of vascular smooth muscle cells....

  6. Some problems associated with radiolabeling surface antigens on helminth parasites: a brief review

    Energy Technology Data Exchange (ETDEWEB)

    Hayunga, E.G. (Division of Tropical Public Health, Department of Preventive Medicine and Biometrics, Uniformed Services University of the Health Sciences, Bethesda, MD (USA)); Murrell, K.D. (Agricultural Research Service, Beltsville, MD (USA))

    1982-06-01

    Recent developments in technology have facilitated substantial advances in the characterization of surface antigens from a wide variety of both normal and neoplastic cells. However, the immunochemistry of parasites has lagged behind. Efforts to apply conventional radiolabeling methods to helminths have not always been successful. Experimental work with Schistosoma mansoni is reviewed to illustrate common problems encountered in surface labeling studies. These findings should provide insight for the future investigation of other helminth species.

  7. Interplay between carbohydrate and lipid in recognition of glycolipid antigens by natural killer T cells.

    Science.gov (United States)

    Pei, Bo; Vela, Jose Luis; Zajonc, Dirk; Kronenberg, Mitchell

    2012-04-01

    Natural killer T (NKT) cells are a T cell subpopulation that were named originally based on coexpression of receptors found on natural killer (NK) cells, cells of the innate immune system, and by T lymphocytes. The maturation and activation of NKT cells requires presentation of glycolipid antigens by CD1d, a cell surface protein distantly related to the major histocompatibility complex (MHC)-encoded antigen presenting molecules. This specificity distinguishes NKT cells from most CD4(+) and CD8(+) T cells that recognize peptides presented by MHC class I and class II molecules. The rapid secretion of a large amount of both Th1 and Th2 cytokines by activated NKT cells endows them with the ability to play a vital role in the host immune defense against various microbial infections. In this review, we summarize progress on identifying the sources of microbe-derived glycolipid antigens recognized by NKT cells and the biochemical basis for their recognition.

  8. Unique lipid anchor attaches Vi antigen capsule to the surface of Salmonella enterica serovar Typhi.

    Science.gov (United States)

    Liston, Sean D; Ovchinnikova, Olga G; Whitfield, Chris

    2016-06-14

    Polysaccharide capsules are surface structures that are critical for the virulence of many Gram-negative pathogenic bacteria. Salmonella enterica serovar Typhi is the etiological agent of typhoid fever. It produces a capsular polysaccharide known as "Vi antigen," which is composed of nonstoichiometrically O-acetylated α-1,4-linked N-acetylgalactosaminuronic acid residues. This glycan is a component of currently available vaccines. The genetic locus for Vi antigen production is also present in soil bacteria belonging to the genus Achromobacter Vi antigen assembly follows a widespread general strategy with a characteristic glycan export step involving an ATP-binding cassette transporter. However, Vi antigen producers lack the enzymes that build the conserved terminal glycolipid characterizing other capsules using this method. Achromobacter species possess a Vi antigen-specific depolymerase enzyme missing in S enterica Typhi, and we exploited this enzyme to isolate acylated Vi antigen termini. Mass spectrometry analysis revealed a reducing terminal N-acetylhexosamine residue modified with two β-hydroxyl acyl chains. This terminal structure resembles one half of lipid A, the hydrophobic portion of bacterial lipopolysaccharides. The VexE protein encoded in the Vi antigen biosynthesis locus shares similarity with LpxL, an acyltransferase from lipid A biosynthesis. In the absence of VexE, Vi antigen is produced, but its physical properties are altered, its export is impaired, and a Vi capsule structure is not assembled on the cell surface. The structure of the lipidated terminus dictates a unique assembly mechanism and has potential implications in pathogenesis and vaccine production.

  9. Flow Cytometric Analysis of T, B, and NK Cells Antigens in Patients with Mycosis Fungoides

    Science.gov (United States)

    Yazıcı, Serkan; Bülbül Başkan, Emel; Budak, Ferah; Oral, Barbaros; Adim, Şaduman Balaban; Ceylan Kalin, Zübeyde; Özkaya, Güven; Aydoğan, Kenan; Saricaoğlu, Hayriye; Tunali, Şükran

    2015-01-01

    We retrospectively analyzed the clinicopathological correlation and prognostic value of cell surface antigens expressed by peripheral blood mononuclear cells in patients with mycosis fungoides (MF). 121 consecutive MF patients were included in this study. All patients had peripheral blood flow cytometry as part of their first visit. TNMB and histopathological staging of the cases were retrospectively performed in accordance with International Society for Cutaneous Lymphomas/European Organization of Research and Treatment of Cancer (ISCL/EORTC) criteria at the time of flow cytometry sampling. To determine prognostic value of cell surface antigens, cases were divided into two groups as stable and progressive disease. 17 flow cytometric analyses of 17 parapsoriasis (PP) and 11 analyses of 11 benign erythrodermic patients were included as control groups. Fluorescent labeled monoclonal antibodies were used to detect cell surface antigens: T cells (CD3+, CD4+, CD8+, TCRαβ+, TCRγδ+, CD7+, CD4+CD7+, CD4+CD7−, and CD71+), B cells (HLA-DR+, CD19+, and HLA-DR+CD19+), NKT cells (CD3+CD16+CD56+), and NK cells (CD3−CD16+CD56+). The mean value of all cell surface antigens was not statistically significant between parapsoriasis and MF groups. Along with an increase in cases of MF stage statistically significant difference was found between the mean values of cell surface antigens. Flow cytometric analysis of peripheral blood cell surface antigens in patients with mycosis fungoides may contribute to predicting disease stage and progression. PMID:26788525

  10. Flow Cytometric Analysis of T, B, and NK Cells Antigens in Patients with Mycosis Fungoides

    Directory of Open Access Journals (Sweden)

    Serkan Yazıcı

    2015-01-01

    Full Text Available We retrospectively analyzed the clinicopathological correlation and prognostic value of cell surface antigens expressed by peripheral blood mononuclear cells in patients with mycosis fungoides (MF. 121 consecutive MF patients were included in this study. All patients had peripheral blood flow cytometry as part of their first visit. TNMB and histopathological staging of the cases were retrospectively performed in accordance with International Society for Cutaneous Lymphomas/European Organization of Research and Treatment of Cancer (ISCL/EORTC criteria at the time of flow cytometry sampling. To determine prognostic value of cell surface antigens, cases were divided into two groups as stable and progressive disease. 17 flow cytometric analyses of 17 parapsoriasis (PP and 11 analyses of 11 benign erythrodermic patients were included as control groups. Fluorescent labeled monoclonal antibodies were used to detect cell surface antigens: T cells (CD3+, CD4+, CD8+, TCRαβ+, TCRγδ+, CD7+, CD4+CD7+, CD4+CD7−, and CD71+, B cells (HLA-DR+, CD19+, and HLA-DR+CD19+, NKT cells (CD3+CD16+CD56+, and NK cells (CD3−CD16+CD56+. The mean value of all cell surface antigens was not statistically significant between parapsoriasis and MF groups. Along with an increase in cases of MF stage statistically significant difference was found between the mean values of cell surface antigens. Flow cytometric analysis of peripheral blood cell surface antigens in patients with mycosis fungoides may contribute to predicting disease stage and progression.

  11. Protection from anti-TCR/CD3-induced apoptosis in immature thymocytes by a signal through thymic shared antigen-1/stem cell antigen-2

    OpenAIRE

    1996-01-01

    During T cell development in the thymus, the expression of thymic shared antigen-1 (TSA-1)/stem cell antigen-2 (Sca-2), a glycosylphosphatidylinositol (GPI)-anchored differentiation antigen, is developmentally regulated. The expression level of TSA-1 is the highest in most immature CD4- CD8- thymocytes, high in CD4+ CD8+ thymocytes, but barely detectable in mature CD4+ CD8- or CD4- CD8- thymocytes and peripheral T cells. We have previously shown that surface TSA-1 expression in peripheral T c...

  12. Impact of Cell-surface Antigen Expression on Target Engagement and Function of an Epidermal Growth Factor Receptor × c-MET Bispecific Antibody*

    Science.gov (United States)

    Jarantow, Stephen W.; Bushey, Barbara S.; Pardinas, Jose R.; Boakye, Ken; Lacy, Eilyn R.; Sanders, Renouard; Sepulveda, Manuel A.; Moores, Sheri L.; Chiu, Mark L.

    2015-01-01

    The efficacy of engaging multiple drug targets using bispecific antibodies (BsAbs) is affected by the relative cell-surface protein levels of the respective targets. In this work, the receptor density values were correlated to the in vitro activity of a BsAb (JNJ-61186372) targeting epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-MET). Simultaneous binding of the BsAb to both receptors was confirmed in vitro. By using controlled Fab-arm exchange, a set of BsAbs targeting EGFR and c-MET was generated to establish an accurate receptor quantitation of a panel of lung and gastric cancer cell lines expressing heterogeneous levels of EGFR and c-MET. EGFR and c-MET receptor density levels were correlated to the respective gene expression levels as well as to the respective receptor phosphorylation inhibition values. We observed a bias in BsAb binding toward the more highly expressed of the two receptors, EGFR or c-MET, which resulted in the enhanced in vitro potency of JNJ-61186372 against the less highly expressed target. On the basis of these observations, we propose an avidity model of how JNJ-61186372 engages EGFR and c-MET with potentially broad implications for bispecific drug efficacy and design. PMID:26260789

  13. Impact of Cell-surface Antigen Expression on Target Engagement and Function of an Epidermal Growth Factor Receptor × c-MET Bispecific Antibody.

    Science.gov (United States)

    Jarantow, Stephen W; Bushey, Barbara S; Pardinas, Jose R; Boakye, Ken; Lacy, Eilyn R; Sanders, Renouard; Sepulveda, Manuel A; Moores, Sheri L; Chiu, Mark L

    2015-10-09

    The efficacy of engaging multiple drug targets using bispecific antibodies (BsAbs) is affected by the relative cell-surface protein levels of the respective targets. In this work, the receptor density values were correlated to the in vitro activity of a BsAb (JNJ-61186372) targeting epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-MET). Simultaneous binding of the BsAb to both receptors was confirmed in vitro. By using controlled Fab-arm exchange, a set of BsAbs targeting EGFR and c-MET was generated to establish an accurate receptor quantitation of a panel of lung and gastric cancer cell lines expressing heterogeneous levels of EGFR and c-MET. EGFR and c-MET receptor density levels were correlated to the respective gene expression levels as well as to the respective receptor phosphorylation inhibition values. We observed a bias in BsAb binding toward the more highly expressed of the two receptors, EGFR or c-MET, which resulted in the enhanced in vitro potency of JNJ-61186372 against the less highly expressed target. On the basis of these observations, we propose an avidity model of how JNJ-61186372 engages EGFR and c-MET with potentially broad implications for bispecific drug efficacy and design.

  14. Antigen specificity of invariant natural killer T-cells

    Directory of Open Access Journals (Sweden)

    Alysia M. Birkholz

    2015-12-01

    Full Text Available Natural killer T-cells, with an invariant T-cell antigen receptor α-chain (iNKT cells, are unique and conserved subset of lymphocytes capable of altering the immune system through their rapid and potent cytokine responses. They are reactive to lipid antigens presented by the CD1d molecule, an antigen-presenting molecule that is not highly polymorphic. iNKT cell responses frequently involve mixtures of cytokines that work against each other, and therefore attempts are underway to develop synthetic antigens that elicit only strong interferon-gamma (IFNγ or only strong interleukin-4 responses but not both. Strong IFNγ responses may correlate with tighter binding to CD1d and prolonged stimulation of iNKT cells, and this may be useful for vaccine adjuvants and for stimulating anti-tumor responses. iNKT cells are self-reactive although the structure of the endogenous antigen is controversial. By contrast, bacterial and fungal lipids that engage the T-cell receptor and activate IFNγ from iNKT cells have been identified from both pathogenic and commensal organisms and the responses are in some cases highly protective from pathogens in mice. It is possible that the expanding knowledge of iNKT cell antigens and iNKT cell activation will provide the basis for therapies for patients suffering from infectious and immune diseases and cancer.

  15. Antigen specificity of invariant natural killer T-cells.

    Science.gov (United States)

    Birkholz, Alysia M; Kronenberg, Mitchell

    2015-12-01

    Natural killer T-cells, with an invariant T-cell antigen receptor α-chain (iNKT cells), are unique and conserved subset of lymphocytes capable of altering the immune system through their rapid and potent cytokine responses. They are reactive to lipid antigens presented by the CD1d molecule, an antigen-presenting molecule that is not highly polymorphic. iNKT cell responses frequently involve mixtures of cytokines that work against each other, and therefore attempts are underway to develop synthetic antigens that elicit only strong interferon-gamma (IFNγ) or only strong interleukin-4 responses but not both. Strong IFNγ responses may correlate with tighter binding to CD1d and prolonged stimulation of iNKT cells, and this may be useful for vaccine adjuvants and for stimulating anti-tumor responses. iNKT cells are self-reactive although the structure of the endogenous antigen is controversial. By contrast, bacterial and fungal lipids that engage the T-cell receptor and activate IFNγ from iNKT cells have been identified from both pathogenic and commensal organisms and the responses are in some cases highly protective from pathogens in mice. It is possible that the expanding knowledge of iNKT cell antigens and iNKT cell activation will provide the basis for therapies for patients suffering from infectious and immune diseases and cancer.

  16. Fibroblasts as Efficient Antigen-Presenting Cells in Lymphoid Organs

    Science.gov (United States)

    Kundig, Thomas M.; Bachmann, Martin F.; Dipaolo, Claudio; Simard, John J. L.; Battegay, Manuel; Lother, Heinz; Gessner, Andre; Kuhlcke, Klaus; Ohashi, Pamela S.; Hengartner, Hans; Zinkernagel, Rolf M.

    1995-06-01

    Only so-called "professional" antigen-presenting cells (APCs) of hematopoietic origin are believed capable of inducing T lymphocyte responses. However, fibroblasts transfected with viral proteins directly induced antiviral cytotoxic T lymphocyte responses in vivo, without involvement of host APCs. Fibroblasts induced T cells only in the milieu of lymphoid organs. Thus, antigen localization affects self-nonself discrimination and cell-based vaccine strategies.

  17. Responses of synovial fluid and peripheral blood mononuclear cells to bacterial antigens and autologous antigen presenting cells.

    Science.gov (United States)

    Klasen, I S; Melief, M J; Swaak, T J; Severijnen, A J; Hazenberg, M P

    1993-01-01

    The specificity of T cells in the inflamed joints of patients with rheumatoid arthritis (RA) has been the subject of much study. Bacterial antigens are suspect in the aetiology of rheumatic diseases. The responsiveness of the mononuclear cell fraction of peripheral blood and synovial fluid of patients with RA and of patients with rheumatic diseases other than RA to bacterial antigens such as cell wall fragments of the anaerobic intestinal flora, cell wall fragments of Streptococcus pyogenes, intestinal flora derived peptidoglycan polysaccharide complexes, the 65 kilodalton protein of Mycobacterium tuberculosis, and muramyldipeptide was investigated. No significant difference in response was found to all these bacterial antigens in the synovial fluid of patients with RA compared with the responses in patients with other rheumatic diseases. The highest responsiveness in the synovial fluid of the patients with RA was to the streptococcal cell wall fragments and to the 65 kilodalton protein. Higher responses to several bacterial antigens in the synovial fluid of patients with RA were found compared with peripheral blood from the same patient group. The antigen presenting cell population of the synovial fluid in patients with RA and the patients with other rheumatic diseases was found to be stimulatory for autologous peripheral blood T cells even in the absence of antigen. This suggests an important role for the synovial antigen presenting cell in the aetiology of inflammatory joint diseases. PMID:8447692

  18. A human T cell clone that mediates the monocyte procoagulant response to specific sensitizing antigen.

    Science.gov (United States)

    Schwartz, B S; Reitnauer, P J; Hank, J A; Sondel, P M

    1985-09-01

    A panel of human purified protein derivative of the tubercle bacillus (PPD)-reactive T cell clones was derived by cloning out of soft agar followed by cultivation on inactivated feeder cells in the presence of interleukin-2. 1 of 4 clones tested was able to mediate an increase in monocyte procoagulant activity (PCA) in response to PPD. All four clones had identical surface marker phenotypes (T4+, T8-) and proliferated in response to antigen. The reactive T cell clone possessed no PCA of its own, but upon being presented with PPD was able to instruct monocytes to increase their expression of PCA. Antigen presentation could be performed only by autologous monocytes; allogeneic monocytes from donors unrelated to the donor of the reactive clone could not present antigen to cells of the clone in a way that would initiate the procoagulant response. Cells of the reactive clone did not mediate increased monocyte PCA in response to Candida, even though peripheral blood mononuclear cells from the donor demonstrated increased PCA to both Candida and PPD. Thus, the PCA response to specific antigen can be mediated by a single clone of cells that shows specificity in the recognition of both antigen and antigen presenting cell.

  19. Immunosensor Based on Surface Plasmon Resonance for Antigen Recognition

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    A novel immunosensor based on surface plasmon resonance(SPR)has been developed for the recognition of antigen.The sensor was designed on the basis of the fixed angle of incidence and measuring the reflected intensities in a wavelength range of 430-750 nm in real-time. An ultra-bright white light-emitting diode(LED)was used as the light source. Molecular self-assembling in solution was used to form the sensing membrane on gold substrate. It has been seen that the sensitivity of the SPR sensor with 3-mercaptopropionic acid(MPA)/protein A(SPA) sensing membrane is considerably higher than that with MPA or SPA modified Sensing membrane. The kinetic processes on the sensing membrane were studied. The human B factor(Bf), an activator of complement 3(C3), was recognized among the other antigens. This sensor can also be used for other antigen/antibody or adaptor/receptor recognition. Under optimized experimental conditions, the sensor has good selectivity, repeatability, and reversibility.

  20. Cancer Patient T Cells Genetically Targeted to Prostate-Specific Membrane Antigen Specifically Lyse Prostate Cancer Cells and Release Cytokines in Response to Prostate-Specific Membrane Antigen

    Directory of Open Access Journals (Sweden)

    Michael C. Gong

    1999-06-01

    Full Text Available The expression of immunoglobulin-based artificial receptors in normal T lymphocytes provides a means to target lymphocytes to cell surface antigens independently of major histocompatibility complex restriction. Such artificial receptors have been previously shown to confer antigen-specific tumoricidal properties in murine T cells. We constructed a novel ζ chain fusion receptor specific for prostate-specific membrane antigen (PSMA termed Pz-1. PSMA is a cell-surface glycoprotein expressed on prostate cancer cells and the neovascular endothelium of multiple carcinomas. We show that primary T cells harvested from five of five patients with different stages of prostate cancer and transduced with the Pz-1 receptor readily lyse prostate cancer cells. Having established a culture system using fibroblasts that express PSMA, we next show that T cells expressing the Pz-1 receptor release cytokines in response to cell-bound PSMA. Furthermore, we show that the cytokine release is greatly augmented by B7.1-mediated costimulation. Thus, our findings support the feasibility of adoptive cell therapy by using genetically engineered T cells in prostate cancer patients and suggest that both CD4+ and CD8+ T lymphocyte functions can be synergistically targeted against tumor cells.

  1. Antigen loading on dendritic cells affects the lell function in stimulating T cells.

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To study the effect of antigen loading on dendritic cells (DC). Methods: DCs collected from peripheral blood monocytes were loaded with a tumor antigen from XG-7 cell line. These DCs were then co-cultured with allogeneic T cells and were compared with those DCs without antigen exposure.

  2. Activation of Type II Cells into Regenerative Stem Cell Antigen-1+ Cells during Alveolar Repair

    Science.gov (United States)

    Kumar, Varsha Suresh; Zhang, Wei; Rehman, Jalees; Malik, Asrar B.

    2015-01-01

    The alveolar epithelium is composed of two cell types: type I cells comprise 95% of the gas exchange surface area, whereas type II cells secrete surfactant, while retaining the ability to convert into type I cells to induce alveolar repair. Using lineage-tracing analyses in the mouse model of Pseudomonas aeruginosa–induced lung injury, we identified a population of stem cell antigen (Sca)-1–expressing type II cells with progenitor cell properties that mediate alveolar repair. These cells were shown to be distinct from previously reported Sca-1–expressing bronchioalveolar stem cells. Microarray and Wnt reporter studies showed that surfactant protein (Sp)-C+Sca-1+ cells expressed Wnt signaling pathway genes, and inhibiting Wnt/β-catenin signaling prevented the regenerative function of Sp-C+Sca-1+ cells in vitro. Thus, P. aeruginosa–mediated lung injury induces the generation of a Sca-1+ subset of type II cells. The progenitor phenotype of the Sp-C+Sca-1+ cells that mediates alveolar epithelial repair might involve Wnt signaling. PMID:25474582

  3. Viral sequestration of antigen subverts cross presentation to CD8(+ T cells.

    Directory of Open Access Journals (Sweden)

    Eric F Tewalt

    2009-05-01

    Full Text Available Virus-specific CD8(+ T cells (T(CD8+ are initially triggered by peptide-MHC Class I complexes on the surface of professional antigen presenting cells (pAPC. Peptide-MHC complexes are produced by two spatially distinct pathways during virus infection. Endogenous antigens synthesized within virus-infected pAPC are presented via the direct-presentation pathway. Many viruses have developed strategies to subvert direct presentation. When direct presentation is blocked, the cross-presentation pathway, in which antigen is transferred from virus-infected cells to uninfected pAPC, is thought to compensate and allow the generation of effector T(CD8+. Direct presentation of vaccinia virus (VACV antigens driven by late promoters does not occur, as an abortive infection of pAPC prevents production of these late antigens. This lack of direct presentation results in a greatly diminished or ablated T(CD8+ response to late antigens. We demonstrate that late poxvirus antigens do not enter the cross-presentation pathway, even when identical antigens driven by early promoters access this pathway efficiently. The mechanism mediating this novel means of viral modulation of antigen presentation involves the sequestration of late antigens within virus factories. Early antigens and cellular antigens are cross-presented from virus-infected cells, as are late antigens that are targeted to compartments outside of the virus factories. This virus-mediated blockade specifically targets the cross-presentation pathway, since late antigen that is not cross-presented efficiently enters the MHC Class II presentation pathway. These data are the first to describe an evasion mechanism employed by pathogens to prevent entry into the cross-presentation pathway. In the absence of direct presentation, this evasion mechanism leads to a complete ablation of the T(CD8+ response and a potential replicative advantage for the virus. Such mechanisms of viral modulation of antigen presentation

  4. Highly sensitive potentiometric immunosensor for hepatitis B surface antigen diagnosis

    Institute of Scientific and Technical Information of China (English)

    YUAN; Ruo; TANG; Dianping; CHAI; Yaqin; ZHANG; Lingyan; LI

    2005-01-01

    A highly sensitive potentiometric immunosensor for the diagnoses of epidemic diseases has been developed by means of self-assembly to immobilize hepatitis B surface antibody (HBsAb) for the detection of hepatitis B surface antigen (HBsAg) as a model. At first, the Nafion containing -SO3- groups was immobilized on a platinum electrode surface to absorb the -NH3+ groups of antibody molecules via the opposite-charged adsorption technique, in the meantime, hepatitis B surface antibodies were adsorbed onto the surface of Au nanoparticles, then hepatitis B surface antibodies and Au nanopartilces were entrapped into polyvinyl butyral on the surface of Nafion film. The modified procedure was further characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The influence and factors influencing the performance of resulting immunosensor were studied in detail. The resulting immunosensor exhibited sigmoid curve with log HBsAg concentrations, high sensitivity, wide linear range from 26 to 1280 ng·mL-1 with a detection limit of 3.1 ng·mL-1, rapid potentiometric response (4 months). Analytical results of clinical samples show that the developed immunoassay is comparable with the enzyme-linked immunosorbent assays (ELISAs) method, implying a promising alternative approach for detecting HBsAg in the clinical diagnosis.

  5. Maturation of recombinant hepatitis B virus surface antigen particles.

    Science.gov (United States)

    Zhao, Qinjian; Wang, Yang; Freed, Daniel; Fu, Tong-Ming; Gimenez, Juan A; Sitrin, Robert D; Washabaugh, Michael W

    2006-01-01

    The major surface antigen of Hepatitis B virus (HBsAg) is a cysteine-rich, lipid-bound protein with 226 amino acids. Recombinant HBsAg (rHBsAg) with associated lipids can self-assemble into 22-nm immunogenic spherical particles, which are used in licensed Hepatitis B vaccines. Little is known about the structural evolvement or maturation upon assembly beyond an elevated level of disulfide formation. In this paper, we further characterized the maturation of HBsAg particles with respect to their degree of cross-linking, morphological changes, and changes in conformational flexibility. The lipid-containing rHBsAg particles undergo KSCN- and heat-induced maturation by formation of additional intra- and inter-molecular disulfide bonds. Direct measurements with atomic force microscopy (AFM) revealed morphological changes upon maturation through KSCN-induced and heat-/storage-incurred oxidative refolding. Particle uniformity and regularity was greatly improved, and protrusions formed by the protein subunits were more prominent on the surface of the mature particles. Decreased conformational flexibility in the mature rHBsAg particles was demonstrated by millisecond-scale unfolding kinetics in the presence of an environment-sensitive conformation probe. Both the accessible hydrophobic cavities under native conditions and the changeable hydrophobic cavities upon denaturant-induced unfolding showed substantial decrease upon maturation of the rHBsAg particles. These changes in the structural properties may be critical for the antigenicity and immuno-genicity of this widely-used vaccine component.

  6. Serum anti-hepatitis B surface antigen in hemodialysis patients

    Directory of Open Access Journals (Sweden)

    Rafieian-Kopaei Mahmoud

    2012-01-01

    Full Text Available To evaluate the immune response to hepatitis B vaccination in stable hemodialysis (HD patients, a retro-prospective investigation was conducted on 68 HD patients. Participants were vaccinated against hepatitis B virus with an intramuscular hepatitis B vaccination schedule, 40 micrograms at 0, 1, and 6 months. The serum antibody level against hepatitis B surface antigen (HBs in HD patients was 35±55. In this study, no significant differences of Anti-HBs antibody between diabetic and non-diabetics or male and female subjects were observed. There were not any significant correlation between antibody against HBs-Ag and serum albumin. There was not significant correlation between anti-HBs antibody and age, proportion of HD, duration of HD or dialysis efficacy. In this study, there was not significant correlation between serum antibody level against hepatitis B surface antigen and some demographic indices of HD patients, however, these findings need to re-test in other centers with more participants.

  7. Segmented filamentous bacteria antigens presented by intestinal dendritic cells drive mucosal Th17 cell differentiation.

    Science.gov (United States)

    Goto, Yoshiyuki; Panea, Casandra; Nakato, Gaku; Cebula, Anna; Lee, Carolyn; Diez, Marta Galan; Laufer, Terri M; Ignatowicz, Leszek; Ivanov, Ivaylo I

    2014-04-17

    How commensal microbiota contributes to immune cell homeostasis at barrier surfaces is poorly understood. Lamina propria (LP) T helper 17 (Th17) cells participate in mucosal protection and are induced by commensal segmented filamentous bacteria (SFB). Here we show that MHCII-dependent antigen presentation of SFB antigens by intestinal dendritic cells (DCs) is crucial for Th17 cell induction. Expression of MHCII on CD11c(+) cells was necessary and sufficient for SFB-induced Th17 cell differentiation. Most SFB-induced Th17 cells recognized SFB in an MHCII-dependent manner. SFB primed and induced Th17 cells locally in the LP and Th17 cell induction occurred normally in mice lacking secondary lymphoid organs. The importance of other innate cells was unveiled by the finding that MHCII deficiency in group 3 innate lymphoid cells (ILCs) resulted in an increase in SFB-independent Th17 cell differentiation. Our results outline the complex role of DCs and ILCs in the regulation of intestinal Th17 cell homeostasis.

  8. Facts on the fragmentation of antigens in presenting cells, on the association of antigen fragments with MHC molecules in cell-free systems, and speculation on the cell biology of antigen processing

    DEFF Research Database (Denmark)

    Werdelin, O; Mouritsen, S; Petersen, B L;

    1988-01-01

    The processing of a protein antigen is a multi-step event taking place in antigen-presenting cells. Processing is a prerequisite for the recognition of most antigens by T lymphocytes. The antigen is ingested by endocytosis, transported to an acid cellular compartment and subjected to proteolytic ...

  9. Antigen-presenting cells in human cutaneous leishmaniasis due to Leishmania major

    DEFF Research Database (Denmark)

    ElHassan, A M; Gaafar, A; Theander, T G

    1995-01-01

    , identified morphologically and by their expression of specific cell markers, included Langerhans cells, macrophages, follicular dendritic cells, and interdigitating reticulum cells of the paracortex of lymph nodes. These cells expressed MHC class II antigens and contained Leishmania antigen. Since some...

  10. Flow cytometric measurement of RNA synthesis based on bromouridine labelling and combined with measurement of DNA content or cell surface antigen

    DEFF Research Database (Denmark)

    Jensen, P O; Larsen, J; Larsen, J K

    1993-01-01

    RNA synthesis can be analysed in nuclei or cells labelled with 5-bromouridine (BrUrd) and stained using cross-reacting anti-bromodeoxyuridine (BrdUrd) antibody. Flow cytometric dual parameter analysis of BrUrd incorporation and DNA content in nuclear suspensions of human blood lymphocytes showed ...... in HL-60 and K-562 cells was measured simultaneous with CD13 expression....

  11. Kinetics of antibody-induced modulation of respiratory syncytial virus antigens in a human epithelial cell line

    Directory of Open Access Journals (Sweden)

    Gómez-Garcia Beatriz

    2007-07-01

    Full Text Available Abstract Background The binding of viral-specific antibodies to cell-surface antigens usually results in down modulation of the antigen through redistribution of antigens into patches that subsequently may be internalized by endocytosis or may form caps that can be expelled to the extracellular space. Here, by use of confocal-laser-scanning microscopy we investigated the kinetics of the modulation of respiratory syncytial virus (RSV antigen by RSV-specific IgG. RSV-infected human epithelial cells (HEp-2 were incubated with anti-RSV polyclonal IgG and, at various incubation times, the RSV-cell-surface-antigen-antibody complexes (RSV Ag-Abs and intracellular viral proteins were detected by indirect immunoflourescence. Results Interaction of anti-RSV polyclonal IgG with RSV HEp-2 infected cells induced relocalization and aggregation of viral glycoproteins in the plasma membrane formed patches that subsequently produced caps or were internalized through clathrin-mediated endocytosis participation. Moreover, the concentration of cell surface RSV Ag-Abs and intracellular viral proteins showed a time dependent cyclic variation and that anti-RSV IgG protected HEp-2 cells from viral-induced death. Conclusion The results from this study indicate that interaction between RSV cell surface proteins and specific viral antibodies alter the expression of viral antigens expressed on the cells surface and intracellular viral proteins; furthermore, interfere with viral induced destruction of the cell.

  12. Detection of DR antigen on leukemic cells from a patient suffering from adult T-cell leukemia and progressive systemic sclerosis.

    Directory of Open Access Journals (Sweden)

    Mizushima,Keiichi

    1983-10-01

    Full Text Available This report concerns an unusual case of adult T cell leukemia (ATL complicated with progressive systemic sclerosis (PSS. The surface markers of peripheral blood mononuclear cells (PBM and lymph node cells, both of which mainly consisted of leukemic cells, were examined. The effect of these cells on the pokeweed mitogen (PWM-induced IgG synthesis by normal PBM also was studied. The leukemic cells formed rosettes with sheep red blood cells (SRBC; E and expressed T cell antigen, Leu-1, and DR antigen. The detection of cell surface antigens was carried out by employing monoclonal antibodies against these antigens. We diagnosed this case as DR positive ATL. In terms of the immunoregulatory function of these leukemic cells, the co-culture experiments showed that these cells had some suppressive effect on the PWM-induced IgG production by allogeneic normal PBM.

  13. The peripheral blood fibrocyte is a potent antigen-presenting cell capable of priming naive T cells in situ.

    Science.gov (United States)

    Chesney, J; Bacher, M; Bender, A; Bucala, R

    1997-06-10

    Recent studies have identified a novel population of blood-borne cells, termed fibrocytes, that have a distinct cell surface phenotype (collagen+/CD13(+)/CD34(+)/CD45(+)), rapidly enter sites of tissue injury, and synthesize connective tissue matrix molecules. We found by flow cytometry that purified human fibrocytes express each of the known surface components that are required for antigen presentation, including class II major histocompatability complex molecules (HLA-DP, -DQ, and -DR), the costimulatory molecules CD80 and CD86, and the adhesion molecules CD11a, CD54, and CD58. Human fibrocytes induced antigen-presenting cell-dependent T cell proliferation when cultured with specific antigen and this proliferative activity was significantly higher than that induced by monocytes and nearly as high as that induced by purified dendritic cells. Mouse fibrocytes also were found to express the surface components required for antigen presentation and to function as potent APCs in vitro. Mouse fibrocytes pulsed in vitro with the HIV-proteins p24 or gp120 and delivered to a site of cutaneous injury were found to migrate to proximal lymph nodes and to specifically prime naive T cells. These data suggest that fibrocytes play an early and important role in the initiation of antigen-specific immunity.

  14. Recognition of antigen-specific B-cell receptors from chronic lymphocytic leukemia patients by synthetic antigen surrogates.

    Science.gov (United States)

    Sarkar, Mohosin; Liu, Yun; Morimoto, Jumpei; Peng, Haiyong; Aquino, Claudio; Rader, Christoph; Chiorazzi, Nicholas; Kodadek, Thomas

    2014-12-18

    In patients with chronic lymphocytic leukemia (CLL), a single neoplastic antigen-specific B cell accumulates and overgrows other B cells, leading to immune deficiency. CLL is often treated with drugs that ablate all B cells, leading to further weakening of humoral immunity, and a more focused therapeutic strategy capable of targeting only the pathogenic B cells would represent a significant advance. One approach to this would be to develop synthetic surrogates of the CLL antigens allowing differentiation of the CLL cells and healthy B cells in a patient. Here, we describe nonpeptidic molecules capable of targeting antigen-specific B cell receptors with good affinity and selectivity using a combinatorial library screen. We demonstrate that our hit compounds act as synthetic antigen surrogates and recognize CLL cells and not healthy B cells. Additionally, we argue that the technology we developed can be used to identify other classes of antigen surrogates.

  15. Construction of Recombinant Modified Vaccinia Ankara (MVA) Expressing Hepatitis B Virus Surface Antigen

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The T lymphocyte response has been shown to be the determinant in the clearance of many viral infections.Hence, therapeutic vaccine candidates against HBV are designed to enhance this response of the immune system.Vaccinia virus vector-based vaccines have been proposed as excellent candidates to elicit long-term and strong T lymphocyte mediated immune responses. In this study, the recombinant MVA expressing HBV surface antigen has been constructed, which can elicit a potent T cell mediated response. The ELISA results for the surface protein in the medium of the recombinant MVA, strongly indicate that the recombinant virus has been successfully obtained.

  16. Epstein-Barr virus nuclear antigen 2 specifically induces expression of the B-cell activation antigen CD23

    Energy Technology Data Exchange (ETDEWEB)

    Wang, F.; Gregory, C.D.; Rowe, M.; Rickinson, A.B.; Wang, D.; Birkenbach, M.; Kikutani, H.; Kishimoto, T.; Kieff, E.

    1987-05-01

    Epstein-Barr virus (EBV) infection of EBV-negative Burkitt lymphoma (BL) cells includes some changes similar to those seen in normal B lymphocytes that have been growth transformed by EBV. The role of individual EBV genes in this process was evaluated by introducing each of the viral genes that are normally expressed in EBV growth-transformed and latently infected lymphoblasts into an EBV-negative BL cell line, using recombinant retrovirus-mediated transfer. Clones of cells were derived that stably express the EBV nuclear antigen 1 (EBNA-1), EBNA-2, EBNA-3, EBNA-leader protein, or EBV latent membrane protein (LMP). These were compared with control clones infected with the retrovirus vector. All 10 clones converted to EBNA-2 expression differed from control clones or clones expressing other EBV proteins by growth in tight clumps and by markedly increased expression of one particular surface marker of B-cell activation, CD23. Other activation antigens were unaffected by EBNA-2 expression, as were markers already expressed on the parent BL cell line. The results indicate that EBNA-2 is a specific direct or indirect trans-activator of CD23. This establishes a link between an EBV gene and cell gene expression. Since CD23 has been implicated in the transduction of B-cell growth signals, its specific induction by EBNA-2 could be important in EBV induction of B-lymphocyte transformation.

  17. T Cells as Antigen Carriers for Anti-tumor Vaccination.

    Science.gov (United States)

    Traversari, Catia; Russo, Vincenzo

    2016-01-01

    The exploitation of the physiologic processing and presenting machinery of dendritic cells (DCs) by in vivo loading of tumor-associated antigens may improve the immunogenic potential and clinical efficacy of DC-based cancer vaccines. The approach developed by our group was based on the clinical observation that some patients treated with the infusion of donor lymphocytes transduced to express the HSV-TK suicide gene for relapse of hematologic malignancies, after allogeneic hematopoietic stem cell transplantation, developed a T cell-mediated immune response specifically directed against the HSV-TK gene product.We demonstrated that lymphocytes genetically modified to express HSV-TK as well as self/tumor antigens, acting as antigen carriers, efficiently target DCs in vivo in tumor-bearing mice. The infusion of TRP-2-transduced lymphocytes induced the establishment of protective immunity and long-term memory in tumor-bearing mice by cross-presentation of the antigen mediated by the CD11c(+)CD8a(+) DCs subset. A similar approach was applied in a clinical setting. Ten patients affected by MAGE-3(+) metastatic melanoma were treated with autologous lymphocytes retrovirally transduced to express the MAGE-3 tumor antigen. In three patients, the treatment led to the increase of MAGE-3 specific CD8+ and CD4+ effectors and the development of long-term memory, which ultimately correlated with a favorable clinical outcome. Transduced lymphocytes represent an efficient way for in vivo loading of tumor-associated antigens of DCs.

  18. Enhancement of antibody production to hepatitis B surface antigen by anti-idiotypic antibody.

    OpenAIRE

    Kakumu, S; Murase, K.; A Tsubouchi; Yoshioka, K.; Sakamoto, N.

    1986-01-01

    Studies were undertaken to determine whether anti-idiotypic antibody (anti-Id) against antibody to hepatitis B surface antigen (anti-HBs) could modulate in vitro anti-HBs production by human peripheral blood mononuclear cells stimulated with pokeweed mitogen. Peripheral blood mononuclear cells from patients positive for serum anti-HBs produced significantly increased amounts of anti-HBs by the addition of IgG fraction of anti-anti-HBs as well as purified HBsAg in a soluble form when compared ...

  19. Biological role of surface Toxoplasma gondii antigen in development of vaccine

    Institute of Scientific and Technical Information of China (English)

    Ke-Yi Liu; Dian-Bo Zhang; Qing-Kuan Wei; Jin Li; Gui-Ping Li; Jin-Zhi Yu

    2006-01-01

    AIM: To analyze the biological role of the surface antigen of Toxoplasma gondii (T gondii) in development of vaccine.METHODS: The surface antigen of Tgondii (SAG1)was expressed in vitro. The immune response of the host to the antigen was investigated by detection of specific antibody reaction to SAG1 and production of cytokines. Mice were immunized with recombinant SAG1and challenged with lethal strain of T gondii RH. The monoclonal antibody to r-SAG1 was prepared and used to study the effects of SAG1 on T gondii tachyzoites under electromicroscope.RESULTS:The mice immunized with recombinant SAG1 delayed death for 60 h compared to the control group.The recombinant SAG1 induced specific high titer of IgG and IgM antibodies as well as IFN-γ, IL-2 and IL-4cytokines in mice. In contrast, IL-12, IL-6 and TNF-αwere undetectable. When T gondii tachyzoites were treated with the monoclonal antibody to r-SAG1, the parasites were gathered together, destroyed, deformed,swollen, and holes and gaps formed on the surface.CONCLUSION: SAG1 may be an excellent vaccine candidate against T gondii. The immune protection induced by SAG1 against Tgondii may be regulated by both hormone- and cell-mediated immune response.

  20. Antigen-induced and non-antigen-induced histamine release from rat mast cells sensitized with mouse antiserum.

    Directory of Open Access Journals (Sweden)

    Kurose,Masao

    1981-10-01

    Full Text Available Marked IgE-mediated histamine release from rat mast cells sensitized in vitro with mouse antiserum occurs in the presence of added Ca++ and phosphatidylserine (PS, although a considerable degree of antigen-induced histamine release which may utilize intracellular or cell-bound calcium is also observed. The decay in the responsiveness to Ca++ of the sensitized cells stimulated by antigen in Ca++-free medium in the presence of PS is relatively slow, and maximum release is produced by Ca++ added 1 min after antigen. Histamine release also occurs when Ca++ is added after PS in the absence of antigen to the sensitized cells suspended in Ca++-free medium. Unlike the antigen-induced release, the intensity of this non-antigen-induced release varies depending on both mast-cell and antiserum pools. A heat-labile factor(s, which is different from antigen-specific IgE antibody and is also contained in normal mouse serum, is involved in this reaction. In the antigen-nondependent (PS + Ca++-induced release, no decay in the responsiveness to Ca++ is observed after PS addition. Both the antigen-induced and non-antigen-induced release are completed fairly rapidly and are dependent of temperature, pH and energy.

  1. Isolation of the phagocytosis-inducing IgG-binding antigen on senescent somatic cells

    Science.gov (United States)

    Kay, Marguerite M. B.

    1981-02-01

    To remove senescent red blood cells (RBCs) from the circulation, macrophages must distinguish them from mature RBCs. That is achieved by a specific recognition system1,2. An antigen that develops on the surface of a senescing RBC is recognized and bound by the Fab region1 of an IgG autoantibody in the serum2. Subsequently the Fc region of the autoantibody is recognized and bound by a macrophage3, which proceeds to phagocytose the RBC. The antigenic molecule can be extracted from senescent but not young RBCs with Triton X-100 (ref. 4), although 10-30% as much antigen can be extracted from middle-aged as from senescent RBCs4. I have now used IgG autoantibodies eluted from senescent RBCs to isolate and purify the IgG-binding antigen on senescent RBCs, andto detect the antigen on other somatic cells. The antigen is a ~=62,000-Mr protein which is present on stored platelets, lymphocytes and neutrophils, and on cultured human adult liver and embryonic kidney cells, as well as senescent RBCs.

  2. Long-term in vivo provision of antigen-specific T cell immunity by programming hematopoietic stem cells

    Science.gov (United States)

    Yang, Lili; Baltimore, David

    2005-03-01

    A method to genetically program mouse hematopoietic stem cells to develop into functional CD8 or CD4 T cells of defined specificity in vivo is described. For this purpose, a bicistronic retroviral vector was engineered that efficiently delivers genes for both and chains of T cell receptor (TCR) to hematopoietic stem cells. When modified cell populations were used to reconstruct the hematopoietic lineages of recipient mice, significant percentages of antigen-specific CD8 or CD4 T cells were observed. These cells expressed normal surface markers and responded to peptide antigen stimulation by proliferation and cytokine production. Moreover, they could mature into memory cells after peptide stimulation. Using TCRs specific for a model tumor antigen, we found that the recipient mice were able to partially resist a challenge with tumor cells carrying the antigen. By combining cells modified with CD8- and CD4-specific TCRs, and boosting with dendritic cells pulsed with cognate peptides, complete suppression of tumor could be achieved and even tumors that had become established would regress and be eliminated after dendritic cell/peptide immunization. This methodology of "instructive immunotherapy" could be developed for controlling the growth of human tumors and attacking established pathogens.

  3. A Rough Energy Landscape to Describe Surface-Linked Antibody and Antigen Bond Formation

    Science.gov (United States)

    Limozin, Laurent; Bongrand, Pierre; Robert, Philippe

    2016-01-01

    Antibodies and B cell receptors often bind their antigen at cell-cell interface while both molecular species are surface-bound, which impacts bond kinetics and function. Despite the description of complex energy landscapes for dissociation kinetics which may also result in significantly different association kinetics, surface-bound molecule (2D) association kinetics usually remain described by an on-rate due to crossing of a single free energy barrier, and few experimental works have measured association kinetics under conditions implying force and two-dimensional relative ligand-receptor motion. We use a new laminar flow chamber to measure 2D bond formation with systematic variation of the distribution of encounter durations between antigen and antibody, in a range from 0.1 to 10 ms. Under physiologically relevant forces, 2D association is 100-fold slower than 3D association as studied by surface plasmon resonance assays. Supported by brownian dynamics simulations, our results show that a minimal encounter duration is required for 2D association; an energy landscape featuring a rough initial part might be a reasonable way of accounting for this. By systematically varying the temperature of our experiments, we evaluate roughness at 2kBT, in the range of previously proposed rough parts of landscapes models during dissociation. PMID:27731375

  4. Self-antigen presentation by dendritic cells in autoimmunity

    Directory of Open Access Journals (Sweden)

    Ann-Katrin eHopp

    2014-02-01

    Full Text Available The operation of both central and peripheral tolerance ensures the prevention of autoimmune diseases. The maintenance of peripheral tolerance requires self-antigen presentation by professional antigen presenting cells (APCs. Dendritic cells (DCs are considered as major APCs involved in this process. The current review discusses the role of DCs in autoimmune diseases, the various factors involved in the induction and maintenance of tolerogenic DC phenotype and pinpoints their therapeutic capacity as well as potential novel targets for future clinical studies.

  5. In Vitro Generation of Antigen-Specific T Cells from Induced Pluripotent Stem Cells of Antigen-Specific T Cell Origin.

    Science.gov (United States)

    Kaneko, Shin

    2016-01-01

    Induced pluripotent stem (iPS) cells derived from T lymphocyte (T-iPS cells) preserve the T cell receptor (TCR) α and β gene rearrangements identical to the original T cell clone. Re-differentiated CD8 single positive αβ T cells from the T-iPS cells exhibited antigen-specific cytotoxicity, improved proliferative response, and elongation of telomere indicating rejuvenation of antigen specific T cell immunity in vitro. To regenerate antigen specific cytotoxic T lymphocytes (CTL), first, we have optimized a method for reprogramming-resistant CD8 T cell clones into T-iPS cells by using sendaiviral vectors. Second, we have optimized stepwise differentiation methods for inducing hematopoietic progenitor cells, T cell progenitors, and functionally matured CD8 single positive CTL. These protocols provide useful in vitro tools and models both for research of antigen-specific T cell immunotherapy and for research of normal and pathological thymopoiesis.

  6. A highly sensitive method for quantitation of mast cell surface antigen-1%肥大细胞表面抗原-1高灵敏度定量分析法的建立

    Institute of Scientific and Technical Information of China (English)

    陈洪淼; 宋振镇; 陈壮荔; 王振; 胡国鹏; 冯魏

    2012-01-01

    Mast cell plays a pivotal role in type I allergy, innate immunity, chronic inflammation and tissue remodeling. The recently cloned mast cell surface antigen-1 (MASA-1) protein is mainly expressed on the surface of activated mast cells, and is useful in the determination of the activation status of mast cells. The aim of this study is to develop a rapid and sensitive method for fhe quantitation of MASA-I. Based previously prepared a mouse monoclonal antibody against human MASA-1, we optimized the conditions including coating concentration, temperature and incubation time, and finally established a sandwich ELISA with a sensitivity of 0.5 ng/ml. By using this method, the MASA-1 levels in the bronchoalveolar lavage fluid of patients with respiratory system disorders are measured to be within the range of 0-130 ng/ml. The MASA-1 level correlates with the cell number of MASA-1 -positive cells, but not with that of cells stained by toluidine blue. Compared with the cell staining technique, the ELISA system is more rapid and accurate, and allows handling of large number of same samples in parallel, therefore is applicable in both research study and clinical test relating to mast cell.%目的 建立肥大细胞表面抗原-1 (MASA- 1)的高灵敏度定量分析方法.方法 首先制备抗人MASA-1的单克隆抗体,然后结合先前制备的多克隆抗体,通过优化包被浓度、温度和反应时间等条件.建立夹心酶联免疫定量方法 结果 成功制备了特异性高的小鼠抗人MASA-1的单克隆抗体;夹心ELISA法的检测灵敏度可达0.5 ng/ml.利用该法测出各种呼吸系统疾病病人的肺清洗液中的MASA-1含量为0~130 ng/ml.MASA-1的浓度与甲苯胺蓝染色阳性细胞数之间不存在相关关系,但与MASA-1染色阳性细胞数之间呈显著正相关关系.结论 相对于MASA-1细胞染色法,本研究开发的酶联免疫法具有快速、准确及能同时分析大量样品的优点,有助于肥大细胞的实验室研究和临床检测.

  7. Effect of hepatitis B immunisation in newborn infants of mothers positive for hepatitis B surface antigen

    DEFF Research Database (Denmark)

    Lee, Chuanfang; Gong, Yan; Brok, Jesper

    2006-01-01

    To evaluate the effects of hepatitis B vaccine and immunoglobulin in newborn infants of mothers positive for hepatitis B surface antigen.......To evaluate the effects of hepatitis B vaccine and immunoglobulin in newborn infants of mothers positive for hepatitis B surface antigen....

  8. Baculovirus surface display of Theileria parva p67 antigen preserves the conformation of sporozoite-neutralizing epitopes

    NARCIS (Netherlands)

    Kaba, S.A.; Hemmes, J.C.; Lent, van J.W.M.; Vlak, J.M.; Nene, V.; Musoke, A.J.; Oers, van M.M.

    2003-01-01

    Theileria parva is an intracellular protozoan parasite that causes East Coast fever, a severe lymphoproliferative disease in cattle. Previous attempts to produce recombinant sporozoite surface antigen (p67) in bacterial or insect cells for vaccine purposes have not resulted in a correctly folded pro

  9. Affinity isolation of antigen-specific circulating B cells for generation of phage display-derived human monoclonal antibodies

    DEFF Research Database (Denmark)

    Ditzel, Henrik

    2009-01-01

    A method is described for affinity isolation of antigen-specific circulating B cells of interest for subsequent generation of immune antibody phage display libraries. This approach should overcome the problem of low yields of monoclonal antibodies of interest in the libraries generated from...... peripheral blood lymphocytes caused by the low abundance of antigen-specific B cells in the circulation. The preselection of B cells is based on the specificity of the surface Ig receptor and is accomplished using the antigen of interest conjugated to magnetic beads. This method should significantly increase...

  10. Regulatory T Cells Are Dispensable for Tolerance to RBC Antigens.

    Science.gov (United States)

    Richards, Amanda L; Kapp, Linda M; Wang, Xiaohong; Howie, Heather L; Hudson, Krystalyn E

    2016-01-01

    Autoimmune hemolytic anemia (AIHA) occurs when pathogenic autoantibodies against red blood cell (RBC) antigens are generated. While the basic disease pathology of AIHA is well studied, the underlying mechanism(s) behind the failure in tolerance to RBC autoantigens are poorly understood. Thus, to investigate the tolerance mechanisms required for the establishment and maintenance of tolerance to RBC antigens, we developed a novel murine model. With this model, we evaluated the role of regulatory T cells (Tregs) in tolerance to RBC-specific antigens. Herein, we show that neither sustained depletion of Tregs nor immunization with RBC-specific proteins in conjunction with Treg depletion led to RBC-specific autoantibody generation. Thus, these studies demonstrate that Tregs are not required to prevent autoantibodies to RBCs and suggest that other tolerance mechanisms are likely involved.

  11. Regulatory T cells are Dispensable for Tolerance to RBC Antigens

    Directory of Open Access Journals (Sweden)

    Amanda L Richards

    2016-09-01

    Full Text Available Autoimmune hemolytic anemia (AIHA occurs when pathogenic autoantibodies against red blood cell (RBC antigens are generated. Whilst the basic disease pathology of AIHA is well studied, the underlying mechanism(s behind the failure in tolerance to RBC autoantigens are poorly understood. Thus, to investigate the tolerance mechanisms required for the establishment and maintenance of tolerance to RBC antigens, we developed a novel murine model. With this model, we evaluated the role of regulatory T cells (Tregs in tolerance to RBC-specific antigens. Herein, we show that neither sustained depletion of Tregs nor immunization with RBC-specific proteins in conjunction with Treg depletion led to RBC-specific autoantibody generation. Thus, these studies demonstrate that Tregs are not required to prevent autoantibodies to RBCs and suggest that other tolerance mechanisms are likely involved.

  12. Stem cell antigen 2 expression in adult and developing mice.

    Science.gov (United States)

    Antica, M; Wu, L; Scollay, R

    1997-01-01

    Stem cell antigen 2 (Sca-2) expression can distinguish the most immature T-lymphocyte precursors in the thymus from the hemopoietic stem cells. Sequence analysis of the Sca-2 protein showed that Sca-2 is a glycosylphosphatidylinositol (GPI) anchored molecule that shares some characteristics with the members of the Ly-6 multigene family, and that it is the same as the thymic shared antigen-1 (TSA-1). Here we extend these studies and critically reassess the expression of the Sca-2/TSA-1 antigen in hematopoietic tissues of adult and developing mice. With more sensitive methods we show that the distribution of Sca-2/TSA-1 differs from existing reports. We find especially high expression of Sca-2/TSA1 at day 14 of fetal development.

  13. Clinical significance of hepatitis B surface antigen mutants

    Institute of Scientific and Technical Information of China (English)

    Nicola; Coppola; Lorenzo; Onorato; Carmine; Minichini; Giovanni; Di; Caprio; Mario; Starace; Caterina; Sagnelli; Evangelista; Sagnelli

    2015-01-01

    Hepatitis B virus(HBV) infection is a major public health problem in many countries, with nearly 300 million people worldwide carrying HBV chronic infection and over 1 million deaths per year due to cirrhosis and liver cancer. Several hepatitis B surface antigen(HBs Ag) mutations have been described, most frequently due to a single amino acid substitution and seldom to a nucleotide deletion. The majority of mutations are located in the S region, but they have also been found in the pre-S1 and pre-S2 regions. Single amino acid substitutions in the major hydrophilic region of HBs Ag, called the "a" determinant, have been associated with immune escape and the consequent failure of HBV vaccination and HBs Ag detection, whereas deletions in the pre-S1 or pre-S2 regions have been associated with the development of hepatocellular carcinoma. This review article will focus on the HBs Ag mutants and their biological and clinical implications.

  14. Chimeric antigen receptor T cells: a novel therapy for solid tumors.

    Science.gov (United States)

    Yu, Shengnan; Li, Anping; Liu, Qian; Li, Tengfei; Yuan, Xun; Han, Xinwei; Wu, Kongming

    2017-03-29

    The chimeric antigen receptor T (CAR-T) cell therapy is a newly developed adoptive antitumor treatment. Theoretically, CAR-T cells can specifically localize and eliminate tumor cells by interacting with the tumor-associated antigens (TAAs) expressing on tumor cell surface. Current studies demonstrated that various TAAs could act as target antigens for CAR-T cells, for instance, the type III variant epidermal growth factor receptor (EGFRvIII) was considered as an ideal target for its aberrant expression on the cell surface of several tumor types. CAR-T cell therapy has achieved gratifying breakthrough in hematological malignancies and promising outcome in solid tumor as showed in various clinical trials. The third generation of CAR-T demonstrates increased antitumor cytotoxicity and persistence through modification of CAR structure. In this review, we summarized the preclinical and clinical progress of CAR-T cells targeting EGFR, human epidermal growth factor receptor 2 (HER2), and mesothelin (MSLN), as well as the challenges for CAR-T cell therapy.

  15. Dissection of T-cell antigen specificity in human melanoma

    DEFF Research Database (Denmark)

    Andersen, Rikke Sick; Albæk Thrue, Charlotte; Junker, Niels

    2012-01-01

    Tumor-infiltrating lymphocytes (TIL) isolated from melanoma patients and expanded in vitro by interleukin (IL)-2 treatment can elicit therapeutic response after adoptive transfer, but the antigen specificities of the T cells transferred have not been determined. By compiling all known melanoma......-associated antigens and applying a novel technology for high-throughput analysis of T-cell responses, we dissected the composition of melanoma-restricted T-cell responses in 63 TIL cultures. T-cell reactivity screens against 175 melanoma-associated epitopes detected 90 responses against 18 different epitopes...... from different fragments of resected melanoma lesions. In summary, our findings provide an initial definition of T-cell populations contributing to tumor recognition in TILs although the specificity of many tumor-reactive TILs remains undefined....

  16. HYBRIDOMA PRODUCTION USING IMMUNE LYMPHOCYTES AGAINST BRUGIA MALAYI ANTIGEN WITH MYELOMA CELLS

    Directory of Open Access Journals (Sweden)

    Soeyoko Soeyoko

    2012-09-01

    Full Text Available Wuchereria bancrofti, Brugia malayi, and Brugia timori are the causative agents of lymphatic hiariasis in Indonesia, but in some endemic areas, Brugia malayi is the most commonly found,Diagnosis of hiariasis is normally based on clinical and parasitological examinations, but both have limitations. Therefore now the immunological examination plays an important role in the diagnosis of filariasis. The discovery of monoclonal antibodies recently may probably give a firm scientific basis in immunology and add a new dimension to the efforts of developing a specific and sensitive immunological test for various stages of filarial infection. In this study, the production of hybridoma cells to develop monoclonal antibodies against B. malayi integrated a number of techniques: preparations of B. malayi surface antigen, immune lymphocyte cells, NSI myeloma cells and macrophage feeder layers, and a fusion of immune lymphocytes with myeloma cells. The results of this study can be concluded in three points: Protein analysis of the surface antigen was examined by Biureet and SDS-PAGE. A total of fourteen examinations were conducted by using 4000 L3 for each experiment. Three were not detected by Biureet method, but showed five protein fractions by SDS-PAGE. The protein concentration of the surface L3 was varied from 85.0/µg/ml to 769.23/µg/ml, with an average of 297.04/µg/ml.The immunoreactivifies of Balb/c mice antibodies to B. malayi L3 surface antigen were tested by ELISA and showed a gradual increase after four times immunizations at two weeks interval. The optical density (OD after four times immunizations was varied from 0.363 to 0.878 each mouse, where as the positive control sera OD was 0.570.Hybridization using immune lymphocytes against B. malayi L3 surface antigen with myeloma cells yielded 60.41% hybrid cells and none of them produced monoclonal antibodies tested of ELISA.

  17. CD8α− Dendritic Cells Induce Antigen-Specific T Follicular Helper Cells Generating Efficient Humoral Immune Responses

    Directory of Open Access Journals (Sweden)

    Changsik Shin

    2015-06-01

    Full Text Available Recent studies on T follicular helper (Tfh cells have significantly advanced our understanding of T cell-dependent B cell responses. However, little is known about the early stage of Tfh cell commitment by dendritic cells (DCs, particularly by the conventional CD8α+ and CD8α− DC subsets. We show that CD8α− DCs localized at the interfollicular zone play a pivotal role in the induction of antigen-specific Tfh cells by upregulating the expression of Icosl and Ox40l through the non-canonical NF-κB signaling pathway. Tfh cells induced by CD8α− DCs function as true B cell helpers, resulting in significantly increased humoral immune responses against various human pathogenic antigens, including Yersinia pestis LcrV, HIV Gag, and hepatitis B surface antigen. Our findings uncover a mechanistic role of CD8α− DCs in the initiation of Tfh cell differentiation and thereby provide a rationale for investigating CD8α− DCs in enhancing antigen-specific humoral immune responses for improving vaccines and therapeutics.

  18. Molecular structure and biological function of proliferating cell nuclear antigen

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Proliferating cell nuclear antigen (PCNA) is the core component of replication complex in eukaryote.As a processive factor of DNA polymerase delta, PCNA coordinates the replication process by interacting with various replication proteins. PCNA appears to play an essential role in many cell events, such as DNA damage repair, cell cycle regulation, and apoptosis, through the coordination or organization of different partners. PCNA is an essential factor in cell proliferation, and has clinical significance in tumor research. In this article we review the functional structure of PCNA, which acts as a function switch in different cell events.

  19. Uptake of antigen-antibody complexes by human dendritic cells.

    Science.gov (United States)

    Fanger, N A; Guyre, P M; Graziano, R F

    2001-01-01

    Fc receptors specific for IgG (FcγR) potentiate the immune response by facilitating the interaction between myeloid cells and antibody-coated targets (1-3). Monocyte and neutrophil FcyR engagement can lead to the induction of lytic-type mechanisms associated with innate responses. FcyR triggering can also play a key role in adaptive immune responses. For example, FcyR-directed capture and uptake of antigens (Ag) by dendritic cells (DC) results in processing and presentation to naive Ag-specific T cells, leading to their expansion and maturation into effector T-cell populations. This chapter describes methodology currently in use to explore and manipulate antigen-antibody (Ag-Ab) uptake by FcyR expressed on DC.

  20. MHC class II antigen presentation by B cells in health and disease

    NARCIS (Netherlands)

    Souwer, Yuri

    2009-01-01

    MHC class II antigen presentation by B cells is important to activate CD4+ T cells that stimulate the B cell to produce antibodies. Besides this, disruption of MHC class II antigen presentation could play a role in immune escape by tumor cells. This thesis describes MHC class II antigen presentation

  1. Ethanol Metabolism Alters Major Histocompatibility Complex Class I-Restricted Antigen Presentation In Liver Cells

    Science.gov (United States)

    Osna, Natalia A.; White, Ronda L.; Thiele, Geoffrey M.; Donohue, Terrence M.

    2009-01-01

    The proteasome is a major enzyme that cleaves proteins for antigen presentation. Cleaved peptides traffic to the cell surface, where they are presented in the context of MHC class I. Recognition of these complexes by cytotoxic T lymphocytes is crucial for elimination of cells bearing “non-self” proteins. Our previous studies revealed that ethanol suppresses proteasome function in ethanol-metabolizing liver cells. We hypothesized that proteasome suppression reduces the hydrolysis of antigenic peptides, thereby decreasing the presentation of the peptide-MHC class I-complexes on the cell surface. To test this, we used the mouse hepatocyte cell line (CYP2E1/ADH-transfected HepB5 cells) or primary mouse hepatocytes, both derived from livers of C57Bl/6 mice, which present the ovalbumin peptide, SIINFEKL, complexed with H2Kb. To induce H2Kb expression, HepB5 cells were treated with interferon gamma (IFNγ) and then exposed to ethanol. In these cells, ethanol metabolism decreased not only proteasome activity, but also hydrolysis of the C-extended peptide, SIINFEKL-TE and the presentation of SIINFEKL-H2Kb complexes measured after the delivery of SIINFEKL-TE to cytoplasm. The suppressive effects of ethanol were, in part, attributed to ethanol-elicited impairment of IFNγ signaling. However, in primary hepatocytes, even in the absence of IFNγ, we observed a similar decline in proteasome activity and antigen presentation after ethanol exposure. We conclude that proteasome function is directly suppressed by ethanol metabolism and indirectly, by preventing the activating effects of IFNγ. Ethanol-elicited reduction in proteasome activity contributes to the suppression of SIINFEKL-H2Kb presentation on the surface of liver cells. Immune response to viral antigens plays a crucial role in the pathogenesis of hepatitis C or B viral infections (HCV and HBV, respectively). Professional antigen-presenting cells (dendritic cells and macrophages) are responsible for priming the

  2. Trafficking of B cell antigen in lymph nodes

    DEFF Research Database (Denmark)

    Gonzalez, Santiago F.; Degn, Søren Egedal; Pitcher, Lisa A.

    2011-01-01

    The clonal selection theory first proposed by Macfarlane Burnet is a cornerstone of immunology ( 1 ). At the time, it revolutionized the thinking of immunologists because it provided a simple explanation for lymphocyte specificity, immunological memory, and elimination of self-reactive clones ( 2......, 5 ) have provided new insights into the trafficking of B cells and their antigen. In this review, we summarize these advances in the context of our current view of B cell circulation and activation....

  3. Adsorption of multimeric T cell antigens on carbon nanotubes

    DEFF Research Database (Denmark)

    Fadel, Tarek R; Li, Nan; Shah, Smith;

    2013-01-01

    Antigen-specific activation of cytotoxic T cells can be enhanced up to three-fold more than soluble controls when using functionalized bundled carbon nanotube substrates ((b) CNTs). To overcome the denaturing effects of direct adsorption on (b) CNTs, a simple but robust method is demonstrated...... to stabilize the T cell stimulus on carbon nanotube substrates through non-covalent attachment of the linker neutravidin....

  4. Existence of a squamous cell carcinoma antigen-immunoglobulin complex causes a deviation between squamous cell carcinoma antigen concentrations determined using two different immunoassays: first report of squamous cell carcinoma antigen coupling with immunoglobulin A.

    Science.gov (United States)

    Mori, Eriko; Kurano, Makoto; Tobita, Akiko; Shimosaka, Hironori; Yatomi, Yutaka

    2017-01-01

    Background Squamous cell carcinoma antigen is used as a tumour marker and is routinely measured in clinical laboratories. We validated two different immunoassays and found three cases in which the squamous cell carcinoma antigen concentrations deviated greatly between the two immunoassays. Here, we aimed to elucidate the mechanisms responsible for these deviations. Methods The squamous cell carcinoma antigen concentrations were determined using the ARCHITECT SCC (CLIA method) and the ST AIA-PACK SCC (FEIA method). We performed polyethylene glycol precipitation and size exclusion chromatography to assess the molecular weight and spike recovery and absorption tests to examine the presence of an autoantibody. Results Both methods exhibited good performances for the measurement of squamous cell carcinoma antigen, although a correlation test showed large differences in the squamous cell carcinoma antigen concentrations measured using the two methods in three cases. The results of polyethylene glycol treatment and size exclusion chromatography indicated the existence of a large molecular weight squamous cell carcinoma antigen in these three cases. The spike recovery tests suggested the possible presence of an autoantibody against squamous cell carcinoma antigen. Moreover, the absorption test revealed that large squamous cell carcinoma antigen complexes were formed by the association of squamous cell carcinoma antigen with IgG in two cases and with both IgG and IgA in one case. Conclusions This study describes the existence of large molecular weight squamous cell carcinoma antigen that has complexed with immunoglobulin in the serum samples. The reason for the deviations between the two immunoassays might be due to differences of their reactivities against the squamous cell carcinoma antigen immune complexes with their autoantibody. To our knowledge, this is the first report to describe the coupling of squamous cell carcinoma antigen with IgA.

  5. Modulation of TCR-mediated signaling pathway by thymic shared antigen-1 (TSA-1)/stem cell antigen-2 (Sca-2).

    Science.gov (United States)

    Saitoh, S; Kosugi, A; Noda, S; Yamamoto, N; Ogata, M; Minami, Y; Miyake, K; Hamaoka, T

    1995-12-15

    Thymic shared antigen-1 (TSA-1) is a glycosyl-phosphatidylinositol (GPI)-anchored differentiation Ag expressed on murine lymphocytes, and is identical to stem cell Ag-2 (Sca-2). Using newly established mAb against TSA-1/Sca-2, we have previously shown that surface TSA-1 expression is induced upon activation in T cells, and that anti-TSA-1 inhibits IL-2 production induced by anti-CD3 stimulation in T cell hybridomas. In the present study, we have analyzed the functional role of TSA-1 during T cell activation using normal T cells, T cell hybridomas, and transfected Jurkat cell lines that expressed either GPI-anchored or transmembrane form of TSA-1. Anti-TSA-1 inhibited IL-2 production from normal T cells stimulated with soluble anti-CD3 plus accessory cells. Anti-TSA-1 exhibited the inhibitory effect on T cells, but not on accessory cells, because anti-TSA-1 inhibited IL-2 production in Jurkat cells transfected with TSA-1 cDNA, but not in control transfectant. A transmembrane form of TSA-1 was expressed in Jurkat cells by fusing the extracellular portion of TSA-1 to the transmembrane and cytoplasmic regions of the class 1 Db. The analysis using this transfectant revealed that anti-TSA-1-mediated inhibition of IL-2 production did not require the GPI anchor of TSA-1. Finally, in addition to the inhibition of IL-2 production, tyrosine phosphorylation of CD3 zeta-chains observed following TCR stimulation, one of the important early activation events, was markedly reduced by anti-TSA-1. These results imply that TSA-1/Sca-2 plays an important regulatory role in the TCR signaling pathway of activated T cells in addition to its role in T cell differentiation.

  6. Artificial neural network accurately predicts hepatitis B surface antigen seroclearance.

    Directory of Open Access Journals (Sweden)

    Ming-Hua Zheng

    Full Text Available BACKGROUND & AIMS: Hepatitis B surface antigen (HBsAg seroclearance and seroconversion are regarded as favorable outcomes of chronic hepatitis B (CHB. This study aimed to develop artificial neural networks (ANNs that could accurately predict HBsAg seroclearance or seroconversion on the basis of available serum variables. METHODS: Data from 203 untreated, HBeAg-negative CHB patients with spontaneous HBsAg seroclearance (63 with HBsAg seroconversion, and 203 age- and sex-matched HBeAg-negative controls were analyzed. ANNs and logistic regression models (LRMs were built and tested according to HBsAg seroclearance and seroconversion. Predictive accuracy was assessed with area under the receiver operating characteristic curve (AUROC. RESULTS: Serum quantitative HBsAg (qHBsAg and HBV DNA levels, qHBsAg and HBV DNA reduction were related to HBsAg seroclearance (P<0.001 and were used for ANN/LRM-HBsAg seroclearance building, whereas, qHBsAg reduction was not associated with ANN-HBsAg seroconversion (P = 0.197 and LRM-HBsAg seroconversion was solely based on qHBsAg (P = 0.01. For HBsAg seroclearance, AUROCs of ANN were 0.96, 0.93 and 0.95 for the training, testing and genotype B subgroups respectively. They were significantly higher than those of LRM, qHBsAg and HBV DNA (all P<0.05. Although the performance of ANN-HBsAg seroconversion (AUROC 0.757 was inferior to that for HBsAg seroclearance, it tended to be better than those of LRM, qHBsAg and HBV DNA. CONCLUSIONS: ANN identifies spontaneous HBsAg seroclearance in HBeAg-negative CHB patients with better accuracy, on the basis of easily available serum data. More useful predictors for HBsAg seroconversion are still needed to be explored in the future.

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

    DEFF Research Database (Denmark)

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

    1999-01-01

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

  8. Toxicities of chimeric antigen receptor T cells: recognition and management.

    Science.gov (United States)

    Brudno, Jennifer N; Kochenderfer, James N

    2016-06-30

    Chimeric antigen receptor (CAR) T cells can produce durable remissions in hematologic malignancies that are not responsive to standard therapies. Yet the use of CAR T cells is limited by potentially severe toxicities. Early case reports of unexpected organ damage and deaths following CAR T-cell therapy first highlighted the possible dangers of this new treatment. CAR T cells can potentially damage normal tissues by specifically targeting a tumor-associated antigen that is also expressed on those tissues. Cytokine release syndrome (CRS), a systemic inflammatory response caused by cytokines released by infused CAR T cells can lead to widespread reversible organ dysfunction. CRS is the most common type of toxicity caused by CAR T cells. Neurologic toxicity due to CAR T cells might in some cases have a different pathophysiology than CRS and requires different management. Aggressive supportive care is necessary for all patients experiencing CAR T-cell toxicities, with early intervention for hypotension and treatment of concurrent infections being essential. Interleukin-6 receptor blockade with tocilizumab remains the mainstay pharmacologic therapy for CRS, though indications for administration vary among centers. Corticosteroids should be reserved for neurologic toxicities and CRS not responsive to tocilizumab. Pharmacologic management is complicated by the risk of immunosuppressive therapy abrogating the antimalignancy activity of the CAR T cells. This review describes the toxicities caused by CAR T cells and reviews the published approaches used to manage toxicities. We present guidelines for treating patients experiencing CRS and other adverse events following CAR T-cell therapy.

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

    Directory of Open Access Journals (Sweden)

    L. E. Esteban

    2013-01-01

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

  10. Genomic Typing of Red Cell Antigens

    Science.gov (United States)

    2011-09-01

    Antigen‐Matched  Red  Cells  for  Sickle  Cell  Anemia   Patients  Using  Molecular Typing to Augment Testing: Meghan Delaney, Prashant Gaur, Askale...Storry JR &  Delaney, M. CASE  REPORT OF TESTING AND MANAGEMENT OF BOMBAY  (OH)  PREGNANCY . XXXIst  International  Congress of the ISBT, Berlin, Germany...JK null Phenotype (Manuscript in preparation).  5. Delaney M et al: A Genetic Impossibility? Case report of Bombay (Oh)  pregnancy  with Group AB

  11. Bystander T cells in human immune responses to dengue antigens

    Directory of Open Access Journals (Sweden)

    Suwannasaen Duangchan

    2010-09-01

    Full Text Available Abstract Background Previous studies of T cell activation in dengue infection have focused on restriction of specific T cell receptors (TCRs and classical MHC molecules. However, bystander T cell activation, which is TCR independent, occurs via cytokines in other viral infections, both in vitro and in vivo, and enables T cells to bypass certain control checkpoints. Moreover, clinical and pathological evidence has pointed to cytokines as the mediators of dengue disease severity. Therefore, we investigated bystander T cell induction by dengue viral antigen. Results Whole blood samples from 55 Thai schoolchildren aged 13-14 years were assayed for in vitro interferon-gamma (IFN-γ induction in response to inactivated dengue serotype 2 antigen (Den2. The contribution of TCR-dependent and independent pathways was tested by treatment with cyclosporin A (CsA, which inhibits TCR-dependent activation of T cells. ELISA results revealed that approximately 72% of IFN-γ production occurred via the TCR-dependent pathway. The major IFN-γ sources were natural killer (NK (mean ± SE = 55.2 ± 3.3, CD4+T (24.5 ± 3.3 and CD8+T cells (17.9 ± 1.5, respectively, as demonstrated by four-color flow cytometry. Interestingly, in addition to these cells, we found CsA-resistant IFN-γ producing T cells (CD4+T = 26.9 ± 3.6% and CD8+T = 20.3 ± 2.1% implying the existence of activated bystander T cells in response to dengue antigen in vitro. These bystander CD4+ and CD8+T cells had similar kinetics to NK cells, appeared after 12 h and were inhibited by anti-IL-12 neutralization indicating cytokine involvement. Conclusions This study described immune cell profiles and highlighted bystander T cell activation in response to dengue viral antigens of healthy people in an endemic area. Further studies on bystander T cell activation in dengue viral infection may reveal the immune mechanisms that protect or enhance pathogenesis of secondary dengue infection.

  12. Autoantibodies to neuronal surface antigens in thyroid antibody-positive and -negative limbic encephalitis

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    Erdem Tuzun

    2011-01-01

    Full Text Available Background : Thyroid antibodies (Thy-Abs are frequently detected in various autoimmune disorders in coexistence with other systemic autoantibodies. In association with an encephalopathy, they are often taken as evidence of Hashimoto′s encephalitis (HE. However, the presence of Thy-Abs in a cohort of limbic encephalitis (LE patients and their association with anti-neuronal autoimmunity has not been explored. Patients and Methods : We investigated thyroid and anti-neuronal antibodies in the sera of 24 LE patients without identified tumors by cell-based assay and radioimmunoassay and evaluated their clinical features. Results : There was a female predominance in Thy-Ab-positive LE patients. Five of the eight Thy-Ab-positive patients and six of the 16 Thy-Ab-negative patients had antibodies to voltage-gated potassium channel (VGKC, N-methyl-D-aspartate receptor (NMDAR or undefined surface antigens on cultured hippocampal neurons. There were trends towards fewer VGKC antibodies (1/8 vs. 5/16, P = 0.159 and more NMDAR antibodies (2/8 vs. 1/16, P = 0.095 among the Thy-Ab-positive LE patients; antibodies to undefined surface antigens were only identified in Thy-Ab-positive patients (2/8 vs. 0/16, P = 0.018. There were no distinguishing clinical features between Thy-Ab-positive patients with and without neuronal antibodies. However, patients with anti-neuronal antibodies showed a better treatment response. Conclusion : Thy-Abs can be found in a high proportion of patients with non-paraneoplastic LE, often in association with antibodies to specific or as yet undefined neuronal surface antigens. These results suggest that acute idiopathic encephalitis patients with Thy-Abs should be closely monitored for ion-channel antibodies and it should not be assumed that they have HE.

  13. Evidence for glycosyl-phosphatidylinositol anchoring of Toxoplasma gondii major surface antigens

    Energy Technology Data Exchange (ETDEWEB)

    Tomavo, S.; Schwarz, R.T.; Dubremetz, J.F. (Institut National de la Recheche Medicale, Villeneuve d' Ascq (France))

    1989-10-01

    The four major surface antigens of Toxoplasma gondii tachyzoites (P43, P35, P30, and P22) were made water soluble by phosphatidylinositol-specific phospholipase C (PI-PLC). These antigens were biosynthetically labeled with {sup 3}H-fatty acids, ({sup 3}H)ethanolamine, and ({sup 3}H)carbohydrates. Treatment of {sup 3}H-fatty-acid-labeled parasite lysates with PI-PLC removed the radioactive label from these antigens. A cross-reacting determinant was exposed on these antigens after PI-PLC treatment.

  14. Nematode-Derived Proteins Suppress Proliferation and Cytokine Production of Antigen-Specific T Cells via Induction of Cell Death

    Science.gov (United States)

    Hartmann, Wiebke; Brenz, Yannick; Kingsley, Manchang Tanyi; Ajonina-Ekoti, Irene; Brattig, Norbert W.; Liebau, Eva; Breloer, Minka

    2013-01-01

    In order to establish long-lasting infections in their mammalian host, filarial nematodes have developed sophisticated strategies to dampen their host’s immune response. Proteins that are actively secreted by the parasites have been shown to induce the expansion of regulatory T cells and to directly interfere with effector T cell function. Here, we analyze the suppressive capacity of Onchocercavolvulus-derived excreted/secreted proteins. Addition of two recombinant O. volvulus proteins, abundant larval transcript-2 (OvALT-2) and novel larval transcript-1 (OvNLT-1) to cell cultures of T cell receptor transgenic CD4+ and CD8+ T cells suppressed antigen-specific stimulation in vitro. Ovalbumin-specific CD4+ DO11.10 and OT-II T cells that had been stimulated with their cognate antigen in the presence of OvALT-2 or OvNLT-1 displayed reduced DNA synthesis quantified by 3H-thymidine incorporation and reduced cell division quantified by CFSE dilution. Furthermore, the IL-2 and IFN-γ response of ovalbumin-specific CD8+ OT-I T cells was suppressed by OvALT-2 and OvNLT-1. In contrast, another recombinant O. volvulus protein, microfilariae surface-associated antigen (Ov103), did not modulate T cell activation, thus serving as internal control for non-ESP-mediated artifacts. Suppressive capacity of the identified ESP was associated with induction of apoptosis in T cells demonstrated by increased exposure of phosphatidylserine on the plasma membrane. Of note, the digestion of recombinant proteins with proteinase K did not abolish the suppression of antigen-specific proliferation although the suppressive capacity of the identified excreted/secreted products was not mediated by low molecular weight contaminants in the undigested preparations. In summary, we identified two suppressive excreted/secreted products from O. volvulus, which interfere with the function of antigen-specific T cells in vitro. PMID:23861729

  15. Nematode-derived proteins suppress proliferation and cytokine production of antigen-specific T cells via induction of cell death.

    Directory of Open Access Journals (Sweden)

    Wiebke Hartmann

    Full Text Available In order to establish long-lasting infections in their mammalian host, filarial nematodes have developed sophisticated strategies to dampen their host's immune response. Proteins that are actively secreted by the parasites have been shown to induce the expansion of regulatory T cells and to directly interfere with effector T cell function. Here, we analyze the suppressive capacity of Onchocercavolvulus-derived excreted/secreted proteins. Addition of two recombinant O. volvulus proteins, abundant larval transcript-2 (OvALT-2 and novel larval transcript-1 (OvNLT-1 to cell cultures of T cell receptor transgenic CD4(+ and CD8(+ T cells suppressed antigen-specific stimulation in vitro. Ovalbumin-specific CD4(+ DO11.10 and OT-II T cells that had been stimulated with their cognate antigen in the presence of OvALT-2 or OvNLT-1 displayed reduced DNA synthesis quantified by (3H-thymidine incorporation and reduced cell division quantified by CFSE dilution. Furthermore, the IL-2 and IFN-γ response of ovalbumin-specific CD8(+ OT-I T cells was suppressed by OvALT-2 and OvNLT-1. In contrast, another recombinant O. volvulus protein, microfilariae surface-associated antigen (Ov103, did not modulate T cell activation, thus serving as internal control for non-ESP-mediated artifacts. Suppressive capacity of the identified ESP was associated with induction of apoptosis in T cells demonstrated by increased exposure of phosphatidylserine on the plasma membrane. Of note, the digestion of recombinant proteins with proteinase K did not abolish the suppression of antigen-specific proliferation although the suppressive capacity of the identified excreted/secreted products was not mediated by low molecular weight contaminants in the undigested preparations. In summary, we identified two suppressive excreted/secreted products from O. volvulus, which interfere with the function of antigen-specific T cells in vitro.

  16. Chimeric Antigen Receptor T Cell Therapy in Hematology.

    Science.gov (United States)

    Ataca, Pınar; Arslan, Önder

    2015-12-01

    It is well demonstrated that the immune system can control and eliminate cancer cells. Immune-mediated elimination of tumor cells has been discovered and is the basis of both cancer vaccines and cellular therapies including hematopoietic stem cell transplantation. Adoptive T cell transfer has been improved to be more specific and potent and to cause less off-target toxicity. Currently, there are two forms of engineered T cells being tested in clinical trials: T cell receptor (TCR) and chimeric antigen receptor (CAR) modified T cells. On 1 July 2014, the United States Food and Drug Administration granted 'breakthrough therapy' designation to anti-CD19 CAR T cell therapy. Many studies were conducted to evaluate the benefits of this exciting and potent new treatment modality. This review summarizes the history of adoptive immunotherapy, adoptive immunotherapy using CARs, the CAR manufacturing process, preclinical and clinical studies, and the effectiveness and drawbacks of this strategy.

  17. Red-blood-cell alloimmunisation in relation to antigens' exposure and their immunogenicity: a cohort study.

    NARCIS (Netherlands)

    Evers, D.; Middelburg, R.A.; Haas, M. de; Zalpuri, S.; Vooght, K.M. De; Kerkhof, D. van de; Visser, O; Pequeriaux, N.C.V.; Hudig, F.; Schonewille, H.; Zwaginga, J.J.; Bom, J.G. Van Der

    2016-01-01

    BACKGROUND: Matching donor red blood cells based on recipient antigens prevents alloimmunisation. Knowledge about the immunogenicity of red-blood-cell antigens can help optimise risk-adapted matching strategies. We set out to assess the immunogenicity of red-blood-cell antigens. METHODS: In an incid

  18. Systemic and mucosal immune response induced by transcutaneous immunization using Hepatitis B surface antigen-loaded modified liposomes.

    Science.gov (United States)

    Mishra, Dinesh; Mishra, Pradyumna Kumar; Dubey, Vaibhav; Nahar, Manoj; Dabadghao, Sunil; Jain, N K

    2008-04-23

    We have evaluated the efficiency of novel modified liposomes (ethosomes) for transcutaneous immunization (TCI) against Hepatitis B. Antigen-loaded ethosomes were prepared and characterized for shape, lamellarity, fluidity, size distribution, and entrapment efficiency. Spectral bio-imaging and flow cytometric studies showed efficient uptake of Hepatitis B surface antigen (HBsAg)-loaded ethosomes by murine dendritic cells (DCs) in vitro, reaching a peak by 180 min. Transcutaneous delivery potential of the antigen-loaded system using human cadaver skin demonstrated a much higher skin permeation of the antigen in comparison to conventional liposomes and soluble antigen preparation. Topically applied HBsAg-loaded ethosomes in experimental mice showed a robust systemic and mucosal humoral immune response compared to intramuscularly administered alum-adsorbed HBsAg suspension, topically applied plain HBsAg solution and hydroethanolic (25%) HBsAg solution. The ability of the antigen-pulsed DCs to stimulate autologous peripheral blood lymphocytes was demonstrated by BrdU assay and a predominantly TH1 type of immune response was observed by multiplex cytometric bead array analysis. HBsAg-loaded ethosomes are able to generate a protective immune response and their ability to traverse and target the immunological milieu of the skin may find a potential application in the development of a transcutaneous vaccine against Hepatitis B virus (HBV).

  19. Molecular characterization of antigen-peptide pulsed dendritic cells: immature dendritic cells develop a distinct molecular profile when pulsed with antigen peptide.

    Directory of Open Access Journals (Sweden)

    Amy X Yang

    Full Text Available As dendritic cells (DCs are the most potent professional antigen-presenting cells, they are being tested as cancer vaccines for immunotherapy of established cancers. Although numerous studies have characterized DCs by their phenotype and function, few have identified potential molecular markers of antigen presentation prior to vaccination of host. In this study we generated pre-immature DC (piDC, immature DC (iDC, and mature DC (mDC from human peripheral blood monocytes (PBMC obtained from HLA-A2 healthy donors, and pulsed them with human papillomavirus E7 peptide (p11-20, a class I HLA-A2 binding antigen. We then characterized DCs for cell surface phenotype and gene expression profile by microarray technology. We identified a set of 59 genes that distinguished three differentiation stages of DCs (piDC, iDC and mDC. When piDC, iDC and mDC were pulsed with E7 peptide for 2 hrs, the surface phenotype did not change, however, iDCs rather than mDCs showed transcriptional response by up-regulation of a set of genes. A total of 52 genes were modulated in iDC upon antigen pulsing. Elongation of pulse time for iDCs to 10 and 24 hrs did not significantly bring further changes in gene expression. The E7 peptide up-modulated immune response (KPNA7, IGSF6, NCR3, TREM2, TUBAL3, IL8, NFKBIA, pro-apoptosis (BTG1, SEMA6A, IGFBP3 and SRGN, anti-apoptosis (NFKBIA, DNA repair (MRPS11, RAD21, TXNRD1, and cell adhesion and cell migration genes (EPHA1, PGF, IL8 and CYR61 in iDCs. We confirmed our results by Q-PCR analysis. The E7 peptide but not control peptide (PADRE induced up-regulation of NFKB1A gene only in HLA-A2 positive iDCs and not in HLA-A2 negative iDCs. These results suggest that E7 up-regulation of genes is specific and HLA restricted and that these genes may represent markers of antigen presentation and help rapidly assess the quality of dendritic cells prior to administration to the host.

  20. Microfluidic squeezing for intracellular antigen loading in polyclonal B-cells as cellular vaccines

    Science.gov (United States)

    Lee Szeto, Gregory; van Egeren, Debra; Worku, Hermoon; Sharei, Armon; Alejandro, Brian; Park, Clara; Frew, Kirubel; Brefo, Mavis; Mao, Shirley; Heimann, Megan; Langer, Robert; Jensen, Klavs; Irvine, Darrell J.

    2015-05-01

    B-cells are promising candidate autologous antigen-presenting cells (APCs) to prime antigen-specific T-cells both in vitro and in vivo. However to date, a significant barrier to utilizing B-cells as APCs is their low capacity for non-specific antigen uptake compared to “professional” APCs such as dendritic cells. Here we utilize a microfluidic device that employs many parallel channels to pass single cells through narrow constrictions in high throughput. This microscale “cell squeezing” process creates transient pores in the plasma membrane, enabling intracellular delivery of whole proteins from the surrounding medium into B-cells via mechano-poration. We demonstrate that both resting and activated B-cells process and present antigens delivered via mechano-poration exclusively to antigen-specific CD8+T-cells, and not CD4+T-cells. Squeezed B-cells primed and expanded large numbers of effector CD8+T-cells in vitro that produced effector cytokines critical to cytolytic function, including granzyme B and interferon-γ. Finally, antigen-loaded B-cells were also able to prime antigen-specific CD8+T-cells in vivo when adoptively transferred into mice. Altogether, these data demonstrate crucial proof-of-concept for mechano-poration as an enabling technology for B-cell antigen loading, priming of antigen-specific CD8+T-cells, and decoupling of antigen uptake from B-cell activation.

  1. Preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens and their use for bacterial detection.

    Science.gov (United States)

    Dykman, Lev A; Staroverov, Sergei A; Guliy, Olga I; Ignatov, Oleg V; Fomin, Alexander S; Vidyasheva, Irina V; Karavaeva, Olga A; Bunin, Viktor D; Burygin, Gennady L

    2012-01-01

    This article reports the first preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens by using a combinatorial phage library of sheep antibodies. The prepared phage antibodies were used for the first time for lipopolysaccharide and flagellin detection by dot assay, electro-optical analysis of cell suspensions, and transmission electron microscopy. Interaction of A. brasilense Sp245 with antilipopolysaccharide and antiflagellin phage-displayed miniantibodies caused the magnitude of the electro-optical signal to change considerably. The electro-optical results were in good agreement with the electron microscopic data. This is the first reported possibility of employing phage-displayed miniantibodies in bacterial detection aided by electro-optical analysis of cell suspensions.

  2. Vaccine adjuvant ginsenoside Rg1 enhances immune responses against hepatitis B surface antigen in mice.

    Science.gov (United States)

    Yuan, Ding; Yuan, Qin; Cui, Qianqian; Liu, Chaoqi; Zhou, Zhiyong; Zhao, Haixia; Dun, Yaoyan; Wang, Ting; Zhang, Changcheng

    2016-06-01

    The adjuvant effect of ginsenoside Rg1 on immune responses against hepatitis B surface antigen (HBsAg) in mice was investigated. Female BALB/c mice were subcutaneously injected with saline or HBsAg antigen with or without Rg1 on days 7 and 21. Samples were collected 2 weeks after the boosting for the detection of anti-HBsAg immunoglobulin G (IgG) isotypes in sera and gamma interferon (IFN-γ) and interleukin-4 (IL-4) produced in splenocytes. The innate and adaptive immune responses were measured in mice immunized as described above. The results showed that ginsenoside Rg1 had adjuvant properties in stimulating IgG, splenocyte proliferation, and mRNA expression of cytokines IFN-γ and IL-4, as well as the expression of cell surface marker TLR4 in the HBsAg-immunized mice. These results indicate that Rg1 enhances both Th1 (IgG2b and IFN-γ) and Th2 (IgG1 and IL-4) responses. In addition, the TLR4 signaling pathway is involved in the adjuvant activities of ginsenoside Rg1.

  3. Going viral: chimeric antigen receptor T-cell therapy for hematological malignancies.

    Science.gov (United States)

    Gill, Saar; June, Carl H

    2015-01-01

    On July 1, 2014, the United States Food and Drug Administration granted 'breakthrough therapy' designation to CTL019, the anti-CD19 chimeric antigen receptor T-cell therapy developed at the University of Pennsylvania. This is the first personalized cellular therapy for cancer to be so designated and occurred 25 years after the first publication describing genetic redirection of T cells to a surface antigen of choice. The peer-reviewed literature currently contains the outcomes of more than 100 patients treated on clinical trials of anti-CD19 redirected T cells, and preliminary results on many more patients have been presented. At last count almost 30 clinical trials targeting CD19 were actively recruiting patients in North America, Europe, and Asia. Patients with high-risk B-cell malignancies therefore represent the first beneficiaries of an exciting and potent new treatment modality that harnesses the power of the immune system as never before. A handful of trials are targeting non-CD19 hematological and solid malignancies and represent the vanguard of enormous preclinical efforts to develop CAR T-cell therapy beyond B-cell malignancies. In this review, we explain the concept of chimeric antigen receptor gene-modified T cells, describe the extant results in hematologic malignancies, and share our outlook on where this modality is likely to head in the near future.

  4. Modulation of T cell responses after cross-talk between antigen presenting cells and T cells: a give-and-take relationship.

    Science.gov (United States)

    Wauben, Marca H M; 't Hoen, Esther N M; Taams, Leonie S

    2003-01-01

    T cells presenting antigens in the context of MHC class II can induce anergy. In rat CD4+ T cell clones we have shown that depending on the depth of anergy other T cell responses can be inhibited in the presence of professional antigen presenting cells (APCs). This inhibition is cell-contact dependent, and APCs recovered from co-cultures with suppressive anergic T cells are modulated in their capacity to activate T cells. No changes in cell surface expression of MHC molecules, B7-1/B7-2, and OX40L were detected. Remarkably cell clusters formed by anergic T cells appeared to be more tight than clusters of activated T cells, and after fluorescent cell surface labelling of T cells, transfer of label was more profound in co-cultures of anergic T cells and APCs compared to activated T cells and APCs. Previously, it has been shown that activated T cells can absorb molecules from APCs in a unidirectional process. We now have evidence that also APCs can absorb cell surface molecules from T cells during APC-T cell co-cultures. We speculate that the quantity and quality of molecule reshuffling during cross-talk between T cells and APCs play a role in the regulation of the T cell response.

  5. Vaccination with trypomastigote surface antigen 1-encoding plasmid DNA confers protection against lethal Trypanosoma cruzi infection.

    Science.gov (United States)

    Wizel, B; Garg, N; Tarleton, R L

    1998-11-01

    DNA vaccination was evaluated with the experimental murine model of Trypanosoma cruzi infection as a means to induce antiparasite protective immunity, and the trypomastigote surface antigen 1 (TSA-1), a target of anti-T. cruzi antibody and major histocompatibility complex (MHC) class I-restricted CD8(+) cytotoxic T-lymphocyte (CTL) responses, was used as the model antigen. Following the intramuscular immunization of H-2(b) and H-2(d) mice with a plasmid DNA encoding an N-terminally truncated TSA-1 lacking or containing the C-terminal nonapeptide tandem repeats, the antibody level, CTL response, and protection against challenge with T. cruzi were assessed. In H-2(b) mice, antiparasite antibodies were induced only by immunization with the DNA construct encoding TSA-1 containing the C-terminal repeats. However, both DNA constructs were efficient in eliciting long-lasting CTL responses against the protective H-2Kb-restricted TSA-1515-522 epitope. In H-2(d) mice, inoculation with either of the two TSA-1-expressing vectors effectively generated antiparasite antibodies and primed CTLs that lysed T. cruzi-infected cells in an antigen-specific, MHC class I-restricted, and CD8(+)-T-cell-dependent manner. When TSA-1 DNA-vaccinated animals were challenged with T. cruzi, 14 of 22 (64%) H-2(b) and 16 of 18 (89%) H-2(d) mice survived the infection. The ability to induce significant murine anti-T. cruzi protective immunity by immunization with plasmid DNA expressing TSA-1 provides the basis for the application of this technology in the design of optimal DNA multicomponent anti-T. cruzi vaccines which may ultimately be used for the prevention or treatment of Chagas' disease.

  6. Cholera Toxin Inhibits the T-Cell Antigen Receptor-Mediated Increases in Inositol Trisphosphate and Cytoplasmic Free Calcium

    Science.gov (United States)

    Imboden, John B.; Shoback, Dolores M.; Pattison, Gregory; Stobo, John D.

    1986-08-01

    The addition of monoclonal antibodies to the antigen receptor complex on the malignant human T-cell line Jurkat generates increases in inositol trisphosphate and in the concentration of cytoplasmic free calcium. Exposure of Jurkat cells to cholera toxin for 3 hr inhibited these receptor-mediated events and led to a selective, partial loss of the antigen receptor complex from the cellular surface. None of the effects of cholera toxin on the antigen receptor complex were mimicked by the B subunit of cholera toxin or by increasing intracellular cAMP levels with either forskolin or 8-bromo cAMP. These results suggest that a cholera toxin substrate can regulate signal transduction by the T-cell antigen receptor.

  7. An Essential Role of the Avidity of T-Cell Receptor in Differentiation of Self-Antigen-reactive CD8+ T Cells.

    Science.gov (United States)

    Kondo, Kenta; Fujiki, Fumihiro; Nakajima, Hiroko; Yatsukawa, Erika; Morimoto, Soyoko; Tatsumi, Naoya; Nishida, Sumiyuki; Nakata, Jun; Oka, Yoshihiro; Tsuboi, Akihiro; Hosen, Naoki; Oji, Yusuke; Sugiyama, Haruo

    2016-04-01

    Many studies demonstrated crucial roles of avidity of T-cell receptor (TCR) in T-cell fate. However, majority of these findings resulted from analysis of non-self-antigen-specific CD8 T cells, and little is known about roles of TCR avidity in the fate of self-antigen-specific CD8 T cells. Wilms tumor gene 1 (WT1) protein is a self-antigen most suitable for addressing this issue because WT1 protein is a highly immunogenic, typical self-antigen. Here, we isolated 2 distinct and functional TCRs, TCR1 and TCR2, from murine WT1 peptide (RMFPNAPYL)-specific cytotoxic T lymphocytes (WT1-CTLs) and generated TCR1-retrogenic (Rg) and TCR2-Rg mice under T and B-cell-deficient and -reconstituted conditions. TCR1-transduced CD8 T (TCR1-T) cells had approximately 2-fold higher avidity to WT1 peptide than TCR2-transduced CD8 T (TCR2-T) cells. Cytokine production profiles and cell surface phenotypes showed that TCR1-T cells were more differentiated than TCR2-T cells under both conditions. Therefore, TCR1-T cells with TCR avidity higher than that of TCR2-T cells are more differentiated compared with TCR2-T cells. Furthermore, TCR1-T cells that developed under T and B-cell-reconstituted conditions displayed cytotoxicity against endogenously WT1-expressing tumor cells, whereas TCR2 T cells that developed under the same conditions did not. Thus, it was demonstrated, for the first time, that TCR avidity played an essential role in differentiation of self-antigen-reactive T cells, through the success of establishment of two distinct WT1-CTLs with a difference in only TCR avidity under the identical genetic background. Present results should provide us with an insight for elucidation of the differentiation mechanisms of self-antigen-reactive T cells, including tumor antigen-reactive T cells.

  8. Comparison of antigen-specific T-cell responses of tuberculosis patients using complex or single antigens of Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Mustafa, A S; Amoudy, H A; Wiker, H G;

    1998-01-01

    GroES, rPstS, rGroEL and rDnaK) antigens of Mycobacterium tuberculosis. The responses of PBMC to these defined antigens were compared with the corresponding results obtained with complex antigens, such as whole-cell M. tuberculosis, M. tuberculosis culture filtrate (MT-CF) and cell wall antigens, as well...... as the vaccine strain, Mycobacterium bovis bacillus Calmette-Guerin (BCG). In addition, M. tuberculosis and MT-CF-induced T-cell lines were tested in the same assays against the panel of purified and complex antigens. The compiled data from PBMC and T-cell lines tested for antigen-induced proliferation and IFN......We have screened peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients for proliferative reactivity and interferon-gamma (IFN-gamma) secretion against a panel of purified recombinant (r) and natural (n) culture filtrate (rESAT-6, nMPT59, nMPT64 and nMPB70) and somatic-derived (r...

  9. Original encounter with antigen determines antigen-presenting cell imprinting of the quality of the immune response in mice.

    Directory of Open Access Journals (Sweden)

    Valérie Abadie

    Full Text Available BACKGROUND: Obtaining a certain multi-functionality of cellular immunity for the control of infectious diseases is a burning question in immunology and in vaccine design. Early events, including antigen shuttling to secondary lymphoid organs and recruitment of innate immune cells for adaptive immune response, determine host responsiveness to antigens. However, the sequence of these events and their impact on the quality of the immune response remain to be elucidated. Here, we chose to study Modified Vaccinia virus Ankara (MVA which is now replacing live Smallpox vaccines and is proposed as an attenuated vector for vaccination strategies against infectious diseases. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed in vivo mechanisms triggered following intradermal (i.d. and intramuscular (i.m. Modified Vaccinia virus Ankara (MVA administration. We demonstrated significant differences in the antigen shuttling to lymphoid organs by macrophages (MPhis, myeloid dendritic cells (DCs, and neutrophils (PMNs. MVA i.d. administration resulted in better antigen distribution and more sustained antigen-presenting cells (APCs recruitment into draining lymph nodes than with i.m. administration. These APCs, which comprise both DCs and MPhis, were differentially involved in T cell priming and shaped remarkably the quality of cytokine-producing virus-specific T cells according to the entry route of MVA. CONCLUSIONS/SIGNIFICANCE: This study improves our understanding of the mechanisms of antigen delivery and their consequences on the quality of immune responses and provides new insights for vaccine development.

  10. Comparison of antigen-specific T-cell responses of tuberculosis patients using complex or single antigens of Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Mustafa, A S; Amoudy, H A; Wiker, H G

    1998-01-01

    We have screened peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients for proliferative reactivity and interferon-gamma (IFN-gamma) secretion against a panel of purified recombinant (r) and natural (n) culture filtrate (rESAT-6, nMPT59, nMPT64 and nMPB70) and somatic-derived (r......GroES, rPstS, rGroEL and rDnaK) antigens of Mycobacterium tuberculosis. The responses of PBMC to these defined antigens were compared with the corresponding results obtained with complex antigens, such as whole-cell M. tuberculosis, M. tuberculosis culture filtrate (MT-CF) and cell wall antigens, as well...... as the vaccine strain, Mycobacterium bovis bacillus Calmette-Guerin (BCG). In addition, M. tuberculosis and MT-CF-induced T-cell lines were tested in the same assays against the panel of purified and complex antigens. The compiled data from PBMC and T-cell lines tested for antigen-induced proliferation and IFN...

  11. Circadian control of antigen-specific T cell responses

    Directory of Open Access Journals (Sweden)

    Nobis CC

    2016-09-01

    Full Text Available Chloé C Nobis,1–3 Nathalie Labrecque,2–4 Nicolas Cermakian1,5–8 1Douglas Mental Health University Institute, 2Maisonneuve-Rosemont Hospital Research Centre, 3Department of Microbiology, Infectious Diseases and Immunology, 4Department of Medicine, University of Montreal, 5Department of Psychiatry, 6Department of Microbiology and Immunology, 7Department of Neurology and Neurosurgery, 8Department of Physiology, McGill University, Montreal, QC, Canada Abstract: The immune system is composed of two arms, the innate and the adaptive immunity. While the innate response constitutes the first line of defense and is not specific for a particular pathogen, the adaptive response is highly specific and allows for long-term memory of the pathogen encounter. T lymphocytes (or T cells are central players in the adaptive immune response. Various aspects of T cell functions vary according to the time of day. Circadian clocks located in most tissues and cell types generate 24-hour rhythms of various physiological processes. These clocks are based on a set of clock genes, and this timing mechanism controls rhythmically the expression of numerous other genes. Clock genes are expressed in cells of the immune system, including T cells. In this review, we provide an overview of the circadian control of the adaptive immune response, with emphasis on T cells, including their development, trafficking, response to antigen, and effector functions. Keywords: circadian clock, adaptive immune response, T lymphocyte, antigen, cytokine, proliferation

  12. Antigenicity and immunogenicity of novel chimeric hepatitis B surface antigen particles with exposed hepatitis C virus epitopes.

    Science.gov (United States)

    Netter, H J; Macnaughton, T B; Woo, W P; Tindle, R; Gowans, E J

    2001-03-01

    The small envelope protein of hepatitis B virus (HBsAg-S) can self-assemble into highly organized virus like particles (VLPs) and induce an effective immune response. In this study, a restriction enzyme site was engineered into the cDNA of HBsAg-S at a position corresponding to the exposed site within the hydrophilic a determinant region (amino acid [aa] 127-128) to create a novel HBsAg vaccine vector allowing surface orientation of the inserted sequence. We inserted sequences of various lengths from hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) E2 protein containing immunodominant epitopes and demonstrated secretion of the recombinant HBsAg VLPs from transfected mammalian cells. A number of different recombinant proteins were synthesized, and HBsAg VLPs containing inserts up to 36 aa were secreted with an efficiency similar to that of wild-type HBsAg. The HVR1 region exposed on the particles retained an antigenic structure similar to that recognized immunologically during natural infection. VLPs containing epitopes from either HCV-1a or -1b strains were produced that induced strain-specific antibody responses in immunized mice. Injection of a combination of these VLPs induced antibodies against both HVR1 epitopes that resulted in higher titers than were achieved by vaccination with the individual VLPs, suggesting a synergistic effect. This may lead to the development of recombinant particles which are able to induce a broad anti-HCV immune response against the HCV quasispecies or other quasispecies-like infectious agents.

  13. Antigenicity and Immunogenicity of Novel Chimeric Hepatitis B Surface Antigen Particles with Exposed Hepatitis C Virus Epitopes†

    Science.gov (United States)

    Netter, Hans J.; Macnaughton, Thomas B.; Woo, Wai-Ping; Tindle, Robert; Gowans, Eric J.

    2001-01-01

    The small envelope protein of hepatitis B virus (HBsAg-S) can self-assemble into highly organized virus like particles (VLPs) and induce an effective immune response. In this study, a restriction enzyme site was engineered into the cDNA of HBsAg-S at a position corresponding to the exposed site within the hydrophilic a determinant region (amino acid [aa] 127–128) to create a novel HBsAg vaccine vector allowing surface orientation of the inserted sequence. We inserted sequences of various lengths from hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) E2 protein containing immunodominant epitopes and demonstrated secretion of the recombinant HBsAg VLPs from transfected mammalian cells. A number of different recombinant proteins were synthesized, and HBsAg VLPs containing inserts up to 36 aa were secreted with an efficiency similar to that of wild-type HBsAg. The HVR1 region exposed on the particles retained an antigenic structure similar to that recognized immunologically during natural infection. VLPs containing epitopes from either HCV-1a or -1b strains were produced that induced strain-specific antibody responses in immunized mice. Injection of a combination of these VLPs induced antibodies against both HVR1 epitopes that resulted in higher titers than were achieved by vaccination with the individual VLPs, suggesting a synergistic effect. This may lead to the development of recombinant particles which are able to induce a broad anti-HCV immune response against the HCV quasispecies or other quasispecies-like infectious agents. PMID:11160717

  14. IL-4Rα-Associated Antigen Processing by B Cells Promotes Immunity in Nippostrongylus brasiliensis Infection

    Science.gov (United States)

    Hoving, Jennifer C.; Nieuwenhuizen, Natalie; McSorley, Henry J.; Ndlovu, Hlumani; Bobat, Saeeda; Kimberg, Matti; Kirstein, Frank; Cutler, Anthony J.; DeWals, Benjamin; Cunningham, Adam F.; Brombacher, Frank

    2013-01-01

    In this study, B cell function in protective TH2 immunity against N. brasiliensis infection was investigated. Protection against secondary infection depended on IL-4Rα and IL-13; but not IL-4. Protection did not associate with parasite specific antibody responses. Re-infection of B cell-specific IL-4Rα−/− mice resulted in increased worm burdens compared to control mice, despite their equivalent capacity to control primary infection. Impaired protection correlated with reduced lymphocyte IL-13 production and B cell MHC class II and CD86 surface expression. Adoptive transfer of in vivo N. brasiliensis primed IL-4Rα expressing B cells into naïve BALB/c mice, but not IL-4Rα or IL-13 deficient B cells, conferred protection against primary N. brasiliensis infection. This protection required MHC class II compatibility on B cells suggesting cognate interactions by B cells with CD4+ T cells were important to co-ordinate immunity. Furthermore, the rapid nature of these protective effects by B cells suggested non-BCR mediated mechanisms, such as via Toll Like Receptors, was involved, and this was supported by transfer experiments using antigen pulsed Myd88−/− B cells. These data suggest TLR dependent antigen processing by IL-4Rα-responsive B cells producing IL-13 contribute significantly to CD4+ T cell-mediated protective immunity against N. brasiliensis infection. PMID:24204255

  15. Studies of the antigenicity and immunogenicity of bromelain-pretreated red blood cells.

    Science.gov (United States)

    Cox, K O; Baddams, H; Evans, A

    1977-02-01

    The effects of the proteolytic enzyme bromelain (Br) on the antigenicity and immunogenicity of sheep and mouse red blood cells (RBC) have been investigated. The results presented support the previous claim that there are antigens present on Br RBC that are not present in an exposed form on untreated RBC and that Br RBC have lost some of the antigens present on the surface of normal RBC. The susceptibility of Br RBC to osmotic lysis was very similar to that of normal RBC, implying that the modified RBC were not more fragile than normal RBC. Injection of mice with Br mouse-RBC did not increase the unusually high "background" number of cells producing IgM antibodies against Br mouse-RBC and mice did not mount delayed-type hypersensitivity reactions against Br mouse-RBC, either before or after sensitizing injections of Br mouse-RBC. However, mouse-RBC and Br mouse-RBC elicited similar antibody responses in rabbits and guinea pigs. Although mice appeared unresponsive to Br mouse-RBC injections, delayed-type hypersensitivity responses and antibody production in primary and secondary responses were of similar levels irrespective of whether sheep-RBC or Br sheep-RBC were used as immunogens. From these studies it appears that mice have B-cells producing antibodies against the "new" antigens on Br mouse-RBC, but there are no T-cells that respond to these antigens by way of "helper" activity in antibody production or by way of cell-mediated immune reactions.

  16. Identifying protective Streptococcus pyogenes vaccine antigens recognized by both B and T cells in human adults and children

    DEFF Research Database (Denmark)

    Mortensen, Rasmus; Nissen, Thomas Nørrelykke; Fredslund, Sine

    2016-01-01

    No commercial vaccine exists against Group A streptococci (GAS; Streptococcus pyogenes) and only little is known about anti-GAS protective immunity. In our effort to discover new protective vaccine candidates, we selected 21 antigens based on an in silico evaluation. These were all well......-conserved among different GAS strains, upregulated in host-pathogen interaction studies, and predicted to be extracellular or associated with the surface of the bacteria. The antigens were tested for both antibody recognition and T cell responses in human adults and children. The antigenicity of a selected group...

  17. A human T cell clone that mediates the monocyte procoagulant response to specific sensitizing antigen.

    OpenAIRE

    Schwartz, B S; Reitnauer, P J; Hank, J A; Sondel, P M

    1985-01-01

    A panel of human purified protein derivative of the tubercle bacillus (PPD)-reactive T cell clones was derived by cloning out of soft agar followed by cultivation on inactivated feeder cells in the presence of interleukin-2. 1 of 4 clones tested was able to mediate an increase in monocyte procoagulant activity (PCA) in response to PPD. All four clones had identical surface marker phenotypes (T4+, T8-) and proliferated in response to antigen. The reactive T cell clone possessed no PCA of its o...

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

    Science.gov (United States)

    1984-06-15

    different VATs. These cDNAs were used to screen the genomic libraries . The cloned probes were examined by nucleotide sequence analysis and used to...examination of the consequence of - antigenic variation on the structure of the 1.1, 1.D and 1.11 VSG -, gene sequences. In addition, all genomic libraries have...immunoprecipitated by heterologous VAt specific antisera (preprint 2, figure 3; preprint 3, figure 1). These data presented in preprints 2 and Genomic

  19. Linearized hepatitis B surface antigen and hepatitis B core-related antigen in the natural history of chronic hepatitis B.

    Science.gov (United States)

    Seto, W-K; Wong, D K-H; Fung, J; Huang, F-Y; Liu, K S-H; Lai, C-L; Yuen, M-F

    2014-11-01

    Changes in two novel HBV serological markers, linearized hepatitis B surface antigen (HQ-HBsAg) and hepatitis B core-related antigen (HBcrAg), in the natural history of chronic hepatitis B (CHB) have not been well characterized. Serum HQ-HBsAg and HBcrAg levels of 404 Asian treatment-naïve CHB patients were analysed in a cross-sectional manner. Patients were categorized into five groups: immune tolerant (IT group, n=52), immune clearance (IC group, n=105), hepatitis B e antigen (HBeAg)-negative hepatitis (ENH group, n=97), HBeAg-negative quiescent group (ENQ group, n=95) and CHB with hepatitis B surface antigen (HBsAg) seroclearance (SC group, n=55). HQ-HBsAg and HBcrAg were measured and correlated with HBV DNA, HBsAg, HBV genotype and clinical parameters. HQ-HBsAg showed good correlation with HBsAg, especially in the ENQ group (r=0.874, pHBcrAg correlated best with HBV DNA in the ENQ group (r=0.537, pHBcrAg; this subgroup of patients, when compared with those with detectable HBcrAg, had significantly lower median HBV DNA (3.17/4.48 log IU/mL, pHBcrAg up to 42 months after HBsAg seroclearance. When comparing anti-HBs positivity and median time after HBsAg seroclearance in the SC group with and without detectable HQ-HBsAg/HBcrAg, there was no significant difference (22.7% and 36.4%, respectively, p 0.284, and 76.5 and 93.2 months, respectively, p 0.245). HQ-HBsAg and HBcrAg showed unique patterns of distribution throughout the five disease phases of CHB, including high detectability rates after HBsAg seroclearance, opening up different possibilities for their applicability.

  20. Autoantibodies in autoimmune thyroid disease promote immune complex formation with self antigens and increase B cell and CD4+ T cell proliferation in response to self antigens

    DEFF Research Database (Denmark)

    Nielsen, Claus Henrik; Hegedüs, Laszlo; Leslie, Robert Graham Quinton

    2004-01-01

    B cells are centrally involved as antigen-presenting cells in certain autoimmune diseases. To establish whether autoantibodies form immune complexes (IC) with self-antigens in autoimmune thyroid disease (AITD) and promote B cell uptake of self-antigen, sera from patients with Hashimoto......'s thyroiditis (HT), Graves' disease (GD) and healthy controls were incubated with human thyroglobulin (Tg) before adding normal peripheral blood mononuclear cells. The deposition of immunoglobulins and C3 fragments on B cells was then assessed. Inclusion of Tg in serum from HT patients promoted B cell capture...

  1. Human leukocyte antigen E contributes to protect tumor cells from lysis by natural killer cells.

    Science.gov (United States)

    Lo Monaco, Elisa; Tremante, Elisa; Cerboni, Cristina; Melucci, Elisa; Sibilio, Leonardo; Zingoni, Alessandra; Nicotra, Maria Rita; Natali, Pier Giorgio; Giacomini, Patrizio

    2011-09-01

    The nonclassic class I human leukocyte antigen E (HLA-E) molecule engages the inhibitory NKG2A receptor on several cytotoxic effectors, including natural killer (NK) cells. Its tissue distribution was claimed to be wider in normal than in neoplastic tissues, and surface HLA-E was undetectable in most tumor cell lines. Herein, these issues were reinvestigated taking advantage of HLA-E-specific antibodies, immunohistochemistry, and biochemical methods detecting intracellular and surface HLA-E regardless of conformation. Contrary to published evidence, HLA-E was detected in a few normal epithelia and in a large fraction (approximately 1/3) of solid tumors, including those derived from HLA-E-negative/low-normal counterparts. Remarkably, HLA-E was detected in 30 of 30 tumor cell lines representative of major lymphoid and nonlymphoid lineages, and in 11 of 11, it was surface-expressed, although in a conformation poorly reactive with commonly used antibodies. Coexpression of HLA-E and HLA class I ligand donors was not required for surface expression but was associated with NKG2A-mediated protection from lysis by the cytotoxic cell line NKL and polyclonal NK cells from healthy donors, as demonstrated by antibody-mediated relief of protection in 10% to 20% of the tested target-effector combinations. NKG2A-mediated protection of additional targets became evident on NK effector blocking with antibodies to activating receptors (DNAM-1, natural cytotoxicity receptors, and NKG2D). Thus, initial evidence that the long-elusive HLA-E molecule is enhanced by malignant transformation and is functional in tumor cells is presented here, although its importance and precise functional role remain to be addressed in the context of a general understanding of the NK ligand-receptor network.

  2. Human Leukocyte Antigen E Contributes to Protect Tumor Cells from Lysis by Natural Killer Cells

    Directory of Open Access Journals (Sweden)

    Elisa Lo Monaco

    2011-09-01

    Full Text Available The nonclassic class I human leukocyte antigen E (HLA-E molecule engages the inhibitory NKG2A receptor on several cytotoxic effectors, including natural killer (NK cells. Its tissue distribution was claimed to be wider in normal than in neoplastic tissues, and surface HLA-E was undetectable in most tumor cell lines. Herein, these issues were reinvestigated taking advantage of HLA-E-specific antibodies, immunohistochemistry, and biochemical methods detecting intracellular and surface HLA-E regardless of conformation. Contrary to published evidence, HLA-E was detected in a few normal epithelia and in a large fraction (approximately 1/3 of solid tumors, including those derived from HLA-E-negative/low-normal counterparts. Remarkably, HLA-E was detected in 30 of 30 tumor cell lines representative of major lymphoid and nonlymphoid lineages, and in 11 of 11, it was surface-expressed, although in a conformation poorly reactive with commonly used antibodies. Coexpression of HLA-E and HLA class I ligand donors was not required for surface expression but was associated with NKG2A-mediated protection from lysis by the cytotoxic cell line NKL and polyclonal NK cells from healthy donors, as demonstrated by antibody-mediated relief of protection in 10% to 20% of the tested target-effector combinations. NKG2A-mediated protection of additional targets became evident on NK effector blocking with antibodies to activating receptors (DNAM-1, natural cytotoxicity receptors, and NKG2D. Thus, initial evidence that the long-elusive HLA-E molecule is enhanced by malignant transformation and is functional in tumor cells is presented here, although its importance and precise functional role remain to be addressed in the context of a general understanding of the NK ligand-receptor network.

  3. Human Leukocyte Antigen E Contributes to Protect Tumor Cells from Lysis by Natural Killer Cells12

    Science.gov (United States)

    Monaco, Elisa Lo; Tremante, Elisa; Cerboni, Cristina; Melucci, Elisa; Sibilio, Leonardo; Zingoni, Alessandra; Nicotra, Maria Rita; Natali, Pier Giorgio; Giacomini, Patrizio

    2011-01-01

    The nonclassic class I human leukocyte antigen E (HLA-E) molecule engages the inhibitory NKG2A receptor on several cytotoxic effectors, including natural killer (NK) cells. Its tissue distribution was claimed to be wider in normal than in neoplastic tissues, and surface HLA-E was undetectable in most tumor cell lines. Herein, these issues were reinvestigated taking advantage of HLA-E-specific antibodies, immunohistochemistry, and biochemical methods detecting intracellular and surface HLA-E regardless of conformation. Contrary to published evidence, HLA-E was detected in a few normal epithelia and in a large fraction (approximately 1/3) of solid tumors, including those derived from HLA-E-negative/low-normal counterparts. Remarkably, HLA-E was detected in 30 of 30 tumor cell lines representative of major lymphoid and nonlymphoid lineages, and in 11 of 11, it was surface-expressed, although in a conformation poorly reactive with commonly used antibodies. Coexpression of HLA-E and HLA class I ligand donors was not required for surface expression but was associated with NKG2A-mediated protection from lysis by the cytotoxic cell line NKL and polyclonal NK cells from healthy donors, as demonstrated by antibody-mediated relief of protection in 10% to 20% of the tested target-effector combinations. NKG2A-mediated protection of additional targets became evident on NK effector blocking with antibodies to activating receptors (DNAM-1, natural cytotoxicity receptors, and NKG2D). Thus, initial evidence that the long-elusive HLA-E molecule is enhanced by malignant transformation and is functional in tumor cells is presented here, although its importance and precise functional role remain to be addressed in the context of a general understanding of the NK ligand-receptor network. PMID:21969815

  4. A method for visualizing surface-exposed and internal PfEMP1 adhesion antigens in Plasmodium falciparum infected erythrocytes

    DEFF Research Database (Denmark)

    Bengtsson, Dominique; Sowa, Kordai M; Salanti, Ali;

    2008-01-01

    BACKGROUND: The insertion of parasite antigens into the host erythrocyte membrane and the structure and distribution of Plasmodium falciparum adhesion receptors on that membrane are poorly understood. Laser scanning confocal microscopy (LSCM) and a novel labelling and fixation method have been used...... to obtain high resolution immuno-fluorescent images of erythrocyte surface PfEMP1 and internal antigens which allow analysis of the accumulation of PfEMP1 on the erythrocyte membrane during asexual development. METHODS: A novel staining technique has been developed which permits distinction between...... erythrocyte surface PfEMP1 and intracellular PfEMP1, in parasites whose nuclear material is exceptionally well resolved. Primary antibody detection by fluorescence is carried out on the live parasitized erythrocyte. The surface labelled cells are then fixed using paraformaldehyde and permeabilized with a non...

  5. Do FY antigens act as minor histocompatibility antigens in the graft-versus-host disease paradigm after human leukocyte antigen-identical sibling hematopoietic stem cell transplantation?

    Science.gov (United States)

    Sellami, Mohamed Hichem; Chaabane, Manel; Kaabi, Houda; Torjemane, Lamia; Ladeb, Saloua; Ben Othmane, Tarek; Hmida, Slama

    2012-03-01

    FY antigens are candidate minor histocompatibility antigens relevant to renal allograft rejection, but no data have been reported about their role in graft-versus-host disease (GVHD) incidence after human leukocyte antigen (HLA)-identical siblings hematopoietic stem cell transplantation (HSCT). The aim of this study was to examine the effect of donor/recipient disparity at FY antigens on the incidence of GVHD in Tunisian patients receiving an HLA-identical HSCT. This work enrolled 105 Tunisian pairs of recipients and their HLA-identical sibling donors of HSCs. FY genotyping was performed with the polymerase chain reaction-sequence-specific primer method and donor/recipient disparity for these antigens was analyzed at two levels: incompatibility and nonidentity. The case-control analyses showed no significant correlation between FY disparity and the incidence of either acute or chronic GVHD. Sample size calculation showed that 572 cases and 1716 controls would be necessary to be able to detect a significant association with 80% power and two-sided type I error level of 5% (α=0.05). The lack of association in the studied cohort may be explained by the low immunogenicity of FY antigens in HSCT context, compared with other antigens such as HA-1 and CD31.

  6. T cells expressing an anti-B-cell maturation antigen chimeric antigen receptor cause remissions of multiple myeloma.

    Science.gov (United States)

    Ali, Syed Abbas; Shi, Victoria; Maric, Irina; Wang, Michael; Stroncek, David F; Rose, Jeremy J; Brudno, Jennifer N; Stetler-Stevenson, Maryalice; Feldman, Steven A; Hansen, Brenna G; Fellowes, Vicki S; Hakim, Frances T; Gress, Ronald E; Kochenderfer, James N

    2016-09-29

    Therapies with novel mechanisms of action are needed for multiple myeloma (MM). B-cell maturation antigen (BCMA) is expressed in most cases of MM. We conducted the first-in-humans clinical trial of chimeric antigen receptor (CAR) T cells targeting BCMA. T cells expressing the CAR used in this work (CAR-BCMA) specifically recognized BCMA-expressing cells. Twelve patients received CAR-BCMA T cells in this dose-escalation trial. Among the 6 patients treated on the lowest 2 dose levels, limited antimyeloma activity and mild toxicity occurred. On the third dose level, 1 patient obtained a very good partial remission. Two patients were treated on the fourth dose level of 9 × 10(6) CAR(+) T cells/kg body weight. Before treatment, the first patient on the fourth dose level had chemotherapy-resistant MM, making up 90% of bone marrow cells. After treatment, bone marrow plasma cells became undetectable by flow cytometry, and the patient's MM entered a stringent complete remission that lasted for 17 weeks before relapse. The second patient on the fourth dose level had chemotherapy-resistant MM making up 80% of bone marrow cells before treatment. Twenty-eight weeks after this patient received CAR-BCMA T cells, bone marrow plasma cells were undetectable by flow cytometry, and the serum monoclonal protein had decreased by >95%. This patient is in an ongoing very good partial remission. Both patients treated on the fourth dose level had toxicity consistent with cytokine-release syndrome including fever, hypotension, and dyspnea. Both patients had prolonged cytopenias. Our findings demonstrate antimyeloma activity of CAR-BCMA T cells. This trial was registered to www.clinicaltrials.gov as #NCT02215967.

  7. Linear antigenic mapping of flagellin (FliC) from Salmonella enterica serovar Enteritidis with yeast surface expression system.

    Science.gov (United States)

    Wang, Gaoling; Shi, Bingtian; Li, Tao; Zuo, Teng; Wang, Bin; Si, Wei; Xin, Jiuqing; Yang, Kongbin; Shi, Xuanlin; Liu, Siguo; Liu, Henggui

    2016-02-29

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of food-borne illness around the world and can have significant health implications in humans, poultry and other animals. Flagellin (FliC) is the primary component of bacterial flagella. It has been shown that the FliC of S. Enteritidis is a significant antigenic structure and can elicit strong humoral responses against S. Enteritidis infection in chickens. Here, we constructed a FliC antigen library using a yeast surface expression system. Yeast cells expressing FliC peptide antigens were labeled with chicken sera against S. Enteritidis and sorted using FACS. The analyses of FliC peptides revealed that the FliC linear antigenicity in chickens resided on three domains which were able to elicit strong humoral responses in vivo. Animal experiments further revealed that the antibodies elicited by these antigenic domains were able to significantly inhibit the invasion of S. Enteritidis into the liver and spleen of chickens. These findings will facilitate our better understanding of the humoral responses elicited by FliC in chickens upon infection by S. Enteritidis.

  8. Differentiation induced by physiological and pharmacological stimuli leads to increased antigenicity of human neuroblastoma cells

    Institute of Scientific and Technical Information of China (English)

    Lena-Maria Carlson; Sven P(a)hlman; Anna De Geer; Per Kogner; Jelena Levitskaya

    2008-01-01

    Sympathetic neuronal differentiation is associated with favorable prognosis of neuroblastoma (NB), the most common extra-cranial solid tumor of early childhood. Differentiation agents have proved useful in clinical protocols of NB treatment, but using them as a sole treatment is not sufficient to induce tumor elimination in patients. Therefore, complementary approaches, such as immunotherapy, are warranted. Here we demonstrate that differentiation of NB cell lines and ex vivo isolated tumor cells in response to physiological or pharmacological stimuli is associated with acquisition of increased antigenicity. This manifests as increased expression of surface major histocompatibility class I complexes and ICAM-1 molecules and translates into increased sensitivity of NB cells to lysis by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. The latter is paralleled by enhanced ability of differentiated cells to form immune conjugates and bind increased amounts of granzyme B to the cell surface. We demonstrate, for the first time, that, regardless of the stimulus applied, the differentiation state in NBs is associated with increased tumor antigenicity that enables more efficient elimination of tumor cells by cytotoxic lymphocytes and paves the way for combined application of differentiation-inducing agents and immunotherapy as an auxiliary approach in NB patients.

  9. Pattern of distribution of blood group antigens on human epidermal cells during maturation

    DEFF Research Database (Denmark)

    Dabelsteen, Erik; Buschard, Karsten; Hakomori, Sen-Itiroh

    1984-01-01

    The distribution in human epidermis of A, B, and H blood group antigens and of a precursor carbohydrate chain, N-acetyl-lactosamine, was examined using immunofluorescence staining techniques. The material included tissue from 10 blood group A, 4 blood group B, and 9 blood group O persons. Murine...... on the lower spinous cells whereas H antigen was seen predominantly on upper spinous cells or on the granular cells. Epithelia from blood group A or B persons demonstrated A or B antigens, respectively, but only if the tissue sections were trypsinized before staining. In such cases A or B antigens were found...... monoclonal antibodies were used to identify H antigen (type 2 chain) and N-acetyl-lactosamine. Human antisera were used to identify A and B antigens. In all groups N-acetyl-lactosamine and H antigen were found on the cell membranes of the spinous cell layer. N-acetyl-lactosamine was present mainly...

  10. Cloning of a major surface-antigen gene of Trypanosoma cruzi and identification of a nonapeptide repeat.

    Science.gov (United States)

    Peterson, D S; Wrightsman, R A; Manning, J E

    The parasitic protozoan Trypanosoma cruzi can establish infection in humans and other vertebrate hosts through direct penetration of host cells by trypomastigotes transmitted by the insect vector. Although the molecular processes involved in trypomastigote interiorization of vertebrate cells are unknown, several studies suggest that surface glycoproteins are involved. It is likely that the proteins involved are specific to the trypomastigote stage of the parasite, since only trypomastigotes found in both the insect vector and the vertebrate host bloodstream are capable of invading vertebrate cells. In contrast, the epimastigote stage, found exclusively in the vector, and the amastigote stage, an intracellular stage in the vertebrate host, cannot penetrate the cell directly. We have therefore concentrated our efforts on trypomastigote surface proteins and, along with others, have identified two trypomastigote-specific surface glycoproteins of relative molecular mass (Mr) 90,000 (90K) and 85,000 (85K). Antibody neutralization experiments indicate that the 85K glycoprotein is necessary for efficient interiorization of trypomastigotes in mammalian cells. Here we describe the molecular cloning of a genomic DNA fragment that encodes antigenic determinants present in the 85K trypomastigote surface antigen. The polypeptide fragment encoded by the cloned DNA is recognized by serum from a T. cruzi-infected host and is inferred by DNA sequence analysis to contain a nonapeptide unit that is tandemly repeated five times. Also, the messenger complementary to the cloned DNA fragment is present only in the trypomastigote stage of the parasite.

  11. Antigen Processing by Autoreactive B Cells Promotes Determinant Spreading

    Institute of Scientific and Technical Information of China (English)

    Yang D.Dai; George Carayanniotis; Eli Sercarz

    2005-01-01

    Acute primary immune responses tend to focus on few immunodominant determinants using a very limited number of T cell clones for expansion, whereas chronic inflammatory responses generally recruit a large number of different T cell clones to attack a broader range of determinants of the invading pathogens or the inflamed tissues.In T cell-mediated organ-specific autoimmune disease, a transition from the acute to the chronic phase contributes to pathogenesis, and the broadening process is called determinant spreading. The cellular components catalyzing the spreading reaction are not identified. It has been suggested that autoreactive B cells may play a central role in diversifying autoreactive T cell responses, possibly through affecting antigen processing and presentation. The clonal identity and diversity of the B cells and antibodies seem critical in regulating T cell activity and subsequent tissue damage or repair. Here, we use two autoimmune animal models, experimental autoimmune thyroiditis (EAT)and type 1 diabetes (T1D), to discuss how autoreactive B cells or antibodies alter the processing and presentation of autoantigens to regulate specific T cell response.

  12. Protection from anti-TCR/CD3-induced apoptosis in immature thymocytes by a signal through thymic shared antigen-1/stem cell antigen-2.

    Science.gov (United States)

    Noda, S; Kosugi, A; Saitoh, S; Narumiya, S; Hamaoka, T

    1996-05-01

    During T cell development in the thymus, the expression of thymic shared antigen-1 (TSA-1)/stem cell antigen-2 (Sca-2), a glycosylphosphatidylinositol (GPI)-anchored differentiation antigen, is developmentally regulated. The expression level of TSA-1 is the highest in most immature CD4- CD8- thymocytes, high in CD4+ CD8+ thymocytes, but barely detectable in mature CD4+ CD8- or CD4- CD8- thymocytes and peripheral T cells. We have previously shown that surface TSA-1 expression in peripheral T cells is induced upon activation and that anti-TSA-1 mAb inhibits the T cell receptor (TCR) signaling pathway in activated T cells. In the present study, we have analyzed a role of TSA-1 in thymic selection events, especially in TCR-mediated apoptosis. In in vitro experiments, anti-TSA-1 blocked anti-CD3-induced cell death of T cell hybridomas. When anti-TSA-1 was injected into newborn mice in vivo together with anti-CD3 epsilon or anti-TCR-beta, TCR/CD3-mediated apoptosis of thymocytes was almost completely blocked. The blockade of apoptosis was defined by the inhibition of, first, the decrease in total number of thymocytes; second, the decrease in percentages of CD4+ CD8+ thymocytes; and third, the induction of DNA fragmentation. However, anti-TSA-1 did not block either steroid- or radiation-induced apoptosis, indicating that a signal via TSA-1 does not inhibit a common pathway of thymocyte apoptosis. Since TCR-mediated apoptosis is pivotal in thymic ontogeny, these results suggest that TSA-1/Sca-2 is an important cell surface molecule regulating the fate of a developing T cell.

  13. Antigen-Specific Inhibition of High-Avidity T Cell Target Lysis by Low-Avidity T Cells via Trogocytosis

    Directory of Open Access Journals (Sweden)

    Brile Chung

    2014-08-01

    Full Text Available Current vaccine conditions predominantly elicit low-avidity cytotoxic T lymphocytes (CTLs, which are non-tumor-cytolytic but indistinguishable by tetramer staining or enzyme-linked immunospot from high-avidity CTLs. Using CTL clones of high or low avidity for melanoma antigens, we show that low-avidity CTLs can inhibit tumor lysis by high-avidity CTLs in an antigen-specific manner. This phenomenon operates in vivo: high-avidity CTLs control tumor growth in animals but not in combination with low-avidity CTLs specific for the same antigen. The mechanism involves stripping of specific peptide-major histocompatibility complexes (pMHCs via trogocytosis by low-avidity melanoma-specific CTLs without degranulation, leading to insufficient levels of specific pMHC on target cell surface to trigger lysis by high-avidity CTLs. As such, peptide repertoire on the cell surface is dynamic and continually shaped by interactions with T cells. These results describe immune regulation by low-avidity T cells and have implications for vaccine design.

  14. Is Stage-Specific Embryonic Antigen 4 a Marker for Human Ductal Stem/Progenitor Cells?

    Science.gov (United States)

    Kayali, Ayse; Lopez, Ana; Hayek, Alberto

    2012-01-01

    Abstract The presence of pancreatic stem cells (PnSCs) has not been firmly demonstrated in the human or animal pancreas. Previous reports have suggested that ductal and acinar structures in the exocrine pancreas can be a potential source of progenitor cells. More recently, immature insulin precursors in the periphery of human islets have been found to self-replicate and differentiate to endocrine cells in vitro. Transplantation of these cells under the kidney capsule improves the diabetic state in mice. The controversy surrounding where PnSCs reside could be resolved if a specific marker were to be found that allowed their identification, purification, and directed differentiation to endocrine cells. We have identified in human pancreas cells positive for the stage-specific embryonic antigen 4 (SSEA4), a stem cell marker. These cells also express ductal, pancreatic progenitor, and stem cell protein markers. Interestingly, some of the SSEA4+ cells scattered in the ducts do not show a ductal cell phenotype. SSEA4+-sorted cells formed aggregate-like spheres in culture and robustly differentiated to pancreatic hormone-expressing cells in conditions of high glucose concentration and B27 supplementation. We hypothesize that SSEA4+ cells or a subpopulation of those cells residing in the pancreatic ducts may be the elusive PnSCs, and in this case, SSEA4 may represent a potential surface antigen marker for human PnSCs. The discovery of specific markers for the identification and purification of human PnSCs would greatly facilitate studies aimed at the expansion of these cells and the development of targeting tools for their potential induction to insulin-producing cells. PMID:23515456

  15. Is stage-specific embryonic antigen 4 a marker for human ductal stem/progenitor cells?

    Science.gov (United States)

    Afrikanova, Ivka; Kayali, Ayse; Lopez, Ana; Hayek, Alberto

    2012-08-01

    The presence of pancreatic stem cells (PnSCs) has not been firmly demonstrated in the human or animal pancreas. Previous reports have suggested that ductal and acinar structures in the exocrine pancreas can be a potential source of progenitor cells. More recently, immature insulin precursors in the periphery of human islets have been found to self-replicate and differentiate to endocrine cells in vitro. Transplantation of these cells under the kidney capsule improves the diabetic state in mice. The controversy surrounding where PnSCs reside could be resolved if a specific marker were to be found that allowed their identification, purification, and directed differentiation to endocrine cells. We have identified in human pancreas cells positive for the stage-specific embryonic antigen 4 (SSEA4), a stem cell marker. These cells also express ductal, pancreatic progenitor, and stem cell protein markers. Interestingly, some of the SSEA4(+) cells scattered in the ducts do not show a ductal cell phenotype. SSEA4(+)-sorted cells formed aggregate-like spheres in culture and robustly differentiated to pancreatic hormone-expressing cells in conditions of high glucose concentration and B27 supplementation. We hypothesize that SSEA4(+) cells or a subpopulation of those cells residing in the pancreatic ducts may be the elusive PnSCs, and in this case, SSEA4 may represent a potential surface antigen marker for human PnSCs. The discovery of specific markers for the identification and purification of human PnSCs would greatly facilitate studies aimed at the expansion of these cells and the development of targeting tools for their potential induction to insulin-producing cells.

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

    DEFF Research Database (Denmark)

    Hviid, Lars

    2010-01-01

    There is substantial immuno-epidemiological evidence that the parasite-encoded, so-called variant surface antigens (VSAs), such as PfEMP1 on the surface of infected erythrocytes (IEs) are important-in some cases probably decisive determinants of clinical outcome of P. falciparum malaria. The evid...

  17. Chemokine programming dendritic cell antigen response: part I - select chemokine programming of antigen uptake even after maturation.

    Science.gov (United States)

    Park, Jaehyung; Wu, Cindy T; Bryers, James D

    2013-05-01

    Here, we report on the successful programming of dendritic cells (DCs) using selectively applied mixtures of chemokines as a novel protocol for engineering vaccine efficiency. Antigen internalization by DCs is a pivotal step in antigen uptake/presentation for bridging innate and adaptive immunity and in exogenous gene delivery used in vaccine strategies. Contrary to most approaches to improve vaccine efficiency, active enhancement of antigen internalization by DCs as a vaccine strategy has been less studied because DCs naturally down-regulate antigen internalization upon maturation. Whereas chemokines are mainly known as signal proteins that induce leucocyte chemotaxis, very little research has been carried out to identify any additional effects of chemokines on DCs following maturation. Here, immature DCs are pre-treated with select chemokines before intentional maturation using lipopolysaccharide (LPS). When pre-treated with a mixture of CCL3 and CCL19 in a 7 : 3 ratio, then matured with LPS, chemokine pre-treated DCs exhibited 36% higher antigen uptake capacity than immature DCs and 27% higher antigen-processing capacity than immature DCs treated only with LPS. Further, CCL3 : CCL19 (7 : 3) pre-treatment of DCs modulated MHC molecule expression and secretion of various cytokines of DCs. Collectively, DC programming was feasible using a specific chemokine combination and these results provide a novel strategy for enhancing DC-based vaccine efficiency. In Part II, we report on the phenotype changes and antigen presentation capacity of chemokine pre-treated murine bone marrow-derived DCs examined in long-term co-culture with antigen-specific CD4(+) T cells.

  18. Enhanced cytotoxicity of natural killer cells following the acquisition of chimeric antigen receptors through trogocytosis.

    Directory of Open Access Journals (Sweden)

    Fu-Nan Cho

    Full Text Available Natural killer (NK cells have the capacity to target tumors and are ideal candidates for immunotherapy. Viral vectors have been used to genetically modify in vitro expanded NK cells to express chimeric antigen receptors (CARs, which confer cytotoxicity against tumors. However, use of viral transduction methods raises the safety concern of viral integration into the NK cell genome. In this study, we used trogocytosis as a non-viral method to modify NK cells for immunotherapy. A K562 cell line expressing high levels of anti-CD19 CARs was generated as a donor cell to transfer the anti-CD19 CARs onto NK cells via trogocytosis. Anti-CD19 CAR expression was observed in expanded NK cells after these cells were co-cultured for one hour with freeze/thaw-treated donor cells expressing anti-CD19 CARs. Immunofluorescence analysis confirmed the localization of the anti-CD19 CARs on the NK cell surface. Acquisition of anti-CD19 CARs via trogocytosis enhanced NK cell-mediated cytotoxicity against the B-cell acute lymphoblastic leukemia (B-ALL cell lines and primary B-ALL cells derived from patients. To our knowledge, this is the first report that describes the increased cytotoxicity of NK cells following the acquisition of CARs via trogocytosis. This novel strategy could be a potential valuable therapeutic approach for the treatment of B-cell tumors.

  19. An Immunohistochemical Study of Langerhans Cells,T-Cells and the HLA Antigen in Human Cornea

    Institute of Scientific and Technical Information of China (English)

    1993-01-01

    The distribution of Langerhans cells (LC),T-cell subsets andHLA antigen in 12 normal and 7 morbid corneas,including 4 of suppurativecorneal ulcer and 3 of uveogenic endophthalmitis,was investigated withmonoclonal antibodies.The results revealed that a small amount of LC andT-cell subsets were present in the limbal region of normal corneas,whilelarge numbers of LC and OKT_4~+ were observed in the corneas of suppurativeulcer.HLA-A,B,C antigens were expressed on the epithelial cells andkeratocytes of the n...

  20. Changes in surface antigens of Hymenolepis nana during differentiation and maturation in mice.

    Science.gov (United States)

    Ito, A; Onitake, K

    1987-06-01

    The surface antigens of oncosphere, cysticercoid, adult scolex and adult strobila (other than scolex) of Hymenolepis nana differ critically from one another. When the oncosphere of H. nana undergoes differentiation and development into the mature tapeworm, the infected mouse first produces anti-oncosphere antibody, followed by anti-cysticercoid, anti-adult scolex and finally anti-strobila (other than scolex region) antibodies of IgG, IgM and IgA isotypes as detected by indirect immunofluorescent antibody test. The parasite changed its surface antigens throughout its differentiation and maturation, and all developmental stages were recognized by the infected mouse host. However, there appeared no further changes in surface antigens during aging after maturation. The antibody responses were always delayed compared with the differentiation and maturation of the parasite.

  1. An Overview of B-1 Cells as Antigen-Presenting Cells

    Science.gov (United States)

    Popi, Ana F.; Longo-Maugéri, Ieda M.; Mariano, Mario

    2016-01-01

    The role of B cells as antigen-presenting cells (APCs) has been extensively studied, mainly in relation to the activation of memory T cells. Considering the B cell subtypes, the role of B-1 cells as APCs is beginning to be explored. Initially, it was described that B-1 cells are activated preferentially by T-independent antigens. However, some reports demonstrated that these cells are also involved in a T-dependent response. The aim of this review is to summarize information about the ability of B-1 cells to play a role as APCs and to briefly discuss the role of the BCR and toll-like receptor signals in this process. Furthermore, some characteristics of B-1 cells, such as natural IgM production and phagocytic ability, could interfere in the participation of these cells in the onset of an adaptive response. PMID:27148259

  2. Dietary antigens limit mucosal immunity by inducing regulatory T cells in the small intestine.

    Science.gov (United States)

    Kim, Kwang Soon; Hong, Sung-Wook; Han, Daehee; Yi, Jaeu; Jung, Jisun; Yang, Bo-Gie; Lee, Jun Young; Lee, Minji; Surh, Charles D

    2016-02-19

    Dietary antigens are normally rendered nonimmunogenic through a poorly understood "oral tolerance" mechanism that involves immunosuppressive regulatory T (Treg) cells, especially Treg cells induced from conventional T cells in the periphery (pTreg cells). Although orally introducing nominal protein antigens is known to induce such pTreg cells, whether a typical diet induces a population of pTreg cells under normal conditions thus far has been unknown. By using germ-free mice raised and bred on an elemental diet devoid of dietary antigens, we demonstrated that under normal conditions, the vast majority of the small intestinal pTreg cells are induced by dietary antigens from solid foods. Moreover, these pTreg cells have a limited life span, are distinguishable from microbiota-induced pTreg cells, and repress underlying strong immunity to ingested protein antigens.

  3. Modes of Antigen Presentation by Lymph Node Stromal Cells and Their Immunological Implications.

    Science.gov (United States)

    Hirosue, Sachiko; Dubrot, Juan

    2015-01-01

    Antigen presentation is no longer the exclusive domain of cells of hematopoietic origin. Recent works have demonstrated that lymph node stromal cell (LNSC) populations, such as fibroblastic reticular cells, lymphatic and blood endothelial cells, not only provide a scaffold for lymphocyte interactions but also exhibit active immunomodulatory roles that are critical to mounting and resolving effective immune responses. Importantly, LNSCs possess the ability to present antigens and establish antigen-specific interactions with T cells. One example is the expression of peripheral tissue antigens, which are presented on major histocompatibility complex (MHC)-I molecules with tolerogenic consequences on T cells. Additionally, exogenous antigens, including self and tumor antigens, can be processed and presented on MHC-I complexes, which result in dysfunctional activation of antigen-specific CD8(+) T cells. While MHC-I is widely expressed on cells of both hematopoietic and non-hematopoietic origins, antigen presentation via MHC-II is more precisely regulated. Nevertheless, LNSCs are capable of endogenously expressing, or alternatively, acquiring MHC-II molecules. Transfer of antigen between LNSC and dendritic cells in both directions has been recently suggested to promote tolerogenic roles of LNSCs on the CD4(+) T cell compartment. Thus, antigen presentation by LNSCs is thought to be a mechanism that promotes the maintenance of peripheral tolerance as well as generates a pool of diverse antigen-experienced T cells for protective immunity. This review aims to integrate the current and emerging literature to highlight the importance of LNSCs in immune responses, and emphasize their role in antigen trafficking, retention, and presentation.

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

    DEFF Research Database (Denmark)

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

    1991-01-01

    Human T cells express HLA class II antigens upon activation. Although activated, class II+ T cells can present alloantigens under certain circumstances, the functional role of class II antigens on activated T cells remains largely unknown. Here, we report that cross-linking of HLA-DR molecules ex...

  5. Chemotactic migration of T cells towards dendritic cells promotes the detection of rare antigens.

    Directory of Open Access Journals (Sweden)

    Renske M A Vroomans

    Full Text Available In many immunological processes chemoattraction is thought to play a role in guiding cells to their sites of action. However, based on in vivo two-photon microscopy experiments in the absence of cognate antigen, T cell migration in lymph nodes (LNs has been roughly described as a random walk. Although it has been shown that dendritic cells (DCs carrying cognate antigen in some circumstances attract T cells chemotactically, it is currently still unclear whether chemoattraction of T cells towards DCs helps or hampers scanning. Chemoattraction towards DCs could on the one hand help T cells to rapidly find DCs. On the other hand, it could be deleterious if DCs become shielded by a multitude of attracted yet non-specific T cells. Results from a recent simulation study suggested that the deleterious effect dominates. We re-addressed the question whether T cell chemoattraction towards DCs is expected to promote or hamper the detection of rare antigens using the Cellular Potts Model, a formalism that allows for dynamic, flexible cellular shapes and cell migration. Our simulations show that chemoattraction of T cells enhances the DC scanning efficiency, leading to an increased probability that rare antigen-specific T cells find DCs carrying cognate antigen. Desensitization of T cells after contact with a DC further improves the scanning efficiency, yielding an almost threefold enhancement compared to random migration. Moreover, the chemotaxis-driven migration still roughly appears as a random walk, hence fine-tuned analysis of cell tracks will be required to detect chemotaxis within microscopy data.

  6. Chemotactic migration of T cells towards dendritic cells promotes the detection of rare antigens.

    Science.gov (United States)

    Vroomans, Renske M A; Marée, Athanasius F M; de Boer, Rob J; Beltman, Joost B

    2012-01-01

    In many immunological processes chemoattraction is thought to play a role in guiding cells to their sites of action. However, based on in vivo two-photon microscopy experiments in the absence of cognate antigen, T cell migration in lymph nodes (LNs) has been roughly described as a random walk. Although it has been shown that dendritic cells (DCs) carrying cognate antigen in some circumstances attract T cells chemotactically, it is currently still unclear whether chemoattraction of T cells towards DCs helps or hampers scanning. Chemoattraction towards DCs could on the one hand help T cells to rapidly find DCs. On the other hand, it could be deleterious if DCs become shielded by a multitude of attracted yet non-specific T cells. Results from a recent simulation study suggested that the deleterious effect dominates. We re-addressed the question whether T cell chemoattraction towards DCs is expected to promote or hamper the detection of rare antigens using the Cellular Potts Model, a formalism that allows for dynamic, flexible cellular shapes and cell migration. Our simulations show that chemoattraction of T cells enhances the DC scanning efficiency, leading to an increased probability that rare antigen-specific T cells find DCs carrying cognate antigen. Desensitization of T cells after contact with a DC further improves the scanning efficiency, yielding an almost threefold enhancement compared to random migration. Moreover, the chemotaxis-driven migration still roughly appears as a random walk, hence fine-tuned analysis of cell tracks will be required to detect chemotaxis within microscopy data.

  7. Antigen-activated dendritic cells ameliorate influenza A infections

    Science.gov (United States)

    Boonnak, Kobporn; Vogel, Leatrice; Orandle, Marlene; Zimmerman, Daniel; Talor, Eyal; Subbarao, Kanta

    2013-01-01

    Influenza A viruses cause significant morbidity and mortality worldwide. There is a need for alternative or adjunct therapies, as resistance to currently used antiviral drugs is emerging rapidly. We tested ligand epitope antigen presentation system (LEAPS) technology as a new immune-based treatment for influenza virus infection in a mouse model. Influenza-J-LEAPS peptides were synthesized by conjugating the binding ligand derived from the β2-microglobulin chain of the human MHC class I molecule (J-LEAPS) with 15 to 30 amino acid–long peptides derived from influenza virus NP, M, or HA proteins. DCs were stimulated with influenza-J-LEAPS peptides (influenza-J-LEAPS) and injected intravenously into infected mice. Antigen-specific LEAPS-stimulated DCs were effective in reducing influenza virus replication in the lungs and enhancing survival of infected animals. Additionally, they augmented influenza-specific T cell responses in the lungs and reduced the severity of disease by limiting excessive cytokine responses, which are known to contribute to morbidity and mortality following influenza virus infection. Our data demonstrate that influenza-J-LEAPS–pulsed DCs reduce virus replication in the lungs, enhance survival, and modulate the protective immune responses that eliminate the virus while preventing excessive cytokines that could injure the host. This approach shows promise as an adjunct to antiviral treatment of influenza virus infections. PMID:23934125

  8. Antibody-antigen-adjuvant conjugates enable co-delivery of antigen and adjuvant to dendritic cells in cis but only have partial targeting specificity

    NARCIS (Netherlands)

    Kreutz, M.; Giquel, B.; Hu, Q.; Abuknesha, R.; Uematsu, S.; Akira, S.; Nestle, F.O.; Diebold, S.S.

    2012-01-01

    Antibody-antigen conjugates, which promote antigen-presentation by dendritic cells (DC) by means of targeted delivery of antigen to particular DC subsets, represent a powerful vaccination approach. To ensure immunity rather than tolerance induction the co-administration of a suitable adjuvant is par

  9. Surface antigens and virulence in Plasmodium falciparum malaria

    OpenAIRE

    Normark, Johan

    2008-01-01

    Plasmodium falciparum is an intracellular protozoan that may cause severe forms of malaria. It is a major world health hazard and reaps the highest toll among the children and pregnant mothers of the developing world. An Anopheles mosquito vector injects the pathogen when taking a blood meal. After multiplication in cells of the liver, the parasite escapes and infects red blood cells in a cyclic manner and this is when the clinical manifestations of malaria as a disease beco...

  10. Identification of Babesia bigemina infected erythrocyte surface antigens containing epitopes conserved among strains.

    Science.gov (United States)

    Shompole, S; McElwain, T F; Jasmer, D P; Hines, S A; Katende, J; Musoke, A J; Rurangirwa, F R; McGuire, T C

    1994-03-01

    The presence of previously uncharacterized antigens (new antigens) on the surface of intact erythrocytes infected with three strains of Babesia bigemina from Kenya and one each from Puerto Rico, Mexico, St. Croix, and Texcoco-Mexico was demonstrated by indirect immunofluorescent antibody (IFA) reactions. These antigens were not strain specific because antibodies in bovine immune serum to either the Mexico or Kenya isolates reacted with all seven strains tested. Homologous and heterologous immune serum antibodies bound a maximum of 83% and 55%, respectively, of intact erythrocytes infected with the Kenya-Ngong strain but not uninfected erythrocytes. Both sera caused agglutination of only infected erythrocytes. Antibodies eluted from the surface of glutaraldehyde (0.25%) fixed infected erythrocytes had IFA reaction patterns among strains similar to those of immune sera before elution. Eluted antibodies were used to determine if these antigens were protein and encoded by B. bigemina. Eluted antibodies bound seven parasite-encoded proteins of 240, 220, 66, 62, 58, 52 and 38 kDa in an erythrocyte surface-specific immunoprecipitation reaction of 35S-methionine labelled proteins. It was concluded that the surface of B. bigemina infected erythrocytes had parasite-encoded proteins and that these proteins had surface exposed epitopes that were conserved among the seven strains examined which were from two continents.

  11. Regulation of NK-cell function by mucins via antigen-presenting cells.

    Science.gov (United States)

    Laskarin, G; Redzovic, A; Medancic, S Srsen; Rukavina, D

    2010-12-01

    Decidual antigen-presenting cells including dendritic cells (DCs) and CD14(+) macrophages, as mediators of the first encounter with fetal antigens, appear to be critically involved in the initiation of primary immune response by regulating innate- and adaptive immunity. Interleukin-15, produced by them, permits the proliferation and differentiation of CD3(-)CD16(-)CD94(+)NKG2A(+)CD56(+bright) decidual NK cells that identify trophoblast cells. These cells are able to kill them after Th1 cytokine overstimulation and by increasing the release of preformed cytotoxic mediators. Thus, the local microenvironment is a potent modulator of antigen-presenting cell functions. Tumor associated glycoprotein-72 (TAG-72) and mucine 1 (MUC-1) are glycoproteins secreted by uterine epithelial cells. Our hypothesis is that TAG-72 and MUC-1 are the natural ligands for carbohydrate recognition domains (CRDs) of endocytic mannose receptor (MR or CD206) and DC-specific ICAM non-integrin (DC-SIGN or CD209) expressed on decidual CD14(+) macrophages and CD1a(+) DCs. They might be able to condition antigen-presenting cells to produce distinct profiles of cyto/chemokines with consequential reduction in NK-cell numbers and cytotoxic potential leading to insufficient control over trophoblast growth. This hypothesis could explain the disappearance of MUC-1 beneath the attached embryo during the process of successful implantation when tight regulation of trophoblast invasion is needed. As IL-15 is the earliest and the most important factor in NK-cell proliferation, differentiation, and maturation, we expected primarily an increase of IL-15 expression in antigen-presenting cells concomitant with the disappearance of mucins and the enhancement in NK cells numbers and of cytotoxic potential after their close contact with early pregnancy decidual antigen-presenting cells. If our hypothesis is correct, it would contribute to the understanding of the role of mucins in the redirection of immune response

  12. IMMUNO-ELECTRON MICROSCOPE ANALYSIS OF THE SURFACE LAYERS OF THE UNFERTILISED SEA URCHIN EGG. II. LOCALISATION OF SURFACE ANTIGENS.

    Science.gov (United States)

    BAXANDALL, J; PERLMANN, P; AFZELIUS, B A

    1964-12-01

    The immunological properties of the surface layers of Paracentrotus lividus eggs have been studied further by using ferritin-labelled antibody to localise specific antigenic sites. In order to detect a wider spectrum of antigenic determinants, several antisera against egg and jelly substance have been employed in combination with absorption procedures using lyophilised antigen. This use of absorbed antisera was made feasible by adding ferritin label in a second antiserum layer of ferritin-anti-gamma-globulin. Eggs were treated with antibody for short periods to detect antigenic sites without incurring structural changes (shown in previous paper) resulting from long antibody treatment. Unspecific ferritin uptake, found in pinocytotic vesicles and yolk granules, is considered in relation to yolk formation. The jelly layer, found to be immunologically heterogeneous, included one component interacting with antijelly gamma-globulin and one with antiegg gamma-globulin. The vitelline membrane proved to be rich in egg antigens (heat-stable and heat-labile). The role of this layer in specificity of fertilisation, parthenogenetic activation, and the possibility of being analogous to a basement membrane are discussed. Few antigenic sites were found on the plasma membrane with antiegg gamma-globulin. This gamma-globulin resulted in some specific labelling of cortical granules and its action is considered in relation to the permeability properties of the egg.

  13. Expression of basement membrane antigens in spindle cell melanoma.

    Science.gov (United States)

    Prieto, V G; Woodruff, J M

    1998-07-01

    Spindle cell melanoma (SCM) is an uncommon form of melanoma that may be confused histologically with other tumors, including malignant peripheral nerve sheath tumors (MPNST). Tumors with neural differentiation and melanocytic nevi may both show basement membrane immunohistochemically and at the ultrastructural level. However, most ultrastructural studies of melanoma have failed to demonstrate well formed basement membrane around tumor cells. The presence of basement membrane has been used by some authors as evidence favoring MPNST, as opposed to SCM. To evaluate this distinction immunohistochemically, 22 primary and metastatic cutaneous melanomas having a spindle cell component (SCM) were studied using monoclonal antibodies against laminin and Type IV collagen. S100 protein and HMB45 antigen expression were also studied. All but one of the SCM were reactive for S100 protein in at least 25% of the cells. Thirteen of 20 tumors (65%) were focally reactive with HMB45. Laminin was expressed in 42% of the tumors (only membranous pattern in 3; cytoplasmic and membranous in 5). Seventeen tumors (77%) expressed type IV collagen (only membranous pattern in 7; cytoplasmic and membranous pattern in 10). Laminin and type IV collagen, known components of basement membrane, are often found in SCM. Therefore, their detection cannot be used to distinguish SCM from MPNST.

  14. Sharing the burden: antigen transport and firebreaks in immune responses

    OpenAIRE

    Handel, Andreas; Yates, Andrew; Pilyugin, Sergei S.; Antia, Rustom

    2008-01-01

    Communication between cells is crucial for immune responses. An important means of communication during viral infections is the presentation of viral antigen on the surface of an infected cell. Recently, it has been shown that antigen can be shared between infected and uninfected cells through gap junctions, connexin-based channels, that allow the transport of small molecules. The uninfected cell receiving antigen can present it on its surface. Cells presenting viral antigen are detected and ...

  15. Antigenic modulation of metastatic breast and ovary carcinoma cells by intracavitary injection of IFN-alpha.

    Science.gov (United States)

    Giacomini, P.; Mottolese, M.; Fraioli, R.; Benevolo, M.; Venturo, I.; Natali, P. G.

    1992-01-01

    Antigenic modulation of major histocompatibility and tumour associated antigens was observed in neoplastic cells obtained from patients with pleural and abdominal effusions of breast and ovary carcinomas following a single intracavitary dose of 18 x 10(6) U recombinant IFN-alpha. This regimen resulted in antigenic modulation in seven out of 11 tested cases, suggesting a potential, although limited, responsiveness of at least a fraction of breast and ovary carcinoma cells to in situ biomodification with IFN-alpha. PMID:1503908

  16. Preparation and testing of a Haemophilus influenzae Type b/Hepatitis B surface antigen conjugate vaccine.

    Science.gov (United States)

    An, So Jung; Woo, Joo Sung; Chae, Myung Hwa; Kothari, Sudeep; Carbis, Rodney

    2015-03-24

    The majority of conjugate vaccines focus on inducing an antibody response to the polysaccharide antigen and the carrier protein is present primarily to induce a T-cell dependent response. In this study conjugates consisting of poly(ribosylribitolphosphate) (PRP) purified from Haemophilus influenzae Type b bound to Hepatitis B virus surface antigen (HBsAg) virus like particles were prepared with the aim of inducing an antibody response to not only the PRP but also the HBsAg. A conjugate consisting of PRP bound to HBsAg via an adipic acid dihydrazide (ADH) spacer induced strong IgG antibodies to both the PRP and HBsAg. When conjugation was performed without the ADH spacer the induction of an anti-PRP response was equivalent to that seen by conjugate with the ADH spacer, however, a negligible anti-HBsAg response was induced. For comparison, PRP was conjugated to diphtheria toxoid (DT) and Vi polysaccharide purified from Salmonella Typhi conjugated to HBsAg both using an ADH spacer. The PRPAH-DT conjugate induced strong anti-PRP and anti-DT responses, the Vi-AHHBsAg conjugate induced a good anti-HBsAg response but not as strong as that induced by the PRPAH-HBsAg conjugate. This study demonstrated that in mice it was possible to induce robust antibody responses to both polysaccharide and carrier protein provided the conjugate has certain physico-chemical properties. A PRPAH-HBsAg conjugate with the capacity to induce anti-PRP and anti-HBsAg responses could be incorporated into a multivalent pediatric vaccine and simplify formulation of such a vaccine.

  17. The effects of Fasciola hepatica tegumental antigens on mast cell function.

    Science.gov (United States)

    Vukman, Krisztina V; Adams, Paul N; Dowling, David; Metz, Martin; Maurer, Marcus; O'Neill, Sandra M

    2013-06-01

    Fasciola hepatica infection is associated with T helper 2/T regulatory immune responses and increased mast cell numbers. The aim of this study was to examine the interaction between F. hepatica tegumental coat antigen and mast cells in vivo and in vitro. Firstly, BALB/C, C57BL/6 or STAT6(-/-) mice were infected with F. hepatica metacercarie or mice were treated with F. hepatica tegumental coat antigen and then mast cells numbers in the peritoneal cavity and/or the liver were quantified. Also, the proliferation, chemotaxis, degranulation and cytokine secretion of mast cells from bone marrow or from peritoneal exudate cells stimulated with F. hepatica tegumental coat antigen were measured. Finally, we tested whether F. hepatica tegumental coat antigen inhibits degranulation of mast cells in vivo in a passive cutaneous and systemic anaphylaxis mouse model. Mast cell numbers increased in the peritoneal cavity and liver of F. hepatica infected mice, and this was mimicked by injection of F. hepatica tegumental coat antigen in a STAT6(-/-) independent manner. The increase in mast cell number was not the result of F. hepatica tegumental coat antigen-induced proliferation; rather F. hepatica tegumental coat antigen indirectly induces mast cell migration by dendritic cell-derived chemokines. Fasciola hepatica tegumental coat antigen interactions with mast cells do not drive T helper 2 or T regulatory immune responses. These studies on mast cell and F. hepatica tegumental coat antigen interaction may help us to understand the function of mast cells in immunity against F. hepatica and the immunomodulatory effect of F. hepatica tegumental coat antigen on these cells.

  18. Survival and signaling changes in antigen presenting cell subsets after radiation

    Science.gov (United States)

    Parker, Jennifer Janell

    examine co-stimulatory receptor activation, pro-inflammatory cytokine release, and T cell proliferation with and without radiation and inhibition of the NFkappaB pathway, demonstrated that NEMO is necessary for the activation, maturation, and enhanced responsiveness of human subsets of antigen presenting cells that occur after radiation. These findings provided insight into the mechanism of action of radiation-enhanced promotion of the antigen presenting cell responses. The methods of analysis employed can be used for monitoring immune changes that impact immune modulation in transplantation and tumor vaccines studies. Furthermore, NFkappaB pathway proteins have the potential to serve as biomarkers for optimal antitumor responses. The NBD peptide may also have usefulness as a therapeutic agent for inhibition of graft versus host disease (GVHD) in patients who have undergone transplantation. While the first set of experiments focused on antigen presenting cell responsiveness, the second set of experiments were designed to enhance our understanding of why antigen presenting cells, specifically monocytes and dendritic cells, are more radioresistant than conventional T cells. Flow cytometric analysis of various surface markers and intracellular signaling markers were used to examine the mechanisms behind the radioresistance of antigen presenting cells. The experiments described here showed a hierarchy of radiosensitivity among T cells, with naive CD8 T cells being the most radiosensitive and CD4 memory T cells being the most radioresistant. Antigen presenting cells were found to be significantly more radioresistant than T cell subsets (<10 fold decrease after radiation), and among APC, monocytes were more radiosensitive than either total or conventional dendritic cells. Furthermore APC expressed lower levels of Bax after radiation than T cells, and APC subsets that expressed high levels were also more sensitive to radiation induced cell death. These results demonstrate that T

  19. Cell-surface expression of Hsp70 on hematopoietic cancer cells after inhibition of HDAC activity

    DEFF Research Database (Denmark)

    Jensen, Helle

    -derived antigenic peptides, a function which is currently explored in immunotherapeutic approaches against cancer. Additionally, membrane-bound Hsp70 can stimulate antigen presenting cells to release proinflammatory cytokines and can provide a target structure for NK cell-mediated lysis. Human cancer cells...... frequently express Hsp70 on their cell surface, whereas the corresponding normal tissues do not. In addition, several clinically applied reagents, such as alkyl-lysophospholipides, chemotherapeutic agents, and anti-inflammatory reagents, have been found to enhance Hsp70 cell surface expression on cancer...... cells. We have found that inhibition of histone deacetylase (HDAC) activity leads to surface expression of Hsp70 on various hematopoietic cancer cells, an occurance that was not observed on naïve or activated peripheral blood cells. HDAC-inhibitor mediated Hsp70 cell surface expression was confined...

  20. Antigenic protein synthesis of Campylobacter jejuni in contact with chicken cells

    DEFF Research Database (Denmark)

    Vegge, Christina Skovgaard; Bang, Dang D.; Li, Yiping

    to the environment of the avian gastrointestinal tract. Consequently, the most important reservoir for C. jejuni is the gut of chickens, which are colonized efficiently without causing disease in the birds. Upon co-cultivation with mammalian cells, C. jejuni secrete specific Cia proteins, which are required...... the synthesis of antigenic C. jejuni proteins upon cultivation with chicken cells. Two strains of C. jejuni (the human isolate NCTC11168 and the chicken isolate DVI-SC11) were incubated with primary intestinal chicken cells and subsequently used to raise antisera in rabbits. Negative controls were carried out...... in parallel. These antisera were tested by Western blotting against C. jejuni total protein as well as periplasmic-, surface- and extracellular protein fractions. A unique antibody reaction was discovered to a protein from samples, which had been cultivated with chicken cells. The identity of this protein...

  1. Analysis of the antibody repertoire of patients with mantle cell lymphoma directed against mantle cell lymphoma-associated antigens

    OpenAIRE

    Zwick, Carsten; Preuss, Klaus-Dieter; Kubuschok, Boris; Held, Gerhard; Ahlgrimm, Manfred; Bittenbring, Joerg; Schubert, Joerg; Neumann, Frank; Pfreundschuh, Michael

    2009-01-01

    Abstract Treatment results of mantle cell lymphomas (MCL) are not satisfactory and novel therapeutic approaches are warranted. Because ?shared? tumor antigens like the group of cancer testis antigens are only rarely expressed in MCL, we applied serological analysis of antigens using recombinant expression cloning (SEREX) to a complementary DNA library derived from five cases of MCL using the sera of eight patients with MCL in order to define MCL-associated antigens that are immunog...

  2. Red blood cells as innovative antigen carrier to induce specific immune tolerance.

    Science.gov (United States)

    Cremel, Magali; Guérin, Nathalie; Horand, Françoise; Banz, Alice; Godfrin, Yann

    2013-02-25

    The route of administration, the dose of antigen as well as the type of antigen-presenting cells (APCs) targeted are important factors to induce immune tolerance. Despite encouraging results obtained in animal models, intravenous injection of soluble antigen is unsuccessful in human clinical trials on autoimmune disease due to inefficient antigen delivery. To improve antigen delivery, we used mouse red blood cells (RBCs) as antigen vehicles to specifically target APCs which are responsible for removal of senescent RBCs after phagocytosis. In this study, we demonstrated that antigen-delivery by RBCs induced a strong decrease in the humoral response compared with the ovalbumin (OVA) free form in mice. In addition, OVA-loaded RBC treated with [bis(sulphosuccinimidyl)] suberate (BS3), a chemical compound known to enhance RBC phagocytosis, induced an inhibition of antigen-specific T cell responses and an increase in the percentage of regulatory T cells. The state of tolerance induced is long lasting, antigen-specific and sufficiently robust to withstand immunization with antigen mixed with cholera toxin adjuvant. This RBC strategy, which does not abolish the immune system, constitutes an attractive approach for induction of tolerance compared to systemic immunosuppressant therapies already in use.

  3. Prevalence of hepatitis B surface antigen (HBsAg) in blood donors from Bombay.

    Science.gov (United States)

    Satoskar, A; Ray, V

    1992-01-01

    Analysis of serum samples from 3104 blood donors from Bombay screened for hepatitis B surface antigen (HBsAg) by ELISA. HBsAg was detected in 4.7% of the subjects. Relatives showed a significantly higher prevalence of HBsAg than volunteer donors. There was no significant association between HBsAg positivity and a particular blood group.

  4. Identification and Characterization of Ixodes scapularis Antigens That Elicit Tick Immunity Using Yeast Surface Display

    NARCIS (Netherlands)

    T.J. Schuijt; S. Narasimhan; S. Daffre; K. Deponte; J.W.R. Hovius; C. van 't Veer; T. van der Poll; K. Bakhtiari; J.C.M. Meijers; E.T. Boder; A.P. van Dam; E. Fikrig

    2011-01-01

    Repeated exposure of rabbits and other animals to ticks results in acquired resistance or immunity to subsequent tick bites and is partially elicited by antibodies directed against tick antigens. In this study we demonstrate the utility of a yeast surface display approach to identify tick salivary a

  5. Stable expression of sialyl-Tn antigen in T47-D cells induces a decrease of cell adhesion and an increase of cell migration.

    Science.gov (United States)

    Julien, Sylvain; Lagadec, Chann; Krzewinski-Recchi, Marie-Ange; Courtand, Gilles; Le Bourhis, Xuefen; Delannoy, Philippe

    2005-03-01

    Sialyl-Tn is a carbohydrate antigen overexpressed in several epithelial cancers including breast cancer, and usually associated with poor prognosis. Sialyl-Tn is synthesized by a CMP-Neu5Ac: GalNAc alpha2,6-sialyltransferase: ST6GalNAc I, which catalyzes the transfer of a sialic acid residue in alpha2,6-linkage to the GalNAcalpha1-O-Ser/Thr structure. The resulting disaccharide (Neu5Acalpha2-6GalNAcalpha1-O-Ser/Thr) cannot be further elongated and sialyl-Tn expression results therefore in a shortening of the O-glycan chains. However, usual breast cancer cell lines express neither ST6GalNAc I nor sialyl-Tn antigen. We have previously shown that stable transfection of MDA-MB-231 cells with the hST6GalNAc I cDNA induces the sialyl-Tn antigen expression at the cell surface and leads to a decreased cell growth and an increased cell migration. We describe herein the generation of new T47-D clones expressing sialyl-Tn antigen after hST6GalNAc I cDNA stable transfection. sialyl-Tn antigen is carried by several high molecular weight membrane bound O-glycoproteins, including MUC1. We show that sialyl-Tn expression induces a decrease of cell growth and adhesion, and an increase of cell migration in sialyl-Tn positive clones compared to mock transfected cells. These observations show that the alteration of the O-glycans pattern is sufficient to modify the biological features of cancer cells. These T47-D sialyl-Tn expressing clones might allow further in vivo investigation to determine precisely the impact of such O-glycosylation modifications on breast cancer development.

  6. Chloroquine inhibits accessory cell presentation of soluble natural and synthetic protein antigens

    DEFF Research Database (Denmark)

    Buus, S; Werdelin, O

    1984-01-01

    was time- and dose-dependent. A brief treatment solely of the accessory cells with the drug compromised their ability to stimulate primed T cells in a subsequent culture provided the accessory cells were treated with chloroquine before their exposure to the antigen. These results suggest that chloroquine......We have studied the in vitro effect of the lysosomotrophic agent, chloroquine, on the presentation of soluble protein antigens by guinea pig accessory cells. Chloroquine inhibited the capacity of antigen-pulsed accessory cells to stimulate proliferation in appropriately primed T cells. The effect...

  7. Detection of Avian Antigen-Specific T Cells Induced by Viral Vaccines

    DEFF Research Database (Denmark)

    Dalgaard, Tina Sørensen; Norup, Liselotte Rothmann; Juul-Madsen, Helle Risdahl

    2016-01-01

    Live attenuated viral vaccines are widely used in commercial poultry production, but the development of new effective inactivated/subunit vaccines is needed. Studies of avian antigen-specific T cells are primarily based on analyses ex vivo after activating the cells with recall antigen. There is ......Live attenuated viral vaccines are widely used in commercial poultry production, but the development of new effective inactivated/subunit vaccines is needed. Studies of avian antigen-specific T cells are primarily based on analyses ex vivo after activating the cells with recall antigen....... There is a particular interest in developing robust high-throughput assays as chicken vaccine trials usually comprise many individuals. In many respects, the avian immune system differs from the mammalian, and T cell assessment protocols must be adjusted accordingly to account for, e.g., differences in leukocyte...... responding to the stimulation. This method has been successfully applied to studies of chicken antigen-specific T cells....

  8. Identification of a highly antigenic linear B cell epitope within Plasmodium vivax apical membrane antigen 1 (AMA-1.

    Directory of Open Access Journals (Sweden)

    Lilian Lacerda Bueno

    Full Text Available Apical membrane antigen 1 (AMA-1 is considered to be a major candidate antigen for a malaria vaccine. Previous immunoepidemiological studies of naturally acquired immunity to Plasmodium vivax AMA-1 (PvAMA-1 have shown a higher prevalence of specific antibodies to domain II (DII of AMA-1. In the present study, we confirmed that specific antibody responses from naturally infected individuals were highly reactive to both full-length AMA-1 and DII. Also, we demonstrated a strong association between AMA-1 and DII IgG and IgG subclass responses. We analyzed the primary sequence of PvAMA-1 for B cell linear epitopes co-occurring with intrinsically unstructured/disordered regions (IURs. The B cell epitope comprising the amino acid sequence 290-307 of PvAMA-1 (SASDQPTQYEEEMTDYQK, with the highest prediction scores, was identified in domain II and further selected for chemical synthesis and immunological testing. The antigenicity of the synthetic peptide was identified by serological analysis using sera from P. vivax-infected individuals who were knowingly reactive to the PvAMA-1 ectodomain only, domain II only, or reactive to both antigens. Although the synthetic peptide was recognized by all serum samples specific to domain II, serum with reactivity only to the full-length protein presented 58.3% positivity. Moreover, IgG reactivity against PvAMA-1 and domain II after depletion of specific synthetic peptide antibodies was reduced by 18% and 33% (P = 0.0001 for both, respectively. These results suggest that the linear epitope SASDQPTQYEEEMTDYQK is highly antigenic during natural human infections and is an important antigenic region of the domain II of PvAMA-1, suggesting its possible future use in pre-clinical studies.

  9. The T cell antigen receptor: the Swiss army knife of the immune system.

    Science.gov (United States)

    Attaf, M; Legut, M; Cole, D K; Sewell, A K

    2015-07-01

    The mammalian T cell receptor (TCR) orchestrates immunity by responding to many billions of different ligands that it has never encountered before and cannot adapt to at the protein sequence level. This remarkable receptor exists in two main heterodimeric isoforms: αβ TCR and γδ TCR. The αβ TCR is expressed on the majority of peripheral T cells. Most αβ T cells recognize peptides, derived from degraded proteins, presented at the cell surface in molecular cradles called major histocompatibility complex (MHC) molecules. Recent reports have described other αβ T cell subsets. These 'unconventional' T cells bear TCRs that are capable of recognizing lipid ligands presented in the context of the MHC-like CD1 protein family or bacterial metabolites bound to the MHC-related protein 1 (MR1). γδ T cells constitute a minority of the T cell pool in human blood, but can represent up to half of total T cells in tissues such as the gut and skin. The identity of the preferred ligands for γδ T cells remains obscure, but it is now known that this receptor can also functionally engage CD1-lipid, or immunoglobulin (Ig) superfamily proteins called butyrophilins in the presence of pyrophosphate intermediates of bacterial lipid biosynthesis. Interactions between TCRs and these ligands allow the host to discriminate between self and non-self and co-ordinate an attack on the latter. Here, we describe how cells of the T lymphocyte lineage and their antigen receptors are generated and discuss the various modes of antigen recognition by these extraordinarily versatile receptors.

  10. Generating a transgenic mouse line stably expressing human MHC surface antigen from a HAC carrying multiple genomic BACs.

    Science.gov (United States)

    Hasegawa, Yoshinori; Ishikura, Tomoyuki; Hasegawa, Takanori; Watanabe, Takashi; Suzuki, Junpei; Nakayama, Manabu; Okamura, Yoshiaki; Okazaki, Tuneko; Koseki, Haruhiko; Ohara, Osamu; Ikeno, Masashi; Masumoto, Hiroshi

    2015-03-01

    The human artificial chromosome (HAC) vector is a promising tool to improve the problematic suppression and position effects of transgene expression frequently seen in transgenic cells and animals produced by conventional plasmid or viral vectors. We generated transgenic mice maintaining a single HAC vector carrying two genomic bacterial artificial chromosomes (BACs) from human HLA-DR loci (DRA and DRB1). Both transgenes on the HAC in transgenic mice exhibited tissue-specific expression in kidney, liver, lung, spleen, lymph node, bone marrow, and thymus cells in RT-PCR analysis. Stable functional expression of a cell surface HLA-DR marker from both transgenes, DRA and DRB1 on the HAC, was detected by flow cytometric analysis of splenocytes and maintained through at least eight filial generations. These results indicate that the de novo HAC system can allow us to manipulate multiple BAC transgenes with coordinated expression as a surface antigen through the generation of transgenic animals.

  11. Detection of 2 immunoreactive antigens in the cell wall of Sporothrix brasiliensis and Sporothrix globosa.

    Science.gov (United States)

    Ruiz-Baca, Estela; Hernández-Mendoza, Gustavo; Cuéllar-Cruz, Mayra; Toriello, Conchita; López-Romero, Everardo; Gutiérrez-Sánchez, Gerardo

    2014-07-01

    The cell wall of members of the Sporothrix schenckii complex contains highly antigenic molecules which are potentially useful for the diagnosis and treatment of sporotrichosis. In this study, 2 immunoreactive antigens of 60 (Gp60) and 70 kDa (Gp70) were detected in the cell wall of the yeast morphotypes of Sporothrix brasiliensis and Sporothrix globosa.

  12. A putative, novel coli surface antigen 8B (CS8B) of enterotoxigenic Escherichia coli.

    Science.gov (United States)

    Njoroge, Samuel M; Boinett, Christine J; Madé, Laure F; Ouko, Tom T; Fèvre, Eric M; Thomson, Nicholas R; Kariuki, Samuel

    2015-10-01

    Enterotoxigenic Escherichia coli (ETEC) strains harbor multiple fimbriae and pili to mediate host colonization, including the type IVb pilus, colonization factor antigen III (CFA/III). Not all colonization factors are well characterized or known in toxin positive ETEC isolates, which may have an impact identifying ETEC isolates based on molecular screening of these biomarkers. We describe a novel coli surface antigen (CS) 8 subtype B (CS8B), a family of CFA/III pilus, in a toxin producing ETEC isolate from a Kenyan collection. In highlighting the existence of this putative CS, we provide the sequence and specific primers, which can be used alongside other ETEC primers previously described.

  13. Vaccination with Trypomastigote Surface Antigen 1-Encoding Plasmid DNA Confers Protection against Lethal Trypanosoma cruzi Infection

    OpenAIRE

    1998-01-01

    DNA vaccination was evaluated with the experimental murine model of Trypanosoma cruzi infection as a means to induce antiparasite protective immunity, and the trypomastigote surface antigen 1 (TSA-1), a target of anti-T. cruzi antibody and major histocompatibility complex (MHC) class I-restricted CD8+ cytotoxic T-lymphocyte (CTL) responses, was used as the model antigen. Following the intramuscular immunization of H-2b and H-2d mice with a plasmid DNA encoding an N-terminally truncated TSA-1 ...

  14. Trypanosoma cruzi: identification of a surface antigen restricted to the flagellar region of the infective form of the parasite.

    Science.gov (United States)

    Saborio, J L; Wrightsman, R A; Kazuko, S G; Granger, B S; Manning, J E

    1990-05-01

    A hybridoma cell line was derived from spleen cells of B6D2 mice infected with the Peru strain of Trypanosoma cruzi. The monoclonal antibody produced by this hybridoma, designated mAb20H1, reacts exclusively with molecular components of trypomastigotes, the infective form of the parasite. The results of indirect immunofluorescence and of immunoelectron microscopy with gold-tagged antibodies indicate that the 20H1 antigen is restricted to the surface of the part of the flagellum in contact with the cell body and to the surface of the cell body in the immediate vicinity of this organelle. Western blot analysis showed that the 20H1 antigen consists of four to five different molecules with sizes between 34 and 41 kDa, and that these molecules are glycoproteins with affinity for concanavalin A. In other strains of T. cruzi, mAb20H1 reacts with glycoproteins with apparent sizes that range between 37 and 43 kDa in the CL, Esmeraldo and Y strains, and between 41 and 45 kDa in the Silvio strain.

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

    Directory of Open Access Journals (Sweden)

    Todd M Brusko

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

  16. A Lipid Based Antigen Delivery System Efficiently Facilitates MHC Class-I Antigen Presentation in Dendritic Cells to Stimulate CD8(+) T Cells.

    Science.gov (United States)

    Maji, Mithun; Mazumder, Saumyabrata; Bhattacharya, Souparno; Choudhury, Somsubhra Thakur; Sabur, Abdus; Shadab, Md; Bhattacharya, Pradyot; Ali, Nahid

    2016-06-02

    The most effective strategy for protection against intracellular infections such as Leishmania is vaccination with live parasites. Use of recombinant proteins avoids the risks associated with live vaccines. However, due to low immunogenicity, they fail to trigger T cell responses particularly of CD8(+) cells requisite for persistent immunity. Previously we showed the importance of protein entrapment in cationic liposomes and MPL as adjuvant for elicitation of CD4(+) and CD8(+) T cell responses for long-term protection. In this study we investigated the role of cationic liposomes on maturation and antigen presentation capacity of dendritic cells (DCs). We observed that cationic liposomes were taken up very efficiently by DCs and transported to different cellular sites. DCs activated with liposomal rgp63 led to efficient presentation of antigen to specific CD4(+) and CD8(+) T cells. Furthermore, lymphoid CD8(+) T cells from liposomal rgp63 immunized mice demonstrated better proliferative ability when co-cultured ex vivo with stimulated DCs. Addition of MPL to vaccine enhanced the antigen presentation by DCs and induced more efficient antigen specific CD8(+) T cell responses when compared to free and liposomal antigen. These liposomal formulations presented to CD8(+) T cells through TAP-dependent MHC-I pathway offer new possibilities for a safe subunit vaccine.

  17. A Lipid Based Antigen Delivery System Efficiently Facilitates MHC Class-I Antigen Presentation in Dendritic Cells to Stimulate CD8+ T Cells

    Science.gov (United States)

    Maji, Mithun; Mazumder, Saumyabrata; Bhattacharya, Souparno; Choudhury, Somsubhra Thakur; Sabur, Abdus; Shadab, Md.; Bhattacharya, Pradyot; Ali, Nahid

    2016-06-01

    The most effective strategy for protection against intracellular infections such as Leishmania is vaccination with live parasites. Use of recombinant proteins avoids the risks associated with live vaccines. However, due to low immunogenicity, they fail to trigger T cell responses particularly of CD8+ cells requisite for persistent immunity. Previously we showed the importance of protein entrapment in cationic liposomes and MPL as adjuvant for elicitation of CD4+ and CD8+ T cell responses for long-term protection. In this study we investigated the role of cationic liposomes on maturation and antigen presentation capacity of dendritic cells (DCs). We observed that cationic liposomes were taken up very efficiently by DCs and transported to different cellular sites. DCs activated with liposomal rgp63 led to efficient presentation of antigen to specific CD4+ and CD8+ T cells. Furthermore, lymphoid CD8+ T cells from liposomal rgp63 immunized mice demonstrated better proliferative ability when co-cultured ex vivo with stimulated DCs. Addition of MPL to vaccine enhanced the antigen presentation by DCs and induced more efficient antigen specific CD8+ T cell responses when compared to free and liposomal antigen. These liposomal formulations presented to CD8+ T cells through TAP-dependent MHC-I pathway offer new possibilities for a safe subunit vaccine.

  18. The uptake of soluble and particulate antigens by epithelial cells in the mouse small intestine.

    Science.gov (United States)

    Howe, Savannah E; Lickteig, Duane J; Plunkett, Kyle N; Ryerse, Jan S; Konjufca, Vjollca

    2014-01-01

    Intestinal epithelial cells (IECs) overlying the villi play a prominent role in absorption of digested nutrients and establish a barrier that separates the internal milieu from potentially harmful microbial antigens. Several mechanisms by which antigens of dietary and microbial origin enter the body have been identified; however whether IECs play a role in antigen uptake is not known. Using in vivo imaging of the mouse small intestine, we investigated whether epithelial cells (enterocytes) play an active role in the uptake (sampling) of lumen antigens. We found that small molecular weight antigens such as chicken ovalbumin, dextran, and bacterial LPS enter the lamina propria, the loose connective tissue which lies beneath the epithelium via goblet cell associated passageways. However, epithelial cells overlying the villi can internalize particulate antigens such as bacterial cell debris and inert nanoparticles (NPs), which are then found co-localizing with the CD11c+ dendritic cells in the lamina propria. The extent of NP uptake by IECs depends on their size: 20-40 nm NPs are taken up readily, while NPs larger than 100 nm are taken up mainly by the epithelial cells overlying Peyer's patches. Blocking NPs with small proteins or conjugating them with ovalbumin does not inhibit their uptake. However, the uptake of 40 nm NPs can be inhibited when they are administered with an endocytosis inhibitor (chlorpromazine). Delineating the mechanisms of antigen uptake in the gut is essential for understanding how tolerance and immunity to lumen antigens are generated, and for the development of mucosal vaccines and therapies.

  19. High throughput functional epitope mapping: revisiting phage display platform to scan target antigen surface.

    Science.gov (United States)

    Rojas, Gertrudis; Tundidor, Yaima; Infante, Yanelys Cabrera

    2014-01-01

    Antibody engineering must be accompanied by mapping strategies focused on identifying the epitope recognized by each antibody to define its unique functional identity. High throughput fine specificity determination remains technically challenging. We review recent experiences aimed at revisiting the oldest and most extended display technology to develop a robust epitope mapping platform, based on the ability to manipulate target-derived molecules (ranging from the whole native antigen to antigen domains and smaller fragments) on filamentous phages. Single, multiple and combinatorial mutagenesis allowed comprehensive scanning of phage-displayed antigen surface that resulted in the identification of clusters of residues contributing to epitope formation. Functional pictures of the epitope(s) were thus delineated in the natural context. Successful mapping of antibodies against interleukin-2, epidermal growth factor and its receptor, and vascular endothelial growth factor showed the versatility of these procedures, which combine the accuracy of site-directed mutagenesis with the high throughput potential of phage display.

  20. Monoclonal Antibodies as Probes for Unique Antigens in Secretory Cells of Mixed Exocrine Organs

    Science.gov (United States)

    Basbaum, C. B.; Mann, J. K.; Chow, A. W.; Finkbeiner, W. E.

    1984-07-01

    In the past, it has been difficult to identify the secretory product and control mechanisms associated with individual cell types making up mixed exocrine organs. This report establishes the feasibility of using immunological methods to characterize both the biochemical constituents and regulatory mechanisms associated with secretory cells in the trachea. Monoclonal antibodies directed against components of tracheal mucus were produced by immunizing mice with dialyzed, desiccated secretions harvested from tracheal organ culture. An immunofluorescence assay revealed that of the total 337 hybridomas screened, 100 produced antibodies recognizing goblet cell granules; 64, gland cell granules; and 3, antigen confined to the ciliated apical surface of the epithelium. The tracheal goblet cell antibody described in this report was strongly cross-reactive with intestinal goblet cells, as well as with a subpopulation of submandibular gland cells, but not with cells of Brunner's glands or the ciliated cell apical membrane. The serous cell antibody was not cross-reactive with goblet, Brunner's gland, or submandibular cells, or the ciliated cell apical membrane. The antibody directed against the apical membrane of ciliated cells did not cross-react with gland or goblet cells or the apical membrane of epithelial cells in the duodenum. Monoclonal antibodies, therefore, represent probes by which products unique to specific cells or parts of cells in the trachea can be distinguished. The antibodies, when used in enzyme immunoassays, can be used to quantitatively monitor secretion by individual cell types under a variety of physiological and pathological conditions. They also provide the means for purification and characterization of cell-specific products by immunoaffinity chromatography.

  1. Identification and characterization of a putative agglutination/immobilization antigen on the surface of Cryptocaryon irritans.

    Science.gov (United States)

    Hatanaka, A; Umeda, N; Yamashita, S; Hirazawa, N

    2007-08-01

    The ciliated protozoan Cryptocaryon irritans, a parasite of seawater fishes, was found to express an antigen that elicits antibodies in rabbits and tiger puffer (Takifugu ruburipes). Serum from rabbits and fish immunized with theronts had agglutination/immobilization activity against theronts in vitro; fish serum antibody levels (measured by enzyme-linked immunosorbent assays: ELISA) correlated with this activity. Anti-theront antibody levels in fish were significantly higher in the immunized group as compared with control fish at 2 weeks after booster immunization (injection of bovine serum albumin; Student's t-test, Pagglutination/immobilization antigen. Indirect immunofluorescence staining of theronts suggested that this 32 kDa antigen was expressed on the surface of cilia. The full-length 32 kDa antigen cDNA contained 1147 basepairs, encoding a 328-amino acid protein including hydrophobic N- and C-termini. As with Tetrahymena and Paramecium spp., TAA and TAG appear to be used as glutamine codons in the 32 kDa antigen gene.

  2. Enteric trimethyl chitosan nanoparticles containing hepatitis B surface antigen for oral delivery.

    Science.gov (United States)

    Farhadian, Asma; Dounighi, Naser Mohammadpour; Avadi, Mohammadreza

    2015-01-01

    Oral vaccination is the preferred route of immunization. However, the degradative condition of the gastrointestinal tract and the higher molecular size of peptides pose major challenges in developing an effective oral vaccination system. One of the most excellent methods used in the development of oral vaccine delivery system relies on the entrapment of the antigen in polymeric nanoparticles. In this work, trimethyl chitosan (TMC) nanoparticles were fabricated using ionic gelation teqnique by interaction hydroxypropyl methylcellulose phthalate (HPMCP), a pH-sensitive polymer, with TMC and the utility of the particles in the oral delivery of hepatitis B surface antigen (HBsAg) was evaluated employing solutions that simulated gastric and intestinal conditions. The particle size, morphology, zeta potential, loading capacity, loading efficiency, in vitro release behavior, structure, and morphology of nanoparticles were evaluated, and the activity of the loaded antigen was assessed. Size of the optimized TMC/HPMCP nanoparticles and that of the antigen-loaded nanoparticles were 85 nm and 158 nm, respectively. Optimum loading capacity (76.75%) and loading efficiency (86.29%) were achieved at 300 µg/mL concentration of the antigen. SEM images revealed a spherical shape as well as a smooth and near-homogenous surface of nanoparticles. Results of the in vitro release studies showed that formulation with HPMCP improved the acid stability of the TMC nanoparticles as well as their capability to preserve the loaded HBsAg from gastric destruction. The antigen showed good activity both before and after loading. The results suggest that TMC/HPMCP nanoparticles could be used in the oral delivery of HBsAg vaccine.

  3. Identification of Anti-tumor Cells Carrying Natural Killer (NK Cell Antigens in Patients With Hematological Cancers

    Directory of Open Access Journals (Sweden)

    Ewelina Krzywinska

    2015-10-01

    Full Text Available Natural killer (NK cells, a cytotoxic lymphocyte lineage, are able to kill tumor cells in vitro and in mouse models. However, whether these cells display an anti-tumor activity in cancer patients has not been demonstrated. Here we have addressed this issue in patients with several hematological cancers. We found a population of highly activated CD56dimCD16+ NK cells that have recently degranulated, evidence of killing activity, and it is absent in healthy donors. A high percentage of these cells expressed natural killer cell p46-related protein (NKp46, natural-killer group 2, member D (NKG2D and killer inhibitory receptors (KIRs and a low percentage expressed NKG2A and CD94. They are also characterized by a high metabolic activity and active proliferation. Notably, we found that activated NK cells from hematological cancer patients have non-NK tumor cell antigens on their surface, evidence of trogocytosis during tumor cell killing. Finally, we found that these activated NK cells are distinguished by their CD45RA+RO+ phenotype, as opposed to non-activated cells in patients or in healthy donors displaying a CD45RA+RO− phenotype similar to naïve T cells. In summary, we show that CD45RA+RO+ cells, which resemble a unique NK population, have recognized tumor cells and degranulate in patients with hematological neoplasias.

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

  5. Intraspecific epitopic variation in a carbohydrate antigen exposed on the surface of Trichostrongylus colubriformis infective L3 larvae.

    Directory of Open Access Journals (Sweden)

    David R Maass

    2009-09-01

    Full Text Available The carbohydrate larval antigen, CarLA, is present on the exposed surface of all strongylid nematode infective L3 larvae tested, and antibodies against CarLA can promote rapid immune rejection of incoming Trichostrongylus colubriformis larvae in sheep. A library of ovine recombinant single chain Fv (scFv antibody fragments, displayed on phage, was prepared from B cell mRNA of field-immune sheep. Phage displaying scFvs that bind to the surface of living exsheathed T. colubriformis L3 larvae were identified, and the majority of worm-binding scFvs recognized CarLA. Characterization of greater than 500 worm surface binding phage resulted in the identification of nine different anti-CarLA scFvs that recognized three distinct T. colubriformis CarLA epitopes based on blocking and additive ELISA. All anti-CarLA scFvs were specific to the T. colubriformis species of nematode. Each of the three scFv epitope classes displayed identical Western blot recognition patterns and recognized the exposed surface of living T. colubriformis exsheathed L3 larvae. Surprisingly, each of the anti-CarLA scFvs was able to bind to only a subset of worms. Double-labelling indirect immunofluorescence revealed that the three classes of anti-CarLA scFvs recognize distinct, non-overlapping, T. colubriformis sub-populations. These results demonstrate that individual T. colubriformis L3 larvae display only one of at least three distinct antigenic forms of CarLA on their surface at any given time, and suggest that antigenic variation within CarLA is likely a mechanism of immune evasion in strongylid nematodes.

  6. SPONGIOTIC DERMATITIS WITH A MIXED INFLAMMATORY INFILTRATE OF LYMPHOCYTES, ANTIGEN PRESENTING CELLS, IMMUNOGLOBULINS AND COMPLEMENT

    Directory of Open Access Journals (Sweden)

    Abreu Velez Ana Maria

    2011-04-01

    Full Text Available Background: The clinical and histological presentation of spongiotic dermatitis and its inflammatory infiltrates warrant further investigation. In this case documentation of a patient with cutaneous spongiotic reactivity, we aim to characterize antigen presenting cells, as well as the skin-specific cutaneous lymphocyte antigen population by multiple techniques. Case report: A 30 year old Caucasian female presented with a two week history of blistering and erosions around the vaginal, rectal and axillary areas. Material and Methods: We utilized hematoxylin and eosin histology, direct immunofluorescence, immunohistochemistry and confocal microscopy methods to evaluate the immune reaction patterns of the cutaneous inflammatory cells. Results: In the primary histologic areas of spongiotic dermatitis, a mixed population of B and T lymphocytes was seen. Ki-67 antigen proliferative index staining was accentuated in these areas, correlating with the presence of large numbers of epidermal and dermal antigen presenting cells. Among the antigen presenting cell population, we detected strong positivities with CD1a, Factor XIIIa, myeloid/hystoid antigen, S100, HAM-56, and CD68. Interestingly, immunoglobulins G, D and M and Complement factors C1q and C3 were also strongly expressed in antigen presenting cell areas, including positivity within the spongiotic epidermis and around dermal vessels. Conclusions: We document a heterogeneous population of B and T lymphocytes and the presence of multiple classes of antigen presenting cells, immunoglobulins and complement in and surrounding histologically spongiotic areas; these findings further correlated with increased levels of expression of Ki-67.

  7. Unique interplay between sugar and lipid in determining the antigenic potency of bacterial antigens for NKT cells.

    Directory of Open Access Journals (Sweden)

    Enrico Girardi

    2011-11-01

    Full Text Available Invariant natural killer T (iNKT cells are an evolutionary conserved T cell population characterized by features of both the innate and adaptive immune response. Studies have shown that iNKT cells are required for protective responses to Gram-positive pathogens such as Streptococcus pneumoniae, and that these cells recognize bacterial diacylglycerol antigens presented by CD1d, a non-classical antigen-presenting molecule. The combination of a lipid backbone containing an unusual fatty acid, vaccenic acid, as well as a glucose sugar that is weaker or not stimulatory when linked to other lipids, is required for iNKT cell stimulation by these antigens. Here we have carried out structural and biophysical studies that illuminate the reasons for the stringent requirement for this unique combination. The data indicate that vaccenic acid bound to the CD1d groove orients the protruding glucose sugar for TCR recognition, and it allows for an additional hydrogen bond of the glucose with CD1d when in complex with the TCR. Furthermore, TCR binding causes an induced fit in both the sugar and CD1d, and we have identified the CD1d amino acids important for iNKT TCR recognition and the stability of the ternary complex. The studies show also how hydrogen bonds formed by the glucose sugar can account for the distinct binding kinetics of the TCR for this CD1d-glycolipid complex. Therefore, our studies illuminate the mechanism of glycolipid recognition for antigens from important pathogens.

  8. Effective Delivery of Antigen-Encapsulin Nanoparticle Fusions to Dendritic Cells Leads to Antigen-Specific Cytotoxic T Cell Activation and Tumor Rejection.

    Science.gov (United States)

    Choi, Bongseo; Moon, Hyojin; Hong, Sung Joon; Shin, Changsik; Do, Yoonkyung; Ryu, Seongho; Kang, Sebyung

    2016-08-23

    In cancer immunotherapy, robust and efficient activation of cytotoxic CD8(+) T cell immune responses is a promising, but challenging task. Dendritic cells (DCs) are well-known professional antigen presenting cells that initiate and regulate antigen-specific cytotoxic CD8(+) T cells that kill their target cells directly as well as secrete IFN-γ, a cytokine critical in tumor rejection. Here, we employed recently established protein cage nanoparticles, encapsulin (Encap), as antigenic peptide nanocarriers by genetically incorporating the OT-1 peptide of ovalbumin (OVA) protein to the three different positions of the Encap subunit. With them, we evaluated their efficacy in activating DC-mediated antigen-specific T cell cytotoxicity and consequent melanoma tumor rejection in vivo. DCs efficiently engulfed Encap and its variants (OT-1-Encaps), which carry antigenic peptides at different positions, and properly processed them within phagosomes. Delivered OT-1 peptides were effectively presented by DCs to naïve CD8(+) T cells successfully, resulting in the proliferation of antigen-specific cytotoxic CD8(+) T cells. OT-1-Encap vaccinations in B16-OVA melanoma tumor bearing mice effectively activated OT-1 peptide specific cytotoxic CD8(+) T cells before or even after tumor generation, resulting in significant suppression of tumor growth in prophylactic as well as therapeutic treatments. A large number of cytotoxic CD8(+) T cells that actively produce both intracellular and secretory IFN-γ were observed in tumor-infiltrating lymphocytes collected from B16-OVA tumor masses originally vaccinated with OT-1-Encap-C upon tumor challenges. The approaches we describe herein may provide opportunities to develop epitope-dependent vaccination systems that stimulate and/or modulate efficient and epitope-specific cytotoxic T cell immune responses in nonpathogenic diseases.

  9. Antigen 43 from Escherichia coli induces inter- and intraspecies cell aggregation and changes in colony morphology of Pseudomonas fluorescens

    DEFF Research Database (Denmark)

    Kjærgaard, Kristian; Schembri, Mark; Hasman, Henrik;

    2000-01-01

    Antigen 43 (Ag43) is a surface-displayed autotransporter protein of Escherichia coli. By virtue of its self-association characteristics, this protein is able to mediate autoaggregation and flocculation off. coli cells in static cultures. Additionally, surface display of Ag43 is associated...... with a distinct frizzy colony morphology in E. coli. Here we show that Ag43 can be expressed in a functional form on the surface of the environmentally important Pseudomonas fluorescens strain SBW25 with ensuing cell aggregation and frizzy colony types. Using green fluorescence protein-tagged cells, we...... demonstrate that Ag43 can be used as a tool to provide interspecies cell aggregation between E. coli and P. fluorescens. Furthermore, Ag43 expression enhances biofilm formation in P. fluorescens to glass surfaces. The versatility of this protein was also reflected in Ag43 surface display in a variety of other...

  10. A mimotope of Pre-S2 region of surface antigen of viral hepatitis Bscreened by phage display

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    To acquire the phage-displayed mimotopes which mimic the specificity of hepatitis B virus surface antigen (HBsAg), a random peptide library expressing linear peptide with 12 amino acids in length were used to screen with the serum from a hepatitis B virus infected patient in the recovery phase. After 3 rounds of biopanning, the positive phages were confirmed by competitive ELISA using HBsAg/P33. Two phagotopes were identified and one of them was confirmed as mimotope by competition experiment. Based on the mimotpe, a multiple antigenic peptide with four branches was synthesized by solid phase peptide synthesis. The antiginicity and specificity of the synthesized antigen was tested in BALB/c mice compared with the native epitope-based antigen. The results showed that the mimotope-based antigen could evoke higher titer of antibodies with the same specificity of the epitope-based antigen. Those findings indicate mimotopes can be used in antigen and vaccine design.

  11. CD80 and CD86 differentially regulate mechanical interactions of T-cells with antigen-presenting dendritic cells and B-cells.

    Directory of Open Access Journals (Sweden)

    Tong Seng Lim

    Full Text Available Functional T-cell responses are initiated by physical interactions between T-cells and antigen-presenting cells (APCs, including dendritic cells (DCs and B-cells. T-cells are activated more effectively by DCs than by B-cells, but little is known about the key molecular mechanisms that underpin the particular potency of DC in triggering T-cell responses. To better understand the influence of physical intercellular interactions on APC efficacy in activating T-cells, we used single cell force spectroscopy to characterize and compare the mechanical forces of interactions between DC:T-cells and B:T-cells. Following antigen stimulation, intercellular interactions of DC:T-cell conjugates were stronger than B:T-cell interactions. DCs induced higher levels of T-cell calcium mobilization and production of IL-2 and IFNγ than were elicited by B-cells, thus suggesting that tight intercellular contacts are important in providing mechanically stable environment to initiate T-cell activation. Blocking antibodies targeting surface co-stimulatory molecules CD80 or CD86 weakened intercellular interactions and dampen T-cell activation, highlighting the amplificatory roles of CD80/86 in regulating APC:T-cell interactions and T-cell functional activation. The variable strength of mechanical forces between DC:T-cells and B:T-cell interactions were not solely dependent on differential APC expression of CD80/86, since DCs were superior to B-cells in promoting strong interactions with T-cells even when CD80 and CD86 were inhibited. These data provide mechanical insights into the effects of co-stimulatory molecules in regulating APC:T-cell interactions.

  12. CD80 and CD86 differentially regulate mechanical interactions of T-cells with antigen-presenting dendritic cells and B-cells.

    Science.gov (United States)

    Lim, Tong Seng; Goh, James Kang Hao; Mortellaro, Alessandra; Lim, Chwee Teck; Hämmerling, Günter J; Ricciardi-Castagnoli, Paola

    2012-01-01

    Functional T-cell responses are initiated by physical interactions between T-cells and antigen-presenting cells (APCs), including dendritic cells (DCs) and B-cells. T-cells are activated more effectively by DCs than by B-cells, but little is known about the key molecular mechanisms that underpin the particular potency of DC in triggering T-cell responses. To better understand the influence of physical intercellular interactions on APC efficacy in activating T-cells, we used single cell force spectroscopy to characterize and compare the mechanical forces of interactions between DC:T-cells and B:T-cells. Following antigen stimulation, intercellular interactions of DC:T-cell conjugates were stronger than B:T-cell interactions. DCs induced higher levels of T-cell calcium mobilization and production of IL-2 and IFNγ than were elicited by B-cells, thus suggesting that tight intercellular contacts are important in providing mechanically stable environment to initiate T-cell activation. Blocking antibodies targeting surface co-stimulatory molecules CD80 or CD86 weakened intercellular interactions and dampen T-cell activation, highlighting the amplificatory roles of CD80/86 in regulating APC:T-cell interactions and T-cell functional activation. The variable strength of mechanical forces between DC:T-cells and B:T-cell interactions were not solely dependent on differential APC expression of CD80/86, since DCs were superior to B-cells in promoting strong interactions with T-cells even when CD80 and CD86 were inhibited. These data provide mechanical insights into the effects of co-stimulatory molecules in regulating APC:T-cell interactions.

  13. Identification and characterization of Ixodes scapularis antigens that elicit tick immunity using yeast surface display.

    Science.gov (United States)

    Schuijt, Tim J; Narasimhan, Sukanya; Daffre, Sirlei; DePonte, Kathleen; Hovius, Joppe W R; Van't Veer, Cornelis; van der Poll, Tom; Bakhtiari, Kamran; Meijers, Joost C M; Boder, Eric T; van Dam, Alje P; Fikrig, Erol

    2011-01-05

    Repeated exposure of rabbits and other animals to ticks results in acquired resistance or immunity to subsequent tick bites and is partially elicited by antibodies directed against tick antigens. In this study we demonstrate the utility of a yeast surface display approach to identify tick salivary antigens that react with tick-immune serum. We constructed an Ixodes scapularis nymphal salivary gland yeast surface display library and screened the library with nymph-immune rabbit sera and identified five salivary antigens. Four of these proteins, designated P8, P19, P23 and P32, had a predicted signal sequence. We generated recombinant (r) P8, P19 and P23 in a Drosophila expression system for functional and immunization studies. rP8 showed anti-complement activity and rP23 demonstrated anti-coagulant activity. Ixodes scapularis feeding was significantly impaired when nymphs were fed on rabbits immunized with a cocktail of rP8, rP19 and rP23, a hall mark of tick-immunity. These studies also suggest that these antigens may serve as potential vaccine candidates to thwart tick feeding.

  14. Identification and characterization of Ixodes scapularis antigens that elicit tick immunity using yeast surface display.

    Directory of Open Access Journals (Sweden)

    Tim J Schuijt

    Full Text Available Repeated exposure of rabbits and other animals to ticks results in acquired resistance or immunity to subsequent tick bites and is partially elicited by antibodies directed against tick antigens. In this study we demonstrate the utility of a yeast surface display approach to identify tick salivary antigens that react with tick-immune serum. We constructed an Ixodes scapularis nymphal salivary gland yeast surface display library and screened the library with nymph-immune rabbit sera and identified five salivary antigens. Four of these proteins, designated P8, P19, P23 and P32, had a predicted signal sequence. We generated recombinant (r P8, P19 and P23 in a Drosophila expression system for functional and immunization studies. rP8 showed anti-complement activity and rP23 demonstrated anti-coagulant activity. Ixodes scapularis feeding was significantly impaired when nymphs were fed on rabbits immunized with a cocktail of rP8, rP19 and rP23, a hall mark of tick-immunity. These studies also suggest that these antigens may serve as potential vaccine candidates to thwart tick feeding.

  15. Blockade of LFA-1 augments in vitro differentiation of antigen-induced Foxp3+ Treg cells

    Science.gov (United States)

    Verhagen, Johan; Wraith, David C.

    2014-01-01

    Adoptive transfer of antigen-specific, in vitro-induced Foxp3+ Treg (iTreg) cells protects against autoimmune disease. To generate antigen-specific iTreg cells at high purity, however, remains a challenge. Whereas polyclonal T cell stimulation with anti-CD3 and anti-CD28 antibody yields Foxp3+ iTreg cells at a purity of 90–95%, antigen-induced iTreg cells typically do not exceed a purity of 65–75%, even in a TCR-transgenic model. In a similar vein to thymic Treg cell selection, iTreg cell differentiation is influenced not only by antigen recognition and the availability of TGF-β but also by co-factors including costimulation and adhesion molecules. In this study, we demonstrate that blockade of the T cell integrin Leukocyte Function-associated Antigen-1 (LFA-1) during antigen-mediated iTreg cell differentiation augments Foxp3 induction, leading to approximately 90% purity of Foxp3+ iTreg cells. This increased efficacy not only boosts the yield of Foxp3+ iTreg cells, it also reduces contamination with activated effector T cells, thus improving the safety of adoptive transfer immunotherapy. PMID:25108241

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

    Directory of Open Access Journals (Sweden)

    Rancés Blanco

    2013-01-01

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

  17. Diminished Memory T-Cell Expansion Due to Delayed Kinetics of Antigen Expression by Lentivectors.

    Directory of Open Access Journals (Sweden)

    Karina Furmanov

    Full Text Available Memory CD8(+ T lymphocytes play a central role in protective immunity. In attempt to increase the frequencies of memory CD8(+ T cells, repeated immunizations with viral vectors are regularly explored. Lentivectors have emerged as a powerful vaccine modality with relatively low pre-existing and anti-vector immunity, thus, thought to be ideal for boosting memory T cells. Nevertheless, we found that lentivectors elicited diminished secondary T-cell responses that did not exceed those obtained by priming. This was not due to the presence of anti-vector immunity, as limited secondary responses were also observed following heterologous prime-boost immunizations. By dissecting the mechanisms involved in this process, we demonstrate that lentivectors trigger exceptionally slow kinetics of antigen expression, while optimal activation of lentivector-induced T cells relays on durable expression of the antigen. These qualities hamper secondary responses, since lentivector-encoded antigen is rapidly cleared by primary cytotoxic T cells that limit its presentation by dendritic cells. Indeed, blocking antigen clearance by cytotoxic T cells via FTY720 treatment, fully restored antigen presentation. Taken together, while low antigen expression is expected during secondary immunization with any vaccine vector, our results reveal that the intrinsic delayed expression kinetics of lentiviral-encoded antigen, further dampens secondary CD8(+ T-cell expansion.

  18. Detecting Antigen-Specific T Cell Responses: From Bulk Populations to Single Cells

    Directory of Open Access Journals (Sweden)

    Chansavath Phetsouphanh

    2015-08-01

    Full Text Available A new generation of sensitive T cell-based assays facilitates the direct quantitation and characterization of antigen-specific T cell responses. Single-cell analyses have focused on measuring the quality and breadth of a response. Accumulating data from these studies demonstrate that there is considerable, previously-unrecognized, heterogeneity. Standard assays, such as the ICS, are often insufficient for characterization of rare subsets of cells. Enhanced flow cytometry with imaging capabilities enables the determination of cell morphology, as well as the spatial localization of the protein molecules within a single cell. Advances in both microfluidics and digital PCR have improved the efficiency of single-cell sorting and allowed multiplexed gene detection at the single-cell level. Delving further into the transcriptome of single-cells using RNA-seq is likely to reveal the fine-specificity of cellular events such as alternative splicing (i.e., splice variants and allele-specific expression, and will also define the roles of new genes. Finally, detailed analysis of clonally related antigen-specific T cells using single-cell TCR RNA-seq will provide information on pathways of differentiation of memory T cells. With these state of the art technologies the transcriptomics and genomics of Ag-specific T cells can be more definitively elucidated.

  19. Presentation of antigen by B cells subsets. Pt. 1. Lyb-5{sup +} and Lyb-5{sup -} B cells differ in ability to stimulate specific T cells

    Energy Technology Data Exchange (ETDEWEB)

    Zimecki, M. [Polska Akademia Nauk, Wroclaw (Poland). Inst. Immunologii i Terapii Doswiadczalnej; Whiteley, P.J. [Merck and Co., Inc., Rahway, NJ (United States); Pierce, C.W.; Kapp, J.A. [Harrington Cancer Center, Amarillo (United States). Dept. of Cellular and Molecular Immunology

    1994-12-31

    We have examined the antigen presenting cell (APC) function of different B cells. Resident, peritoneal B cells from normal mice were more efficient than splenic B cells in presenting antigen to CD4{sup +} T cell lines. Peritoneal B cells from X-linked immunodeficient (Xid) mice, by contrast, stimulated no detectable responses. Xid splenic B cells were much less efficient APC than normal splenic B cells. B cells from neonatal mice also were very poor APC until the mice were 3 to 4 weeks old. Xid B cells presented antigen to T cell hybridomas as well as normal B cells showing that they process antigen normally. Thus, the defect is most likely in providing secondary signals. The ability of B cells to present antigen efficiency correlates with the percentage of B cells reported to express the Lyb-5 antigen. Anti-Lyb-5 serum and complement abrogated the APC activity of B cells suggesting that Lyb-5{sup +}, but not Lyb-5{sup -} cells are efficient APC. We also found that activated and resting normal splenic B cells, separated by buoyant density, presented antigen equally. Both populations also contained Lyb-5{sup +} B cells although they were a larger fraction of the activated cells. Lyb-5 is now thought to be an activation antigen rather than a differentiation antigen. If this idea is correct, then our data indicate that anti-Lyb-5 more cleanly separates activated and resting B cells than buoyant density techniques. (author). 38 refs, 7 figs, 1 tab.

  20. Bacterial cell surface structures in Yersinia enterocolitica.

    Science.gov (United States)

    Białas, Nataniel; Kasperkiewicz, Katarzyna; Radziejewska-Lebrecht, Joanna; Skurnik, Mikael

    2012-06-01

    Yersinia enterocolitica is a widespread member of the family of Enterobacteriaceae that contains both non-virulent and virulent isolates. Pathogenic Y. enterocolitica strains, especially belonging to serotypes O:3, O:5,27, O:8 and O:9 are etiologic agents of yersiniosis in animals and humans. Y. enterocolitica cell surface structures that play a significant role in virulence have been subject to many investigations. These include outer membrane (OM) glycolipids such as lipopolysaccharide (LPS) and enterobacterial common antigen (ECA) and several cell surface adhesion proteins present only in virulent Y. enterocolitica, i.e., Inv, YadA and Ail. While the yadA gene is located on the Yersinia virulence plasmid the Ail, Inv, LPS and ECA are chromosomally encoded. These structures ensure the correct architecture of the OM, provide adhesive properties as well as resistance to antimicrobial peptides and to host innate immune response mechanisms.

  1. Antibody-antigen-adjuvant conjugates enable co-delivery of antigen and adjuvant to dendritic cells in cis but only have partial targeting specificity.

    Directory of Open Access Journals (Sweden)

    Martin Kreutz

    Full Text Available Antibody-antigen conjugates, which promote antigen-presentation by dendritic cells (DC by means of targeted delivery of antigen to particular DC subsets, represent a powerful vaccination approach. To ensure immunity rather than tolerance induction the co-administration of a suitable adjuvant is paramount. However, co-administration of unlinked adjuvant cannot ensure that all cells targeted by the antibody conjugates are appropriately activated. Furthermore, antigen-presenting cells (APC that do not present the desired antigen are equally strongly activated and could prime undesired responses against self-antigens. We, therefore, were interested in exploring targeted co-delivery of antigen and adjuvant in cis in form of antibody-antigen-adjuvant conjugates for the induction of anti-tumour immunity. In this study, we report on the assembly and characterization of conjugates consisting of DEC205-specific antibody, the model antigen ovalbumin (OVA and CpG oligodeoxynucleotides (ODN. We show that such conjugates are more potent at inducing cytotoxic T lymphocyte (CTL responses than control conjugates mixed with soluble CpG. However, our study also reveals that the nucleic acid moiety of such antibody-antigen-adjuvant conjugates alters their binding and uptake and allows delivery of the antigen and the adjuvant to cells partially independently of DEC205. Nevertheless, antibody-antigen-adjuvant conjugates are superior to antibody-free antigen-adjuvant conjugates in priming CTL responses and efficiently induce anti-tumour immunity in the murine B16 pseudo-metastasis model. A better understanding of the role of the antibody moiety is required to inform future conjugate vaccination strategies for efficient induction of anti-tumour responses.

  2. Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8 T cells.

    Directory of Open Access Journals (Sweden)

    Lynette Beattie

    2010-03-01

    Full Text Available Kupffer cells (KCs represent the major phagocytic population within the liver and provide an intracellular niche for the survival of a number of important human pathogens. Although KCs have been extensively studied in vitro, little is known of their in vivo response to infection and their capacity to directly interact with antigen-specific CD8(+ T cells. Here, using a combination of approaches including whole mount and thin section confocal microscopy, adoptive cell transfer and intra-vital 2-photon microscopy, we demonstrate that KCs represent the only detectable population of mononuclear phagocytes within granulomas induced by Leishmania donovani infection that are capable of presenting parasite-derived peptide to effector CD8(+ T cells. This restriction of antigen presentation to KCs within the Leishmania granuloma has important implications for the identification of new candidate vaccine antigens and for the design of novel immuno-therapeutic interventions.

  3. Biotin avidin amplified magnetic immunoassay for hepatitis B surface antigen detection using GoldMag nanoparticles

    Science.gov (United States)

    Yu, An; Geng, Tingting; Fu, Qiang; Chen, Chao; Cui, Yali

    2007-04-01

    Using GoldMag (Fe3O4/Au) nanoparticles as a carrier, a biotin-avidin amplified ELISA was developed to detect hepatitis B surface antigen (HBsAg). A specific antibody was labeled with biotin and then used to detect the antigen with an antibody coated on GoldMag nanoparticles by a sandwich ELISA assay. The results showed that 5 mol of biotin were surface bound per mole of antibody. The biotin-avidin amplified ELISA assay has a higher sensitivity than that of the direct ELISA assay. There is 5-fold difference between HBsAg positive and negative serum even at dilution of 1:10000, and the relative standard deviation of the parallel positive serum at dilution of 1:4000 is 5.98% (n=11).

  4. Towards efficient cancer immunotherapy: advances in developing artificial antigen-presenting cells

    NARCIS (Netherlands)

    Eggermont, L.J.; Paulis, L.E.M.; Tel, J.; Figdor, C.G.

    2014-01-01

    Active anti-cancer immune responses depend on efficient presentation of tumor antigens and co-stimulatory signals by antigen-presenting cells (APCs). Therapy with autologous natural APCs is costly and time-consuming and results in variable outcomes in clinical trials. Therefore, development of artif

  5. Axotomy induces MHC class I antigen expression on rat nerve cells

    DEFF Research Database (Denmark)

    Maehlen, J; Schröder, H D; Klareskog, L;

    1988-01-01

    Immunomorphological staining demonstrates that class I major histocompatibility complex (MHC)-coded antigen expression can be selectively induced on otherwise class I-negative rat nerve cells by peripheral axotomy. Induction of class I as well as class II antigen expression was simultaneously see...

  6. Size-dependent accumulation of particles in lysosomes modulates dendritic cell function through impaired antigen degradation

    Directory of Open Access Journals (Sweden)

    Seydoux E

    2014-08-01

    Full Text Available Emilie Seydoux,1,2 Barbara Rothen-Rutishauser,1,3 Izabela M Nita,1 Sandor Balog,3 Amiq Gazdhar,1 Philip A Stumbles,4,5 Alke Petri-Fink,3,6 Fabian Blank,1,* Christophe von Garnier1,*1Department of Respiratory Medicine, Inselspital, Bern University Hospital, Department of Clinical Research, University of Bern, 2Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland; 3Adolphe Merkle Institute, University of Fribourg, Fribourg, Switzerland; 4School of Veterinary and Life Sciences, Molecular and Biomedical Sciences, Murdoch University, Perth, WA, Australia; 5Telethon Kids Institute, Perth, WA, Australia; 6Department of Chemistry, University of Fribourg, Fribourg, Switzerland*These authors contributed equally to the manuscriptIntroduction: Nanosized particles may enable therapeutic modulation of immune responses by targeting dendritic cell (DC networks in accessible organs such as the lung. To date, however, the effects of nanoparticles on DC function and downstream immune responses remain poorly understood. Methods: Bone marrow–derived DCs (BMDCs were exposed in vitro to 20 or 1,000 nm polystyrene (PS particles. Particle uptake kinetics, cell surface marker expression, soluble protein antigen uptake and degradation, as well as in vitro CD4+ T-cell proliferation and cytokine production were analyzed by flow cytometry. In addition, co-localization of particles within the lysosomal compartment, lysosomal permeability, and endoplasmic reticulum stress were analyzed. Results: The frequency of PS particle–positive CD11c+/CD11b+ BMDCs reached an early plateau after 20 minutes and was significantly higher for 20 nm than for 1,000 nm PS particles at all time-points analyzed. PS particles did not alter cell viability or modify expression of the surface markers CD11b, CD11c, MHC class II, CD40, and CD86. Although particle exposure did not modulate antigen uptake, 20 nm PS particles decreased the capacity

  7. Functional expression of CD95/Fas Antigen and Bcl-2 on Cord blood Hematopoietic Progenitor Cells

    Institute of Scientific and Technical Information of China (English)

    马艳萍; 邹萍

    2002-01-01

    The cell-surface expression and functional status of the CD95/Fas antigen on primitive hematopoietic progenitors isolated from human cord blood (CB) were studied. The CD34+ cells freshly isolated from CB displayed low CD95 expression. The combinations of cytokines such as SCF+FL could up-regulate the expression of CD95 in vitro culture and tumor necrosis factor-e (TNF-α) and interon-γ (IFN-γ) further increased the CD95 expression induced by positive cytokines. The functional status of CD95-mediated apoptosis were analyzed by incubation of CD34+CB cells in the presence of anti-CD95 monoclonal antibodies (McAbs). The effects of anti-CD95 McAbs were measured by viable cell counting, flow cytometry, LTIC and CFU-C assays. A decrease of viable cells, CFUC and LTIC numbers were observed in the presence of anti-CD95 McAbs and TNF-α or IFN-γ.However, growth factor deprivation or the early-acting cytokine such as SCF and FL cross-linking to CD95 caused low apoptosis of CD34+ cells. The correlation of increased intracytoplasmic levels of bcl2 and the presence of CD95 on fresh CB CD34+ cells suggested that bcl-2 might be involved in protecting against CD95-mediated apoptosis of CB CD34+ cells.

  8. Retinoic acid induction of CD38 antigen expression on normal and leukemic human myeloid cells: relationship with cell differentiation.

    Science.gov (United States)

    Prus, Eugenia; Fibach, Eitan

    2003-04-01

    Differentiation in the hematopoietic system involves, among other changes, altered expression of antigens, including the CD34 and CD38 surface antigens. In normal hematopoiesis, the most immature stem cells have the CD34 + CD34 - phenotype. In acute myeloid leukemia (AML), although blasts from most patients are CD38 +, some are CD38 - . AML blasts are blocked at early stages of differentiation; in some leukemic cells this block can be overcome by a variety of agents, including retinoids, that induce maturation into macrophages and granulocytes both in vitro and in vivo. Retinoids can also induce CD38 expression. In the present study, we investigated the relationship between induction of CD38 expression and induction of myeloid differentiation by retinoic acid (RA) in normal and leukemic human hematopoietic cells. In the promyelocytic (PML) CD34 - cell lines, HL60 and CB-1, as well as in normal CD34 + CD34 - hematopietic progenitor cells RA induced both CD38 expression as well as morphological and functional myeloid differentiation that resulted in loss of self-renewal. In contrast, in the myeloblastic CD34 + leukemic cell lines, ML-1 and KG-1a, as well as in primary cultures of cells derived from CD34 + -AML (M0 and M1) patients, RA caused an increase in CD38 + that was not associated with significant differentiation. Yet, long exposure of ML-1, but not KG-1, cells to RA resulted in loss of self-renewal. The results suggest that while in normal hematopoietic cells and in PML CD34 - cells induction of CD38 antigen expression by RA results in terminal differentiation along the myeloid lineage, in early myeloblastic leukemic CD34 + cells, induction of CD38 and differentiation are not functionally related. Since, several lines of evidence suggest that the CD38 - cells are the targets of leukemic transformation, transition of these cellsinto CD38 + phenotype by RA or other drugs may have therapeutic effect, either alone or in conjunction with cytotoxic drugs, regardless

  9. How T-cells use large deviations to recognize foreign antigens

    CERN Document Server

    Zint, Natali; Hollander, Frank den

    2008-01-01

    A stochastic model for the activation of T-cells is analysed. T-cells are part of the immune system and recognize foreign antigens against a background of the body's own molecules. The model under consideration is a slight generalization of a model introduced by Van den Berg, Rand and Burroughs in 2001, and is capable of explaining how this recognition works on the basis of rare stochastic events. With the help of a refined large deviation theorem and numerical evaluation it is shown that, for a wide range of parameters, T-cells can distinguish reliably between foreign antigens and self-antigens.

  10. Role of TLR-2 and fungal surface antigens on innate immune response against Sporothrix schenckii.

    Science.gov (United States)

    Negrini, Thais de C; Ferreira, Lucas S; Alegranci, Pâmela; Arthur, Rodrigo A; Sundfeld, Pedro P; Maia, Danielle C G; Spolidorio, Luis C; Carlos, Iracilda Z

    2013-01-01

    Sporotrichosis is an infection caused by the dimorphic fungus Sporothrix schenckii. Toll-like receptors (TLRs) play an important role in immunity, since they bind to pathogen surface antigens and initiate the immune response. However, little is known about the role of TLR-2 and fungal surface antigens in the recognition of S. schenckii and in the subsequent immune response. This study aimed to evaluate the involvement of TLR-2 and fungal surface soluble (SolAg) and lipidic (LipAg) antigens in phagocytosis of S. schenckii and production of immune mediators by macrophages obtained from WT and TLR-2(-/-) animals. The results showed that TLR-2(-/-) animals had had statistical lower percentage of macrophages with internalized yeasts compared to WT. SolAg and LipAg impaired phagocytosis and immunological mediator production for both WT and TLR-2(-/-). The absence of TLR-2 led to lower production of the cytokines TNF-α, IL-1β, IL-12 and IL-10 compared to WT animals. These results suggest a new insight in relation to how the immune system, through TLR-2, recognizes and induces the production of mediators in response to the fungus S. schenckii.

  11. Red Blood Cell Antigen Genotyping for Sickle Cell Disease, Thalassemia, and Other Transfusion Complications.

    Science.gov (United States)

    Fasano, Ross M; Chou, Stella T

    2016-10-01

    Since the discovery of the ABO blood group in the early 20th century, more than 300 blood group antigens have been categorized among 35 blood group systems. The molecular basis for most blood group antigens has been determined and demonstrates tremendous genetic diversity, particularly in the ABO and Rh systems. Several blood group genotyping assays have been developed, and 1 platform has been approved by the Food and Drug Administration as a "test of record," such that no phenotype confirmation with antisera is required. DNA-based red blood cell (RBC) phenotyping can overcome certain limitations of hemagglutination assays and is beneficial in many transfusion settings. Genotyping can be used to determine RBC antigen phenotypes in patients recently transfused or with interfering allo- or autoantibodies, to resolve discrepant serologic typing, and/or when typing antisera are not readily available. Molecular RBC antigen typing can facilitate complex antibody evaluations and guide RBC selection for patients with sickle cell disease (SCD), thalassemia, and autoimmune hemolytic anemia. High-resolution RH genotyping can identify variant RHD and RHCE in patients with SCD, which have been associated with alloimmunization. In the future, broader access to cost-efficient, high-resolution RBC genotyping technology for both patient and donor populations may be transformative for the field of transfusion medicine.

  12. Selective culling of high avidity antigen-specific CD4+ T cells after virulent Salmonella infection.

    Science.gov (United States)

    Ertelt, James M; Johanns, Tanner M; Mysz, Margaret A; Nanton, Minelva R; Rowe, Jared H; Aguilera, Marijo N; Way, Sing Sing

    2011-12-01

    Typhoid fever is a persistent infection caused by host-adapted Salmonella strains adept at circumventing immune-mediated host defences. Given the importance of T cells in protection, the culling of activated CD4+ T cells after primary infection has been proposed as a potential immune evasion strategy used by this pathogen. We demonstrate that the purging of activated antigen-specific CD4+ T cells after virulent Salmonella infection requires SPI-2 encoded virulence determinants, and is not restricted only to cells with specificity to Salmonella-expressed antigens, but extends to CD4+ T cells primed to expand by co-infection with recombinant Listeria monocytogenes. Unexpectedly, however, the loss of activated CD4+ T cells during Salmonella infection demonstrated using a monoclonal population of adoptively transferred CD4+ T cells was not reproduced among the endogenous repertoire of antigen-specific CD4+ T cells identified with MHC class II tetramer. Analysis of T-cell receptor variable segment usage revealed the selective loss and reciprocal enrichment of defined CD4+ T-cell subsets after Salmonella co-infection that is associated with the purging of antigen-specific cells with the highest intensity of tetramer staining. Hence, virulent Salmonella triggers the selective culling of high avidity activated CD4+ T-cell subsets, which re-shapes the repertoire of antigen-specific T cells that persist later after infection.

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

    DEFF Research Database (Denmark)

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

    2004-01-01

    The slow acquisition of protection against Plasmodium falciparum malaria probably reflects the extensive diversity of important antigens. The variant surface antigens (VSA) that mediate parasite adhesion to a range of host molecules are regarded as important targets of acquired protective immunit...

  14. Effects of laboratory maintenance on the nature of surface reactive antigens of Neisseria gonorrhoeae.

    Science.gov (United States)

    Arko, R J; Bullard, J C; Duncan, W P

    1976-01-01

    The extensive in vitro cultivation methods used in propagating and maintaining gonococcal cells were found to affect their virulence, antigenicity, and ultrastructure. Adapting a laboratory-maintained strain of gonococci to animal virulence resulted in two lines of pilated cells with similar colonial morphologies. The animal-adapted cells, however, had a greater amount of extracellular pili and a more prominent peptidoglycan cell wall layer. They were also more resistant to the bactericidal effects of guinea-pig complement and more reactive in macroagglutination and bactericidal tests with strain-specific gonococcal antibody. In comparative guinea-pig protection trials, formalin-fixed cells of the animal-adapted cell line were 500 times more effective as immunogens than the laboratory-maintained cell line. Images PMID:825184

  15. Simulation and Theory of Antibody Binding to Crowded Antigen-Covered Surfaces.

    Directory of Open Access Journals (Sweden)

    Cristiano De Michele

    2016-03-01

    Full Text Available In this paper we introduce a fully flexible coarse-grained model of immunoglobulin G (IgG antibodies parametrized directly on cryo-EM data and simulate the binding dynamics of many IgGs to antigens adsorbed on a surface at increasing densities. Moreover, we work out a theoretical model that allows to explain all the features observed in the simulations. Our combined computational and theoretical framework is in excellent agreement with surface-plasmon resonance data and allows us to establish a number of important results. (i Internal flexibility is key to maximize bivalent binding, flexible IgGs being able to explore the surface with their second arm in search for an available hapten. This is made clear by the strongly reduced ability to bind with both arms displayed by artificial IgGs designed to rigidly keep a prescribed shape. (ii The large size of IgGs is instrumental to keep neighboring molecules at a certain distance (surface repulsion, which essentially makes antigens within reach of the second Fab always unoccupied on average. (iii One needs to account independently for the thermodynamic and geometric factors that regulate the binding equilibrium. The key geometrical parameters, besides excluded-volume repulsion, describe the screening of free haptens by neighboring bound antibodies. We prove that the thermodynamic parameters govern the low-antigen-concentration regime, while the surface screening and repulsion only affect the binding at high hapten densities. Importantly, we prove that screening effects are concealed in relative measures, such as the fraction of bivalently bound antibodies. Overall, our model provides a valuable, accurate theoretical paradigm beyond existing frameworks to interpret experimental profiles of antibodies binding to multi-valent surfaces of different sorts in many contexts.

  16. Simulation and Theory of Antibody Binding to Crowded Antigen-Covered Surfaces

    Science.gov (United States)

    De Michele, Cristiano; De Los Rios, Paolo; Foffi, Giuseppe; Piazza, Francesco

    2016-01-01

    In this paper we introduce a fully flexible coarse-grained model of immunoglobulin G (IgG) antibodies parametrized directly on cryo-EM data and simulate the binding dynamics of many IgGs to antigens adsorbed on a surface at increasing densities. Moreover, we work out a theoretical model that allows to explain all the features observed in the simulations. Our combined computational and theoretical framework is in excellent agreement with surface-plasmon resonance data and allows us to establish a number of important results. (i) Internal flexibility is key to maximize bivalent binding, flexible IgGs being able to explore the surface with their second arm in search for an available hapten. This is made clear by the strongly reduced ability to bind with both arms displayed by artificial IgGs designed to rigidly keep a prescribed shape. (ii) The large size of IgGs is instrumental to keep neighboring molecules at a certain distance (surface repulsion), which essentially makes antigens within reach of the second Fab always unoccupied on average. (iii) One needs to account independently for the thermodynamic and geometric factors that regulate the binding equilibrium. The key geometrical parameters, besides excluded-volume repulsion, describe the screening of free haptens by neighboring bound antibodies. We prove that the thermodynamic parameters govern the low-antigen-concentration regime, while the surface screening and repulsion only affect the binding at high hapten densities. Importantly, we prove that screening effects are concealed in relative measures, such as the fraction of bivalently bound antibodies. Overall, our model provides a valuable, accurate theoretical paradigm beyond existing frameworks to interpret experimental profiles of antibodies binding to multi-valent surfaces of different sorts in many contexts. PMID:26967624

  17. Immune Responses of Dendritic Cells Loaded with Antigens from Apoptotic Cholangiocarcinoma Cells Caused by γ-Irradation

    Institute of Scientific and Technical Information of China (English)

    WUGang; HANBenli; PEIXuetao

    2002-01-01

    Objective:To investigate the induction cytotoxic T cells(CTLs) with antitumor activity and therapeutic efficacy after dendritic cells(DCs) acquired antigen from apoptotic cholangiocarcinoma cells caused by γ-irradiation. Methods:DCs from peripheral blood mononuclear cells (PBMC) that maintain the antigen capturing and processing capacity charateristic of immature cells have been established in vitro, using granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). Then, in cholangiocarcinoma cells apoptosis was induced by γ-irradiation. The experimental groups were as follows:(1)coculture of DCs and apoptotic cancer cells and T cells;(2)coculture of DCs and necrotic cancer cells and T cells;(3)coculture of DCs, cultured cancer cell and T cells. They are cocultured for 7 days.DCs and T cells were riched, isolated and their antitumor response was tested. Results:The cells had typical dendritic morphology, expressed high levels of CDla and B7, acquired antigen from apoptotic cells caused by γ-irradiation and induced an increased T cell stimulatory capacity in mixed lymphocyte reactions (MLR). Conclusion:DCs obtained from PBMCs using GM-CSF and IL-4 can efficiently present antigen derived from apoptotic cells caused by γ-irradiation and efficiently induce T cells.This strategy, therefore, may present an effective approach to transduce DCs with antigen.

  18. Chimeric Antigen Receptor T Cell (Car T Cell Therapy In Hematology

    Directory of Open Access Journals (Sweden)

    Pinar Ataca

    2015-12-01

    Full Text Available It is well demonstrated that immune system can control and eliminate cancer cells. Immune-mediated elimination of tumor cells has been discovered and is the basis of both cancer vaccines and cellular therapies including hematopoietic stem cell transplantation (HSCT. Adoptive T cell transfer has been improved to be more specific and potent and cause less off-target toxicities. Currently, there are two forms of engineered T cells being tested in clinical trials: T cell receptor (TCR and chimeric antigen receptor (CAR modified T cells. On July 1, 2014, the United States Food and Drug Administration granted ‘breakthrough therapy’ designation to anti-CD19 CAR T cell therapy. Many studies were conducted to evaluate the beneficiaries of this exciting and potent new treatment modality. This review summarizes the history of adoptive immunotherapy, adoptive immunotherapy using CARs, the CAR manufacturing process, preclinical-clinical studies, effectiveness and drawbacks of this strategy.

  19. Docking of B-cell epitope antigen to specific hepatitis B antibody

    Indian Academy of Sciences (India)

    R Rajkannan; E J Padma Malar

    2007-09-01

    The interaction of pres1 region of hepatitis B virus B-cell epitope antigen with specific hepatitis B neutralizing monoclonal antibody was examined by docking study. We modelled the 3D complex structure of B-cell epitope antigen residues CTTPAQGNSMFPSCCCTKPTDGNCY by homology modelling and docked it with the crystal structure of monoclonal antibody specific for the pres1 region of the hepatitis B virus. At the optimized docked conformation, the interactions between the amino acids of antigen and antibody were examined. It is found that the docked complex is stabilized by 59.3 kcal/mol. The stability of the docked antigen-antibody complex is due to hydrogen bonding and van der Waals interactions. The amino acids of the antigen and antibody responsible for the interaction were identified.

  20. In vivo requirement for Atg5 in antigen presentation by dendritic cells.

    Science.gov (United States)

    Lee, Heung Kyu; Mattei, Lisa M; Steinberg, Benjamin E; Alberts, Philipp; Lee, Yun Hee; Chervonsky, Alexander; Mizushima, Noboru; Grinstein, Sergio; Iwasaki, Akiko

    2010-02-26

    Autophagy is known to be important in presentation of cytosolic antigens on MHC class II (MHC II). However, the role of autophagic process in antigen presentation in vivo is unclear. Mice with dendritic cell (DC)-conditional deletion in Atg5, a key autophagy gene, showed impaired CD4(+) T cell priming after herpes simplex virus infection and succumbed to rapid disease. The most pronounced defect of Atg5(-/-) DCs was the processing and presentation of phagocytosed antigens containing Toll-like receptor stimuli for MHC class II. In contrast, cross-presentation of peptides on MHC I was intact in the absence of Atg5. Although induction of metabolic autophagy did not enhance MHC II presentation, autophagic machinery was required for optimal phagosome-to-lysosome fusion and subsequent processing of antigen for MHC II loading. Thus, our study revealed that DCs utilize autophagic machinery to optimally process and present extracellular microbial antigens for MHC II presentation.

  1. Focused specificity of intestinal TH17 cells towards commensal bacterial antigens.

    Science.gov (United States)

    Yang, Yi; Torchinsky, Miriam B; Gobert, Michael; Xiong, Huizhong; Xu, Mo; Linehan, Jonathan L; Alonzo, Francis; Ng, Charles; Chen, Alessandra; Lin, Xiyao; Sczesnak, Andrew; Liao, Jia-Jun; Torres, Victor J; Jenkins, Marc K; Lafaille, Juan J; Littman, Dan R

    2014-06-05

    T-helper-17 (TH17) cells have critical roles in mucosal defence and in autoimmune disease pathogenesis. They are most abundant in the small intestine lamina propria, where their presence requires colonization of mice with microbiota. Segmented filamentous bacteria (SFB) are sufficient to induce TH17 cells and to promote TH17-dependent autoimmune disease in animal models. However, the specificity of TH17 cells, the mechanism of their induction by distinct bacteria, and the means by which they foster tissue-specific inflammation remain unknown. Here we show that the T-cell antigen receptor (TCR) repertoire of intestinal TH17 cells in SFB-colonized mice has minimal overlap with that of other intestinal CD4(+) T cells and that most TH17 cells, but not other T cells, recognize antigens encoded by SFB. T cells with antigen receptors specific for SFB-encoded peptides differentiated into RORγt-expressing TH17 cells, even if SFB-colonized mice also harboured a strong TH1 cell inducer, Listeria monocytogenes, in their intestine. The match of T-cell effector function with antigen specificity is thus determined by the type of bacteria that produce the antigen. These findings have significant implications for understanding how commensal microbiota contribute to organ-specific autoimmunity and for developing novel mucosal vaccines.

  2. Enhanced interferon-γ secretion and antitumor activity of T-lymphocytes activated by dendritic cells loaded with glycoengineered myeloma antigens

    Institute of Scientific and Technical Information of China (English)

    XIONG Hong; WU Qiu-ye; HU Hong-gang; LIU Ban; GUO Zhong-wu; Daniel Man-yuan Sze; HOU Jian

    2007-01-01

    Background Immunotherapy is emerging as a promising cure for cancer. However, a severe problem in this area is the immune tolerance to tumor cells and tumor-associated antigens, as evidenced by the ability of cancer to escape immune surveillance. To overcome this problem this work examined the potential of improving the antigenicity of myeloma by metabolic engineering of its cell surface carbohydrate antigens (i.e., glycoengineering) and presentation of the modified tumor antigens by dendritic cells (DCs) to generate cytotoxic T-lymphocytes (CTLs).Methods CD138+ myeloma cells were isolated from 11 multipe myeloma (MM) patients by the immunomagnetic bead method. The MM cells were treated with N-propionyl-D-mannosamine (ManNPr), a synthetic analog of N-acetyl-D-mannosamine (ManNAc), the natural biosynthetic precursor of N-acetyl sialic acid (NeuNAc), to express unnatural N-propionylated sialoglycans. The giycoengineered cells were then induced to apoptosis, and the apoptotic products were added to cultured functional DCs that could present the unnatural carbohydrate antigens to autologous T-lymphocytes.Results It was found that the resultant DCs could activate CD4+ and CD8+ T-lymphocytes, resulting in increased expression of T cell surface markers, including CD8CD28 and CD4CD29. Moreover, upon stimulation by glycoengineered MM cells, these DC-activated T-lymphocytes could release significantly higher levels of IFN-γ (P<0.05).Lactate dehydrogenase (LDH) assays further showed that the stimulated T-lymphocytes were cytotoxic to glycoengineered MM cells.Conclusions This work demonstrated that glycoengineered myeloma cells were highly antigenic and the CTLs induced by the DCs loaded with the unnatural myeloma antigens were specifically cytotoxic to the glycoengineered myeloma.This may provide a new strategy for overcoming the problem of immune tolerance for the development of effective immunotherapies for MM.

  3. Flow cytometric assessment of antigen-specific proliferation in peripheral chicken T cells by CFSE dilution

    DEFF Research Database (Denmark)

    Dalgaard, Tina; Norup, Liselotte Rothmann; Rubbenstroth, Dennis;

    2010-01-01

    peripheral mononuclear blood cells (PBMC) and to evaluate and optimize its performance in relation to detection of vaccine-induced chicken T cells specific for Newcastle disease virus (NDV). The method was based on analysis of CFSE dilution upon ex vivo recall stimulation with whole vaccine antigen. Analysis...... of proliferation was combined with the use of monoclonal antibodies directed against the lymphocyte surface markers CD4 and CD8 in order to phenotype the responding cells. Problems with nonspecific background proliferation especially in the CD8 compartment were significantly reduced by replacing medium containing...... fetal calf serum with serum-free medium. It was rendered probable that antigen-specific cellular immunity can be assessed by this method as NDV-vaccinated chickens showed a significantly higher proliferative capacity than age-matched naïve controls. Furthermore it was shown that the recall stimulation...

  4. Strategy for eliciting antigen-specific CD8+ T cell-mediated immune response against a cryptic CTL epitope of merkel cell polyomavirus large T antigen

    Directory of Open Access Journals (Sweden)

    Gomez Bianca P

    2012-10-01

    Full Text Available Abstract Background Merkel cell carcinoma (MCC is a relatively new addition to the expanding category of oncovirus-induced cancers. Although still comparably rare, the number of cases has risen dramatically in recent years. Further complicating this trend is that MCC is an extremely aggressive neoplasm with poor patient prognosis and limited treatment options for advanced disease. The causative agent of MCC has been identified as the merkel cell polyomavirus (MCPyV. The MCPyV-encoded large T (LT antigen is an oncoprotein that is theorized to be essential for virus-mediated tumorigenesis and is therefore, an excellent MCC antigen for the generation of antitumor immune responses. As a foreign antigen, the LT oncoprotein avoids the obstacle of immune tolerance, which normally impedes the development of antitumor immunity. Ergo, it is an excellent target for anti-MCC immunotherapy. Since tumor-specific CD8+ T cells lead to better prognosis for MCC and numerous other cancers, we have generated a DNA vaccine that is capable of eliciting LT-specific CD8+ T cells. The DNA vaccine (pcDNA3-CRT/LT encodes the LT antigen linked to a damage-associated molecular pattern, calreticulin (CRT, as it has been demonstrated that the linkage of CRT to antigens promotes the induction of antigen-specific CD8+ T cells. Results The present study shows that DNA vaccine-induced generation of LT-specific CD8+ T cells is augmented by linking CRT to the LT antigen. This is relevant since the therapeutic effects of the pcDNA3-CRT/LT DNA vaccine is mediated by LT-specific CD8+ T cells. Mice vaccinated with the DNA vaccine produced demonstrably more LT-specific CD8+ T cells. The DNA vaccine was also able to confer LT-specific CD8+ T cell-mediated protective and therapeutic effects to prolong the survival of mice with LT-expressing tumors. In the interest of determining the LT epitope which most MCC-specific CD8+ T cells recognize, we identified the amino acid sequence of the

  5. Proliferating cell nuclear antigen and Ki-67 immunohistochemistry of oligodendrogliomas with special reference to prognosis

    DEFF Research Database (Denmark)

    HEEGAARD, S.; Sommer, Helle Mølgaard; BROHOLM, H.;

    1995-01-01

    Background. The biologic behavior of oligodendrogliomas is somewhat unpredictable. A supplementary prognostic factor is, therefore, desirable. Methods. Thirty-two pure supratentorial oligodendrogliomas were investigated using proliferating cell nuclear antigen (PCNA) and Ki-67 immunohistochemical...

  6. Antigen selection in B-cell lymphomas--tracing the evidence.

    Science.gov (United States)

    Sutton, Lesley-Ann; Agathangelidis, Andreas; Belessi, Chrysoula; Darzentas, Nikos; Davi, Frederic; Ghia, Paolo; Rosenquist, Richard; Stamatopoulos, Kostas

    2013-12-01

    While signaling through the B cell receptor (BcR) facilitates B cell development and maintenance, it also carries intertwined risks for the development of lymphomas since malignant B cells can exploit these pathways in order to trigger and fuel clonal expansion. This corruption of the normal B cell response to antigens, leading to sustained BcR signaling, has given great impulse to investigate in detail the role of antigen in lymphomas. Suffice it to conclude from such studies, largely immunogenetics based, that the evidence implicating antigens (exogenous or self) in lymphoma development is substantial and that lymphomagenesis is functionally driven and dynamic, rather than a simple stochastic process. As the paradigm of antigen-driven lymphoma evolves, further investigation will be paramount to the identification of the inciting agent(s) that may be responsible for immunoproliferative neoplasms and also for the development of therapeutic agents targeting effectors of the BcR signaling pathway.

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

    Science.gov (United States)

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

    2017-02-17

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

  8. Tandem CAR T cells targeting HER2 and IL13Rα2 mitigate tumor antigen escape.

    Science.gov (United States)

    Hegde, Meenakshi; Mukherjee, Malini; Grada, Zakaria; Pignata, Antonella; Landi, Daniel; Navai, Shoba A; Wakefield, Amanda; Fousek, Kristen; Bielamowicz, Kevin; Chow, Kevin K H; Brawley, Vita S; Byrd, Tiara T; Krebs, Simone; Gottschalk, Stephen; Wels, Winfried S; Baker, Matthew L; Dotti, Gianpietro; Mamonkin, Maksim; Brenner, Malcolm K; Orange, Jordan S; Ahmed, Nabil

    2016-08-01

    In preclinical models of glioblastoma, antigen escape variants can lead to tumor recurrence after treatment with CAR T cells that are redirected to single tumor antigens. Given the heterogeneous expression of antigens on glioblastomas, we hypothesized that a bispecific CAR molecule would mitigate antigen escape and improve the antitumor activity of T cells. Here, we created a CAR that joins a HER2-binding scFv and an IL13Rα2-binding IL-13 mutein to make a tandem CAR exodomain (TanCAR) and a CD28.ζ endodomain. We determined that patient TanCAR T cells showed distinct binding to HER2 or IL13Rα2 and had the capability to lyse autologous glioblastoma. TanCAR T cells exhibited activation dynamics that were comparable to those of single CAR T cells upon encounter of HER2 or IL13Rα2. We observed that TanCARs engaged HER2 and IL13Rα2 simultaneously by inducing HER2-IL13Rα2 heterodimers, which promoted superadditive T cell activation when both antigens were encountered concurrently. TanCAR T cell activity was more sustained but not more exhaustible than that of T cells that coexpressed a HER2 CAR and an IL13Rα2 CAR, T cells with a unispecific CAR, or a pooled product. In a murine glioblastoma model, TanCAR T cells mitigated antigen escape, displayed enhanced antitumor efficacy, and improved animal survival. Thus, TanCAR T cells show therapeutic potential to improve glioblastoma control by coengaging HER2 and IL13Rα2 in an augmented, bivalent immune synapse that enhances T cell functionality and reduces antigen escape.

  9. Engineering antigen-specific T cells from genetically modified human hematopoietic stem cells in immunodeficient mice.

    Directory of Open Access Journals (Sweden)

    Scott G Kitchen

    Full Text Available There is a desperate need for effective therapies to fight chronic viral infections. The immune response is normally fastidious at controlling the majority of viral infections and a therapeutic strategy aimed at reestablishing immune control represents a potentially powerful approach towards treating persistent viral infections. We examined the potential of genetically programming human hematopoietic stem cells to generate mature CD8+ cytotoxic T lymphocytes that express a molecularly cloned, "transgenic" human anti-HIV T cell receptor (TCR. Anti-HIV TCR transduction of human hematopoietic stem cells directed the maturation of a large population of polyfunctional, HIV-specific CD8+ cells capable of recognizing and killing viral antigen-presenting cells. Thus, through this proof-of-concept we propose that genetic engineering of human hematopoietic stem cells will allow the tailoring of effector T cell responses to fight HIV infection or other diseases that are characterized by the loss of immune control.

  10. Purification and characterization of a major human Pneumocystis carinii surface antigen

    DEFF Research Database (Denmark)

    Lundgren, B; Lipschik, G Y; Kovacs, J A

    1991-01-01

    with zymolyase followed by molecular sieve and ion exchange chromatography. The native proteins had an apparent mol wt of 290,000 or greater, based on molecular sieve studies as well as cross-linking studies. Both proteins were glycoproteins; treatment with endoglycosidase H resulted in a 9% decrease in mol wt......Previous studies of Pneumocystis carinii have identified the major surface antigen of rat and human isolates as proteins of 116,000 and 95,000 mol wt, respectively, that are antigenically not identical. In this study both rat and human P. carinii proteins were purified by solubilization....... The carbohydrate composition of the rat P. carinii glycoprotein was distinct from the human isolate; glucose, mannose, galactose, and glucosamine occurred in approximately equimolar ratios in the human P. carinii protein, whereas glucose and mannose were the predominant sugars of the rat P. carinii protein...

  11. Methionine enkephalin (MENK) improved the functions of bone marrow-derived dendritic cells (BMDCs) loaded with antigen.

    Science.gov (United States)

    Li, Weiwei; Meng, Jingjuan; Li, Xuan; Hua, Hui; Yiming, Meng; Wang, Qiushi; Wang, Enhua; Shan, Fengping

    2012-09-01

    The aim of this investigation is to look at whether MENK could improve antitumor effect of CD8+T cell elicited by BMDCs. We investigated the effects of MENK on the differentiation, maturation, and functions of murine BMDC loaded with Rac-1 antigens (RG) and CTL of tumor specific immune response elicited by the BMDC in vitro and in vivo. The production of cytokine IL-12 and TNF-α secreted by BMDCs in the presence of MENK was assayed with ELISA and key surface markers of CD40, CD86, CD83 and MHC-II on the BMDCs were analyzed with use of flow cytometry (FCM). In addition, the activities to induce CD8+ T cell proliferation, along with displayed cytotoxicity of the CD8+T cells(CTL) by the BMDCs after treatment with MENK were determined with use of FCM as well as MTS. Our results indicated that MENK induced phenotypic and functional maturation of BMDC loaded with RG antigen, as evidenced by higher level of expression of key surface markers and more production of cytokines. Subsequently, the BMDC activated by MENK intensified immune responses mounted by CTL, resulting in stronger antitumor activity. Our results suggest that MENK could be working as an effective immune adjuvant in vaccine preparation for cancer fight and other immune related diseases. We concluded that MENK could be a positive immune modulator in the improved functions of BMDCs loaded with antigen as well as in CD8+T cell mediated anti-tumor responses.

  12. Quantitative detection of the tumor-associated antigen large external antigen in colorectal cancer tissues and cells using quantum dot probe

    Directory of Open Access Journals (Sweden)

    Wang S

    2016-01-01

    Full Text Available Shuo Wang, Wanming Li, Dezheng Yuan, Jindan Song, Jin Fang Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang, People’s Republic of China Abstract: The large external antigen (LEA is a cell surface glycoprotein that has been proven to be highly expressed in colorectal cancer (CRC as a tumor-associated antigen. To evaluate and validate the relationship between LEA expression and clinical characteristics of CRC with high efficiency, LEA expression levels were detected in 85 tissue blocks from CRC patients by quantum dot-based immunohistochemistry (QD-IHC combined with imaging quantitative analysis using quantum dots with a 605 nm emission wavelength (QD605 conjugated to an ND-1 monoclonal antibody against LEA as a probe. Conventional IHC was performed in parallel for comparison. Both QD-IHC and conventional IHC showed that LEA was specifically expressed in CRC, but not in non-CRC tissues, and high LEA expression was significantly associated with a more advanced T-stage (P<0.05, indicating that LEA is likely to serve as a CRC prognostic marker. Compared with conventional IHC, receiver operating characteristic analysis revealed that QD-IHC possessed higher sensitivity, resulting in an increased positive detection rate of CRC, from 70.1% to 89.6%. In addition, a simpler operation, objective analysis of results, and excellent repeatability make QD-IHC an attractive alternative to conventional IHC in clinical practice. Furthermore, to explore whether the QD probes can be utilized to quantitatively detect living cells or single cells, quantum dot-based immunocytochemistry (QD-ICC combined with imaging quantitative analysis was developed to evaluate LEA expression in several CRC cell lines. It was demonstrated that QD-ICC could also predict the correlation between LEA expression and the T-stage characteristics of

  13. Effect of BSA Antigen Sensitization during the Acute Phase of Influenza A Viral Infection on CD11c+ Pulmonary Antigen Presenting Cells

    Directory of Open Access Journals (Sweden)

    Fumitaka Sato

    2009-01-01

    Conclusions: BSA antigen sensitization during the acute phase of influenza A viral infection enhanced IL-10 production from naive CD4+ T cell interaction with CD11c+ pulmonary APCs. The IL-10 secretion evoked Th2 responses in the lungs with downregulation of Th1 responses and was important for the eosinophil recruitment into the lungs after BSA antigen challenge.

  14. T cell re-targeting to EBV antigens following TCR gene transfer: CD28-containing receptors mediate enhanced antigen-specific IFNgamma production

    NARCIS (Netherlands)

    N. van der Schaft (Niels); B. Lankiewicz (Birgit); H.A. Drexhage (Hemmo); C.A. Berrevoets (Cor); D.J. Moss (Denis); V. Levitsky (Victor); M. Bonneville (Marc); S.P. Lee (Steven); A.J. McMichael (Andrew); J.W. Gratama (Jan-Willem); R.L.H. Bolhuis (Reinder); R.A. Willemsen (Ralph); J.E.M.A. Debets (Reno)

    2006-01-01

    textabstractAbstract EBV is associated with a broad range of malignancies. Adoptive immunotherapy of these tumors with EBV-specific CTL proved useful. We generated a panel of primary human T cells specific to various EBV antigens (i.e. Epstein-Barr nuclear antigen 3A, 3B and BamHI-M leftward reading

  15. Antigen-Specific lgA B Memory Cell Responses to Shigella Antigens Elicited in Volunteers Immunized with Live Attenuated Shigella flexneri 2a Oral Vaccine Candidates

    Science.gov (United States)

    2011-01-01

    cell responses to Shigella antigens elicited in volunteers immunized with live attenuated Shigella flexneri 2a oral vaccine candidates J.K. Simona,b... Shigella ;. B cell memory; Immunoglobulin lgA; Mucosal immunity Abstract We studied the induction of antigen-specific lgA memory B cells (BM) in...volunteers who received live attenuated Shigella flexneri 2a vaccines. Subjects ingested a single oral dose of 107 , 108 or 109 CFU of S. flexneri 2a with

  16. Bacteroides gingivalis antigens and bone resorbing activity in root surface fractions of periodontally involved teeth

    Energy Technology Data Exchange (ETDEWEB)

    Patters, M.R.; Landsberg, R.L.; Johansson, L.A.; Trummel, C.L.; Robertson, P.R. (Department of Periodontology, University of Connecticut, School of Dental Medicine, Farmington, Connecticut, U.S.A.)

    1982-01-01

    Bone resorbing activity and the presence of antigens of Bacteroides gingivalis were assessed in plaque, calculus, cementum, and dentin obtained from roots of teeth previously exposed to periodontitis. Each fraction was obtained by scaling the root surface. The fraction were extracted by stirring and sonication, and the soluble centrifuged, sterilized, dialyzed, and adjusted to equivalent protein concentrations. Cementum and dentin extracts from impacted teeth were prepared similarly and served as controls. Stimulation of bone resorption by each extract was assessed in organ cultures of fetal rat bones by measurement of release of previously-incorporated /sup 45/Ca from the bone into the medium. In some groups of teeth, calculus and cementum were treated with acid prior to scaling. Citric acid washes were recovered and dialyzed. An enzyme-linked immunosorbent assay (ELISA) was used to assess the extracts for the presence of antigens reactive with an antiserum to B. gingivalis. Significant stimulation of bone resorption was found in all calculus and periodontally-involved cementum preparations. ELISA showed significant levels of B.gingivalis antigens in plaque, calculus, and cementum of periodontally-involved teeth, but not in involved dentin nor in cementum or dentin of impact teeth. Treatment with citric acid removed essentially all B.gingivalis antigens from cementum but not calculus. The results suggest that substances which stimulate bone resorption and substances which react with B. gingivalis antiserum are present in surface plaque, calculus, and cementum or periodontally-involved teeth. These substances are not present in cementum and dentin of impacted teeth nor in dentin of periodontally-involved teeth. Treatment by both scaling and citric demineralization will remove most of these substances from cementum of teeth previously exposed to periodontitis.

  17. Combinational targeting offsets antigen escape and enhances effector functions of adoptively transferred T cells in glioblastoma.

    Science.gov (United States)

    Hegde, Meenakshi; Corder, Amanda; Chow, Kevin K H; Mukherjee, Malini; Ashoori, Aidin; Kew, Yvonne; Zhang, Yi Jonathan; Baskin, David S; Merchant, Fatima A; Brawley, Vita S; Byrd, Tiara T; Krebs, Simone; Wu, Meng Fen; Liu, Hao; Heslop, Helen E; Gottschalk, Stephen; Gottachalk, Stephen; Yvon, Eric; Ahmed, Nabil

    2013-11-01

    Preclinical and early clinical studies have demonstrated that chimeric antigen receptor (CAR)-redirected T cells are highly promising in cancer therapy. We observed that targeting HER2 in a glioblastoma (GBM) cell line results in the emergence of HER2-null tumor cells that maintain the expression of nontargeted tumor-associated antigens. Combinational targeting of these tumor-associated antigens could therefore offset this escape mechanism. We studied the single-cell coexpression patterns of HER2, IL-13Rα2, and EphA2 in primary GBM samples using multicolor flow cytometry and immunofluorescence, and applied a binomial routine to the permutations of antigen expression and the related odds of complete tumor elimination. This mathematical model demonstrated that cotargeting HER2 and IL-13Rα2 could maximally expand the therapeutic reach of the T cell product in all primary tumors studied. Targeting a third antigen did not predict an added advantage in the tumor cohort studied. We therefore generated bispecific T cell products from healthy donors and from GBM patients by pooling T cells individually expressing HER2 and IL-13Rα2-specific CARs and by making individual T cells to coexpress both molecules. Both HER2/IL-13Rα2-bispecific T cell products offset antigen escape, producing enhanced effector activity in vitro immunoassays (against autologous glioma cells in the case of GBM patient products) and in an orthotopic xenogeneic murine model. Further, T cells coexpressing HER2 and IL-13Rα2-CARs exhibited accentuated yet antigen-dependent downstream signaling and a particularly enhanced antitumor activity.

  18. The uptake of soluble and particulate antigens by epithelial cells in the mouse small intestine.

    Directory of Open Access Journals (Sweden)

    Savannah E Howe

    Full Text Available Intestinal epithelial cells (IECs overlying the villi play a prominent role in absorption of digested nutrients and establish a barrier that separates the internal milieu from potentially harmful microbial antigens. Several mechanisms by which antigens of dietary and microbial origin enter the body have been identified; however whether IECs play a role in antigen uptake is not known. Using in vivo imaging of the mouse small intestine, we investigated whether epithelial cells (enterocytes play an active role in the uptake (sampling of lumen antigens. We found that small molecular weight antigens such as chicken ovalbumin, dextran, and bacterial LPS enter the lamina propria, the loose connective tissue which lies beneath the epithelium via goblet cell associated passageways. However, epithelial cells overlying the villi can internalize particulate antigens such as bacterial cell debris and inert nanoparticles (NPs, which are then found co-localizing with the CD11c+ dendritic cells in the lamina propria. The extent of NP uptake by IECs depends on their size: 20-40 nm NPs are taken up readily, while NPs larger than 100 nm are taken up mainly by the epithelial cells overlying Peyer's patches. Blocking NPs with small proteins or conjugating them with ovalbumin does not inhibit their uptake. However, the uptake of 40 nm NPs can be inhibited when they are administered with an endocytosis inhibitor (chlorpromazine. Delineating the mechanisms of antigen uptake in the gut is essential for understanding how tolerance and immunity to lumen antigens are generated, and for the development of mucosal vaccines and therapies.

  19. Expression of Immunoglobulin Receptors with Distinctive Features Indicating Antigen Selection by Marginal Zone B Cells from Human Spleen

    Science.gov (United States)

    Colombo, Monica; Cutrona, Giovanna; Reverberi, Daniele; Bruno, Silvia; Ghiotto, Fabio; Tenca, Claudya; Stamatopoulos, Kostas; Hadzidimitriou, Anastasia; Ceccarelli, Jenny; Salvi, Sandra; Boccardo, Simona; Calevo, Maria Grazia; De Santanna, Amleto; Truini, Mauro; Fais, Franco; Ferrarini, Manlio

    2013-01-01

    Marginal zone (MZ) B cells, identified as surface (s)IgMhighsIgDlowCD23low/−CD21+CD38− B cells, were purified from human spleens, and the features of their V(D)J gene rearrangements were investigated and compared with those of germinal center (GC), follicular mantle (FM) and switched memory (SM) B cells. Most MZ B cells were CD27+ and exhibited somatic hypermutations (SHM), although to a lower extent than SM B cells. Moreover, among MZ B-cell rearrangements, recurrent sequences were observed, some of which displayed intraclonal diversification. The same diversifying sequences were detected in very low numbers in GC and FM B cells and only when a highly sensitive, gene-specific polymerase chain reaction was used. This result indicates that MZ B cells could expand and diversify in situ and also suggested the presence of a number of activation-induced cytidine deaminase (AID)-expressing B cells in the MZ. The notion of antigen-driven expansion/selection in situ is further supported by the VH CDR3 features of MZ B cells with highly conserved amino acids at specific positions and by the finding of shared (“stereotyped”) sequences in two different spleens. Collectively, the data are consistent with the notion that MZ B cells are a special subset selected by in situ antigenic stimuli. PMID:23877718

  20. Cytotoxicity of tumor antigen specific human T cells is unimpaired by arginine depletion.

    Directory of Open Access Journals (Sweden)

    Markus Munder

    Full Text Available Tumor-growth is often associated with the expansion of myeloid derived suppressor cells that lead to local or systemic arginine depletion via the enzyme arginase. It is generally assumed that this arginine deficiency induces a global shut-down of T cell activation with ensuing tumor immune escape. While the impact of arginine depletion on polyclonal T cell proliferation and cytokine secretion is well documented, its influence on chemotaxis, cytotoxicity and antigen specific activation of human T cells has not been demonstrated so far. We show here that chemotaxis and early calcium signaling of human T cells are unimpaired in the absence of arginine. We then analyzed CD8(+ T cell activation in a tumor peptide as well as a viral peptide antigen specific system: (i CD8(+ T cells with specificity against the MART-1aa26-35*A27L tumor antigen expanded with in vitro generated dendritic cells, and (ii clonal CMV pp65aa495-503 specific T cells and T cells retrovirally transduced with a CMV pp65aa495-503 specific T cell receptor were analyzed. Our data demonstrate that human CD8(+ T cell antigen specific cytotoxicity and perforin secretion are completely preserved in the absence of arginine, while antigen specific proliferation as well as IFN-γ and granzyme B secretion are severely compromised. These novel results highlight the complexity of antigen specific T cell activation and demonstrate that human T cells can preserve important activation-induced effector functions in the context of arginine deficiency.

  1. Successive Administration of Streptococcus Type 5 Group A Antigens and S. typhimurium Antigenic Complex Corrects Elevation of Serum Cytokine Concentration and Number of Bone Marrow Stromal Pluripotent Cells in CBA Mice Induced by Each Antigen Separately.

    Science.gov (United States)

    Gorskaya, Yu F; Danilova, T A; Grabko, V I; Nesterenko, V G

    2015-12-01

    Administration of bacterial antigens to CBA mice induced an increase in serum concentration of virtually all cytokines with a peak in 4 h after administration of S. typhimurium antigens and in 7 h after administration of streptococcus antigens. In 20 h, cytokine concentrations returned to the control level or were slightly below it. In 4 h after administration of S. typhimurium antigens preceded 3 h before by administration of streptococcus antigens, we observed a significant decrease in serum concentrations of IFN-γ, IL-10, GM-CSF, IL-12, and TNF-α, in comparison with injection S. typhimurium antigens alone and IL-5, IL-10, GM-CSF, and TNF-α in comparison with injection of streptococcus antigens alone; the concentrations of IL-2 and IFN-γ, in contrast, increased by 1.5 times in this case. In 20 h after administration of S. typhimurium antigens, the number of multipotential stromal cells (MSC) in the bone marrow and their cloning efficiency (ECF-MSC) increased by 4.8 and 4.4 times, respectively, in comparison with the control, while after administration of streptococcus antigens by 2.6 and 2.4 times, respectively. In 20 h after administration of S. typhimurium antigens preceded 3 h before by administration of streptococcus antigens, these parameters increased by 3.2 and 2.9 times, respectively, in comparison with the control, i.e. the observed increase in the level of MSC count and ECF-MSC is more consistent with the response of the stromal tissue to streptococcus antigens. Thus, successive administration of two bacterial antigens corrected both serum cytokine profiles and MSC response to administration of each antigen separately, which indicates changeability of the stromal tissue in response to changes in the immune response.

  2. MetQ of Neisseria gonorrhoeae Is a Surface-Expressed Antigen That Elicits Bactericidal and Functional Blocking Antibodies

    Science.gov (United States)

    Semchenko, Evgeny A.; Day, Christopher J.

    2016-01-01

    ABSTRACT Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection (STI) gonorrhea, is a growing public health threat for which a vaccine is urgently needed. We characterized the functional role of the gonococcal MetQ protein, which is the methionine binding component of an ABC transporter system, and assessed its potential as a candidate antigen for inclusion in a gonococcal vaccine. MetQ has been found to be highly conserved in all strains investigated to date, it is localized on the bacterial surface, and it binds l-methionine with a high affinity. MetQ is also involved in gonococcal adherence to cervical epithelial cells. Mutants lacking MetQ have impaired survival in human monocytes, macrophages, and serum. Furthermore, antibodies raised against MetQ are bactericidal and are able to block gonococcal adherence to epithelial cells. These data suggest that MetQ elicits both bactericidal and functional blocking antibodies and is a valid candidate antigen for additional investigation and possible inclusion in a vaccine for prevention of gonorrhea. PMID:27895130

  3. Defective signaling through the B cell antigen receptor in Epstein-Barr virus-transformed ataxia-telangiectasia cells.

    Science.gov (United States)

    Khanna, K K; Yan, J; Watters, D; Hobson, K; Beamish, H; Spring, K; Shiloh, Y; Gatti, R A; Lavin, M F

    1997-04-04

    A characteristic series of immunological abnormalities are observed in the human genetic disorder ataxia-telangiectasia (A-T). The recent cloning of a gene mutated in this syndrome provides additional evidence for a defect in intracellular signaling in A-T. We have investigated the possibility that signaling through the B cell antigen receptor is one manifestation of the A-T defect. In response to cross-linking of the B cell receptor, several A-T cell lines were defective in their mitogenic response; in addition Ca2+ mobilization from internal stores was either absent or considerably reduced in these cell lines in response to cross-linking. The defect in signaling was not due to difference in expression of surface immunoglobulin. The defective response in A-T cells was also evident in several arms of the intracellular cascade activated by B cell cross-linking. Tyrosine phosphorylation of phospholipase Cgamma1, a key step in activation of the enzyme, was reduced or negligible in some A-T cell lines. This defect in signaling was also seen at the level of Lyn tyrosine kinase activation and its association with and activation of phosphatidylinositol 3-kinase. Our results provide evidence for a role for the ATM gene product in intracellular signaling which may account at least in part for the abnormalities in B cell function in A-T.

  4. A new human cholangiocellular carcinoma cell line (HuCC-T1) producing carbohydrate antigen 19/9 in serum-free medium.

    Science.gov (United States)

    Miyagiwa, M; Ichida, T; Tokiwa, T; Sato, J; Sasaki, H

    1989-06-01

    A human cholangiocellular carcinoma cell line, HuCC-T1, was established in vitro from the malignant cells of ascites of a 56-yr-old patient. Histologic findings of the primary liver tumor revealed a moderately differentiated adenocarcinoma. Tumor cells from the ascites have been cultured with RPMI 1640 medium containing 0.2% lactalbumin hydrolysate and the cultured cells grew as monolayers with a population doubling time of 74 h during exponential growth at Passage 25. They had an epithelial-like morphology and were positive for mucine staining. Ultrastructural studies revealed the presence of microvilli on the cell surface and poorly developed organelles in the cytoplasm. The HuCC-T1 cell was tumorigenic in nude mice. The number of chromosomes in HuCC-T1 ranged from 61 to 80. These human cholangiocellular carcinoma cells in serum-free medium secreted several tumor markers, including carbohydrate antigen 19/9, carbohydrate antigen 125, carcinoembryonic antigen, and tissue polypeptide antigen. The carbohydrate antigen 19/9 secretion level of HuCC-T1 cells cultured in RPMI 1640 medium with 1% fetal bovine serum was sixfold higher than that with 0.2% lactalbumin hydrolysate. These findings suggest that HuCC-T1 will provide useful information to clarify the mechanism of tumor marker secretion and tumor cell growth in the human cholangiocellular carcinoma.

  5. Antigen-induced regulatory T cells in HBV chronically infected patients.

    Science.gov (United States)

    Barboza, Luisa; Salmen, Siham; Goncalves, Loredana; Colmenares, Melisa; Peterson, Darrell; Montes, Henry; Cartagirone, Raimondo; Gutiérrez, Maria del Carmen; Berrueta, Lisbeth

    2007-11-10

    T cell response against HBV is vigorous in patients with acute hepatitis who clear the virus, whereas it is weak and narrowly focused in patients with chronic disease. We report that following incubation with HBcAg, a population of CD4+FoxP3+ cells expressing phenotypic markers of both natural and induced Tregs, can be antigen-induced from peripheral mononuclear cells. Conversely, naive and naturally immune subjects did not increase CD4+FoxP3+ Tregs following stimulation with HBcAg, supporting the idea that natural Tregs are able to respond specifically to HBV antigen. Furthermore, increased frequencies of antigen-induced CD4+FoxP3+IL-10+ Tregs correlated with viral load, suggesting that antigen-induced Tregs could contribute to an inadequate response against the virus, leading to chronic infection and support the view that specific natural Tregs may be implicated in host immune tolerance during HBV infection.

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

    Directory of Open Access Journals (Sweden)

    Jennifer Gordon

    Full Text Available JC virus (JCV, a common human polyomavirus, is the etiological agent of the demyelinating disease, progressive multifocal leukoencephalopathy (PML. In addition to its role in PML, studies have demonstrated the transforming ability of the JCV early protein, T-antigen, and its association with some human cancers. JCV infection occurs in childhood and latent virus is thought to be maintained within the bone marrow, which harbors cells of hematopoietic and non-hematopoietic lineages. Here we show that non-hematopoietic mesenchymal stem cells (MSCs isolated from the bone marrow of JCV T-antigen transgenic mice give rise to JCV T-antigen positive cells when cultured under neural conditions. JCV T-antigen positive cells exhibited neural crest characteristics and demonstrated p75, SOX-10 and nestin positivity. When cultured in conditions typical for mesenchymal cells, a population of T-antigen negative cells, which did not express neural crest markers arose from the MSCs. JCV T-antigen positive cells could be cultured long-term while maintaining their neural crest characteristics. When these cells were induced to differentiate into neural crest derivatives, JCV T-antigen was downregulated in cells differentiating into bone and maintained in glial cells expressing GFAP and S100. We conclude that JCV T-antigen can be stably expressed within a fraction of bone marrow cells differentiating along the neural crest/glial lineage when cultured in vitro. These findings identify a cell population within the bone marrow permissible for JCV early gene expression suggesting the possibility that these cells could support persistent viral infection and thus provide clues toward understanding the role of the bone marrow in JCV latency and reactivation. Further, our data provides an excellent experimental model system for studying the cell-type specificity of JCV T-antigen expression, the role of bone marrow-derived stem cells in the pathogenesis of JCV-related diseases

  7. Enhancing humoral responses to a malaria antigen with nanoparticle vaccines that expand Tfh cells and promote germinal center induction.

    Science.gov (United States)

    Moon, James J; Suh, Heikyung; Li, Adrienne V; Ockenhouse, Christian F; Yadava, Anjali; Irvine, Darrell J

    2012-01-24

    For subunit vaccines, adjuvants play a key role in shaping immunological memory. Nanoparticle (NP) delivery systems for antigens and/or molecular danger signals are promising adjuvants capable of promoting both cellular and humoral immune responses, but in most cases the mechanisms of action of these materials are poorly understood. Here, we studied the immune response elicited by NPs composed of multilamellar "stapled" lipid vesicles carrying a recombinant Plasmodium vivax circumsporozoite antigen, VMP001, both entrapped in the aqueous core and anchored to the lipid bilayer surfaces. Immunization with these particles and monophosphoryl lipid A (MPLA), a US Food and Drug Administration-approved immunostimulatory agonist for Toll-like receptor-4, promoted high-titer, high-avidity antibody responses against VMP001, lasting more than 1 y in mice at 10-fold lower doses than conventional adjuvants. Compared to soluble VMP001 mixed with MPLA, VMP001-NPs promoted broader humoral responses, targeting multiple epitopes of the protein and a more balanced Th1/Th2 cytokine profile from antigen-specific T cells. To begin to understand the underlying mechanisms, we examined components of the B-cell response and found that NPs promoted robust germinal center (GC) formation at low doses of antigen where no GC induction occurred with soluble protein immunization, and that GCs nucleated near depots of NPs accumulating in the draining lymph nodes over time. In parallel, NP vaccination enhanced the expansion of antigen-specific follicular helper T cells (T(fh)), compared to vaccinations with soluble VMP001 or alum. Thus, NP vaccines may be a promising strategy to enhance the durability, breadth, and potency of humoral immunity by enhancing key elements of the B-cell response.

  8. Interaction between antigen presenting cells and autoreactive T cells derived from BXSB mice with murine lupus

    Institute of Scientific and Technical Information of China (English)

    Peng Yang; Bo Li; Ping Lv; Yan Zhang; XiaoMing Gao

    2007-01-01

    Systemic lupus erythematosus (SLE) is a typical autoimmune disease involving multiple systems and organs. Ample evidence suggests that autoreactive T cells play a pivotal role in the development of this autoimmune disorder. This study was undertaken to investigate the mechanisms of interaction between antigen presenting cells (APCs) and an autoreactive T cell (ATL1) clone obtained from lupus-prone BXSB mice. ATL1 cells, either before or after γ-ray irradiation, were able to activate naive B cells, as determined by B cell proliferation assays. Macrophages from BXSB mice were able to stimulate the proliferation of resting ATL1 cells at a responder/stimulator (R/S) ratio of 1/2.5. Dendritic cells (DCs) were much more powerful stimulators for ATL1 cells on a per cell basis. The T cell stimulating ability of macrophages and B cells, but not DCs, was sensitive toγ-ray irradiation. Monoclonal antibodies against mouse MHC-Ⅱand CD4 were able to block DC-mediated stimulation of ATL1 proliferation, indicating cognate recognition between ATL1 and APCs. Our data suggest that positive feedback loops involving macrophages, B cells and autoreactive T cells may play a pivotal role in keeping the momentum of autoimmune responses leading to autoimmune diseases.

  9. Expression of tumor antigens on primary ovarian cancer cells compared to established ovarian cancer cell lines

    Science.gov (United States)

    Kloudová, Kamila; Hromádková, Hana; Partlová, Simona; Brtnický, Tomáš; Rob, Lukáš; Bartůňková, Jiřina; Hensler, Michal; Halaška, Michael J.; Špíšek, Radek; Fialová, Anna

    2016-01-01

    In order to select a suitable combination of cancer cell lines as an appropriate source of antigens for dendritic cell-based immunotherapy of ovarian cancer, we analyzed the expression level of 21 tumor associated antigens (BIRC5, CA125, CEA, DDX43, EPCAM, FOLR1, Her-2/neu, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MUC-1, NY-ESO-1, PRAME, p53, TPBG, TRT, WT1) in 4 established ovarian cancer cell lines and in primary tumor cells isolated from the high-grade serous epithelial ovarian cancer tissue. More than 90% of tumor samples expressed very high levels of CA125, FOLR1, EPCAM and MUC-1 and elevated levels of Her-2/neu, similarly to OVCAR-3 cell line. The combination of OV-90 and OVCAR-3 cell lines showed the highest overlap with patients' samples in the TAA expression profile. PMID:27323861

  10. In vitro expansion of antigen-specific CD8(+) T cells distorts the T-cell repertoire.

    Science.gov (United States)

    Koning, Dan; Costa, Ana I; Hasrat, Raiza; Grady, Bart P X; Spijkers, Sanne; Nanlohy, Nening; Keşmir, Can; van Baarle, Debbie

    2014-03-01

    Short-term in vitro expansion of antigen-specific T cells is an appreciated assay for the analysis of small memory T-cell populations. However, how well short-term expanded T cells represent the direct ex vivo situation remains to be elucidated. In this study we compared the clonality of Epstein-Barr virus (EBV) and cytomegalovirus (CMV)-specific CD8(+) T cells directly ex vivo and after in vitro stimulation with antigen. Our data show that the antigen-specific T cell repertoire significantly alters after in vitro culture. Clear shifts in clonotype hierarchy were observed, with the most dominant ex vivo clonotype decreasing after stimulation at the expense of several previously subdominant clonotypes. Notably, these alterations were more pronounced in polyclonal T-cell populations compared to mono- or oligoclonal repertoires. Furthermore, TCR diversity significantly increased after culture with antigen. These results suggest that the T-cell repertoire is highly subjective to variation after in vitro stimulation with antigen. Hence, although short-term expansion of T cells provides a simple and efficient tool to examine antigen-specific immune responses, caution is required if T-cell populations are expanded prior to detailed, clonotypic analyses or other repertoire-based investigations.

  11. The T-cell anergy induced by Leishmania amazonensis antigens is related with defective antigen presentation and apoptosis

    Directory of Open Access Journals (Sweden)

    Roberta O. Pinheiro

    2004-09-01

    Full Text Available Leishmania amazonensis is the main agent of diffuse cutaneous leishmaniasis, a disease associated with anergic immune responses. In this study we show that the crude antigen of Leishmania amazonensis (LaAg but not L. braziliensis promastigotes (LbAg contains substances that suppress mitogenic and spontaneous proliferative responses of T cells. The suppressive substances in LaAg are thermoresistant (100ºC/1h and partially dependent on protease activity. T cell anergy was not due to a decreased production of growth factors as it was not reverted by addition of exogenous IL-2, IL-4, IFN-gamma or IL-12. LaAg did not inhibit anti-CD3-induced T cell activation, suggesting that anergy was due to a defect in antigen presentation. It was also not due to cell necrosis, but was accompanied by expressive DNA fragmentation in lymph node cells, indicative of apoptosis. Although pre-incubation of macrophages with LaAg prevented their capacity to present antigens, this effect was not due to apoptosis of the former. These results suggest that the T cell anergy found in diffuse leishmaniasis may be the result of parasite antigen-driven apoptosis of those cells following defective antigen presentation.A Leishmania amazonensis é o principal agente etiológico da leishmaniose cutânea difusa, uma doença associada a respostas imunes anérgicas. Neste estudo nós mostramos que o extrato bruto de promastigotas de Leishmania amazonensis (LaAg, mas não de L. braziliensis (LbAg, contém substâncias que suprimem respostas proliferativas, espontâneas e mitogênicas, de células T. As substâncias supressoras no LaAg são termo-resistentes (100°C/1h e parcialmente dependentes da atividade de proteases. A anergia de células T não foi devida à diminuição na produção de fatores de crescimento, uma vez que não foi revertida pela adição de: IL-2, IL-4, IFN-gama ou IL-12. O LaAg não inibiu a ativação de células T induzida por anti-CD3, sugerindo que a anergia

  12. Presentation of antigen by B cell subsets. Pt. 4. Defective T-B cell signalling causes inability to present antigen by B cells from immunodeficient mice

    Energy Technology Data Exchange (ETDEWEB)

    Zimecki, Michal [Polish Academy of Sciences, Wroclaw (Poland). Institute of Immunology and Experimental Therapy; Kapp, Judith A. [Emory Univ., Atlanta, GA (United States). School of Medicine

    1994-12-31

    The purpose of this study was to investigate differences in T-B cell signalling between B cells from normal and immunodeficient mice. B cell blasts from normal and immunodeficient mice expressed comparable levels of membrane-associated IL-1. B cells from normal, but not immunodeficient mice, prefixed with glutar-aldehyde and cultured with thymocytes or a T cell line BK33, induce in T cells production of a factor which causes release of IL-1 by macrophages. This factor, preincubated with B cells from immunodeficient mice significantly enhances their APC function. Furthermore, this cytokine induces expression of Lyb-5 alloantigen on B cells from immunodeficient mice. This effect could be blocked by neutralizing antibodies to IL-6 but not to IL-2, IL-4 or GM-CSF. We conclude that immature B cells from immunodeficient (CBA/N x BALB/c)F{sub 1} mice are unable to stimulate interacting T cells to produce IL-6 and therefore are inefficient antigen presenting cells. (author). 30 refs, 2 figs, 3 tabs.

  13. Studies on mechanism of Sialy Lewis-X antigen in liver metastases of human colorectal carcinoma

    Institute of Scientific and Technical Information of China (English)

    Xiao Wei Li; Yan Qing Ding; Jun Jie Cai; Shao Qing Yang; Lian Bing An; Dong Fang Qiao

    2001-01-01

    @@INTRODUCTION Sialyl Lewis-X antigen ,correlated with carcinoma, is a group of carbohydrate antigen containing oligosaccharide expressed of embryonic tisue and glycoproteins on cell surface of embryonic tissue[1].The SLeX antigen located on cell surface is synthesized principally by two enzymes ,al ,3fucosyltransfrease and a2, 3sialyctransferase.In adults ,SLeX antigen is expressed principally on the surfaces of granulocytic cells and some tumor cells .

  14. A new Kupffer cell receptor mediating plasma clearance of carcinoembryonic antigen by the rat.

    Science.gov (United States)

    Toth, C A; Thomas, P; Broitman, S A; Zamcheck, N

    1982-05-15

    Native human carcinoembryonic antigen is rapidly removed from the circulation by the rat liver Kupffer cell after intravenous injection. The molecule is subsequently transferred to the hepatocyte in an immunologically identifiable form. Carcinoembryonic antigen has a circulatory half-life of 3.7 (+/- 0.8) min, and cellular entry is by receptor-mediated endocytosis. Non-specific fluid pinocytosis and phagocytosis can be excluded as possible mechanisms by the kinetics of clearance and failure of colloidal carbon to inhibit uptake. Substances with known affinity for the hepatic receptors for mannose, N-acetylglucosamine, fucose and galactose all fail to inhibit carcinoembryonic antigen clearance. After two cycles of the Smith degradation, carcinoembryonic antigen is still able to inhibit clearance of the native molecule. Receptor specificity is apparently not dependent on those non-reducing terminal sugars of the native molecule. Performic acid-oxidized carcinoembryonic antigen also inhibits clearance of carcinoembryonic antigen in vivo. Receptor binding is not dependent on tertiary protein conformation. Non-specific cross-reacting antigen, a glycoprotein structurally similar to carcinoembryonic antigen, is cleared by the same mechanism.

  15. Vitamin D controls T cell antigen receptor signaling and activation of human T cells

    DEFF Research Database (Denmark)

    von Essen, Marina Rode; Kongsbak-Wismann, Martin; Schjerling, Peter

    2010-01-01

    Phospholipase C (PLC) isozymes are key signaling proteins downstream of many extracellular stimuli. Here we show that naive human T cells had very low expression of PLC-gamma1 and that this correlated with low T cell antigen receptor (TCR) responsiveness in naive T cells. However, TCR triggering...... led to an upregulation of approximately 75-fold in PLC-gamma1 expression, which correlated with greater TCR responsiveness. Induction of PLC-gamma1 was dependent on vitamin D and expression of the vitamin D receptor (VDR). Naive T cells did not express VDR, but VDR expression was induced by TCR...... signaling via the alternative mitogen-activated protein kinase p38 pathway. Thus, initial TCR signaling via p38 leads to successive induction of VDR and PLC-gamma1, which are required for subsequent classical TCR signaling and T cell activation....

  16. The Murine Humoral Immune Response to Hepatitis B Surface Antigen: Idiotype Network Pathways.

    Science.gov (United States)

    Schick, Michael Roy

    Recognition of a wide spectrum in disease outcomes following Hepatitis B Virus (HBV) infection has led to the suggestion that individual differences may be due to characteristics of the immune response. HBV, a hepatotropic virus, is not directly cytopathic to the host hepatocytes but the cellular damage which does not occur may be due to the host's own immune response. It is this variety in immune response capabilities following natural infection or vaccination which led to the present study in which the murine humoral immune response to hepatitis B surface antigen (HBsAg) was examined. Following immunization with purified HBsAg an anti-HBs response could be detected in 19 inbred strains of mice. The response, which varied among the strains, was linked to the major histocompatibility complex (MHC). Among high responders to HBsAg were two strains in which a poor response to a single epitope could be detected. Although quantitatively serum from these strains resembled serum from other high responders, there was a major difference in the qualitative aspects. Included within this study was the role of idotype networks within the murine anti-HBs response. By directly targeting HBsAg-specific B cells within the framework of an idiotype network by an Ab-2, it was possible to circumvent T cell-dependent regulation of an immune response. In each of five inbred strains of mice immunized with a polyclonal rabbit Ab-2 an Ab-3 population with HBsAg-specificity (Ab -1^') was induced. These mice were also immunized with HBsAg resulting in a higher anti-HBs response as compared to HBsAg immunization alone in all of the strains tested except for one. The response in this strain, normally a low responder to HBsAg, indicated that the mechanisms for genetic restriction of the anti -HBs response was still active, although it was not apparent during anti-Id immunization. The effects of an anti-Id on the murine antibody response to HBsAg may lead to insights on the presence of idiotype

  17. Epigenetic mechanisms regulate MHC and antigen processing molecules in human embryonic and induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Beatriz Suárez-Alvarez

    Full Text Available BACKGROUND: Human embryonic stem cells (hESCs are an attractive resource for new therapeutic approaches that involve tissue regeneration. hESCs have exhibited low immunogenicity due to low levels of Mayor Histocompatibility Complex (MHC class-I and absence of MHC class-II expression. Nevertheless, the mechanisms regulating MHC expression in hESCs had not been explored. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the expression levels of classical and non-classical MHC class-I, MHC class-II molecules, antigen-processing machinery (APM components and NKG2D ligands (NKG2D-L in hESCs, induced pluripotent stem cells (iPSCs and NTera2 (NT2 teratocarcinoma cell line. Epigenetic mechanisms involved in the regulation of these genes were investigated by bisulfite sequencing and chromatin immunoprecipitation (ChIP assays. We showed that low levels of MHC class-I molecules were associated with absent or reduced expression of the transporter associated with antigen processing 1 (TAP-1 and tapasin (TPN components in hESCs and iPSCs, which are involved in the transport and load of peptides. Furthermore, lack of beta2-microglobulin (beta2m light chain in these cells limited the expression of MHC class I trimeric molecule on the cell surface. NKG2D ligands (MICA, MICB were observed in all pluripotent stem cells lines. Epigenetic analysis showed that H3K9me3 repressed the TPN gene in undifferentiated cells whilst HLA-B and beta2m acquired the H3K4me3 modification during the differentiation to embryoid bodies (EBs. Absence of HLA-DR and HLA-G expression was regulated by DNA methylation. CONCLUSIONS/SIGNIFICANCE: Our data provide fundamental evidence for the epigenetic control of MHC in hESCs and iPSCs. Reduced MHC class I and class II expression in hESCs and iPSCs can limit their recognition by the immune response against these cells. The knowledge of these mechanisms will further allow the development of strategies to induce tolerance and improve stem cell

  18. Hepatitis C virus and ethanol alter antigen presentation in liver cells

    Institute of Scientific and Technical Information of China (English)

    Natalia A Osna

    2009-01-01

    Alcoholic patients have a high incidence of hepatitis Cvirus (HCV) infection. Alcohol consumption enhances the severity of the HCV disease course and worsens the outcome of chronic hepatitis C. The accumulation of virally infected cells in the liver is related to the HCVinduced inability of the immune system to recognizeinfected cells and to develop the immune responses. This review covers the effects of HCV proteins and ethanol on major histocompatibility complex (MHC) classⅠ- and class Ⅱ-restricted antigen presentation. Here, we discuss the liver which functions as an immune privilege organ; factors, which affect cleavage and loading of antigenic peptides onto MHC classⅠand class Ⅱ in hepatocytes and dendritic cells, and the modulating effects of ethanol and HCV on antigen presentation by liver cells. Altered antigen presentation in the liver limits the ability of the immune system to clear HCV and infected cells and contributes to disease progression. HCV by itself affects dendritic cell function, switching their cytokine profile to the suppressive phenotype of interleukin-10 (IL-10) and transforming growth factor beta (TGFβ) predominance,preventing cell maturation and allostimulation capacity.The synergistic action of ethanol with HCV results in the suppression of MHC class Ⅱ-restricted antigen presentation. In addition, ethanol metabolism and HCV proteins reduce proteasome function and interferon signaling, thereby suppressing the generation of peptides for MHC classⅠ-restricted antigen presentation.Collectively, ethanol exposure further impairs antigen presentation in HCV-infected liver cells, which may provide a partial explanation for exacerbations and the poor outcome of HCV infection in alcoholics.

  19. Diverse endogenous antigens for mouse NKT cells: self-antigens that are not glycosphingolipids.

    Science.gov (United States)

    Pei, Bo; Speak, Anneliese O; Shepherd, Dawn; Butters, Terry; Cerundolo, Vincenzo; Platt, Frances M; Kronenberg, Mitchell

    2011-02-01

    NKT cells with an invariant Ag receptor (iNKT cells) represent a highly conserved and unique subset of T lymphocytes having properties of innate and adaptive immune cells. They have been reported to regulate a variety of immune responses, including the response to cancers and the development of autoimmunity. The development and activation of iNKT cells is dependent on self-Ags presented by the CD1d Ag-presenting molecule. It is widely believed that these self-Ags are glycosphingolipids (GSLs), molecules that contain ceramide as the lipid backbone. In this study, we used a variety of methods to show that mammalian Ags for mouse iNKT cells need not be GSLs, including the use of cell lines deficient in GSL biosynthesis and an inhibitor of GSL biosynthesis. Presentation of these Ags required the expression of CD1d molecules that could traffic to late endosomes, the site where self-Ag is acquired. Extracts of APCs contain a self-Ag that could stimulate iNKT cells when added to plates coated with soluble, rCD1d molecules. The Ag(s) in these extracts are resistant to sphingolipid-specific hydrolase digestion, consistent with the results using live APCs. Lyosphosphatidylcholine, a potential self-Ag that activated human iNKT cell lines, did not activate mouse iNKT cell hybridomas. Our data indicate that there may be more than one type of self-Ag for iNKT cells, that the self-Ags comparing mouse and human may not be conserved, and that the search to identify these molecules should not be confined to GSLs.

  20. Rheumatoid arthritis and its association with HLA-DR antigens. I. Cell mediated immune response against connective tissue antigens.

    Science.gov (United States)

    Vullo, C M; Pesoa, S A; Onetti, C M; Riera, C M

    1987-04-01

    HLA-DR antigens and cellular sensitivity to native bovine type I and type II collagen and proteoglycans were examined in patients with classic rheumatoid arthritis (RA) and normal individuals. Fifty eight percent of patients with RA (n = 88) and 28% of normals (n = 52) were DR4+ (pc less than 0.01). DR4 phenotype was significantly increased in patients with severe disease stages (III-IV), as defined by the ARA criteria, in contrast to those showing mild disease stages (I-II) (p less than 0.05). Furthermore, peripheral blood mononuclear cells from 55 patients and 30 controls were evaluated for the in vitro production of leukocyte inhibitory factor in response to native type I and type II collagen and proteoglycans. By using this assay, cells from the arthritic group exhibited a statistically significant response when stimulated with native type I collagen and proteoglycans. The cellular immune response was not associated with any particular HLA-DR antigens, or to the disease stage or severity.

  1. Antigen-presenting cells in parotid glands contain cystatin D originating from acinar cells.

    Science.gov (United States)

    Nashida, Tomoko; Sato, Ritsuko; Haga-Tsujimura, Maiko; Yoshie, Sumio; Yoshimura, Ken; Imai, Akane; Shimomura, Hiromi

    2013-02-01

    Cystatin D encoded by Cst5 is a salivary classified type II cystatin. We investigated the dynamism of cystatin D by examining the distribution of cystatin D protein and mRNA in rats, to identify novel functions. The simultaneous expression of Cst5 and cystatin D was observed in parotid glands, however in situ hybridization showed that only acinar cells produced cystatin D. Synthesized cystatin D was localized in small vesicles and secreted from the apical side to the saliva, and from the basolateral side to the extracellular region, a second secretory pathway for cystatin D. We also identified antigen-presenting cells in the parotid glands that contained cystatin D without the expression of Cst5, indicating the uptake of cystatin D from the extracellular region. Cystatin D was detected in blood serum and renal tubular cells with megalin, indicating the circulation of cystatin D through the body and uptake by renal tubular cells. Thus, the novel dynamism of cystatin D was shown and a function for cystatin D in the regulation of antigen-presenting cell activity was proposed.

  2. Low antigenicity of hematopoietic progenitor cells derived from human ES cells

    Directory of Open Access Journals (Sweden)

    Eun-Mi Kim

    2010-02-01

    Full Text Available Eun-Mi Kim1, Nicholas Zavazava1,21Department of Internal Medicine, University of Iowa and Veterans Affairs Medical Center, Iowa City, Iowa, USA; 2Immunology Graduate Program, University of Iowa, Iowa City, Iowa, USAAbstract: Human embryonic stem (hES cells are essential for improved understanding of diseases and our ability to probe new therapies for use in humans. Currently, bone marrow cells and cord blood cells are used for transplantation into patients with hematopoietic malignancies, immunodeficiencies and in some cases for the treatment of autoimmune diseases. However, due to the high immunogenicity of these hematopoietic cells, toxic regimens of drugs are required for preconditioning and prevention of rejection. Here, we investigated the efficiency of deriving hematopoietic progenitor cells (HPCs from the hES cell line H13, after co-culturing with the murine stromal cell line OP9. We show that HPCs derived from the H13 ES cells poorly express major histocompatibility complex (MHC class I and no detectable class II antigens (HLA-DR. These characteristics make hES cell-derived hematopoietic cells (HPCs ideal candidates for transplantation across MHC barriers under minimal immunosuppression.Keywords: human embryonic stem cells, H13, hematopoiesis, OP9 stromal cells, immunogenicity

  3. Production of Exocytic Vesicular Antigens by Primary Liver Cell Cultures

    Science.gov (United States)

    1990-05-08

    microbial symbionts which occur naturally in the gut and on mucous membranes. Another method invclves the use of synthetic peptides which mimic...Streptococcus pneumoniae, hepatitis B virus, Plasmodium spp. and dengue virus, which are creating tremendous burdens worldwide [32]. Most of the...place in the immune system when an antibody’s unique antigen-binding peptide sequence (the idiotype) stimulates production of another antibody directed

  4. Antigen-activated dendritic cells ameliorate influenza A infections

    OpenAIRE

    2013-01-01

    Influenza A viruses cause significant morbidity and mortality worldwide. There is a need for alternative or adjunct therapies, as resistance to currently used antiviral drugs is emerging rapidly. We tested ligand epitope antigen presentation system (LEAPS) technology as a new immune-based treatment for influenza virus infection in a mouse model. Influenza-J-LEAPS peptides were synthesized by conjugating the binding ligand derived from the β2-microglobulin chain of the human MHC class I molecu...

  5. Antigen-bound C3b and C4b enhance antigen-presenting cell function in activation of human T-cell clones.

    Science.gov (United States)

    Arvieux, J; Yssel, H; Colomb, M G

    1988-10-01

    The effect of complement fragments C3b and C4b, on the triggering of antigen-specific human T-cell clones by Epstein-Barr virus-transformed human lymphoblastoid B cells (LCL) when these fragments are covalently coupled to the antigen tetanus toxin (TT) is described. TT was chemically cross-linked to purified C3b [(TT-C3b)n], C4b [(TT-C4b)n] or bovine serum albumin [(TT-BSA)n] as a control. T-cell activation was quantified by tritiated thymidine incorporation and 51Cr release. (TT-C3b)n and (TT-C4b)n induced proliferative responses comparable to (TT-BSA)n but at 18-25 and 4-6 lower concentrations, respectively. This enhancing effect required the covalent cross-linking of the complement fragments to the antigen and involved intracellular processing of the latter by LCL. Antigen presentation was similarly enhanced when measuring the cytotoxic activity of a helper T-cell clone against LCL previously pulsed with (TT-C3b)n or (TT-C4b)n compared with (TT-BSA)n. Binding studies, carried out on LCL using TT radiolabelled with 125I before cross-linking, indicated that (TT-C3b)n and (TT-C4b)n gave three- to four-fold more binding than (TT-BSA)n. Addition of antibodies against CR1 and CR2 or proteolytic removal of these complement receptors with trypsin inhibited by about 60% the enhancing effect of TT-bound C3b and C4b in both binding and functional assays. These results indicate that binding of C3b or C4b to antigen enhances antigen-specific proliferative and cytotoxic responses of T cells by targeting opsonized antigen onto complement receptors CR1 and CR2 of LCL. The putative significance of these findings in terms of regulation of immune responses by complement is discussed.

  6. A yeast surface display system for the discovery of ligands that trigger cell activation.

    Science.gov (United States)

    Cho, B K; Kieke, M C; Boder, E T; Wittrup, K D; Kranz, D M

    1998-11-01

    Opposing cells often communicate signalling events using multivalent interactions between receptors present on their cell surface. For example, T cells are typically activated when the T cell receptor (TCR) and its associated costimulatory molecules are multivalently engaged by the appropriate ligands present on an antigen presenting cell. In this report, yeast expressing high cell-surface levels of a TCR ligand (a recombinant antibody to the TCR Vbeta domain) were shown to act as 'pseudo' antigen presenting cells and induce T cell activation as monitored by increased levels of CD25 and CD69 and by downregulation of cell surface TCR. Similar levels of T cell activation could occur even when a 30-fold excess of irrelevant yeast was present, suggesting that such a yeast display system, by virtue of its ability to present ligands multivalently, may be used in highly sensitive procedures to identify novel polypeptides that interact multivalently with cell surface receptors and thereby trigger specific cellular responses.

  7. Self-assembled PEG-b-PDPA-b-PGEM copolymer nanoparticles as protein antigen delivery vehicles to dendritic cells: preparation, characterization and cellular uptake

    Science.gov (United States)

    Li, Pan; Zhou, Junhui; Huang, Pingsheng; Zhang, Chuangnian; Wang, Weiwei; Li, Chen; Kong, Deling

    2017-01-01

    Antigen uptake by dendritic cells (DCs) is a key step for initiating antigen-specific T cell immunity. In the present study, novel synthetic polymeric nanoparticles were prepared as antigen delivery vehicles to improve the antigen uptake by DCs. Well-defined cationic and acid-responsive copolymers, monomethoxy poly(ethylene glycol)-block-poly(2-(diisopropyl amino) ethyl methacrylate)-block-poly(2-(guanidyl) ethyl methacrylate) (mPEG-b-PDPA-b-PGEM, PEDG) were synthesized by reversible addition-fragmentation chain transfer polymerization of 2-(diisopropylamino)ethyl methacrylate) and N-(tert-butoxycarbonyl) amino ethyl methacrylate monomers, followed by deprotection of tert-butyl protective groups and guanidinylation of obtained primary amines. 1H NMR, 13C NMR and GPC results indicated the successful synthesis of well-defined PEDG copolymers. PEDG copolymers could self-assemble into nanoparticles in aqueous solution, which were of cationic surface charges and showed acid-triggered disassembly contributed by PGEM and PDPA moieties, respectively. Significantly, PEDG nanoparticles could effectively condense with negatively charged model antigen ovalbumin (OVA) to form OVA/PEDG nanoparticle formulations with no influence on its secondary and tertiary structures demonstrating by far-UV circular dichroism and UV–vis spectra. In vitro antigen cellular uptake by bone marrow DCs (BMDCs) indicated using PEDG nanoparticles as antigen delivery vehicles could significantly improve the antigen uptake efficiency of OVA compared with free OVA or the commercialized Alum adjuvant. Moreover, as the surface cationic charges of OVA/PEDG nanoparticle formulations reduced, the uptake efficiency decreased correspondingly. Collectively, our work suggests that guanidinylated, cationic and acid-responsive PEDG nanoparticles represent a new kind of promising antigen delivery vehicle to DCs and hold great potential to serve as immunoadjuvants in the development of vaccines. PMID:28149525

  8. Prospects for adoptive immunotherapy of pancreatic cancer using chimeric antigen receptor-engineered T-cells.

    Science.gov (United States)

    Alrifai, Doraid; Sarker, Debashis; Maher, John

    2016-01-01

    Adoptive immunotherapy using chimeric antigen receptor (CAR) engineered T-cells is emerging as a powerful new approach to cancer immunotherapy. CARs are fusion molecules that couple the antibody-like binding of a native cell surface target to the delivery of a bespoke T-cell activating signal. Recent studies undertaken by several centers have demonstrated highly compelling efficacy in patients with acute and chronic B-cell malignancies. However, comparable therapeutic activity has not been achieved in solid tumors. Modern management of pancreatic ductal adenocarcinoma (PDAC) remains ineffective, reflected in the virtual equivalence of annual incidence and mortality statistics for this tumor type. Increasing evidence indicates that these tumors are recognized by the immune system, but deploy powerful evasion strategies that limit natural immune surveillance and render efforts at immunotherapy challenging. Here, we review preclinical and clinical studies that have been initiated or completed in an effort to develop CAR-based immunotherapy for PDAC. We also consider the hurdles to the effective clinical development of this exciting new therapeutic modality.

  9. A new and robust method of tethering IgG surrogate antigens on lipid bilayer membranes to facilitate the TIRFM based live cell and single molecule imaging experiments.

    Directory of Open Access Journals (Sweden)

    Shaosen Zhang

    Full Text Available Our understanding of cell-cell interactions has been significantly improved in the past years with the help of Total Internal Reflection Fluorescence Microscope (TIRFM in combination with an antigen presenting system supported by planar lipid bilayer (PLB membranes, which are used to mimic the extensive receptor and ligand interactions within cell-cell contact interface. In TIRFM experiments, it is a challenge to uniformly present ligand molecules in monomeric format on the surface of PLB membranes. Here, we introduce a new and robust method of tethering IgG surrogate antigen ligands on the surface of Ni(2+-containing PLB membranes. In this method, we use a modified D domain from staphylococcal protein A molecule that is fused with an N-terminus polyhistidine tag (H12-D-domain to tether IgG surrogate antigens on Ni(2+-containing PLB membranes. We systematically assessed the specificity and capability of H12-D-domain construct to capture IgG molecules from different species through live cell and single molecule TIRFM imaging. We find that these IgG surrogate antigens tethered by H12-D-domain show better lateral mobility and are more uniformly distributed on PLB membranes than the ones tethered by streptavidin. Neither IgM molecules, nor Fab or F(ab'2 fragments of IgG molecules can be tethered on PLB membranes by H12-D-domain construct. These tethered IgG surrogate antigens strongly induce the formation and accumulation of signaling active antigen receptor microclusters within the immunological synapse in B or T lymphocyte cells. Thus our method provides a new and robust method to tether IgG surrogate antigens or other molecules fused with IgG Fc portion on PLB membranes for TIRFM based molecule imaging experiments.

  10. Different patterns of glycolipid antigens are expressed following differentiation of TERA-2 human embryonal carcinoma cells induced by retinoic acid, hexamethylene bisacetamide (HMBA) or bromodeoxyuridine (BUdR).

    Science.gov (United States)

    Andrews, P W; Nudelman, E; Hakomori, S; Fenderson, B A

    1990-04-01

    NTERA-2 cl.D1 human embryonal carcinoma (EC) cells were induced to differentiate by either bromodeoxyuridine (BUdR) or hexamethylene bisacetamide (HMBA), and also by retinoic acid. Following exposure to each of these inducers, the globoseries glycolipid antigens stage-specific embryonic antigens -3 and -4 (SSEA-3 and -4) and the glycoprotein antigen TRA-1-60, all characteristic of the human EC cell surface, underwent a marked reduction in expression within about 7 days. At the same time, the lactoseries glycolipid antigen SSEA-1, and ganglioseries antigens A2B5 (GT3) and ME311 (9-0-acetyl GD3) were induced in BUdR- and retinoic acid-treated cells. However, these antigens did not appear during the first 7-14 days of HMBA-induced differentiation. The observations of cell surface antigen expression were paralleled by analysis of glycolipids isolated from the cells by thin-layer chromatography. This analysis, in which the new monoclonal antibodies VINIS-56 and VIN-2PB-22 were included, also revealed expression of gangliosides GD3 and GD2 in all differentiated cultures, albeit at much lower levels following HMBA exposure than following retinoic acid or BUdR-exposure. Further, disialylparagloboside was detected in retinoic acid and BUdR-induced, but not HMBA-induced, cultures. Taken with morphological observations, the results suggest that HMBA induces differentiation of NTERA-2 cl.D1 EC cells along a pathway distinct from the pathway(s) induced by retinoic acid and BUdR.

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

    DEFF Research Database (Denmark)

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

    2001-01-01

    Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a variant antigen expressed on the surface of infected erythrocytes. Each parasite genome contains about 40 PfEMP1 genes, but only 1 PfEMP1 gene is expressed at a given time. PfEMP1 serves as a parasite-sequestering ligand...... to endothelial cells and enables the parasites to avoid splenic passage. PfEMP1 antibodies may protect from disease by inhibiting sequestration, thus facilitating the destruction of infected erythrocytes in the spleen. In this study, we have measured antibodies in Ghanaian children to a conserved region of Pf......EMP1 by enzyme-linked immunosorbent assay and antibodies to variant molecules on erythrocytes infected with field isolates of P. falciparum by flow cytometry. Based on close clinical monitoring, the children were grouped into those who did (susceptible) and those who did not (protected) have malaria...

  12. Macrophages transfer antigens to dendritic cells by releasing exosomes containing dead-cell-associated antigens partially through a ceramide-dependent pathway to enhance CD4(+) T-cell responses.

    Science.gov (United States)

    Xu, Yingping; Liu, Yi; Yang, Chunqing; Kang, Li; Wang, Meixiang; Hu, Jingxia; He, Hao; Song, Wengang; Tang, Hua

    2016-10-01

    Defects in rapid clearance of apoptotic cells lead to an accumulation of dead cells (late apoptotic or secondary necrotic cells), which results in an aberrant immune response. However, little is known about whether and how macrophages (Mφs) cooperate with dendritic cells (DCs) in the presentation of dead-cell-associated antigens in this process. By transferring high numbers of dead cells to mimic a failure of apoptotic cell clearance in vivo, we found that Mφs and neutrophils were the predominant phagocytes in the uptake of dead cells in the spleen. Moreover, both Mφs and DCs were required for an optimal CD4(+) T-cell response triggered by dead-cell-associated antigens. Importantly, although Mφs alone had a poor capacity for antigen presentation, they could transfer phagocytosed antigens to DCs for potent antigen presentation to enhance T-cell responses. Finally, we found that exosomes released from Mφs acted as a transmitter to convey antigens to DCs partially in a ceramide-dependent manner, since treatment with the neutral sphingomyelinase inhibitor GW4869 and spiroepoxide resulted in a significant reduction of T-cell proliferation in vitro and in vivo. These findings point to a novel pathway of cross-talk between Mφs and DCs, which will be helpful to explain possible mechanisms for autoimmune diseases characterized by increased rates of apoptosis.

  13. Development of an algorithm for production of inactivated arbovirus antigens in cell culture.

    Science.gov (United States)

    Goodman, C H; Russell, B J; Velez, J O; Laven, J J; Nicholson, W L; Bagarozzi, D A; Moon, J L; Bedi, K; Johnson, B W

    2014-11-01

    Arboviruses are medically important pathogens that cause human disease ranging from a mild fever to encephalitis. Laboratory diagnosis is essential to differentiate arbovirus infections from other pathogens with similar clinical manifestations. The Arboviral Diseases Branch (ADB) reference laboratory at the CDC Division of Vector-Borne Diseases (DVBD) produces reference antigens used in serological assays such as the virus-specific immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Antigen production in cell culture has largely replaced the use of suckling mice; however, the methods are not directly transferable. The development of a cell culture antigen production algorithm for nine arboviruses from the three main arbovirus families, Flaviviridae, Togaviridae, and Bunyaviridae, is described here. Virus cell culture growth and harvest conditions were optimized, inactivation methods were evaluated, and concentration procedures were compared for each virus. Antigen performance was evaluated by the MAC-ELISA at each step of the procedure. The antigen production algorithm is a framework for standardization of methodology and quality control; however, a single antigen production protocol was not applicable to all arboviruses and needed to be optimized for each virus.

  14. DNA-based nanoparticle tension sensors reveal that T-cell receptors transmit defined pN forces to their antigens for enhanced fidelity.

    Science.gov (United States)

    Liu, Yang; Blanchfield, Lori; Ma, Victor Pui-Yan; Andargachew, Rakieb; Galior, Kornelia; Liu, Zheng; Evavold, Brian; Salaita, Khalid

    2016-05-17

    T cells are triggered when the T-cell receptor (TCR) encounters its antigenic ligand, the peptide-major histocompatibility complex (pMHC), on the surface of antigen presenting cells (APCs). Because T cells are highly migratory and antigen recognition occurs at an intermembrane junction where the T cell physically contacts the APC, there are long-standing questions of whether T cells transmit defined forces to their TCR complex and whether chemomechanical coupling influences immune function. Here we develop DNA-based gold nanoparticle tension sensors to provide, to our knowledge, the first pN tension maps of individual TCR-pMHC complexes during T-cell activation. We show that naïve T cells harness cytoskeletal coupling to transmit 12-19 pN of force to their TCRs within seconds of ligand binding and preceding initial calcium signaling. CD8 coreceptor binding and lymphocyte-specific kinase signaling are required for antigen-mediated cell spreading and force generation. Lymphocyte function-associated antigen 1 (LFA-1) mediated adhesion modulates TCR-pMHC tension by intensifying its magnitude to values >19 pN and spatially reorganizes the location of TCR forces to the kinapse, the zone located at the trailing edge of migrating T cells, thus demonstrating chemomechanical crosstalk between TCR and LFA-1 receptor signaling. Finally, T cells display a dampened and poorly specific response to antigen agonists when TCR forces are chemically abolished or physically "filtered" to a level below ∼12 pN using mechanically labile DNA tethers. Therefore, we conclude that T cells tune TCR mechanics with pN resolution to create a checkpoint of agonist quality necessary for specific immune response.

  15. Surface swarming motility by Pectobacterium atrosepticum is a latent phenotype that requires O antigen and is regulated by quorum sensing.

    Science.gov (United States)

    Bowden, Steven D; Hale, Nicola; Chung, Jade C S; Hodgkinson, James T; Spring, David R; Welch, Martin

    2013-11-01

    We describe a previously cryptic phenotype associated with the opportunistic phytopathogen Pectobacterium atrosepticum (Pca): surface swarming. We found that when Pca was spotted onto plates containing <0.5% (w/v) agar, the culture produced copious amounts of extracellular matrix material containing highly motile cells. Once produced, this 'slime layer' spread rapidly across the plate either as an advancing front or as tendrils. Transposon mutagenesis was used to identify mutants that were affected in swarming. Hypo-swarmer mutants mostly carried insertions in a horizontally acquired island (HAI5), which encodes a cluster of genes involved in O antigen biosynthesis. Hyper-swarmer mutants mostly carried insertions in hexY, a known antagonist of the class I flagellar master regulator, FlhD4C2. In addition, we found that the nucleoid protein, histone-like nuclear structuring protein 2 (H-NS2), also regulated swarming behaviour. A mutant in which hns2 was overexpressed displayed a hyper-swarming phenotype, whereas a mutant in which the hns2 ORF was inactivated had a hypo-swarming phenotype. Swarming was also regulated by quorum sensing (QS) and by the carbon source being utilized. We show, using a range of epistasis experiments, that optimal swarming requires both motility and O antigen biosynthesis, and that H-NS2 and QS both promote swarming through their effects on motility.

  16. Nucleotide sequence and transcription of a trypomastigote surface antigen gene of Trypanosoma cruzi.

    Science.gov (United States)

    Fouts, D L; Ruef, B J; Ridley, P T; Wrightsman, R A; Peterson, D S; Manning, J E

    1991-06-01

    In previous studies we identified a 500-bp segment of the gene, TSA-1, which encodes an 85-kDa trypomastigote-specific surface antigen of the Peru strain of Trypanosoma cruzi. TSA-1 was shown to be located at a telomeric site and to contain a 27-bp tandem repeat unit within the coding region. This repeat unit defines a discrete subset of a multigene family and places the TSA-1 gene within this subset. In this study, we present the complete nucleotide sequence of the TSA-1 gene from the Peru strain. By homology matrix analysis, fragments of two other trypomastigote specific surface antigen genes, pTt34 and SA85-1.1, are shown to have extensive sequence homology with TSA-1 indicating that these genes are members of the same gene family as TSA-1. The TSA-1 subfamily was also found to be active in two other strains of T. cruzi, one of which contains multiple telomeric members and one of which contains a single non-telomeric member, suggesting that transcription is not necessarily dependent on the gene being located at a telomeric site. Also, while some of the sequences found in this gene family are present in 2 size classes of poly(A)+ RNA, others appear to be restricted to only 1 of the 2 RNA classes.

  17. Yeast surface display of a noncovalent MHC class II heterodimer complexed with antigenic peptide.

    Science.gov (United States)

    Boder, Eric T; Bill, Jerome R; Nields, Andrew W; Marrack, Philippa C; Kappler, John W

    2005-11-20

    Microbial protein display technologies have enabled directed molecular evolution of binding and stability properties in numerous protein systems. In particular, dramatic improvements to antibody binding affinity and kinetics have been accomplished using these tools in recent years. Examples of successful application of display technologies to other immunological proteins have been limited to date. Herein, we describe the expression of human class II major histocompatibility complex allele (MHCII) HLA-DR4 on the surface of Saccharomyces cerevisiae as a noncovalently associated heterodimer. The yeast-displayed MHCII is fully native as assessed by binding of conformationally specific monoclonal antibodies; failure of antibodies specific for empty HLA-DR4 to bind yeast-displayed protein indicates antigenic peptide is bound. This report represents the first example of a noncovalent protein dimer displayed on yeast and of successful display of wild-type MHCII. Results further point to the potential for using yeast surface display for engineering and analyzing the antigen binding properties of MHCII.

  18. A sharp T-cell antigen receptor signaling threshold for T-cell proliferation

    Science.gov (United States)

    Au-Yeung, Byron B.; Zikherman, Julie; Mueller, James L.; Ashouri, Judith F.; Matloubian, Mehrdad; Cheng, Debra A.; Chen, Yiling; Shokat, Kevan M.; Weiss, Arthur

    2014-01-01

    T-cell antigen receptor (TCR) signaling is essential for activation, proliferation, and effector function of T cells. Modulation of both intensity and duration of TCR signaling can regulate these events. However, it remains unclear how individual T cells integrate such signals over time to make critical cell-fate decisions. We have previously developed an engineered mutant allele of the critical T-cell kinase zeta-chain-associated protein kinase 70 kDa (Zap70) that is catalytically inhibited by a small molecule inhibitor, thereby blocking TCR signaling specifically and efficiently. We have also characterized a fluorescent reporter Nur77–eGFP transgenic mouse line in which T cells up-regulate GFP uniquely in response to TCR stimulation. The combination of these technologies unmasked a sharp TCR signaling threshold for commitment to cell division both in vitro and in vivo. Further, we demonstrate that this threshold is independent of both the magnitude of the TCR stimulus and Interleukin 2. Similarly, we identify a temporal threshold of TCR signaling that is required for commitment to proliferation, after which T cells are able to proliferate in a Zap70 kinase-independent manner. Taken together, our studies reveal a sharp threshold for the magnitude and duration of TCR signaling required for commitment of T cells to proliferation. These results have important implications for understanding T-cell responses to infection and optimizing strategies for immunomodulatory drug delivery. PMID:25136127

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

    Directory of Open Access Journals (Sweden)

    Yu-Chuen Huang

    2013-01-01

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

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

    DEFF Research Database (Denmark)

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

    1983-01-01

    Fetal hematopoietic cells that express the common acute lymphoblastic leukemia antigen (CALLA) were purified from both fetal liver and fetal bone marrow by immune rosetting with sheep erythrocytes coated with rabbit anti-mouse immunoglobulin and by fluorescence-activated cell sorting. Dual...... antigen. Furthermore, using methanol-fixed cells, it could be shown that approximately 20% contained intracytoplasmic mu chains (cyto-mu) and that approximately 15% were positive for the terminal transferase enzyme (TdT) marker. The CALLA+ fetal cells thus closely resemble the childhood acute...... that these cells are relatively immature lymphoid cells, CALLA+ cells do not appear to contain either myeloid precursor cells (CFU-G/M) or the earliest lymphoid stem cells. Udgivelsesdato: 1983-Jan-1...

  1. Comparison of microglia and infiltrating CD11c+ cells as antigen presenting cells for T cell proliferation and cytokine response

    DEFF Research Database (Denmark)

    Wlodarczyk, Agnieszka; Løbner, Morten; Cédile, Oriane

    2014-01-01

    BACKGROUND: Tissue-resident antigen-presenting cells (APC) exert a major influence on the local immune environment. Microglia are resident myeloid cells in the central nervous system (CNS), deriving from early post-embryonic precursors, distinct from adult hematopoietic lineages. Dendritic cells...... but detectable levels of all these cytokines. Transforming growth factor beta expression was similar in all three populations. Although CNS-resident and blood-derived CD11c+ cells showed equivalent ability to induce proliferation of myelin oligodendrocyte glycoprotein-immunised CD4+ T cells, CD11c+ microglia...

  2. Kinetics of Hepatitis B Surface Antigen Level in Chronic Hepatitis B Patients who Achieved Hepatitis B Surface Antigen Loss during Pegylated Interferon Alpha-2a Treatment

    Science.gov (United States)

    Li, Ming-Hui; Zhang, Lu; Qu, Xiao-Jing; Lu, Yao; Shen, Ge; Wu, Shu-Ling; Chang, Min; Liu, Ru-Yu; Hu, Lei-Ping; Li, Zhen-Zhen; Hua, Wen-Hao; Song, Shu-Jing; Xie, Yao

    2017-01-01

    Background: Hepatitis B surface antigen (HBsAg) loss/seroconversion is considered to be the ideal endpoint of antiviral therapy and the ultimate treatment goal in chronic hepatitis B (CHB). This study aimed to assess the patterns of HBsAg kinetics in CHB patients who achieved HBsAg loss during the treatment of pegylated interferon (PEG-IFN) α-2a. Methods: A total of 150 patients were enrolled, composing of 83 hepatitis B envelope antigen (HBeAg)-positive and 67 HBeAg-negative patients. Patients were treated with PEG-IFN α-2a180 μg/week until HBsAg loss/seroconversion was achieved, which occurred within 96 weeks. Serum hepatitis B virus deoxyribonucleic acid and serological indicators (HBsAg, anti-HBs, HBeAg, and anti-HBe) were determined before and every 3 months during PEG-IFN α-2a treatment. Biochemical markers and peripheral blood neutrophil and platelet counts were tested every 1–3 months. Results: Baseline HBsAg levels were 2.5 ± 1.3 log IU/ml, and decreased rapidly at 12 and 24 weeks by 48.3% and 88.3%, respectively. The mean time to HBsAg loss was 54.2 ± 30.4 weeks, though most patients needed extended treatment and 30.0% of HBsAg loss occurred during 72–96 weeks. Baseline HBsAg levels were significantly higher in HBeAg-positive patients (2.9 ± 1.1 log IU/ml) compared with HBeAg-negative patients (2.0 ± 1.3 log IU/ml; t = 4.733, P < 0.001), but the HBsAg kinetics were similar. Patients who achieved HBsAg loss within 48 weeks had significantly lower baseline HBsAg levels and had more rapid decline of HBsAg at 12 weeks compared to patients who needed extended treatment to achieve HBsAg loss. Conclusions: Patients with lower baseline HBsAg levels and more rapid decline during early treatment with PEG-IFN are more likely to achieve HBsAg loss during 96 weeks of treatment, and extended therapy longer than 48 weeks may be required to achieve HBsAg loss. PMID:28229987

  3. Cationic liposomes promote antigen cross-presentation in dendritic cells by alkalizing the lysosomal pH and limiting the degradation of antigens.

    Science.gov (United States)

    Gao, Jie; Ochyl, Lukasz J; Yang, Ellen; Moon, James J

    2017-01-01

    Cationic liposomes (CLs) have been widely examined as vaccine delivery nanoparticles since they can form complexes with biomacromolecules, promote delivery of antigens and adjuvant molecules to antigen-presenting cells (APCs), and mediate cellular uptake of vaccine components. CLs are also known to trigger antigen cross-presentation - the process by which APCs internalize extracellular protein antigens, degrade them into minimal CD8(+) T-cell epitopes, and present them in the context of major histocompatibility complex-I (MHC-I). However, the precise mechanisms behind CL-mediated induction of cross-presentation and cross-priming of CD8(+) T-cells remain to be elucidated. In this study, we have developed two distinct CL systems and examined their impact on the lysosomal pH in dendritic cells (DCs), antigen degradation, and presentation of peptide:MHC-I complexes to antigen-specific CD8(+) T-cells. To achieve this, we have used 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as the prototypical components of CLs with tertiary amine groups and compared the effect of CLs and anionic liposomes on lysosomal pH, antigen degradation, and cross-presentation by DCs. Our results showed that CLs, but not anionic liposomes, elevated the lysosomal pH in DCs and reduced antigen degradation, thereby promoting cross-presentation and cross-priming of CD8(+) T-cell responses. These studies shed new light on CL-mediated cross-presentation and suggest that intracellular fate of vaccine components and subsequent immunological responses can be controlled by rational design of nanomaterials.

  4. Cationic liposomes promote antigen cross-presentation in dendritic cells by alkalizing the lysosomal pH and limiting the degradation of antigens

    Science.gov (United States)

    Gao, Jie; Ochyl, Lukasz J; Yang, Ellen; Moon, James J

    2017-01-01

    Cationic liposomes (CLs) have been widely examined as vaccine delivery nanoparticles since they can form complexes with biomacromolecules, promote delivery of antigens and adjuvant molecules to antigen-presenting cells (APCs), and mediate cellular uptake of vaccine components. CLs are also known to trigger antigen cross-presentation – the process by which APCs internalize extracellular protein antigens, degrade them into minimal CD8+ T-cell epitopes, and present them in the context of major histocompatibility complex-I (MHC-I). However, the precise mechanisms behind CL-mediated induction of cross-presentation and cross-priming of CD8+ T-cells remain to be elucidated. In this study, we have developed two distinct CL systems and examined their impact on the lysosomal pH in dendritic cells (DCs), antigen degradation, and presentation of peptide:MHC-I complexes to antigen-specific CD8+ T-cells. To achieve this, we have used 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as the prototypical components of CLs with tertiary amine groups and compared the effect of CLs and anionic liposomes on lysosomal pH, antigen degradation, and cross-presentation by DCs. Our results showed that CLs, but not anionic liposomes, elevated the lysosomal pH in DCs and reduced antigen degradation, thereby promoting cross-presentation and cross-priming of CD8+ T-cell responses. These studies shed new light on CL-mediated cross-presentation and suggest that intracellular fate of vaccine components and subsequent immunological responses can be controlled by rational design of nanomaterials. PMID:28243087

  5. Detection of Avian Antigen-Specific T Cells Induced by Viral Vaccines.

    Science.gov (United States)

    Dalgaard, Tina Sørensen; Norup, Liselotte Rothmann; Juul-Madsen, Helle Risdahl

    2016-01-01

    Live attenuated viral vaccines are widely used in commercial poultry production, but the development of new effective inactivated/subunit vaccines is needed. Studies of avian antigen-specific T cells are primarily based on analyses ex vivo after activating the cells with recall antigen. There is a particular interest in developing robust high-throughput assays as chicken vaccine trials usually comprise many individuals. In many respects, the avian immune system differs from the mammalian, and T cell assessment protocols must be adjusted accordingly to account for, e.g., differences in leukocyte subsets.The carboxyfluorescein succinimidyl ester (CFSE) method described in this chapter has been adapted to chicken cells. In this test, cells of interest are stained with CFSE. The succinimidyl ester group covalently binds to cellular amines forming fluorescent conjugates that are retained in the cells even throughout division. This leads to daughter cells containing half the fluorescence of their parents. When lymphocytes are loaded with CFSE prior to ex vivo stimulation with specific antigen, the measurement of serial halving of its fluorescence by flow cytometry identifies the cells responding to the stimulation. This method has been successfully applied to studies of chicken antigen-specific T cells.

  6. The Plant Cell Surface

    Institute of Scientific and Technical Information of China (English)

    Anne-Mie C.Emons; Kurt V.Fagerstedt

    2010-01-01

    @@ Multicellular organization and tissue construction has evolved along essentially different lines in plants and animals. Since plants do not run away, but are anchored in the soil, their tissues are more or less firm and stiff. This strength stems from the cell walls, which encase the fragile cytoplasm, and protect it.

  7. Expression and immunological characterisation of Eimeria tenella glycosylphosphatidylinositol-anchored surface antigen-5

    Science.gov (United States)

    Ho, Sue-Kim; Nathan, Sheila; Wan, Kiew-Lian

    2016-11-01

    Eimeria tenella is the most pathogenic of the Eimeria species that infect chickens and causes huge economic losses to the poultry industry. The glycosylphosphatidylinositol-anchored surface antigen-5 (SAG5) found on the surface of the parasite has been shown to activate the chicken's immune system. In this study, recombinant SAG5 was expressed, purified and used to investigate the immune-inducing characteristics of the molecule. Chickens were immunized with purified recombinant SAG5 and sera were subjected to Enzyme-linked Immunosorbant Assay (ELISA). Results indicated that specific antibodies against rSAG5 were produced, with IgG detected at a higher level compared to IgA and IgM. Information on the immunological responses elicited by SAG5 provides essential knowledge that will contribute towards the effort to develop more effective strategies against coccidiosis.

  8. Pros and Cons of Antigen-Presenting Cell Targeted Tumor Vaccines

    Directory of Open Access Journals (Sweden)

    Cleo Goyvaerts

    2015-01-01

    Full Text Available In therapeutic antitumor vaccination, dendritic cells play the leading role since they decide if, how, when, and where a potent antitumor immune response will take place. Since the disentanglement of the complexity and merit of different antigen-presenting cell subtypes, antitumor immunotherapeutic research started to investigate the potential benefit of targeting these subtypes in situ. This review will discuss which antigen-presenting cell subtypes are at play and how they have been targeted and finally question the true meaning of targeting antitumor-based vaccines.

  9. In vivo targeting of antigens to maturing dendritic cells via the DEC-205 receptor improves T cell vaccination.

    Science.gov (United States)

    Bonifaz, Laura C; Bonnyay, David P; Charalambous, Anna; Darguste, Dara I; Fujii, Shin-Ichiro; Soares, Helena; Brimnes, Marie K; Moltedo, Bruno; Moran, Thomas M; Steinman, Ralph M

    2004-03-15

    The prevention and treatment of prevalent infectious diseases and tumors should benefit from improvements in the induction of antigen-specific T cell immunity. To assess the potential of antigen targeting to dendritic cells to improve immunity, we incorporated ovalbumin protein into a monoclonal antibody to the DEC-205 receptor, an endocytic receptor that is abundant on these cells in lymphoid tissues. Simultaneously, we injected agonistic alpha-CD40 antibody to mature the dendritic cells. We found that a single low dose of antibody-conjugated ovalbumin initiated immunity from the naive CD4+ and CD8+ T cell repertoire. Unexpectedly, the alphaDEC-205 antigen conjugates, given s.c., targeted to dendritic cells systemically and for long periods, and ovalbumin peptide was presented on MHC class I for 2 weeks. This was associated with stronger CD8+ T cell-mediated immunity relative to other forms of antigen delivery, even when the latter was given at a thousand times higher doses. In parallel, the mice showed enhanced resistance to an established rapidly growing tumor and to viral infection at a mucosal site. By better harnessing the immunizing functions of maturing dendritic cells, antibody-mediated antigen targeting via the DEC-205 receptor increases the efficiency of vaccination for T cell immunity, including systemic and mucosal resistance in disease models.

  10. A Single Subset of Dendritic Cells Controls the Cytokine Bias of Natural Killer T Cell Responses to Diverse Glycolipid Antigens

    Science.gov (United States)

    Arora, Pooja; Baena, Andres; Yu, Karl O.A.; Saini, Neeraj K.; Kharkwal, Shalu S.; Goldberg, Michael F.; Kunnath-Velayudhan, Shajo; Carreño, Leandro J.; Venkataswamy, Manjunatha M.; Kim, John; Lazar-Molnar, Eszter; Lauvau, Gregoire; Chang, Young-tae; Liu, Zheng; Bittman, Robert; Al-Shamkhani, Aymen; Cox, Liam R.; Jervis, Peter J.; Veerapen, Natacha; Besra, Gurdyal S.; Porcelli, Steven A.

    2014-01-01

    Summary Many hematopoietic cell types express CD1d and are capable of presenting glycolipid antigens to invariant natural killer T cells (iNKT cells). However, the question of which cells are the principal presenters of glycolipid antigens in vivo remains controversial, and it has been suggested that this might vary depending on the structure of a particular glycolipid antigen. Here we have shown that a single type of cell, the CD8α+ DEC-205+ dendritic cell, was mainly responsible for capturing and presenting a variety of different glycolipid antigens, including multiple forms of α-galactosylceramide that stimulate widely divergent cytokine responses. After glycolipid presentation, these dendritic cells rapidly altered their expression of various costimulatory and coinhibitory molecules in a manner that was dependent on the structure of the antigen. These findings show flexibility in the outcome of two-way communication between CD8α+ dendritic cells and iNKT cells, providing a mechanism for biasing toward either proinflammatory or anti-inflammatory responses. PMID:24412610

  11. A single subset of dendritic cells controls the cytokine bias of natural killer T cell responses to diverse glycolipid antigens.

    Science.gov (United States)

    Arora, Pooja; Baena, Andres; Yu, Karl O A; Saini, Neeraj K; Kharkwal, Shalu S; Goldberg, Michael F; Kunnath-Velayudhan, Shajo; Carreño, Leandro J; Venkataswamy, Manjunatha M; Kim, John; Lazar-Molnar, Eszter; Lauvau, Gregoire; Chang, Young-tae; Liu, Zheng; Bittman, Robert; Al-Shamkhani, Aymen; Cox, Liam R; Jervis, Peter J; Veerapen, Natacha; Besra, Gurdyal S; Porcelli, Steven A

    2014-01-16

    Many hematopoietic cell types express CD1d and are capable of presenting glycolipid antigens to invariant natural killer T cells (iNKT cells). However, the question of which cells are the principal presenters of glycolipid antigens in vivo remains controversial, and it has been suggested that this might vary depending on the structure of a particular glycolipid antigen. Here we have shown that a single type of cell, the CD8α(+) DEC-205(+) dendritic cell, was mainly responsible for capturing and presenting a variety of different glycolipid antigens, including multiple forms of α-galactosylceramide that stimulate widely divergent cytokine responses. After glycolipid presentation, these dendritic cells rapidly altered their expression of various costimulatory and coinhibitory molecules in a manner that was dependent on the structure of the antigen. These findings show flexibility in the outcome of two-way communication between CD8α(+) dendritic cells and iNKT cells, providing a mechanism for biasing toward either proinflammatory or anti-inflammatory responses.

  12. Cell-surface expression of Hsp70 on hematopoietic cancer cells after inhibition of HDAC activity

    DEFF Research Database (Denmark)

    Jensen, Helle; Andresen, Lars; Hansen, Karen Aagaard

    , membrane-bound Hsp70 can stimulate antigen presenting cells (APCs) to release proinflammatory cytokines and can provide a target structure for NK cell-mediated lysis. Human cancer cells frequently express Hsp70 on their cell surface, whereas the corresponding normal tissues do not. In addition, several...... clinically applied reagents, such as alkyl-lysophospholipides, chemotherapeutic agents, and anti-inflammatory reagents, have been found to enhance Hsp70 surface expression on cancer cells. We have found that inhibition of histone deacetylase (HDAC) activity leads to surface expression of Hsp70 on various...... hematopoietic cancer cells, an occurance that was not observed on naïve or activated peripheral blood cells. HDAC-inhibitor mediated Hsp70 surface expression was confined to the apoptotic Annexin V positive cells and blocked by inhibition of apoptosis. Other chemotherapeutic inducers of apoptosis...

  13. Targeting proliferating cell nuclear antigen and its protein interactions induces apoptosis in multiple myeloma cells.

    Directory of Open Access Journals (Sweden)

    Rebekka Müller

    Full Text Available Multiple myeloma is a hematological cancer that is considered incurable despite advances in treatment strategy during the last decade. Therapies targeting single pathways are unlikely to succeed due to the heterogeneous nature of the malignancy. Proliferating cell nuclear antigen (PCNA is a multifunctional protein essential for DNA replication and repair that is often overexpressed in cancer cells. Many proteins involved in the cellular stress response interact with PCNA through the five amino acid sequence AlkB homologue 2 PCNA-interacting motif (APIM. Thus inhibiting PCNA's protein interactions may be a good strategy to target multiple pathways simultaneously. We initially found that overexpression of peptides containing the APIM sequence increases the sensitivity of cancer cells to contemporary therapeutics. Here we have designed a cell-penetrating APIM-containing peptide, ATX-101, that targets PCNA and show that it has anti-myeloma activity. We found that ATX-101 induced apoptosis in multiple myeloma cell lines and primary cancer cells, while bone marrow stromal cells and primary healthy lymphocytes were much less sensitive. ATX-101-induced apoptosis was caspase-dependent and cell cycle phase-independent. ATX-101 also increased multiple myeloma cells' sensitivity against melphalan, a DNA damaging agent commonly used for treatment of multiple myeloma. In a xenograft mouse model, ATX-101 was well tolerated and increased the anti-tumor activity of melphalan. Therefore, targeting PCNA by ATX-101 may be a novel strategy in multiple myeloma treatment.

  14. HIV-1 Trans Infection of CD4+ T Cells by Professional Antigen Presenting Cells

    Directory of Open Access Journals (Sweden)

    Charles R. Rinaldo

    2013-01-01

    Full Text Available Since the 1990s we have known of the fascinating ability of a complex set of professional antigen presenting cells (APCs; dendritic cells, monocytes/macrophages, and B lymphocytes to mediate HIV-1 trans infection of CD4+ T cells. This results in a burst of virus replication in the T cells that is much greater than that resulting from direct, cis infection of either APC or T cells, or trans infection between T cells. Such APC-to-T cell trans infection first involves a complex set of virus subtype, attachment, entry, and replication patterns that have many similarities among APC, as well as distinct differences related to virus receptors, intracellular trafficking, and productive and nonproductive replication pathways. The end result is that HIV-1 can sequester within the APC for several days and be transmitted via membrane extensions intracellularly and extracellularly to T cells across the virologic synapse. Virus replication requires activated T cells that can develop concurrently with the events of virus transmission. Further research is essential to fill the many gaps in our understanding of these trans infection processes and their role in natural HIV-1 infection.

  15. Reassessing the role of HLA-DRB3 T-cell responses: evidence for significant expression and complementary antigen presentation.

    Science.gov (United States)

    Faner, Rosa; James, Eddie; Huston, Laurie; Pujol-Borrel, Ricardo; Kwok, William W; Juan, Manel

    2010-01-01

    In humans, several HLA-DRB loci (DRB1/3/4/5) encode diverse beta-chains that pair with alpha-chains to form DR molecules on the surface of APC. While DRB1 and DRB5 have been extensively studied, the role of DRB3/4 products of DR52/DR53 haplotypes has been largely neglected. To clarify the relative expression of DRB3, we quantified DRB3 mRNA levels in comparison with DRB1 mRNA from the same haplotype in both B cells and monocytes, observing quantitatively significant DRB3 synthesis. In CD19+ cells, DRB1*03/11/13 was 3.5-fold more abundant than DRB3, but in CD14+ this difference was only two-fold. Monocytes also had lower overall levels of DR mRNA compared with B cells, which was confirmed by cell surface staining of DRB1 and DRB3. To evaluate the functional role of DRB3, tetramer-guided epitope mapping was used to detect T cells against tetanus toxin and several influenza antigens presented by DRB3*0101/0202 or DRB1*03/11/13. None of the epitopes discovered were shared among any of the DR molecules. Quantitative assessment of DRB3-tetanus toxin specific T cells revealed that they are present at similar frequencies as those observed for DRB1. These results suggest that DRB3 plays a significant role in antigen presentation with different epitopic preferences to DRB1. Therefore, DRB3, like DRB5, serves to extend and complement the peptide repertoire of DRB1 in antigen presentation.

  16. Particle-based transcutaneous administration of HIV-1 p24 protein to human skin explants and targeting of epidermal antigen presenting cells.

    Science.gov (United States)

    Rancan, Fiorenza; Amselgruber, Sarah; Hadam, Sabrina; Munier, Sevérine; Pavot, Vincent; Verrier, Bernard; Hackbarth, Steffen; Combadiere, Behazine; Blume-Peytavi, Ulrike; Vogt, Annika

    2014-02-28

    Transcutaneous immunization is a promising vaccination strategy for the treatment of infectious diseases and cancer. In this study, we investigate the combination of cyanoacrylate skin surface stripping (CSSS) and particle-based antigen delivery to target the HIV-1 p24 protein to skin antigen presenting cells (APC). The CSSS treatment pre-activates skin APC and opens hair follicles, where protein-loaded particles accumulate and allow for sustained delivery of the loaded antigen to perifollicular APC. We found that poly-lactic acid (PLA) and polystyrene (PS) particles targeted the adsorbed HIV-1 p24 protein to the hair follicles. Small amounts of PS and PLA particles were found to translocate to the epidermis and be internalized by skin cells, whereas most of the particles aggregated in the hair follicle canal, where they released the loaded antigen. The p24 protein diffused to the epidermis and dermis and was detected in skin cells, especially in Langerhans cells and dermal dendritic cells. Furthermore, the combination of CSSS and particle-based delivery resulted in activation and maturation of Langerhans cells (HLA-DR, CD80 and CD83). We conclude that particle-based antigen delivery across partially disrupted skin barrier is a feasible and effective approach to needle-free transcutaneous vaccination.

  17. The clinical value of squamous cell carcinoma antigen in cancer of the uterine cervix

    NARCIS (Netherlands)

    de Bruijn, HWA; Duk, JM; van der Zee, AGJ; Pras, E; Willemse, PHB; Hollema, H; Mourits, MJE; de Vries, EGE; Aalders, JG; Boonstra, J.

    1998-01-01

    A review is given of the clinical use and interpretation of serum tumor marker levels during the treatment of patients with cancer of the uterine cervix, Pretreatment serum squamous cell carcinoma (SCC) antigen provides a new prognostic factor in early stage squamous cell carcinoma of the uterine ce

  18. Robust and Accurate Discrimination of Self/Non-Self Antigen Presentations by Regulatory T Cell Suppression

    Science.gov (United States)

    Furusawa, Chikara; Yamaguchi, Tomoyuki

    2016-01-01

    The immune response by T cells usually discriminates self and non-self antigens, even though the negative selection of self-reactive T cells is imperfect and a certain fraction of T cells can respond to self-antigens. In this study, we construct a simple mathematical model of T cell populations to analyze how such self/non-self discrimination is possible. The results demonstrate that the control of the immune response by regulatory T cells enables a robust and accurate discrimination of self and non-self antigens, even when there is a significant overlap between the affinity distribution of T cells to self and non-self antigens. Here, the number of regulatory T cells in the system acts as a global variable controlling the T cell population dynamics. The present study provides a basis for the development of a quantitative theory for self and non-self discrimination in the immune system and a possible strategy for its experimental verification. PMID:27668873

  19. Diversification of the antigen-specific T cell receptor repertoire after varicella zoster vaccination.

    Science.gov (United States)

    Qi, Qian; Cavanagh, Mary M; Le Saux, Sabine; NamKoong, Hong; Kim, Chulwoo; Turgano, Emerson; Liu, Yi; Wang, Chen; Mackey, Sally; Swan, Gary E; Dekker, Cornelia L; Olshen, Richard A; Boyd, Scott D; Weyand, Cornelia M; Tian, Lu; Goronzy, Jörg J

    2016-03-30

    Diversity and size of the antigen-specific T cell receptor (TCR) repertoire are two critical determinants for successful control of chronic infection. Varicella zoster virus (VZV) that establishes latency during childhood can escape control mechanisms, in particular with increasing age. We examined the TCR diversity of VZV-reactive CD4 T cells in individuals older than 50 years by studying three identical twin pairs and three unrelated individuals before and after vaccination with live attenuated VZV. Although all individuals had a small number of dominant T cell clones, the breadth of the VZV-specific repertoire differed markedly. A genetic influence was seen for the sharing of individual TCR sequences from antigen-reactive cells but not for repertoire richness or the selection of dominant clones. VZV vaccination favored the expansion of infrequent VZV antigen-reactive TCRs, including those from naïve T cells with lesser boosting of dominant T cell clones. Thus, vaccination does not reinforce the in vivo selection that occurred during chronic infection but leads to a diversification of the VZV-reactive T cell repertoire. However, a single-booster immunization seems insufficient to establish new clonal dominance. Our results suggest that repertoire analysis of antigen-specific TCRs can be an important readout to assess whether a vaccination was able to generate memory cells in clonal sizes that are necessary for immune protection.

  20. T Cell Receptors that Recognize the Tyrosinase Tumor Antigen | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    The National Cancer Institute, Surgery Branch, Tumor Immunology Section, is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize T Cells Attacking Cancer: T Cell Receptors that Recognize the Tyrosinase Tumor Antigen

  1. Squamous cell carcinoma antigen isoforms in serum from cervical cancer patients

    NARCIS (Netherlands)

    Roijer, E; de Bruijn, HWA; Dahlen, U; ten Hoor, K; Lundin, M; Nilsson, K; Soderstrom, K; Nilsson, O

    2006-01-01

    Squamous cell carcinoma antigen (SCCA) is a serological marker of squamous cell carcinomas (SCC). To study whether any of the SCCA isoforms would provide additional and more specific/sensitive clinical information than total SCCA, immunoassays specific for the different forms of SCCA (free SCCA2, to

  2. Discovering naturally processed antigenic determinants that confer protective T cell immunity

    DEFF Research Database (Denmark)

    Gilchuk, Pavlo; Spencer, Charles T; Conant, Stephanie B;

    2013-01-01

    CD8+ T cells (TCD8) confer protective immunity against many infectious diseases, suggesting that microbial TCD8 determinants are promising vaccine targets. Nevertheless, current T cell antigen identification approaches do not discern which epitopes drive protective immunity during active infectio...

  3. The B cell antigen receptor and overexpression of MYC can cooperate in the genesis of B cell lymphomas.

    Directory of Open Access Journals (Sweden)

    Yosef Refaeli

    2008-06-01

    Full Text Available A variety of circumstantial evidence from humans has implicated the B cell antigen receptor (BCR in the genesis of B cell lymphomas. We generated mouse models designed to test this possibility directly, and we found that both the constitutive and antigen-stimulated state of a clonal BCR affected the rate and outcome of lymphomagenesis initiated by the proto-oncogene MYC. The tumors that arose in the presence of constitutive BCR differed from those initiated by MYC alone and resembled chronic B cell lymphocytic leukemia/lymphoma (B-CLL, whereas those that arose in response to antigen stimulation resembled large B-cell lymphomas, particularly Burkitt lymphoma (BL. We linked the genesis of the BL-like tumors to antigen stimulus in three ways. First, in reconstruction experiments, stimulation of B cells by an autoantigen in the presence of overexpressed MYC gave rise to BL-like tumors that were, in turn, dependent on both MYC and the antigen for survival and proliferation. Second, genetic disruption of the pathway that mediates signaling from the BCR promptly killed cells of the BL-like tumors as well as the tumors resembling B-CLL. And third, growth of the murine BL could be inhibited by any of three distinctive immunosuppressants, in accord with the dependence of the tumors on antigen-induced signaling. Together, our results provide direct evidence that antigenic stimulation can participate in lymphomagenesis, point to a potential role for the constitutive BCR as well, and sustain the view that the constitutive BCR gives rise to signals different from those elicited by antigen. The mouse models described here should be useful in exploring further the pathogenesis of lymphomas, and in preclinical testing of new therapeutics.

  4. Antigen-Experienced T cells Limit the Priming of Naïve T cells During Infection with Leishmania major1

    Science.gov (United States)

    Gray, Peter M.; Reiner, Steven L.; Smith, Deborah F.; Kaye, Paul M.; Scott, Phillip

    2009-01-01

    One mechanism to control immune responses following infection is to rapidly down regulate antigen presentation, which has been observed in acute viral and bacterial infections. Here we describe experiments designed to address whether antigen presentation is decreased after an initial response to Leishmania major. Naïve α-β-Leishmania-specific (ABLE) T cell receptor transgenic T cells were adoptively transferred into mice at various times after L. major infection to determine the duration of presentation of parasite-derived antigens. ABLE T cells responded vigorously at the initiation of infection, but the ability to prime these cells quickly diminished, independent of IL-10, regulatory T cells or antigen load. However, antigen-experienced clonal and polyclonal T cell populations could respond, indicating that the diminution in naïve ABLE cell responses was not due to lack of antigen presentation. Since naïve T cell priming could be restored by removal of the endogenous T cell population, or adoptive transfer of antigen pulsed dendritic cells, it appears that T cells that have previously encountered antigen during infection compete with naïve antigen-specific T cells. These results suggest that during L. major infection antigen-experienced T cells, rather than naïve T cells, may be primarily responsible for sustaining the immune response. PMID:16818747

  5. Cell surface profiling using high-throughput flow cytometry : a platform for biomarker discovery and analysis of cellular heterogeneity

    NARCIS (Netherlands)

    Gedye, Craig A; Hussain, Ali; Paterson, Joshua; Smrke, Alannah; Saini, Harleen; Sirskyj, Danylo; Pereira, Keira; Lobo, Nazleen; Stewart, Jocelyn; Go, Christopher; Ho, Jenny; Medrano, Mauricio; Hyatt, Elzbieta; Yuan, Julie; Lauriault, Stevan; Meyer, Mona; Kondratyev, Maria; van den Beucken, Twan; Jewett, Michael; Dirks, Peter; Guidos, Cynthia J; Danska, Jayne; Wang, Jean; Wouters, Bradly; Neel, Benjamin; Rottapel, Robert; Ailles, Laurie E

    2014-01-01

    Cell surface proteins have a wide range of biological functions, and are often used as lineage-specific markers. Antibodies that recognize cell surface antigens are widely used as research tools, diagnostic markers, and even therapeutic agents. The ability to obtain broad cell surface protein profil

  6. In Vivo Targeting of Antigens to Maturing Dendritic Cells via the DEC-205 Receptor Improves T Cell Vaccination

    Science.gov (United States)

    Bonifaz, Laura C.; Bonnyay, David P.; Charalambous, Anna; Darguste, Dara I.; Fujii, Shin-Ichiro; Soares, Helena; Brimnes, Marie K.; Moltedo, Bruno; Moran, Thomas M.; Steinman, Ralph M.

    2004-01-01

    The prevention and treatment of prevalent infectious diseases and tumors should benefit from improvements in the induction of antigen-specific T cell immunity. To assess the potential of antigen targeting to dendritic cells to improve immunity, we incorporated ovalbumin protein into a monoclonal antibody to the DEC-205 receptor, an endocytic receptor that is abundant on these cells in lymphoid tissues. Simultaneously, we injected agonistic α-CD40 antibody to mature the dendritic cells. We found that a single low dose of antibody-conjugated ovalbumin initiated immunity from the naive CD4+ and CD8+ T cell repertoire. Unexpectedly, the αDEC-205 antigen conjugates, given s.c., targeted to dendritic cells systemically and for long periods, and ovalbumin peptide was presented on MHC class I for 2 weeks. This was associated with stronger CD8+ T cell–mediated immunity relative to other forms of antigen delivery, even when the latter was given at a thousand times higher doses. In parallel, the mice showed enhanced resistance to an established rapidly growing tumor and to viral infection at a mucosal site. By better harnessing the immunizing functions of maturing dendritic cells, antibody-mediated antigen targeting via the DEC-205 receptor increases the efficiency of vaccination for T cell immunity, including systemic and mucosal resistance in disease models. PMID:15024047

  7. Isolation and characterization of antigen-Ia complexes involved in T cell recognition

    DEFF Research Database (Denmark)

    Buus, S; Sette, A; Colon, S M

    1986-01-01

    Using equilibrium dialysis, it has been previously demonstrated that immunogenic peptides bind specifically to the Ia molecules serving as restriction elements in the immune response to these antigens. Using gel filtration to study the formation of ovalbumin (OVA) peptide-I-Ad complexes, it is he......Using equilibrium dialysis, it has been previously demonstrated that immunogenic peptides bind specifically to the Ia molecules serving as restriction elements in the immune response to these antigens. Using gel filtration to study the formation of ovalbumin (OVA) peptide-I-Ad complexes...... with glutaraldehyde revealed that the ovalbumin peptide was cross-linked solely to the alpha chain of I-Ad. Planar membranes containing I-Ad-OVA complexes stimulated a T cell response with 2 X 10(4) less antigen than required when uncomplexed antigen was used, thus demonstrating the biologic importance...

  8. Comparative study of the role of professional versus semiprofessional or nonprofessional antigen presenting cells in the rejection of vascularized organ allografts.

    Science.gov (United States)

    Sundstrom, J B; Ansari, A A

    1995-12-01

    The immune systems of transplant recipients are progressively challenged with exposure to the multiple lineages of donor cells that comprise the vascularized organ allograft. Each lineage of such donor tissue constitutively expresses or can be induced to express varying densities of MHC antigens ranging from no expression of MHC to MHC class I only to both MHC class I and class II. In addition, the cell surface expression of a diverse assortment of costimulatory and cell adhesion molecules also varies in density in a tissue specific fashion within the allograft. The MHC class I/II molecules displayed on the donor cells contain within their clefts a constellation of processed protein antigens in the form of peptides derived from intracellular and to some extent extracellular sources. Therefore, the potential for each cell lineage to induce alloactivation and serve as a target for allospecific immune responses is dependent on the diversity and density of peptide-bearing MHC molecules, costimulatory molecules, and cell adhesion molecules. In addition, the T cell receptor repertoire of the recipient also contributes to the magnitude of the allogeneic response. Consequently, the variety of clinical outcomes following organ transplantation even with the institution of potent immunosuppressive (drug) therapies is not surprising, as it appears reasonable for such therapies to influence the allogeneic response against distinct lineages differentially. Our failure to prevent chronic human allograft rejection may therefore be due to our limited appreciation of the full spectrum of alloactivating experiences encountered by host T cells as they interact with donor cells of diverse tissue lineages. Investigations by our laboratory of the immunopathogenesis of chronic cardiac allograft rejection have revealed an intrinsic inability of human cardiac myocytes to process and present antigens, not only for primary but also for secondary alloimmune responses. One obvious explanation

  9. Efficient expression of histidine-tagged large hepatitis delta antigen in baculovirus-transduced baby hamster kidney cells

    Institute of Scientific and Technical Information of China (English)

    Ying-Wei Chiang; Jaw-Chin Wu; Kuei-Chun Wang; Chia-Wei Lai; Yao-Chi Chung; Yu-Chen Hu

    2006-01-01

    AIM: To study the baculovirus/mammalian cell system for efficient expression of functional large hepatitis delta antigen (L-HDAg).METHODS: A recombinant baculovirus expressing histidine-tagged L-HDAg (L-HDAgH) was constructed to transduce baby hamster kidney (BHK) cells by a simplified transduction protocol.RESULTS: The recombinant baculovirus transduced BHK cells with efficiencies higher than 90% as determined by flow cytometry. The expression level was significantly higher than that obtained by plasmid transfection and was further enhanced 3-fold to around 19 pg/cell by the addition of 10 mmol/L sodium butyrate. Importantly,the expressed L-HDAgH was localized to the cell nucleus and correctly isoprenylated as determined by immunofluorescence labeling and confocal microscopy.Moreover, L-HDAgH interacted with hepatitis B surface antigen to form virus-like particles.CONCLUSION: The fusion with histidine tags as well as overexpression of L-HDAgH in the baculovirus-transduced BHK cells does not impair the biological functions. Taken together, the baculovirus/mammalian cell system offers an attractive alternative for high level expression of L-HDAgH or other proteins that require extensive posttranslational modifications.

  10. Antigen-engaged B cells undergo chemotaxis toward the T zone and form motile conjugates with helper T cells.

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    Takaharu Okada

    2005-06-01

    Full Text Available Interactions between B and T cells are essential for most antibody responses, but the dynamics of these interactions are poorly understood. By two-photon microscopy of intact lymph nodes, we show that upon exposure to antigen, B cells migrate with directional preference toward the B-zone-T-zone boundary in a CCR7-dependent manner, through a region that exhibits a CCR7-ligand gradient. Initially the B cells show reduced motility, but after 1 d, motility is increased to approximately 9 microm/min. Antigen-engaged B cells pair with antigen-specific helper T cells for 10 to more than 60 min, whereas non-antigen-specific interactions last less than 10 min. B cell-T cell conjugates are highly dynamic and migrate extensively, being led by B cells. B cells occasionally contact more than one T cell, whereas T cells are strictly monogamous in their interactions. These findings provide evidence of lymphocyte chemotaxis in vivo, and they begin to define the spatiotemporal cellular dynamics associated with T cell-dependent antibody responses.

  11. Expression of I-A and I-E,C region-coded Ia antigens on functional B cell subpopulations.

    Science.gov (United States)

    Frelinger, J A; Hibbler, F J; Hill, S W

    1978-12-01

    Ia antigens from specific subregions have been examined on functional B cell populations. Expression of both I-A and I-E,C region antigens was demonstrated on cells required for both lipopolysaccharide mitogenesis and polyclonal activation. Similar I-A and I-E,C subregion expression was found on cells required for response to the T-independent antigen, polyvinylpyrrolidone. TNP-specific IgM and hen egg lysozyme-specific IgG plaque-forming cells also express I-A and I-E,C region antigens. No evidence was found for an Ia- population responsive in the systems tested. Further, no evidence of preferential expression of I-A or I-E,C region antigens was observed in any system examined. Therefore, it appears that B cells express both I-A and I-E,C region-coded Ia antigens.

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

    Directory of Open Access Journals (Sweden)

    Harjeet Singh

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

  13. The impact of T cell intrinsic antigen adaptation on peripheral immune tolerance.

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    Nevil J Singh

    2006-10-01

    Full Text Available Overlapping roles have been ascribed for T cell anergy, clonal deletion, and regulation in the maintenance of peripheral immunological tolerance. A measurement of the individual and additive impacts of each of these processes on systemic tolerance is often lacking. In this report we have used adoptive transfer strategies to tease out the unique contribution of T cell intrinsic receptor calibration (adaptation in the maintenance of tolerance to a systemic self-antigen. Adoptively transferred naïve T cells stably calibrated their responsiveness to a persistent self-antigen in both lymphopenic and T cell-replete hosts. In the former, this state was not accompanied by deletion or suppression, allowing us to examine the unique contribution of adaptation to systemic tolerance. Surprisingly, adapting T cells could chronically help antigen-expressing B cells, leading to polyclonal hypergammaglobulinemia and pathology, in the form of mild arthritis. The helper activity mediated by CD40L and cytokines was evident even if the B cells were introduced after extended adaptation of the T cells. In contrast, in the T cell-replete host, neither arthritis nor autoantibodies were induced. The containment of systemic pathology required host T cell-mediated extrinsic regulatory mechanisms to synergize with the cell intrinsic adaptation process. These extrinsic mechanisms prevented the effector differentiation of the autoreactive T cells and reduced their precursor frequency, in vivo.

  14. SIV antigen immunization induces transient antigen-specific T cell responses and selectively activates viral replication in draining lymph nodes in retroviral suppressed rhesus macaques

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    Barry Peter A

    2011-07-01

    Full Text Available Abstract Background HIV infection causes a qualitative and quantitative loss of CD4+ T cell immunity. The institution of anti-retroviral therapy (ART restores CD4+ T cell responses to many pathogens, but HIV-specific responses remain deficient. Similarly, therapeutic immunization with HIV antigens of chronically infected, ART treated subjects results in poor induction of HIV-specific CD4 responses. In this study, we used a macaque model of ART treatment during chronic infection to study the virologic consequences of SIV antigen stimulation in lymph nodes early after immunization. Rhesus CMV (RhCMV seropositive, Mamu A*01 positive rhesus macaques were chronically infected with SIVmac251 and treated with ART. The immune and viral responses to SIV gag and RhCMV pp65 antigen immunization in draining lymph nodes and peripheral blood were analyzed. Animals were immunized on contralateral sides with SIV gag and RhCMV pp65 encoding plasmids, which allowed lymph nodes draining each antigen to be obtained at the same time from the same animal for direct comparison. Results We observed that both SIV and RhCMV immunizations stimulated transient antigen-specific T cell responses in draining lymph nodes. The RhCMV-specific responses were potent and sustained (50 days post-immunization in the periphery, while the SIV-specific responses were transient and extinguished quickly. The SIV antigen stimulation selectively induced transient SIV replication in draining lymph nodes. Conclusions The data are consistent with a model whereby viral replication in response to SIV antigen stimulation limits the generation of SIV antigen-specific responses and suggests a potential mechanism for the early loss and poor HIV-specific CD4+ T cell response observed in HIV-infected individuals.

  15. Comparison of dengue infection in human mononuclear leukocytes with mosquito C6/36 and mammalian Vero cells using flow cytometry to detect virus antigen

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    Sydow Farid FO von

    2000-01-01

    Full Text Available Fluorescent activated cell sorter (FACS analysis is useful for the detection of cellular surface antigens and intracellular proteins. We used this methodology in order to detect and quantify dengue antigens in highly susceptible cells such as clone C6/36 (Aedes albopictus and Vero cells (green monkey kidney. Additionally, we analyzed the infection in vitro of human peripheral blood mononuclear leukocytes (PBML. FACS analysis turned out to be a reliable technique to quantify virus growth in traditional cell cultures of C6/36 as well as Vero cells. High rates of infection were achieved with a good statistical correlation between the virus amount used in infection and the percentage of dengue antigen containing cells detected in infected cultures. We also showed that human monocytes (CD14+ are preferred target cells for in vitro dengue infection among PBML. Monocytes were much less susceptible to virus infection than cell lines but they displayed dengue antigens detected by FACS five days after infection. In contrast, lymphocytes showed no differences in their profile for dengue specific immunofluorescence. Without an animal model to reproduce dengue disease, alternative assays have been sought to correlate viral virulence with clinical manifestations and disease severity. Study of in vitro interaction of virus and host cells may highlight this relationship.

  16. HCA520, A NOVEL TUMOR ASSOCIATED ANTIGEN, INVOLVED IN CELL PROLIFERATION AND APOPTOSIS

    Institute of Scientific and Technical Information of China (English)

    杨美香; 曲迅; 刘福利; 郑广娟

    2003-01-01

    Objective: Tumor associated antigen encoding gene HCA520 (AF146019) was identified by screening a human hepatocellular carcinoma expressing cDNA library using SEREX technique. In this experiment we studied the effect of HCA520 on cell proliferation and apoptosis. Methods: Gene HCA520 was gained by PCR and transfected into 293 cells. The stable expression cells were obtained by G418 selection. The cell proliferation was measured by [3H]-TdR uptake and apoptosis assay was measured by FACS. Results: Eukaryotic expression plasmid pcDNA3-HCA520 was constructed and its stable transfectants were obtained. Overexpression of HCA520 inhibited the cell proliferation and enhanced cell apoptosis after serum deprivation. Conclusion: HCA520 is a novel tumor associated antigen that can affect cell proliferation and apoptosis.

  17. The perivascular phagocyte of the mouse pineal gland: An antigen-presenting cell

    DEFF Research Database (Denmark)

    Møller, Morten; Rath, Martin F; Klein, David C

    2006-01-01

    The perivascular space of the rat pineal gland is known to contain phagocytic cells that are immunoreactive for leukocyte antigens, and thus they appear to belong to the macrophage/microglial cell line. These cells also contain MHC class II proteins. We investigated this cell type in the pineal...... gland of mice. Actively phagocytosing cells with a prominent lysosomal system were found in the pericapillary spaces of the mouse pineal gland following intravenous injection of horseradish peroxidase. The cells also exhibited strong acid phosphatase activity. Perivascular cells were immunopositive...... for MHC class II protein and for CD68, a marker of monocytes/phagocytes. This study verifies that perivascular phagocytes with antigen-presenting properties are present in the mouse pineal gland....

  18. Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses.

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    Shayla K Shorter

    Full Text Available T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL, have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4 are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant.

  19. Hepatitis B surface antigen nanoparticles coated with chitosan and trimethyl chitosan: Impact of formulation on physicochemical and immunological characteristics.

    Science.gov (United States)

    Tafaghodi, Mohsen; Saluja, Vinay; Kersten, Gideon F A; Kraan, Heleen; Slütter, Bram; Amorij, Jean-Pierre; Jiskoot, Wim

    2012-08-03

    Mucosal immunization offers various advantages over parenteral vaccination, but typically requires potent delivery systems and/or adjuvants to result in protective immunity. Here we report on the preparation of trimethylated chitosan (TMC) and chitosan (CHT) nanoparticles (NPs) loaded with hepatitis B surface antigen (HB), by a simple and scalable method. TMC:HB and CHT:HB NPs were prepared by direct coating of antigen by polymer. The impact of buffer, pH and tonicity of the dispersion medium on NPs' polydispersity, zeta potential and association percentage of polymer with antigen was evaluated. Moreover, biological properties of both NPs were addressed in vitro by studying their effect on cell viability, transepithelial electrical resistance (TEER) and dendritic cell (DC) maturation. Finally, immunogenicity was assessed by evaluating IgG, IgG1, IgG2a, IgA titers and sIgA after both mucosal (nasal) as well intramuscular (i.m.) vaccination in a murine model. TMC:HB and CHT:HB NPs, prepared in acetate buffer pH 6.7 of three different tonicities, had comparable size, polydispersity, zeta potential and association percentage. TMC:HB NPs, but not CHT:HB NPs, had a mild negative effect on cell viability and TEER, and a considerable positive effect on DC maturation. After nasal and i.m. immunization, TMC:HB NPs in hypotonic medium and CHT:HB NPs in all media induced higher serum and nasal antibody titers compared with HB solution (P<0.001). After i.m. injection, both TMC:HB and CHT:HB NPs induced higher IgG and IgG2a titers compared with alum adsorbed HB (P<0.001). For CHT:HB NPs, the tonicity of the dispersion medium did not affect the mucosal and systemic immune responses. In conclusion, TMC NPs and CHT NPs are similarly potent mucosal immunoadjuvants for HB. Moreover, both polymers are potent immunoadjuvants for i.m. administered isotonic HB, resulting in higher IgG2a/IgG1 ratios compared with alum adjuvanted HB.

  20. Virosomes for antigen and DNA delivery

    NARCIS (Netherlands)

    Daemen, T; de Mare, A; Bungener, L; de Jonge, J; Huckriede, A; Wilschut, J

    2005-01-01

    Specific targeting and delivery as well as the display of antigens on the surface of professional antigen-presenting cells (APCs) are key issues in the design and development of new-generation vaccines aimed at the induction of both humoral and cell-mediated immunity. Prophylactic vaccination agains

  1. Whole tumor antigen vaccination using dendritic cells: Comparison of RNA electroporation and pulsing with UV-irradiated tumor cells

    Directory of Open Access Journals (Sweden)

    Benencia Fabian

    2008-04-01

    Full Text Available Abstract Because of the lack of full characterization of tumor associated antigens for solid tumors, whole antigen use is a convenient approach to tumor vaccination. Tumor RNA and apoptotic tumor cells have been used as a source of whole tumor antigen to prepare dendritic cell (DC based tumor vaccines, but their efficacy has not been directly compared. Here we compare directly RNA electroporation and pulsing of DCs with whole tumor cells killed by ultraviolet (UV B radiation using a convenient tumor model expressing human papilloma virus (HPV E6 and E7 oncogenes. Although both approaches led to DCs presenting tumor antigen, electroporation with tumor cell total RNA induced a significantly higher frequency of tumor-reactive IFN-gamma secreting T cells, and E7-specific CD8+ lymphocytes compared to pulsing with UV-irradiated tumor cells. DCs electroporated with tumor cell RNA induced a larger tumor infiltration by T cells and produced a significantly stronger delay in tumor growth compared to DCs pulsed with UV-irradiated tumor cells. We conclude that electroporation with whole tumor cell RNA and pulsing with UV-irradiated tumor cells are both effective in eliciting antitumor immune response, but RNA electroporation results in more potent tumor vaccination under the examined experimental conditions.

  2. Epithelium Expressing the E7 Oncoprotein of HPV16 Attracts Immune-Modulatory Dendritic Cells to the Skin and Suppresses Their Antigen-Processing Capacity.

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    Janin Chandra

    Full Text Available Antigen presenting cells (APCs in skin can promote either antigen-specific effector functions or antigen tolerance, and thus determine clearance or persistence of cutaneous viral infections. Human papillomavirus (HPV infections can persist in squamous epithelium in immunocompetent individuals, and some persisting HPV infections, particularly with HPV16, promote malignant epithelial transformation. Here, we investigate whether local expression of the HPV16 protein most associated with malignant transformation, HPV16-E7, affects the phenotype and function of APC subsets in the skin. We demonstrate an expanded population of Langerhans cells in HPV16-E7 transgenic skin with distinct cell surface markers which express immune-modulatory enzymes and cytokines not expressed by cells from non transgenic skin. Furthermore, HPV16-E7 transgene expression in keratinocytes attracts new APC subsets to the epidermis. In vivo migration and transport of antigen to the draining lymph node by these APCs is markedly enhanced in HPV16-E7 expressing skin, whereas antigen-processing, as measured by proteolytic cleavage of DQ-OVA and activation of T cells in vivo by APCs, is significantly impaired. These data suggest that local expression of HPV16-E7 in keratinocytes can contribute to persisting infection with this oncogenic virus, by altering the phenotype and function of local APCs.

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens play a major role in cytoadhesion of infected erythrocytes (IE), antigenic variation, and immunity to malaria. The current consensus on control of variant surface antigen expression is that only one PfEMP1 encoded by one var...

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

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    Warren L Denning

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

  5. A novel hepatitis B virus mutant with A-to-G at nt551 in the surface antigen gene

    Institute of Scientific and Technical Information of China (English)

    Hua-Biao Chen; De-Xing Fang; Fa-Qing Li; Hui-Ying Jing; Wei-Guo Tan; Su-Qin Li

    2003-01-01

    AIM: Hepatitis B surface antigen (HBsAg) mutant of hepatitisB virus (HBV) is one of the important factors that result inimmune escape and cause failure of immunization. In thisstudy we reported and characterized a novel HBV mutantwith A-to-G at nt551 and intended to provide theoreticaldata for prevention of HBV infection in China.METHODS: A methodology comprising polymerase chainreaction (PCR) amplifying, M13 bacteriophage cloning andnucleotide sequencing was used to analyze the sera of thepediatric patient who was hepatitis B (HB) immune failure.Expression plasmids containing the mutant S gene and awild-type (adr) S gene were constructed respectively andthe recombinant HBsAg were expressed in COS-7 cells underthe regulation of SV40 early promoter. The recombinantproteins were investigated for their immunological reactivitywith different monoclonal antibodies (mAb) against 'a'determinant and vaccine-raised human neutralizingantibodies.RESULTS: It was found that there was a new point mutationat nt551 of the HBV (adr) genome from A to G, leading to asubstitution of methionine (Met) to valine (Val) at position133 in the 'a' determinant of HBsAg. Compared to the wild-type HBsAg, the binding activity of the muant HBsAg tomAbs (A6, A11 and S17) and to vaccine-raised human anti-hepatitis B surface antibody (anti-HBs) decreased significantly.CONCLUSION: According to the facts that the patient hasbeen immunized with HB vaccine and that the serum is anti-HBs positive and HBsAg negative, and based on thenucleotide sequence analysis of the mutant HBV S geneand its alteration of antigenicity, the HBV is considered tobe a new vaccine-induced immune escape mutant differentfrom the known ones.

  6. Cloning of a cDNA encoding a surface antigen of Schistosoma mansoni schistosomula recognized by sera of vassinated mice

    Energy Technology Data Exchange (ETDEWEB)

    Dalton, J.P.; Tom, T.D.; Strand, M.

    1987-06-01

    Spleen cells of mice vaccinated with radiation-attenuated Schistosoma mansoni cercariae were used to produce monoclonal antibodies directed against newly transformed schistosomular surface antigens. One of these monoclonal antibodies recognized a polypeptide of 18 kDa. Binding was measured by radioimmunoassay. This glycoprotein was purified by monoclonal antibody immunoaffinity chromatography and a polyclonal antiserum was prepared against it. Immunofluorescence assays showed that the polyclonal antiserum bound to the surface of newly transformed schistosomula and lung-stage organisms but not to the surface of liver-stage and adult worms. Using this polyclonal antiserum we isolated recombinant clones from an adult worm cDNA expression library constructed in lambdagt11. Clone 654.2 contained an insert of 0.52 kilobase and hybridized to a 1.2-kilobase mRNA species from adult worms. Most importantly, clone 654.2 produced a fusion protein of 125 kDa that was reactive with sera of vaccinated mice that are capable of transferring resistance. This result encourages future vaccination trials with the fusion protein.

  7. Cord Blood Derived CD4+CD25high T Cells Become Functional Regulatory T Cells upon Antigen Encounter

    Science.gov (United States)

    Mayer, Elisabeth; Bannert, Christina; Gruber, Saskia; Klunker, Sven; Spittler, Andreas; Akdis, Cezmi A.

    2012-01-01

    Background: Upon antigen exposure, cord blood derived T cells respond to ubiquitous environmental antigens by high proliferation. To date it remains unclear whether these “excessive” responses relate to different regulatory properties of the putative T regulatory cell (Treg) compartment or even expansion of the Treg compartment itself. Methods: Cord blood (>37 week of gestation) and peripheral blood (healthy controls) were obtained and different Treg cell subsets were isolated. The suppressive potential of Treg populations after antigen exposure was evaluated via functional inhibition assays ([3H]thymidine incorporation assay and CFSE staining) with or without allergen stimulation. The frequency and markers of CD4+CD25highFoxP3+ T cells were characterized by mRNA analysis and flow cytometry. Results: Cord blood derived CD4+CD25high cells did not show substantial suppressor capacity upon TCR activation, in contrast to CD4+CD25high cells freshly purified from adult blood. This could not be explained by a lower frequency of FoxP3+CD4+CD25highcells or FOXP3 mRNA expression. However, after antigen-specific stimulation in vitro, these cells showed strong proliferation and expansion and gained potent suppressive properties. The efficiency of their suppressive capacity can be enhanced in the presence of endotoxins. If T-cells were sorted according to their CD127 expression, a tiny subset of Treg cells (CD4+CD25+CD127low) is highly suppressive even without prior antigen exposure. Conclusion: Cord blood harbors a very small subset of CD4+CD25high Treg cells that requires antigen-stimulation to show expansion and become functional suppressive Tregs. PMID:22272233

  8. A pluripotent human stem-cell clone isolated from the TERA-2 teratocarcinoma line lacks antigens SSEA-3 and SSEA-4 in vitro, but expresses these antigens when grown as a xenograft tumor.

    Science.gov (United States)

    Andrews, P W; Damjanov, I; Simon, D; Dignazio, M

    1985-01-01

    Human embryonal carcinoma (EC) cells generally express the cell-surface, stage-specific embryonic antigens 3 and 4 (SSEA-3 and SSEA-4), the epitopes of which are defined by two monoclonal antibodies that recognize different portions of an extended globoseries oligosaccharide. To examine further the relationship between these epitopes and the human EC phenotype, we investigated the properties of two newly isolated clones from the human teratocarcinoma cell line, TERA-2. One clone expresses SSEA-3 and SSEA-4; the other does not. Nevertheless, these clones otherwise resemble one another, and based upon their morphology, their expression of other cell-surface antigens, and their ability to form xenograft tumors containing a variety of cell types, we conclude that both clones are composed of pluripotent human EC cells. When exposed to retinoic acid in vitro, neither clone differentiates as extensively as other clones that we have previously derived from TERA-2. These observations indicate heterogeneity among stem cells derived from a single human teratocarcinoma, and suggest that SSEA-3 and SSEA-4 are not necessarily integral features of the human EC phenotype. On the other hand, EC cells in xenograft tumors derived from the SSEA-3- and SSEA-4-negative clone re-express these epitopes. Further, this re-expression is stable, since EC cell lines that are SSEA-3- and SSEA-4-positive grow out when the tumors are explanted in vitro. We conclude that the expression of these globoseries epitopes can be modulated by environmental influences.

  9. Expression of hepatitis B surface antigen gene (HBsAg) in Laminaria japonica (Laminariales, Phaeophyta)

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A transformation model for Laminaria japonica was established from 1993 to 1998, on the basis of which the transgenic kelp with heterologous gene encoding hepatitis B surface antigen (HBsAg) was obtained by using the micro- particle bombardment transformation method. Results of quantitative ELISA showed that HBsAg in transgenic kelp was 0.529 μg/mg soluble proteins on average and the highest value was 2.497 μg/mg, implying that recombinant HBsAg had natural epitope. Further support for the integration of HBsAg gene into kelp genome was obtained by PCR- Southern and total DNA hybridization. Prospect of kelp bioreactor producing high value materials such as edible HBV vaccine was discussed as well.

  10. Expression of Human Hepatitis B Virus Surface Antigen Gene in Transgenic Tobacco

    Institute of Scientific and Technical Information of China (English)

    刘玉乐; 王晋芳; 邱并生; 赵淑珍; 田波

    1994-01-01

    Expression of Human hepatitis B virus surface antigen (HBsAg) gene in plant was reported for the first time. The recombinant plasmid pRoKⅡ-HBsAg was constructed by inserting HBsAg gene into the downstream of CaMV 35S promoter of binary vector pRoKⅡ and then introduced into Agrobacterium tumefaciens LBA4404. The kanamycin-resistant plants were obtained by Agrobacterium-mediated transformation system. It was shown that HBsAg gene was expressed in transgenic tobacco plants and their progenies by ELISA. The spherical particles of ψ 22 nm in the leaf extract of trangenic tobacco were observed by immunosorbent electron microscopy.

  11. Detection of dengue NS1 antigen using long-range surface plasmon waveguides.

    Science.gov (United States)

    Wong, Wei Ru; Sekaran, Shamala Devi; Adikan, Faisal Rafiq Mahamd; Berini, Pierre

    2016-04-15

    The non-structural 1 (NS1) protein of the dengue virus circulates in infected patients' blood samples and can be used for early diagnosis of dengue infection. In this paper, we present the detection of naturally-occurring dengue NS1 antigen in infected patient blood plasma using straight long-range surface plasmon waveguides. Three commercially-available anti-NS1 monoclonal antibodies were used for recognition and their performance was compared and discussed. A similar figure of merit to the one used in conventional dengue NS1 capture using an enzyme-linked immunosorbent assay (ELISA) was applied to our results. In general, the positive patient samples can be clearly differentiated from the negative ones and the results agree with those obtained using ELISA. The largest signal-to-noise ratio observed during the experiments was 356 and the best detection limit observed is estimated as 5.73 pg/mm(2).

  12. Glycan elongation beyond the mucin associated Tn antigen protects tumor cells from immune-mediated killing.

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    Caroline B Madsen

    Full Text Available Membrane bound mucins are up-regulated and aberrantly glycosylated during malignant transformation in many cancer cells. This results in a negatively charged glycoprotein coat which may protect cancer cells from immune surveillance. However, only limited data have so far demonstrated the critical steps in glycan elongation that make aberrantly glycosylated mucins affect the interaction between cancer cells and cytotoxic effector cells of the immune system. Tn (GalNAc-Ser/Thr, STn (NeuAcα2-6GalNAc-Ser/Thr, T (Galβ1-3GalNAc-Ser/Thr, and ST (NeuAcα2-6Galβ1-3GalNAc-Ser/Thr antigens are recognized as cancer associated truncated glycans, and are expressed in many adenocarcinomas, e.g. breast- and pancreatic cancer cells. To investigate the role of the cancer associated glycan truncations in immune-mediated killing we created glyco-engineered breast- and pancreatic cancer cells expressing only the shortest possible mucin-like glycans (Tn and STn. Glyco-engineering was performed by zinc finger nuclease (ZFN knockout (KO of the Core 1 enzyme chaperone COSMC, thereby preventing glycan elongation beyond the initial GalNAc residue in O-linked glycans. We find that COSMC KO in the breast and pancreatic cancer cell lines T47D and Capan-1 increases sensitivity to both NK cell mediated antibody-dependent cellular-cytotoxicity (ADCC and cytotoxic T lymphocyte (CTL-mediated killing. In addition, we investigated the association between total cell surface expression of MUC1/MUC16 and NK or CTL mediated killing, and observed an inverse correlation between MUC16/MUC1 expression and the sensitivity to ADCC and CTL-mediated killing. Together, these data suggest that up-regulation of membrane bound mucins protects cells from immune mediated killing, and that particular glycosylation steps, as demonstrated for glycan elongation beyond Tn and STn, can be important for fine tuning of the immune escape mechanisms in cancer cells.

  13. Antibodies to new beta cell antigen ICA12 in Latvian diabetes patients.

    Science.gov (United States)

    Shtauvere-Brameus, A; Hagopian, W; Rumba, I; Sanjeevi, C B

    2002-04-01

    In Latvia diabetes mellitus is diagnosed using the WHO's clinical criteria, and assays for the detection of autoantibodies are not available. In consequence, slowly progressive autoimmune diabetes or LADA is likely to be missed. Antibodies to GAD65 and IA-2 are the major immunological markers in autoimmune diabetes. Recently, a new beta cell antigen, called ICA12, has been identified, which has a homology to the SOX family of transcription factors. The aim of the study was to analyze the prevalence of ICA12 antibodies in diabetes mellitus patients and controls from Latvia and to see whether this antigen is important in revealing autoimmunity when antibodies against major antigens are not present. We studied 88 IDDM patients and 100 NIDDM patients as well as controls for the prevalence of GAD65, IA-2, and ICA12 antibodies by radioligand binding assay (RIA) using (35)S-labeled islet antigens. We found ICA12Abs in 26 of 88 IDDM patients (30%) vs. 4% in healthy controls (4/100) and in 9 of 100 NIDDM patients (9%) vs. 2% controls (2/100). ICA12Abs alone are present in only 3% (3/88) of the patients with IDDM and 1% (1/100) of the NIDDM patients. We conclude that ICA12 represents the minor antigens in autoimmune diabetes and that, as a minor antigen, ICA12 alone does not contribute significantly in revealing new cases of autoimmunity.

  14. Expression of the Gastrin-Releasing Peptide Receptor, the Prostate Stem Cell Antigen and the Prostate-Specific Membrane Antigen in Lymph Node and Bone Metastases of Prostate Cancer

    NARCIS (Netherlands)

    Ananias, Hildo J. K.; van den Heuvel, Marius C.; Helfrich, Wijnand; de Jong, Igle J.

    2009-01-01

    OBJECTIVE. Cell membrane antigens like the gastrin-releasing peptide receptor (GRPR), the prostate stem cell antigen (PSCA), and the prostate-specific membrane antigen (PSMA), expressed in prostate cancer, are attractive targets for new therapeutic and diagnostic applications. Therefore, we investig

  15. Evaluation of germ-cell kinetics in infertile patients with proliferating cell nuclear antigen proliferating index

    Institute of Scientific and Technical Information of China (English)

    Li ZENG; Xiang-Tian KONG; Jin-Wei SU; Tong-Li XIA; Yan-Qun NA; Ying-Lu GUO

    2001-01-01

    To explore the usefulness of proliferating cell nuclear antigen proliferating index (PCNA PI) in the pathological diagnosis and treatment of male infertility. Methods: Testicular biopsy specimen obtained from 48 cases of male infertility and 2 normal controls were fixed and embedded. The sections were stained with anti-PCNA monoclonal antibodies or haematoxylin/eosin. Proliferating index (PI), expressed as the percentage of germ-cell nuclei positively stained with PCNA antibody, was assessed from more than 20 seminiferous tubules or 600 germ-cells. Results: The infertile patients were divided into 4 groups: Group 1, normal spermatogenesis ( 14 cases); Group 2, hypospermatogenesis (16 cases); Group 3, germinal arrest (10 cases); Group 4, Sertoli cell only syndrome (8 cases). The PCNA PI of normal control testis was 86.5% (mean value). Group 3 had a significantly lower PCNA PI (29.8%) than normal testis; Group 1 and 2 had similar Pis (82.3% and 82.3%, respectively) as the control testis. PI of the negative control (Group 4) was 0 as no germ-cells were found. Conclusion: PCNA PI is useful for assessing germ-cell kinetics, especially for pathological diagnosis of germinal arrest which is difficult to differentiate by routine HE staining technique. In germinal arrest, there is a significantly lowered PCNA PI, which is an indication of DNA synthesis deterioration, suggesting the use of therapies be different from those for hypospermatogenesis.

  16. Functional prostate-specific membrane antigen is enriched in exosomes from prostate cancer cells.

    Science.gov (United States)

    Liu, Tiancheng; Mendes, Desiree E; Berkman, Clifford E

    2014-03-01

    Developing simple and effective approaches to detect tumor markers will be critical for early diagnosis or prognostic evaluation of prostate cancer treatment. Prostate‑specific membrane antigen (PSMA) has been validated as an important tumor marker for prostate cancer progression including angiogenesis and metastasis. As a type II membrane protein, PSMA can be constitutively internalized from the cell surface into endosomes. Early endosomes can fuse with multivesicular bodies (MVB) to form and secrete exosomes (40-100 nm) into the extracellular environment. Herein, we tested whether some of the endosomal PSMA could be transferred to exosomes as an extracellular resource for PSMA. Using PSMA-positive LNCaP cells, the secreted exosomes were collected and isolated from the cultured media. The vesicular structures of exosomes were identified by electron microscopy, and exosomal marker protein CD9 and tumor susceptibility gene (TSG 101) were confirmed by western blot analysis. Our present data demonstrate that PSMA can be enriched in exosomes, exhibiting a higher content of glycosylation and partial proteolysis in comparison to cellular PSMA. An in vitro enzyme assay further confirmed that exosomal PSMA retains functional enzymatic activity. Therefore, our data may suggest a new role for PSMA in prostate cancer progression, and provide opportunities for developing non-invasive approaches for diagnosis or prognosis of prostate cancer.

  17. Cell-mediated immune responses to Plasmodium falciparum purified soluble antigens in sickle-cell trait subjects

    DEFF Research Database (Denmark)

    Bayoumi, R A; Abu-Zeid, Y A; Abdulhadi, N H

    1990-01-01

    endemic area 300 km south of Khartoum. Antibodies to ring-infected erythrocyte surface antigen (Pf155/RESA) and to circumsporozoite (CS) protein (anti-NANP40) indicated equal exposure to falciparum malaria. Peripheral blood mononuclear cells (BMNCs) from 20/35 (57%) Hb AS subjects compared with 10/24 (42......To determine the possible differences in the immune response to Plasmodium falciparum between sickle-cell trait (Hb AS) and normal haemoglobin (Hb AA) individuals, we examined 35 Hb AS and 24 Hb AA subjects matched for age and microenvironment. Their age was 2-55 years and all lived in a malaria......%) Hb AA subjects, responded to affinity-purified P. falciparum soluble antigens (SPAg). Of those responding to SPAg, 9 (26%) Hb AS subjects and only two (8%) Hb AA subjects had high responses. The mean proliferative response to SPAg of BMNCs from Hb AS individuals was significantly higher than in Hb AA...

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

    DEFF Research Database (Denmark)

    Hokland, M; Heron, I; Berg, K

    1982-01-01

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

  19. Leishmania-specific surface antigens show sub-genus sequence variation and immune recognition.

    Directory of Open Access Journals (Sweden)

    Daniel P Depledge

    Full Text Available BACKGROUND: A family of hydrophilic acylated surface (HASP proteins, containing extensive and variant amino acid repeats, is expressed at the plasma membrane in infective extracellular (metacyclic and intracellular (amastigote stages of Old World Leishmania species. While HASPs are antigenic in the host and can induce protective immune responses, the biological functions of these Leishmania-specific proteins remain unresolved. Previous genome analysis has suggested that parasites of the sub-genus Leishmania (Viannia have lost HASP genes from their genomes. METHODS/PRINCIPAL FINDINGS: We have used molecular and cellular methods to analyse HASP expression in New World Leishmania mexicana complex species and show that, unlike in L. major, these proteins are expressed predominantly following differentiation into amastigotes within macrophages. Further genome analysis has revealed that the L. (Viannia species, L. (V. braziliensis, does express HASP-like proteins of low amino acid similarity but with similar biochemical characteristics, from genes present on a region of chromosome 23 that is syntenic with the HASP/SHERP locus in Old World Leishmania species and the L. (L. mexicana complex. A related gene is also present in Leptomonas seymouri and this may represent the ancestral copy of these Leishmania-genus specific sequences. The L. braziliensis HASP-like proteins (named the orthologous (o HASPs are predominantly expressed on the plasma membrane in amastigotes and are recognised by immune sera taken from 4 out of 6 leishmaniasis patients tested in an endemic region of Brazil. Analysis of the repetitive domains of the oHASPs has shown considerable genetic variation in parasite isolates taken from the same patients, suggesting that antigenic change may play a role in immune recognition of this protein family. CONCLUSIONS/SIGNIFICANCE: These findings confirm that antigenic hydrophilic acylated proteins are expressed from genes in the same chromosomal

  20. Small-angle neutron scattering study of recombinant yeast-derived human hepatitis B virus surface antigen vaccine particle

    Science.gov (United States)

    Sato, M.; Ito, Y.; Kameyama, K.; Imai, M.; Ishikawa, N.; Takagi, T.

    1995-02-01

    The overall and internal structure of recombinant yeast-derived human hepatitis B virus surface antigen vaccine particles was investigated by small-angle neutron scattering using the contrast variation method. The vaccine is a nearly spherical particle, and its contrast-matching point was determined to be at about 24% D 2O content, indicating that a large part of the vaccine particle is occupied by lipids and carbohydrates from the yeast. The Stuhrmann plot suggests that the surface antigens exist predominantly in the peripheral region of the particle, which is favorable to the induction of anti-virus antibodies.

  1. Intratumoral IL-12 Gene Therapy Results in the Crosspriming of Tc1 Cells Reactive Against Tumor-associated Stromal Antigens

    Science.gov (United States)

    Zhao, Xi; Bose, Anamika; Komita, Hideo; Taylor, Jennifer L; Kawabe, Mayumi; Chi, Nina; Spokas, Laima; Lowe, Devin B; Goldbach, Christina; Alber, Sean; Watkins, Simon C; Butterfield, Lisa H; Kalinski, Pawel; Kirkwood, John M; Storkus, Walter J

    2011-01-01

    HLA-A2 transgenic mice bearing established HLA-A2neg B16 melanomas were effectively treated by intratumoral (i.t.) injection of syngeneic dendritic cells (DCs) transduced to express high levels of interleukin (IL)-12, resulting in CD8+ T cell-dependent antitumor protection. In this model, HLA-A2-restricted CD8+ T cells do not directly recognize tumor cells and therapeutic benefit was associated with the crosspriming of HLA-A2-restricted type-1 CD8+ T cells reactive against antigens expressed by stromal cells [i.e., pericytes and vascular endothelial cells (VEC)]. IL-12 gene therapy-induced CD8+ T cells directly recognized HLA-A2+ pericytes and VEC flow-sorted from B16 tumor lesions based on interferon (IFN)-γ secretion and translocation of the lytic granule-associated molecule CD107 to the T cell surface after coculture with these target cells. In contrast, these CD8+ T effector cells failed to recognize pericytes/VEC isolated from the kidneys of tumor-bearing HHD mice. The tumor-associated stromal antigen (TASA)-derived peptides studied are evolutionarily conserved and could be recognized by CD8+ T cells harvested from the blood of HLA-A2+ normal donors or melanoma patients after in vitro stimulation. These TASA and their derivative peptides may prove useful in vaccine formulations against solid cancers, as well as, in the immune monitoring of HLA-A2+ cancer patients receiving therapeutic interventions, such as IL-12 gene therapy. PMID:21189473

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

    DEFF Research Database (Denmark)

    Schirmbeck, Reinhold; Dikopoulos, Nektarios; Kwissa, Marcin;

    2003-01-01

    Processing exogenous hepatitis B surface antigen (HBsAg) of the hepatitis B virus (HBV) generates the K(b)-binding S(208-215) epitope 1; processing endogenous HBsAg generates the K(b)-binding S(190-197) epitope 2. Cross-reactive CD8(+) T cell responses were primed to epitope 1 but not epitope 2...... HBs-tg mice showed reduced antigenemia. Hence, vaccination with natural HBsAg variants from different HBV sero/genotypes can prime cross-reactive, specific CD8(+) T cell immunity that breaks tolerance to HBsAg....

  3. Structural characteristics of an antigen required for its interaction with Ia and recognition by T cells

    DEFF Research Database (Denmark)

    Sette, A; Buus, S; Colon, S;

    1987-01-01

    A detailed analysis of the residues within an immunogenic peptide that endow it with the capacity to interact with Ia and to be recognized by T cells is presented. Ia interacts with only a few of the peptide residues and overall exhibits a very broad specificity. Some residues appear to interact...... both with Ia and with T cells, leading to a model in which a peptide antigen is 'sandwiched' between Ia and the T-cell receptor....

  4. Nanoscale Patterning of Antigen on Silicon Substrate to Examine Mast Cell Activation

    Science.gov (United States)

    2002-04-01

    Examine Mast Cell Activation DISTRIBUTION: Approved for public release, distribution unlimited This paper is part of the following report: TITLE...Materials Research Society N4.3 Nanoscale Patterning of Antigen on Silicon Substrate to Examine Mast Cell Activation Reid N. Orth", Min Wu2 , Theodore G...nanometer scale to spatially control the stimulation of specific immunoreceptors on RBL mast cells . This work was motivated by previous research to

  5. Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Rocha, C.D.; Trombone, A.P.F.; Lorenzi, J.C.C.; Almeida, L.P.; Gembre, A.F.; Padilha, E. [Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Ramos, S.G. [Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Silva, C.L.; Coelho-Castelo, A.A.M. [Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil)

    2012-09-21

    In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.

  6. Mapping of B-Cell Epitopes in a Trypanosoma cruzi Immunodominant Antigen Expressed in Natural Infections

    Science.gov (United States)

    Lesénéchal, Mylène; Becquart, Laurence; Lacoux, Xavier; Ladavière, Laurent; Baida, Renata C. P.; Paranhos-Baccalà, Glaucia; da Silveira, José Franco

    2005-01-01

    Tc40 is an immunodominant antigen present in natural Trypanosoma cruzi infections. This immunogen was thoroughly mapped by using overlapping amino acid sequences identified by gene cloning and chemical peptide synthesis. To map continuous epitopes of the Tc40 antigen, an epitope expression library was constructed and screened with sera from human chagasic patients. A major, linear B-cell epitope spanning residues 403 to 426 (PAKAAAPPAA) was identified in the central domain of Tc40. A synthetic peptide spanning this region reacted strongly with 89.8% of the serum samples from T. cruzi-infected individuals. This indicates that the main antigenic site is defined by the linear sequence of the peptide rather than a conformation-dependent structure. The major B-cell epitope of Tc40 shares a high degree of sequence identity with T. cruzi ribosomal and RNA binding proteins, suggesting the existence of cross-reactivity among these molecules. PMID:15699429

  7. The dendritic cell-specific C-type lectin DC-SIGN is a receptor for Schistosoma mansoni egg antigens and recognizes the glycan antigen Lewis x.

    NARCIS (Netherlands)

    Die, van I.M.; Vliet, van SJ; Nyame, AK; Cummings, RD; Bank, CM; Appelmelk, B.J.; Geijtenbeek, T.B.H.; Kooijk, van Y.

    2003-01-01

    Schistosoma mansoni soluble egg antigens (SEAs) are crucially involved in modulating the host immune response to infection by S. mansoni. We report that human dendritic cells bind SEAs through the C-type lectin dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN). Monoclonal antibodies agai

  8. Dichotomy of the human T cell response to Leishmania antigens. I. Th1-like response to Leishmania major promastigote antigens in individuals recovered from cutaneous leishmaniasis

    DEFF Research Database (Denmark)

    Kemp, M; Hey, A S; Kurtzhals, J A

    1994-01-01

    The T cell response to antigens from Leishmania major promastigotes was investigated in peripheral blood mononuclear cells from Sudanese individuals with a history of cutaneous leishmaniasis (CL), Sudanese individuals with positive DTH reaction in the leishmanin skin test but with no history...

  9. Large, but not small, antigens require time- and temperature-dependent processing in accessory cells before they can be recognized by T cells

    DEFF Research Database (Denmark)

    Buus, S; Werdelin, O

    1986-01-01

    of antigen presentation we used the proliferative response of appropriately primed T cells during a co-culture with the paraformaldehyde-fixed and antigen-exposed presenting cells. We demonstrate that the large synthetic polypeptide antigen, dinitrophenyl-poly-L-lysine, requires processing. After an initial...... time-lag of 30 min this antigen is fully processed within 2 to 4 of culture at 37 degrees C. In contrast, the immunogenic heptapeptide, angiotensin III, can be presented by pre-fixed accessory cells, viz. without any prior processing. Antigen processing was found to be temperature...

  10. Phenotype of Antigen Unexperienced TH Cells in the Inflamed Central Nervous System in Experimental Autoimmune Encephalomyelitis.

    Science.gov (United States)

    Franck, Sophia; Paterka, Magdalena; Birkenstock, Jerome; Zipp, Frauke; Siffrin, Volker; Witsch, Esther

    2016-11-10

    Multiple sclerosis is a chronic, disseminated inflammation of the central nervous system which is thought to be driven by autoimmune T cells. Genetic association studies in multiple sclerosis and a large number of studies in the animal model of the disease support a role for effector/memory T helper cells. However, the mechanisms underlying relapses, remission and chronic progression in multiple sclerosis or the animal model experimental autoimmune encephalomyelitis, are not clear. In particular, there is only scarce information on the role of central nervous system-invading naive T helper cells in these processes. By applying two-photon laser scanning microscopy we could show in vivo that antigen unexperienced T helper cells migrated into the deep parenchyma of the inflamed central nervous system in experimental autoimmune encephalomyelitis, independent of their antigen specificity. Using flow cytometric analyses of central nervous system-derived lymphocytes we found that only antigen-specific, formerly naive T helper cells became activated during inflammation of the central nervous system encountering their corresponding antigen.

  11. Thymic-shared antigen-1 (TSA-1). A lymphostromal cell membrane Ly-6 superfamily molecule with a putative role in cellular adhesion.

    Science.gov (United States)

    Classon, B J; Boyd, R L

    1998-01-01

    The seeding and colonization of the thymus by bone marrow stem cells and the maturation of these cells into mature T lymphocytes are dependent on cell-surface recognition events between different cell lineages within the thymic microenvironment. Positive and negative selection processes within the thymus produce a peripheral T-cell repertoire capable of recognizing peptides derived from foreign antigen bound to self MHCmolecules. In addition to the TCR/MHC-peptide interaction, many other cell-surface molecules act in concert to regulate the kinetics of cellular interactions and intracellular signaling events during thymopoiesis. We have investigated the complexity of the thymic stroma by using monoclonal antibodies to clone cell-membrane molecules of thymic stromal cells. Thymic-shared antigen-1 (TSA-1) is a molecule of interest because it is expressed by both immature thymocytes and stromal cells. We report herein the structural and evolutionary relationships between TSA-1 and molecules of the Ly-6 superfamily (Ly-6SF), and present evidence that TSA-1 functions as a cell-surface receptor by binding a cognate cell target molecule on the surface of a subset of thymocytes.

  12. Antigen-induced recruitment of eosinophils: importance of CD4+ T cells, IL5, and mast cells.

    Science.gov (United States)

    Hom, J T; Estridge, T

    1994-12-01

    Eosinophils of sensitized mice readily recruit to the site of antigen challenge. In the present study, experiments were performed to determine the involvement of different cell types in the antigen-induced recruitment of eosinophils. We demonstrated that a single treatment with anti-L3T4 monoclonal antibody (mAb) on the day of allergen challenge significantly decreased antigen-induced recruitment of eosinophils. Treatments with anti-L3T4 mAb during the sensitization period also caused a substantial reduction in the migration of eosinophils into the site of challenge with antigen. Thus, it appears that both stages of eosinophil recruitment, sensitization and antigen-challenge, are dependent upon the presence of L3T4+ T cells. Moreover, while treatments with anti-IL5 mAb blocked eosinophil migration, anti-IL2 mAb failed to alter the antigen-induced recruitment of eosinophils. In addition, significant numbers of eosinophils from the mast-cell-deficient mice were found to migrate into the peritoneal cavities upon allergen challenge. Eosinophil migration was also observed in several mouse strains of different H-2 haplotypes. The present findings suggest that CD4+ T cells and IL5 but not IL2 may play important roles in modulating the recruitment of eosinophils. Moreover, the involvement of mast cells does not appear to be essential for eosinophil migration. Finally, the development of antigen-induced recruitment of eosinophils is probably not under the immunogenetic regulation by genes within the H-2 complex.

  13. Stable solid-phase Rh antigen.

    Science.gov (United States)

    Yared, M A; Moise, K J; Rodkey, L S

    1997-12-01

    Numerous investigators have attempted to isolate the Rh antigens in a stable, immunologically reactive form since the discovery of the Rh system over 56 years ago. We report here a successful and reproducible approach to solubilizing and adsorbing the human Rh antigen(s) to a solid-phase matrix in an antigenically active form. Similar results were obtained with rabbit A/D/F red blood cell antigens. The antigen preparation was made by dissolution of the red blood cell membrane lipid followed by fragmentation of the residual cytoskeleton in an EDTA solution at low ionic strength. The antigenic activity of the soluble preparations was labile in standard buffers but was stable in zwitterionic buffers for extended periods of time. Further studies showed that the antigenic activity of these preparations was enhanced, as was their affinity for plastic surfaces, in the presence of acidic zwitterionic buffers. Adherence to plastic surfaces at low pH maintained antigenic reactivity and specificity for antibody was retained. The data show that this approach yields a stable form of antigenically active human Rh D antigen that could be used in a red blood cell-free assay for quantitative analysis of Rh D antibody and for Rh D antibody immunoadsorption and purification.

  14. Modulation of Immune Responses by Exosomes Derived from Antigen-Presenting Cells

    Science.gov (United States)

    Shenoda, Botros B.; Ajit, Seena K.

    2016-01-01

    Exosome-mediated signaling is important in mediating the inflammatory response. To exert their biological or pathophysiological functions in the recipient cells, exosomes deliver a diverse array of biomacromolecules including long and short coding and non-coding RNAs, proteins, and lipids. Exosomes secreted by antigen-presenting cells can confer therapeutic benefits by attenuating or stimulating the immune response. Exosomes play a crucial role in carrying and presenting functional major histocompatibility peptide complexes to modulate antigen-specific T cell responses. Exosomes from Dendritic Cells (DCs) can activate T and B cells and have been explored for their immunostimulatory properties in cancer therapy. The immunosuppressive properties of exosomes derived from macrophages and DCs can reduce inflammation in animal models for several inflammatory disorders. This review focuses on the protective role of exosomes in attenuating inflammation or augmenting immune response, emphasizing studies on exosomes derived from DCs and macrophages. PMID:27660518

  15. Efficient induction of CD25- iTreg by co-immunization requires strongly antigenic epitopes for T cells

    Directory of Open Access Journals (Sweden)

    Li Jinyao

    2011-05-01

    Full Text Available Abstract Background We previously showed that co-immunization with a protein antigen and a DNA vaccine coding for the same antigen induces CD40low IL-10high tolerogenic DCs, which in turn stimulates the expansion of antigen-specific CD4+CD25-Foxp3+ regulatory T cells (CD25- iTreg. However, it was unclear how to choose the antigen sequence to maximize tolerogenic antigen presentation and, consequently, CD25- iTreg induction. Results In the present study, we demonstrated the requirement of highly antigenic epitopes for CD25- iTreg induction. Firstly, we showed that the induction of CD25- iTreg by tolerogenic DC can be blocked by anti-MHC-II antibody. Next, both the number and the suppressive activity of CD25- iTreg correlated positively with the overt antigenicity of an epitope to activate T cells. Finally, in a mouse model of dermatitis, highly antigenic epitopes derived from a flea allergen not only induced more CD25- iTreg, but also more effectively prevented allergenic reaction to the allergen than did weakly antigenic epitopes. Conclusions Our data thus indicate that efficient induction of CD25- iTreg requires highly antigenic peptide epitopes. This finding suggests that highly antigenic epitopes should be used for efficient induction of CD25- iTreg for clinical applications such as flea allergic dermatitis.

  16. Evaluation of three recombinant multi-antigenic vaccines composed of surface and secretory antigens of Toxoplasma gondii in murine models of experimental toxoplasmosis.

    Science.gov (United States)

    Dziadek, Bozena; Gatkowska, Justyna; Brzostek, Anna; Dziadek, Jaroslaw; Dzitko, Katarzyna; Grzybowski, Marcin; Dlugonska, Henryka

    2011-01-17

    The great clinical and economical impact of Toxoplasma gondii infections makes the development of an effective vaccine for controlling toxoplasmosis an extremely important aim. In the presented study, we evaluate the protective and immunogenic properties of three recombinant subunit vaccines composed of rROP2+rGRA4+rSAG1, rROP2+rROP4+rGRA4 and rROP2+rROP4+rSAG1 proteins of T. gondii in an experimental toxoplasmosis model in the C3H/HeJ and C57BL/6 mouse strains. All three recombinant vaccines induced partial protection as measured by the reduction of brain cyst burden following challenge with five tissue cysts of the low virulence DX T. gondii strain. The level of protection was dependent on the antigen composition of the vaccine and the genetic background of the laboratory animals. The strongest protection against chronic toxoplasmosis was induced in both C3H/HeJ and C57BL/6 mice by the mixture of rhoptry proteins rROP2 and rROP4 combined with tachyzoite major protein rSAG1. The average parasite burden in these groups of mice was reduced by 71% and 90%, respectively, compared to non-vaccinated mice. The observed protective effect was related to the vaccine-induced cellular and humoral immune responses, as measured by the antigen-induced release of the Th1 cytokines IFN-γ and IL-2, the antigen-stimulated proliferation of spleen cells of vaccinated animals in comparison to control animals and the development of systemic antigen-specific IgG1 and IgG2a (C3H/HeJ) or IgG2c (C57BL/6) antibodies. Our studies show that recombinant rROP2, rROP4, rGRA4 and rSAG1 antigens may be promising candidates for a subunit vaccine against toxoplasmosis. Additionally, we demonstrate that the ideal composition of vaccine antigens can be equally effective in mice with different genetic backgrounds and variable levels of innate resistance to toxoplasmosis, resulting in strong protection against T. gondii invasion.

  17. Antigen-sensitized CD4+CD62Llow memory/effector T helper 2 cells can induce airway hyperresponsiveness in an antigen free setting

    Directory of Open Access Journals (Sweden)

    Nagatani Katsuya

    2005-05-01

    Full Text Available Abstract Background Airway hyperresponsiveness (AHR is one of the most prominent features of asthma, however, precise mechanisms for its induction have not been fully elucidated. We previously reported that systemic antigen sensitization alone directly induces AHR before development of eosinophilic airway inflammation in a mouse model of allergic airway inflammation, which suggests a critical role of antigen-specific systemic immune response itself in the induction of AHR. In the present study, we examined this possibility by cell transfer experiment, and then analyzed which cell source was essential for this process. Methods BALB/c mice were immunized with ovalbumin (OVA twice. Spleen cells were obtained from the mice and were transferred in naive mice. Four days later, AHR was assessed. We carried out bronchoalveolar lavage (BAL to analyze inflammation and cytokine production in the lung. Fluorescence and immunohistochemical studies were performed to identify T cells recruiting and proliferating in the lung or in the gut of the recipient. To determine the essential phenotype, spleen cells were column purified by antibody-coated microbeads with negative or positive selection, and transferred. Then, AHR was assessed. Results Transfer of spleen cells obtained from OVA-sensitized mice induced a moderate, but significant, AHR without airway antigen challenge in naive mice without airway eosinophilia. Immunization with T helper (Th 1 elicited antigen (OVA with complete Freund's adjuvant did not induce the AHR. Transferred cells distributed among organs, and the cells proliferated in an antigen free setting for at least three days in the lung. This transfer-induced AHR persisted for one week. Interleukin-4 and 5 in the BAL fluid increased in the transferred mice. Immunoglobulin E was not involved in this transfer-induced AHR. Transfer of in vitro polarized CD4+ Th2 cells, but not Th1 cells, induced AHR. We finally clarified that CD4+CD62Llow memory

  18. Antigen-Specific Th17 Cells Are Primed by Distinct and Complementary Dendritic Cell Subsets in Oropharyngeal Candidiasis.

    Directory of Open Access Journals (Sweden)

    Kerstin Trautwein-Weidner

    2015-10-01

    Full Text Available Candida spp. can cause severe and chronic mucocutaneous and systemic infections in immunocompromised individuals. Protection from mucocutaneous candidiasis depends on T helper cells, in particular those secreting IL-17. The events regulating T cell activation and differentiation toward effector fates in response to fungal invasion in different tissues are poorly understood. Here we generated a Candida-specific TCR transgenic mouse reactive to a novel endogenous antigen that is conserved in multiple distant species of Candida, including the clinically highly relevant C. albicans and C. glabrata. Using TCR transgenic T cells in combination with an experimental model of oropharyngeal candidiasis (OPC we investigated antigen presentation and Th17 priming by different subsets of dendritic cells (DCs present in the infected oral mucosa. Candida-derived endogenous antigen accesses the draining lymph nodes and is directly presented by migratory DCs. Tissue-resident Flt3L-dependent DCs and CCR2-dependent monocyte-derived DCs collaborate in antigen presentation and T cell priming during OPC. In contrast, Langerhans cells, which are also present in the oral mucosa and have been shown to prime Th17 cells in the skin, are not required for induction of the Candida-specific T cell response upon oral challenge. This highlights the functional compartmentalization of specific DC subsets in different tissues. These data provide important new insights to our understanding of tissue-specific antifungal immunity.

  19. Three-day dendritic cells for vaccine development: Antigen uptake, processing and presentation

    Directory of Open Access Journals (Sweden)

    Schendel Dolores J

    2010-09-01

    Full Text Available Abstract Background Antigen-loaded dendritic cells (DC are capable of priming naïve T cells and therefore represent an attractive adjuvant for vaccine development in anti-tumor immunotherapy. Numerous protocols have been described to date using different maturation cocktails and time periods for the induction of mature DC (mDC in vitro. For clinical application, the use of mDC that can be generated in only three days saves on the costs of cytokines needed for large scale vaccine cell production and provides a method to produce cells within a standard work-week schedule in a GMP facility. Methods In this study, we addressed the properties of antigen uptake, processing and presentation by monocyte-derived DC prepared in three days (3d mDC compared with conventional DC prepared in seven days (7d mDC, which represent the most common form of DC used for vaccines to date. Results Although they showed a reduced capacity for spontaneous antigen uptake, 3d mDC displayed higher capacity for stimulation of T cells after loading with an extended synthetic peptide that requires processing for MHC binding, indicating they were more efficient at antigen processing than 7d DC. We found, however, that 3d DC were less efficient at expressing protein after introduction of in vitro transcribed (ivtRNA by electroporation, based on published procedures. This deficit was overcome by altering electroporation parameters, which led to improved protein expression and capacity for T cell stimulation using low amounts of ivtRNA. Conclusions This new procedure allows 3d mDC to replace 7d mDC for use in DC-based vaccines that utilize long peptides, proteins or ivtRNA as sources of specific antigen.

  20. Preparation of tumor antigen-loaded mature dendritic cells for immunotherapy.

    Science.gov (United States)

    Sabado, Rachel Lubong; Miller, Elizabeth; Spadaccia, Meredith; Vengco, Isabelita; Hasan, Farah; Bhardwaj, Nina

    2013-08-01

    While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors, their clinical efficacy remains to be established. The method described below, prepared in accordance with Good Manufacturing Process (GMP) guidelines, is an optimization of the most common ex vivo preparation method for generating large numbers of DCs for clinical studies. Our method utilizes the synthetic TLR 3 agonist Polyinosinic-Polycytidylic Acid-poly-L-lysine Carboxymethylcellulose (Poly-ICLC) to stimulate the DCs. Our previous study established that Poly-ICLC is the most potent individual maturation stimulus for human DCs as assessed by an upregulation of CD83 and CD86, induction of interleukin-12 (IL-12), tumor necrosis factor (TNF), interferon gamma-induced protein 10 (IP-10), interleukmin 1 (IL-1), and type I interferons (IFN), and minimal interleukin 10 (IL-10) production. DCs are differentiated from frozen peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis. PBMCs are isolated by Ficoll gradient centrifugation and frozen in aliquots. On Day 1, PBMCs are thawed and plated onto tissue culture flasks to select for monocytes which adhere to the plastic surface after 1-2 hr incubation at 37 °C in the tissue culture incubator. After incubation, the lymphocytes are washed off and the adherent monocytes are cultured for 5 days in the presence of interleukin-4 (IL-4) and granulocyte macrophage-colony stimulating factor (GM-CSF) to differentiate to immature DCs. On Day 6, immature DCs are pulsed with the keyhole limpet hemocyanin (KLH) protein which serves as a control for the quality of the vaccine and may boost the immunogenicity of the vaccine. The DCs are stimulated to mature, loaded with peptide antigens, and incubated overnight. On Day 7, the cells are washed, and frozen in 1 ml aliquots containing 4-20 x 10(6) cells using a controlled-rate freezer. Lot release testing for the batches of DCs is performed and must meet minimum

  1. Activated human nasal epithelial cells modulate specific antibody response against bacterial or viral antigens.

    Directory of Open Access Journals (Sweden)

    Chiou-Yueh Yeh

    Full Text Available Nasal mucosa is an immune responsive organ evidenced by eliciting both specific local secretory IgA and systemic IgG antibody responses with intra-nasal administration of antigens. Nevertheless, the role of nasal epithelial cells in modulating such responses is unclear. Human nasal epithelial cells (hNECs obtained from sinus mucosa of patients with chronic rhinosinusitis were cultured in vitro and firstly were stimulated by Lactococcus lactis bacterium-like particles (BLPs in order to examine their role on antibody production. Secondly, both antigens of immunodominant protein IDG60 from oral Streptococcus mutans and hemagglutinin (HA from influenza virus were tested to evaluate the specific antibody response. Stimulated hNECs by BLPs exhibited a significant increase in the production of interleukin-6 (IL-6, and thymic stromal lymphopoietin (TSLP. Conditioned medium of stimulated hNECs has effects on enhancing the proliferation of CD4+ T cells together with interferon-γ and IL-5 production, increasing the costimulatory molecules on dendritic cells and augmenting the production of IDG60 specific IgA, HA specific IgG, IgA by human peripheral blood lymphocytes. Such production of antigen specific IgG and IgA is significantly counteracted in the presence of IL-6 and TSLP neutralizing antibodies. In conclusion, properly stimulated hNECs may impart immuno-modulatory effects on the antigen-specific antibody response at least through the production of IL-6 and TSLP.

  2. Identification of Theileria lestoquardi Antigens Recognized by CD8+ T Cells

    Science.gov (United States)

    Ngugi, Daniel; Lizundia, Regina; Hostettler, Isabel; Woods, Kerry; Ballingall, Keith; MacHugh, Niall D.; Morrison, W. Ivan; Weir, Willie; Shiels, Brian; Werling, Dirk

    2016-01-01

    As part of an international effort to develop vaccines for Theileria lestoquardi, we undertook a limited screen to test T. lestoquardi orthologues of antigens recognised by CD8+ T lymphocyte responses against T. annulata and T. parva in cattle. Five MHC defined sheep were immunized by live T. lestoquardi infection and their CD8+ T lymphocyte responses determined. Thirteen T. lestoquardi orthologues of T. parva and T. annulata genes, previously shown to be targets of CD8+ T lymphocyte responses of immune cattle, were expressed in autologous fibroblasts and screened for T cell recognition using an IFNγ assay. Genes encoding T. lestoquardi antigens Tl8 (putative cysteine proteinase, 349 aa) or Tl9 (hypothetical secreted protein, 293 aa) were recognise by T cells from one animal that displayed a unique MHC class I genotype. Antigenic 9-mer peptide epitopes of Tl8 and Tl9 were identified through peptide scans using CD8+ T cells from the responding animal. These experiments identify the first T. lestoquardi antigens recognised by CD8+ T cell responses linked to specific MHC class I alleles. PMID:27611868

  3. MHC-based detection of antigen-specific CD8(+) T cell responses

    DEFF Research Database (Denmark)

    Hadrup, Sine Reker; Schumacher, Nana Maria Pii

    2010-01-01

    The hallmark of adaptive immunity is its ability to recognise a wide range of antigens and technologies that capture this diversity are therefore of substantial interest. New methods have recently been developed that allow the parallel analysis of T cell reactivity against vast numbers of differe...

  4. Aspergillus galactomannan antigen levels in allogeneic haematopoietic stem cell transplant recipients given total parenteral nutrition.

    NARCIS (Netherlands)

    Blijlevens, N.M.A.; Donnelly, J.P.; Meis, J.F.G.M.; Verweij, P.E.; Pauw, B.E. de

    2002-01-01

    False-positive tests for Aspergillus galactomannan have been reported in neutropenic patients. We failed to detect any circulating antigen during the 2 weeks following allogeneic haematopoietic stem cell transplantation of 12 patients who had severe mucositis but were unable to eat.

  5. Defining the HLA class I-associated viral antigen repertoire from HIV-1-infected human cells

    DEFF Research Database (Denmark)

    Ternette, Nicola; Yang, Hongbing; Partridge, Thomas

    2016-01-01

    % of the identified sequences originated from viral protein regions for which T-cell responses have previously been reported but for which the precise HLA class I-binding sequences have not yet been defined. These results validate and expand the current knowledge of virus-specific antigenic peptide presentation...

  6. Tolerization of an established αb-crystallin-reactive T-cell response by intravenous antigen

    NARCIS (Netherlands)

    Verbeek, R.; Mark, K. van der; Wawrousek, E.F.; Plomp, A.C.; Noort, J.M. van

    2007-01-01

    Tolerance induction to prevent activation of a naïve T-cell repertoire has been well documented in rodents and can be readily achieved by intravenous, oral or intranasal administration of antigen in the absence of adjuvants. In autoimmune diseases such as multiple sclerosis (MS) the presence of an e

  7. CLIC1 regulates dendritic cell antigen processing and presentation by modulating phagosome acidification and proteolysis

    Directory of Open Access Journals (Sweden)

    Kanin Salao

    2016-05-01

    Full Text Available Intracellular chloride channel protein 1 (CLIC1 participates in inflammatory processes by regulating macrophage phagosomal functions such as pH and proteolysis. Here, we sought to determine if CLIC1 can regulate adaptive immunity by actions on dendritic cells (DCs, the key professional antigen presenting cells. To do this, we first generated bone marrow-derived DCs (BMDCs from germline CLIC1 gene-deleted (CLIC1−/− and wild-type (CLIC1+/+ mice, then studied them in vitro and in vivo. We found phagocytosis triggered cytoplasmic CLIC1 translocation to the phagosomal membrane where it regulated phagosomal pH and proteolysis. Phagosomes from CLIC1−/− BMDCs displayed impaired acidification and proteolysis, which could be reproduced if CLIC1+/+, but not CLIC1−/− cells, were treated with IAA94, a CLIC family ion channel blocker. CLIC1−/− BMDC displayed reduced in vitro antigen processing and presentation of full-length myelin oligodendrocyte glycoprotein (MOG and reduced MOG-induced experimental autoimmune encephalomyelitis. These data suggest that CLIC1 regulates DC phagosomal pH to ensure optimal processing of antigen for presentation to antigen-specific T-cells. Further, they indicate that CLIC1 is a novel therapeutic target to help reduce the adaptive immune response in autoimmune diseases.

  8. Expression Patterns of Cancer-Testis Antigens in Human Embryonic Stem Cells and Their Cell Derivatives Indicate Lineage Tracks

    OpenAIRE

    Nadya Lifantseva; Anna Koltsova; Tatyana Krylova; Tatyana Yakovleva; Galina Poljanskaya; Olga Gordeeva

    2011-01-01

    Pluripotent stem cells can differentiate into various lineages but undergo genetic and epigenetic changes during long-term cultivation and, therefore, require regular monitoring. The expression patterns of cancer-testis antigens (CTAs) MAGE-A2, -A3, -A4, -A6, -A8, -B2, and GAGE were examined in undifferentiated human embryonic stem (hES) cells, their differentiated derivatives, teratocarcinoma (hEC) cells, and cancer cell lines of neuroectodermal and mesodermal origin. Undifferentiated hES ce...

  9. Affinity for self antigen selects Treg cells with distinct functional properties.

    Science.gov (United States)

    Wyss, Lena; Stadinski, Brian D; King, Carolyn G; Schallenberg, Sonja; McCarthy, Nicholas I; Lee, Jun Young; Kretschmer, Karsten; Terracciano, Luigi M; Anderson, Graham; Surh, Charles D; Huseby, Eric S; Palmer, Ed

    2016-09-01

    The manner in which regulatory T cells (Treg cells) control lymphocyte homeostasis is not fully understood. We identified two Treg cell populations with differing degrees of self-reactivity and distinct regulatory functions. We found that GITR(hi)PD-1(hi)CD25(hi) (Triple(hi)) Treg cells were highly self-reactive and controlled lympho-proliferation in peripheral lymph nodes. GITR(lo)PD-1(lo)CD25(lo) (Triple(lo)) Treg cells were less self-reactive and limited the development of colitis by promoting the conversion of CD4(+) Tconv cells into induced Treg cells (iTreg cells). Although Foxp3-deficient (Scurfy) mice lacked Treg cells, they contained Triple(hi)-like and Triple(lo)-like CD4(+) T cells zsuper> T cells infiltrated the skin, whereas Scurfy Triple(lo)CD4(+) T cells induced colitis and wasting disease. These findings indicate that the affinity of the T cell antigen receptor for self antigen drives the differentiation of Treg cells into distinct subsets with non-overlapping regulatory activities.

  10. cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

    Energy Technology Data Exchange (ETDEWEB)

    Torian, B.E.; Stroeher, V.L.; Stamm, W.E. (Univ. of Washington, Seattle (USA)); Flores, B.M. (Louisiana State Univ. Medical Center, New Orleans (USA)); Hagen, F.S. (Zymogenetics Incorporated, Seattle, WA (USA))

    1990-08-01

    A {lambda}gt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically {sup 35}S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-{sup 125}I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4{degree}C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested.

  11. Circulating hepatitis B surface antigen particles carry hepatocellular microRNAs.

    Directory of Open Access Journals (Sweden)

    Luisa Novellino

    Full Text Available Hepatitis B virus (HBV produces high quantities of subviral surface antigen particles (HBsAg which circulate in the blood outnumbering virions of about 1\\10(3-6 times. In individuals coinfected with the defective hepatitis Delta virus (HDV the small HDV-RNA-genome and Delta antigen circulate as ribonucleoprotein complexes within HBsAg subviral particles. We addressed the question whether subviral HBsAg particles may carry in the same way cellular microRNAs (miRNAs which are released into the bloodstream within different subcellular forms such as exosomes and microvescicles. Circulating HBsAg particles were isolated from sera of 11 HBsAg carriers by selective immunoprecipitation with monoclonal anti-HBs-IgG, total RNA was extracted and human miRNAs were screened by TaqMan real-time quantitative PCR Arrays. Thirty-nine human miRNAs were found to be significantly associated with the immunoprecipitated HBsAg, as determined by both comparative DDCT analysis and non-parametric tests (Mann-Whitney, p<0.05 with respect to controls. Moreover immunoprecipitated HBsAg particles contained Ago2 protein that could be revealed in ELISA only after 0.5% NP40. HBsAg associated miRNAs were liver-specific (most frequent = miR-27a, miR-30b, miR-122, miR-126 and miR-145 as well as immune regulatory (most frequent = miR-106b and miR-223. Computationally predicted target genes of HBsAg-associated miRNAs highlighted molecular pathways dealing with host-pathogen. The finding that HBsAg particles carry selective pools of hepatocellular miRNAs opens new avenues of research to disentangle the complex interactions between host and HBV and provides a non invasive tool to study the physiopathology of liver epigenetics.

  12. Identification of new meningococcal serogroup B surface antigens through a systematic analysis of neisserial genomes.

    Science.gov (United States)

    Pajon, Rolando; Yero, Daniel; Niebla, Olivia; Climent, Yanet; Sardiñas, Gretel; García, Darién; Perera, Yasser; Llanes, Alejandro; Delgado, Maité; Cobas, Karem; Caballero, Evelin; Taylor, Stephen; Brookes, Charlotte; Gorringe, Andrew

    2009-12-11

    The difficulty of inducing an effective immune response against the Neisseria meningitidis serogroup B capsular polysaccharide has lead to the search for vaccines for this serogroup based on outer membrane proteins. The availability of the first meningococcal genome (MC58 strain) allowed the expansion of high-throughput methods to explore the protein profile displayed by N. meningitidis. By combining a pan-genome analysis with an extensive experimental validation to identify new potential vaccine candidates, genes coding for antigens likely to be exposed on the surface of the meningococcus were selected after a multistep comparative analysis of entire Neisseria genomes. Eleven novel putative ORF annotations were reported for serogroup B strain MC58. Furthermore, a total of 20 new predicted potential pan-neisserial vaccine candidates were produced as recombinant proteins and evaluated using immunological assays. Potential vaccine candidate coding genes were PCR-amplified from a panel of representative strains and their variability analyzed using maximum likelihood approaches for detecting positive selection. Finally, five proteins all capable of inducing a functional antibody response vs N. meningitidis strain CU385 were identified as new attractive vaccine candidates: NMB0606 a potential YajC orthologue, NMB0928 the neisserial NlpB (BamC), NMB0873 a LolB orthologue, NMB1163 a protein belonging to a curli-like assembly machinery, and NMB0938 (a neisserial specific antigen) with evidence of positive selection appreciated for NMB0928. The new set of vaccine candidates and the novel proposed functions will open a new wave of research in the search for the elusive neisserial vaccine.

  13. Circulating hepatitis B surface antigen particles carry hepatocellular microRNAs.

    Science.gov (United States)

    Novellino, Luisa; Rossi, Riccardo L; Bonino, Ferruccio; Cavallone, Daniela; Abrignani, Sergio; Pagani, Massimiliano; Brunetto, Maurizia R

    2012-01-01

    Hepatitis B virus (HBV) produces high quantities of subviral surface antigen particles (HBsAg) which circulate in the blood outnumbering virions of about 1\\10(3-6) times. In individuals coinfected with the defective hepatitis Delta virus (HDV) the small HDV-RNA-genome and Delta antigen circulate as ribonucleoprotein complexes within HBsAg subviral particles. We addressed the question whether subviral HBsAg particles may carry in the same way cellular microRNAs (miRNAs) which are released into the bloodstream within different subcellular forms such as exosomes and microvescicles. Circulating HBsAg particles were isolated from sera of 11 HBsAg carriers by selective immunoprecipitation with monoclonal anti-HBs-IgG, total RNA was extracted and human miRNAs were screened by TaqMan real-time quantitative PCR Arrays. Thirty-nine human miRNAs were found to be significantly associated with the immunoprecipitated HBsAg, as determined by both comparative DDCT analysis and non-parametric tests (Mann-Whitney, p<0.05) with respect to controls. Moreover immunoprecipitated HBsAg particles contained Ago2 protein that could be revealed in ELISA only after 0.5% NP40. HBsAg associated miRNAs were liver-specific (most frequent = miR-27a, miR-30b, miR-122, miR-126 and miR-145) as well as immune regulatory (most frequent = miR-106b and miR-223). Computationally predicted target genes of HBsAg-associated miRNAs highlighted molecular pathways dealing with host-pathogen. The finding that HBsAg particles carry selective pools of hepatocellular miRNAs opens new avenues of research to disentangle the complex interactions between host and HBV and provides a non invasive tool to study the physiopathology of liver epigenetics.

  14. Immunochemical Investigations of Cell Surface Antigens of Anaerobic Bacteria.

    Science.gov (United States)

    1976-01-15

    Peptococcus micros 5.8 Fusobacterium varium 5.2 Clostridium sartagoformum 5.2 Clostridium tyrobutyricum 5.0 Bacteriological studies were also performed on...4.5 Non-sooreforming Gram- Corynebacterium sp. 4.4 positive rod 6.3 c-hemolytic streptococcus 4.0 Clostridium perfringens 6.1 Proteus mirabilis 4.0... Clostridium paraputrificum 6.0 Proteus morganii 3.9 Clostridium species 6.0 Moraxella sp. 3.1 Bacteroides fragilis 5.8 Peptococcus morbillorum 5.7

  15. Inflammatory environment and oxidized LDL convert circulating human proangiogenic cells into functional antigen-presenting cells.

    Science.gov (United States)

    Vinci, Maria Cristina; Piacentini, Luca; Chiesa, Mattia; Saporiti, Federica; Colombo, Gualtiero I; Pesce, Maurizio

    2015-09-01

    The function of human circulating PACs has been described extensively. However, little focus has been placed on understanding how these cells differ in their functions in the presence of microenvironments mimicking vascular inflammation. We hypothesized that exposure to proinflammatory cytokines or the oxLDL, an autoantigen abundant in advanced atherosclerotic plaques, converts PACs into immune-modulating/proinflammatory cells. Hence, we examined the effect of oxLDL and inflammatory stimuli on their phenotype by use of a functional genomics model based on secretome and whole genome transcriptome profiling. PACs obtained from culturing a PBMC fraction in angiogenic medium were primed with DC differentiation cytokines and then exposed to proinflammatory cytokines or oxLDL. Under these conditions, PACs converted into APCs, expressed maturation markers CD80 and CD83, and showed an increased up-regulation of CD86. APCcy and APCox induced a robust T cell BrdU incorporation. Despite a similar ability to induce lymphocyte proliferation, APCcy and APCox differed for the secretory pathway and mRNA expression. Analysis of the differentially expressed genes identified 4 gene "clusters," showing reciprocal modulation in APCcy vs. APCox, justifying, according to functional genomics analyses, a different putative function of the cells in antigen processing. Together, these data show that treatment with inflammatory cytokines or oxLDL converts human PAC phenotypes and functions into that of APCs with similar lymphocyte-activating ability but distinct maturation degree and paracrine functions.

  16. Immunological evaluation of colonic delivered Hepatitis B surface antigen loaded TLR-4 agonist modified solid fat nanoparticles.

    Science.gov (United States)

    Sahu, Kantrol Kumar; Pandey, Ravi Shankar

    2016-10-01

    Hepatitis B is one of the leading liver diseases and remains a major global health problem. Currently available vaccines provide protection but often results in weaker/minimal mucosal immunity. Thus the present study is devoted to the development and in-vivo exploration of the colonically delivered biomimetic nanoparticles which capably enhance humoral as well as cellular immune response. In present work, Hepatitis B surface