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Sample records for cell nuclear antigen

  1. Proliferating cell nuclear antigen in neutrophil fate.

    Science.gov (United States)

    Witko-Sarsat, Véronique; Ohayon, Delphine

    2016-09-01

    The life span of a neutrophil is a tightly regulated process as extended survival is beneficial for pathogen elimination and cell death necessary to prevent cytotoxic content release from activated neutrophils at the inflammatory site. Therefore, the control between survival and death must be a dynamic process. We have previously described that proliferating cell nuclear antigen (PCNA) which is known as a nuclear protein pivotal in DNA synthesis, is a key element in controlling neutrophil survival through its association with procaspases. Contrary to the dogma which asserted that PCNA has a strictly nuclear function, in mature neutrophils, PCNA is present exclusively within the cytosol due to its nuclear export at the end of the granulocytic differentiation. More recent studies are consistent with the notion that the cytosolic scaffold of PCNA is aimed at modulating neutrophil fate rather than simply preventing death. Ultimately, targeting neutrophil survival might have important applications not just in the field of immunology and inflammation, but also in hematology and transfusion. The neutrophil emerges as a unique and powerful cellular model to unravel the basic mechanisms governing the cell cycle-independent functions of PCNA and should be considered as a leader of the pack. PMID:27558345

  2. 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.

  3. Nuclear localization of Merkel cell polyomavirus large T antigen in Merkel cell carcinoma

    International Nuclear Information System (INIS)

    To clarify whether mutations in the large T gene encoded by Merkel cell polyomavirus affect the expression and function of large T antigen in Merkel cell carcinoma cases, we investigated the expression of large T antigen in vitro and in vivo. Immunohistochemistry using a rabbit polyclonal antibody revealed that large T antigen was expressed in the nuclei of Merkel cell carcinoma cells with Merkel cell polyomavirus infection. Deletion mutant analyses identified an Arg-Lys-Arg-Lys sequence (amino acids 277-280) as a nuclear localization signal in large T antigen. Sequence analyses revealed that there were no mutations in the nuclear localization signal in any of the eleven Merkel cell polyomavirus strains examined. Furthermore, stop codons were not observed in the upstream of the nuclear localization signal in any of the Merkel cell carcinoma cases examined. These data suggest that the nuclear localization signal is highly conserved and functional in Merkel cell carcinoma cases.

  4. Changes in distribution of nuclear matrix antigens during the mitotic cell cycle.

    Science.gov (United States)

    Chaly, N; Bladon, T; Setterfield, G; Little, J E; Kaplan, J G; Brown, D L

    1984-08-01

    We examined the distribution of nonlamin nuclear matrix antigens during the mitotic cell cycle in mouse 3T3 fibroblasts. Four monoclonal antibodies produced against isolated nuclear matrices were used to characterize antigens by the immunoblotting of isolated nuclear matrix preparations, and were used to localize the antigens by indirect immunofluorescence. For comparison, lamins and histones were localized using human autoimmune antibodies. At interphase, the monoclonal antibodies recognized non-nucleolar and nonheterochromatin nuclear components. Antibody P1 stained the nuclear periphery homogeneously, with some small invaginations toward the interior of the nucleus. Antibody I1 detected an antigen distributed as fine granules throughout the nuclear interior. Monoclonals PI1 and PI2 stained both the nuclear periphery and interior, with some characteristic differences. During mitosis, P1 and I1 were chromosome-associated, whereas PI1 and PI2 dispersed in the cytoplasm. Antibody P1 heavily stained the periphery of the chromosome mass, and we suggest that the antigen may play a role in maintaining interphase and mitotic chromosome order. With antibody I1, bright granules were distributed along the chromosomes and there was also some diffuse internal staining. The antigen to I1 may be involved in chromatin/chromosome higher-order organization throughout the cell cycle. Antibodies PI1 and PI2 were redistributed independently during prophase, and dispersed into the cytoplasm during prometaphase. Antibody PI2 also detected antigen associated with the spindle poles. PMID:6378926

  5. Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells.

    OpenAIRE

    Zuber, M; Tan, E M; Ryoji, M

    1989-01-01

    Proliferating cell nuclear antigen (PCNA) (also called cyclin) is known to stimulate the activity of DNA polymerase delta but not the other DNA polymerases in vitro. We injected a human autoimmune antibody against PCNA into unfertilized eggs of Xenopus laevis and examined the effects of this antibody on the replication of injected plasmid DNA as well as egg chromosomes. The anti-PCNA antibody inhibited plasmid replication by up to 67%, demonstrating that PCNA is involved in plasmid replicatio...

  6. Proliferating cell nuclear antigen: a marker for hepatocellular proliferation in rodents.

    OpenAIRE

    Eldrige, S R; Butterworth, B E; Goldsworthy, T L

    1993-01-01

    Two different markers for quantitating cell proliferation were evaluated in livers of control and chemically treated mice and rats. Proliferating cell nuclear antigen (PCNA), an endogenous cell replication marker, and bromodeoxyuridine (BrdU), an exogenously administered DNA precursor label, were detected in formalin-fixed, paraffin-embedded tissues using immunohistochemical techniques. The percentage of cells in S phase (labeling indexes, LI) evaluated as PCNA- or BrdU-positive hepatocellula...

  7. Dietary influences over proliferating cell nuclear antigen expression in the locust midgut

    OpenAIRE

    Zudaire, E. (Enrique); Simpson, S J; Illa, I.; Montuenga, L M

    2004-01-01

    We have studied the influence of variations in dietary protein (P) and digestible carbohydrate (C), the quantity of food eaten, and insect age during the fifth instar on the expression of the proliferating cell nuclear antigen (PCNA) in the epithelial cells of the midgut (with special reference to the midgut caeca) in the African migratory locust, Locusta migratoria. Densitometric analysis of PCNA-immunostained cells was used as an indirect measure of the levels of expression of PCNA, and a P...

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

    International Nuclear Information System (INIS)

    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

  9. 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.

  10. Abnormal expressions of proliferating cell nuclear antigen and P27 protein in brain glioma

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    BACKGROUND: Both proliferating cell nuclear antigen and P27 protein are important factors to regulate cell cycle. While, the combination of them can provide exactly objective markers to evaluate prognosis of patients with brain glioma needs to be further studied based on pathological level.OBJECTIVE: To observe the expressions of proliferating cell nuclear antigen and P27 protein in both injured and normal brain glioma tissues and analyze the effect of them on onset and development of brain glioma.DESIGN: Case contrast observation.SETTING: Department of Neurosurgery, the Second Affiliated Hospital of Xi'an Jiaotong University.PARTICIPANTS: A total of 63 patients with brain glioma were selected from Department of Neurosurgery,the Second Affiliated Hospital of Xi'an Jiaotong University from July 1996 to June 2000. There were 38 males and 25 females and their ages ranged from 23 to 71 years. Based on pathological classification and grading standards of brain glioma, patients were divided into grade Ⅰ - tⅡ (n =30) and grade Ⅲ - Ⅳ (n =33). All cases received one operation but no radiotherapy and chemiotherapy before operation. Sample tissues were collected from tumor parenchyma. Non-neoplastic brain tissues were collected from another 12 non-tumor subjects who received craniocerebral trauma infra-decompression and regarded as the control group. There were 10 males and 2 females and their ages ranged from 16 to 54 years. The experiment had got confirmed consent from local ethic committee and the collection was provided confirmed consent from patients and their relatives. All samples were restained with HE staining so as to diagnose as the brain glioma.While, all patients with brain glioma received radiotherapy after operation and their survival periods were followed up.METHODS: Primary lesion wax of brain glioma was cut into serial sections and stained with S-P immunohistochemical staining. Brown substance which was observed in tumor nucleus was regarded as the

  11. Crystallization and X-ray diffraction studies of crustacean proliferating cell nuclear antigen

    International Nuclear Information System (INIS)

    Proliferating cell nuclear antigen from Litopenaeus vannamei was recombinantly expressed, purified and crystallized. Diffraction data were obtained and processed to 3 Å. Proliferating cell nuclear antigen (PCNA), a member of the sliding clamp family of proteins, interacts specifically with DNA replication and repair proteins through a small peptide motif called the PCNA-interacting protein or PIP box. PCNA is recognized as one of the key proteins involved in DNA metabolism. In the present study, the recombinant PCNA from Litopenaeus vannamei (LvPCNA) was heterologously overexpressed and purified using metal ion-affinity chromatography. Crystals suitable for diffraction grew overnight using the hanging-drop vapour-diffusion method. LvPCNA crystals belong to space group C2 with unit-cell parameters a = 144.6, b = 83.4, c = 74.3 Å, β = 117.6°. One data set was processed to 3 Å resolution, with an overall Rmeas of 0.09 and a completeness of 93.3%. Initial phases were obtained by molecular replacement using a homology model of LvPCNA as the search model. Refinement and structural analysis are underway. This report is the first successful crystallographic analysis of a marine crustacean decapod shrimp (L. vannamei) proliferating cell nuclear antigen

  12. Architecture of the DNA polymerase B-proliferating cell nuclear antigen (PCNA)-DNA ternary complex

    OpenAIRE

    Mayanagi, Kouta; Kiyonari, Shinichi; Nishida, Hirokazu; Saito, Mihoko; Kohda, Daisuke; Ishino, Yoshizumi; Shirai, Tsuyoshi; Morikawa, Kosuke

    2011-01-01

    DNA replication in archaea and eukaryotes is executed by family B DNA polymerases, which exhibit full activity when complexed with the DNA clamp, proliferating cell nuclear antigen (PCNA). This replication enzyme consists of the polymerase and exonuclease moieties responsible for DNA synthesis and editing (proofreading), respectively. Because of the editing activity, this enzyme ensures the high fidelity of DNA replication. However, it remains unclear how the PCNA-complexed enzyme temporally ...

  13. Targeting cytosolic proliferating cell nuclear antigen (PCNA in neutrophil-dominated inflammation

    Directory of Open Access Journals (Sweden)

    Alessia eDe Chiara

    2012-10-01

    Full Text Available New therapeutic approaches that can accelerate neutrophil apoptosis under inflammatory conditions to enhance the resolution of inflammation are now under study. Neutrophils are deprived of proliferative capacity and have a tightly controlled lifespan to avoid their persistence at the site of injury. We have recently described that the proliferating cell nuclear antigen (PCNA, a nuclear factor involved in DNA replication and repair of proliferating cells is a key regulator of neutrophil survival. In this review, we will try to put into perspective the physiologic relevance of PCNA in neutrophils. We will discuss key issues such as molecular structure, post-translational modifications, based on our knowledge of nuclear PCNA, assuming that similar principles governing its function are conserved between nuclear and cytosolic PCNA. The example of cystic fibrosis that features one of the most intense neutrophil-dominated pulmonary inflammation will be discussed. We believe that through an intimate comprehension of the cytosolic PCNA scaffold based on nuclear PCNA knowledge, novel pathways regulating neutrophil survival can be unraveled and innovative agents can be developed to dampen inflammation where it proves detrimental.

  14. Dynamics of beta and proliferating cell nuclear antigen sliding clamps in traversing DNA secondary structure.

    Science.gov (United States)

    Yao, N; Hurwitz, J; O'Donnell, M

    2000-01-14

    Chromosomal replicases of cellular organisms utilize a ring shaped protein that encircles DNA as a mobile tether for high processivity in DNA synthesis. These "sliding clamps" have sufficiently large linear diameters to encircle duplex DNA and are perhaps even large enough to slide over certain DNA secondary structural elements. This report examines the Escherichia coli beta and human proliferating cell nuclear antigen clamps for their ability to slide over various DNA secondary structures. The results show that these clamps are capable of traversing a 13-nucleotide ssDNA loop, a 4-base pair stem-loop, a 4-nucleotide 5' tail, and a 15-mer bubble within the duplex. However, upon increasing the size of these structures (20-nucleotide loop, 12-base pair stem-loop, 28-nucleotide 5' tail, and 20-nucleotide bubble) the sliding motion of the beta and proliferating cell nuclear antigen over these elements is halted. Studies of the E. coli replicase, DNA polymerase III holoenzyme, in chain elongation with the beta clamp demonstrate that upon encounter with an oligonucleotide annealed in its path, it traverses the duplex and resumes synthesis on the 3' terminus of the oligonucleotide. This sliding and resumption of synthesis occurs even when the oligonucleotide contains a secondary structure element, provided the beta clamp can traverse the structure. However, upon encounter with a downstream oligonucleotide containing a large internal secondary structure, the holoenzyme clears the obstacle by strand displacing the oligonucleotide from the template. Implications of these protein dynamics to DNA transactions are discussed. PMID:10625694

  15. Cell proliferation-associated nuclear antigen defined by antibody Ki-67: a new kind of cell cycle-maintaining proteins

    International Nuclear Information System (INIS)

    A decade of studies on the human nuclear antigen defined by monoclonal antibody Ki-67 (the 'Ki-67 proteins') has made it abundantly clear that this structure is strictly associated with human cell proliferation and the expression of this protein can be used to access the growth fraction of a given cell population. Until recently the Ki-67 protein was described as a nonhistone protein that is highly susceptible to protease treatment. We have isolated and sequenced cDNAs encoding for this antigen and found two isoforms of the full length cDNA of 11.5 and 12.5 kb, respectively, sequence and structure of which are thus far unique. The gene encoding the Ki-67 protein is organized in 15 exons and is localized on chromosome 10. The center of this gene is formed by an extraordinary 6845 bp exon containing 16 successively repeated homologous segments of 366 bp ('Ki-67 repeats'), each containing a highly conserved new motif of 66 bp ('Ki-67 motif'). The deduced peptide sequence of this central exon possesses 10 ProGluSerThr (PEST) motifs which are associated with high turnover proteins such as other cell cycle-related proteins, oncogenes and transcription factors, etc. Like the latter proteins the Ki-67 antigen plays a pivotal role in maintaining cell proliferation because Ki-67 protein antisense oligonucleotides significantly inhibit 3H-thymidine incorporation in permanent human tumor cell lines in a dose-dependent manner. (author). 30 refs, 2 figs

  16. 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...... analyses. The correlation of PCNA and Ki-67 labeling index (LI) with prognosis were studied, and the correlation of LI with clinical data was evaluated. Results. The PCNA LI had a range of 0-17% (mean, 5.27%; standard deviation [SD] = 4.65), and the Ki-67 LI had a range of 0-29% (mean, 4.19%; SD = 5.......66). In general, the PCNA LI seemed to be higher than the Ki-67 LI. The mean survival time was 4.4 years, and 5- and 10-year survival rates were 38% and 19%, respectively. Ki-67 and PCNA staining indicated that patients with a high LI (>3% and >4%, respectively) had a significantly higher mortality, with...

  17. EXPRESSION OF P53 PROTEIN AND PROLIFERATING CELL NUCLEAR ANTIGEN IN HUMAN GESTATION TROPHOBLASTIC DISEASE

    Institute of Scientific and Technical Information of China (English)

    黄铁军; 王志忠; 方光光; 刘志恒

    2004-01-01

    Objective: To study the relationship between p53 protein, proliferating cell nuclear antigen (PCNA) expression and benign or malignant gestational trophoblastic disease (MGTD). Methods: The histotomic sections of 48 patients with gestational trophoblastic disease and 24 patients of normal chorionic villi were stained using immunohistochemistry. The monoclonal antibodies were used to determine p53 protein and PCNA. Results: The frequency of p53 and PCNA positive expression were significantly different among the chorionic villi of normal pregnancy, hydratidiform mole (HM) and MGTD. But neither p53 nor PCNA has any relation with the clinical staging or metastasis of MGTD. Conclusion: Both P53 and PCNA are valuable in diagnosis of human gestational trophoblastic disease.

  18. Phenobarbital-induced hepatocellular proliferation: anti-bromodeoxyuridine and anti-proliferating cell nuclear antigen immunocytochemistry.

    Science.gov (United States)

    Jones, H B; Clarke, N A; Barrass, N C

    1993-01-01

    We report modifications to immunocytochemical detection procedures for proliferating cell nuclear antigen (PCNA) which permit its identification in liver samples previously fixed for BrdU immunocytochemistry. Both methods have been used for the assessment of phenobarbital-induced cell proliferation in rat liver. The difficulties associated with the hitherto unsuccessful application of PCNA immunocytochemical methods to tissues fixed in formalin for BrdU visualization were overcome by epitope unmasking with acid hydrolysis, extension of primary antiserum (PC10) incubation, and employment of streptavidin-ABC-HRP. BrdU delivery via osmotic minipumps for 48 hr before euthanasia, followed by fixation in cold formalin for 14 days, yielded reliable and reproducible hepatocellular labeling and a peak of cell proliferation in all lobes on Day 3 (i.e., labeling during Days 1-3) of dosing with 80 mg/kg/day phenobarbital. Labeling indices (LI) of both control and phenobarbital-treated liver were lower in the left and right median lobes as compared with the lateral lobes. In sections of the left lateral lobe from the same liver, PCNA immunocytochemistry revealed a peak of proliferative activity (about one third of the maximum LI generated by BrdU incorporation) on Day 1. These findings, together with the advantages and disadvantages of both techniques, are discussed in the context of their applications to different investigative requirements. PMID:8093255

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-02-19

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

  20. Human NTH1 physically interacts with p53 and proliferating cell nuclear antigen

    International Nuclear Information System (INIS)

    Thymine glycol (Tg) is one of predominant oxidative DNA lesions caused by ionizing radiation and other oxidative stresses. Human NTH1 is a bifunctional enzyme with DNA glycosylase and AP lyase activities and removes Tg as the first step of base excision repair (BER). We have searched for the factors interacting with NTH1 by using a pull-down assay and found that GST-NTH1 fusion protein precipitates proliferating cell nuclear antigen (PCNA) and p53 as well as XPG from human cell-free extracts. GST-NTH1 also bound to recombinant FLAG-tagged XPG, PCNA, and (His)6-tagged p53 proteins, indicating direct protein-protein interaction between those proteins. Furthermore, His-p53 and FLAG-XPG, but not PCNA, stimulated the Tg DNA glycosylase/AP lyase activity of GST-NTH1 or NTH1. These results provide an insight into the positive regulation of BER reaction and also suggest a possible linkage between BER of Tg and other cellular mechanisms

  1. Epidermal growth factor receptor and proliferating cell nuclear antigen in astrocytomas

    Directory of Open Access Journals (Sweden)

    Maiti Arpan

    2008-01-01

    Full Text Available Aims: The involvement of various growth factors, growth factor receptors and proliferative markers in the molecular pathogenesis of astrocytic neoplasms are being studied extensively. Epidermal Growth Factor Receptor (EGFR gene overexpression occurs in nearly 50% of cases of glioblastoma. Since EGFR and proliferating cell nuclear antigen (PCNA are involved in mitogenic signal transduction and cellular proliferation pathway, we have studied the correlation between the expression of EGFR and PCNA labeling index in astrocytic tumors. Materials and Methods: We investigated the immunohistochemical expression of EGFR and PCNA using the appropriate monoclonal antibodies in 40 cases of astrocytic tumors of which 21 cases were glioblastoma, eight cases were Grade III or anaplastic astrocytomas and six cases were Grade II or diffuse astrocytomas and five cases were Grade I or pilocytic astrocytomas. Results: Both the EGFR expression and PCNA labeling index increase with increasing grades of astrocytomas with a significantly high percentage of cells showing positive staining for both EGFR and PCNA in GBM and Grade III astrocytomas compared to Grade II astrocytomas. The expression levels of both EGFR and PCNA were low in Grade I or pilocytic astrocytomas. Conclusions: A significant correlation was found between EGFR overexpression and PCNA labeling index in Grade III and Grade II astrocytomas and glioblastoma. These suggest that the tumor proliferation, at least in higher grades of astrocytomas is dependent in some measure on EGF and EGFR-related signaling pathways.

  2. A Peptide Mimicking a Region in Proliferating Cell Nuclear Antigen Specific to Key Protein Interactions Is Cytotoxic to Breast Cancer

    OpenAIRE

    Smith, Shanna J.; Gu, Long; Phipps, Elizabeth A.; Lacey E Dobrolecki; Mabrey, Karla S.; Gulley, Pattie; Dillehay, Kelsey L; Dong, Zhongyun; Fields, Gregg B.; Chen, Yun-Ru; Ann, David; Hickey, Robert J.; Malkas, Linda H.

    2015-01-01

    Proliferating cell nuclear antigen (PCNA) is a highly conserved protein necessary for proper component loading during the DNA replication and repair process. Proteins make a connection within the interdomain connector loop of PCNA, and much of the regulation is a result of the inherent competition for this docking site. If this target region of PCNA is modified, the DNA replication and repair process in cancer cells is potentially altered. Exploitation of this cancer-associated region has imp...

  3. Proliferating cell nuclear antigen, p53 and micro vessel density: Grade II vs. Grade III astrocytoma

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    Malhan Priya

    2010-01-01

    Full Text Available Histological classification and grading are prime procedures in the management of patients with astrocytoma, providing vital data for therapeutic decision making and prognostication. However, it has limitations in assessing biological tumor behavior. This can be overcome by using newer immunohistochemical techniques. This study was carried out to compare proliferative indices using proliferating cell nuclear antigen (PCNA, extent of p53 expression and micro vessel morphometric parameters in patients with low grade and anaplastic astrocytoma. Twenty-five patients, each of grade II and grade III astrocytoma were evaluated using monoclonal antibodies to PCNA, p53 protein and factor VIII related antigen. PCNA, p53-labeling indices were calculated along with micro vessel morphometric analysis using Biovis Image plus Software. Patients with grade III astrocytoma had higher PCNA and p53 labeling indices as compared with grade II astrocytoma (29.14 plus/minus 9.87% vs. 16.84 plus/minus 6.57%, p 0.001; 18.18 plus/minus 6.14% vs. 6.14 plus/minus 7.23%, p 0.001, respectively. Micro vessel percentage area of patients with grade III astrocytoma was also (4.26 plus/minus 3.70 vs. 1.05 plus/minus 0.56, p 0.001, higher along with other micro vessel morphometric parameters. Discordance between histology and one or more IHC parameters was seen in 5/25 (20% of patients with grade III astrocytoma and 9/25 (36% of patients with grade II disease. PCNA and p53 labeling indices were positively correlated with Pearson′s correlation, p less than 0.001 for both. Increased proliferative fraction, genetic alterations and neovascularization mark biological aggressiveness in astrocytoma. Immunohistochemical evaluation scores over meet the challenge of accurate prognostication of this potentially fatal malignancy.

  4. Immunochemical expression of proliferative cell nuclear antigen in aging cultured astrocytes

    Directory of Open Access Journals (Sweden)

    M. C. Vanzani

    2003-08-01

    Full Text Available Cell differentiation degree and mitotic activity were sequentially assessed by immunoperoxidase labeling of glial fibrillary acidic protein (GFAP and proliferative cell nuclear antigen (PCNA, respectively, in rat brain cultured astrocytes maintained up to 60 days in vitro (DIV of first subculture, or weekly passaged until their 12th subculture. Cell count was performed through a 0.01 mm2 section reticule and morphometric analysis with a stereological grid. The number of double immunoreactive cells peaked by 2 DIV to achieve its lowest value at 60 DIV. At 24 hs of cell seeding of successive passages, such values peaked by the 6th subculture to gradually decrease thereafter. Increasing cell hypertrophy was found during the long-term first subculture but not after passaging. At the end of the observation period, doubly immunolabeled astrocytes were still recorded, thus evidencing retention of proliferative potential despite aging.El grado de diferenciación celular y la actividad mitótica fueron secuencialmente determinados mediante marcación por inmunoperoxidasa de la proteína gliofibrilar ácida (GFAP y del antígeno nuclear de proliferación celular (PCNA, respectivamente, en cultivos astrocitarios obtenidos de encéfalo de rata y mantenidos hasta 60 días in vitro (DIV de su primer subcultivo, o mediante pasajes semanales hasta el 12do subcultivo. El conteo celular se realizó mediante una retícula de 0.01-mm2 de sección y el análisis morfométrico con una grilla estereológica. El número de células doblemente inmunorreactivas alcanzó valores máximos a los 2 DIV para descender a los menores a los 60 DIV. A las 24 hs de sembrado celular de los sucesivos pasajes, esos valores ascendieron hacia el 6to subcultivo para luego declinar. En cuanto a la hipertrofia celular, se observó en todo el curso del primer subcultivo, pero no durante los posteriores pasajes. Al final del período de observación, todavía se continuaban detectando

  5. Growth inhibiting effects of antisense eukaryotic expression vector of proliferating cell nuclear antigen gene on human bladder cancer cells

    Institute of Scientific and Technical Information of China (English)

    童强松; 曾甫清; 林晨; 赵军; 鲁功成

    2003-01-01

    Objective To explore the growth inhibiting effects on human bladder cancer by antisense RNA targeting the proliferating cell nuclear antigen (PCNA) gene. Methods The eukaryotic expression vector for antisense PCNA cDNA was constructed and transferred into a bladder cancer EJ cell line. The PCNA expression in the cancer cells was detected by RT-PCR and Western blotting assays. The in vitro proliferation activities of the transferred cells were observed by growth curve, tetrazolium bromide (MTT) colorimetry, tritiated thymidine (3H-TdR)incorporation, flow cytometry and clone formation testing, while its in vivo anti-tumor effects were detected on nude mice allograft models.Results After the antisense vector, pLAPSN, was transferred, cellular PCNA expression was inhibited at both protein and mRNA levels. The growth rates of EJ cells were reduced from 27.91% to 62.07% (P<0.01), with an inhibition of DNA synthesis rate by 52.31% (P<0.01). Transferred cells were blocked at G0/G1 phases in cell-cycle assay, with the clone formation ability decreased by 50.81% (P<0.01). The in vivo carcinogenic abilities of the transferred cancer cells were decreased by 54.23% (P<0.05). Conclusions Antisense PCNA gene transfer could inhibit the growth of bladder cancer cells in vitro and in vivo, which provided an ideal strategy for gene therapy of human cancers.

  6. Tight coevolution of proliferating cell nuclear antigen (PCNA)-partner interaction networks in fungi leads to interspecies network incompatibility.

    OpenAIRE

    Zamir, L.; Zaretsky, M.; Fridman, Y; Ner-Gaon, H.; Rubin, E; Aharoni, A.

    2012-01-01

    The structure and connectivity of protein-protein interaction (PPI) networks are maintained throughout evolution by coordinated changes (coevolution) of network proteins. Despite extensive research, relatively little is known regarding the molecular basis and functional implications of the coevolution of PPI networks. Here, we used proliferating cell nuclear antigen, a hub protein that mediates DNA replication and repair in eukaryotes, as a model system to study the coevolution of PPI network...

  7. Interaction of Proliferating Cell Nuclear Antigen With DNA at the Single Molecule Level

    KAUST Repository

    Raducanu, Vlad-Stefan

    2016-05-01

    Proliferating cell nuclear antigen (PCNA) is a key factor involved in Eukaryotic DNA replication and repair, as well as other cellular pathways. Its importance comes mainly from two aspects: the large numbers of interacting partners and the mechanism of facilitated diffusion along the DNA. The large numbers of interacting partners makes PCNA a necessary factor to consider when studying DNA replication, either in vitro or in vivo. The mechanism of facilitated diffusion along the DNA, i.e. sliding along the duplex, reduces the six degrees of freedom of the molecule, three degrees of freedom of translation and three degrees of freedom of rotation, to only two, translation along the duplex and rotational tracking of the helix. Through this mechanism PCNA can recruit its partner proteins and localize them to the right spot on the DNA, maybe in the right spatial orientation, more effectively and in coordination with other proteins. Passive loading of the closed PCNA ring on the DNA without free ends is a topologically forbidden process. Replication factor C (RFC) uses energy of ATP hydrolysis to mechanically open the PCNA ring and load it on the dsDNA. The first half of the introduction gives overview of PCNA and RFC and the loading mechanism of PCNA on dsDNA. The second half is dedicated to a diffusion model and to an algorithm for analyzing PCNA sliding. PCNA and RFC were successfully purified, simulations and a mean squared displacement analysis algorithm were run and showed good stability and experimental PCNA sliding data was analyzed and led to parameters similar to the ones in literature.

  8. Myeloid Cell Nuclear Differentiation Antigen (MNDA) Expression Distinguishes Extramedullary Presentations of Myeloid Leukemia From Blastic Plasmacytoid Dendritic Cell Neoplasm.

    Science.gov (United States)

    Johnson, Ryan C; Kim, Jinah; Natkunam, Yasodha; Sundram, Uma; Freud, Aharon G; Gammon, Bryan; Cascio, Michael J

    2016-04-01

    Myeloid neoplasms constitute one of the most common malignancies in adults. In most cases these proliferations initially manifest in the blood and marrow; however, extramedullary involvement may precede blood or marrow involvement in a subset of cases, making a definitive diagnosis challenging by morphologic and immunohistochemical assessment alone. Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive entity that frequently presents in extramedullary sites and can show morphologic and immunophenotypic overlap with myeloid neoplasms. Given that BPDCN and myeloid neoplasms may both initially present in extramedullary sites and that novel targeted therapies may be developed that exploit the unique molecular signature of BPDCN, new immunophenotypic markers that can reliably separate myeloid neoplasms from BPDCN are desirable. We evaluated the utility of myeloid cell nuclear differentiation antigen (MNDA) expression in a series of extramedullary myeloid leukemias (EMLs) and BPDCN. Forty biopsies containing EML and 19 biopsies containing BPDCN were studied by MNDA immunohistochemistry. The majority of myeloid neoplasms showed nuclear expression of MNDA (65%). In contrast, all cases of BPDCN lacked MNDA expression. These findings show that MNDA is expressed in the majority of EMLs and support the inclusion of MNDA immunohistochemistry in the diagnostic evaluation of blastic hematopoietic infiltrates, particularly when the differential diagnosis is between myeloid leukemia and BPDCN. PMID:26796502

  9. Low power laser irradiation stimulates cell proliferation via proliferating cell nuclear antigen and Ki-67 expression during tissue repair

    Science.gov (United States)

    Prabhu, Vijendra; Rao, Bola Sadashiva Satish; Mahato, Krishna Kishore

    2015-03-01

    Low power laser irradiation (LPLI) is becoming an increasingly popular and fast growing therapeutic modality in dermatology to treat various ailments without any reported side effects. In the present study an attempt was made to investigate the proliferative potential of red laser light during tissue repair in Swiss albino mice. To this end, full thickness excisional wounds of diameter 15 mm created on mice were exposed to single dose of Helium-Neon laser (632.8 nm; 7 mW; 4.02 mWcm-2; Linear polarization) at 2 Jcm-2 and 10 Jcm-2 along with un-illuminated controls. The granulation tissues from all the respective experimental groups were harvested on day 10 post-wounding following euthanization. Subsequently, tissue regeneration potential of these laser doses under study were evaluated by monitoring proliferating cell nuclear antigen and Ki-67 following the laser treatment and comparing it with the un-illuminated controls. The percentages of Ki-67 or PCNA positive cells were determined by counting positive nuclei (Ki-67/PCNA) and total nuclei in five random fields per tissue sections. Animal wounds treated with single exposure of the 2 Jcm-2 indicated significant elevation in PCNA (Ptested experimental groups as evidenced by the microscopy results in the study. In summary, the findings of the present study have clearly demonstrated the regulation of cell proliferation by LPLI via PCNA and Ki-67 expression during tissue regeneration.

  10. Low power laser irradiation stimulates cell proliferation via proliferating cell nuclear antigen and Ki-67 expression during tissue repair

    Science.gov (United States)

    Prabhu, Vijendra; Rao, Bola Sadashiva Satish; Mahato, Krishna Kishore

    2015-03-01

    Low power laser irradiation (LPLI) is becoming an increasingly popular and fast growing therapeutic modality in dermatology to treat various ailments without any reported side effects. In the present study an attempt was made to investigate the proliferative potential of red laser light during tissue repair in Swiss albino mice. To this end, full thickness excisional wounds of diameter 15 mm created on mice were exposed to single dose of Helium-Neon laser (632.8 nm; 7 mW; 4.02 mWcm-2; Linear polarization) at 2 Jcm-2 and 10 Jcm-2 along with un-illuminated controls. The granulation tissues from all the respective experimental groups were harvested on day 10 post-wounding following euthanization. Subsequently, tissue regeneration potential of these laser doses under study were evaluated by monitoring proliferating cell nuclear antigen and Ki-67 following the laser treatment and comparing it with the un-illuminated controls. The percentages of Ki-67 or PCNA positive cells were determined by counting positive nuclei (Ki-67/PCNA) and total nuclei in five random fields per tissue sections. Animal wounds treated with single exposure of the 2 Jcm-2 indicated significant elevation in PCNA (PJcm-2) expression as compared to other tested experimental groups as evidenced by the microscopy results in the study. In summary, the findings of the present study have clearly demonstrated the regulation of cell proliferation by LPLI via PCNA and Ki-67 expression during tissue regeneration.

  11. An Egr-1-specific DNAzyme regulates Egr-1 and proliferating cell nuclear antigen expression in rat vascular smooth muscle cells

    Science.gov (United States)

    ZHANG, JUNBIAO; GUO, CHANGLEI; WANG, RAN; HUANG, LULI; LIANG, WANQIAN; LIU, RUNNAN; SUN, BING

    2013-01-01

    The aim of the present study was to transfect rat aortic smooth muscle cells with an early growth response factor-1 (Egr-1)-specific DNAzyme (ED5), to observe its effect on Egr-1 and proliferating cell nuclear antigen (PCNA) expression and to elucidate the mechanism of ED5-mediated inhibition of vascular smooth muscle cell (VSMC) proliferation. VSMCs in primary culture obtained by tissue block adhesion were identified by morphological observation and α smooth muscle actin (α-SM-actin) immunocytochemistry. The cells were then transfected with ED5 or scrambled ED5 (ED5SCR). The three groups of cells used in the present study were the control group, ED5 group and ED5SCR group. The expression levels of Egr-1 and PCNA protein were detected following transfection by analyzing and calculating the integral optical density value in each group. Primary culture of VSMCs and transfection of ED5 and ED5SCR were successfully accomplished. Following stimulation with 10% fetal calf serum, the Egr-1 protein was expressed most strongly at 1 h and demonstrated a declining trend over time; the expression of PCNA protein began at 4 h, peaked at 24 h and then demonstrated a slightly declining trend over time. Compared with the control group and the ED5SCR group, ED5 inhibited the expression of Egr-1 and PCNA (P<0.05). ED5 was able to inhibit the expression of Egr-1 and PCNA proteins in VSMCs to a certain extent and VSMC proliferation in vitro. DNAzyme gene therapy may be useful as a new method for treating vascular proliferative diseases, including atherosclerosis and restenosis. PMID:23737882

  12. Appearance of an inhibitory cell nuclear antigen in rat and human serum during variable degrees of hepatic regenerative activity

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    AIM To determine whether proliferating cell nuclear antigen (PCNA) is present in the peripheral circulation and whether PCNA levels correlate with enhanced regenerative activity.METHODS In animal studies, adult male Sprague-Dawley rats (n=3-4/ group) were sacrificed at 0, 12, 24, 36, 48, 72 and 96 hours following 70% partial hepatectomy. At each interval, sera were analyzed by Western blot for PCNA by two monoclonal antibodies (PC-10 and 19F-4). In human studies, sera from 4 patients with liver cirrhosis and 4 healthy controls were tested in a similar manner.RESULTS The PC-10 monoclonal antibody identified a protein with a molecular mass of 120 KD which remained stable in rat sera for 24 hours following partial hepatectomy, then increased 1.5-fold at 48 hours prior to returning to baseline at 96 hours after partial hepatectomy. However, it was not detected in the sera of patients with or without liver disease. In the 19F-4 monoclonal antibody, a protein with a molecular mass of approximately 46 KD was found. which was present in rat sera prior to partial hepatectomy and for 12 hours after surgery. Thereafter, levels fell by approximately 50% at 24 hours, 65% at 36 hours and 75% at 48 hours where they remained until 96 hours after partial hepatectomy. The decrease in levels correlated with the extent of partial hepatectomy. In human sera, the appearance of this inhibitory cell nuclear antigen (ICNA) was higher in the sera of patients with cirrhosis than in healthy controls.CONCLUSION The PC-10 monoclonal antibody can detect a protein in the circulation when active hepatic regenerative activity is taking place. The 19F-4 monoclonal antibody, however, identifies a protein in both rat and human sera that inversely correlates with hepatic regenerative activity. This protein which is tentatively referred to as inhibitory cell nuclear antigen (ICNA) may be used in documenting the extent of suppression of hepatic regeneration.

  13. Insulin, pioglitazone and Zingiber officinale administrations improve proliferating cell nuclear antigen immunostaining effects on diabetic and insulin resistant rat testis

    OpenAIRE

    DARAMOLA, Adetola Olubunmi; OLATUNJI-BELLO, Ibiyemi Ibitola; OBIKA, Leonard Fidelis

    2013-01-01

    This study accessed the effects of hypoglycaemic drugs on spermatogenesis in diabetic and insulin resistant rat testis following proliferating cell nuclear antigen (PCNA) immunostaining. Male adult Sprague-Dawley rats (120-140 g) were randomly divided into 5 groups. Group 1 served as control group; fed on normal rat pellets. Group 2 served as streptozotocin-insulin treated group; received a single dose IP Injection of streptozotocin 45 mg/kg BW in Na+ citrate buffer pH 4.5 and treated with in...

  14. Expression, purification and preliminary X-ray analysis of proliferating cell nuclear antigen from the archaeon Thermococcus thioreducens

    International Nuclear Information System (INIS)

    The proliferating cell nuclear antigen (PCNA) from a novel hyperthermophilic archaeon Thermococcus thioreducens has been crystallized, and diffraction data have been collected to 1.86 Å. Proliferating cell nuclear antigen (PCNA) is a DNA sliding clamp which confers processivity on replicative DNA polymerases. PCNA also acts as a sliding platform that enables the association of many DNA-processing proteins with DNA in a non-sequence-specific manner. In this investigation, the PCNA from the hyperthermophilic archaeon Thermococcus thioreducens (TtPCNA) was cloned, overexpressed in Escherichia coli and purified to greater than 90% homogeneity. TtPCNA crystals were obtained by sitting-drop vapor-diffusion methods and the best ordered crystal diffracted to 1.86 Å resolution using synchrotron radiation. The crystals belonged to the hexagonal space group P63, with unit-cell parameters a = b = 89.0, c = 62.8 Å. Crystals of TtPCNA proved to be amenable to complete X-ray analysis and future structure determination

  15. The Effect of the LysoPC-induced Endothelial Cell Conditioned Medium on Proliferating Cell Nuclear Antigen Expression of the Calf Thoracic Aorta Smooth Muscle Cells

    Institute of Scientific and Technical Information of China (English)

    周洪莲; 姚济华; 余枢

    2002-01-01

    In order to study the effect of and mechanism of lysophosphatidylcholine (LysoPC) on proliferation of the calf thoracic aorta smooth muscle cells (ASMCs), the ASMCs were used to observe the effects of LysoPC-induced endothelial cell conditioned medium on the DNA content and proliferating cell nuclear antigen (PCNA) expression in the calf thoracic ASMCs by flow cytometry and Western Blot technique. It was found that LysoPC-induced endothelial cell conditioned medium could significantly promote PCNA expression of the calf ASMCs, induce the converting of ASMCs from G0/G1 phase to S phase of DNA synthesis, and increase the tyrosine phosphorylation protein expression. Tyrosine protein kinase inhibitor (TPKi) RG50864 could obviously inhibit proliferation of LysoPC-induced ASMCs in a dose-dependence manner. The results indicated that the effect of LysoPC promoting the proliferation of ASMCs is partly evoked by endothelial cell derived growth factors such as PDGF and so on.

  16. Pro-recombination Role of Srs2 Protein Requires SUMO (Small Ubiquitin-like Modifier) but Is Independent of PCNA (Proliferating Cell Nuclear Antigen) Interaction

    DEFF Research Database (Denmark)

    Kolesar, Peter; Altmannova, Veronika; Silva, Sonia;

    2016-01-01

    -interacting motif (SIM) of Srs2 is important for the interaction with several recombination factors. Lack of SIM, but not proliferating cell nuclear antigen (PCNA)-interacting motif (PIM), leads to increased cell death under circumstances requiring homologous recombination for DNA repair. Simultaneous mutation of...

  17. Influence of radiotherapy on expression of the proliferating cell nuclear antigen (PCNA) and c-fos in human cervical cancer

    International Nuclear Information System (INIS)

    Objective: To investigate changes of proliferating cell nuclear antigen (PCNA) expression in human cervical cancer following irradiation. Methods: Immunohistochemical staining for PCNA was performed in frozen sections of formalin-fixed cervical cancer biopsy tissues. Results: The majority of the cancer cells showed PCNA-immunoreactivity before irradiation. Following irradiation (30-40 Gy/15-20 f) PCNA-immuno-positive staining was hardly detectable in most of the cancer cells. The PCNA-immunoreactivity, however, increased after radiotherapy, and moderate or heavy immuno-positive staining for PCNA was seen in irradiated mesenchymal tissue cells. On the other hand, after irradiation Fos-immunoreactivity decreased remarkably, and Fos-immuno-positive staining was hardly detectable in most of cancer cells. No obvious change in Fos-immuno-reactivity, however, was seen in mesenchymal connective tissue following irradiation. Conclusion: Irradiation inhibits PCNA and c-fos expression in cervical cancer cells whereas it induces the expression of PCNA in mesenchymal tissue cells. The present results suggest that expression of PCNA and c-fos may be regarded as a molecular marker for evaluating the cancer cell proliferation and mesenchymal tissue repair during radiotherapy of human cervical cancer

  18. Proliferating cell nuclear antigen (PCNA) allows the automatic identification of follicles in microscopic images of human ovarian tissue

    CERN Document Server

    Kelsey, Thomas W; Castillo, Luis; Wallace, W Hamish B; Gonzálvez, Francisco Cóppola; 10.2147/PLMI.S11116

    2010-01-01

    Human ovarian reserve is defined by the population of nongrowing follicles (NGFs) in the ovary. Direct estimation of ovarian reserve involves the identification of NGFs in prepared ovarian tissue. Previous studies involving human tissue have used hematoxylin and eosin (HE) stain, with NGF populations estimated by human examination either of tissue under a microscope, or of images taken of this tissue. In this study we replaced HE with proliferating cell nuclear antigen (PCNA), and automated the identification and enumeration of NGFs that appear in the resulting microscopic images. We compared the automated estimates to those obtained by human experts, with the "gold standard" taken to be the average of the conservative and liberal estimates by three human experts. The automated estimates were within 10% of the "gold standard", for images at both 100x and 200x magnifications. Automated analysis took longer than human analysis for several hundred images, not allowing for breaks from analysis needed by humans. O...

  19. Expression and significance of C-fos and proliferating cell nuclear antigen in the small intestinal tissue of human fetus

    Directory of Open Access Journals (Sweden)

    Xue-hong LIU

    2011-02-01

    Full Text Available Objective To explore the expression rule of proliferating cell nuclear antigen(PCNA,C-fos proteins and apoptosis genes in the small intestinal tissue of human fetus.Methods At the second-to fourth-month of gestation,the expressions of cell proliferation and apoptosis were observed in 16 specimens of human fetal small intestinal tissue by using the immunohistochemical methods and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling(TUNEL.Results At the second to fourth month of gestation,all the PCNA and C-fos proteins were positively expressed in the small intestinal tissues and cells of human fetus.With the increase in gestational period,the positive cell number and average intensity(AI of PCNA protein increased gradually(P < 0.01.The positive cell number of C-fos protein increased first,and then decreased,while the AI of C-fos protein stably increased in the small intestinal tissues and cells of human fetus(P < 0.01.At the second to fourth month of gestation,TUNEL positive cells were seen to distribute in each layer of the small intestinal tissues of human fetus.With the increase of age,all the positive cell number and AI of TUNEL positive cells showed a tendency of decrease following increase in the small intestine of human fetus(P < 0.01.Conclusions PCNA,C-fos and apoptosis gene participate in adjusting the growth and development of the cells and tissues in the small intestine of human fetus.In the third month of gestation,especially,proliferation and apoptosis are significantly increased in the small intestinal tissue of human fetus,which may be the key period of intestinal tissue development.

  20. Phosphorylation at tyrosine 114 of Proliferating Cell Nuclear Antigen (PCNA) is required for adipogenesis in response to high fat diet

    Energy Technology Data Exchange (ETDEWEB)

    Lo, Yuan-Hung; Ho, Po-Chun; Chen, Min-Shan; Hugo, Eric; Ben-Jonathan, Nira [Department of Cancer Biology, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267-0521 (United States); Department of Environmental Health, University of Cincinnati College of Medicine, 3223 Eden Avenue, Kettering Laboratory, Cincinnati, OH 45267-0056 (United States); Wang, Shao-Chun, E-mail: shao-chun.wang@uc.edu [Department of Cancer Biology, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267-0521 (United States); Department of Environmental Health, University of Cincinnati College of Medicine, 3223 Eden Avenue, Kettering Laboratory, Cincinnati, OH 45267-0056 (United States)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Proliferating Cell Nuclear Antigen (PCNA) is phosphorylated at Y114. Black-Right-Pointing-Pointer Phospho-Y114 of PCNA is not required for cell proliferation for normal growth. Black-Right-Pointing-Pointer MCE during adipogenesis is abolished in the lack of the phosphorylation. Black-Right-Pointing-Pointer Homozygous Y114F mice are resistant to high fat diet induced obesity. Black-Right-Pointing-Pointer Our results shed light on the interface between proliferation and differentiation. -- Abstract: Clonal proliferation is an obligatory component of adipogenesis. Although several cell cycle regulators are known to participate in the transition between pre-adipocyte proliferation and terminal adipocyte differentiation, how the core DNA synthesis machinery is coordinately regulated in adipogenesis remains elusive. PCNA (Proliferating Cell Nuclear Antigen) is an indispensable component for DNA synthesis during proliferation. Here we show that PCNA is subject to phosphorylation at the highly conserved tyrosine residue 114 (Y114). Replacing the Y114 residue with phenylalanine (Y114F), which is structurally similar to tyrosine but cannot be phosphorylated, does not affect normal animal development. However, when challenged with high fat diet, mice carrying homozygous Y114F alleles (PCNA{sup F/F}) are resistant to adipose tissue enlargement in comparison to wild-type (WT) mice. Mouse embryonic fibroblasts (MEFs) harboring WT or Y114F mutant PCNA proliferate at similar rates. However, when subjected to adipogenesis induction in culture, PCNA{sup F/F} MEFs are not able to re-enter the cell cycle and fail to form mature adipocytes, while WT MEFs undergo mitotic clonal expansion in response to the adipogenic stimulation, accompanied by enhanced Y114 phosphorylation of PCNA, and differentiate to mature adipocytes. Consistent with the function of Y114 phosphorylation in clonal proliferation in adipogenesis, fat tissues isolated from WT

  1. Phosphorylation at tyrosine 114 of Proliferating Cell Nuclear Antigen (PCNA) is required for adipogenesis in response to high fat diet

    International Nuclear Information System (INIS)

    Highlights: ► Proliferating Cell Nuclear Antigen (PCNA) is phosphorylated at Y114. ► Phospho-Y114 of PCNA is not required for cell proliferation for normal growth. ► MCE during adipogenesis is abolished in the lack of the phosphorylation. ► Homozygous Y114F mice are resistant to high fat diet induced obesity. ► Our results shed light on the interface between proliferation and differentiation. -- Abstract: Clonal proliferation is an obligatory component of adipogenesis. Although several cell cycle regulators are known to participate in the transition between pre-adipocyte proliferation and terminal adipocyte differentiation, how the core DNA synthesis machinery is coordinately regulated in adipogenesis remains elusive. PCNA (Proliferating Cell Nuclear Antigen) is an indispensable component for DNA synthesis during proliferation. Here we show that PCNA is subject to phosphorylation at the highly conserved tyrosine residue 114 (Y114). Replacing the Y114 residue with phenylalanine (Y114F), which is structurally similar to tyrosine but cannot be phosphorylated, does not affect normal animal development. However, when challenged with high fat diet, mice carrying homozygous Y114F alleles (PCNAF/F) are resistant to adipose tissue enlargement in comparison to wild-type (WT) mice. Mouse embryonic fibroblasts (MEFs) harboring WT or Y114F mutant PCNA proliferate at similar rates. However, when subjected to adipogenesis induction in culture, PCNAF/F MEFs are not able to re-enter the cell cycle and fail to form mature adipocytes, while WT MEFs undergo mitotic clonal expansion in response to the adipogenic stimulation, accompanied by enhanced Y114 phosphorylation of PCNA, and differentiate to mature adipocytes. Consistent with the function of Y114 phosphorylation in clonal proliferation in adipogenesis, fat tissues isolated from WT mice contain significantly more adipocytes than those isolated from PCNAF/F mice. This study identifies a critical role for PCNA in adipose

  2. The Relationship between Apoptosis and the Expression of Proliferating Cell Nuclear Antigen and the Clinical Stages in Gastric Carcinoma

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The relationship between the apoptosis and the expression of proliferating cell nuclear antigen (PCNA) and the clinical stages in gastric cancers was studied. By using terminal deoxynucleotidyl transferase-mediated nick end labelling (TUNEL) technique and PCNA immunohistochemical staining, the apoptosis and the expression of PCNA in tissue of gastric carcinoma were assayed in situ, the index of apoptosis (AI), index of PCNA (PI) and the rate of AI/PI were calculated. AI and PI in gastric cancer tissues were (6.5±3.7) % and (49.8±15.9) % respectively, and the rate of AI/PI was 0.13±0.05, which were obviously different from those of normal gastric mucosa in paragastric cancer (P<0.01). With the advanced TNM stages of gastric carcinoma, the AI was decreased, PI was increased and the rate of AI/PI decreased in gastric carcinoma. There was significant difference in them between the gastric cancer tissues and normal gastric mucosa in pericarcinoma in TNM stage Ⅱ to Ⅳ (P<0.05). It was suggested that the decreased apoptotic cells and the increased proliferating cells were obviously related to the tumor genesis and tumor progression in gastric carcinoma. The AI, PI and the rate of AI/PI would become the prognostic factors in advanced gastric carcinoma.

  3. A Peptide mimicking a region in proliferating cell nuclear antigen specific to key protein interactions is cytotoxic to breast cancer.

    Science.gov (United States)

    Smith, Shanna J; Gu, Long; Phipps, Elizabeth A; Dobrolecki, Lacey E; Mabrey, Karla S; Gulley, Pattie; Dillehay, Kelsey L; Dong, Zhongyun; Fields, Gregg B; Chen, Yun-Ru; Ann, David; Hickey, Robert J; Malkas, Linda H

    2015-02-01

    Proliferating cell nuclear antigen (PCNA) is a highly conserved protein necessary for proper component loading during the DNA replication and repair process. Proteins make a connection within the interdomain connector loop of PCNA, and much of the regulation is a result of the inherent competition for this docking site. If this target region of PCNA is modified, the DNA replication and repair process in cancer cells is potentially altered. Exploitation of this cancer-associated region has implications for targeted breast cancer therapy. In the present communication, we characterize a novel peptide (caPeptide) that has been synthesized to mimic the sequence identified as critical to the cancer-associated isoform of PCNA. This peptide is delivered into cells using a nine-arginine linking mechanism, and the resulting peptide (R9-cc-caPeptide) exhibits cytotoxicity in a triple-negative breast cancer cell line, MDA-MB-436, while having less of an effect on the normal counterparts (MCF10A and primary breast epithelial cells). The novel peptide was then evaluated for cytotoxicity using various in vivo techniques, including ATP activity assays, flow cytometry, and clonogenetic assays. This cytotoxicity has been observed in other breast cancer cell lines (MCF7 and HCC1937) and other forms of cancer (pancreatic and lymphoma). R9-cc-caPeptide has also been shown to block the association of PCNA with chromatin. Alanine scanning of the peptide sequence, combined with preliminary in silico modeling, gives insight to the disruptive ability and the molecular mechanism of action of the therapeutic peptide in vivo. PMID:25480843

  4. Monoclonal immunoglobulin M antibody to Japanese encephalitis virus that can react with a nuclear antigen in mammalian cells.

    OpenAIRE

    Gould, E A; Chanas, A C; Buckley, A.; Clegg, C S

    1983-01-01

    An immunoglobulin M (IgM) class monoclonal antibody raised against Japanese encephalitis virus reacted with an epitope on the nonstructural virus protein P74 (NV4 in the old nomenclature) of several flaviviruses and also with an antigen present in the nuclei of a variety of mammalian cell types. This antigen had a characteristic granular distribution by immunofluorescence and may correspond to a polypeptide of molecular weight 56,000 seen in nitrocellulose transfers of sodium dodecyl sulfate-...

  5. Expression of Ki-67 nuclear antigen in B and T cell lymphoproliferative disorders.

    Science.gov (United States)

    de Melo, N.; Matutes, E.; Cordone, I.; Morilla, R.; Catovksy, D.

    1992-01-01

    AIMS: To determine whether the proliferation rates of tumour cells may relate to prognosis and reflect disease activity. METHODS: Blood mononuclear cells from 155 patients with B cell (n = 120) or T cell (n = 35) chronic lymphoproliferative disorders were tested with the monoclonal antibody Ki-67 by indirect immunoperoxidase or immunoalkaline phosphatase techniques. B cell diseases included chronic lymphocytic leukaemia (CLL), CLL in prolymphocytic transformation (CLL/PL), prolymphocytic leukaemia (B-PLL) and non-Hodgkin's lymphoma (B-NHL) in leukaemic phase. The T cell diseases comprised large granular lymphocyte (LGL) leukaemia, T-PLL, and T-NHL. RESULTS: These showed significantly higher proportions of Ki-67 positive cells in T cell (11.2%) than in B cell (2.9%) disorders (p < 0.001). The highest values were found in NHL of both B and T cell types, particularly when low grade disease transformed to high grade. The lowest percentages of Ki-67 positive cells were found in CLL (1.4%) and LGL leukaemia (1.7%); intermediate values were seen in B PLL (3.3%) and T PLL (5.8%). CONCLUSIONS: There is a positive correlation between prognosis and proliferation rates in chronic B and T cell lymphoproliferative disorders. Estimation of Ki-67 in circulating leukaemic cells could be used to determine prognosis in low grade malignancies. Images PMID:1401173

  6. Proliferating cell nuclear antigen is required for loading of the SMCX/KMD5C histone demethylase onto chromatin

    Directory of Open Access Journals (Sweden)

    Liang Zhihui

    2011-10-01

    Full Text Available Abstract Background Histone methylation is regulated by a large number of histone methyltransferases and demethylases. The recently discovered SMCX/KMD5C demethylase has been shown to remove methyl residues from lysine 4 of histone H3 (H3K4, and constitutes an important component of the regulatory element-1-silencing transcription factor (REST protein complex. However, little is known about the cellular mechanisms that control SMCX activity and intracellular trafficking. Results In this study, we found that small interfering RNA-mediated knockdown of proliferating cell nuclear antigen (PCNA resulted in the reduction of the chromatin-bound SMCX fraction. We identified a PCNA-interaction protein motif (PIP box in the SMCX protein. Using site-directed mutagenesis, we found that the amino acids of the SMCX PIP box are involved in the association of SMCX with PCNA and its interaction with chromatin. Conclusions Our data indicate that the intracellular trafficking of SMCX is controlled by its association with PCNA.

  7. Expression of intestinal trefoil factor, proliferating cell nuclear antigen and histological changes in intestine of rats after intrauterine asphyxia

    Institute of Scientific and Technical Information of China (English)

    Ling-Fen Xu; Jun Li; Mei Sun; Hong-Wei Sun

    2005-01-01

    AIM: To study the expressions of intestinal trefoil factor (ITF) and proliferating cell nuclear antigen (PCNA) and histologic changes in intestine, to investigate the relationship between ITF and intestinal damage and repair after intrauterine hypoxia so as to understand the mechanism of intestinal injury and to find a new way to prevent and treat gastrointestinal diseases.METHODS: Wistar rats, pregnant for 21 d, were used to establish animal models of intrauterine asphyxia by clamping one side of vessels supplying blood to uterus for 20 min, another side was regarded as sham operation group. Intestinal tissues were taken away at 0, 24, 48and 72 h after birth and stored in different styles. ITF mRNA was detected by RT-PCR. PCNA expression was measured by immunohistochemistry. Intestinal tissues were studied histologically by HE staining in order to observe the areas and degree of injury and to value the intestinal mucosa injury index (IMDI).RESULTS: ITF mRNA appeared in full-term rats and increased with age. After ischemia, ITF mRNA was decreased to the minimum (0.59±0.032) 24 h after birth, then began to increase higher after 72 h than it was in the control group (P<0.01). PCNA positive staining located in goblet cell nuclei. The PCNA level had a remarkable decline (53.29±1.97) 48 h after ischemia. Structure changes were obvious in 48-h group, IMDI (3.40±0.16) was significantly increased. Correlation analyses showed that IMDI had a negative correlation with ITF mRNA and PCNA (r = -0.543,P<0.05; r = -0.794, P<0.01, respectively).CONCLUSION: Intrauterine ischemia can result in an early decrease of ITF mRNA expression. ITF and PCNA may play an important role in the damage and repair of intestinal mucosa.

  8. 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

  9. Proliferating cell nuclear antigen (PCNA) activity in hepatocellular carcinoma, benign peri-neoplastic and normal liver.

    Science.gov (United States)

    Mun, Kein-Seong; Cheah, Phaik-Leng; Baharudin, Nurul Bahiyah; Looi, Lai-Meng

    2006-12-01

    Hepatocellular carcinoma (HCC) is among the ten most common cancers in Malaysian males. As cellular proliferation is an important feature of malignant transformation, we studied the proliferation pattern of normal and benign perineoplastic liver versus hepatocellular carcinoma in an attempt to further understand the tumour transformation process. 39 HCC (21 with accompanying and 18 without cirrhosis) histologically diagnosed at the Department of Pathology, University of Malaya Medical Centre between January 1992 and December 2003 were immunohistochemically studied using a monoclonal antibody to PCNA (Clone PC10: Dako). 20 livers from cases who had succumbed to traumatic injuries served as normal liver controls (NL). PCNA labeling index (PCNA-LI) was determined by counting the number of immunopositive cells in 1000 contiguous HCC, benign cirrhotic perineoplastic liver (BLC), benign perineoplastic non-cirrhotic (BLNC) and NL cells and conversion to a percentage. The PCNA-LI was also expressed as Ojanguren et al's grades. PCNA was expressed in 10% NL, 38.9% BLNC, 76.2% BLC and 71.8% HCC with BLNC, BLC and HCC showing significantly increased (p cells being immunopositive) immunoreactivity on Ojanguren et al's grading system and only HCC demonstrated immunoreactivity which ranged up to grade 3 (75% of cells). From this study, there appears to be a generally increasing trend of proliferative activity from NL to BLNC to BLC and HCC. Nonetheless, BLNC and BLC, like NL, retained low PCNA-LI and only HCC had a significantly increased PCNA-LI compared with the benign categories. This is probably related to the malignant nature of HCC and may reflect the uncontrolled proliferation of the neoplastic hepatocytes. PMID:18376794

  10. Proliferating cell nuclear antigen (PCNA interactions in solution studied by NMR.

    Directory of Open Access Journals (Sweden)

    Alfredo De Biasio

    Full Text Available PCNA is an essential factor for DNA replication and repair. It forms a ring shaped structure of 86 kDa by the symmetric association of three identical protomers. The ring encircles the DNA and acts as a docking platform for other proteins, most of them containing the PCNA Interaction Protein sequence (PIP-box. We have used NMR to characterize the interactions of PCNA with several other proteins and fragments in solution. The binding of the PIP-box peptide of the cell cycle inhibitor p21 to PCNA is consistent with the crystal structure of the complex. A shorter p21 peptide binds with reduced affinity but retains most of the molecular recognition determinants. However the binding of the corresponding peptide of the tumor suppressor ING1 is extremely weak, indicating that slight deviations from the consensus PIP-box sequence dramatically reduce the affinity for PCNA, in contrast with a proposed less stringent PIP-box sequence requirement. We could not detect any binding between PCNA and the MCL-1 or the CDK2 protein, reported to interact with PCNA in biochemical assays. This suggests that they do not bind directly to PCNA, or they do but very weakly, with additional unidentified factors stabilizing the interactions in the cell. Backbone dynamics measurements show three PCNA regions with high relative flexibility, including the interdomain connector loop (IDCL and the C-terminus, both of them involved in the interaction with the PIP-box. Our work provides the basis for high resolution studies of direct ligand binding to PCNA in solution.

  11. Site-specific mutagenesis of Drosophila proliferating cell nuclear antigen enhances its effects on calf thymus DNA polymerase δ

    Directory of Open Access Journals (Sweden)

    Miller Holly

    2004-08-01

    Full Text Available Abstract Background We and others have shown four distinct and presumably related effects of mammalian proliferating cell nuclear antigen (PCNA on DNA synthesis catalyzed by mammalian DNA polymerase δ(pol δ. In the presence of homologous PCNA, pol δ exhibits 1 increased absolute activity; 2 increased processivity of DNA synthesis; 3 stable binding of synthetic oligonucleotide template-primers (t1/2 of the pol δ•PCNA•template-primer complex ≥2.5 h; and 4 enhanced synthesis of DNA opposite and beyond template base lesions. This last effect is potentially mutagenic in vivo. Biochemical studies performed in parallel with in vivo genetic analyses, would represent an extremely powerful approach to investigate further, both DNA replication and repair in eukaryotes. Results Drosophila PCNA, although highly similar in structure to mammalian PCNA (e.g., it is >70% identical to human PCNA in amino acid sequence, can only substitute poorly for either calf thymus or human PCNA (~10% as well in affecting calf thymus pol δ. However, by mutating one or only a few amino acids in the region of Drosophila PCNA thought to interact with pol δ, all four effects can be enhanced dramatically. Conclusions Our results therefore suggest that all four above effects depend at least in part on the PCNA-pol δ interaction. Moreover unlike mammals, Drosophila offers the potential for immediate in vivo genetic analyses. Although it has proven difficult to obtain sufficient amounts of homologous pol δ for parallel in vitro biochemical studies, by altering Drosophila PCNA using site-directed mutagenesis as suggested by our results, in vitro biochemical studies may now be performed using human and/or calf thymus pol δ preparations.

  12. Kaposi sarcoma herpes virus latency associated nuclear antigen protein release the G2/M cell cycle blocks by modulating ATM/ATR mediated checkpoint pathway.

    Directory of Open Access Journals (Sweden)

    Amit Kumar

    Full Text Available The Kaposi's sarcoma-associated herpesvirus infects the human population and maintains latency stage of viral life cycle in a variety of cell types including cells of epithelial, mesenchymal and endothelial origin. The establishment of latent infection by KSHV requires the expression of an unique repertoire of genes among which latency associated nuclear antigen (LANA plays a critical role in the replication of the viral genome. LANA regulates the transcription of a number of viral and cellular genes essential for the survival of the virus in the host cell. The present study demonstrates the disruption of the host G2/M cell cycle checkpoint regulation as an associated function of LANA. DNA profile of LANA expressing human B-cells demonstrated the ability of this nuclear antigen in relieving the drug (Nocodazole induced G2/M checkpoint arrest. Caffeine suppressed nocodazole induced G2/M arrest indicating involvement of the ATM/ATR. Notably, we have also shown the direct interaction of LANA with Chk2, the ATM/ATR signalling effector and is responsible for the release of the G2/M cell cycle block.

  13. GAGE cancer-germline antigens are recruited to the nuclear envelope by germ cell-less (GCL)

    DEFF Research Database (Denmark)

    Gjerstorff, Morten F; Rösner, Heike I; Pedersen, Christina B;

    2012-01-01

    metazoan transcriptional regulator, Germ cell-less (GCL), as an interaction partner of GAGE12I. GCL directly binds LEM-domain proteins (LAP2β, emerin, MAN1) at the nuclear envelope, and we found that GAGE proteins were recruited to the nuclear envelope inner membrane by GCL. Based on yeast two...

  14. A tumor necrosis factor α- and interleukin 6-inducible protein that interacts with the small subunit of DNA polymerase δ and proliferating cell nuclear antigen

    OpenAIRE

    He, Hua; Tan, Cheng-Keat; Downey, Kathleen M.; So, Antero G.

    2001-01-01

    A cDNA encoding a protein of 36 kDa, polymerase delta-interacting protein 1 (PDIP1), that interacts with the small subunit (p50) of DNA polymerase δ (pol δ) was identified in a two-hybrid screen of a HepG2 cDNA library by using p50 as bait. The interaction of PDIP1 with p50 was confirmed by pull-down assays, and a similar assay was used to demonstrate that PDIP1 interacts directly with the proliferating cell nuclear antigen (PCNA). PCNA and p50 bound to PDIP1 simultaneously, and PDIP1 stimula...

  15. Stoichiometric complex formation by proliferating cell nuclear antigen (PCNA) and its interacting protein: purification and crystallization of the DNA polymerase and PCNA monomer mutant complex from Pyrococcus furiosus

    International Nuclear Information System (INIS)

    A stable stoichiometric complex of archaeal DNA polymerase with proliferating cell nuclear antigen (PCNA) was formed using a PCNA monomer mutant and the complex was successfully crystallized. Replicative DNA polymerase interacts with processivity factors, the β-subunit of DNA polymerase III or proliferating cell nuclear antigen (PCNA), in order to function with a long template DNA. The archaeal replicative DNA polymerase from Pyrococcus furiosus interacts with PCNA via its PCNA-interacting protein (PIP) motif at the C-terminus. The PCNA homotrimeric ring contains one PIP interacting site on each monomer and since the ring can accommodate up to three molecules simultaneously, formation of a stable stoichiometric complex of PCNA with its interacting protein has been difficult to control in vitro. A stable complex of the DNA polymerase with PCNA, using a PCNA monomer mutant, has been purified and crystallized. The best ordered crystal diffracted to 3.0 Å resolution using synchrotron radiation. The crystals belong to space group P21212, with unit-cell parameters a = 225.3, b = 123.3, c = 91.3 Å

  16. Functions of replication factor C and proliferating-cell nuclear antigen: Functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T4

    International Nuclear Information System (INIS)

    The proliferating-cell nuclear antigen (PCNA) and the replication factors A and C (RF-A and RF-C) are cellular proteins essential for complete elongation of DNA during synthesis from the simian virus 40 origin of DNA replication in vitro. All three cooperate to stimulate processive DNA synthesis by DNA polymerase δ on a primed single-stranded M13 template DNA and as such can be categorized as DNA polymerase accessory proteins. Biochemical analyses with highly purified RF-C and PCNA have demonstrated functions that are completely analogous to the functions of bacteriophage T4 DNA polymerase accessory proteins. A primer-template-specific DNA binding activity and a DNA-dependent ATPase activity copurified with the multisubunit protein RF-C and are similar to the functions of the phage T4 gene 44/62 protein complex. Furthermore, PCNA stimulated the RF-C ATPase activity and is, therefore, analogous to the phage T4 gene 45 protein, which stimulates the ATPase function of the gene 44/62 protein complex. Indeed, some primary sequence similarities between human PCNA and the phage T4 gene 45 protein could be detected. These results demonstrate a striking conservation of the DNA replication apparatus in human cells and bacteriophage T4

  17. Expression of cell cycle regulator p57kip2, cyclinE protein and proliferating cell nuclear antigen in human pancreatic cancer: An immunohistochemical study

    Institute of Scientific and Technical Information of China (English)

    Hui Yue; Hui-Yong Jiang

    2005-01-01

    AIM: To investigate the effects of p57kip2, cyclinE protein and proliferating cell nuclear antigen (PCNA) on occurrence and progression of human pancreatic cancer.METHODS: The expression of p57kip2, cyclinE protein and PCNA in tumor tissues and adjacent tissues from 32patients with pancreatic cancer was detected by SP immunohistochemical technique.RESULTS: The positive expression rate of p57kip2 protein in tumor tissues was 46.9%, which was lower than that in adjacent pancreatic tissues (x2 = 5.317, P<0.05). P57kip2protein positive expression remarkably correlated with tumor cell differentiation (P<0.05), but not with lymph node metastasis (P>0.05). The positive expression rate of cyclinE protein in tumor tissues was 68.8%, which was higher than that in adjacent pancreatic tissues (x2 = 4.063,P<0.05). CyclinE protein positive expression significantly correlated with tumor cell differentiation and lymph node metastasis (P<0.05). The positive expression rate of PCNA in the tumor tissues was 71.9%, which was higher than that in adjacent pancreatic tissues (x2 = 5.189, P<0.05).PCNA positive expression remarkably correlated with tumor cell differentiation and lymph node metastasis (P<0.05).CONCLUSION: The decreased expression of p57kip2 and/or overexpression of cyclinE protein and PCNA may contribute to the occurrence and progression of pancreatic cancer.p57kip2, cyclinE protein, and PCNA play an important role in occurrence and progression of pancreatic cancer.

  18. Histocompatibility antigens on astrocytoma cells.

    OpenAIRE

    Hirschberg, H.; Endresen, L I; Wikeby, P

    1982-01-01

    Biopsies tumour cells from astrocytoma-bearing patients were grown in primary culture for 3-5 days. Both low and high grade tumours were represented in the study. The cultured cells could be shown to express the HLA-A and -B antigens using a multispecific allo-antiserum and a rabbit anti-beta-2 microglobulin antibody. The tumour cells were negative for the HLA-DR determinants when tested with either rabbit anti-Ia-like antisera or specific anti-HLA-DR allo-antisera. They also failed to stimul...

  19. Rapid effect of heat shock on two heterogeneous nuclear ribonucleoprotein-associated antigens in HeLa cells

    OpenAIRE

    1989-01-01

    During severe heat shock, which known to interrupt both splicing of RNA transcripts and nucleocytoplasmic transport, it is to be expected that the substructure of heterogeneous nuclear ribonucleoproteins (hnRNP) is altered in some way. Recently, we have shown that such a stress actually induces rapid alterations at the level of individual proteins (Lutz, Y., M. Jacob, and J.-P. Fuchs. 1988 Exp. Cell Res. 175:109-124). Here we report further investigations on two related 72.5-74-kD hnRNP prote...

  20. Proliferating cell nuclear antigen in gonad and associated storage tissue of the Pacific oyster Crassostrea gigas: seasonal immunodetection and expression in laser microdissected tissues.

    Science.gov (United States)

    Franco, Alban; Jouaux, Aude; Mathieu, Michel; Sourdaine, Pascal; Lelong, Christophe; Kellner, Kristell; Heude Berthelin, Clothilde

    2010-04-01

    To understand the processes involved in tissue remodeling associated with the seasonal reproductive cycle of the oyster Crassostrea gigas, we used immunodetection and expression measurements of proliferating cell nuclear antigen (PCNA). The expression of the PCNA gene was measured by real-time polymerase chain reaction in the whole gonadal area compared with laser microdissected gonad and storage tissue. Results underlined the advantage of the laser microdissection approach to detect expression, mainly for early stages of spermatogenesis. In the storage tissue, PCNA expression was reduced in the gonadal tubules, but immunolabeled hemocytes and vesicular cells were detected when the storage tissue was being restored. In the gonadal tubules, the PCNA gene was more highly expressed in males than in females. As soon as spermatogenesis was initiated, PCNA expression showed a high and constant level. In females, the expression level increased gradually until the ripe stage. The immunological approach established the involvement of peritubular cells in gonadal tubule expansion during early gametogenesis. In both sexes, gonial mitosis was immunodetected throughout the reproductive cycle. In males, the occurrence of two types of spermatogonia was ascertained by differential immunolabeling, and intragonadal somatic cell proliferation was noted. As expected, immunolabeling was never observed from stage II spermatocytes to spermatozoa. In females, positively stained cells were detected from oogonia to growing oocytes with various labeled intracellular locations. PMID:20151153

  1. Harnessing Dendritic Cells for Tumor Antigen Presentation

    International Nuclear Information System (INIS)

    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+ and CD4+ T cells; the in vitro loading of DCs with tumor antigens

  2. Urinary bladder lesions after the chernobyl accident. Immunohistochemical assessment of p53, proliferating cell nuclear antigen, cyclin D1 and p21WAF1/Cip1

    International Nuclear Information System (INIS)

    During the 11-year period subsequent to the Chernobyl accident, the incidence of urinary bladder cancer in Ukraine has increased from 26.2 to 36.1 per 100,000 population. Cesium-137 (137Cs) accounts for 80-90% of the incorporated radioactivity in this population, which has been exposed to long-term, low-dose ionizing radiation, and 80% of the more labile pool of cesium is excreted via the urine. The present study was performed to evaluate the histopathological features and the immunohistochemical status of p53, p21WAF1/Cip1, cyclin D1 and PCNA (proliferating cell nuclear antigen) in urinary bladder mucosa of 55 males (49-92 years old) with benign prostatic hyperplasia who underwent surgery in Kiev, Ukraine, in 1995 and 1996. Group I (28 patients) inhabiting radiocontaminated areas of the country, group II (17 patients) from Kiev city with less radiocontamination and a control group III (10 patients) living in so-called ''clean'' areas of Ukraine were compared. In groups I and II, an increase in multiple areas of moderate or severe dysplasia or carcinoma in situ was seen in 42 (93%) of 45 cases. In addition, two small transitional cell carcinomas were found in one patient in each of groups I and II. Nuclear accumulation of p53, PCNA, cyclin D1, and to a lesser extent p21WAF1/Cip1, was significantly increased in both groups I and II as compared with the control group III, indicating possible transformation events or enhancement of repair activities, that may precede the defect in the regulatory pathway itself, at least in the G1 phase of the cell cycle. Our results suggest that early malignant transformation is taking place in the bladder urothelium of people in the radiocontaminated areas of Ukraine and that this could possibly lead sometime in the future to an increased incidence of urinary bladder cancer. (author)

  3. Establishment of Human Embryonic Stem Cell Line Stably Expressing Epstein-Barr Virus-Encoded Nuclear Antigen 1

    Institute of Scientific and Technical Information of China (English)

    Cai-Ping REN; Ming ZHAO; Wen-Jiao SHAN; Xu-Yu YANG; Zhi-Hua YIN; Xing-Jun JIANG; Hong-Bo ZHANG; Kai-Tai YAO

    2005-01-01

    Human embryonic stem (hES) cells have the capability of unlimited undifferentiated proliferation,yet maintain the potential to form perhaps any cell type in the body. Based on the high efficiency of the Epstein-Barr virus-based episomal vector in introducing exogenous genes of interest into mammalian cells,we applied this system to hES cells, expecting that this would resolve the problem of poor transfection efficiency existing in current hES cell research. Therefore, the first step was to establish EBNAl-positive hES cells. Using the Fugene 6 transfection reagent, we transfected hES cells with the EBNA1 expression vector and subsequently generated hES cell clones that stably expressed EBNA 1 under drug selection. These clones were confirmed to express EBNA1 mRNA by RT-PCR and to express EBNA1 protein by Western blotting. Furthermore, luciferase reporter gene analysis was performed on the EBNA1 clones and revealed that the expressed EBNA1 protein was functional. When the EBNAl-positive cells were injected into severe combined immunodeficient (SCID) mice, they formed teratoma tissues containing all three embryonic germ layers and EBNA1 protein was detected in these teratoma tissues by Western blotting. All the results show that we have successfully created stable EBNA1-hES cells, thus laying a good foundation for further research.

  4. EBV Nuclear Antigen 3C Mediates Regulation of E2F6 to Inhibit E2F1 Transcription and Promote Cell Proliferation.

    Science.gov (United States)

    Pei, Yonggang; Banerjee, Shuvomoy; Sun, Zhiguo; Jha, Hem Chandra; Saha, Abhik; Robertson, Erle S

    2016-08-01

    Epstein-Barr virus (EBV) is considered a ubiquitous herpesvirus with the ability to cause latent infection in humans worldwide. EBV-association is evidently linked to different types of human malignancies, mainly of epithelial and lymphoid origin. Of interest is the EBV nuclear antigen 3C (EBNA3C) which is critical for EBV-mediated immortalization. Recently, EBNA3C was shown to bind the E2F1 transcription regulator. The E2F transcription factors have crucial roles in various cellular functions, including cell cycle, DNA replication, DNA repair, cell mitosis, and cell fate. Specifically, E2F6, one of the unique E2F family members, is known to be a pRb-independent transcription repressor of E2F-target genes. In our current study, we explore the role of EBNA3C in regulating E2F6 activities. We observed that EBNA3C plays an important role in inducing E2F6 expression in LCLs. Our study also shows that EBNA3C physically interacts with E2F6 at its amino and carboxy terminal domains and they form a protein complex in human cells. In addition, EBNA3C stabilizes the E2F6 protein and is co-localized in the nucleus. We also demonstrated that both EBNA3C and E2F6 contribute to reduction in E2F1 transcriptional activity. Moreover, E2F1 forms a protein complex with EBNA3C and E2F6, and EBNA3C competes with E2F1 for E2F6 binding. E2F6 is also recruited by EBNA3C to the E2F1 promoter, which is critical for EBNA3C-mediated cell proliferation. These results demonstrate a critical role for E2F family members in EBV-induced malignancies, and provide new insights for targeting E2F transcription factors in EBV-associated cancers as potential therapeutic intervention strategies. PMID:27548379

  5. 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.

  6. Antigen dynamics of follicular dendritic cells

    NARCIS (Netherlands)

    Heesters, B.A.

    2015-01-01

    Stromal-derived follicular dendritic cells (FDCs) are a major depot for antigen that are essential for formation of germinal centers, the site where memory and effector B cells differentiate and high-affinity antibody production takes place. Historically, FDCs have been characterized as ‘accessory’

  7. Effects of Antisense Oligodeoxynucleotide to Follicle-stimulating Hormone Receptor on the Expression of Proliferating Cell Nuclear Antigen and Vascular Endothelial Growth Factor in Primary Culture Cells Derived from Human Ovarian Mucinous Cystadenocarcino

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The effects of antisense oligodeoxynucleotide (antisense ODN) to follicle-stimulating hormone receptor (FSHR) and follicle-stimulating hormone (FSH) on the expression of proliferating cell nuclear antigen (PCNA) and vascular endothelial growth factor (VEGF) were studied in primary culture cells derived from human ovarian mucinous cystadenocarcinoma (OMC). The prlmary OMC cells were cultured with the enzyme digestion method, and the expression of pan Keratin protein and FSHR mRNA was detected for identification of the cells. OMC cells were co-cultured with antisense ODN, nonsense ODN and FSH with different concentrations for 48 h and 72 h. The expression of PCNA and VEGF was detected by using SP immunohistochemistry. Compared with that in the control group, the PCNA and VEGF expression was increased obviously in FSH groups (P<0.05 or P< 0.01), while decreased significantly in antisense ODN groups (P<0. 05 or P<0.01) and unchanged in nonsense ODN groups, respectively. Meanwhile, antisense ODN could antagonize the increased expression of PCNA and VEGF caused by FSH significantly (P<0.01). It was suggested that FSH might promotethe development of OMC to some extent. Antisense ODN could inhibit the proliferative activity of OMC cells and the promoting proliferative activity enhanced by FSH.

  8. The response of proliferating cell nuclear antigen (PCNA) to ionizing radiation in human lymphoblastoid cell lines with different p53 status

    International Nuclear Information System (INIS)

    Purpose/Objective: PCNA is a 36kD auxiliary protein for DNA polymerase δ involved in DNA replication and nucleotide excision repair. There are two intranuclear fractions: a detergent-extractable, soluble fraction (SOL) and a tightly DNA bound fraction (BF). To function, PCNA monomers form a trimeric sliding clamp, which is loaded onto DNA. To better understand the role of the p53/p21 pathway in the regulation of PCNA after radiation in vivo, we studied three closely related human lymphoblastoid cell lines, WTK1 (p53mt), TK6 (p53wt) and TK6E6, a HPV16E6 transfected line, that differ in p53 status, radiosensitivity and susceptibility to radiation-induced apoptosis. Materials and Methods: The total cellular PCNA content was divided into the two intranuclear fractions with detergent pretreatment (EDTA 2% containing 2.5 μl/ml Triton-X 100, 5 μl/ml BSA, 15 min, on ice). Time-dependent changes in PCNA monomer and trimer levels were measured after irradiation (0 - 4 Gy) in the different nuclear fractions (BF, SOL) by western blot analysis after protein cross-linking (glutaraldehyde 0.02%, 10 min, room temperature) as an assay for PCNA dependent repair activity. The results were compared to those with human diploid fibroblasts (GM6419) studied under different growth conditions (log-phase, density inhibition) yielding differing p21 levels. Residual damage was assessed by the micronucleus assay. Micronuclei were counted in binucleate cells only, and at least two slides per data point (at least 500 binucleate cells were scored per slide) were examined under the fluorescence microscope. Results: There was no change in total cellular PCNA protein levels up to 48h after irradiation, consistent with predominantly post-translational regulation. Changes in intranuclear distribution and complex formation were observed in a p53/p21 dependent manner. After irradiation, there was an increase in BF PCNA levels up to 2 - 3 times baseline level with a maximum at 30 - 60 min, which could

  9. Epstein-Barr nuclear antigen 1 induces expression of the cellular microRNA hsa-miR-127 and impairing B-cell differentiation in EBV-infected memory B cells. New insights into the pathogenesis of Burkitt lymphoma

    International Nuclear Information System (INIS)

    Epstein-Barr Virus (EBV) is a γ-herpesvirus that infects >90% of the human population. Although EBV persists in its latent form in healthy carriers, the virus is also associated with several human cancers. EBV is strongly associated with Burkitt lymphoma (BL), even though there is still no satisfactory explanation of how EBV participates in BL pathogenesis. However, new insights into the interplay between viruses and microRNAs (miRNAs) have recently been proposed. In particular, it has been shown that B-cell differentiation in EBV-positive BL is impaired at the post-transcriptional level by altered expression of hsa-miR-127. Here, we show that the overexpression of hsa-miR-127 is due to the presence of the EBV-encoded nuclear antigen 1 (EBNA1) and give evidence of a novel mechanism of direct regulation of the human miRNA by this viral product. Finally, we show that the combinatorial expression of EBNA1 and hsa-miR-127 affects the expression of master B-cell regulators in human memory B cells, confirming the scenario previously observed in EBV-positive BL primary tumors and cell lines. A good understanding of these mechanisms will help to clarify the complex regulatory networks between host and pathogen, and favor the design of more specific treatments for EBV-associated malignancies

  10. Antígeno nuclear de proliferação celular em tumores de adrenal Proliferating cell nuclear antigen in adrenal tumors

    Directory of Open Access Journals (Sweden)

    Rodrigo A.R. Falconi

    2000-01-01

    Full Text Available Fez-se um estudo imunohistoquímico do antígeno nuclear de proliferação celular (PCNA em 26 adenomas e 24 carcinomas de adrenal através da técnica da avidina-biotina-peroxidase. O índice de marcação (IP do PCNA, definido com o número de células marcadas/1000 contadas, foi em média de 77,4± 66,1 (mediana - 63,5 para os adenomas enquanto que para os carcinomas foi 215,8± 56,0 (mediana - 217,5 (p<0,0001. Estabelecendo-se o IP de 100 o marcador (para a discriminação de carcinomas dos adenomas o marcador exibiu sensibilidade, especificidade e valor preditivo positivo seguintes, respectivamente: 100%, 69% e 75%.

  11. Galactosylated LDL nanoparticles: a novel targeting delivery system to deliver antigen to macrophages and enhance antigen specific T cell responses

    OpenAIRE

    Wu, Fang; Wuensch, Sherry A.; Azadniv, Mitra; Ebrahimkhani, Mohammad R.; Crispe, I. Nicholas

    2009-01-01

    We aim to define the role of Kupffer cells in intrahepatic antigen presentation, using the selective delivery of antigen to Kupffer cells rather than other populations of liver antigen-presenting cells. To achieve this we developed a novel antigen delivery system that can target antigens to macrophages, based on a galactosylated low-density lipoprotein nano-scale platform. Antigen was delivered via the galactose particle receptor (GPr), internalized, degraded and presented to T cells. The con...

  12. Antigen expression on recurrent meningioma cells

    International Nuclear Information System (INIS)

    Meningiomas are intracranial brain tumours that frequently recur. Recurrence rates up to 20% in 20 years for benign meningiomas, up to 80% for atypical meningiomas and up to 100% for malignant meningiomas, have been reported. The most important prognostic factors for meningioma recurrence are meningioma grade, meningioma invasiveness and radicality of neurosurgical resection. The aim of our study was to evaluate the differences in antigenic expression on the surface of meningioma cells between recurrent and non-recurrent meningiomas. 19 recurrent meningiomas and 35 non-recurrent meningiomas were compared regarding the expression of MIB-1 antigen, progesterone receptors, cathepsin B and cathepsin L, using immunohistochemistry. MIB-1 antigen expression was higher in the recurrent meningioma group (p=0.001). No difference in progesterone receptor status between recurrent and non-recurrent meningiomas was confirmed. Immunohistochemical intensity scores for cathepsin B (p= 0.007) and cathepsin L (p<0.001) were both higher in the recurrent than in the non-recurrent meningioma group. MIB-1 antigen expression is higher in recurrent compared to non-recurrent meningiomas. There is no difference in expression of progesterone receptors between recurrent and non-recurrent meningiomas. Cathepsins B and L are expressed more in recurrent meningiomas

  13. 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.

  14. Expressions of Proliferating Cell Nuclear Antigen and Wheat Germ Agglutinin Receptor in Human Bladder Carcinoma%膀胱癌增殖细胞核抗原与麦胚凝集素受体的相关关系

    Institute of Scientific and Technical Information of China (English)

    张士文; 葛根; 金伯涛

    2001-01-01

    [Purpose]To probe the relation of proliferating cell nuclear antigen (PCNA) and wheat germ agglutinin (WGA) receptors expressed in human bladder transitional cell carcinoma (TCC).[Methods]PCNA and WGA receptors were detected by immunohistochemical method (ABC method) in 63 specimens of TCC.[Results]We found that the distributions of PCNA and WGA receptors were increased with increase of histopathological grade in TCC (P<0.01).There was a higher expression in invasive tumors than that in superficial tumors (P<0.005),and there was a positive relation between PCNA and WGA receptors also.[Conclusion]It is shown that PCNA and WGA can be used as tumor markers for bladder cancer.%[目的 ]探讨增殖细胞核抗原 (proliferating cell nuclear antigen,PCNA)和麦胚凝集素 (wheat germ agglutinin,WGA)在膀胱移行细胞癌 (TCC)中表达的相关关系。 [方法 ]采用免疫组织化学 ABC法对 63例 TCC标本进行 PCNA和 WGA受体检测。 [结果 ]PCNA与 WGA的强阳性表达随着肿瘤的病理分级升高而增高;浸润性肿瘤中的 WGA受体的强阳性表达显著高于浅表性肿瘤 (P<0.05); PCNA与 WGA受体表达一致性良好,呈显著性相关 (P<0.005)。 [结论 ]我们认为 PCNA和 WGA受体均可作为 TCC的肿瘤标记物,证明了 TCC细胞的增殖活性增强将改变其细胞膜的抗原性。

  15. Demonstration of cytoplasmic and nuclear antigens in acute leukaemia using flow cytometry.

    Science.gov (United States)

    Farahat, N; van der Plas, D; Praxedes, M; Morilla, R; Matutes, E; Catovsky, D

    1994-01-01

    AIMS--To detect cytoplasmic and nuclear antigens using flow cytometry in acute leukaemia and to use this technique for double marker combinations. METHODS--Cytoplasmic staining was carried out in samples from 40 cases of acute leukaemia with monoclonal antibodies against the myeloid antigen CD13, the lymphoid antigens CD3, CD22, mu chain and the enzymes terminal deoxynucleotidyl transferase (TdT) and myeloperoxidase (MPO). The cells were fixed with paraformaldehyde and permeabilised with Tween 20 and Becton Dickinson's FACS lysing solution. Flow cytometry results were compared in the same cases with immunocytochemistry results using the alkaline phosphatase anti-alkaline phosphatase method. RESULTS--The gentle permeabilisation induced by this method permitted preservation of the membrane antigens and the size and morphology of the cells. The results using flow cytometry were comparable with those obtained using immunocytochemistry, with nearly complete concordance in most cases. CONCLUSIONS--This technique is simple, rapid, sensitive and reproducible and it is suitable for double staining procedures, such as nuclear and cytoplasmic, nuclear and membrane, or cytoplasmic and membrane. It therefore provides a powerful tool for extending the use of immunophenotyping for the diagnosis and follow up of acute leukaemia. It could also be used for the investigation of minimal residual disease. PMID:7962655

  16. Proliferating cell nuclear antigen (PCNA) interacts with a meiosis-specific RecA homologues, Lim15/Dmc1, but does not stimulate its strand transfer activity

    International Nuclear Information System (INIS)

    PCNA is a multi-functional protein that is involved in various nuclear events. Here we show that PCNA participates in events occurring during early meiotic prophase. Analysis of protein-protein interactions using surface plasmon resonance indicates that Coprinus cinereus PCNA (CoPCNA) specifically interacts with a meiotic specific RecA-like factor, C. cinereus Lim15/Dmc1 (CoLim15) in vitro. The binding efficiency increases with addition of Mg2+ ions, while ATP inhibits the interaction. Co-immunoprecipitation experiments indicate that the CoLim15 protein interacts with the CoPCNA protein in vitro and in the cell extracts. Despite the interaction between these two factors, no enhancement of CoLim15-dependent strand transfer activity by CoPCNA was found in vitro. We propose that the interaction between Lim15/Dmc1 and PCNA mediates the recombination-associated DNA synthesis during meiosis

  17. Immunoblotting profiles in 55 systemic lupus erythematosus sera lacking precipitating antibodies to extractable nuclear antigens.

    OpenAIRE

    Meyer, O.; Bourgeois, P; Aeschlimann, A.; Haim, T; Mery, J P; Kahn, M F

    1989-01-01

    Serum samples from 55 patients with systemic lupus erythematosus (SLE) were selected for the absence of anti-extractable nuclear antigen antibodies after routine immunodiffusion tests. These sera were immunoblotted for anti-Sm and anti-RNP antibodies on a HeLa cell nuclear extract. Ten (18%) were negative and 45 (82%) produced complex patterns: 10 (18%) suggestive of anti-Sm, three (5%) anti-RNP, and 32 (58%) a combination of anti-Sm and anti-RNP antibodies. These data were very similar to th...

  18. Antigen

    Science.gov (United States)

    An antigen is any substance that causes your immune system to produce antibodies against it. This means your immune ... and is trying to fight it off. An antigen may be a substance from the environment, such ...

  19. Photoaffinity labeling demonstrates binding between Ia and antigen on antigen-presenting cells

    International Nuclear Information System (INIS)

    Antigen-presenting cells (APCs) bind and present antigens to immunocompetent T lymphocytes in the context of Ia molecules: however, the molecular nature of the immunogenic complexes on the surface of these cells is unknown. They have used radioiodinated photoreactive Beef insulin (BI) derivatized in the B29 position with (n-[4-(4'-azido-3'-[125]iodophenylazo)benzoyl]-3-aminopropyl-n-oxy-succinimide) (B29-AZAP) as antigen to examine the nature of these molecular complexes. The probe was reacted with either of two B hybridoma APCs, TA3 (Ia/sup k/d/) and LB(Ia/sup d/b/) which present insulin on I-A/sup d/ and I-A/sub b/ respectively, to appropriately restricted, BI specific T helper lymphocytes (T/sub H/). Samples were photolyzed, solubilized and then analyzed by SDS-PAGE. Two protein bands of 36-kDa and 27-kDa were specifically labeled on TA3 and LB cells. Treatment of these bands with dithiothreitol or endo-N-β-glycosidase F demonstrates that each is composed of a single glycoprotein. These bands are immunoprecipitable with haplotype specific but not control anti-Ia antibodies. This identifies the labeled bands as the α- and β- subunits of class II MHC antigens. They conclude that a molecular complex may form between Ia and antigen on APCs and that formation of this complex does not require the presence of an antigen specific T/sub H/ cell receptor

  20. Neutrophil elastase enhances antigen presentation by upregulating human leukocyte antigen class I expression on tumor cells.

    Science.gov (United States)

    Chawla, Akhil; Alatrash, Gheath; Philips, Anne V; Qiao, Na; Sukhumalchandra, Pariya; Kerros, Celine; Diaconu, Iulia; Gall, Victor; Neal, Samantha; Peters, Haley L; Clise-Dwyer, Karen; Molldrem, Jeffrey J; Mittendorf, Elizabeth A

    2016-06-01

    Neutrophil elastase (NE) is an innate immune cell-derived inflammatory mediator that we have shown increases the presentation of tumor-associated peptide antigens in breast cancer. In this study, we extend these observations to show that NE uptake has a broad effect on enhancing antigen presentation by breast cancer cells. We show that NE increases human leukocyte antigen (HLA) class I expression on the surface of breast cancer cells in a concentration and time-dependent manner. HLA class I upregulation requires internalization of enzymatically active NE. Western blots of NE-treated breast cancer cells confirm that the expression of total HLA class I as well as the antigen-processing machinery proteins TAP1, LMP2, and calnexin does not change following NE treatment. This suggests that NE does not increase the efficiency of antigen processing; rather, it mediates the upregulation of HLA class I by stabilizing and reducing membrane recycling of HLA class I molecules. Furthermore, the effects of NE extend beyond breast cancer since the uptake of NE by EBV-LCL increases the presentation of HLA class I-restricted viral peptides, as shown by their increased sensitivity to lysis by EBV-specific CD8+ T cells. Together, our results show that NE uptake increases the responsiveness of breast cancer cells to adaptive immunity by broad upregulation of membrane HLA class I and support the conclusion that the innate inflammatory mediator NE enhances tumor cell recognition and increases tumor sensitivity to the host adaptive immune response. PMID:27129972

  1. Dendritic cell function and antigen presentation in malaria.

    Science.gov (United States)

    Cockburn, Ian A; Zavala, Fidel

    2016-06-01

    Due to the diverse roles T cells play in protection against malaria as well as pathogenesis it is critical to know which cells present antigen and the nature of the antigens they present. During pre-erythrocytic stages of infection, cutting-edge imaging studies have shown how Plasmodium antigens are presented during both the priming and effector phases of the protective CD8+ T cell response. During blood stages, pathology is in part due to the loss of DC function and the action of pathogenic T cells in the brain. Recently endothelial cells presenting malaria antigen to cognate T cells have emerged as critical players in malaria pathogenesis. Manipulating these processes may inform both vaccine design and the development of therapies for cerebral malaria. PMID:26845735

  2. The effect of Chinese herbal medicine"heche assisted pregnancy recipe"on endometrial estrogen and progesterone receptor, proliferating cell nuclear antigen and vascular endothelial growth factor in the patients with infertility

    Institute of Scientific and Technical Information of China (English)

    刘效群; 阚国英; 彭玉梅; 樊瑞琴; 齐惠敏; 焦妹芬; 李忠; 石彬; 尹桂然; 董锡月

    2003-01-01

    Objectives:To investigate the effect of Chinese herbal medicine"heche assisted preg-nancy recipe (HCAPR)" on estrogen receptor(ER), progesterone receptor (PR), pro-lifierating cell nuclear antigen(PCNA) and vascular endothelial growth factor (VEGF)in endometrium of infertile women.Methods: The S-P immunohistochemical assay was used to observe expression ofER, PR , PCNA and VEGF in late proliferative phase before and after the HCAPR treat-ment.Results: After the treatment, the expression of ER,PR,PCNA and VEGF in nucleiof glandular epithelium and stromal cells was significantly stronger (all P<0. 001) re-spectively than that before treatment , especially the expression of PCNA and VEGF.Conclusions: These results suggest that traditional Chinese medicine HCAPR oftonifying kidney and regulating menstruation increased the synthesis of ER,PR, PCNAand VEGF, which may promote normal growth and development of the endometrium ,improve the micro-environment of the endometrium, and enhance uterine receptivity.The evidence may provide theoretical basis for therapy infertility with Chinese herbalmedicine.

  3. Carbohydrate-functionalized nanovaccines preserve HIV-1 antigen stability and activate antigen presenting cells.

    Science.gov (United States)

    Vela Ramirez, J E; Roychoudhury, R; Habte, H H; Cho, M W; Pohl, N L B; Narasimhan, B

    2014-01-01

    The functionalization of polymeric nanoparticles with ligands that target specific receptors on immune cells offers the opportunity to tailor adjuvant properties by conferring pathogen mimicking attributes to the particles. Polyanhydride nanoparticles are promising vaccine adjuvants with desirable characteristics such as immunomodulation, sustained antigen release, activation of antigen presenting cells (APCs), and stabilization of protein antigens. These capabilities can be exploited to design nanovaccines against viral pathogens, such as HIV-1, due to the important role of dendritic cells (DCs) and macrophages in viral spread. In this work, an optimized process was developed for carbohydrate functionalization of HIV-1 antigen-loaded polyanhydride nanoparticles. The carbohydrate-functionalized nanoparticles preserved antigenic properties upon release and also enabled sustained antigen release kinetics. Particle internalization was observed to be chemistry-dependent with positively charged nanoparticles being taken up more efficiently by DCs. Up-regulation of the activation makers CD40 and CD206 was demonstrated with carboxymethyl-α-d-mannopyranosyl-(1,2)-d-mannopyranoside functionalized nanoparticles. The secretion of the cytokines IL-6 and TNF-α was shown to be chemistry-dependent upon stimulation with carbohydrate-functionalized nanoparticles. These results offer important new insights upon the interactions between carbohydrate-functionalized nanoparticles and APCs and provide foundational information for the rational design of targeted nanovaccines against HIV-1. PMID:25068589

  4. Photoaffinity labeling demonstrates binding between Ia molecules and nominal antigen on antigen-presenting cells.

    OpenAIRE

    Phillips, M L; Yip, C C; Shevach, E M; Delovitch, T L

    1986-01-01

    We have used radioiodinated photoreactive bovine insulin as antigen to examine the molecular nature of immunogenic complexes that form on antigen-presenting cells. The probe was allowed to bind to either insulin-presenting B-hybridoma cells, lipopolysaccharide-stimulated blasts, or bovine insulin-specific helper-T-hybridoma cells in the dark. Samples were then exposed to light to induce crosslinkage, solubilized, and analyzed by gel electrophoresis. Two protein bands at about 36 kDa and 27 kD...

  5. Distribution of primed T cells and antigen-loaded antigen presenting cells following intranasal immunization in mice.

    Directory of Open Access Journals (Sweden)

    Annalisa Ciabattini

    Full Text Available Priming of T cells is a key event in vaccination, since it bears a decisive influence on the type and magnitude of the immune response. T-cell priming after mucosal immunization via the nasal route was studied by investigating the distribution of antigen-loaded antigen presenting cells (APCs and primed antigen-specific T cells. Nasal immunization studies were conducted using the model protein antigen ovalbumin (OVA plus CpG oligodeoxynucleotide adjuvant. Trafficking of antigen-specific primed T cells was analyzed in vivo after adoptive transfer of OVA-specific transgenic T cells in the presence or absence of fingolimod, a drug that causes lymphocytes sequestration within lymph nodes. Antigen-loaded APCs were observed in mediastinal lymph nodes, draining the respiratory tract, but not in distal lymph nodes. Antigen-specific proliferating T cells were first observed within draining lymph nodes, and later in distal iliac and mesenteric lymph nodes and in the spleen. The presence at distal sites was due to migration of locally primed T cells as shown by fingolimod treatment that caused a drastic reduction of proliferated T cells in non-draining lymph nodes and an accumulation of extensively divided T cells within draining lymph nodes. Homing of nasally primed T cells in distal iliac lymph nodes was CD62L-dependent, while entry into mesenteric lymph nodes depended on both CD62L and α4β7, as shown by in vivo antibody-mediated inhibition of T-cell trafficking. These data, elucidating the trafficking of antigen-specific primed T cells to non-draining peripheral and mucosa-associated lymph nodes following nasal immunization, provide relevant insights for the design of vaccination strategies based on mucosal priming.

  6. The role of antigen in the development of B-cell chronic lymphocytic leukemia

    OpenAIRE

    Hoogeboom, R.

    2013-01-01

    These studies strongly suggest that MALT-lymphomas and M-CLL in majority are highly selected for single extrinsic antigens and that these antigens can be both self-antigens and exo-antigens. Our finding that primary CLL cells are responsive to stimulation with their cognate antigen suggests that antigen-dependent BCR signaling may drive CLL expansion in vivo.

  7. Delayed type hypersensitivity to allogeneic mouse epidermal cell antigens, 2

    International Nuclear Information System (INIS)

    A low dose of ultraviolet B radiation impairs the effectiveness of epidermal cell antigens. We studied the effect of ultraviolet B radiation on the delayed type hypersensitivity induced by allogeneic epidermal cell antigen. The delayed type hypersensitivity response was assayed by footpad swelling in mice. When epidermal cells were exposed to ultraviolet B radiation (660 J/m2), their ability to induce T cells of delayed type hypersensitivity activation was markedly inhibited in any combination of recipient mice and allogeneic epidermal cells. The effect of ultraviolet B radiation on epidermal cells was observed before immunization and challenge. Ultraviolet B treated epidermal cells did not induce suppressor T cells in mice. These results indicate that ultraviolet B radiation destroys the antigenicity of epidermal cells. (author)

  8. Control of T cell antigen reactivity via programmed TCR downregulation.

    Science.gov (United States)

    Gallegos, Alena M; Xiong, Huizhong; Leiner, Ingrid M; Sušac, Bože; Glickman, Michael S; Pamer, Eric G; van Heijst, Jeroen W J

    2016-04-01

    The T cell antigen receptor (TCR) is unique in that its affinity for ligand is unknown before encounter and can vary by orders of magnitude. How the immune system regulates individual T cells that display very different reactivity to antigen remains unclear. Here we found that activated CD4(+) T cells, at the peak of clonal expansion, persistently downregulated their TCR expression in proportion to the strength of the initial antigen recognition. This programmed response increased the threshold for cytokine production and recall proliferation in a clone-specific manner and ultimately excluded clones with the highest antigen reactivity. Thus, programmed downregulation of TCR expression represents a negative feedback mechanism for constraining T cell effector function with a suitable time delay to thereby allow pathogen control while avoiding excess inflammatory damage. PMID:26901151

  9. Modulatory Effect of Taurine on 7,12-Dimethylbenz(a)Anthracene-Induced Alterations in Detoxification Enzyme System, Membrane Bound Enzymes, Glycoprotein Profile and Proliferative Cell Nuclear Antigen in Rat Breast Tissue.

    Science.gov (United States)

    Vanitha, Manickam Kalappan; Baskaran, Kuppusamy; Periyasamy, Kuppusamy; Selvaraj, Sundaramoorthy; Ilakkia, Aruldoss; Saravanan, Dhiravidamani; Venkateswari, Ramachandran; Revathi Mani, Balasundaram; Anandakumar, Pandi; Sakthisekaran, Dhanapal

    2016-08-01

    The modulatory effect of taurine on 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast cancer in rats was studied. DMBA (25 mg/kg body weight) was administered to induce breast cancer in rats. Protein carbonyl levels, activities of membrane bound enzymes (Na(+) /K(+) ATPase, Ca(2+) ATPase, and Mg(2+) ATPase), phase I drug metabolizing enzymes (cytochrome P450, cytochrome b5, NADPH cytochrome c reductase), phase II drug metabolizing enzymes (glutathione-S-transferase and UDP-glucuronyl transferase), glycoprotein levels, and proliferative cell nuclear antigen (PCNA) were studied. DMBA-induced breast tumor bearing rats showed abnormal alterations in the levels of protein carbonyls, activities of membrane bound enzymes, drug metabolizing enzymes, glycoprotein levels, and PCNA protein expression levels. Taurine treatment (100 mg/kg body weight) appreciably counteracted all the above changes induced by DMBA. Histological examination of breast tissue further supported our biochemical findings. The results of the present study clearly demonstrated the chemotherapeutic effect of taurine in DMBA-induced breast cancer. PMID:27091720

  10. Phosphorylation of the Epstein-Barr virus nuclear antigen 2

    DEFF Research Database (Denmark)

    Grässer, F A; Göttel, S; Haiss, P;

    1992-01-01

    A major in vivo phosphorylation site of the Epstein-Barr virus nuclear antigen 2 (EBNA-2) was found to be localized at the C-terminus of the protein. In vitro phosphorylation studies using casein kinase 1 (CK-1) and casein kinase 2 (CK-2) revealed that EBNA-2 is a substrate for CK-2, but not for CK......-1. The CK-2 specific phosphorylation site was localized in the 140 C-terminal amino acids using a recombinant trpE-C-terminal fusion protein. In a similar experiment, the 58 N-terminal amino acids expressed as a recombinant trpE-fusion protein were not phosphorylated. Phosphorylation of a synthetic...

  11. Galactosylated LDL nanoparticles: a novel targeting delivery system to deliver antigen to macrophages and enhance antigen specific T cell responses.

    Science.gov (United States)

    Wu, Fang; Wuensch, Sherry A; Azadniv, Mitra; Ebrahimkhani, Mohammad R; Crispe, I Nicholas

    2009-01-01

    We aim to define the role of Kupffer cells in intrahepatic antigen presentation, using the selective delivery of antigen to Kupffer cells rather than other populations of liver antigen-presenting cells. To achieve this we developed a novel antigen delivery system that can target antigens to macrophages, based on a galactosylated low-density lipoprotein nanoscale platform. Antigen was delivered via the galactose particle receptor (GPr), internalized, degraded and presented to T cells. The conjugation of fluoresceinated ovalbumin (FLUO-OVA) and lactobionic acid with LDL resulted in a substantially increased uptake of FLUO-OVA by murine macrophage-like ANA1 cells in preference to NIH3T3 cells, and by primary peritoneal macrophages in preference to primary hepatic stellate cells. Such preferential uptake led to enhanced proliferation of OVA specific T cells, showing that the galactosylated LDL nanoscale platform is a successful antigen carrier, targeting antigen to macrophages but not to all categories of antigen presenting cells. This system will allow targeted delivery of antigen to macrophages in the liver and elsewhere, addressing the question of the role of Kupffer cells in liver immunology. It may also be an effective way of delivering drugs or vaccines directly at macrophages. PMID:19637876

  12. 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 thyroidi......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...

  13. CD8+ T cell priming by dendritic cell vaccines requires antigen transfer to endogenous antigen presenting cells.

    Directory of Open Access Journals (Sweden)

    Alice W Yewdall

    Full Text Available Immunotherapeutic strategies to stimulate anti-tumor immunity are promising approaches for cancer treatment. A major barrier to their success is the immunosuppressive microenvironment of tumors, which inhibits the functions of endogenous dendritic cells (DCs that are necessary for the generation of anti-tumor CD8+ T cells. To overcome this problem, autologous DCs are generated ex vivo, loaded with tumor antigens, and activated in this non-suppressive environment before administration to patients. However, DC-based vaccines rarely induce tumor regression.We examined the fate and function of these DCs following their injection using murine models, in order to better understand their interaction with the host immune system. Contrary to previous assumptions, we show that DC vaccines have an insignificant role in directly priming CD8+ T cells, but instead function primarily as vehicles for transferring antigens to endogenous antigen presenting cells, which are responsible for the subsequent activation of T cells.This reliance on endogenous immune cells may explain the limited success of current DC vaccines to treat cancer and offers new insight into how these therapies can be improved. Future approaches should focus on creating DC vaccines that are more effective at directly priming T cells, or abrogating the tumor induced suppression of endogenous DCs.

  14. Epstein-Barr Virus nuclear antigen 1 (EBNA1) confers resistance to apoptosis in EBV-positive B-lymphoma cells through up-regulation of survivin

    International Nuclear Information System (INIS)

    Resistance to apoptosis is an important component of the overall mechanism which drives the tumorigenic process. EBV is a ubiquitous human gamma-herpesvirus which preferentially establishes latent infection in viral infected B-lymphocytes. EBNA1 is typically expressed in most forms of EBV-positive malignancies and is important for replication of the latent episome in concert with replication of the host cells. Here, we investigate the effects of EBNA1 on survivin up-regulation in EBV-infected human B-lymphoma cells. We present evidence which demonstrates that EBNA1 forms a complex with Sp1 or Sp1-like proteins bound to their cis-element at the survivin promoter. This enhances the activity of the complex and up-regulates survivin. Knockdown of survivin and EBNA1 showed enhanced apoptosis in infected cells and thus supports a role for EBNA1 in suppressing apoptosis in EBV-infected cells. Here, we suggest that EBV encoded EBNA1 can contribute to the oncogenic process by up-regulating the apoptosis suppressor protein, survivin in EBV-associated B-lymphoma cells.

  15. Antigen-Specific CD4+ T Cells Recognize Epitopes of Protective Antigen following Vaccination with an Anthrax Vaccine

    OpenAIRE

    Laughlin, Elsa M.; Miller, Joseph D.; James, Eddie; Fillos, Dimitri; Ibegbu, Chris C.; Mittler, Robert S.; Akondy, Rama; Kwok, William; Ahmed, Rafi; Nepom, Gerald,

    2007-01-01

    Detection of antigen-specific CD4+ T cells is facilitated by the use of fluorescently labeled soluble peptide-major histocompatibility complex (MHC) multimers which mirror the antigen specificity of T-cell receptor recognition. We have used soluble peptide-MHC class II tetramers containing peptides from the protective antigen (PA) of Bacillus anthracis to detect circulating T cells in peripheral blood of subjects vaccinated with an anthrax vaccine. PA-specific HLA class II-restricted T lympho...

  16. T Cells Expressing CD19/CD20 Bispecific Chimeric Antigen Receptors Prevent Antigen Escape by Malignant B Cells.

    Science.gov (United States)

    Zah, Eugenia; Lin, Meng-Yin; Silva-Benedict, Anne; Jensen, Michael C; Chen, Yvonne Y

    2016-06-01

    The adoptive transfer of T cells expressing anti-CD19 chimeric antigen receptors (CARs) has shown remarkable curative potential against advanced B-cell malignancies, but multiple trials have also reported patient relapses due to the emergence of CD19-negative leukemic cells. Here, we report the design and optimization of single-chain, bispecific CARs that trigger robust cytotoxicity against target cells expressing either CD19 or CD20, two clinically validated targets for B-cell malignancies. We determined the structural parameters required for efficient dual-antigen recognition, and we demonstrate that optimized bispecific CARs can control both wild-type B-cell lymphoma and CD19(-) mutants with equal efficiency in vivo To our knowledge, this is the first bispecific CAR capable of preventing antigen escape by performing true OR-gate signal computation on a clinically relevant pair of tumor-associated antigens. The CD19-OR-CD20 CAR is fully compatible with existing T-cell manufacturing procedures and implementable by current clinical protocols. These results present an effective solution to the challenge of antigen escape in CD19 CAR T-cell therapy, and they highlight the utility of structure-based rational design in the development of receptors with higher-level complexity. Cancer Immunol Res; 4(6); 498-508. ©2016 AACRSee related Spotlight by Sadelain, p. 473. PMID:27059623

  17. Functional Development of the T Cell Receptor for Antigen

    Science.gov (United States)

    Ebert, Peter J.R.; Li, Qi-Jing; Huppa, Johannes B.; Davis, Mark M.

    2016-01-01

    For over three decades now, the T cell receptor (TCR) for antigen has not ceased to challenge the imaginations of cellular and molecular immunologists alike. T cell antigen recognition transcends every aspect of adaptive immunity: it shapes the T cell repertoire in the thymus and directs T cell-mediated effector functions in the periphery, where it is also central to the induction of peripheral tolerance. Yet, despite its central position, there remain many questions unresolved: how can one TCR be specific for one particular peptide-major histocompatibility complex (pMHC) ligand while also binding other pMHC ligands with an immunologically relevant affinity? And how can a T cell’s extreme specificity (alterations of single methyl groups in their ligand can abrogate a response) and sensitivity (single agonist ligands on a cell surface are sufficient to trigger a measurable response) emerge from TCR–ligand interactions that are so low in affinity? Solving these questions is intimately tied to a fundamental understanding of molecular recognition dynamics within the many different contexts of various T cell–antigen presenting cell (APC) contacts: from the thymic APCs that shape the TCR repertoire and guide functional differentiation of developing T cells to the peripheral APCs that support homeostasis and provoke antigen responses in naïve, effector, memory, and regulatory T cells. Here, we discuss our recent findings relating to T cell antigen recognition and how this leads to the thymic development of foreign-antigen-responsive αβT cells. PMID:20800817

  18. Typing of murine cell-surface antigens by cellular radioimmunoassay

    International Nuclear Information System (INIS)

    A cellular radioimmunoassay utilizing 125I-labelled Protein A was used for detecting antigen-antibody complexes on gultaraldehyde fixed cells attached to microtiter plates. This method is rapid, sensitive and specific for revealing H-2 private and public specificities as well as Ia and Lyt antigens. As plates may be kept for months, several reactivities can be tested in one step on a large panel rendering a regular supply of animals unnecessary. (Auth.)

  19. Reassessing target antigens for adoptive T cell therapy

    Science.gov (United States)

    Hinrichs, Christian S.; Restifo, Nicholas P.

    2014-01-01

    Adoptive T cell therapy can target and kill widespread malignant cells thereby inducing durable clinical responses in melanoma and selected other malignances. However, many commonly targeted tumor antigens are also expressed by healthy tissues, and T cells do not distinguish between benign and malignant tissues if both express the target antigen. As such, autoimmune toxicity from T-cell-mediated destruction of normal tissue has limited the development and adoption of this otherwise promising type of cancer therapy. A review of the unique biology of T-cell therapy and of recent clinical experience compels a reassessment of target antigens that traditionally have been viewed from the perspective of weaker immunotherapeutic modalities. In selecting target antigens for adoptive T-cell therapy, expression by tumors and not by essential healthy tissues is of paramount importance. The risk of autoimmune adverse events can be further mitigated by generating antigen receptors using strategies that reduce the chance of cross-reactivity against epitopes in unintended targets. In general, a circumspect approach to target selection and thoughtful preclinical and clinical studies are pivotal to the ongoing advancement of these promising treatments. PMID:24142051

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

    Directory of Open Access Journals (Sweden)

    Koh Yih Yih

    2014-01-01

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

  1. 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.

  2. The relationship between sirtuin 1 (SIRT1 expression and tumor size, Proliferating Cell Nuclear Antigen (PCNA expression and histological grading in rat breast carcinoma induced by dimethylbenz(anthracene (DMBA

    Directory of Open Access Journals (Sweden)

    Novrita Padauleng Dewajani Purnomosari, Sri Herwiyanti Harjadi, Irianiwati, Sitarina Widyarini

    2014-08-01

    Full Text Available Controversy regarding the role of SIRT1 in pathology of cancers exists and is still under debate.SIRT1 could act as either a tumor supressor or tumor promotor. This study was conducted toevaluate the relationship between SIRT1 expression and tumor size, Proliferating Cell NuclearAntigen (PCNA expression and histological grading in rat breast carcinoma induced bydimethylbenz(áanthracene (DMBA. Thirty female Sprague Dawley rats were randomly allocatedinto three groups with 10 rats in each group. Group 1 as negative control was just fed thestandard food. Group 2 as vehicle control was fed the standard food and corn oil. Group 3 asinduction group was fed the standard food and induced with DMBA at dose of 20 mg/kg bodyweight (BW in corn oil twice a week for five weeks. All rats were palpated weekly to determinethe appearance, size and location of tumors. Sixteen weeks after DMBA induction rats weresacrified and histological preparations of the breast carcinoma tissue were then processed forSIRT1 and PCNA expression examination as well as histological grading. The result showed thatSIRT1 expression was significantly higher in breast carcinoma tissue compared to normal gland(26.12 vs 0.05; p = 0.004. SIRT1-positive was observed mostly in poor histological gradecarcinomas (56.2%, and it was not observed in good histological grade carcinomas. However,there was no significantly difference between SIRT1 and histological grading (p = 0.097; r =0.285. A significant correlation between SIRT1 expression and the tumor size (p =0.009; r=0.877, as well as PCNA expression (p =0.000; r =0.790 was observed. In conclusion, thereis relationship between SIRT1 expression and tumor size as well as PCNA expression in rat breastcarcinoma induced by DMBA.

  3. Chimeric Antigen Receptors Modified T-Cells for Cancer Therapy.

    Science.gov (United States)

    Dai, Hanren; Wang, Yao; Lu, Xuechun; Han, Weidong

    2016-07-01

    The genetic modification and characterization of T-cells with chimeric antigen receptors (CARs) allow functionally distinct T-cell subsets to recognize specific tumor cells. The incorporation of costimulatory molecules or cytokines can enable engineered T-cells to eliminate tumor cells. CARs are generated by fusing the antigen-binding region of a monoclonal antibody (mAb) or other ligand to membrane-spanning and intracellular-signaling domains. They have recently shown clinical benefit in patients treated with CD19-directed autologous T-cells. Recent successes suggest that the modification of T-cells with CARs could be a powerful approach for developing safe and effective cancer therapeutics. Here, we briefly review early studies, consider strategies to improve the therapeutic potential and safety, and discuss the challenges and future prospects for CAR T-cells in cancer therapy. PMID:26819347

  4. CD4+ and CD8+ T-Cell Responses to Latent Antigen EBNA-1 and Lytic Antigen BZLF-1 during Persistent Lymphocryptovirus Infection of Rhesus Macaques

    OpenAIRE

    Leskowitz, R. M.; Zhou, X. Y.; Villinger, F; Fogg, M. H.; Kaur, A; Lieberman, P. M.; Wang, F.; Ertl, H. C.

    2013-01-01

    Epstein-Barr virus (EBV) infection leads to lifelong viral persistence through its latency in B cells. EBV-specific T cells control reactivations and prevent the development of EBV-associated malignancies in most healthy carriers, but infection can sometimes cause chronic disease and malignant transformation. Epstein-Barr nuclear antigen 1 (EBNA-1) is the only viral protein consistently expressed during all forms of latency and in all EBV-associated malignancies and is a promising target for ...

  5. Antigenicity and immunogenicity of an extract from the cell wall and cell membrane of Histoplasma capsulatum yeast cells.

    OpenAIRE

    Gómez, A M; Rhodes, J C; Deepe, G S

    1991-01-01

    In order to identify T-cell antigens from Histoplasma capsulatum yeast cells, we prepared a detergent extract of the cell wall and cell membrane of yeast-phase H. capsulatum G217B and analyzed its antigenicity and immunogenicity. Mice injected with viable H. capsulatum yeast cells or with 500 or 1,000 micrograms of the extract mounted a delayed-type hypersensitivity response to solubilized cell wall and cell membrane. Vaccination with this antigenic preparation conferred a protective immune r...

  6. 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...... fragmentation. Some of the antigen fragments bind to MHC class II molecules and are transported to the surface of the antigen-presenting cell where the actual presentation to T lymphocytes occurs. The nature of the processed antigen, how and where it is derived and subsequently becomes associated with MHC...... molecules are the questions discussed in this review. To us, the entire concept of processing has appeal not only because it explains some hitherto well-established, but poorly understood, phenomena such as the fact that T lymphocytes focus their attention entirely upon antigens on other cells. It has...

  7. Cross-presentation of cell-associated antigens by MHC class I in dendritic cell subsets

    Directory of Open Access Journals (Sweden)

    Enric eGutiérrez-Martínez

    2015-07-01

    Full Text Available Dendritic cells have the unique ability to pick up dead cells carrying antigens in tissue and migrate to the lymph nodes where they can cross-present cell-associated antigens by MHC class I to CD8+ T cells. There is strong in vivo evidence that the mouse XCR1+ dendritic cells subset acts as a key player in this process. The intracellular processes underlying cross-presentation remain controversial and several pathways have been proposed. Indeed, a wide number of studies have addressed the cellular process of cross-presentation in vitro using a variety of sources of antigen and antigen presenting cells. Here we review the in vivo and in vitro evidence supporting the current mechanistic models and disscuss their physiological relevance to the cross-presentation of cell-associated antigens by dendritic cells subsets

  8. Labeling and use of monoclonal antibodies in immunofluorescence: protocols for cytoskeletal and nuclear antigens.

    Science.gov (United States)

    Bauer, Christoph R

    2014-01-01

    Antibodies are widely used to target and label specifically extra- or intracellular antigens within cells and tissues. Most protocols follow an indirect approach implying the successive incubation with primary and secondary antibodies. In these protocols the primary antibodies are specifically targeted against the antigen in question and are normally not labeled. The secondary antibodies come from a different species and are in contrast fluorescently labeled. The idea is that the primary antibodies specifically bind to their targets but cannot be visualized directly. Only binding of the secondary (fluorescent) antibodies to the constant region of the primary antibodies allows consecutively the visualization in a fluorescent microscope.Primary antibodies can be either of monoclonal (normally produced in mouse) or of polyclonal origin (normally produced in rabbit, goat, sheep, or donkey). Using (primary) monoclonal antibodies has the clear advantage that all antibodies used are identical in origin and behavior and should thus give a more clear-cut labeling result. On the other hand the demands towards labeling protocols might be concomitantly higher: Binding of primary antibodies will only occur if fixation and labeling protocols preserve the antigen sufficiently to keep its specific and unique target structure available. One could imagine that for polyclonal antibodies this demand is slightly lower as there is a pool of antibodies with varying specificities against multiple parts of their target antigens. Certain fractions of this pool might thus tolerate a larger variety of conditions, and consequently a larger variety of protocols might still result in successful labeling.Each step in a labeling protocol can be decisive for the outcome of an experiment especially if monoclonal antibodies are used. Especially critical are choice of buffer and fixation and permeabilization parameters of the protocol.In this chapter we discuss and detail proven protocols using

  9. Development of antibodies to human embryonic stem cell antigens

    OpenAIRE

    Stanley Marisa; Rao Mahendra S; Olson Judith M; Cai Jingli; Taylor Eva; Ni Hsiao-Tzu

    2005-01-01

    Abstract Background Using antibodies to specific protein antigens is the method of choice to assign and identify cell lineage through simultaneous analysis of surface molecules and intracellular markers. Embryonic stem cell research can be benefited from using antibodies specific to transcriptional factors/markers that contribute to the "stemness" phenotype or critical for cell lineage. Results In this report, we have developed and validated antibodies (either monoclonal or polyclonal) specif...

  10. Defective antigen-presenting cell function in human neonates

    OpenAIRE

    Velilla, Paula A.; Rugeles, Maria T.; Chougnet, Claire A.

    2006-01-01

    Immaturity of the immune system has been suggested as an underlying factor for the high rate of morbidity and mortality from infections in newborns. Functional impairment of neonatal T cells is frequently quoted as the main underlying mechanism for such immaturity. However, recent studies suggest that neonatal antigen-presenting cells (APCs) also exhibit functional alterations, which could lead to secondary defects of adaptive T cell responses. In this review, we summarize what is known on th...

  11. Radioimmunoassay for Epstein-Barr Virus (EBV)-associated Nuclear Antigen (EBNA). Binding of iodinated antibodies to antigen immobilized in polyacrylamide gel

    International Nuclear Information System (INIS)

    A solid-phase radioimmunoassay was developed for the EBV-associated nuclear antigen (EBNA). Total homogenates of EBV-DNA and EBNA positive or negative cells were polymerized in polyacrylamide gel and compared for their ability to bind 125I-IgG prepared from anti-EBNA positive and anti-EBNA negative sera. EBNA specific binding was demonstrated and confirmed by serological and cellular specificity controls. The assay allows the quantitation of antigen or antibody even in the presence of detergents and is suitable for biochemical characterization of the antigen. Reciprocal blocking studies with extracts from different cell lines showed quantitative and qualitative differences. One part of the EBNA specificiti(es) present in the human Burkitt lymphoma derived lines RAJI, DAUDI and AW-RAMOS was lacking in B96-8, a marmoset line carrying EBV derived from a human infectious mononucleosis line. This result may reflect differences in the viral genomes derived from Burkitt lymphoma and infectious mononucleosis lines or differences in the host cells. (author)

  12. Antigen-specific T cell activation independently of the MHC: chimeric antigen receptor (CAR-redirected T cells.

    Directory of Open Access Journals (Sweden)

    Hinrich eAbken

    2013-11-01

    Full Text Available Adoptive T cell therapy has recently shown powerful in initiating a lasting anti-tumor response with spectacular therapeutic success in some cases. Specific T cell therapy, however, is limited since a number of cancer cells are not recognized by T cells due to various mechanisms including the limited availability of tumor-specific T cells and deficiencies in antigen processing or major histocompatibility complex (MHC expression of cancer cells. To make adoptive cell therapy applicable for the broad variety of cancer entities, patient's T cells are engineered ex vivo with pre-defined specificity by a recombinant chimeric antigen receptor (CAR which consists in the extracellular part of an antibody-derived domain for binding with a tumor-associated antigen and in the intracellular part of a TCR-derived signaling moiety for T cell activation. The specificity of CAR mediated T cell recognition is defined by the antibody domain, is independent of MHC presentation and can be extended to any target for which an antibody is available. We discuss the advantages and limitations of MHC-independent T cell targeting by an engineered CAR and review most significant progress recently made in early stage clinical trials to treat cancer.

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

    DEFF Research Database (Denmark)

    Andersen, Rikke Sick; Albæk Thrue, Charlotte; Junker, Niels; Skou, Rikke Birgitte Lyngaa; Donia, Marco; Ellebæk, Eva; Svane, Inge Marie; Schumacher, Ton N; Thor Straten, Per; Hadrup, Sine Reker

    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....

  14. Localization of latency-associated nuclear antigen (LANA) on mitotic chromosomes.

    Science.gov (United States)

    Rahayu, Retno; Ohsaki, Eriko; Omori, Hiroko; Ueda, Keiji

    2016-09-01

    In latent infection of Kaposi's sarcoma-associated herpesvirus (KSHV), viral gene expression is extremely limited and copy numbers of viral genomes remain constant. Latency-associated nuclear antigen (LANA) is known to have a role in maintaining viral genome copy numbers in growing cells. Several studies have shown that LANA is localized in particular regions on mitotic chromosomes, such as centromeres/pericentromeres. We independently examined the distinct localization of LANA on mitotic chromosomes during mitosis, using super-resolution laser confocal microscopy and correlative fluorescence microscopy-electron microscopy (FM-EM) analyses. We found that the majority of LANA were not localized at particular regions such as telomeres/peritelomeres, centromeres/pericentromeres, and cohesion sites, but at the bodies of condensed chromosomes. Thus, LANA may undergo various interactions with the host factors on the condensed chromosomes in order to tether the viral genome to mitotic chromosomes and realize faithful viral genome segregation during cell division. PMID:27254595

  15. [GnRH analogues containing SV-40 virus T-antigen nuclear localization sequence].

    Science.gov (United States)

    Burov, S V; Iablokova, T V; Dorosh, M Iu; Kriviziuk, E V; Efremov, A M; Orlov, S V

    2010-01-01

    To improve the efficiency of anticancer drugs due to their delivery to intracellular targets a set of GnRH analogues containing nuclear localization signal (NLS) of SV-40 virus large T-antigen have been synthesized. NLS was attached to the parent molecule via ε-amino group of D-Lysine in position 1 or 6 of peptide sequence using orthogonal protection strategy. The biological activity studies revealed that incorporation of NLS moiety significantly increases cytotoxic activity of palmitoyl-containing GnRH analogues in vitro. The influence of tested peptides on tumor cells does not accompanied by the destruction of cell membrane, as confirmed in experiments with normal fibroblasts, used as a control. PMID:21063449

  16. Immunochemical properties of antigen-specific monkey T-cell suppressor factor induced with a Streptococcus mutans antigen.

    OpenAIRE

    Lamb, J R; Zanders, E D; Kontiainen, S; Lehner, T.

    1980-01-01

    Antigen-specific suppressor factor could be released from monkey suppressor T cells induced in vitro with a protein antigen isolated from the carcinogenic bacterium Streptococcus mutans. The suppressor activity was due to the factor itself and not to carryover of free antigen. Characterization of the monkey factor revealed it to have a molecular weight of ca. 70,000, and to contain a constant region and determinants encoded by the major histocompatibility complex. The presence of immunoglobul...

  17. Human leukocyte antigen-DO regulates surface presentation of human leukocyte antigen class II-restricted antigens on B cell malignancies

    NARCIS (Netherlands)

    Kremer, A.N.; Meijden, E.D. van der; Honders, M.W.; Pont, M.J.; Goeman, J.J.; Falkenburg, J.H.F.; Griffioen, M.

    2014-01-01

    Hematological malignancies often express surface HLA class II, making them attractive targets for CD4+ T cell therapy. We previously demonstrated that HLA class II ligands can be divided into DM-resistant and DM-sensitive antigens. In contrast to presentation of DM-resistant antigens, presentation o

  18. Current Concepts and Future Directions for the Assessment of Autoantibodies to Cellular Antigens Referred to as Anti-Nuclear Antibodies

    OpenAIRE

    Michael Mahler; Pier-Luigi Meroni; Xavier Bossuyt; Fritzler, Marvin J.

    2014-01-01

    The detection of autoantibodies that target intracellular antigens, commonly termed anti-nuclear antibodies (ANA), is a serological hallmark in the diagnosis of systemic autoimmune rheumatic diseases (SARD). Different methods are available for detection of ANA and all bearing their own advantages and limitations. Most laboratories use the indirect immunofluorescence (IIF) assay based on HEp-2 cell substrates. Due to the subjectivity of this diagnostic platform, automated digital reading syste...

  19. Hepatitis B virus antigens impair NK cell function.

    Science.gov (United States)

    Yang, Yinli; Han, Qiuju; Zhang, Cai; Xiao, Min; Zhang, Jian

    2016-09-01

    An inadequate immune response of the host is thought to be a critical factor causing chronic hepatitis B virus (CHB) infection. Natural killer (NK) cells, as one of the key players in the eradication and control of viral infections, were functionally impaired in CHB patients, which might contribute to viral persistence. Here, we reported that HBV antigens HBsAg and HBeAg directly inhibited NK cell function. HBsAg and/or HBeAg blocked NK cell activation, cytokine production and cytotoxic granule release in human NK cell-line NK-92 cells, which might be related to the downregulation of activating receptors and upregulation of inhibitory receptor. Furthermore, the underlying mechanisms likely involved the suppression of STAT1, NF-κB and p38 MAPK pathways. These findings implicated that HBV antigen-mediated inhibition of NK cells might be an efficient strategy for HBV evasion, targeting the early antiviral responses mediated by NK cells and resulting in the establishment of chronic virus infection. Therefore, this study revealed the relationship between viral antigens and human immune function, especially a potential important interaction between HBV and innate immune responses. PMID:27341035

  20. 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....

  1. Trafficking of B cell antigen in lymph nodes

    DEFF Research Database (Denmark)

    Gonzalez, Santiago F.; Degn, Søren Egedal; Pitcher, Lisa A.; Woodruff, Matthew; Heesters, Balthasar A.; Carroll, Michael C.

    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...... microscopy ( 4, 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....

  2. Cutaneous lymphocyte antigen expression on human effector B cells depends on the site and on the nature of antigen encounter.

    Science.gov (United States)

    Kantele, Anu; Savilahti, Erkki; Tiimonen, Heidi; Iikkanen, Katja; Autio, Soile; Kantele, Jussi M

    2003-12-01

    In contrast to T cells, information on skin-homing B cells expressing the cutaneous lymphocyte antigen (CLA) is sparse. CLA expression on human B cells was investigated among circulating immunoglobulin-secreting cells (ISC) and among antigen-specific antibody-secreting cells (ASC) elicited by parenteral, oral or rectal primary immunization, or by parenteral or oral secondary immunization with Salmonella typhi Ty21a. CLA expression was examined by combining cell sorting with an enzyme-linked immunospot assay. Among all ISC, the proportion of CLA(+) cells was 13-21%. Parenteral immunization induced antigen-specific ASC of which 13% were CLA(+), while oral and rectal immunizations were followed by only 1% of CLA(+) ASC (p<0.001). Oral re-immunization was followed by an up-regulation of CLA (34-48%) regardless of the route of priming. Parenteral re-immunization elicited ASC of which 9-14% were CLA(+). In conclusion, the expression of CLA on human effector B cells depends on the site of antigen encounter: intestinal stimulation elicits cells with no CLA, while parenteral encounter elicits significant numbers of CLA(+) cells. Even though primary antigen encounter in the intestine failed to stimulate CLA expression, up-regulation of CLA was found upon intestinal antigen re-encounter. These findings may be of relevance in the pathogenesis of some cutaneous disorders. PMID:14635035

  3. 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. PMID:27207799

  4. Analysis of expression profiles of MAGE-A antigens in oral squamous cell carcinoma cell lines

    Directory of Open Access Journals (Sweden)

    Reichert Torsten E

    2009-04-01

    Full Text Available Abstract Background The immunological response to solid tumours is insufficient. Therefore, tumour specific antigens have been explored to facilitate the activation of the immune system. The cancer/testis antigen class of MAGE-A antigens is a possible target for vaccination. Their differential expression profiles also modulate the course of the cancer disease and its response to antineoplastic drugs. Methods The expression profiles of MAGE-A2, -A3, -A4, -A6 and -A10 in five own oral squamous cell carcinoma cell lines were characterised by rt-PCR, qrt-PCR and immunocytochemistry with a global MAGE-A antibody (57B and compared with those of an adult keratinocyte cell line (NHEK. Results All tumour cell lines expressed MAGE-A antigens. The antigens were expressed in groups with different preferences. The predominant antigens expressed were MAGE-A2, -A3 and -A6. MAGE-A10 was not expressed in the cell lines tested. The MAGE-A gene products detected in the adult keratinocyte cell line NHEK were used as a reference. Conclusion MAGE-A antigens are expressed in oral squamous cell carcinomas. The expression profiles measured facilitate distinct examinations in forthcoming studies on responses to antineoplastic drugs or radiation therapy. MAGE-A antigens are still an interesting aim for immunotherapy.

  5. A nuclear proliferation antigen in chronic lymphoproliferative disease

    International Nuclear Information System (INIS)

    To determine whether the proliferation rates of tumour cells, in chronic lymphoproliferative disorders may reflect disease activity and relate to prognosis, we studied the expression of Ki-67% (a nuclear proliferation marker) by alkaline phosphatase anti-alkaline phosphatase technique (APAAP), in peripheral blood and bone marrow mononuclear cells (separated on ficoll-hypaque) and in lymph-node biopsies, from patients with chronic lymphatic leukemia (Cll), chronic lymphatic leukemia/prolymphocytic leukemia (CLL/PLL), prolymphocytic leukemia (PLL) and non-hodgkin's lymphoma with leukemic plase. The proliferation rate was determined for these patients at presentation and again two months after therapy (to detect any change with therapy). We found that the highest rate of proliferation in each group was parallel to the degree of malignancy i.e. PLL showed higher proliferation than CLL/PLL, and CLL/PLL showed higher proliferation than CLL. In the NHL group the highest proliferation rate was found in the high high-grade NHL, followed by intermediate grade NHL then the low grade NHL. Lymph node biopsies also showed the same relation between proliferation rates and degree of malignancy. Bone marrow cells did not show a particular pattern probably due to interference from the erythroid element and contamination by peripheral blood. Ki-67% was compared to other proliferation markers serum B2 microglobulin and lactate dehydrogenase. It was found to be an independent marker of proliferation it is unaffected by hepatic, renal and gastrointestinal elements and thus its specificity for the tumour proliferation

  6. 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.

  7. Serological identification of tumor antigens of esophageal squamous cell carcinoma.

    Science.gov (United States)

    Shimada, Hideaki; Nakashima, Kazue; Ochiai, Takenori; Nabeya, Yoshihiro; Takiguchi, Masaki; Nomura, Fumio; Hiwasa, Takaki

    2005-01-01

    Autoantibodies are often detected in the patients with esophageal cancer. We applied serological analysis of recombinant cDNA expression libraries (SEREX) to a case of esophageal squamous cell carcinoma in order to identify tumor antigens. A cDNA library derived from an esophageal cancer cell line was bacterially expressed and screened for interaction with antibodies in five allogeneic sera of patients with esophageal squamous cell carcinoma. To examine the specific immunoreactivity of the antigens, sera from 16 more patients with esophageal squamous cell carcinoma, 16 patients with gastric cancer, 16 patients with colon cancer, 16 patients with breast cancer and 37 healthy volunteers were screened. We identified 11 independent cDNA clones that potentially encoded esophageal cancer tumor antigens. The identified cDNA clones were SURF1, HOOK2, CENP-F, ZIC2, hCLA-iso, Ki-1/57, enigma, HCA25a, SPK and two EST clones named LOC146223 and AGENCOURT_7565913. The sero-positive rates of antibodies against SURF1 (48%), LOC146223 (38%), HOOK2 (14%) and AGENCOURT_7565913 (14%) were significantly higher in esophageal cancer patients than in healthy controls. At least one of these antibodies was detected in 18 (86%) of 21 sera from esophageal cancer patients. A disease-specific humoral immune response against SURF1, LOC146223, HOOK2 or AGENCOURT_7565913 was observed in most patients with esophageal squamous cell carcinoma. Antibodies against these SEREX antigens may represent a pool of candidates for serum tumor markers of esophageal squamous cell carcinoma. PMID:15586227

  8. Human epidermal Langerhans cells cointernalize by receptor-mediated endocytosis "nonclassical" major histocompatibility complex class I molecules (T6 antigens) and class II molecules (HLA-DR antigens).

    OpenAIRE

    Hanau, D.; Fabre, M.; Schmitt, D A; Garaud, J C; Pauly, G; Tongio, M M; Mayer, S.; Cazenave, J. P.

    1987-01-01

    HLA-DR and T6 surface antigens are expressed only by Langerhans cells and indeterminate cells in normal human epidermis. We have previously demonstrated that T6 antigens are internalized in Langerhans cells and indeterminate cells by receptor-mediated endocytosis. This process is induced by the binding of BL6, a monoclonal antibody directed against T6 antigens. In the present study, using a monoclonal antibody directed against HLA-DR antigens, on human epidermal cells in suspension, we show t...

  9. A Population Dynamics Analysis of the Interaction between Adaptive Regulatory T Cells and Antigen Presenting Cells

    OpenAIRE

    Fouchet, David; Regoes, Roland

    2008-01-01

    Background Regulatory T cells are central actors in the maintenance of tolerance of self-antigens or allergens and in the regulation of the intensity of the immune response during infections by pathogens. An understanding of the network of the interaction between regulatory T cells, antigen presenting cells and effector T cells is starting to emerge. Dynamical systems analysis can help to understand the dynamical properties of an interaction network and can shed light on the different tasks t...

  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; Abal, A T; Ravn, P; Oftung, F; Andersen, P

    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......-induced proliferation and IFN-gamma secretion showed that the most frequently recognized antigen was ESAT-6, followed by MPT59, GroES, MPB70, MPT64, DnaK, GroEL and PstS. The frequency of ESAT-6 responders, as measured both by proliferation (18/19) and secretion of IFN-gamma (16/19) was comparable to the results...

  11. Structure and biochemical characterization of proliferating cellular nuclear antigen from a parasitic protozoon

    Energy Technology Data Exchange (ETDEWEB)

    Cardona-Felix, Cesar S.; Lara-Gonzalez, Samuel; Brieba, Luis G. (LNLS)

    2012-02-08

    Proliferating cellular nuclear antigen (PCNA) is a toroidal-shaped protein that is involved in cell-cycle control, DNA replication and DNA repair. Parasitic protozoa are early-diverged eukaryotes that are responsible for neglected diseases. In this work, a PCNA from a parasitic protozoon was identified, cloned and biochemically characterized and its crystal structure was determined. Structural and biochemical studies demonstrate that PCNA from Entamoeba histolytica assembles as a homotrimer that is able to interact with and stimulate the activity of a PCNA-interacting peptide-motif protein from E. histolytica, EhDNAligI. The data indicate a conservation of the biochemical mechanisms of PCNA-mediated interactions between metazoa, yeast and parasitic protozoa.

  12. Modification of the immunogenicity and antigenicity of rat hepatoma cells

    International Nuclear Information System (INIS)

    γ-irradiated rat hepatoma cells are immunogenic in syngeneic WAB/Not rats, so that immunized animals are protected against tumour-cell challenge and circulating tumour-specific antibody is produced. Treatment of the immunizing cells with glutaraldehyde at concentrations of 0.001% or greater rendered these cells non-protective and unable to induce significant formation of specific antibody. However, tumour-specific antigens were shown to be expressed upon treated cells; they specifically bound tumour-specific antibody from syngeneic immune sera assessed in indirect membrane-immunofluoresence tests. Also, these cells specifically absorbed antibody from immune or tumour-bearer sera, as demonstrated in the indirect membrane-immunofluorescence test or a complement-dependent 51Cr-release test. Alloantigen expression was not influenced by a glutaraldehyde treatment, although glutaraldehyde-treated hepatoma cells failed to induce alloantibody formation in KX/Not rats. Polyacrylamide-gel electrophoresis of treated cells, surface-labelled with 125I, indicated that extensive cross-linking of the surface protein occurred as a result of glutaraldehyde treatment. These results establish that although the expression of a tumour-specific antigen is necessary for the induction of immuno-protection against tumour-cell challenge, this alone is not a sufficient condition for eliciting tumour immunity. (author)

  13. Isolation of additional monoclonal antibodies directed against cell surface antigens of Myxococcus xanthus cells undergoing submerged development.

    OpenAIRE

    Gill, J.S.; Dworkin, M

    1988-01-01

    Thirteen additional monoclonal antibodies directed against cell surface antigens of Myxococcus xanthus cells undergoing submerged development were isolated and partially characterized. As measured by quantitative enzyme-linked immunosorbent assay, 10 of these antibodies recognized antigens common to both vegetatively growing cells and cells undergoing submerged development; 3 antibodies recognized antigens specific to developing cells. Five antigens were revealed as single bands on Western bl...

  14. Internalization and presentation of myelin antigens by the brain endothelium guides antigen-specific T cell migration

    Science.gov (United States)

    Lopes Pinheiro, Melissa A; Kamermans, Alwin; Garcia-Vallejo, Juan J; van het Hof, Bert; Wierts, Laura; O'Toole, Tom; Boeve, Daniël; Verstege, Marleen; van der Pol, Susanne MA; van Kooyk, Yvette; de Vries, Helga E; Unger, Wendy WJ

    2016-01-01

    Trafficking of myelin-reactive CD4+ T-cells across the brain endothelium, an essential step in the pathogenesis of multiple sclerosis (MS), is suggested to be an antigen-specific process, yet which cells provide this signal is unknown. Here we provide direct evidence that under inflammatory conditions, brain endothelial cells (BECs) stimulate the migration of myelin-reactive CD4+ T-cells by acting as non-professional antigen presenting cells through the processing and presentation of myelin-derived antigens in MHC-II. Inflamed BECs internalized myelin, which was routed to endo-lysosomal compartment for processing in a time-dependent manner. Moreover, myelin/MHC-II complexes on inflamed BECs stimulated the trans-endothelial migration of myelin-reactive Th1 and Th17 2D2 cells, while control antigen loaded BECs did not stimulate T-cell migration. Furthermore, blocking the interaction between myelin/MHC-II complexes and myelin-reactive T-cells prevented T-cell transmigration. These results demonstrate that endothelial cells derived from the brain are capable of enhancing antigen-specific T cell recruitment. DOI: http://dx.doi.org/10.7554/eLife.13149.001 PMID:27336724

  15. Analysis of speckled fluorescent antinuclear antibody test antisera using electrofocused nuclear antigens.

    OpenAIRE

    Okarma, T B; Krueger, J A; Holman, H. R.

    1982-01-01

    Antibodies to different components of the extractable nuclear antigen (ENA) have been thought to be serological markers for clinical subsets of rheumatic diseases. However, incomplete characterization and standardization of antigenic components such as ribonucleoprotein (RNP), Sm, and SS-B (Ha), and the multiplicity of autoantibodies produced by different patients have confounded correlations between autoantibody specificity and disease subsets. This study describes the preparative separation...

  16. Germinal center B cells recognize antigen through a specialized immune synapse architecture.

    Science.gov (United States)

    Nowosad, Carla R; Spillane, Katelyn M; Tolar, Pavel

    2016-07-01

    B cell activation is regulated by B cell antigen receptor (BCR) signaling and antigen internalization in immune synapses. Using large-scale imaging across B cell subsets, we found that, in contrast with naive and memory B cells, which gathered antigen toward the synapse center before internalization, germinal center (GC) B cells extracted antigen by a distinct pathway using small peripheral clusters. Both naive and GC B cell synapses required proximal BCR signaling, but GC cells signaled less through the protein kinase C-β-NF-κB pathway and produced stronger tugging forces on the BCR, thereby more stringently regulating antigen binding. Consequently, GC B cells extracted antigen with better affinity discrimination than naive B cells, suggesting that specialized biomechanical patterns in B cell synapses regulate T cell-dependent selection of high-affinity B cells in GCs. PMID:27183103

  17. Solid phase radioimmunoassay for detection of antibodies to extractable nuclear antigens

    Energy Technology Data Exchange (ETDEWEB)

    Whittingham, S. (Walter and Eliza Hall Inst. of Medical Research, Parkville (Australia))

    1983-06-24

    A solid phase radioimmunoassay is described for the detection of autoantibodies to the saline-soluble extractable nuclear antigens, ribonucleoprotein (RNP) and SS-B (or La). This assay depends on enrichment of antigens from a crude, commercially available (Pel Freez, USA) extract of rabbit thymus by absorption to the F(ab)/sub 2/ fraction of specific high titre antibody attached to a microtitre plate. Serum antibody reactive with this antigen is then detected by /sup 125/I-labelled Protein A. The assay is simple and is more sensitive than the gel diffusion assays in general use for detecting such antibodies.

  18. A solid phase radioimmunoassay for detection of antibodies to extractable nuclear antigens

    International Nuclear Information System (INIS)

    A solid phase radioimmunoassay is described for the detection of autoantibodies to the saline-soluble extractable nuclear antigens, ribonucleoprotein (RNP) and SS-B (or La). This assay depends on enrichment of antigens from a crude, commercially available (Pel Freez, USA) extract of rabbit thymus by absorption to the F(ab)2 fraction of specific high titre antibody attached to a microtitre plate. Serum antibody reactive with this antigen is then detected by 125I-labelled Protein A. The assay is simple and is more sensitive than the gel diffusion assays in general use for detecting such antibodies. (Auth.)

  19. Interferon-gamma-like molecule induces Ia antigens on cultured mast cell progenitors.

    OpenAIRE

    Wong, G H; Clark-lewis, I.; McKimm-Breschkin, J L; Schrader, J W

    1982-01-01

    Persisting (P) cells (murine cells that resemble mast cells and grow continuously in vitro for prolonged periods in the presence of a specific growth factor) did not express detectable levels of Ia antigens (murine class II major histocompatibility antigens) when their growth was supported by partially purified P cell-stimulating factor. However, when these Ia-negative P cells were transferred to medium conditioned by concanavalin A-stimulated spleen cells, Ia antigens appeared within 24 hr. ...

  20. 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; Abal, A T; Ravn, P; Oftung, F; Andersen, P

    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...

  1. 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.

  2. An antigen-specific, four-color, B-cell FluoroSpot assay utilizing tagged antigens for detection.

    Science.gov (United States)

    Jahnmatz, Peter; Bengtsson, Theresa; Zuber, Bartek; Färnert, Anna; Ahlborg, Niklas

    2016-06-01

    The FluoroSpot assay, a variant of ELISpot utilizing fluorescent detection, has so far been used primarily for assessment of T cells, where simultaneous detection of several cytokines has allowed a more qualitative analysis of functionally distinct T cells. The potential to measure multiple analytes also presents several advantages when analyzing B cells. Our aim was to develop a B-cell FluoroSpot assay adaptable to studies of a variety of antigens. The assay utilizes anti-IgG antibodies immobilized in 96-well filter membrane plates. During cell culture, IgG antibodies secreted by antibody-secreting cells (ASCs) are captured in the vicinity of each of these cells and the specificity of single ASCs is defined using antigens for detection. The antigens were labeled with biotin or peptide tags enabling secondary detection with fluorophore-conjugated streptavidin or tag-specific antibodies. The assay, utilizing up to four different tag systems and fluorophores simultaneously, was evaluated using hybridomas and immunized splenocytes as ASCs. Assay variants were developed that could: i) identify multiple ASCs with different antigen specificities; ii) detect ASCs showing cross-reactivity with different but related antigens; and iii) define the antigen-specificity and, by including anti-IgG subclass detection reagents, simultaneously determine the IgG subclass of antibodies secreted by ASCs. As demonstrated here, the B-cell FluoroSpot assay using tag-based detection systems provides a versatile and powerful tool to investigate antibody responses by individual cells that can be readily adapted to studies of a variety of antigen-specific ASCs. PMID:26930550

  3. Cell Wall Anchoring of the Campylobacter Antigens to Lactococcus lactis.

    Science.gov (United States)

    Kobierecka, Patrycja A; Olech, Barbara; Książek, Monika; Derlatka, Katarzyna; Adamska, Iwona; Majewski, Paweł M; Jagusztyn-Krynicka, Elżbieta K; Wyszyńska, Agnieszka K

    2016-01-01

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

  4. Ribosome Protein L4 is essential for Epstein-Barr Virus Nuclear Antigen 1 function.

    Science.gov (United States)

    Shen, Chih-Lung; Liu, Cheng-Der; You, Ren-In; Ching, Yung-Hao; Liang, Jun; Ke, Liangru; Chen, Ya-Lin; Chen, Hong-Chi; Hsu, Hao-Jen; Liou, Je-Wen; Kieff, Elliott; Peng, Chih-Wen

    2016-02-23

    Epstein-Barr Virus (EBV) Nuclear Antigen 1 (EBNA1)-mediated origin of plasmid replication (oriP) DNA episome maintenance is essential for EBV-mediated tumorigenesis. We have now found that EBNA1 binds to Ribosome Protein L4 (RPL4). RPL4 shRNA knockdown decreased EBNA1 activation of an oriP luciferase reporter, EBNA1 DNA binding in lymphoblastoid cell lines, and EBV genome number per lymphoblastoid cell line. EBV infection increased RPL4 expression and redistributed RPL4 to cell nuclei. RPL4 and Nucleolin (NCL) were a scaffold for an EBNA1-induced oriP complex. The RPL4 N terminus cooperated with NCL-K429 to support EBNA1 and oriP-mediated episome binding and maintenance, whereas the NCL C-terminal K380 and K393 induced oriP DNA H3K4me2 modification and promoted EBNA1 activation of oriP-dependent transcription. These observations provide new insights into the mechanisms by which EBV uses NCL and RPL4 to establish persistent B-lymphoblastoid cell infection. PMID:26858444

  5. FK506 inhibits antigen receptor-mediated induction of c-rel in B and T lymphoid cells

    OpenAIRE

    1995-01-01

    Stimulation of B and T cells via the antigen receptor, by phorbol ester or by phorbol ester and ionomycin, leads to nuclear translocation of the inducible transcription factor NF-kappa B, comprising the p50 and p65 rel-related polypeptides. In this report we show that c-rel is a component of the antigen receptor-induced kappa B binding proteins in both B and T cells. Whereas NF-kappa B can be induced by phorbol ester alone, optimal induction of c-rel requires stimulation by both phorbol ester...

  6. Memory and effector T cells modulate subsequently primed immune responses to unrelated antigens

    OpenAIRE

    Tian, Jide D; LU, Y. X.; Hanssen, L.; Dang, H.; Kaufman, D L

    2003-01-01

    Memory and effector T cells modulate subsequently primed T cell responses to the same antigen. However, little is known about the impact of pre-existing memory and effector T cell immunity on subsequently primed immune responses to unrelated antigens. Here, we show that an antigen-primed first wave of Th1 and Th2 immunity enhanced or inhibited the subsequently primed T cell immunity to an unrelated Antigen, depending on whether the second antigen was administered in the same or opposite type ...

  7. T-cell recognition of a cross-reactive antigen(s) in erythrocyte stages of Plasmodium falciparum and Plasmodium yoelii: inhibition of parasitemia by this antigen(s).

    OpenAIRE

    Lucas, B.; Engels, A; Camus, D; Haque, A.

    1993-01-01

    In the current study, we investigated the presence of a cross-reactive antigen(s) in the erythrocyte stage from Plasmodium yoelii (265 BY strain) and Plasmodium falciparum through recognition by T cells primed in vivo with antigens from each of these parasites. BALB/c mice are naturally resistant to P. falciparum but are susceptible to P. yoelii infection. Mice that had recovered from P. yoelii primary infection became resistant to a second infection. A higher in vitro proliferative response ...

  8. Modulation of Epstein–Barr Virus Nuclear Antigen 2-dependent transcription by protein arginine methyltransferase 5

    International Nuclear Information System (INIS)

    Highlights: ► Catalytic active PRMT5 substantially binds to the EBNA2 RG domain. ► PRMT5 augments the EBNA2-dependent transcription. ► PRMT5 triggers the symmetric dimethylation of the EBNA2 RG domain. ► PRMT5 enhances the promoter occupancy of EBNA2 on its target promoters. -- Abstract: Epstein–Barr Virus Nuclear Antigen (EBNA) 2 features an Arginine–Glycine repeat (RG) domain at amino acid positions 335–360, which is a known target for protein arginine methyltransferaser 5 (PRMT5). In this study, we performed protein affinity pull-down assays to demonstrate that endogenous PRMT5 derived from lymphoblastoid cells specifically associated with the protein bait GST-E2 RG. Transfection of a plasmid expressing PRMT5 induced a 2.5- to 3-fold increase in EBNA2-dependent transcription of both the LMP1 promoter in AKATA cells, which contain the EBV genome endogenously, and a Cp-Luc reporter plasmid in BJAB cells, which are EBV negative. Furthermore, we showed that there was a 2-fold enrichment of EBNA2 occupancy in target promoters in the presence of exogenous PRMT5. Taken together, we show that PRMT5 triggers the symmetric dimethylation of EBNA2 RG domain to coordinate with EBNA2-mediated transcription. This modulation suggests that PRMT5 may play a role in latent EBV infection

  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. Detection of cytomegalovirus in shell vial cultures by using a DNA probe and early nuclear antigen monoclonal antibody.

    OpenAIRE

    Scott, A A; K. A. Walker; Hennigar, L M; Williams, C H; Manos, J P; Gansler, T

    1988-01-01

    An in situ biotinylated DNA probe assay was evaluated as an adjunct to anti-cytomegalovirus early nuclear antigen indirect immunofluorescence and cytopathic effect on cytomegalovirus-infected monolayers in shell vial cultures. Viral infection was detected by early nuclear antigen indirect immunofluorescence at 24 h and by DNA probe assay and shell vial cytopathic effect at 5 days.

  11. Serological analysis of cell surface antigens of null cell acute lymphocytic leukemia by mouse monoclonal antibodies.

    OpenAIRE

    Ueda, R; Tanimoto, M; Takahashi, T.; Ogata, S; Nishida, K; Namikawa, R.; Nishizuka, Y; Ota, K.

    1982-01-01

    Nine antigens systems were defined. Two were related to HLA-A,B,C and to Ia-like antigens; the others could be grouped into three categories. (i) NL-22, NL-1: NL-22 antibody reacted with leukemia cells from 12 to 16 cases of null cell acute lymphocytic leukemia (null-ALL) but not with any other type of leukemia tested or with lymphoid cells of various origins. Among cultured cell lines tested, one (NALM-6) of three null-ALL cell lines was positive, the others were negative. Absorption analysi...

  12. Multivalent glycopeptide dendrimers for the targeted delivery of antigens to dendritic cells

    NARCIS (Netherlands)

    J.J. García-Vallejo; M. Ambrosini; A. Overbeek; W.E. van Riel; K. Bloem; W.W.J. Unger; F. Chiodo; J.G. Bolscher; K. Nazmi; H. Kalay; Y. van Kooyk

    2013-01-01

    Dendritic cells are the most powerful type of antigen presenting cells. Current immunotherapies targeting dendritic cells have shown a relative degree of success but still require further improvement. One of the most important issues to solve is the efficiency of antigen delivery to dendritic cells

  13. Selective transport of internalized antigens to the cytosol for MHC class I presentation in dendritic cells

    NARCIS (Netherlands)

    Rodriguez, A; Regnault, A; Kleijmeer, M; Ricciardi-Castagnoli, P; Amigorena, S

    1999-01-01

    In order for cytotoxic T cells to initiate immune responses, peptides derived from internalized antigens must be presented to the cytotoxic T cells on major histocompatibility complex (MHC) class I molecules. Here we show that dendritic cells, the only antigen-presenting cells that initiate immune r

  14. 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.

  15. A population dynamics analysis of the interaction between adaptive regulatory T cells and antigen presenting cells.

    Directory of Open Access Journals (Sweden)

    David Fouchet

    Full Text Available BACKGROUND: Regulatory T cells are central actors in the maintenance of tolerance of self-antigens or allergens and in the regulation of the intensity of the immune response during infections by pathogens. An understanding of the network of the interaction between regulatory T cells, antigen presenting cells and effector T cells is starting to emerge. Dynamical systems analysis can help to understand the dynamical properties of an interaction network and can shed light on the different tasks that can be accomplished by a network. METHODOLOGY AND PRINCIPAL FINDINGS: We used a mathematical model to describe a interaction network of adaptive regulatory T cells, in which mature precursor T cells may differentiate into either adaptive regulatory T cells or effector T cells, depending on the activation state of the cell by which the antigen was presented. Using an equilibrium analysis of the mathematical model we show that, for some parameters, the network has two stable equilibrium states: one in which effector T cells are strongly regulated by regulatory T cells and another in which effector T cells are not regulated because the regulatory T cell population is vanishingly small. We then simulate different types of perturbations, such as the introduction of an antigen into a virgin system, and look at the state into which the system falls. We find that whether or not the interaction network switches from the regulated (tolerant state to the unregulated state depends on the strength of the antigenic stimulus and the state from which the network has been perturbed. CONCLUSION/SIGNIFICANCE: Our findings suggest that the interaction network studied in this paper plays an essential part in generating and maintaining tolerance against allergens and self-antigens.

  16. Simple solid-phase radioimmunoassay for human leukemia-associated cell membrane antigens

    International Nuclear Information System (INIS)

    In the present study, a simple solid-phase radioimmunoassay was developed to determine detergent-extracted human leukemia-associated cell membrane antigens. In the assay, 96-well microtiter plates are coated with human leukemia cell membrane antigens containing a T cell leukemia or a non-T cell leukemia antigen in the presence of a detergent, and treated with 1.6% bovine serum albumin solution. The coated antigens were reacted with an appropriate murine monoclonal antibody (mAb). The bound mAb is determined by a second reaction with 125I-labeled F(ab')2 of goat anti-mouse Ig. The best antigen dose-dependent antibody binding results were obtained using the plates coated with antigens in the presence of taurocholate. In addition, the usefulness of the present assay with taurocholate during the purification of the antigens was demonstrated. (Auth.)

  17. MONOCLONAL-ANTIBODIES TO HUMAN EMBRYONAL CARCINOMA-CELLS - ANTIGENIC RELATIONSHIPS OF GERM-CELL TUMORS

    NARCIS (Netherlands)

    DEWIT, TFR; WILSON, L; VANDENELSEN, PJ; THIELEN, F; BREKHOFF, D; OOSTERHUIS, JW; PERA, MF; STERN, PL

    1991-01-01

    Fifteen monoclonal antibodies (mAb) that show specificity for human embryonal carcinoma cells are described. C57BL/6 mice were immunized with Tera-2 embryonal carcinoma cells, and hybridomas were isolated and tested versus a set of human developmental tumor cell lines. The antigens exhibit relativel

  18. From the Deep Sea to Everywhere: Environmental Antigens for iNKT Cells.

    Science.gov (United States)

    Wingender, Gerhard

    2016-08-01

    Invariant natural killer T (iNKT) cells are a unique subset of innate T cells that share features with innate NK cells and adaptive memory T cells. The first iNKT cell antigen described was found 1993 in a marine sponge and it took over 10 years for other, bacterial antigens to be described. Given the paucity of known bacterial iNKT cell antigens, it appeared as if iNKT cells play a very specialist role in the protection against few, rare and unusual pathogenic bacteria. However, in the last few years several publications painted a very different picture, suggesting that antigens for iNKT cells are found almost ubiquitous in the environment. These environmental iNKT cell antigens can shape the distribution, phenotype and function of iNKT cells. Here, these recent findings will be reviewed and their implications for the field will be outlined. PMID:26703211

  19. Epstein-Barr Virus Nuclear Antigen 3C Augments Mdm2-Mediated p53 Ubiquitination and Degradation by Deubiquitinating Mdm2▿

    OpenAIRE

    Saha, Abhik; Murakami, Masanao; Kumar, Pankaj; Bajaj, Bharat; Sims, Karen; Erle S Robertson

    2009-01-01

    Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is one of the essential latent antigens for primary B-cell transformation. Previous studies established that EBNA3C facilitates degradation of several vital cell cycle regulators, including the retinoblastoma (pRb) and p27KIP proteins, by recruitment of the SCFSkp2 E3 ubiquitin ligase complex. EBNA3C was also shown to be ubiquitinated at its N-terminal residues. Furthermore, EBNA3C can bind to and be degraded in vitro by purified 20S protea...

  20. The role of class I histocompatibility antigens in the regulation of T-cell activation.

    OpenAIRE

    Dasgupta, J D; Cemach, K; Dubey, D P; Yunis, E J; Amos, D. B.

    1987-01-01

    Class I major histocompatibility antigens in humans (HLA antigens) were found to participate in the regulation of T-cell activation and proliferation induced by phytohemagglutinin. W6/32, a monomorphic antibody directed against class I HLA-A,B,C antigens, significantly inhibited the phytohemagglutinin-induced cell proliferation of peripheral blood lymphocytes. Almost complete suppression of cell activation was achieved on a subfraction of peripheral blood lymphocytes enriched in Mo1+ monocyte...

  1. Infected site-restricted Foxp3+ natural regulatory T cells are specific for microbial antigens

    OpenAIRE

    Suffia, Isabelle J.; Reckling, Stacie K.; Piccirillo, Ciriaco A; Goldszmid, Romina S.; Belkaid, Yasmine

    2006-01-01

    Natural regulatory T (T reg) cells are involved in control of the immune response, including response to pathogens. Previous work has demonstrated that the repertoire of natural T reg cells may be biased toward self-antigen recognition. Whether they also recognize foreign antigens and how this recognition contributes to their function remain unknown. Our studies addressed the antigenic specificity of natural T reg cells that accumulate at sites of chronic infection with Leishmania major in mi...

  2. Targeting of antigens to B cells augments antigen-specific T-cell responses and breaks immune tolerance to tumor-associated antigen MUC1

    Science.gov (United States)

    Ding, Chuanlin; Wang, Li; Marroquin, Jose

    2008-01-01

    B cells are antibody (Ab)–secreting cells as well as potent antigen (Ag)–presenting cells that prime T-cell activation, which evokes great interest in their use for vaccine development. Here, we targeted ovalbumin (OVA) to B cells via CD19 and found that a single low dose of anti–CD19-OVA conjugates, but not isotype mAb-OVA, stimulated augmented CD4 and CD8 T-cell proliferation and expansion. Administration of TLR9 agonist CpG could significantly enhance long-term T-cell survival. Similar results were obtained when the tumor-associated Ag MUC1 was delivered to B cells. MUC1 transgenic (Tg) mice were previously found to lack effective T-cell help and produce low-titer of anti-MUC1 Abs after vaccination. Targeting MUC1 to B cells elicited high titer of anti-MUC1 Abs with different isotypes, predominantly IgG2a and IgG2b, in MUC1 Tg mice. The isotype switching of anti-MUC1 Ab was CD4 dependent. In addition, IFN-γ–producing CD8 T cells and in vivo cytolytic activity were significantly increased in these mice. The mice also showed significant resistance to MUC1+ lymphoma cell challenge both in the prophylactic and therapeutic settings. We conclude that Ags targeting to B cells stimulate CD4 and CD8 T-cell responses as well as Th-dependent humoral immune responses. PMID:18669871

  3. Mast cells modulate transport of CD23/IgE/antigen complex across human intestinal epithelial barrier

    Directory of Open Access Journals (Sweden)

    Ping-Chang Yang

    2009-06-01

    Full Text Available Background: Food allergy and chronic intestinal inflammation are common in western countries. The complex of antigen/IgE is taken up into the body from the gut lumen with the aid of epithelial cell-derived CD23 (low affinity IgE receptor II that plays an important role in the pathogenesis of intestinal allergy. This study aimed to elucidate the role of mast cell on modulation of antigen/IgE complex transport across intestinal epithelial barrier. Methods: Human intestinal epithelial cell line HT29 cell monolayer was used as a study platform. Transepithelial electric resistance (TER and permeability to ovalbumin (OVA were used as the markers of intestinal epithelial barrier function that were recorded in response to the stimulation of mast cell-derived chemical mediators. Results: Conditioned media from naïve mast cell line HMC-1 cells or monocyte cell line THP-1 cells significantly upregulated the expression of CD23 and increased the antigen transport across the epithelium. Treatment with stem cell factor (SCF, nerve growth factor (NGF, retinoic acid (RA or dimethyl sulphoxide (DMSO enhanced CD23 expression in HT29 cells. Conditioned media from SCF, NGF or RA-treated HMC-1 cells, and SCF, NGF, DMSO or RA-treated THP-1 cells enhanced immune complex transport via enhancing the expression of the CD23 in HT29 cells and the release of inflammatory mediator TNF-α. Nuclear factor kappa B inhibitor, tryptase and TNF-α inhibited the increase in CD23 in HT29 cells and prevents the enhancement of epithelial barrier permeability. Conclusions: Mast cells play an important role in modulating the intestinal CD23 expression and the transport of antigen/IgE/CD23 complex across epithelial barrier.

  4. Flow cytometric assay detecting cytotoxicity against human endogenous retrovirus antigens expressed on cultured multiple sclerosis cells

    DEFF Research Database (Denmark)

    Møller-Larsen, A; Brudek, T; Petersen, T; Petersen, E L; Aagaard, M; Hansen, Dorte; Christensen, T

    2013-01-01

    expressing increased amounts of human endogenous retrovirus antigens. MS patients also have increased antibody levels to these antigens. The target cells are spontaneously growing peripheral blood mononuclear cells (PBMCs) of B cell lineage, expressing human endogenous retrovirus HERV epitopes on their...

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

    DEFF Research Database (Denmark)

    Buus, S; Werdelin, O

    1984-01-01

    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...

  6. Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

    Energy Technology Data Exchange (ETDEWEB)

    Diaz, Jason; Wang, Xin; Tsang, Sabrina H. [Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 (United States); Jiao, Jing [Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104 (United States); You, Jianxin, E-mail: jianyou@mail.med.upenn.edu [Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 (United States)

    2014-07-08

    Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT’s ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells.

  7. Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

    International Nuclear Information System (INIS)

    Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT’s ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells

  8. Enhanced T cell responses to antigenic peptides targeted to B cell surface Ig, Ia, or class I molecules

    OpenAIRE

    1988-01-01

    The helper T cell recognition of soluble globular protein antigens requires that the proteins be processed by an APC, releasing a peptide that is transported to and held on the APC surface where it is recognized by the specific T cell in conjunction with Ia. When cellular processing functions are blocked, APC lose their ability to present native antigens while retaining the capacity to activate T cells when provided with a cognate peptide fragment that contains the T cell antigenic determinan...

  9. Rationally designed inhibitor targeting antigen-trimming aminopeptidases enhances antigen presentation and cytotoxic T-cell responses.

    Science.gov (United States)

    Zervoudi, Efthalia; Saridakis, Emmanuel; Birtley, James R; Seregin, Sergey S; Reeves, Emma; Kokkala, Paraskevi; Aldhamen, Yasser A; Amalfitano, Andrea; Mavridis, Irene M; James, Edward; Georgiadis, Dimitris; Stratikos, Efstratios

    2013-12-01

    Intracellular aminopeptidases endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2), and as well as insulin-regulated aminopeptidase (IRAP) process antigenic epitope precursors for loading onto MHC class I molecules and regulate the adaptive immune response. Their activity greatly affects the antigenic peptide repertoire presented to cytotoxic T lymphocytes and as a result can regulate cytotoxic cellular responses contributing to autoimmunity or immune evasion by viruses and cancer cells. Therefore, pharmacological regulation of their activity is a promising avenue for modulating the adaptive immune response with possible applications in controlling autoimmunity, in boosting immune responses to pathogens, and in cancer immunotherapy. In this study we exploited recent structural and biochemical analysis of ERAP1 and ERAP2 to design and develop phosphinic pseudopeptide transition state analogs that can inhibit this family of enzymes with nM affinity. X-ray crystallographic analysis of one such inhibitor in complex with ERAP2 validated our design, revealing a canonical mode of binding in the active site of the enzyme, and highlighted the importance of the S2' pocket for achieving inhibitor potency. Antigen processing and presentation assays in HeLa and murine colon carcinoma (CT26) cells showed that these inhibitors induce increased cell-surface antigen presentation of transfected and endogenous antigens and enhance cytotoxic T-cell responses, indicating that these enzymes primarily destroy epitopes in those systems. This class of inhibitors constitutes a promising tool for controlling the cellular adaptive immune response in humans by modulating the antigen processing and presentation pathway. PMID:24248368

  10. 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

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

    OpenAIRE

    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 dif...

  12. γδ T cells recognize a microbial encoded B cell antigen to initiate a rapid antigen specific Interleukin 17 response

    Science.gov (United States)

    Zeng, Xun; Wei, Yu-ling; Huang, Jun; Newell, Evan W.; Yu, Hongxiang; Kidd, Brian A.; Kuhns, Michael S.; Waters, Ray W.; Davis, Mark M.; Weaver, Casey T.; Chien, Yueh-hsiu

    2012-01-01

    Summary γδ T cells contribute uniquely to host immune defense. However, how they function remains an enigma. Although it is unclear what most γδ T cells recognize, common dogma asserts that they recognize self-antigens. While they are the major initial Interleukin-17 (IL-17) producers in infections, it is unclear what is required to trigger these cells to act. Here, we report that a noted B cell antigen, the algae protein-phycoerythrin (PE) is an antigen for murine and human γδ T cells. PE also stained specific bovine γδ T cells. Employing this specificity, we demonstrated that antigen recognition, but not extensive clonal expansion, was required to activate naïve γδ T cells to make IL-17. In this activated state, γδ T cells gained the ability to respond to cytokine signals that perpetuated the IL-17 production. These results underscore the adaptability of lymphocyte antigen receptors and suggest a previously unrecognized antigen-driven rapid response in protective immunity prior to the maturation of classical adaptive immunity. PMID:22960222

  13. γδ T cells recognize a microbial encoded B cell antigen to initiate a rapid antigen-specific interleukin-17 response.

    Science.gov (United States)

    Zeng, Xun; Wei, Yu-Ling; Huang, Jun; Newell, Evan W; Yu, Hongxiang; Kidd, Brian A; Kuhns, Michael S; Waters, Ray W; Davis, Mark M; Weaver, Casey T; Chien, Yueh-hsiu

    2012-09-21

    γδ T cells contribute uniquely to immune competence. Nevertheless, how they function remains an enigma. It is unclear what most γδ T cells recognize, what is required for them to mount an immune response, and how the γδ T cell response is integrated into host immune defense. Here, we report that a noted B cell antigen, the algae protein phycoerythrin (PE), is a murine and human γδ T cell antigen. Employing this specificity, we demonstrated that antigen recognition activated naive γδ T cells to make interleukin-17 and respond to cytokine signals that perpetuate the response. High frequencies of antigen-specific γδ T cells in naive animals and their ability to mount effector response without extensive clonal expansion allow γδ T cells to initiate a swift, substantial response. These results underscore the adaptability of lymphocyte antigen receptors and suggest an antigen-driven rapid response in protective immunity prior to the maturation of classical adaptive immunity. PMID:22960222

  14. Stratification of Antigen-presenting Cells within the Normal Cornea

    Directory of Open Access Journals (Sweden)

    Jared E. Knickelbein

    2009-11-01

    Full Text Available The composition and location of professional antigen presenting cells (APC varies in different mucosal surfaces. The cornea, long considered an immune-privileged tissue devoid of APCs, is now known to host a heterogeneous network of bone marrow-derived cells. Here, we utilized transgenic mice that express enhanced green fluorescent protein (EGFP from the CD11c promoter (pCD11c in conjunction with immunohistochemical staining to demonstrate an interesting stratification of APCs within non-inflamed murine corneas. pCD11c+ dendritic cells (DCs reside in the basal epithelium, seemingly embedded in the basement membrane. Most DCs express MHC class II on at least some dendrites, which extend up to 50 µm in length and traverse up 20 µm tangentially towards the apical surface of the epithelium. The DC density diminishes from peripheral to central cornea. Beneath the DCs and adjacent to the stromal side of the basement membrane reside pCD11c-CD11b+ putative macrophages that express low levels of MHC class II. Finally, MHC class IIpCD11c-CD11b+ cells form a network throughout the remainder of the stroma. This highly reproducible stratification of bone marrow-derived cells is suggestive of a progression from an APC function at the exposed corneal surface to an innate immune barrier function deeper in the stroma.

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

    DEFF Research Database (Denmark)

    Heron, I; Hokland, M; Berg, K

    1979-01-01

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

  16. Dendritic Cells in the Periphery Control Antigen-Specific Natural and Induced Regulatory T Cells

    OpenAIRE

    Yamazaki, Sayuri; Morita, Akimichi

    2013-01-01

    Dendritic cells (DCs) are specialized antigen-presenting cells that regulate both immunity and tolerance. DCs in the periphery play a key role in expanding naturally occurring Foxp3+ CD25+ CD4+ regulatory T cells (Natural T-regs) and inducing Foxp3 expression (Induced T-regs) in Foxp3− CD4+ T cells. DCs are phenotypically and functionally heterogeneous, and further classified into several subsets depending on distinct marker expression and their location. Recent findings indicate the presence...

  17. Low dose antigen promotes induction of FOXP3 in human CD4+ T cells

    OpenAIRE

    Long, S. Alice; Rieck, Mary; Tatum, Megan; Bollyky, Paul L.; Wu, Rebecca P.; Muller, Isabelle; Ho, Jhon-Chun; Shilling, Heather G.; Buckner, Jane H.

    2011-01-01

    Low antigen dose promotes induction and persistence of Treg in mice, yet few studies have addressed the role of antigen dose in the induction of adaptive CD4+FOXP3+ Treg in humans. To this end, we examined the level of FOXP3 expression in human CD4+CD25− T cells upon activation with autologous antigen presenting cells and varying doses of peptide. Antigen specific T cells expressing FOXP3 were identified by flow cytometry using MHC Class II tetramer (Tmr). We found an inverse relationship bet...

  18. Circulating human basophils lack the features of professional antigen presenting cells

    OpenAIRE

    Sharma, Meenu; Hegde, Pushpa; Aimanianda, Vishukumar; Beau, Remi; Sénéchal, Helene; Poncet, Pascal; Latgé, Jean-Paul; Kaveri, Srini V; Bayry, Jagadeesh

    2013-01-01

    Recent reports in mice demonstrate that basophils function as antigen presenting cells (APC). They express MHC class II and co-stimulatory molecules CD80 and CD86, capture and present soluble antigens or IgE-antigen complexes and polarize Th2 responses. Therefore, we explored whether human circulating basophils possess the features of professional APC. We found that unlike dendritic cells (DC) and monocytes, steady-state circulating human basophils did not express HLA-DR and co-stimulatory mo...

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

    International Nuclear Information System (INIS)

    The polyomavirus small T-antigen gene, together with the polyomavirus promoter, was inserted into retrovirus vector pGV16 which contains the Moloney sarcoma virus long terminal repeat and neomycin resistance gene driven by the simian virus 40 promoter. This expression vector, pGVST, was packaged into retrovirus particles by transfection of PSI2 cells which harbor packaging-defective murine retrovirus genome. NIH 3T3 cells were infected by this replication-defective retrovirus containing pGVST. Of the 15 G418-resistant cell clones, 8 express small T antigen at various levels as revealed by immunoprecipitation. A cellular protein with an apparent molecular weight of about 32,000 coprecipitates with small T antigen. Immunofluorescent staining shows that small T antigen is mainly present in the nuclei. Morphologically, cells expressing small T antigen are indistinguishable from parental NIH 3T3 cells and have a microfilament pattern similar to that in parental NIH 3T3 cells. Cells expressing small T antigen form a flat monolayer but continue to grow beyond the saturation density observed for parental NIH 3T3 cells and eventually come off the culture plate as a result of overconfluency. There is some correlation between the level of expression of small T antigen and the growth rate of the cells. Small T-antigen-expressing cells form small colonies in soft agar. However, the proportion of cells which form these small colonies is rather small. A clone of these cells tested did not form tumors in nude mice within 3 months after inoculation of 106 cells per animal. Thus, present studies establish that the small T antigen of polyomavirus is a second nucleus-localized transforming gene product of the virus (the first one being large T antigen) and by itself has a function which is to stimulate the growth of NIH 3T3 cells beyond their saturation density in monolayer culture

  20. Epstein–Barr virus latent antigens EBNA3C and EBNA1 modulate epithelial to mesenchymal transition of cancer cells associated with tumor metastasis

    OpenAIRE

    Gaur, Nivedita; Gandhi, Jaya; Erle S Robertson; Verma, Subhash C.; Kaul, Rajeev

    2014-01-01

    Epithelial–mesenchymal transition is an important mechanism in cancer invasiveness and metastasis. We had previously reported that cancer cells expressing Epstein–Barr virus (EBV) latent viral antigens EBV nuclear antigen EBNA3C and/ or EBNA1 showed higher motility and migration potential and had a propensity for increased metastases when tested in nude mice model. We now show that both EBNA3C and EBNA1 can modulate cellular pathways critical for epithelial to mesenchymal transition of cancer...

  1. CD13 Regulates Dendritic Cell Cross-presentation and T Cell Responses by Inhibiting Receptor-Mediated Antigen Uptake

    OpenAIRE

    Ghosh, Mallika; McAuliffe, Beata; Subramani, Jaganathan; Basu, Sreyashi; Shapiro, Linda H.

    2012-01-01

    Dendritic cell (DC) antigen cross-presentation is generally associated with immune responses to tumors and viral antigens and enhancing this process is a focus of tumor vaccine design. In this study, we found that the myeloid cell surface peptidase CD13 is highly and specifically expressed on the subset of DCs responsible for cross-presentation, the CD8+ murine splenic DCs. In vivo studies indicated that lack of CD13 significantly enhanced T cell responses to soluble OVA antigen, although dev...

  2. Evaluation of Multiplexed Fluorescent Microsphere Immunoassay for Detection of Autoantibodies to Nuclear Antigens

    OpenAIRE

    Martins, Thomas B; Burlingame, Rufus; von Mühlen, Carlos A.; Jaskowski, Troy D; Litwin, Christine M.; Hill, Harry R.

    2004-01-01

    Antibodies to extractable nuclear antigens (ENA) are found in a variety of collagen vascular diseases. Determining the individual specificities of these antibodies is extremely useful in establishing the disease diagnosis and in some cases the prognosis. With a multiplexed fluorescent microsphere immunoassay, reactivity to five of the most diagnostically useful ENA was measured in 249 serum samples, including samples from 56 patients previously documented to have systemic lupus erythematosus ...

  3. Antigen sampling by intestinal M cells is the principal pathway initiating mucosal IgA production to commensal enteric bacteria

    Science.gov (United States)

    Rios, D; Wood, M B; Li, J; Chassaing, B; Gewirtz, A T; Williams, I R

    2016-01-01

    Secretory IgA (SIgA) directed against gut resident bacteria enables the mammalian mucosal immune system to establish homeostasis with the commensal gut microbiota after weaning. Germinal centers (GCs) in Peyer's patches (PPs) are the principal inductive sites where naive B cells specific for bacterial antigens encounter their cognate antigens and receive T-cell help driving their differentiation into IgA-producing plasma cells. We investigated the role of antigen sampling by intestinal M cells in initiating the SIgA response to gut bacteria by developing mice in which receptor activator of nuclear factor-κB ligand (RANKL)-dependent M-cell differentiation was abrogated by conditional deletion of Tnfrsf11a in the intestinal epithelium. Mice without intestinal M cells had profound delays in PP GC maturation and emergence of lamina propria IgA plasma cells, resulting in diminished levels of fecal SIgA that persisted into adulthood. We conclude that M-cell-mediated sampling of commensal bacteria is a required initial step for the efficient induction of intestinal SIgA. PMID:26601902

  4. Genetic disruption of KSHV major latent nuclear antigen LANA enhances viral lytic transcriptional program

    International Nuclear Information System (INIS)

    Following primary infection, KSHV establishes a lifelong persistent latent infection in the host. The mechanism of KSHV latency is not fully understood. The latent nuclear antigen (LANA or LNA) encoded by ORF73 is one of a few viral genes expressed during KSHV latency, and is consistently detected in all KSHV-related malignancies. LANA is essential for KSHV episome persistence, and regulates the expression of viral lytic genes through epigenetic silencing, and inhibition of the expression and transactivation function of the key KSHV lytic replication initiator RTA (ORF50). In this study, we used a genetic approach to examine the role of LANA in regulating KSHV lytic replication program. Deletion of LANA did not affect the expression of its adjacent genes vCyclin (ORF72) and vFLIP (ORF71). In contrast, the expression levels of viral lytic genes including immediate-early gene RTA, early genes MTA (ORF57), vIL-6 (ORF-K2) and ORF59, and late gene ORF-K8.1 were increased before and after viral lytic induction with 12-O-tetradecanoyl-phorbol-13-acetate and sodium butyrate. This enhanced expression of viral lytic genes was also observed following overexpression of RTA with or without simultaneous chemical induction. Consistent with these results, the LANA mutant cells produced more infectious virions than the wild-type virus cells did. Furthermore, genetic repair of the mutant virus reverted the phenotypes to those of wild-type virus. Together, these results have demonstrated that, in the context of viral genome, LANA contributes to KSHV latency by regulating the expression of RTA and its downstream genes

  5. Gamma delta T cells recognize a microbial encoded B Cell antigen to initiate a rapid antigen-specific Interleukin-17 response

    Science.gov (United States)

    Gamma delta T cells contribute uniquely to host immune defense, but the way in which they do so remains an enigma. Here we show that an algae protein, phycoerythrin (PE) is recognized by gamma delta T cells from mice, bovine and humans and binds directly to specific gamma delta T cell antigen recept...

  6. Antigen-specific murine T cell clones produce soluble interleukin 2 receptor on stimulation with specific antigens

    International Nuclear Information System (INIS)

    In this study, monoclonal antibodies were used to the murine IL 2 receptor (IL 2R) termed 3C7 and 7D4, which bind to different epitopes on the murine IL 2R, to develop an ELISA to measure soluble murine IL 2R. Surprisingly, stimulated murine spleen cells not only expressed cell-associated IL 2R, but also produced a considerable level of cellfree IL 2R in the culture supernatant fluid. To assess the fine specificity of this response, myoglobin-immune murine T cell clones were stimulated with appropriate or inappropriate antigen and syngeneic or allogeneic presenting cells. Proliferation, measured by [3H] thymidine incorporation, and levels of soluble IL 2R were determined at day 4. The production of soluble IL2R displayed the same epitope fine specificity, genetic restriction, and antigen dose-response as the proliferative response. Indeed, in some cases there was sharper discrimination of epitope specificity and genetic restriction with the soluble IL 2R levels. There was also reproducible clone-to-clone variation in the amount of soluble receptor produced in response to antigen among 12 T cell clones and lines tested. In time course experiments, proliferation was greatest at day 3, whereas soluble IL 2R levels continued to rise in subsequent days. To the authors' knowledge, this is the first demonstration of release of secretion of soluble IL 2R by murine T cells, and the first demonstration of the fine specificity and genetic restriction of the induction of soluble IL 2R by specific antigen

  7. Small molecule and peptide-mediated inhibition of Epstein-Barr virus nuclear antigen 1 dimerization

    International Nuclear Information System (INIS)

    Highlights: ► Evidence that targeting EBNA1 dimer, an EBV onco-antigen, can be achievable. ► A small molecule and a peptide as EBNA1 dimerization inhibitors identified. ► Both inhibitors associated with EBNA1 and blocked EBNA1 DNA binding activity. ► Also, prevented its dimerization, and repressed viral gene transcription. -- Abstract: Latent Epstein-Barr virus (EBV) infection is associated with human B cell lymphomas and certain carcinomas. EBV episome persistence, replication, and gene expression are dependent on EBV-encoded nuclear antigen 1 (EBNA1)’s DNA binding domain (DBD)/dimerization domain (DD)-mediated sequence-specific DNA binding activity. Homodimerization of EBNA1 is essential for EBNA1 DNA binding and transactivation. In this study, we characterized a novel small molecule EBNA1 inhibitor EiK1, screened from the previous high throughput screening (HTS). The EiK1 compound specifically inhibited the EBNA1-dependent, OriP-enhanced transcription, but not EBNA1-independent transcription. A Surface Plasmon Resonance Biacore assay revealed that EiK1 associates with EBNA1 amino acid 459–607 DBD/DD. Consistent with the SPR data, in vitro gel shift assays showed that EiK1 suppressed the activity of EBNA1 binding to the cognate familial repeats (FR) sequence, but not control RBP-Jκ binding to the Jκ site. Subsequently, a cross-linker-mediated in vitro multimerization assay and EBNA1 homodimerization-dependent yeast two-hybrid assay showed that EiK1 significantly inhibited EBNA1 dimerization. In an attempt to identify more highly specific peptide inhibitors, small peptides encompassing the EBNA1 DBD/DD were screened for inhibition of EBNA1 DBD-mediated DNA binding function. The small peptide P85, covering EBNA1 a.a. 560–574, significantly blocked EBNA1 DNA binding activity in vitro, prevented dimerization in vitro and in vivo, associated with EBNA1 in vitro, and repressed EBNA1-dependent transcription in vivo. Collectively, this study describes two

  8. Small molecule and peptide-mediated inhibition of Epstein-Barr virus nuclear antigen 1 dimerization

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sun Young; Song, Kyung-A [Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Samsung Biomedical Research Institute (SBRI), Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Kieff, Elliott [Department of Medicine, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA 02115 (United States); Kang, Myung-Soo, E-mail: mkang@skku.edu [Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Samsung Biomedical Research Institute (SBRI), Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Department of Medicine, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA 02115 (United States)

    2012-07-27

    Highlights: Black-Right-Pointing-Pointer Evidence that targeting EBNA1 dimer, an EBV onco-antigen, can be achievable. Black-Right-Pointing-Pointer A small molecule and a peptide as EBNA1 dimerization inhibitors identified. Black-Right-Pointing-Pointer Both inhibitors associated with EBNA1 and blocked EBNA1 DNA binding activity. Black-Right-Pointing-Pointer Also, prevented its dimerization, and repressed viral gene transcription. -- Abstract: Latent Epstein-Barr virus (EBV) infection is associated with human B cell lymphomas and certain carcinomas. EBV episome persistence, replication, and gene expression are dependent on EBV-encoded nuclear antigen 1 (EBNA1)'s DNA binding domain (DBD)/dimerization domain (DD)-mediated sequence-specific DNA binding activity. Homodimerization of EBNA1 is essential for EBNA1 DNA binding and transactivation. In this study, we characterized a novel small molecule EBNA1 inhibitor EiK1, screened from the previous high throughput screening (HTS). The EiK1 compound specifically inhibited the EBNA1-dependent, OriP-enhanced transcription, but not EBNA1-independent transcription. A Surface Plasmon Resonance Biacore assay revealed that EiK1 associates with EBNA1 amino acid 459-607 DBD/DD. Consistent with the SPR data, in vitro gel shift assays showed that EiK1 suppressed the activity of EBNA1 binding to the cognate familial repeats (FR) sequence, but not control RBP-J{kappa} binding to the J{kappa} site. Subsequently, a cross-linker-mediated in vitro multimerization assay and EBNA1 homodimerization-dependent yeast two-hybrid assay showed that EiK1 significantly inhibited EBNA1 dimerization. In an attempt to identify more highly specific peptide inhibitors, small peptides encompassing the EBNA1 DBD/DD were screened for inhibition of EBNA1 DBD-mediated DNA binding function. The small peptide P85, covering EBNA1 a.a. 560-574, significantly blocked EBNA1 DNA binding activity in vitro, prevented dimerization in vitro and in vivo, associated

  9. Human antibodies targeting cell surface antigens overexpressed by the hormone refractory metastatic prostate cancer cells: ICAM-1 is a tumor antigen that mediates prostate cancer cell invasion

    OpenAIRE

    Conrad, Fraser; Zhu, Xiaodong; Zhang, Xin; Chalkley, Robert J.; Burlingame, Alma L; Marks, James D.; Liu, Bin

    2009-01-01

    Transition from hormone-sensitive to hormone-refractory metastatic tumor types poses a major challenge for prostate cancer treatment. Tumor antigens that are differentially expressed during this transition are likely to play important roles in imparting prostate cancer cells with the ability to grow in a hormone-deprived environment and to metastasize to distal sites such as the bone and thus, are likely targets for therapeutic intervention. To identify those molecules and particularly cell s...

  10. Target antigen expression on a professional antigen-presenting cell induces superior proliferative antitumor T-cell responses via chimeric T-cell receptors.

    Science.gov (United States)

    Rossig, Claudia; Bär, Annette; Pscherer, Sibylle; Altvater, Bianca; Pule, Martin; Rooney, Cliona M; Brenner, Malcolm K; Jürgens, Heribert; Vormoor, Josef

    2006-01-01

    Human T cells expressing tumor antigen-specific chimeric receptors fail to sustain their growth and activation in vivo, which greatly reduces their therapeutic value. The defective proliferative response to tumor cells in vitro can partly be overcome by concomitant CD28 costimulatory signaling. We investigated whether T-cell activation via chimeric receptors (chRec) can be further improved by ligand expression on antigen-presenting cells of B-cell origin. We generated Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) expressing a CD19-specific chRec. These CTLs are provided with native receptor stimulation by autologous EBV-transformed B-lymphoblastoid cell lines (LCLs) but exclusively with chRec (CD19-specific) stimulation by allogeneic, human leukocyte antigen (HLA)-mismatched CD19+ LCLs. CD19zeta-transduced EBV-specific CTLs specifically lysed both allogeneic EBV targets and CD19+ tumor cells through the chRec in a major histocompatibility complex-independent manner, while maintaining their ability to recognize autologous EBV targets through the native T-cell receptor. The transduced CTLs failed to proliferate in response to CD19+ tumor targets even in the presence of CD28 costimulatory signaling. By contrast, CD19 expressed on HLA-mismatched LCL-induced T-cell activation and long-term proliferation that essentially duplicated the result from native receptor stimulation with autologous LCLs, suggesting that a deficit of costimulatory molecules on target cells in addition to CD28 is indeed responsible for inadequate chRec-mediated T-cell function. Hence, effective tumor immunotherapy may be favored if engagement of the chRec on modified T cells is complemented by interaction with multiple costimulator molecules. The use of T cells with native specificity for EBV may be one means of attaining this objective. PMID:16365597

  11. Epithelial membrane antigen in cells from the uterine cervix: immunocytochemical staining of cervical smears.

    OpenAIRE

    Valkova, B; Ormerod, M G; Moncrieff, D.; Coleman, D V

    1984-01-01

    Smears made from cervical scrapes have been stained immunocytochemically for epithelial membrane antigen using a polyclonal antiserum and two monoclonal antibodies. With the polyclonal antiserum malignant cells and those showing dysplasia consistently expressed the antigen. Normal cells were generally negative, with the exception of some metaplastic cells. The monoclonal antibodies, although they stained the abnormal cells less consistently, gave the same pattern of staining. All three antibo...

  12. Germ tube-specific antigens of Candida albicans cell walls

    International Nuclear Information System (INIS)

    Studies were performed to characterize the surface differences between blastospores and germ tubes of the pathogenic, dimorphic yeast, Candida albicans, and to identify components of yeast cells responsible for these differences. Investigation of surfaces differences of the two growth forms was facilitated by the production of rabbit antiserum prepared against Formalin-treated yeast possessing germ tubes. To prepare antiserum specific for germ tubes, this serum was adsorbed with stationary phase blastospores. Whereas the unadsorbed antiserum reacted with both blastospore and germ tube forms by immunofluorescence and Enzyme-Linked Immunosorbent Assay, the adsorbed antiserum did not react with blastospores but detected germ tube-specific antigens in hyphal forms. The differences between blastospores and germ tubes of Candida albicans, were further studied by comparing enzymatic digests of cell walls of both growth forms in radiolabeled organisms. Organisms were labeled either on the surface with 125I, or metabolically with [35S] methionine or [3H] mannose. Three-surface-located components (as shown by antibody adsorption and elution experiments) were precipitated from Zymolase digests. All three components were mannoproteins as shown by their ability to bind Concanavalin A, and to be labeled in protein labeling procedures, and two of these (200,000 and 155,000 molecular weight) were germ tube specific, as shown by their ability to be precipitated by germ tube-specific antiserum. Monoclonal antibodies were prepared to C. albicans, using blastospores bearing germ tubes as immunogen

  13. Common polysaccharide antigens from the cell envelope of Clostridium perfringens type A.

    OpenAIRE

    Dayalu, K I; Cherniak, R; Hatheway, C L

    1981-01-01

    Soluble antigens were obtained by extracting five serotype strains of Clostridium perfringens type A with water at 100 degrees C. The type-specific polysaccharides were precipitated with ethanol, and the common antigens were recovered from the ethanol supernatants by concentration, dialysis, and lyophilization. Refluxing the water-extracted cell residues with 1% acetic acid followed by concentration, dialysis, and lyophilization gave additional common antigen fractions. A comprehensive, side-...

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

    International Nuclear Information System (INIS)

    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+ 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+, but not Lyb-5- 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+ 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

  15. Cell Wall-Associated Protein Antigens of Streptococcus salivarius: Purification, Properties, and Function in Adherence

    Science.gov (United States)

    Weerkamp, Anton H.; Jacobs, Ton

    1982-01-01

    Three cell wall-associated protein antigens (antigens b, c, and d) were isolated from mutanolysin-solubilized cell walls of Streptococcus salivarius HB and purified to apparent homogeneity by a combination of ion-exchange chromatography, gel filtration, and immunoadsorption chromatography. Antigens b and c were also isolated from culture supernatants. Antigen b consisted of more than 80% protein and had an apparent molecular weight as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 320,000. Antigen c consisted of 57% protein, about 30% neutral sugar, and about 13% amino sugar, and its glycoprotein nature was confirmed by specific staining techniques. During sodium dodecyl sulfate-polyacrylamide gel electrophoresis antigen c resolved into two or more bands, depending on the source or the isolation procedure, in the molecular weight range from 220,000 to 280,000. Antigen d consisted of 95% protein and was observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as two bands with molecular weights of 129,000 and 121,000. Under nondenaturing conditions all three antigens had molecular weights in the range from 1 × 106 to 3 × 106 as determined by gel filtration. The amino acid compositions of antigens b, c, and d were characterized by low amounts of basic amino acids and relatively high levels of nonpolar amino acids. Among oral streptococcal species antigens b and c were virtually restricted to strains of S. salivarius and most often to serotype I strains. Antigen b was recognized as the factor that mediates coaggregation of S. salivarius with Veillonella strains. The purified protein retained its biological activity. Antigen c could be linked to functions relating to adhesion of the streptococci to host tissues on the basis of its absence in mutant strains and blocking by specific antisera. The purified molecule had no detectable biological activity. Antigen d could not be linked to an established adhesion function. Images

  16. 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.

  17. Molecular signals in antigen presentation. II. Activation of cytolytic cells in vitro after ultraviolet radiation or combined gamma and ultraviolet radiation treatment of antigen-presenting cells

    International Nuclear Information System (INIS)

    Murine low-density spleen cells have potent antigen-presenting ability in a hapten-specific cytolytic T lymphocyte (CTL) system using the hapten azobenzenearsonate (ABA). Exposure of these cells to 0.33 KJ/m2 of ultraviolet radiation (UVR) after coupling to hapten results in markedly inhibited antigen-presenting function that can be substantially corrected or bypassed by interleukin 1 (IL 1). These results have been interpreted to reflect an inhibition of Lyt-1+ T cell activation by UVR-treated APC. Treatment of these cells sequentially with 1500 rad of γ-radiation (GR) prior to hapten coupling, followed by 0.33 KJ/m2 of UVR radiation after coupling, results in an antigen-resenting defect only minimally improved by IL 1. However, partially purified interleukin 2 (IL 2) can completely bypass or correct this defect. Thus, combined Cr and UVR induces a different or more profound defect in APC function when compared to UVR alone. However, these cells do provide a signal(s) other than hapten necessary for CTL activation because ABA-coupled high density spleen cells do not activate CTL cells, even with the addition of IL 2. Fluorescence-activated cell sorter analysis demonstrates that exposure of these low density spleen cells to GP or UVR results in decreased I-A antigen expression at 24 hr; exposure to both GR and UVR results in a greater decrease in I-A antigen expression at 24 hr than either alone. The addition of nonhapten-coupled low-density APC partially reconstitutes the ability of combined GR/UVR-treated LD-APC to present antigen, and this effect is enhanced by the administration of exogenous IL 1

  18. Enhanced immune stimulation by a therapeutic lymphoma tumor antigen vaccine produced in insect cells involves mannose receptor targeting to antigen presenting cells.

    Science.gov (United States)

    Betting, David J; Mu, Xi Y; Kafi, Kamran; McDonnel, Desmond; Rosas, Francisco; Gold, Daniel P; Timmerman, John M

    2009-01-01

    Therapeutic vaccination of lymphoma patients with tumor-specific immunoglobulin (idiotype, Id) coupled to the carrier protein keyhole limpet hemocyanin (Id-KLH) is undergoing clinical investigation, and methods to improve the immunogenicity of these and other protein tumor antigen vaccines are being sought. Id proteins can be produced via tumor-myeloma hybridomas or recombinant methods in mammalian, bacteria, or insect cells. We now demonstrate that terminal mannose residues, characteristic of recombinant proteins produced in insect cells, yield Id proteins with significantly enhanced immunostimulatory properties compared to Id proteins derived from mammalian cells. Recombinant baculovirus-infected insect cell-derived Id showed higher binding to and activation of human dendritic cells mediated by mannose receptors. In vivo, insect cell-derived Id elicited higher levels of tumor-specific CD8+ cytotoxic T lymphocyte (CTL) and improved eradication of pre-established murine lymphoma. Insect cell and mammalian Id generated similar levels of tumor-specific antibodies, showing no impairment in antibody responses to native tumor antigen despite the glycoslylation differences in the immunogen. Combining insect cell production and maleimide-based KLH conjugation offered the highest levels of anti-tumor immunity. Our data comparing sources of recombinant Id protein tumor antigens used in therapeutic cancer vaccines demonstrate that insect cell-derived antigens can offer several immunologic advantages over proteins derived from mammalian sources. PMID:19000731

  19. CD4+ and CD8+ T-cell responses to latent antigen EBNA-1 and lytic antigen BZLF-1 during persistent lymphocryptovirus infection of rhesus macaques.

    Science.gov (United States)

    Leskowitz, R M; Zhou, X Y; Villinger, F; Fogg, M H; Kaur, A; Lieberman, P M; Wang, F; Ertl, H C

    2013-08-01

    Epstein-Barr virus (EBV) infection leads to lifelong viral persistence through its latency in B cells. EBV-specific T cells control reactivations and prevent the development of EBV-associated malignancies in most healthy carriers, but infection can sometimes cause chronic disease and malignant transformation. Epstein-Barr nuclear antigen 1 (EBNA-1) is the only viral protein consistently expressed during all forms of latency and in all EBV-associated malignancies and is a promising target for a therapeutic vaccine. Here, we studied the EBNA-1-specific immune response using the EBV-homologous rhesus lymphocryptovirus (rhLCV) infection in rhesus macaques. We assessed the frequency, phenotype, and cytokine production profiles of rhLCV EBNA-1 (rhEBNA-1)-specific T cells in 15 rhesus macaques and compared them to the lytic antigen of rhLCV BZLF-1 (rhBZLF-1). We were able to detect rhEBNA-1-specific CD4(+) and/or CD8(+) T cells in 14 of the 15 animals screened. In comparison, all 15 animals had detectable rhBZLF-1 responses. Most peptide-specific CD4(+) T cells exhibited a resting phenotype of central memory (TCM), while peptide-specific CD8(+) T cells showed a more activated phenotype, belonging mainly to the effector cell subset. By comparing our results to the human EBV immune response, we demonstrate that the rhLCV model is a valid system for studying chronic EBV infection and for the preclinical development of therapeutic vaccines. PMID:23698300

  20. Microsomal triglyceride transfer protein lipidation and control of CD1d on antigen-presenting cells

    OpenAIRE

    Dougan, Stephanie K.; Salas, Azucena; Rava, Paul; Agyemang, Amma; Kaser, Arthur; Morrison, Jamin; Khurana, Archana; Kronenberg, Mitchell; Johnson, Caroline; Exley, Mark; Hussain, M. Mahmood; Blumberg, Richard S.

    2005-01-01

    Microsomal triglyceride transfer protein (MTP), an endoplasmic reticulum (ER) chaperone that loads lipids onto apolipoprotein B, also regulates CD1d presentation of glycolipid antigens in the liver and intestine. We show MTP RNA and protein in antigen-presenting cells (APCs) by reverse transcription–polymerase chain reaction and by immunoblotting of mouse liver mononuclear cells and mouse and human B cell lines. Functional MTP, demonstrated by specific triglyceride transfer activity, is prese...

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

    OpenAIRE

    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 fluorescence techniques disclosed that these cells were heterogenous with respect to the expression of a series of differentiation and activation antigens defined by monoclonal antibodies. Thus, whereas all...

  2. Formaldehyde treatment of proteins can constrain presentation to T cells by limiting antigen processing.

    OpenAIRE

    Di Tommaso, A; De Magistris, M T; Bugnoli, M.; Marsili, I; Rappuoli, R; Abrignani, S.

    1994-01-01

    Proteins to be used as vaccines are frequently treated with formaldehyde, although little is known about the effects of this treatment on protein antigenicity. To investigate the effect of formaldehyde treatment on antigen recognition by T cells, we compared the in vitro T-cell response to proteins that have been formaldehyde treated with the response to untreated proteins. We found that peripheral blood mononuclear cells from individuals vaccinated with three formaldehyde-treated proteins (p...

  3. Variability in expression of cell surface antigens of Candida albicans during morphogenesis.

    OpenAIRE

    Brawner, D L; Cutler, J. E.

    1986-01-01

    The location and expression of two different cell surface antigens on germinating and nongerminating Candida albicans cells was examined by using transmission electron microscopy after labeling with monoclonal antibodies (H9 or C6) and immunocolloidal gold. Immunodeterminant expression of the two carbohydrate antigens was followed from early germination events through 20 h of development. The determinant detected by H9 antibody, which was initially lost from the mother cell surface and prefer...

  4. Batf3-Dependent Dendritic Cells in the Renal Lymph Node Induce Tolerance against Circulating Antigens

    OpenAIRE

    Gottschalk, Catherine; Damuzzo, Vera; Gotot, Janine; Kroczek, Richard A.; Yagita, Hideo; Murphy, Kenneth M.; Knolle, Percy A.; Ludwig-Portugall, Isis; Kurts, Christian

    2013-01-01

    Although the spleen is a major site where immune tolerance to circulating innocuous antigens occurs, the kidney also contributes. Circulating antigens smaller than albumin are constitutively filtered and concentrated in the kidney and reach the renal lymph node by lymphatic drainage, where resident dendritic cells (DCs) capture them and induce tolerance of specific cytotoxic T cells through unknown mechanisms. Here, we found that the coinhibitory cell surface receptor programmed death 1 (PD-1...

  5. Scaffolded Antigens in Yeast Cell Particle Vaccines Provide Protection against Systemic Polyoma Virus Infection

    OpenAIRE

    Tipper, Donald J.; Szomolanyi-Tsuda, Eva

    2016-01-01

    Background. U65, a self-aggregating peptide scaffold, traps fused protein antigens in yeast cells. Conversion to Yeast Cell Particle (YCP) vaccines by partial removal of surface mannoproteins exposes β-glucan, mediating efficient uptake by antigen-presenting cells (APCs). YCP vaccines are inexpensive, capable of rapid large-scale production and have potential for both parenteral and oral use. Results. YCP processing by alkaline hydrolysis exposes up to 20% of the glucan but converts scaffolde...

  6. Regulation of murine macrophage Ia-antigen expression by products of activated spleen cells

    OpenAIRE

    1980-01-01

    This investigation examined the effects of mediators derived form activated spleen cells on macrophage Ia-antigen expression and function. Incubation of adherent thioglycollate-induced murine peritoneal macrophages(> 90% Ia-) with concanavalin A (Con A)- stimulated spleen cell supernate (Con A sup) resulted in a dose- dependent increase in the percentage of Ia-containing (Ia+) phagocytic cells, as detected by antiserum-and-complement-mediated cytotoxicity. The Ia-antigen expression of macroph...

  7. Design and Development of Therapies using Chimeric Antigen Receptor-Expressing T cells

    OpenAIRE

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

    2014-01-01

    Investigators developed chimeric antigen receptors (CARs) for expression on T cells more than 25 years ago. When the CAR is derived from an antibody, the resultant cell should combine the desirable targeting features of an antibody (e.g. lack of requirement for major histocompatibility complex recognition, ability to recognize non-protein antigens) with the persistence, trafficking and effector functions of a T-cell. This article describes how the past two decades have seen a crescendo of res...

  8. Merkel Cell Polyomavirus Large T Antigen Has Growth-Promoting and Inhibitory Activities

    OpenAIRE

    Cheng, Jingwei; Rozenblatt-Rosen, Orit; Paulson, Kelly G.; Nghiem, Paul; DeCaprio, James A.

    2013-01-01

    Merkel cell carcinoma (MCC) is a rare and aggressive form of skin cancer. In at least 80% of all MCC, Merkel cell polyomavirus (MCPyV) DNA has undergone clonal integration into the host cell genome, and most tumors express the MCPyV large and small T antigens. In all cases of MCC reported to date, the integrated MCPyV genome has undergone mutations in the large T antigen. These mutations result in expression of a truncated large T antigen that retains the Rb binding or LXCXE motif but deletes...

  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. Stimulation of T-cell activation by UV-treated, antigen-pulsed macrophages: evidence for a requirement for antigen processing and interleukin 1 secretion

    International Nuclear Information System (INIS)

    The nature of the defect(s) in the ability of UV-treated guinea pig macrophages to stimulate the proliferative response of guinea pig T cells to soluble protein antigens was investigated. T cells proliferated vigorously when cultured with peritoneal exudate cells (PEC) which had been pulsed with soluble protein antigens, but failed to proliferate when cultured with soluble antigen or with antigen-pulsed, UV-treated PEC. UV-treated macrophages were unable to secrete interleukin 1 (IL-1). Addition of IL-1 partially restored the T-cell proliferative response stimulated by antigen-pulsed, UV-treated PEC. However, IL-1 was able to restore such a response only when the PEC were pulsed with antigen before being exposed to UV. Similar results were obtained when antigen-pulsed PEC were used to stimulate T cells to secrete interleukin 2 (IL-2). These results demonstrate that UV-treated macrophages are defective both in their ability to properly process and present antigen for T-cell recognition and in their ability to secrete IL-1

  11. Human parvovirus B19 induced apoptotic bodies contain altered self-antigens that are phagocytosed by antigen presenting cells.

    Directory of Open Access Journals (Sweden)

    Kanoktip Thammasri

    Full Text Available Human parvovirus B19 (B19V from the erythrovirus genus is known to be a pathogenic virus in humans. Prevalence of B19V infection has been reported worldwide in all seasons, with a high incidence in the spring. B19V is responsible for erythema infectiosum (fifth disease commonly seen in children. Its other clinical presentations include arthralgia, arthritis, transient aplastic crisis, chronic anemia, congenital anemia, and hydrops fetalis. In addition, B19V infection has been reported to trigger autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. However, the mechanisms of B19V participation in autoimmunity are not fully understood. B19V induced chronic disease and persistent infection suggests B19V can serve as a model for viral host interactions and the role of viruses in the pathogenesis of autoimmune diseases. Here we investigate the involvement of B19V in the breakdown of immune tolerance. Previously, we demonstrated that the non-structural protein 1 (NS 1 of B19V induces apoptosis in non-permissive cells lines and that this protein can cleave host DNA as well as form NS1-DNA adducts. Here we provide evidence that through programmed cell death, apoptotic bodies (ApoBods are generated by B19V NS1 expression in a non-permissive cell line. Characterization of purified ApoBods identified potential self-antigens within them. In particular, signature self-antigens such as Smith, ApoH, DNA, histone H4 and phosphatidylserine associated with autoimmunity were present in these ApoBods. In addition, when purified ApoBods were introduced to differentiated macrophages, recognition, engulfment and uptake occurred. This suggests that B19V can produce a source of self-antigens for immune cell processing. The results support our hypothesis that B19V NS1-DNA adducts, and nucleosomal and lysosomal antigens present in ApoBods created in non-permissive cell lines, are a source of self-antigens.

  12. Pollen-induced antigen presentation by mesenchymal stem cells and T cells from allergic rhinitis.

    Science.gov (United States)

    Desai, Mauli B; Gavrilova, Tatyana; Liu, Jianjun; Patel, Shyam A; Kartan, Saritha; Greco, Steven J; Capitle, Eugenio; Rameshwar, Pranela

    2013-10-01

    Mesenchymal stem cells (MSCs) are promising cellular suppressor of inflammation. This function of MSCs is partly due to their licensing by inflammatory mediators. In cases with reduced inflammation, MSCs could become immune-enhancer cells. MSCs can suppress the inflammatory response of antigen-challenged lymphocytes from allergic asthma. Although allergic rhinitis (AR) is also an inflammatory response, it is unclear if MSCs can exert similar suppression. This study investigated the immune effects (suppressor vs enhancer) of MSCs on allergen-stimulated lymphocytes from AR subjects (grass or weed allergy). In contrast to subjects with allergic asthma, MSCs caused a significant (Pcells (antigen-presenting cells (APCs)). This correlated with increased production of inflammatory cytokines from T cells, and increased expressions of major histocompatibility complex (MHC)-II and CD86 on MSCs. The specificity of APC function was demonstrated in APC assay using MSCs that were knocked down for the master regulator of MHC-II transcription, CIITA. The difference in the effects of MSCs on allergic asthma and AR could not be explained by the sensitivity to the allergen, based on skin tests. Thus, we deduced that the contrasting immune effects of MSCs for antigen-challenged lymphocytes on AR and allergic asthma could be disease specific. It is possible that the enhanced inflammation from asthma might be required to license the MSCs to become suppressor cells. This study underscores the need for robust preclinical studies to effectively translate MSCs for any inflammatory disorder. PMID:25505949

  13. Serum antibodies to whole-cell and recombinant antigens of Borrelia burgdorferi in cottontail rabbits.

    Science.gov (United States)

    Magnarelli, Louis A; Norris, Steven J; Fikrig, Erol

    2012-01-01

    Archived serum samples, from 95 eastern cottontail rabbits (Sylvilagus floridanus) captured in New York, New York, USA and Millbrook, New York, USA, during 1985-86, were analyzed in solid-phase enzyme-linked immunosorbent assays (ELISA) for total and class-specific immunoglobulin (Ig) M antibodies to whole-cell or recombinant antigens of Borrelia burgdorferi sensu stricto. Using a polyvalent conjugate, rabbit sera contained antibodies to whole-cell and recombinant antigens (protein [p]35, p37, or VlsE) during different seasons, but there was no reactivity to outer surface protein (Osp)A or OspB. Seventy-six of the 102 sera (75%) analyzed were reactive with one or more of the antigens; 61 of the positive samples (80%) reacted to whole-cell antigens, followed by results for the p35 (58%, 44/76), VlsE (43%, 33/76), and p37 (29%, 22/ 76) antigens. Fifty-eight sera (76%) contained antibodies to the VlsE or p35 antigens with or without reactivity to whole-cell antigens. High antibody titers (≥1:2,560) recorded for 52 sera indicate robust antibody production. In analyses for IgM antibodies in an ELISA containing whole-cell antigens, there were 30 positive sera; titers ranged from 1:160 to 1:640. There was minimal cross-reactivity when rabbit antisera to Treponema pallidum or four serovars of Leptospira interrogans were screened against B. burgdorferi antigens. Based on more-specific results, VlsE and p35 antigens appear to be useful markers for detecting possible B. burgdorferi infections. PMID:22247369

  14. Monoclonal antibodies to cell surface antigens of human melanoma

    International Nuclear Information System (INIS)

    The authors have worked with three human melanoma antigens which have been defined by monoclonal mouse antibodies: p97, a glycoprotein that is structurally related to transferrin, a proteoglycan, and a GD3 ganglioside that is slightly different from the GD3 of normal brain. All three antigens can be detected in frozen sections of melanoma, using immunohistological techniques. Antibodies and Fab fragments, specific for either p97 or the proteoglycan antigen, have been radiolabelled with 131I and successfully used for tumor imaging, and Phase I therapeutic trails are underway, using 131I-labelled Fab fragments, specific for p97 or the proteoglycan antigen, to localize a potentially therapeutic dose of radiation into tumors. It may be feasible to use the same monoclonal antibodies, or antibody fragments, as carriers of neutron capturers, such as boron, for possible use in tumor therapy. The initial experiments on this are best carried out by using nude mice (or rats) carrying human melanoma xenografts

  15. 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

  16. 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.

  17. Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B antigen

    International Nuclear Information System (INIS)

    A U2 small nuclear RNA-associated protein, designated B'', was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies enabled them to isolate cDNA clone λHB''-1 from a phage λgt11 expression library. This clone appeared to code for the B'' protein as established by in vitro translation of hybrid-selected mRNA. The identity of clone λHB''-1 was further confirmed by partial peptide mapping and analysis of the reactivity of the recombinant antigen with monospecific and monoclonal antibodies. Analysis of the nucleotide sequence of the 1015-base-pair cDNA insert of clone λHB''-1 revealed a large open reading frame of 800 nucleotides containing the coding sequence for a polypeptide of 25,457 daltons. In vitro transcription of the λHB''-1 cDNA insert and subsequent translation resulted in a protein product with the molecular size of the B'' protein. These data demonstrate that clone λHB''-1 contains the complete coding sequence of this antigen. The deduced polypeptide sequence contains three very hydrophilic regions that might constitute RNA binding sites and/or antigenic determinants. These findings might have implications both for the understanding of the pathogenesis of rheumatic diseases as well as for the elucidation of the biological function of autoimmune antigens

  18. 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.

  19. Biochemical basis of synergy between antigen and T-helper (Th) cell-mediated activation of resting human B cells.

    OpenAIRE

    Chartash, E K; Crow, M K; Friedman, S M

    1989-01-01

    We have utilized CD23 expression as a marker for B cell activation in order to investigate the biochemical basis for synergy between antigen and T helper (Th) cells in the activation of resting human B cells. Our results confirm that while ligation of surface immunoglobulin (sIg) receptors by antigen analogues (e.g., F(ab')2 goat anti-human IgM) does not lead to CD23 expression, this stimulus markedly enhances CD23 expression induced during antigen specific Th-B cell interaction or by rIL-4. ...

  20. Merkel cell polyomavirus large T antigen has growth-promoting and inhibitory activities.

    Science.gov (United States)

    Cheng, Jingwei; Rozenblatt-Rosen, Orit; Paulson, Kelly G; Nghiem, Paul; DeCaprio, James A

    2013-06-01

    Merkel cell carcinoma (MCC) is a rare and aggressive form of skin cancer. In at least 80% of all MCC, Merkel cell polyomavirus (MCPyV) DNA has undergone clonal integration into the host cell genome, and most tumors express the MCPyV large and small T antigens. In all cases of MCC reported to date, the integrated MCPyV genome has undergone mutations in the large T antigen. These mutations result in expression of a truncated large T antigen that retains the Rb binding or LXCXE motif but deletes the DNA binding and helicase domains. However, the transforming functions of full-length and truncated MCPyV large T antigen are unknown. We compared the transforming activities of full-length, truncated, and alternatively spliced 57kT forms of MCPyV large T antigen. MCPyV large T antigen could bind to Rb but was unable to bind to p53. Furthermore, MCPyV-truncated large T antigen was more effective than full-length and 57kT large T antigen in promoting the growth of human and mouse fibroblasts. In contrast, expression of the MCPyV large T antigen C-terminal 100 residues could inhibit the growth of several different cell types. These data imply that the deletion of the C terminus of MCPyV large T antigen found in MCC serves not only to disrupt viral replication but also results in the loss of a distinct growth-inhibitory function intrinsic to this region. PMID:23514892

  1. Structural analysis of antigen-specific Ia-bearing regulatory T-cell factors: gel electrophoretic analysis of the antigen-specific augmenting T -cell factor.

    OpenAIRE

    Miyatani, S; Hiramatsu, K; Nakajima, P B; Owen, F L; Tada, T

    1983-01-01

    An antigen-specific T-cell factor (TaF) that specifically augments the antibody response was purified and biochemically analyzed by NaDodSO4/polyacrylamide gel electrophoresis and isoelectric focusing. Biosynthetically labeled TaF was separated from the Nonidet P-40 extract of T-cell hybridoma FL10, which produces a keyhole limpet hemocyanin-specific TaF, by affinity chromatography either with antigen or with monoclonal anti-I-A antibodies. The material thus obtained was composed of two diffe...

  2. 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

    In this study biopsies from skin lesions and draining lymph nodes of patients suffering from cutaneous leishmaniasis caused by Leishmania major were examined by immunohistochemistry, and by light and electron microscopy to identify the types of antigen-presenting cells (APC) and their location. APC......, 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...... keratinocytes and endothelial cells also showed these characteristics, they may also act as APC. By examining tissue samples from skin lesions and draining lymph nodes it was possible to follow the probable route of trafficking of various inflammatory cells between the skin lesion and lymph nodes. Leishmania...

  3. In situ Delivery of Tumor Antigen- and Adjuvant-Loaded Liposomes Boosts Antigen-Specific T-Cell Responses by Human Dermal Dendritic Cells.

    Science.gov (United States)

    Boks, Martine A; Bruijns, Sven C M; Ambrosini, Martino; Kalay, Hakan; van Bloois, Louis; Storm, Gert; de Gruijl, Tanja; van Kooyk, Yvette

    2015-11-01

    Dendritic cells (DCs) have an important role in tumor control via the induction of tumor-specific T-cell responses and are therefore an ideal target for immunotherapy. The human skin is an attractive site for tumor vaccination as it contains various DC subsets. The simultaneous delivery of tumor antigen with an adjuvant is beneficial for cross-presentation and the induction of tumor-specific T-cell responses. We therefore developed liposomes that contain the melanoma-associated antigen glycoprotein 100280-288 peptide and Toll-like receptor 4 (TLR4) ligand monophosphoryl lipid A (MPLA) as adjuvant. These liposomes are efficiently taken up by monocyte-derived DCs, and antigen presentation to CD8(+) T cells was significantly higher with MPLA-modified liposomes as compared with non-modified liposomes or the co-administration of soluble MPLA. We used a human skin explant model to evaluate the efficiency of intradermal delivery of liposomes. Liposomes were efficiently taken up by CD1a(+) and especially CD14(+) dermal DCs. Induction of CD8(+) T-cell responses by emigrated dermal DCs was significantly higher when MPLA was incorporated into the liposomes as compared with non-modified liposomes or co-administration of soluble MPLA. Thus, the modification of antigen-carrying liposomes with TLR ligand MPLA significantly enhances tumor-specific T-cell responses by dermal DCs and is an attractive vaccination strategy in human skin. PMID:26083554

  4. 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. PMID:27427982

  5. Vaccine delivery by penetratin: mechanism of antigen presentation by dendritic cells.

    Science.gov (United States)

    Pouniotis, Dodie; Tang, Choon-Kit; Apostolopoulos, Vasso; Pietersz, Geoffrey

    2016-08-01

    Cell-penetrating peptides (CPP) or membrane-translocating peptides such as penetratin from Antennapedia homeodomain or TAT from human immunodeficiency virus are useful vectors for the delivery of protein antigens or their cytotoxic (Tc) or helper (Th) T cell epitopes to antigen-presenting cells. Mice immunized with CPP containing immunogens elicit antigen-specific Tc and/or Th responses and could be protected from tumor challenges. In the present paper, we investigate the mechanism of class I and class II antigen presentation of ovalbumin covalently linked to penetratin (AntpOVA) by bone marrow-derived dendritic cells with the use of biochemical inhibitors of various pathways of antigen processing and presentation. Results from our study suggested that uptake of AntpOVA is via a combination of energy-independent (membrane fusion) and energy-dependent pathways (endocytosis). Once internalized by either mechanism, multiple tap-dependent or independent antigen presentation pathways are accessed while not completely dependent on proteasomal processing but involving proteolytic trimming in the ER and Golgi compartments. Our study provides an understanding on the mechanism of antigen presentation mediated by CPP and leads to greater insights into future development of vaccine formulations. PMID:27138940

  6. 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.

  7. Tracking antigen-specific T-cells during clinical tolerance induction in humans.

    Directory of Open Access Journals (Sweden)

    Aamir Aslam

    Full Text Available Allergen immunotherapy presents an opportunity to define mechanisms of induction of clinical tolerance in humans. Significant progress has been made in our understanding of changes in T cell responses during immunotherapy, but existing work has largely been based on functional T cell assays. HLA-peptide-tetrameric complexes allow the tracking of antigen-specific T-cell populations based on the presence of specific T-cell receptors and when combined with functional assays allow a closer assessment of the potential roles of T-cell anergy and clonotype evolution. We sought to develop tools to facilitate tracking of antigen-specific T-cell populations during wasp-venom immunotherapy in people with wasp-venom allergy. We first defined dominant immunogenic regions within Ves v 5, a constituent of wasp venom that is known to represent a target antigen for T-cells. We next identified HLA-DRB1*1501 restricted epitopes and used HLA class II tetrameric complexes alongside cytokine responses to Ves v 5 to track T-cell responses during immunotherapy. In contrast to previous reports, we show that there was a significant initial induction of IL-4 producing antigen-specific T-cells within the first 3-5 weeks of immunotherapy which was followed by reduction of circulating effector antigen-specific T-cells despite escalation of wasp-venom dosage. However, there was sustained induction of IL-10-producing and FOXP3 positive antigen-specific T cells. We observed that these IL-10 producing cells could share a common precursor with IL-4-producing T cells specific for the same epitope. Clinical tolerance induction in humans is associated with dynamic changes in frequencies of antigen-specific T-cells, with a marked loss of IL-4-producing T-cells and the acquisition of IL-10-producing and FOXP3-positive antigen-specific CD4+ T-cells that can derive from a common shared precursor to pre-treatment effector T-cells. The development of new approaches to track antigen

  8. Survival and antigenic profile of irradiated malarial sporozoites in infected liver cells

    Energy Technology Data Exchange (ETDEWEB)

    Suhrbier, A.; Winger, L.A.; Castellano, E.; Sinden, R.E. (Imperial College, London (England))

    1990-09-01

    Exoerythrocytic (EE) stages of Plasmodium berghei derived from irradiated sporozoites were cultured in vitro in HepG2 cells. They synthesized several antigens, predominantly but not exclusively those expressed by normal early erythrocytic schizonts. After invasion, over half the intracellular sporozoites, both normal and irradiated, appeared to die. After 24 h, in marked contrast to the normal parasites, EE parasites derived from irradiated sporozoites continued to break open, shedding their antigens into the cytoplasm of the infected host cells. Increasing radiation dosage, which has previously been shown to reduce the ability of irradiated sporozoites to protect animals, correlated with reduced de novo antigen synthesis by EE parasites derived from irradiated sporozoites.

  9. Location of T cell and major histocompatibility complex antigens in the human thymus

    OpenAIRE

    1980-01-01

    A series of monoclonal antibodies were used to study the intrathymic distribution of T cell-specific antigens, Ia antigens, and beta 2- microglobulin in frozen sections of human thymus by immunofluorescence and immunoperoxidase techniques. Most of the cortical thymocytes reacted with anti-T4, anti-T5, anti-T6, anti-T8, and anti-T10 antibodies, thus indicating coexpression of multiple antigens on cortical lymphocytes. The staining of cells in the medulla was most satisfactorily judged in secti...

  10. Quantitative interrelations of Lewis antigens in normal mucosa and transitional cell bladder carcinomas.

    OpenAIRE

    Limas, C

    1991-01-01

    The factors regulating the expression of the Lewis blood group related antigens in tissues have yet to be clarified. In an attempt to resolve some of the existing controversies the quantitative interrelationship of the Le(a), Le(b), X and Y antigens in normal urothelium and transitional cell carcinomas (TCC) was studied using biopsy specimens derived from 22 patients whose ABO and Lewis red blood cell phenotype was known. A quantitative scale was devised to encompass both the extent and inten...

  11. SERUM ANTIBODIES TO WHOLE-CELL AND RECOMBINANT ANTIGENS OF BORRELIA BURGDORFERI IN COTTONTAIL RABBITS

    OpenAIRE

    Magnarelli, Louis A.; Norris, Steven J; Fikrig, Erol

    2012-01-01

    Archived serum samples, from 95 eastern cottontail rabbits (Sylvilagus floridanus) captured in New York, New York, USA and Millbrook, New York, USA, during 1985–86, were analyzed in solid-phase enzyme-linked immunosorbent assays (ELISA) for total and class-specific immunoglobulin (Ig) M antibodies to whole-cell or recombinant antigens of Borrelia burgdorferi sensu stricto. Using a polyvalent conjugate, rabbit sera contained antibodies to whole-cell and recombinant antigens (protein [p]35, p37...

  12. Distinctive localization of antigen-presenting cells in human lymph nodes

    OpenAIRE

    Angel, Catherine E.; Chen, Chun-Jen J.; Horlacher, Oliver C.; Winkler, Sintia; John, Thomas; Browning, Judy; MacGregor, Duncan; Cebon, Jonathan; Dunbar, P. Rod

    2009-01-01

    Professional antigen-presenting cells (APCs) are sentinel cells of the immune system that present antigen to T lymphocytes and mediate an appropriate immune response. It is therefore surprising that knowledge of the professional APCs in human lymph nodes is limited. Using 3-color immunohistochemistry, we have identified APCs in human lymph nodes, excluding plasmacytoid APCs, that fall into 2 nonoverlapping classes: (1) CD209+ APCs, coexpressing combinations of CD206, CD14, and CD68, that occu...

  13. Immunohistochemical localization of granzyme B antigen in cytotoxic cells in human tissues.

    OpenAIRE

    Hameed, A.; Truong, L D; Price, V; Kruhenbuhl, O.; Tschopp, J

    1991-01-01

    Human granzyme B antigen is expressed in cytoplasmic granules of activated cytotoxic T lymphocytes and natural killer cells. Recombinant granzyme B was generated using a prokaryotic expression vector under the control of T7 transcription and translation signals. The 25-kd recombinant protein (granzyme B) was used to develop a rabbit polyclonal antiserum. Purified anti-granzyme B antibodies were used to detect the antigen expression in cytotoxic cells in human tissues. Using the avidin-biotin-...

  14. Evasion of peptide, but not lipid antigen presentation, through pathogen-induced dendritic cell maturation

    OpenAIRE

    Hava, David L.; van der Wel, Nicole ,; Cohen, Nadia; Dascher, Christopher C.; Houben, Diane; León, Luis; Agarwal, Sandeep; Sugita, Masahiko; van Zon, Maaike; Kent, Sally C.; Shams, Homayoun; Peters, Peter J.; Brenner, Michael B.

    2008-01-01

    Dendritic cells (DC) present lipid and peptide antigens to T cells on CD1 and MHC Class II (MHCII), respectively. The relative contribution of these systems during the initiation of adaptive immunity after microbial infection is not characterized. MHCII molecules normally acquire antigen and rapidly traffic from phagolysosomes to the plasma membrane as part of DC maturation, whereas CD1 molecules instead continually recycle between these sites before, during, and after DC maturation. We find ...

  15. Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells

    OpenAIRE

    Frigault, Matthew J.; Lee, Jihyun; Basil, Maria Ciocca; Carpenito, Carmine; Motohashi, Shinichiro; Scholler, John; Kawalekar, Omkar U.; Guedan, Sonia; McGettigan, Shannon E; Posey, Avery D; Ang, Sonny; Cooper, Laurence J. N.; Platt, Jesse M.; Johnson, F. Brad; Paulos, Chrystal M.

    2015-01-01

    This study compared second generation chimeric antigen receptors encoding signaling domains composed of CD28, ICOS and 4-1BB. Here we report that certain CARs endow T cells with the ability to undergo long-term autonomous proliferation. Transduction of primary human T-cell with lentiviral vectors encoding some of the CARs resulted in sustained proliferation for up to three months following a single stimulation through the TCR. Sustained numeric expansion was independent of cognate antigen and...

  16. Current concepts and future directions for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies.

    Science.gov (United States)

    Mahler, Michael; Meroni, Pier-Luigi; Bossuyt, Xavier; Fritzler, Marvin J

    2014-01-01

    The detection of autoantibodies that target intracellular antigens, commonly termed anti-nuclear antibodies (ANA), is a serological hallmark in the diagnosis of systemic autoimmune rheumatic diseases (SARD). Different methods are available for detection of ANA and all bearing their own advantages and limitations. Most laboratories use the indirect immunofluorescence (IIF) assay based on HEp-2 cell substrates. Due to the subjectivity of this diagnostic platform, automated digital reading systems have been developed during the last decade. In addition, solid phase immunoassays using well characterized antigens have gained widespread adoption in high throughput laboratories due to their ease of use and open automation. Despite all the advances in the field of ANA detection and its contribution to the diagnosis of SARD, significant challenges persist. This review provides a comprehensive overview of the current status on ANA testing including automated IIF reading systems and solid phase assays and suggests an approach to interpretation of results and discusses meeting the problems of assay standardization and other persistent challenges. PMID:24868563

  17. Current Concepts and Future Directions for the Assessment of Autoantibodies to Cellular Antigens Referred to as Anti-Nuclear Antibodies

    Directory of Open Access Journals (Sweden)

    Michael Mahler

    2014-01-01

    Full Text Available The detection of autoantibodies that target intracellular antigens, commonly termed anti-nuclear antibodies (ANA, is a serological hallmark in the diagnosis of systemic autoimmune rheumatic diseases (SARD. Different methods are available for detection of ANA and all bearing their own advantages and limitations. Most laboratories use the indirect immunofluorescence (IIF assay based on HEp-2 cell substrates. Due to the subjectivity of this diagnostic platform, automated digital reading systems have been developed during the last decade. In addition, solid phase immunoassays using well characterized antigens have gained widespread adoption in high throughput laboratories due to their ease of use and open automation. Despite all the advances in the field of ANA detection and its contribution to the diagnosis of SARD, significant challenges persist. This review provides a comprehensive overview of the current status on ANA testing including automated IIF reading systems and solid phase assays and suggests an approach to interpretation of results and discusses meeting the problems of assay standardization and other persistent challenges.

  18. Pulse labeling of small nuclear ribonucleoproteins in vivo reveals distinct patterns of antigen recognition by human autoimmune antibodies.

    OpenAIRE

    Fisher, D E; Reeves, W H; Conner, G E; Blobel, G; Kunkel, H. G.

    1984-01-01

    Antibodies directed against small nuclear ribonucleoprotein ( snRNP ) particles are found in the Sm and RNP autoimmune sera from numerous patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). These two reactivities differ in disease distribution as well as antigen specificity. Although sera from both of these autoimmune syndromes contain snRNP reactive antibodies, distinction in antigen binding specificity have been difficult to define because of the par...

  19. Common Ewing sarcoma-associated antigens fail to induce natural T cell responses in both patients and healthy individuals.

    Science.gov (United States)

    Altvater, Bianca; Kailayangiri, Sareetha; Theimann, Nadine; Ahlmann, Martina; Farwick, Nicole; Chen, Christiane; Pscherer, Sibylle; Neumann, Ilka; Mrachatz, Gabriele; Hansmeier, Anna; Hardes, Jendrik; Gosheger, Georg; Juergens, Heribert; Rossig, Claudia

    2014-10-01

    Disseminated or relapsed Ewing sarcoma (EwS) has remained fatal in the majority of patients. A promising approach to preventing relapse after conventional therapy is to establish tumor antigen-specific immune control. Efficient and specific T cell memory against the tumor depends on the expansion of rare T cells with native specificity against target antigens overexpressed by the tumor. Candidate antigens in EwS include six-transmembrane epithelial antigen of the prostate-1 (STEAP1), and the human cancer/testis antigens X-antigen family member 1 (XAGE1) and preferentially expressed antigen in melanoma (PRAME). Here, we screened normal donors and EwS patients for the presence of circulating T cells reactive with overlapping peptide libraries of these antigens by IFN-γ Elispot analysis. The majority of 22 healthy donors lacked detectable memory T cell responses against STEAP1, XAGE1 and PRAME. Moreover, ex vivo detection of T cells specific for these antigens in both blood and bone marrow were limited to a minority of EwS patients and required nonspecific T cell prestimulation. Cytotoxic T cells specific for the tumor-associated antigens were efficiently and reliably generated by in vitro priming using professional antigen-presenting cells and optimized cytokine stimulation; however, these T cells failed to interact with native antigen processed by target cells and with EwS cells expressing the antigen. We conclude that EwS-associated antigens fail to induce efficient T cell receptor (TCR)-mediated antitumor immune responses even under optimized conditions. Strategies based on TCR engineering could provide a more effective means to manipulating T cell immunity toward targeted elimination of tumor cells. PMID:24973179

  20. Secretion, interaction and assembly of two O-glycosylated cell wall antigens from Candida albicans.

    Science.gov (United States)

    Pavia, J; Aguado, C; Mormeneo, S; Sentandreu, R

    2001-07-01

    The mechanisms of incorporation of two antigens have been determined using a monoclonal antibody (3A10) raised against the material released from the mycelial cell wall by zymolyase digestion and retained on a concanavalin A column. One of the hybridomas secreted an IgG that reacted with two bands in Western blots. Indirect immunofluorescence showed that the antigens were located on the surfaces of mycelial cells, but within the cell walls of yeasts. These antigens were detected in a membrane preparation, in the SDS-soluble material and in the material released by a 1,3-beta-glucanase and chitinase from the cell walls of yeast and mycelial cells. In the latter three samples, an additional high-molecular-mass, highly polydispersed band was also detected. Beta-elimination of each fraction resulted in the disappearance of all antigen bands, suggesting that they are highly O-glycosylated. In addition, the electrophoretic mobility of the high-molecular-mass, highly polydispersed bands increased after digestion with endoglycosidase H, indicating that they are also N-glycosylated. New antigen bands were released when remnants of the cell walls extracted with 1,3-beta-glucanase or chitinase were digested with chitinase or 1,3-beta-glucanase. These results are consistent with the notion that, after secretion, parts of the O-glycosylated antigen molecules are transferred to an N-glycosylated protein(s). This molecular complex, as well as the remaining original 70 and 80 kDa antigen molecules, next bind to 1,3-beta-glucan or chitin, probably via 1,6-beta-glucan, and, in an additional step, to chitin or 1,3-beta-glucan. This process results in the final molecular product of each antigen, and their distribution in the cell walls. PMID:11429475

  1. Co-delivery of antigen and a lipophilic anti-inflammatory drug to cells via a tailorable nanocarrier emulsion.

    Science.gov (United States)

    Chuan, Yap Pang; Zeng, Bi Yun; O'Sullivan, Brendan; Thomas, Ranjeny; Middelberg, Anton P J

    2012-02-15

    Nanotechnology promises new drug carriers that can be tailored to specific applications. Here we report a new approach to drug delivery based on tailorable nanocarrier emulsions (TNEs), motivated by a need to co-deliver a protein antigen and a lipophilic drug for specific inhibition of nuclear factor kappa B (NF-κB) in antigen presenting cells (APCs). Co-delivery for NF-κB inhibition holds promise as a strategy for the treatment of rheumatoid arthritis. We used a highly surface-active peptide (SAP) to prepare a nanosized emulsion having defined surface properties predictable from the SAP sequence. Incorporating the lipophilic drug into the oil phase at the time of emulsion formation enabled its facile packaging. The SAP is depleted from bulk during emulsification, allowing simple subsequent addition of the drug-loaded oil-in-water emulsion to a solution of protein antigen. Decoration of emulsion surface with antigen was achieved via electrostatic deposition. In vitro data showed that the TNE prepared this way was internalized and well-tolerated by model APCs, and that good suppression of NF-κB expression was achieved. This work reports a new type of nanotechnology-based carrier, a TNE, which can potentially be tailored for co-delivery of multiple therapeutic components, and can be made using simple methods using only biocompatible materials. PMID:22153851

  2. 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.

  3. Discovery of T cell antigens by high-throughput screening of synthetic minigene libraries.

    Directory of Open Access Journals (Sweden)

    Brian D Hondowicz

    Full Text Available The identification of novel T cell antigens is central to basic and translational research in autoimmunity, tumor immunology, transplant immunology, and vaccine design for infectious disease. However, current methods for T cell antigen discovery are low throughput, and fail to explore a wide range of potential antigen-receptor interactions. To overcome these limitations, we developed a method in which programmable microarrays are used to cost-effectively synthesize complex libraries of thousands of minigenes that collectively encode the content of hundreds of candidate protein targets. Minigene-derived mRNA are transfected into autologous antigen presenting cells and used to challenge complex populations of purified peripheral blood CD8+ T cells in multiplex, parallel ELISPOT assays. In this proof-of-concept study, we apply synthetic minigene screening to identify two novel pancreatic islet autoantigens targeted in a patient with Type I Diabetes. To our knowledge, this is the first successful screen of a highly complex, synthetic minigene library for identification of a T cell antigen. In principle, responses against the full protein complement of any tissue or pathogen can be assayed by this approach, suggesting that further optimization of synthetic libraries holds promise for high throughput antigen discovery.

  4. Human sunlight-induced basal-cell-carcinoma-associated dendritic cells are deficient in T cell co-stimulatory molecules and are impaired as antigen-presenting cells.

    OpenAIRE

    Nestle, F.O.; Burg, G.; Fäh, J; Wrone-Smith, T; Nickoloff, B. J.

    1997-01-01

    Immune surveillance of skin cancer involves the stimulation of effector T cells by tumor-derived antigens and antigen-presenting cells (APCs). An effective APC must not only display processed antigen in the context of MHC molecules but also express co-stimulatory molecules that are required to fully activate T cells. One of the most common cutaneous neoplasms is basal cell carcinoma. To investigate expression of the co-stimulatory molecules CD80 (B7-1) and CD86 (B7-2) on tumor-associated dend...

  5. Phenotypic Studies of Natural Killer Cell Subsets in Human Transporter Associated with Antigen Processing Deficiency

    OpenAIRE

    Zimmer, Jacques; Bausinger, Huguette; Andrès, Emmanuel; Donato, Lionel; Hanau, Daniel; Hentges, François; Moretta, Alessandro; de la Salle, Henri

    2007-01-01

    Peripheral blood natural killer (NK) cells from patients with transporter associated with antigen processing (TAP) deficiency are hyporesponsive. The mechanism of this defect is unknown, but the phenotype of TAP-deficient NK cells is almost normal. However, we noticed a high percentage of CD56bright cells among total NK cells from two patients. We further investigated TAP-deficient NK cells in these patients and compared them to NK cells from two other TAP-deficient patients with no clinical ...

  6. Functional Cooperation of Epstein-Barr Virus Nuclear Antigen 2 and the Survival Motor Neuron Protein in Transactivation of the Viral LMP1 Promoter

    OpenAIRE

    Voss, Marc D.; Hille, Annette; Barth, Stephanie; Spurk, Andreas; Hennrich, Frank; Holzer, Daniela; Mueller-Lantzsch, Nikolaus; Kremmer, Elisabeth; Grässer, Friedrich A.

    2001-01-01

    Epstein-Barr virus nuclear antigen 2 (EBNA2) is essential for viral transformation of B cells and transactivates cellular and viral target genes by binding RBPJκ tethered to cognate promoter elements. EBNA2 interacts with the DEAD-box protein DP103 (DDX20/Gemin3), which in turn is complexed to the survival motor neuron (SMN) protein. SMN is implicated in RNA processing, but a role in transcriptional regulation has also been suggested. Here, we show that DP103 and SMN are complexed in B cells ...

  7. Immune responses of dendritic cells after acquiring antigen from apoptotic hepatocholangioma cells caused by γ-ray

    International Nuclear Information System (INIS)

    Objective: To investigate the induction of cytotoxic T lymphocytes (CTLs) in antitumor responsiveness and therapeutic effects after dendritic cells (DCs) acquired antigen from apoptotic hepatocholangioma cells. Methods: DCs from blood mononuclear cells that maintain the characteristics of immaturity-anti-gen-capturing and-processing capacity were established in vitro by using granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-4. Then, apoptosis in hepatocholangioma cells was induced with γ-radiation. The experimental groups included (1) co-culture of DCs, and apoptotic cancer cells and T cells; (2) co-culture of DCs necrotic cancer cells and T cells; (3) co-culture of DCs-cultured cancer cell and T cells. These cells were co-cultured for 7 days. DCs and T cell were enriched separately. Finally, antitumor response test was carried out. Results: These cells had typical dendritic morphology, expressed high levels of CD1a, B7 and acquired antigen from apoptotic cells caused by γ-rays and induced an increased T cell-stimulatory capacity in MLR. Conclusions: DCs obtained from blood mononuclear cells using GM-CSF and IL-4 and DCs can efficiently present antigen driven from apoptotic cells caused by γ-rays and induce T cells increasing obviously. It can probably become an effective approach of DC transduction with antigen

  8. Interferon-induced changes in expression of antigens defined by monoclonal antibodies on malignant and nonmalignant mononuclear hematopoietic cells

    DEFF Research Database (Denmark)

    Hokland, M; Ritz, J; Hokland, P

    1983-01-01

    HLA-antigens detected by beta 2-Microglobulin (beta 2-M) could be demonstrated for peripheral blood mononuclear cells, non-T cells, Null cells, activated T cells, fetal thymocytes, adherent cells, and on four malignant non-T lymphoblastoid cell lines. In contrast, no significant differences were...... number as well as the amount of lymphocytes expressing the T10 antigen. It thus seems that the enhancing effect of IFN on resting cells of the immune system is highly selective. On the four lymphoblastoid cell lines, the expression of the common acute lymphoblastic leukemia antigen (CALLA) was...... significantly decreased concomitantly with the increase in MHC-antigens. On the other hand, the density of both a HLA-D related Ia antigen (I2) and a B-lymphocyte differentiation antigen (B1) remained unaltered following IFN treatment. The implications of these findings are discussed. Udgivelsesdato: 1983-null...

  9. 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

  10. Vitamin D controls T cell antigen receptor signaling and activation of human T cells

    DEFF Research Database (Denmark)

    von Essen, Marina Rode; Kongsbak, 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....

  11. 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.

  12. 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...... of skin lesions, and in Danes without known exposure to Leishmania parasites. Proliferation and production of interferon-gamma (IFN-gamma) and IL-4 in antigen-stimulated cultures was measured. Lymphocytes from individuals with a history of CL proliferated vigorously and produced IFN-gamma after...... the unexposed Danes were not activated by gp63. The cells from Danish donors produced either IFN-gamma or IL-4, but not both cytokines after incubation with the crude preparation of L. major antigens. The data show that the T cell response to Leishmania antigens in humans who have had uncomplicated CL...

  13. Inhibition of Ly-6A antigen expression prevents T cell activation

    OpenAIRE

    1990-01-01

    Antisense oligonucleotides complementary to the 5' end of the mRNA encoding the Ly-6A protein were used to block the expression of that protein. Using this approach we could inhibit the expression of Ly-6A by 60-80% in antigen-primed lymph node (LN) T cells as well as in the D10 T cell clone. Inhibition of Ly-6 expression resulted in the inability to restimulate in vitro, antigen-primed T cells. It also blocked the activation of normal spleen cells by Con A, monoclonal antibody (mAb) to CD3, ...

  14. 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

  15. 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.

  16. 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...... (DC) and macrophages infiltrate the CNS during experimental autoimmune encephalomyelitis (EAE). Microglia are not considered to be as effective APC as DC or macrophages. METHODS: In this work we compared the antigen presenting capacity of CD11c+ and CD11c- microglia subsets with infiltrating CD11c......+ APC, which include DC. The microglial subpopulations (CD11c- CD45dim CD11b+ and CD11c+ CD45dim CD11b+) as well as infiltrating CD11c+ CD45high cells were sorted from CNS of C57BL/6 mice with EAE. Sorted cells were characterised by flow cytometry for surface phenotype and by quantitative real-time PCR...

  17. Antigen-specific monoclonal antibodies isolated from B cells expressing constitutively active STAT5.

    Directory of Open Access Journals (Sweden)

    Ferenc A Scheeren

    Full Text Available BACKGROUND: Fully human monoclonal antibodies directed against specific pathogens have a high therapeutic potential, but are difficult to generate. METHODOLOGY/PRINCIPAL FINDINGS: Memory B cells were immortalized by expressing an inducible active mutant of the transcription factor Signal Transducer and Activator of Transcription 5 (STAT5. Active STAT5 inhibits the differentiation of B cells while increasing their replicative life span. We obtained cloned B cell lines, which produced antibodies in the presence of interleukin 21 after turning off STAT5. We used this method to obtain monoclonal antibodies against the model antigen tetanus toxin. CONCLUSIONS/SIGNIFICANCE: Here we describe a novel and relatively simple method of immortalizing antigen-specific human B cells for isolation of human monoclonal antibodies. These results show that STAT5 overexpression can be employed to isolate antigen specific antibodies from human memory B cells.

  18. A rapid method for the detection of antibodies to cell surface antigens: a solid phase radioimmunoassay using cell membranes

    International Nuclear Information System (INIS)

    Cell membranes isolated from murine lymphocytes or ascites tumors bind tightly to the surface of flexible plastic microtiter plates in the absence of additional proteins. This allows the detection of membrane associated molecules by specific antibodies and thus forms the basis for a rapid and sensitive radioimmunoassay for antibodies to membrane-bound components. The assay compares favourably with a variety of methods currently used to detect antibodies to cell surface antigens. The assay detects a variety of well characterized murine cell surface antigens (H-2, I-A, T-200, Thy-1.2, Ig). The level of antibody binding to membranes on plates correlates well with antigen density on intact cells. A modification of the assay involving competition between cross-reacting antibodies allows detection and resolution of closely spaced antigenic determinants. (Auth.)

  19. Identification of a novel SEREX antigen family, ECSA, in esophageal squamous cell carcinoma

    OpenAIRE

    Murakami Akihiro; Hachiya Takahisa; Kurei Shunsuke; Nishimori Takanori; Yasuraoka Mari; Nakashima-Fujita Kazue; Kuboshima Mari; Shiratori Tooru; Shimada Hideaki; Kagaya Akiko; Tamura Yutaka; Nomura Fumio; Ochiai Takenori; Matsubara Hisahiro; Takiguchi Masaki

    2011-01-01

    Abstract Background Diagnosis of esophageal squamous cell carcinoma (SCC) may improve with early diagnosis. Currently it is difficult to diagnose SCC in the early stage because there is a limited number of tumor markers available. Results Fifty-two esophageal SCC SEREX antigens were identified by SEREX (serological identification of antigens by recombinant cDNA expression cloning) using a cDNA phage library and sera of patients with esophageal SCC. Sequence analysis revealed that three of the...

  20. Human T cell responses to dengue virus antigens. Proliferative responses and interferon gamma production.

    OpenAIRE

    Kurane, I; Innis, B L; Nisalak, A; Hoke, C; Nimmannitya, S; Meager, A.; Ennis, F A

    1989-01-01

    The severe complications of dengue virus infections, hemorrhagic manifestations and shock, are more commonly observed during secondary dengue virus infections than during primary infections. It has been speculated that these complications are mediated by cross-reactive host-immune responses. We have begun to analyze human T cell responses to dengue antigens in vitro to explain the possible role of T lymphocytes in the pathogenesis of these complications. Dengue antigens induce proliferative r...

  1. Immunofluorescence of bovine virus diarrhea viral antigen in white blood cells from experimentally infected immunocompetent calves.

    OpenAIRE

    Bezek, D M; Baker, J. C.; Kaneene, J B

    1988-01-01

    A study to evaluate the detection of bovine virus diarrhea viral antigen using immunofluorescence testing of white blood cells was conducted. Five colostrum-deprived calves were inoculated intravenously with a cytopathic strain of the virus. Lymphocyte and buffy coat smears were prepared daily for direct immunofluorescent staining for detection of antigen. Lymphocytes were separated from heparinized blood using a Ficoll density procedure. Buffy coat smears were prepared from centrifuged blood...

  2. Degranulation of human mast cells induces an endothelial antigen central to leukocyte adhesion.

    OpenAIRE

    Klein, L M; Lavker, R M; Matis, W L; Murphy, G F

    1989-01-01

    To understand better the role of mast cell secretory products in the genesis of inflammation, a system was developed for in vitro degranulation of human mast cells in skin organ cultures. Within 2 hr after morphine sulfate-induced degranulation, endothelial cells lining microvessels adjacent to affected mast cells expressed an activation antigen important for endothelial-leukocyte adhesion. Identical results were obtained when other mast cell secretagogues (anti-IgE, compound 48/80, and calci...

  3. The Epstein-Barr virus nuclear antigen-1 reprograms transcription by mimicry of high mobility group A proteins.

    Science.gov (United States)

    Coppotelli, Giuseppe; Mughal, Nouman; Callegari, Simone; Sompallae, Ramakrishna; Caja, Laia; Luijsterburg, Martijn S; Dantuma, Nico P; Moustakas, Aristidis; Masucci, Maria G

    2013-03-01

    Viral proteins reprogram their host cells by hijacking regulatory components of protein networks. Here we describe a novel property of the Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA1) that may underlie the capacity of the virus to promote a global remodeling of chromatin architecture and cellular transcription. We found that the expression of EBNA1 in transfected human and mouse cells is associated with decreased prevalence of heterochromatin foci, enhanced accessibility of cellular DNA to micrococcal nuclease digestion and decreased average length of nucleosome repeats, suggesting de-protection of the nucleosome linker regions. This is a direct effect of EBNA1 because targeting the viral protein to heterochromatin promotes large-scale chromatin decondensation with slow kinetics and independent of the recruitment of adenosine triphosphate-dependent chromatin remodelers. The remodeling function is mediated by a bipartite Gly-Arg rich domain of EBNA1 that resembles the AT-hook of High Mobility Group A (HMGA) architectural transcription factors. Similar to HMGAs, EBNA1 is highly mobile in interphase nuclei and promotes the mobility of linker histone H1, which counteracts chromatin condensation and alters the transcription of numerous cellular genes. Thus, by regulating chromatin compaction, EBNA1 may reset cellular transcription during infection and prime the infected cells for malignant transformation. PMID:23358825

  4. Antigen-activated dendritic cells ameliorate influenza A infections

    OpenAIRE

    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 molecu...

  5. Antigen dynamics govern the induction of CD4(+) T cell tolerance during autoimmunity.

    Science.gov (United States)

    Challa, Dilip K; Mi, Wentao; Lo, Su-Tang; Ober, Raimund J; Ward, E Sally

    2016-08-01

    Antigen-specific T cell tolerance holds great promise for the treatment of autoimmune diseases. However, strategies to induce durable tolerance using high doses of soluble antigen have to date been unsuccessful, due to lack of efficacy and the risk of hypersensitivity. In the current study we have overcome these limitations by developing a platform for tolerance induction based on engineering the immunoglobulin Fc region to modulate the dynamic properties of low doses (1 μg/mouse; ∼50 μg/kg) of Fc-antigen fusions. Using this approach, we demonstrate that antigen persistence is a dominant factor governing the elicitation of tolerance in the model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE), induced by immunizing B10.PL mice with the N-terminal epitope of myelin basic protein. Unexpectedly, our analyses reveal a stringent threshold of antigen persistence for both prophylactic and therapeutic treatments, although distinct mechanisms lead to tolerance in these two settings. Importantly, the delivery of tolerogenic Fc-antigen fusions during ongoing disease results in the downregulation of T-bet and CD40L combined with amplification of Foxp3(+) T cell numbers. The generation of effective, low dose tolerogens using Fc engineering has potential for the regulation of autoreactive T cells. PMID:27236506

  6. Leishmania chagasi T-cell antigens identified through a double library screen.

    Science.gov (United States)

    Martins, Daniella R A; Jeronimo, Selma M B; Donelson, John E; Wilson, Mary E

    2006-12-01

    Control of human visceral leishmaniasis in regions where it is endemic is hampered in part by limited accessibility to medical care and emerging drug resistance. There is no available protective vaccine. Leishmania spp. protozoa express multiple antigens recognized by the vertebrate immune system. Since there is not one immunodominant epitope recognized by most hosts, strategies must be developed to optimize selection of antigens for prevention and immunodiagnosis. For this reason, we generated a cDNA library from the intracellular amastigote form of Leishmania chagasi, the cause of South American visceral leishmaniasis. We employed a two-step expression screen of the library to systematically identify T-cell antigens and T-dependent B-cell antigens. The first step was aimed at identifying the largest possible number of clones producing an epitope-containing polypeptide by screening with a pool of sera from Brazilians with documented visceral leishmaniasis. After removal of clones encoding heat shock proteins, positive clones underwent a second-step screen for their ability to cause proliferation and gamma interferon responses in T cells from immune mice. Six unique clones were selected from the second screen for further analysis. The corresponding antigens were derived from glutamine synthetase, a transitional endoplasmic reticulum ATPase, elongation factor 1gamma, kinesin K39, repetitive protein A2, and a hypothetical conserved protein. Humans naturally infected with L. chagasi mounted both cellular and antibody responses to these proteins. Preparations containing multiple antigens may be optimal for immunodiagnosis and protective vaccines. PMID:17000724

  7. Purification, characterization and docking studies of the HIN domain of human myeloid nuclear differentiation antigen (MNDA).

    Science.gov (United States)

    Li, He; Wang, Zhi-Xin; Wu, Jia-Wei

    2014-05-01

    The HIN domain of myeloid nuclear differentiation antigen (MNDA) was expressed and purified as a monomer using E. coli JM109 as host. The protein interacted with double-stranded DNA at a Kd of 3.15 μM and did not recognize the termini of double-stranded DNA. Isothermal titration calorimetry indicated that the interaction between the protein and double-stranded DNA is mainly mediated by electrostatic attractions and hydrogen bonding. We developed a model to analyze the potential DNA binding site of the MNDA HIN domain. Based on the model, molecular docking and mutation studies suggest that the double-stranded DNA binding site of the protein is different from other HIN-DNA structures. This work facilitates the design of specific drugs against pathogens detected by human MNDA. PMID:24557068

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

    International Nuclear Information System (INIS)

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

  9. Self-antigen recognition by TGFβ1-deficient T cells causes their activation and systemic inflammation

    OpenAIRE

    Bommireddy, Ramireddy; Pathak, Leena J; Martin, Jennifer; Ormsby, Ilona; Engle, Sandra J; Gregory P. Boivin; Babcock, George F.; Eriksson, Anna U.; Singh, Ram R; DOETSCHMAN, THOMAS

    2006-01-01

    To investigate whether the multifocal inflammatory disease in TGFβ1-deficient mice is caused by self-antigen (self-Ag)-specific autoreactive T cells, or whether it is caused by antigen independent, spontaneous hyperactivation of T cells, we have generated Tgfb1−/− and Tgfb1−/− Rag1−/− mice expressing the chicken OVA-specific TCR transgene (DO11.10). On a Rag1-sufficient background, Tgfb1−/− DO11.10 mice develop a milder inflammation than do Tgfb1−/− mice, and their T cells display a less acti...

  10. 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.

  11. Modulation of innate antigen-presenting cell function by pre-patent schistosome infection.

    Directory of Open Access Journals (Sweden)

    Christine E Ferragine

    Full Text Available Schistosomes are intravascular helminths that infect over 200 million people worldwide. Deposition of eggs by adult schistosomes stimulates Th2 responses to egg antigens and induces granulomatous pathology that is a hallmark of schistosome infection. Paradoxically, schistosomes require host immune function for their development and reproduction and for egress of parasite eggs from the host. To identify potential mechanisms by which immune cells might influence parasite development prior to the onset of egg production, we assessed immune function in mice infected with developing schistosomes. We found that pre-patent schistosome infection is associated with a loss of T cell responsiveness to other antigens and is due to a diminution in the ability of innate antigen-presenting cells to stimulate T cells. Diminution of stimulatory capacity by schistosome worms specifically affected CD11b(+ cells and did not require concomitant adaptive responses. We could not find evidence for production of a diffusible inhibitor of T cells by innate cells from infected mice. Rather, inhibition of T cell responsiveness by accessory cells required cell contact and only occurred when cells from infected mice outnumbered competent APCs by more than 3∶1. Finally, we show that loss of T cell stimulatory capacity may in part be due to suppression of IL-12 expression during pre-patent schistosome infection. Modulation of CD4(+ T cell and APC function may be an aspect of host immune exploitation by schistosomes, as both cell types influence parasite development during pre-patent schistosome infection.

  12. M cell-derived vesicles suggest a unique pathway for trans-epithelial antigen delivery.

    Science.gov (United States)

    Sakhon, Olivia S; Ross, Brittany; Gusti, Veronica; Pham, An Joseph; Vu, Kathy; Lo, David D

    2015-01-01

    M cells are a subset of mucosal epithelial cells with specialized capability to transport antigens across the mucosal barrier, but there is limited information on antigen transfer in the subepithelial zone due to the challenges in tracking microparticles and antigens that are transcytosed by this unique cell. Using transgenic reporter mice expressing dsRed in the cytoplasm of M cells and EGFP in myeloid cells, we observed that the M cell basolateral pocket hosts a close interaction between B lymphocytes and dendritic cells. Interestingly, we identified a population of previously undescribed M cell-derived vesicles (MCM) that are constitutively shed into the subepithelial space and readily taken up by CX3CR1(+)CD11b(+) CD11c(+) dendritic cells. These MCM are characterized by their cytoplasmic dsRed confirming their origin from the M cell cytoplasm. MCM showed preferential colocalization in dendritic cells with transcytosed bacteria but not transcytosed polystyrene beads, indicating a selective sorting of cargo fate in the subepithelial zone. The size and number of MCM were found to be upregulated by bacterial transcytosis and soluble toll-like receptor 2 (TLR2) agonist, further pointing to dynamic regulation of this mechanism. These results suggest that MCM provide a unique function by delivering to dendritic cells, various materials such as M cell-derived proteins, effector proteins, toxins, and particles found in the M cell cytoplasm during infection or surveillance. PMID:25838974

  13. Immunocapture and Identification of Cell Membrane Protein Antigenic Targets of Serum Autoantibodies*

    Science.gov (United States)

    Littleton, Edward; Dreger, Mathias; Palace, Jackie; Vincent, Angela

    2009-01-01

    There is increasing interest in the role of antibodies targeting specific membrane proteins in neurological and other diseases. The target(s) of these pathogenic antibodies is known in a few diseases, usually when candidate cell surface proteins have been tested. Approaches for identifying new antigens have mainly resulted in the identification of antibodies to intracellular proteins, which are often very useful as diagnostic markers for disease but unlikely to be directly involved in disease pathogenesis because they are not accessible to circulating antibodies. To identify cell surface antigens, we developed a “conformational membrane antigen isolation and identification” strategy. First, a cell line is identified that reacts with patient sera but not with control sera. Second, intact cells are exposed to sera to allow the binding of presumptive autoantibodies to their cell surface targets. After washing off non-bound serum components, the cells are lysed, and immune complexes are precipitated. Third, the bound surface antigen is identified by mass spectrometry. As a model system we used a muscle cell line, TE671, that endogenously expresses muscle-specific tyrosine receptor kinase (MuSK) and sera or plasmas from patients with a subtype of the autoimmune disease myasthenia gravis in which patients have autoantibodies against MuSK. MuSK was robustly detected as the only membrane protein in immunoprecipitates from all three patient samples tested and not from the three MuSK antibody-negative control samples processed in parallel. Of note, however, there were many intracellular proteins found in the immunoprecipitates from both patients and controls, suggesting that these were nonspecifically immunoprecipitated from cell extracts. The conformational membrane antigen isolation and identification technique should be of value for the detection of highly relevant antigenic targets in the growing number of suspected antibody-mediated autoimmune disorders. The

  14. Restoration of proliferative response to M. leprae antigens in lepromatous T cells against candidate antileprosy vaccines.

    Science.gov (United States)

    Mustafa, A S

    1996-09-01

    Several studies conducted in the last decade suggest that Mycobacterium lepraereactive T cells exist in lepromatous patients, but their number may be too few to yield a detectable response in cell-mediated immunity (CMI) assays. Immunizations with candidate antileprosy vaccines and stimulation of T cells with M. leprae + interleukin-2 restore the M. leprae-induced CMI response in lepromatous leprosy patients. These immunizations and stimulation may enrich the pre-existing M. leprae-responsive T cells in lepromatous patients and, thereby, induce a detectable CMI response to M. leprae antigens upon repeat testing. To verify this proposition, we carried out a study in a group of 10 lepromatous leprosy patients. Peripheral blood mononuclear cells (PBMC) obtained from these patients were anergic to M. leprae antigens in proliferative assays, but they responded to the antigens of candidate antileprosy vaccines, i.e., M. bovis BCG, M. bovis BCG + M. leprae, and Mycobacterium w. The enrichment of M. leprae-responsive T cells was performed by establishing T-cell lines from the PBMC after in vitro stimulation with M. leprae, M. bovis BCG, M. bovis BCG + M. leprae, and Mycobacterium w. When tested for their proliferative responses, 1/10, 3/10, 6/10 and 2/10 T-cell lines established against M. leprae, M. bovis BCG, M. bovis BCG + M. leprae, and Mycobacterium w, respectively, responded to M. leprae. These results suggest that enrichment of pre-existing M. leprae-responsive T cells may contribute to the restoration of the T-cell response to M. leprae in some lepromatous patients. Four of the 10 M. leprae-induced T-cell lines proliferated in response to the 65 kDa, 36 kDa, 28 kDa, and 12 kDa recombinant antigens of M. leprae, suggesting that the nonresponsiveness of T cells in some lepromatous patients may be overcome by using recombinant antigens of M. leprae. PMID:8862259

  15. Expression of MHC class II antigens in human B-cell leukaemia and non-Hodgkin's lymphoma.

    OpenAIRE

    Guy, K.; Krajewski, A S; Dewar, A E

    1986-01-01

    In this review we have summarized our experiences of serological analysis of MHC class II antigen expression in human B cell malignant disease. Cells from a large number of cases of B-cell chronic lymphocytic leukaemia (CLL) and non-Hodgkin's lymphoma (NHL) have been examined for expression of class II antigens. Using a number of monoclonal antibodies which in some cases are specific for class II subregion products (DP, DQ and DR), MHC class II antigens were detected by indirect immunofluores...

  16. CD28 and T cell antigen receptor signal transduction coordinately regulate interleukin 2 gene expression in response to superantigen stimulation

    OpenAIRE

    1992-01-01

    Activation of an immune response requires intercellular contact between T lymphocytes and antigen-presenting cells (APC). Interaction of the T cell antigen receptor (TCR) with antigen in the context of major histocompatibility molecules mediates signal transduction, but T cell activation appears to require the induction of a second costimulatory signal transduction pathway. Recent studies suggest that interaction of CD28 with B7 on APC might deliver such a costimulatory signal. To investigate...

  17. Serum squamous cell carcinoma antigen and CYFRA 21-1 in cervical cancer treatment

    International Nuclear Information System (INIS)

    Purpose: To analyze whether serum squamous cell carcinoma (SCC) antigen and cytokeratin-19 fragments (CYFRA) levels can assist in selecting patients with locally advanced cervical cancer who will benefit from combined treatment or additive surgery. Methods and Materials: Of 114 patients with cervical cancer Stage IB-IV, the first 39 patients received radiotherapy, the following 75 patients received identical radiotherapy plus concomitant chemotherapy (3 cycles of carboplatin and 5-fluorouracil). SCC antigen and CYFRA 21-1 serum levels were measured before treatment, after therapy, and during follow-up. Baseline tumor markers were related to tumor stage and size and clinical outcome. Results: Before treatment, SCC antigen was elevated (>1.9 μg/L) in 60% and CYFRA 21-1 (>2.2 μg/L) in 46% of patients. For all patients, disease-free survival (DFS) was better after combined treatment (67% vs. 43%, p<0.0005). For patients with elevated baseline SCC antigen, DFS was better after combination therapy (67% vs. 27%, p=0.001) which resulted more frequently in a normal SCC antigen (93% vs. 65%, p=0.004). In contrast, in those with a normal baseline CYFRA 21-1, combined therapy resulted in a better DFS (p=0.04). Patients who achieved a normal SCC antigen or CYFRA 21-1 after treatment had a better DFS (respectively 63 vs. 17% and 64 vs. 30%). Elevated SCC antigen posttreatment indicated residual tumor in 11/12 patients (92%), elevated CYFRA 21-1 in 7/10 patients (70%). Forty-seven patients had a tumor recurrence. At recurrence, SCC antigen was raised in 70% and CYFRA 21-1 in 69%. Conclusions: In patients with an elevated pretreatment SCC antigen, SCC antigen normalized more frequently with combined treatment and those patients had a better DFS. Elevated SCC antigen or CYFRA 21-1 levels after treatment completion indicated residual tumor in respectively 92% and 70%. The presence of elevated posttreatment levels of SCC antigen or CYFRA 21-1 indicates the need for additional

  18. 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.

  19. 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...... lymphoblastic leukemia cell with respect to surface marker phenotype. A population of CALLA- cells devoid of mature erythroid and myeloid surface markers was found to contain higher numbers of TdT+ cells but lower numbers of cyto-mu, B1, and Ia+ cells than the CALLA+ subset. In vitro analysis of normal...

  20. A rendezvous before rejection: Where do T cells meet transplant antigens?

    OpenAIRE

    Briscoe, David M.; Sayegh, Mohamed H.

    2002-01-01

    Interactions between recipient T cells and donor endothelial graft cells may be an important mechanism for both acute and chronic rejection of vascularized allografts. This finding provides a starting point for investigations to develop novel ways of inducing long-lasting immunologic tolerance to donor antigens.

  1. Cell density related gene expression: SV40 large T antigen levels in immortalized astrocyte lines

    Directory of Open Access Journals (Sweden)

    Jacobberger James W

    2002-04-01

    Full Text Available Abstract Background Gene expression is affected by population density. Cell density is a potent negative regulator of cell cycle time during exponential growth. Here, we asked whether SV40 large T antigen (Tag levels, driven by two different promoters, changed in a predictable and regular manner during exponential growth in clonal astrocyte cell lines, immortalized and dependent on Tag. Results Expression and cell cycle phase fractions were measured and correlated using flow cytometry. T antigen levels did not change or increased during exponential growth as a function of the G1 fraction and increasing cell density when Tag was transcribed from the Moloney Murine Leukemia virus (MoMuLV long terminal repeat (LTR. When an Rb-binding mutant T antigen transcribed from the LTR was tested, levels decreased. When transcribed from the herpes thymidine kinase promoter, Tag levels decreased. The directions of change and the rates of change in Tag expression were unrelated to the average T antigen levels (i.e., the expression potential. Conclusions These data show that Tag expression potential in these lines varies depending on the vector and clonal variation, but that the observed level depends on cell density and cell cycle transit time. The hypothetical terms, expression at zero cell density and expression at minimum G1 phase fraction, were introduced to simplify measures of expression potential.

  2. 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

  3. Serological survey of normal humans for natural antibody to cell surface antigens of melanoma.

    Science.gov (United States)

    Houghton, A N; Taormina, M C; Ikeda, H; Watanabe, T; Oettgen, H F; Old, L J

    1980-01-01

    Sera of 106 normal adult men were tested for antibodies reacting with cell surface antigens of three established lines of cultured malignant melanoma. Positive reactions with a protein A assay for IgG antibodies were extremely rare (1-2%). The frequency of positive reactions with assays for IgM antibodies was higher: 5-15% in immune adherence assays and 55-82% in anti-C3 mixed hemadsorption assays. After low-titered sera and sera reacting with fetal calf serum components, conventional alloantigens, and widely distributed class 3 antigens were excluded, sera from seven individuals (one with IgG antibody and six with IgM antibodies) were selected for detailed analysis. The serum containing the IgG antibody came from a healthy 65-year-old Caucasian man; titers of antibody in his serum ranged from < 1/10 to 1/40,000 in tests with different melanoma cell lines. This IgG antibody identifies a differentiation antigen of melanocytes, provisionally designated Mel 1, that distinguishes two classes of melanomas: 22 melanoma cell lines typed Mel 1+ and 17 types Mel 1-. Mel 1 is expressed by fetal fibroblasts but not adult fibroblasts and can be found on a proportion of cultured epithelial cancer cell lines (5 out of 23) but not on glioma or B-cell lines. The melanoma antigens detected by the naturally occurring IgM antibodies are serologically unrelated to Mel 1 but, like Mel 1, appear to be differentiation antigens that distinguish subsets of melanoma. These IgM antibodies detect antigens that are identical or closely related to the AH antigen, a melanoma surface antigen that was initially defined by autologous antibody in a patient with melanoma. In view of the immunogenicity of both Mel 1 and the AH antigens in humans and their occurrence on more than 50% of melanomas, it remains to be seen whether antibody to these antigens can be elicited by specific vaccination of seronegative melanoma patients and whether this will have an influence on the clinical course of the disease

  4. Comparison of melanoma antigens in whole tumor vaccine to those from IIB-MEL-J cells.

    Science.gov (United States)

    McGee, J M; Patten, M R; Malnar, K F; Price, J A; Mayes, J S; Watson, G H

    1999-06-01

    Immunotherapy for melanoma shows promise. Our previous whole tumor (WT) vaccine was noted to have positive clinical effects. We have now developed a new, safer melanoma vaccine that is derived from IIB-MEL-J tissue culture (TC) cells. In this study, we compare by Western blot analyses the antigens in the WT vaccine to antigens in the TC vaccine. Sera from 12 WT vaccine recipients, 8 melanoma patients who received no immunotherapy, and 8 controls served as a source of antibodies to investigate potential antigens in the vaccines. Three major antigenic peptides with approximate molecular weighs of 46, 40, and 36 kDA were present in both vaccines, while two other antigenic peptides with approximate molecular weighs of 68 and 48 kDA were present only in the TC vaccine. The reaction was similar between the patients who received the WT vaccine and those who did not receive the vaccine. Some of the individuals who did not have melanoma showed some reaction, but not to the extent of the melanoma patients. The intensity of immunostaining was greater for the TC vaccine when compared to the WT vaccine, indicating that these proteins are in a higher concentration in the TC vaccine. This new vaccine from IIB-MEL-J tissue culture cells provides a higher yield and a much more consistent source of potentially clinically relevant antigens without risk of infection or contamination by other irrelevant materials. PMID:10850304

  5. Antigen presentation by murine epidermal langerhans cells and its alteration by ultraviolet B light

    International Nuclear Information System (INIS)

    Mice that are chronically exposed in vivo to ultraviolet B light (UV-B) display altered immunologic reactivity to various antigenic stimuli. A possible mode of UV-B action is that it exerts adverse effects on antigen-presenting cell function. Because the epidermis is the only tissue that is naturally subject to UV exposure we investigated if murine epidermal cells (EC) could perform an antigen presentation function and, if so, could this function be altered by UV-B irradiation. For this purpose, T cells immune to purified protein derivative of tuberculin (PPD) and dinitrophenylated ovalbumin (DNP6-OVA) from either BALB/c or C3H/He mice were incubated with syngeneic, semisyngeneic, or allogeneic EC or, for control purposes, with peritoneal exudate cells (PEC) that had been pulse-exposed to either the immunizing antigens or, as controls, left unpulsed, or pulsed to human serum albumin (HSA). After 4 days of culture, T cell proliferation was assessed by 3H-thymidine incorporation. PPD- and DNP/6-OVA pulsed, but not HSA-pulsed EC and PEC, induced vigorous proliferation of syngeneic and semisyngeneic, but not allogeneic, immune T cells. Pretreatment of stimulator cells with specific anti-Ia serum and complement virtually abolished this response, which indicated that among EC, Ia-bearing Langerhans cells are the critical stimulators. Exposure of EC either before or after pulsing to UV-B resulted in a dose-dependent impairment of antigen-specific T cell proliferation; the T proliferative response was abolished after administration of 20 mJ/cm2 UV-B. UV-B in the dose range employed did not produce immediate lethal cell damage, premature death of cultured EC, or toxic factors inhibitory for T cell proliferation

  6. Self-antigen presentation by dendritic cells and lymphoid stroma and its implications for autoimmunity

    OpenAIRE

    Lukacs-Kornek, Veronika; Turley, Shannon J.

    2010-01-01

    The induction and maintenance of T cell tolerance is essential to prevent autoimmunity. A combination of central and peripheral mechanisms acts to control autoreactive T cells. In secondary lymphoid organs, dendritic cells (DCs) presenting self-antigen were thought to play a major role in the induction of peripheral T cell tolerance. Multiple recent studies have demonstrated that DCs are not absolutely essential to induce and maintain tolerance. Furthermore, it has also been recently shown th...

  7. Merkel Cell Polyomavirus Small T Antigen Targets the NEMO Adaptor Protein To Disrupt Inflammatory Signaling

    OpenAIRE

    Griffiths, David A.; Abdul-Sada, Hussein; Knight, Laura M.; Jackson, Brian R.; Richards, Kathryn; Prescott, Emma L.; Peach, A. Howard S.; Blair, G. Eric; MacDonald, Andrew; Whitehouse, Adrian

    2013-01-01

    Merkel cell carcinoma (MCC) is a highly aggressive nonmelanoma skin cancer arising from epidermal mechanoreceptor Merkel cells. In 2008, a novel human polyomavirus, Merkel cell polyomavirus (MCPyV), was identified and is strongly implicated in MCC pathogenesis. Currently, little is known regarding the virus-host cell interactions which support virus replication and virus-induced mechanisms in cellular transformation and metastasis. Here we identify a new function of MCPyV small T antigen (ST)...

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    Adoptive cell therapy with engineered T cells expressing chimeric antigen receptors (CARs) originated from antibodies is a promising strategy in cancer immunotherapy. Several unsuccessful trials, however, highlight the need for alternative conventional binding domains and the better combination of...... costimulatory endodomains for CAR construction to improve the effector functions of the engineered T cells. Camelid single-domain antibodies (VHHs), which are the smallest single domain antibodies, can endow great targeting ability to CAR-engineered T cells....

  9. cDNA cloning and transient expression of the Epstein-Barr virus-determined nuclear antigen EBNA3B in human cells and identification of novel transcripts from its coding region.

    OpenAIRE

    Kerdiles, B; Walls, D; Triki, H.; Perricaudet, M; Joab, I

    1990-01-01

    Recombinant plasmids containing sequences from the BamHI-E rightward reading frames 2a and 2b (BERF2a and 2b) of the Epstein-Barr virus (EBV) genome were isolated from a library of cDNA clones which had been previously made from the EBV B95-8 lymphoblastoid cell line (M. Bodescot, O. Brison, and M. Perricaudet, Nucleic Acids Res. 14:7103-7114, 1986). The characterization of these clones in combination with RNase mapping experiments led to the identification of one leftward and several rightwa...

  10. Tumorigenic activity of Merkel cell polyomavirus T antigens expressed in the stratified epithelium of mice

    Science.gov (United States)

    Spurgeon, Megan E.; Cheng, Jingwei; Bronson, Roderick T.; Lambert, Paul F.; DeCaprio, James A.

    2015-01-01

    Merkel cell polyomavirus (MCPyV) is frequently associated with Merkel cell carcinoma (MCC), a highly aggressive neuroendocrine skin cancer. Most MCC tumors contain integrated copies of the viral genome with persistent expression of the MCPyV large T (LT) and small T (ST) antigen. MCPyV isolated from MCC typically contain wild type ST but truncated forms of LT that retain the N-terminus but delete the C-terminus and render LT incapable of supporting virus replication. To determine the oncogenic activity of MCC tumor-derived T antigens in vivo, a conditional, tissue-specific mouse model was developed. Keratin 14-mediated Cre recombinase expression induced expression of MCPyV T antigens in stratified squamous epithelial cells and Merkel cells of the skin epidermis. Mice expressing MCPyV T antigens developed hyperplasia, hyperkeratosis, and acanthosis of the skin with additional abnormalities in whisker pads, footpads and eyes. Nearly half of the mice also developed cutaneous papillomas. Evidence for neoplastic progression within stratified epithelia included increased cellular proliferation, unscheduled DNA synthesis, increased E2F-responsive genes levels, disrupted differentiation, and presence of a DNA damage response. These results indicate that MCPyV T antigens are tumorigenic in vivo, consistent with their suspected etiological role in human cancer. PMID:25596282

  11. Tumorigenic activity of merkel cell polyomavirus T antigens expressed in the stratified epithelium of mice.

    Science.gov (United States)

    Spurgeon, Megan E; Cheng, Jingwei; Bronson, Roderick T; Lambert, Paul F; DeCaprio, James A

    2015-03-15

    Merkel cell polyomavirus (MCPyV) is frequently associated with Merkel cell carcinoma (MCC), a highly aggressive neuroendocrine skin cancer. Most MCC tumors contain integrated copies of the viral genome with persistent expression of the MCPyV large T (LT) and small T (ST) antigen. MCPyV isolated from MCC typically contains wild-type ST but truncated forms of LT that retain the N-terminus but delete the C-terminus and render LT incapable of supporting virus replication. To determine the oncogenic activity of MCC tumor-derived T antigens in vivo, a conditional, tissue-specific mouse model was developed. Keratin 14-mediated Cre recombinase expression induced expression of MCPyV T antigens in stratified squamous epithelial cells and Merkel cells of the skin epidermis. Mice expressing MCPyV T antigens developed hyperplasia, hyperkeratosis, and acanthosis of the skin with additional abnormalities in whisker pads, footpads, and eyes. Nearly half of the mice also developed cutaneous papillomas. Evidence for neoplastic progression within stratified epithelia included increased cellular proliferation, unscheduled DNA synthesis, increased E2F-responsive genes levels, disrupted differentiation, and presence of a DNA damage response. These results indicate that MCPyV T antigens are tumorigenic in vivo, consistent with their suspected etiologic role in human cancer. PMID:25596282

  12. Equine infectious anemia virus-infected dendritic cells retain antigen presentation capability

    International Nuclear Information System (INIS)

    To determine if equine monocyte-derived dendritic cells (DC) were susceptible to equine infectious anemia virus (EIAV) infection, ex vivo-generated DC were infected with virus in vitro. EIAV antigen was detected by immunofluorescence 3 days post-infection with maximum antigen being detected on day 4, whereas there was no antigen detected in DC incubated with the same amount of heat-inactivated EIAV. No cytolytic activity was observed after EIAVWSU5 infection of DC. These monocyte-derived DC were more effective than macrophages and B cells in stimulating allogenic T lymphocytes. Both infected macrophages and DC stimulated similar levels of memory CTL responses in mixtures of CD8+ and CD4+ cells as detected with 51Cr-release assays indicating that EIAV infection of DC did not alter antigen presentation. However, EIAV-infected DC were more effective than infected macrophages when used to stimulate memory CTL in isolated CD8+ cells. The maintenance of antigen processing and presenting function by EIAV-infected DC in vitro suggests that this function is maintained during in vivo infection

  13. Localization of the simian virus 40 small t antigen in the nucleus and cytoplasm of monkey and mouse cells.

    OpenAIRE

    Ellman, M; Bikel, I; Figge, J; Roberts, T; Schlossman, R; Livingston, D M

    1984-01-01

    Monkey and mouse cells producing simian virus 40 small t antigen in the absence of clearly detectable intact or truncated large T antigens were subjected to indirect immunofluorescence and biochemical cell compartment analyses. Results revealed specific immunofluorescence and small t polypeptide in both the nucleus and cytoplasm of these cells.

  14. Anticancer chemotherapy-induced intratumoral recruitment and differentiation of antigen-presenting cells.

    Science.gov (United States)

    Ma, Yuting; Adjemian, Sandy; Mattarollo, Stephen R; Yamazaki, Takahiro; Aymeric, Laetitia; Yang, Heng; Portela Catani, João Paulo; Hannani, Dalil; Duret, Helene; Steegh, Kim; Martins, Isabelle; Schlemmer, Frederic; Michaud, Mickaël; Kepp, Oliver; Sukkurwala, Abdul Qader; Menger, Laurie; Vacchelli, Erika; Droin, Nathalie; Galluzzi, Lorenzo; Krzysiek, Roman; Gordon, Siamon; Taylor, Philip R; Van Endert, Peter; Solary, Eric; Smyth, Mark J; Zitvogel, Laurence; Kroemer, Guido

    2013-04-18

    The therapeutic efficacy of anthracyclines relies on antitumor immune responses elicited by dying cancer cells. How chemotherapy-induced cell death leads to efficient antigen presentation to T cells, however, remains a conundrum. We found that intratumoral CD11c(+)CD11b(+)Ly6C(hi) cells, which displayed some characteristics of inflammatory dendritic cells and included granulomonocytic precursors, were crucial for anthracycline-induced anticancer immune responses. ATP released by dying cancer cells recruited myeloid cells into tumors and stimulated the local differentiation of CD11c(+)CD11b(+)Ly6C(hi) cells. Such cells efficiently engulfed tumor antigens in situ and presented them to T lymphocytes, thus vaccinating mice, upon adoptive transfer, against a challenge with cancer cells. Manipulations preventing tumor infiltration by CD11c(+)CD11b(+)Ly6C(hi) cells, such as the local overexpression of ectonucleotidases, the blockade of purinergic receptors, or the neutralization of CD11b, abolished the immune system-dependent antitumor activity of anthracyclines. Our results identify a subset of tumor-infiltrating leukocytes as therapy-relevant antigen-presenting cells. PMID:23562161

  15. Antigen uptake, processing and presentation to T-cells is still functional in dendritic cells surviving photodynamic treatment

    International Nuclear Information System (INIS)

    Full text: The effect of photodynamic therapy (PDT) on anti-tumoral immune reactions is still discussed controversially. Several studies have demonstrated that PDT is able to activate immune reactions against tumor antigens. However, there is also evidence that PDT exerts immunosuppressive effects. Dendritic cells (DC) are professional antigen presenting cells and play an important role in, both, the induction of immune reactions as well as the induction and maintenance of immunologic tolerance. Therefore, we investigated the effect of hypericin-mediated PDT on the capability of bone marrow-derived DC for antigen uptake, processing and presentation to CD4+ T lymphocytes. Using beta-galactosidase as model antigens we found that, under sublethal PDT conditions, antigen is still incorporated and degraded by surviving DC. PDT-treated DC, in the presence of beta-galactosidase, were still able to re-activate splenic T cells from immunized but not from naive mice. Similarly, naive allogenic T cells were activated in an antigen-independent manner by PDT-treated DC, albeit at lower efficiency as compared to untreated DC. Based on these data, we hypothesize that DC localized in PDT-treated tumor lesions could play a role in the regulation of anti-tumor immune reactions. (author)

  16. Chimeric Antigen Receptor-Engineered T Cells for Immunotherapy of Cancer

    Directory of Open Access Journals (Sweden)

    Marc Cartellieri

    2010-01-01

    Full Text Available CD4+ and CD8+ T lymphocytes are powerful components of adaptive immunity, which essentially contribute to the elimination of tumors. Due to their cytotoxic capacity, T cells emerged as attractive candidates for specific immunotherapy of cancer. A promising approach is the genetic modification of T cells with chimeric antigen receptors (CARs. First generation CARs consist of a binding moiety specifically recognizing a tumor cell surface antigen and a lymphocyte activating signaling chain. The CAR-mediated recognition induces cytokine production and tumor-directed cytotoxicity of T cells. Second and third generation CARs include signal sequences from various costimulatory molecules resulting in enhanced T-cell persistence and sustained antitumor reaction. Clinical trials revealed that the adoptive transfer of T cells engineered with first generation CARs represents a feasible concept for the induction of clinical responses in some tumor patients. However, further improvement is required, which may be achieved by second or third generation CAR-engrafted T cells.

  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. 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.

  19. 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.

  20. Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses

    Science.gov (United States)

    Shorter, Shayla K.; Schnell, Frederick J.; McMaster, Sean R.; Pinelli, David F.; Andargachew, Rakieb; Evavold, Brian D.

    2016-01-01

    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. PMID:26915099

  1. Monitoring Antigen-Specific T Cell Responses Using Real-Time PCR

    Science.gov (United States)

    Lowe, Devin B.; Taylor, Jennifer L.; Storkus, Walter J.

    2016-01-01

    Flow cytometry-, ELISA-, and ELISpot-based in vitro assays have played important roles in assessing the frequencies and functional competence of antigen-specific T cells in the setting of infectious disease and cancer. Such methods have helped in the development of antigen-specific vaccines for human disease prevention/treatment and have also served as a foundation for the monitoring of patients’ immune responsiveness based on antigen-induced T cell expression of effector molecules (such as cytokines, chemokines, or proteins associated with cytolysis) as a consequence of therapeutic intervention. The following method outlines a protocol employing quantitative real-time PCR (qRT-PCR) with SYBR® green technology to examine antigen-specific CD8+ T cell responses based on their rapid up-regulation of IFN-γ mRNA transcription following in vitro stimulation with peptide (antigen)-loaded, autologous peripheral blood mononuclear cells (PBMCs). The advantages of the current qRT-PCR approach over protein-based detection methods include the sensitivity to distinguish resident CD8+ T cell responses against multiple antigens without the need to artificially pre-expand T cell numbers ex vivo, as is commonly required for the latter in vitro assay systems. Following qRT-PCR setup and run, the level of human IFN-γ transcript is normalized to CD8 transcript expression level, with data reported as the relative fold change in this index versus a patient-matched PBMC sample stimulated with a negative control peptide (e.g., HIV NEF). PMID:25149303

  2. Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses.

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. ImmunoChip Study Implicates Antigen Presentation to T Cells in Narcolepsy

    DEFF Research Database (Denmark)

    Faraco, Juliette; Lin, Ling; Kornum, Birgitte Rahbek;

    2013-01-01

    with hypocretin-deficient narcolepsy and 10,421 controls, all of European ancestry, using a custom genotyping array (ImmunoChip). Three loci located outside the Human Leukocyte Antigen (HLA) region on chromosome 6 were significantly associated with disease risk. In addition to a strong signal in the T...... cell receptor alpha (TRA@), variants in two additional narcolepsy loci, Cathepsin H (CTSH) and Tumor necrosis factor (ligand) superfamily member 4 (TNFSF4, also called OX40L), attained genome-wide significance. These findings underline the importance of antigen presentation by HLA Class II to T cells...

  5. Evidence that a glycolipid tail anchors antigen 117 to the plasma membrane of Dictyostelium discoideum cells

    International Nuclear Information System (INIS)

    The authors describe the biochemical features of the putative cell cohesion molecule antigen 117, indicating that it is anchored to the plasma membrane by a glycolipid tail. Antigen 117 can be radiolabeled with [3H]myristate, [3H]palmitate, and [14C]ethanolamine. The fatty acid label is removed by periodate oxidation and nitrous acid deamination, indicating that the fatty acid is attached to the protein by a structure containing carbohydrate and an unsubstituted glucosamine. As cells develop aggregation competence, the antigen is released from the cell surface in a soluble form that can still be radiolabeled with [14C]ethanolamine but not with [3H]myristate of [3H]-palmitate. The molecular weight of the released antigen is similar to that found in the plasma membrane, but it preferentially partitions in Triton X-114 as a hydrophilic, as opposed to a hydrophobic, protein. Plasma membranes contain the enzyme activity responsible for the release of the antigen in a soluble form

  6. 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.

  7. Thymus cell antigen 1 (Thy1, CD90) is expressed by lymphatic vessels and mediates cell adhesion to lymphatic endothelium

    OpenAIRE

    Jurisic, Giorgia; Iolyeva, Maria; Proulx, Steven T; Halin, Cornelia; Detmar, Michael

    2010-01-01

    The lymphatic vascular system plays an important role in inflammation and cancer progression, although the molecular mechanisms involved are poorly understood. As determined by comparative transcriptional profiling studies of ex vivo isolated mouse intestinal lymphatic endothelial cells versus blood vascular endothelial cells, thymus cell antigen 1 (Thy1, CD90) was expressed at much higher levels in lymphatic endothelial cells than in blood vascular endothelial cells. These findings were conf...

  8. Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos

    International Nuclear Information System (INIS)

    Embryonic stem cells were isolated from rabbit blastocysts derived from fertilization (conventional rbES cells), parthenogenesis (pES cells) and nuclear transfer (ntES cells), and propagated in a serum-free culture system. Rabbit ES (rbES) cells proliferated for a prolonged time in an undifferentiated state and maintained a normal karyotype. These cells grew in a monolayer with a high nuclear/cytoplasm ratio and contained a high level of alkaline phosphate activity. In addition, rbES cells expressed the pluripotent marker Oct-4, as well as EBAF2, FGF4, TDGF1, but not antigens recognized by antibodies against SSEA-1, SSEA-3, SSEA-4, TRA-1-10 and TRA-1-81. All 3 types of ES cells formed embryoid bodies and generated teratoma that contained tissue types of all three germ layers. rbES cells exhibited a high cloning efficiency, were genetically modified readily and were used as nuclear donors to generate a viable rabbit through somatic cell nuclear transfer. In combination with genetic engineering, the ES cell technology should facilitate the creation of new rabbit lines

  9. Sialic acid-modified antigens impose tolerance via inhibition of T-cell proliferation and de novo induction of regulatory T cells.

    Science.gov (United States)

    Perdicchio, Maurizio; Ilarregui, Juan M; Verstege, Marleen I; Cornelissen, Lenneke A M; Schetters, Sjoerd T T; Engels, Steef; Ambrosini, Martino; Kalay, Hakan; Veninga, Henrike; den Haan, Joke M M; van Berkel, Lisette A; Samsom, Janneke N; Crocker, Paul R; Sparwasser, Tim; Berod, Luciana; Garcia-Vallejo, Juan J; van Kooyk, Yvette; Unger, Wendy W J

    2016-03-22

    Sialic acids are negatively charged nine-carbon carboxylated monosaccharides that often cap glycans on glycosylated proteins and lipids. Because of their strategic location at the cell surface, sialic acids contribute to interactions that are critical for immune homeostasis via interactions with sialic acid-binding Ig-type lectins (siglecs). In particular, these interactions may be of importance in cases where sialic acids may be overexpressed, such as on certain pathogens and tumors. We now demonstrate that modification of antigens with sialic acids (Sia-antigens) regulates the generation of antigen-specific regulatory T (Treg) cells via dendritic cells (DCs). Additionally, DCs that take up Sia-antigen prevent formation of effector CD4(+) and CD8(+)T cells. Importantly, the regulatory properties endowed on DCs upon Sia-antigen uptake are antigen-specific: only T cells responsive to the sialylated antigen become tolerized. In vivo, injection of Sia-antigen-loaded DCs increased de novo Treg-cell numbers and dampened effector T-cell expansion and IFN-γ production. The dual tolerogenic features that Sia-antigen imposed on DCs are Siglec-E-mediated and maintained under inflammatory conditions. Moreover, loading DCs with Sia-antigens not only inhibited the function of in vitro-established Th1 and Th17 effector T cells but also significantly dampened ex vivo myelin-reactive T cells, present in the circulation of mice with experimental autoimmune encephalomyelitis. These data indicate that sialic acid-modified antigens instruct DCs in an antigen-specific tolerogenic programming, enhancing Treg cells and reducing the generation and propagation of inflammatory T cells. Our data suggest that sialylation of antigens provides an attractive way to induce antigen-specific immune tolerance. PMID:26941238

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

    Directory of Open Access Journals (Sweden)

    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.

  11. Distinct roles for histone methyltransferases G9a and GLP in cancer germline antigen gene regulation in human cancer cells and murine ES cells

    OpenAIRE

    Link, Petra A.; Gangisetty, Omkaram; James, Smitha R.; Woloszynska-Read, Anna; Tachibana, Makoto; Shinkai, Yoichi; Karpf, Adam R.

    2009-01-01

    The H3K9me2 histone methyltransferases G9a and GLP repress Mage-a class cancer germline (CG) antigen gene expression in murine ES cells but the role of these enzymes in CG antigen gene regulation in human cancer cells is unknown. Here we show that while independent or dual knockdown of G9a and GLP in human cancer cells leads to reduced global and CG antigen promoter-associated H3K9me2 levels it does not activate CG antigen gene expression. Moreover, CG antigen gene repression is maintained fo...

  12. Effect on Retinoic Acid Receptor and Proliferating Cell Nuclear Antigen of Retinoic Acid in Colorectal Carcinoma%肠癌组织维甲酸受体的测定和维甲酸对其表达的影响

    Institute of Scientific and Technical Information of China (English)

    樊卫; 卫洪波; 韩晓燕

    2001-01-01

    Objective To investigate the effect on retinoic acid receptor (RAR) of retinoic acid (RA) in colorectal carcinoma. Methods 160 cases of health male Wistar rats were divided into 4 groups, and each group was of 40 cases.80 rats in group 1 and 2 induced by dimethylhydrazine (DMH) (20mg/kg,once a week,injected subdermally) for 7~13 weeks;after that,rats in group 2 and 3 were treated with RA (50mg/kg,every day,orally) for 8 weeks;others were was control. In 7th\14th\21th week, 8 rats were killed in each group. The others were killed in 28th week.Colorectal tumors in mucosa were examined. The RAR concentration was studied. Results The incidence of colorectal carcinoma induced by DMH between group 1and 2 was different significantly (p<0.05),and higher than group 3 and 4.The content of RAR in cancer groups was lower than normal one (p<0.05).RA may increase RAR concentration of cancer tissue progressively (p<0.05).Conclusions RA could decrease the incidence of colorectal carcinoma induced by DMH. Colorectal cancer tissue existed abnormal expression of RAR,RA could regulate RAR concentrations of intestine cells.%目的用放射配体结合分析法测定肠癌组织细胞内维甲酸受体 (retinoic acid receptor, RAR)含量,探讨维甲酸 (Retinoic acid, RA)对肠癌组织内RAR表达的影响.方法取Wistar大鼠160只,随机分4组,每组40只,其中第1、2组二甲基肼诱癌,第3、4组用生理盐水注射;于第7周开始,第2、3组按50mg/kg体重灌服维甲酸,每日1次,共8周.于7,14, 21周每组处死8只,于28周全部处死大鼠.测定RAR的含量.结果第1组肠癌发生率100%,第2组20%,差异有显著性(p<0.01);结肠组织细胞核内存在丰富的RAR,而肠癌组织RAR含量明显减少,并主要表现为数量的减少; RA对正常组织、肠癌组织内的RAR表达有显著性影响,差别有统计学意义(p<0.05).结论 RA可以减少二甲基肼诱发肠癌的发生;肠癌组织

  13. Mitochondrial H2O2 in Lung Antigen-Presenting Cells Blocks NF-κB Activation to Prevent Unwarranted Immune Activation

    Directory of Open Access Journals (Sweden)

    Anupriya Khare

    2016-05-01

    Full Text Available Inhalation of environmental antigens such as allergens does not always induce inflammation in the respiratory tract. While antigen-presenting cells (APCs, including dendritic cells and macrophages, take up inhaled antigens, the cell-intrinsic molecular mechanisms that prevent an inflammatory response during this process, such as activation of the transcription factor NF-κB, are not well understood. Here, we show that the nuclear receptor PPARγ plays a critical role in blocking NF-κB activation in response to inhaled antigens to preserve immune tolerance. Tolerance induction promoted mitochondrial respiration, generation of H2O2, and suppression of NF-κB activation in WT, but not PPARγ-deficient, APCs. Forced restoration of H2O2 in PPARγ-deficient cells suppressed IκBα degradation and NF-κB activation. Conversely, scavenging reactive oxygen species from mitochondria promoted IκBα degradation with loss of regulatory and promotion of inflammatory T cell responses in vivo. Thus, communication between PPARγ and the mitochondria maintains immune quiescence in the airways.

  14. 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.

  15. Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis

    International Nuclear Information System (INIS)

    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

  16. Release of carcinoembryonic antigen from human colon cancer cells by phosphatidylinositol-specific phospholipase C.

    OpenAIRE

    Sack, T L; Gum, J R; Low, M G; Y. S. Kim

    1988-01-01

    Carcinoembryonic antigen (CEA) is released from colon cancer cells into the circulation where it is monitored clinically as an indicator of the recurrence or progression of cancer. We have studied the mechanism of CEA membrane attachment and release using the human colonic adenocarcinoma cell line LS-174T, specimens of human colon cancers, and serum from colon cancer patients. CEA release by cells in vitro and in vivo is associated with the conversion of CEA from a membrane-bound, hydrophobic...

  17. Cryopreservation of MHC Multimers: Recommendations for Quality Assurance in Detection of Antigen Specific T Cells

    OpenAIRE

    Hadrup, Sine Reker; Maurer, Dominik; Laske, Karoline; Frøsig, Thomas Mørch; Andersen, Sofie Ramskov; Britten, Cedrik M.; Sjoerd H van der Burg; Walter, Steffen; Gouttefangeas, Cécile

    2014-01-01

    Fluorescence-labeled peptide-MHC class I multimers serve as ideal tools for the detection of antigen-specific T cells by flow cytometry, enabling functional and phenotypical characterization of specific T cells at the single cell level. While this technique offers a number of unique advantages, MHC multimer reagents can be difficult to handle in terms of stability and quality assurance. The stability of a given fluorescence-labeled MHC multimer complex depends on both the stability of the pep...

  18. Milk-induced eczema is associated with the expansion of T cells expressing cutaneous lymphocyte antigen.

    OpenAIRE

    Abernathy-Carver, K J; Sampson, H A; Picker, L. J.; Leung, D Y

    1995-01-01

    The extravasation of T cells at sites of inflammation is critically dependent on the activity of homing receptors (HR) involved in endothelial cell recognition and binding. Two such HR (the cutaneous lymphocyte antigen [CLA] and L-selectin) have been shown to be selectively involved in T cell migration to skin and peripheral lymph nodes, respectively. This study was designed to assess the relationship between the organ specificity of an allergic reaction to food and the expression of HR on T ...

  19. Bypassing antibiotic selection: positive screening of genetically modified cells with an antigen-dependent proliferation switch

    OpenAIRE

    Kawahara, Masahiro; Ueda, Hiroshi; Morita, Sumiyo; Tsumoto, Kouhei; Kumagai, Izumi; Nagamune, Teruyuki

    2003-01-01

    While antibiotic selection has been routinely used for the selection of genetically modified cells, administration of cytotoxic drugs often leads to deleterious effects not only to inert cells but also to transfected or transduced ones. In this study, we propose an Antigen-MEdiated Genetically modified cell Amplification (AMEGA) system employing antibody/receptor chimeras without antibiotic selection. Based on a rational design where the extracellular domains of dimeric erythropoietin recepto...

  20. 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.

  1. 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....

  2. Indirect 125I-labeled protein A assay for monoclonal antibodies to cell surface antigens

    International Nuclear Information System (INIS)

    An assay for detection of monoclonal hybridoma antibodies against cell surface antigens is described. Samples of spent medium from the hybridoma cultures are incubated in microtest wells with cells, either as adherent monolayers or in suspension. Antibodies bound to surface antigens are detected by successive incubations with rabbit anti-immunoglobulin serum and 125I-labeled protein A from Staphylococcus aureus, followed by autoradiography of the microtest plate or scintillation counting of the individual wells. Particular advantages of this assay for screening hybridomas are: (1) commercially available reagents are used, (2) antibodies of any species and of any immunoglobulin class or subclass can be detected, and (3) large numbers of samples can be screened rapidly and inexpensively. The assay has been used to select hybridomas producing monoclonal antibodies to surface antigens of human melanomas and mouse sarcomas. (Auth.)

  3. Synthetic Peptide Ligands of the Antigen Binding Receptor Induce Programmed Cell Death in a Human B-Cell Lymphoma

    Science.gov (United States)

    Renschler, Markus F.; Bhatt, Ramesh R.; Dower, William J.; Levy, Ronald

    1994-04-01

    Peptide ligands for the antigen binding site of the surface immunoglobulin receptor of a human B-cell lymphoma cell line were identified with the use of filamentous phage libraries displaying random 8- and 12-amino acid peptides. Corresponding synthetic peptides bound specifically to the antigen binding site of this immunoglobulin receptor and blocked the binding of an anti-idiotype antibody. The ligands, when conjugated to form dimers or tetramers, induced cell death by apoptosis in vitro with an IC50 between 40 and 200 nM. This effect was associated with specific stimulation of intracellular protein tyrosine phosphorylation.

  4. 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

  5. Regulation of delayed-type hypersensitivity: VI. Antigen-specific suppressor T cells and suppressor factor for delayed-type hypersensitivity to histocompatibility antigens

    International Nuclear Information System (INIS)

    Mice develop highly significant levels of delayed-type hypersensitivity (DTH) to major and minor histocompatibility antigens when injected s.c. with lymphoid cells from X-irradiated allogeneic donors. However, when mice are inoculated i.v. with a high dose of X-irradiated allogeneic lymphoid cells, they not only fail to develop DTH to the allogeneic cells, but their ability to respond to an immunogenic challenge of the alloantigens is also significantly depressed. This suppression is adoptively transferable by antigen-specific suppressor T cells and not by immune serum. Cell surface phenotypic analysis shows that the primary suppressor cells for alloantigens are Thy-1+, Lyt-1+2-, and Ia-, whereas the secondary suppressor cells appearing after boosting injection are Thy-+, Lyt-1+2+, and Ia-. These suppressor T (Ts) cells localize in the lymphoid organs shortly after their induction and are largely absent from the spleen or lymph node 1 month later.However, ''suppressor memory'' can be recalled by an immunogenic dose of alloantigens which would normally induce DTH effector cells rather than suppressor cells in naive mice. When the suppressor cells were cultured in vitro for 48 hr, the supernatant contained suppressive activity. It appears likely that the manifestation of the suppressor cells is via soluble, antigen-specific suppressor factor(s), the production of which is dependent on viable T cells

  6. Complex Minigene Library Vaccination for Discovery of Pre-Erythrocytic Plasmodium T Cell Antigens

    Science.gov (United States)

    Stone, Brad C.; Kas, Arnold; Billman, Zachary P.; Fuller, Deborah H.; Fuller, James T.; Shendure, Jay; Murphy, Sean C.

    2016-01-01

    Development of a subunit vaccine targeting liver-stage Plasmodium parasites requires the identification of antigens capable of inducing protective T cell responses. However, traditional methods of antigen identification are incapable of evaluating T cell responses against large numbers of proteins expressed by these parasites. This bottleneck has limited development of subunit vaccines against Plasmodium and other complex intracellular pathogens. To address this bottleneck, we are developing a synthetic minigene technology for multi-antigen DNA vaccines. In an initial test of this approach, pools of long (150 bp) antigen-encoding oligonucleotides were synthesized and recombined into vectors by ligation-independent cloning to produce two DNA minigene library vaccines. Each vaccine encoded peptides derived from 36 (vaccine 1) and 53 (vaccine 2) secreted or transmembrane pre-erythrocytic P. yoelii proteins. BALB/cj mice were vaccinated three times with a single vaccine by biolistic particle delivery (gene gun) and screened for interferon-γ-producing T cell responses by ELISPOT. Library vaccination induced responses against four novel antigens. Naïve mice exposed to radiation-attenuated sporozoites mounted a response against only one of the four novel targets (PyMDH, malate dehydrogenase). The response to PyMDH could not be recalled by additional homologous sporozoite immunizations but could be partially recalled by heterologous cross-species sporozoite exposure. Vaccination against the dominant PyMDH epitope by DNA priming and recombinant Listeria boosting did not protect against sporozoite challenge. Improvements in library design and delivery, combined with methods promoting an increase in screening sensitivity, may enable complex minigene screening to serve as a high-throughput system for discovery of novel T cell antigens. PMID:27070430

  7. Antigenic variation of pilin regulates adhesion of Neisseria meningitidis to human epithelial cells.

    Science.gov (United States)

    Nassif, X; Lowy, J; Stenberg, P; O'Gaora, P; Ganji, A; So, M

    1993-05-01

    Pili have been shown to play an essential role in the adhesion of Neisseria meningitidis to epithelial cells. However, among piliated strains, both inter- and intrastrain variability exist with respect to their degree of adhesion to epithelial cells in vitro (Virji et al., 1992). This suggests that factors other than the presence of pili per se are involved in this process. The N. meningitidis pilin subunit undergoes extensive antigenic variation. Piliated low- and high-adhesive derivatives of the same N. meningitidis strain were selected and the nucleotide sequence of the pilin gene expressed in each was determined. The highly adhesive derivatives had the same pilin sequence. The alleles encoding the pilin subunit of the low-adhesive derivatives were completely different from the one found in the high-adhesive isolates. Using polyclonal antibodies raised against one hyperadhesive variant, it was confirmed that the low-adhesive piliated derivatives expressed pilin variants antigenically different from the highly adhesive strains. The role of antigenic variation in the adhesive process of N. meningitidis was confirmed by performing allelic exchanges of the pilE locus between low- and high-adhesive isolates. Antigenic variation has been considered a means by which virulent bacteria evade the host immune system. This work provides genetic proof that a bacterial pathogen, N. meningitidis, can use antigenic variation to modulate their degree of virulence. PMID:8332064

  8. Parallel detection of antigen-specific T cell responses by combinatorial encoding of MHC multimers

    DEFF Research Database (Denmark)

    Andersen, Rikke Sick; Kvistborg, Pia; Frøsig, Thomas Mørch;

    2012-01-01

    -dimensional combinatorial matrix, these eight fluorochromes are combined to generate 28 unique two-color codes. By the use of combinatorial encoding, a large number of different T cell populations can be detected in a single sample. The method can be used for T cell epitope mapping, and also for the monitoring of CD8......Fluorescently labeled multimeric complexes of peptide-MHC, the molecular entities recognized by the T cell receptor, have become essential reagents for detection of antigen-specific CD8(+) T cells by flow cytometry. Here we present a method for high-throughput parallel detection of antigen......-specific T cells by combinatorial encoding of MHC multimers. Peptide-MHC complexes are produced by UV-mediated MHC peptide exchange and multimerized in the form of streptavidin-fluorochrome conjugates. Eight different fluorochromes are used for the generation of MHC multimers and, by a two...

  9. TIM-4, expressed by medullary macrophages, regulates respiratory tolerance by mediating phagocytosis of antigen-specific T cells

    OpenAIRE

    Albacker, Lee A; Yu, Sanhong; Bedoret, Denis; Lee, Wan-Ling; Umetsu, Sarah E.; Monahan, Sheena; Freeman, Gordon J.; Umetsu, Dale T.; DeKruyff, Rosemarie H.

    2012-01-01

    Respiratory exposure to antigen induces T cell tolerance via several overlapping mechanisms that limit the immune response. While the mechanisms involved in the development of Treg cells have received much attention, those that result in T cell deletion are largely unknown. Herein, we show that F4/80+ lymph node medullary macrophages expressing TIM-4, a phosphatidylserine receptor, remove antigen-specific T cells during respiratory tolerance, thereby reducing secondary T cell responses. Block...

  10. A comparative autoradiographic study demonstrating differential intratumor localization of monoclonal antibodies to cell surface (Lym-1) and intracellular (TNT-1) antigens

    International Nuclear Information System (INIS)

    Autoradiography was utilized to explore the patterns of distribution of two different monoclonal antibodies (Lym-1 and TNT-1) in tumor-bearing nude mice. Lym-1 is an antibody against a cell surface B-cell antigen. In comparison, TNT-1 represents a novel approach and is an antibody against an intracellular (nuclear) antigen that is selectively revealed in degenerating tumor cells. Experimentally iodine-125-(125I) labeled Lym-1 or TNT-1 was injected intravenously into nude mice bearing either the Raji lymphoma or the ME-180 human cervical carcinoma. Qualitative autoradiographic analyses performed after injection revealed that Lym-1 accumulated at the periphery of the target tumor where vascular permeability is marked and where Lym-1 positive cells are first encountered. By contrast, TNT-1 lost its initial peripheral distribution and demonstrated progressive concentration in the center of the tumor where binding to its nuclear antigen is facilitated by the presence of cell degeneration and necrosis. These studies confirm the ability of TNT-1 to bind areas deep within tumor that traditionally are considered inaccessible to antibodies administered for imaging and therapy

  11. 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

  12. Expression Patterns of Cancer-Testis Antigens in Human Embryonic Stem Cells and Their Cell Derivatives Indicate Lineage Tracks

    OpenAIRE

    Olga Gordeeva; Tatyana Yakovleva; Galina Poljanskaya; Tatyana Krylova; Anna Koltsova; Nadya Lifantseva

    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...

  13. 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...

  14. 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.

  15. Generation of Large Numbers of Antigen-Expressing Human Dendritic Cells Using CD14-ML Technology

    Science.gov (United States)

    Imamura, Yuya; Haruta, Miwa; Tomita, Yusuke; Matsumura, Keiko; Ikeda, Tokunori; Yuno, Akira; Hirayama, Masatoshi; Nakayama, Hideki; Mizuta, Hiroshi; Nishimura, Yasuharu; Senju, Satoru

    2016-01-01

    We previously reported a method to expand human monocytes through lentivirus-mediated introduction of cMYC and BMI1, and we named the monocyte-derived proliferating cells, CD14-ML. CD14-ML differentiated into functional DC (CD14-ML-DC) upon addition of IL-4, resulting in the generation of a large number of DC. One drawback of this method was the extensive donor-dependent variation in proliferation efficiency. In the current study, we found that introduction of BCL2 or LYL1 along with cMYC and BMI1 was beneficial. Using the improved method, we obtained CD14-ML from all samples, regardless of whether the donors were healthy individuals or cancer patients. In vitro stimulation of peripheral blood T cells with CD14-ML-DC that were loaded with cancer antigen-derived peptides led to the establishment of CD4+ and CD8+ T cell lines that recognized the peptides. Since CD14-ML was propagated for more than 1 month, we could readily conduct genetic modification experiments. To generate CD14-ML-DC that expressed antigenic proteins, we introduced lentiviral antigen-expression vectors and subjected the cells to 2 weeks of culture for drug-selection and expansion. The resulting antigen-expressing CD14-ML-DC successfully induced CD8+ T cell lines that were reactive to CMVpp65 or MART1/MelanA, suggesting an application in vaccination therapy. Thus, this improved method enables the generation of a sufficient number of DC for vaccination therapy from a small amount of peripheral blood from cancer patients. Information on T cell epitopes is not necessary in vaccination with cancer antigen-expressing CD14-ML-DC; therefore, all patients, irrespective of HLA type, will benefit from anti-cancer therapy based on this technology. PMID:27050553

  16. Cell surface antigens of radiation leukemia virus-induced BALB/c leukemias defined by syngeneic cytotoxic T lymphocytes

    International Nuclear Information System (INIS)

    Two cell surface antigens of mouse leukemias were defined by BALB/c cytotoxic T lymphocytes (CTL) generated against syngeneic radiation leukemia virus (RadLV)-induced leukemia, BALBRV1 or BALBRVD. Hyperimmunization of BALB/c mice with irradiated leukemias followed by in vitro sensitization of primed spleen cells resulted in the generation of CTL with high killing activity. The specificity of CTL was examined by direct cytotoxicity assays and competitive inhibition assays. A shared cell surface antigen, designated as BALBRV1 antigen, was detected by BALB/c anti-BALBRV1 CTL. BALBRV1 antigen was expressed not only on RadLV-induced BALB/c leukemias except for BALBRVD, but also on spontaneous or X-ray-induced BALB/c leukemias, chemically-induced leukemias with the H-2d haplotype and some chemically-induced BALB/c sarcomas. In contrast, a unique cell surface antigen, designated as BALBRVD antigen, was detected by BALB/c anti-BALBRVD CTL. BALBRVD antigen was expressed only on BALBRVD, but not on thirty-nine normal lymphoid or tumor cells. These two antigens could be distinguished from those previously defined on Friend, Moloney, Rauscher or Gross murine leukemia virus (MuLV) leukemias, or MuLV-related antigens. Both cytotoxic responses were blocked by antisera against H-2Kd, but not H-2Dd. The relationship of BALBRV1 antigen and BALBRVD antigen to endogenous MuLV is discussed with regard to the antigenic distribution on tumor cell lines. (author)

  17. 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

  18. Increased prevalence of late stage T cell activation antigen (VLA-1) in active juvenile chronic arthritis

    DEFF Research Database (Denmark)

    Ødum, Niels; Morling, Niels; Platz, P; Hofmann, B; Ryder, L P; Heilmann, C; Pedersen, F K; Nielsen, L P; Friis, J; Svejgaard, A

    1987-01-01

    the various HLA class II antigens was observed between the groups. Similarly, no significant differences in stimulatory capability in secondary mixed lymphocyte culture (MLC) were seen. The distribution of T helper/inducer (CD4+), T suppressor/cytotoxic (CD8+), and NK cells was similar in active JCA...

  19. CLIC1 regulates dendritic cell antigen processing and presentation by modulating phagosome acidification and proteolysis

    Science.gov (United States)

    Salao, Kanin; Jiang, Lele; Li, Hui; Tsai, Vicky W.-W.; Husaini, Yasmin; Curmi, Paul M. G.; Brown, Louise J.; Brown, David A.

    2016-01-01

    ABSTRACT 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. PMID:27113959

  20. Interaction of dendritic cells with antigen-containing liposomes: effect of bilayer composition

    DEFF Research Database (Denmark)

    Foged, Camilla; Arigita, Carmen; Sundblad, Anne; Jiskoot, Wim; Storm, Gert; Frøkjær, Sven

    Vaccine efficacy might be improved by exploiting the potent antigen presenting properties of dendrite cells (DCs), since their ability to stimulate specific major histocompatibility complex-restricted immune responses has been well documented during the recent years. In that light, we investigated...

  1. 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 different...

  2. Improved poliovirus D-antigen yields by application of different Vero cell cultivation methods.

    Science.gov (United States)

    Thomassen, Yvonne E; Rubingh, Olaf; Wijffels, René H; van der Pol, Leo A; Bakker, Wilfried A M

    2014-05-19

    Vero cells were grown adherent to microcarriers (Cytodex 1; 3 g L(-1)) using animal component free media in stirred-tank type bioreactors. Different strategies for media refreshment, daily media replacement (semi-batch), continuous media replacement (perfusion) and recirculation of media, were compared with batch cultivation. Cell densities increased using a feed strategy from 1×10(6) cells mL(-1) during batch cultivation to 1.8, 2.7 and 5.0×10(6) cells mL(-1) during semi-batch, perfusion and recirculation, respectively. The effects of these different cell culture strategies on subsequent poliovirus production were investigated. Increased cell densities allowed up to 3 times higher D-antigen levels when compared with that obtained from batch-wise Vero cell culture. However, the cell specific D-antigen production was lower when cells were infected at higher cell densities. This cell density effect is in good agreement with observations for different cell lines and virus types. From the evaluated alternative culture methods, application of a semi-batch mode of operations allowed the highest cell specific D-antigen production. The increased product yields that can easily be reached using these higher cell density cultivation methods, showed the possibility for better use of bioreactor capacity for the manufacturing of polio vaccines to ultimately reduce vaccine cost per dose. Further, the use of animal-component-free cell- and virus culture media shows opportunities for modernization of human viral vaccine manufacturing. PMID:24583004

  3. Antigenic deletion and malignant enhancement induced in lymphoma cells by passage through X-irradiated hosts

    International Nuclear Information System (INIS)

    Studies are reported in which lymphoma cells were induced to delete strong virus-associated membrane antigens, and as a result considerably increase their capacity for metastasis, by X-irradiation of the hosts. The studies involved injecting rats at birth with leukaemia virus cells. The cells expressed strong murine leukaemia virus surface antigens and were consistently rejected when transplanted into normal adult syngeneic rats. When the rats were given 300 to 350 R total body X-irradiation, however, lymphoma cells transplanted within 24 hours subcutaneously or intraperitoneally grow progressively at the site of the graft, occasionally spread to distant sites and eventually cause death of the hosts. Examined under the electron microscope the transplanted lymphoma cells appeared devoid of both mature and immature virus particles. The loss of surface antigens was consistently accompanied by increased malignancy of the lymphoma cells. Explanations for the results are offered. Implications for radiotherapy in man are discussed, and it is suggested that whilst such treatment might be effective in the control of local recurrences, it could possibly induce an increase in the number of distant metastases. Some fluorescence studies of the cells are also described. (U.K.)

  4. Intravacuolar Membranes Regulate CD8 T Cell Recognition of Membrane-Bound Toxoplasma gondii Protective Antigen.

    Science.gov (United States)

    Lopez, Jodie; Bittame, Amina; Massera, Céline; Vasseur, Virginie; Effantin, Grégory; Valat, Anne; Buaillon, Célia; Allart, Sophie; Fox, Barbara A; Rommereim, Leah M; Bzik, David J; Schoehn, Guy; Weissenhorn, Winfried; Dubremetz, Jean-François; Gagnon, Jean; Mercier, Corinne; Cesbron-Delauw, Marie-France; Blanchard, Nicolas

    2015-12-15

    Apicomplexa parasites such as Toxoplasma gondii target effectors to and across the boundary of their parasitophorous vacuole (PV), resulting in host cell subversion and potential presentation by MHC class I molecules for CD8 T cell recognition. The host-parasite interface comprises the PV limiting membrane and a highly curved, membranous intravacuolar network (IVN) of uncertain function. Here, using a cell-free minimal system, we dissect how membrane tubules are shaped by the parasite effectors GRA2 and GRA6. We show that membrane association regulates access of the GRA6 protective antigen to the MHC I pathway in infected cells. Although insertion of GRA6 in the PV membrane is key for immunogenicity, association of GRA6 with the IVN limits presentation and curtails GRA6-specific CD8 responses in mice. Thus, membrane deformations of the PV regulate access of antigens to the MHC class I pathway, and the IVN may play a role in immune modulation. PMID:26628378

  5. Analysis of antigen specific T cells in diabetes - Lessons from pre-clinical studies and early clinical trials.

    Science.gov (United States)

    Krishnamurthy, Balasubramanian; Selck, Claudia; Chee, Jonathan; Jhala, Guarang; Kay, Thomas W H

    2016-07-01

    Antigen-specific immune tolerance promises to provide safe and effective therapies to prevent type 1 diabetes (T1D). Antigen-specific therapy requires two components: well-defined, clinically relevant autoantigens; and safe approaches to inducing tolerance in T cells specific for these antigens. Proinsulin is a critical autoantigen in both NOD mice, based on knockout mouse studies and induction of immune tolerance to proinsulin preventing disease whereas most antigens cannot, and also in human T1D based on proinsulin-specific T cells being found in the islets of affected individuals and the early appearance of insulin autoantibodies. Effective antigen-specific therapies that prevent T1D in humans have not yet been developed although doubt remains about the best molecular form of the antigen, the dose and the route of administration. Preclinical studies suggest that antigen specific therapy is most useful when administered before onset of autoimmunity but this time-window has not been tested in humans until the recent "pre-point" study. There may be a 'window of opportunity' during the neonatal period when 'vaccine' like administration of proinsulin for a short period may be sufficient to prevent diabetes. After the onset of autoimmunity, naive antigen-specific T cells have differentiated into antigen-experienced memory cells and the immune responses have spread to multiple antigens. Induction of tolerance at this stage becomes more difficult although recent studies have suggested generation of antigen-specific TR1 cells can inhibit memory T cells. Preclinical studies are required to identify additional 'help' that is required to induce tolerance to memory T cells and develop protocols for effective therapy in individuals with established autoimmunity. PMID:27083395

  6. In vitro generation of antigen-specific hemolytic plaque-forming cells from human peripheral blood mononuclear cells

    International Nuclear Information System (INIS)

    We have described a culture and assay system for the sensitization of human peripheral blood mononuclear cells with a T cell-dependent antigen, sheep erythrocytes, in the absence of nonspecific stimulatory agents and with the subsequent generation of macroscopic hemolytic plaques. We have shown that the antibody produced by the plaque-forming cells generated in this culture system is specific for the sensitizing antigen, and that the plaques created are not false plaques because their formation is inhibited by cycloheximide. The success of this system can be attributed to several critical factors including large numbers of peripheral blood mononuclear cells (5 x 10(6) culture), a prolonged period of incubation (10-11 d), continuous rocking during the entire period of incubation, culturing in large (35-mm) flat-bottomed culture dishes in the presence of human plasma, and the appropriate antigen concentration (5 x 10(6) sheep erythrocytes/culture). Furthermore, the generation of macroscopic hemolytic plaques requires plaquing sensitized peripheral blood mononuclear cells in target cell monolayers fixed in an agarose matrix with an incubation period of 2-3 h. We have further shown that the antigen-specific response measured by this system is dependent on adherent cells and T lymphocytes. At least one population of the helper T cells is sensitive to 2,000 rad irradiation. This system is simple, sensitive, and should serve as an effective tool for the analysis of cellular interactions involved in the generation of human antigen-specific plaque-forming cells, the genetic control the human immune response, and the pathophysiology of altered immunoregulation in disease

  7. The induction of cytotoxic T cells and tumor regression by soluble antigen formulation.

    Science.gov (United States)

    Hariharan, K; Braslawsky, G; Black, A; Raychaudhuri, S; Hanna, N

    1995-08-15

    CTLs specific for tumor antigens play a major role in the immunity against cancer. We have shown that class I-restricted CTLs can be induced by injecting soluble antigens mixed in an antigen formulation (AF) that consists of squalane, Tween 80, and Pluronic L121 (S. Raychaudhuri et al., Proc. Natl. Acad. Sci. USA, 89: 8308-8312, 1992). In this study, using ovalbumin and the ovalbumin-expressing transfectoma (EG7) as a tumor model system, we examined the in vivo antitumor effect of antigen-AF mixture. Vaccination of mice with ovalbumin in AF 2 or 3 days after EG7 tumor challenge showed significant inhibition of tumor growth compared to mice vaccinated with ovalbumin in alum or in saline. Depletion of CD8+ cells at the time of immunization completely abrogated the AF-induced tumor protection, indicating that CD8+ T cells are the major effectors in tumor protection in vivo. Depletion of CD4+ cells led to a marginal loss of tumor protection, which may be the result of inhibition of ovalbumin-specific CTL response due to the lack of T-helper activity. Our results demonstrate that AF can be used in subunit vaccines to stimulate CTLs and tumor regression in vivo. PMID:7627951

  8. Production and characterization of monoclonal antibodies to cell wall antigens of Aspergillus fumigatus.

    OpenAIRE

    Ste-Marie, L; Sénéchal, S; Boushira, M; Garzon, S.; Strykowski, H; Pedneault, L; de Repentigny, L

    1990-01-01

    Two murine monoclonal antibodies (MAbs) against Aspergillus fumigatus were produced and characterized. Splenocytes from cell wall-immunized BALB/c mice were fused with SP2/0 myeloma cells. The hybridomas were screened with a cold alkali (CA) extract of mycelium containing protein, mannose, and galactose, and two MAbs of the immunoglobulin M class were purified from ascites fluid. MAbs 1 and 40 were characterized by double immunodiffusion against CA antigen, indirect enzyme immunoassay with ma...

  9. A Major Cell Surface Antigen of Coccidioides immitis Which Elicits Both Humoral and Cellular Immune Responses

    OpenAIRE

    Hung, Chiung-Yu; Ampel, Neil M.; Christian, Lara; Seshan, Kalpathi R.; Cole, Garry T.

    2000-01-01

    Multinucleate parasitic cells (spherules) of Coccidioides immitis isolates produce a membranous outer wall component (SOW) in vitro which has been reported to be reactive with antibody from patients with coccidioidal infection, elicits a potent proliferative response of murine immune T cells, and has immunoprotective capacity in a murine model of coccidioidomycosis. To identify the antigenic components of SOW, the crude wall material was first subjected to Triton X-114 extraction, and a water...

  10. Mapping of T cell epitopes using recombinant antigens and synthetic peptides.

    OpenAIRE

    Lamb, J R; Ivanyi, J.; Rees, A D; Rothbard, J B; Howland, K; Young, R. A.; Young, D B

    1987-01-01

    Two complementary approaches were used to determine the epitope specificity of clonal and polyclonal human T lymphocytes reactive with the 65-kd antigen of Mycobacterium leprae. A recombinant DNA sublibrary constructed from portions of the 65-kd gene was used to map T cell determinants within amino acid sequences 101-146 and 409-526. Independently, potential T cell epitopes within the protein were predicted based on an empirical analysis of specific patterns in the amino acid sequence. Of six...

  11. Identification of a region of simian virus 40 large T antigen required for cell transformation.

    OpenAIRE

    Chen, S.; Paucha, E

    1990-01-01

    A series of replication-competent simian virus 40 (SV40) large T antigens with point and deletion mutations in the amino acid sequence between residues 105 and 115 were examined for the ability to immortalize primary cultures of mouse and rat cells. The results show that certain mutants, including one that deletes the entire region, are able to immortalize. However, consistent with previous data, the immortalized cells are not fully transformed, as judged by doubling time, sensitivity to conc...

  12. Expression of p21waf1, p53 and proliferating cell nuclear antigen in lung cancer%p21waf1和p53及增殖细胞核抗原在肺癌组织中的表达

    Institute of Scientific and Technical Information of China (English)

    丁续红; 吴小军; 田素梅; 杨炯; 李清泉

    2005-01-01

    蛋白作用不协调.%BACKGROUND: The different level of proteins regulating cell cycle and theircorrelation is the main criteria to differentiate the benign and malignant cellular proliferation.OBJECTIVE: To investigate the expression status of p21waf1 and p53 in lung cancer as well as proliferating cell nuclear antigen (PCNA)DESIGN:A case-control study.SETTING: Department of Respiratory Medicine, Renmin Hospital ,Wuhan UniversityPAITICIPANTS:This case-control study involved 135 patients who underwent lobectomy or fiberoptic bronchoscopy for primary lung cancer or benign chronic pulmonary diseases at Renmin Hospital of Wuhan University from October 1996 through May 1999. They were divided into two groups: lung cancer group (76 patients, including 56 men and 20 women,aged 18-74 years of age) and chronic pulmonary diseases group (59 cases,including 42 men and 17 women, aged 16-70 years of age).METHODS: Phosphate buffer solution replaced the first antibody as the negative control. Immunohistochemistry was performed using a modified streptavidin-biotinylated peroxidase technique according to the manufacturer's recommendations (Maxim Corporation). For p21waf1 staining, we used hydrated autoclaving as a pretreatment. Antigen retrieval was performed in a standard microwave unit for p53 staining. PCNA staining did not need The ratio of the positive cells indicated by yellowish brown nucleus due to staining was counted for 5 successive high-fold microscopic fields: when it was≥ 10%, it was taken as positive; when it was <10%, it was regarded as high-fold microscopic fields for the percentage of the positive cells indicated by yellowish brown nucleus due to staining in each field, and the average value of the five fields was taken as labeling index (LI) for proliferated nuclear antigens.MAIN OUTCOME MEASURES: The expression leyels of p21waf1, p53and PCNA in lung cancer.cancer were 75% (57/76) and 47%(36/76) respectively. The labeling index of PCNA in lung cancer group was significantly higher than that of

  13. Minor histocompatibility antigens on canine hemopoietic progenitor cells.

    Science.gov (United States)

    Weber, Martin; Lange, Claudia; Günther, Wolfgang; Franz, Monika; Kremmer, Elisabeth; Kolb, Hans-Jochem

    2003-06-15

    Adoptive immunotherapy with CTL against minor histocompatibility Ags (mHA) provides a promising way to treat leukemia relapse in allogeneic chimeras. Here we describe the in vitro generation of CTL against mHA in the dog. We tested their inhibitory effect on the growth of hemopoietic progenitor cells stimulated by hemopoietic growth factors in a 4-day suspension culture. CTL were produced by coculture of donor PBMC with bone marrow-derived dendritic cells (DCs). These DCs were characterized by morphology, high expression of MHC class II and CD1a, and the absence of the monocyte-specific marker CD14. Characteristically these cells stimulated allogeneic lymphocytes (MLR) and, after pulsing with a foreign Ag (keyhole limpet hemocyanin), autologous T cells. CTL were generated either ex vivo by coculture with DCs of DLA-identical littermates or in vivo by immunization of the responder with DCs obtained from a DLA-identical littermate. In suspension culture assays the growth of hemopoietic progenitor cells was inhibited in 53% of DLA-identical littermate combinations. In canine families mHA segregated with DLA as restriction elements. One-way reactivity against mHA was found in five littermate combinations. In two cases mHA might be Y chromosome associated, in three cases autosomally inherited alleles were detected. We conclude that CTL can be produced in vitro and in vivo against mHA on canine hemopoietic progenitor cells using bone marrow-derived DCs. PMID:12794111

  14. Coupling of HIV-1 Antigen to the Selective Autophagy Receptor SQSTM1/p62 Promotes T-Cell-Mediated Immunity

    Science.gov (United States)

    Andersen, Aram Nikolai; Landsverk, Ole Jørgen; Simonsen, Anne; Bogen, Bjarne; Corthay, Alexandre; Øynebråten, Inger

    2016-01-01

    Vaccines aiming to promote T-cell-mediated immune responses have so far showed limited efficacy, and there is a need for novel strategies. Studies indicate that autophagy plays an inherent role in antigen processing and presentation for CD4+ and CD8+ T cells. Here, we report a novel vaccine strategy based on fusion of antigen to the selective autophagy receptor sequestosome 1 (SQSTM1)/p62. We hypothesized that redirection of vaccine antigen from proteasomal degradation into the autophagy pathway would increase the generation of antigen-specific T cells. A hybrid vaccine construct was designed in which the antigen is fused to the C-terminus of p62, a signaling hub, and a receptor that naturally delivers ubiquitinated cargo for autophagic degradation. Fusion of the human immunodeficiency virus-1 antigen Gagp24 to p62 resulted in efficient antigen delivery into the autophagy pathway. Intradermal immunization of mice revealed that, in comparison to Gagp24 delivered alone, fusion to p62 enhanced the number of Gagp24-specific interferon-γ-producing T cells, including CD8+ T cells. The strategy may also have the potential to modulate the antigenic peptide repertoire. Because p62 and autophagy are highly conserved between species, we anticipate this strategy to be a candidate for the development of T-cell-based vaccines in humans.

  15. Dendritic cells engineered to express defined allo-HLA peptide complexes induce antigen-specific cytotoxic T cells efficiently killing tumour cells

    DEFF Research Database (Denmark)

    Stronen, E; Abrahamsen, I W; Gaudernack, G; Wälchli, S; Munthe, E; Buus, S; Johansen, F-E; Lund-Johansen, F; Olweus, J

    2009-01-01

    , efficiently present externally loaded peptides from the antigen, Melan-A/MART-1 to T cells from HLA-A*0201-negative donors. CD8(+) T cells binding HLA-A*0201/MART-1 pentamers were detected already after 12 days of co-culture in 11/11 donors. The majority of cells from pentamer(+) cell lines were CTL and...... efficiently killed HLA-A*0201(+) melanoma cells, whilst sparing HLA-A*0201(+) B-cells. Allo-restricted CTL specific for peptides from the leukaemia-associated antigens CD33 and CD19 were obtained with comparable efficiency. Collectively, the results show that dendritic cells engineered to express defined allo...

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

    Science.gov (United States)

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

    2014-01-01

    Investigators developed chimeric antigen receptors (CARs) for expression on T cells more than 25 years ago. When the CAR is derived from an antibody, the resultant cell should combine the desirable targeting features of an antibody (e.g. lack of requirement for major histocompatibility complex recognition, ability to recognize non-protein antigens) with the persistence, trafficking, and effector functions of a T cell. This article describes how the past two decades have seen a crescendo of research which has now begun to translate these potential benefits into effective treatments for patients with cancer. We describe the basic design of CARs, describe how antigenic targets are selected, and the initial clinical experience with CAR-T cells. Our review then describes our own and other investigators' work aimed at improving the function of CARs and reviews the clinical studies in hematological and solid malignancies that are beginning to exploit these approaches. Finally, we show the value of adding additional engineering features to CAR-T cells, irrespective of their target, to render them better suited to function in the tumor environment, and discuss how the safety of these heavily modified cells may be maintained. PMID:24329793

  17. Lewis (y) Antigen Overexpression Increases the Expression of MMP-2 and MMP-9 and Invasion of Human Ovarian Cancer Cells

    OpenAIRE

    Shulan Zhang; Masao Iwamori; Changzhi Wang; Yifei Wang; Chuan Liu; Song Gao; Lili Gao; Bei Lin; Limei Yan

    2010-01-01

    Lewis (y) antigen is a difucosylated oligosaccharide present on the plasma membrane, and its overexpression is frequently found in human cancers and has been shown to be associated with poor prognosis. Our previous studies have shown that Lewis (y) antigen plays a positive role in the process of invasion and metastasis of ovarian cancer cells. However, the mechanisms by which Lewis (y) antigen enhances the invasion and tumor metastasis are still unknown. In this study, we established a stable...

  18. Antigen Presentation and T-Cell Activation Are Critical for RBP4-Induced Insulin Resistance.

    Science.gov (United States)

    Moraes-Vieira, Pedro M; Castoldi, Angela; Aryal, Pratik; Wellenstein, Kerry; Peroni, Odile D; Kahn, Barbara B

    2016-05-01

    Adipose tissue (AT) inflammation contributes to impaired insulin action, which is a major cause of type 2 diabetes. RBP4 is an adipocyte- and liver-derived protein with an important role in insulin resistance, metabolic syndrome, and AT inflammation. RBP4 elevation causes AT inflammation by activating innate immunity, which elicits an adaptive immune response. RBP4-overexpressing mice (RBP4-Ox) are insulin resistant and glucose intolerant and have increased AT macrophages and T-helper 1 cells. We show that high-fat diet-fed RBP4(-/-) mice have reduced AT inflammation and improved insulin sensitivity versus wild type. We also elucidate the mechanism for RBP4-induced macrophage antigen presentation and subsequent T-cell activation. In RBP4-Ox, AT macrophages display enhanced c-Jun N-terminal kinase, extracellular signal-related kinase, and p38 phosphorylation. Inhibition of these pathways and of NF-κB reduces activation of macrophages and CD4 T cells. MyD88 is an adaptor protein involved in proinflammatory signaling. In macrophages from MyD88(-/-) mice, RBP4 fails to stimulate secretion of tumor necrosis factor, IL-12, and IL-6 and CD4 T-cell activation. In vivo blockade of antigen presentation by treating RBP4-Ox mice with CTLA4-Ig, which blocks costimulation of T cells, is sufficient to reduce AT inflammation and improve insulin resistance. Thus, MyD88 and downstream mitogen-activated protein kinase and NF-κB pathways are necessary for RBP4-induced macrophage antigen presentation and subsequent T-cell activation. Also, blocking antigen presentation with CTLA4-Ig improves RBP4-induced insulin resistance and macrophage-induced T-cell activation. PMID:26936962

  19. Effects of 60Co γ-ray irradiation on expression of surface antigens in endothelial cells of human umbilical veins

    International Nuclear Information System (INIS)

    Culture of endothelial cells of human umbilical veins and avidin-biotin peroxidase complex (ABC) immunochemical technique were used in the experiment to detect the surface antigens in endothelial cells. Endothelial cells separated from five umbilical cords in original culture were divided into two groups, irradiated and non-irradiated. The cells were irradiated with 15 Gy of 60Co γ-rays at dose rates of 21.78 cGy/min. Then antigens RBC A, HLA-ABC, HLA-DR, CD4 and CD8 were assayed for both groups by the method of ABC. The results showed that the values of integrated optical density (IOD) for the surface antigens in the irradiated cells were lower than those in the non-irradiated cells with the difference in antigen expression in endothelial cells being significant (P<0.05) between the two groups

  20. Antigen-oriented T cell migration contributes to myelin peptide induced-EAE and immune tolerance.

    Science.gov (United States)

    Zheng, Peiguo; Fu, Hanxiao; Wei, Gaohui; Wei, Zhongwei; Zhang, Junhua; Ma, Xuehan; Rui, Dong; Meng, Xianchun; Ming, Liang

    2016-08-01

    Treatment with soluble myelin peptide can efficiently and specifically induce tolerance to demyelination autoimmune diseases including multiple sclerosis, however the mechanism underlying this therapeutic effect remains to be elucidated. In actively induced mouse model of experimental autoimmune encephalomyelitis (EAE) we analyzed T cell and innate immune cell responses in the central nervous system (CNS) and spleen after intraperitoneal (i.p.) infusion of myelin oligodendrocyte glycoprotein (MOG). We found that i.p. MOG infusion blocked effector T cell recruitment to the CNS and protected mice from EAE and lymphoid organ atrophy. Innate immune CD11b(+) cells preferentially recruited MOG-specific effector T cells, particularly when activated to become competent antigen presenting cells (APCs). During EAE development, mature APCs were enriched in the CNS rather than in the spleen, attracting effector T cells to the CNS. Increased myelin antigen exposure induced CNS-APC maturation, recruiting additional effector T cells to the CNS, causing symptoms of disease. MOG triggered functional maturation of splenic APCs. MOG presenting APCs interacted with MOG-specific T cells in the spleen, aggregating to cluster around CD11b(+) cells, and were trapped in the periphery. This process was MHC II dependent as an MHC II directed antibody blocked CD4(+) T cell cluster formation. These findings highlight the role of myelin peptide-loaded APCs in myelin peptide-induced EAE and immune tolerance. PMID:27327113

  1. Chimeric antigen receptor T cell therapy: 25years in the making.

    Science.gov (United States)

    Gill, Saar; Maus, Marcela V; Porter, David L

    2016-05-01

    Chimeric antigen receptor (CAR) T cell therapy of cancer is generating enormous enthusiasm. Twenty-five years after the concept was first proposed, major advances in molecular biology, virology, and good manufacturing practices (GMP)-grade cell production have transformed antibody-T cell chimeras from a scientific curiosity to a fact of life for academic cellular immunotherapy researchers and, increasingly, for patients. In this review, we explain the preclinical concept, outline how it has been translated to the clinic, and draw lessons from the first years of CAR T cell therapy for the practicing clinician. PMID:26574053

  2. Improving therapy of chronic lymphocytic leukemia with chimeric antigen receptor T cells.

    Science.gov (United States)

    Fraietta, Joseph A; Schwab, Robert D; Maus, Marcela V

    2016-04-01

    Adoptive cell immunotherapy for the treatment of chronic lymphocytic leukemia (CLL) has heralded a new era of synthetic biology. The infusion of genetically engineered, autologous chimeric antigen receptor (CAR) T cells directed against CD19 expressed by normal and malignant B cells represents a novel approach to cancer therapy. The results of recent clinical trials of CAR T cells in relapsed and refractory CLL have demonstrated long-term disease-free remissions, underscoring the power of harnessing and redirecting the immune system against cancer. This review will briefly summarize T-cell therapies in development for CLL disease. We discuss the role of T-cell function and phenotype, T-cell culture optimization, CAR design, and approaches to potentiate the survival and anti-tumor effects of infused lymphocytes. Future efforts will focus on improving the efficacy of CAR T cells for the treatment of CLL and incorporating adoptive cell immunotherapy into standard medical management of CLL. PMID:27040708

  3. Chimeric Antigen Receptor Therapy for B-cell Malignancies

    Directory of Open Access Journals (Sweden)

    David L Porter, Michael Kalos, Zhaohui Zheng, Bruce Levine, Carl June

    2011-01-01

    Full Text Available We presented data showing that the CART-19 cells expressing the 4-1BB signaling domain can have unprecedented and massive in-vivo expansion, traffic to tumor sites, persist long term in vivo, and induce rapid and potent anti-tumor activity in chemotherapy refractory CLL patients.

  4. Chimeric Antigen Receptor Therapy for B-cell Malignancies

    OpenAIRE

    Porter, David L.; Kalos, Michael; Zheng, Zhaohui; Levine, Bruce; June, Carl

    2011-01-01

    We presented data showing that the CART-19 cells expressing the 4-1BB signaling domain can have unprecedented and massive in-vivo expansion, traffic to tumor sites, persist long term in vivo, and induce rapid and potent anti-tumor activity in chemotherapy refractory CLL patients.

  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

    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...... major brands migrating at apparent Mr of 38,000, 45,000, and 80,000, in addition to many minor bands between Mr 45,000 and 97,000, including Mr 52,000. The Mr 45,000 and 38,000 were associated with the cell membrane and Mr 52,000 as well as Mr 38,000 were associated with the lysosomes. The 1B10...

  6. Osteopontin promotes dendritic cell maturation and function in response to HBV antigens

    Directory of Open Access Journals (Sweden)

    Cui GY

    2015-06-01

    Full Text Available Guangying Cui,1,2 Jianing Chen,1,2 Jianqin He,1,2 Chong Lu,1,2 Yingfeng Wei,1,2 Lin Wang,1,2 Xuejun Xu,3 Lanjuan Li,1,2 Toshimitsu Uede,4 Hongyan Diao1,2 1State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, 2Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, 3Department of Oral Orthodontics, Affiliated Stomatology Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, People’s Republic of China; 4Molecular Immunology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Purpose: Dendritic cells (DCs play critical roles in promoting innate and adaptive immunity in microbial infection. Functional impairment of DCs may mediate the suppression of viral-specific T-cell immune response in chronic hepatitis B (CHB patients. Osteopontin (OPN is involved in several liver diseases and infectious diseases. However, whether OPN affects DC function in hepatitis B virus (HBV infection is unknown.Methods: Twenty CHB patients and 20 healthy volunteers were recruited. OPN secreted by DCs was compared. Peripheral blood mononuclear cells cultured with OPN antibody were examined to study the costimulatory molecular expression and interleukin (IL-12 production of DCs after HBV antigenic stimulation. OPN-deficient mice were used to investigate the influence of OPN on DC maturation and function after HBV antigenic stimulation in vitro and in vivo. Exogenous OPN was administrated to further verify the functioning of DCs from CHB patients upon HBV antigenic stimulation.Results: We found that OPN production of DCs from CHB patients was significantly lower than those from healthy volunteers. The absence of OPN impaired IL-12 production and costimulatory molecular expression of DCs upon stimulation with HBV antigens. Defective DC function led to reduced activation of Th1 response to

  7. Dendritic cells induce antigen-specific regulatory T cells that prevent graft versus host disease and persist in mice

    OpenAIRE

    Sela, Uri; Olds, Peter; Park, Andrew; Schlesinger, Sarah J.; Steinman, Ralph M.

    2011-01-01

    Foxp3+ regulatory T cells (T reg cells) effectively suppress immunity, but it is not determined if antigen-induced T reg cells (iT reg cells) are able to persist under conditions of inflammation and to stably express the transcription factor Foxp3. We used spleen cells to stimulate the mixed leukocyte reaction (MLR) in the presence of transforming growth factor β (TGF-β) and retinoic acid. We found that the CD11chigh dendritic cell fraction was the most potent at inducing high numbers of allo...

  8. Fishing Fish Stem Cells and Nuclear Transplants

    OpenAIRE

    Yunhan Hong

    2011-01-01

    Fish has been the subject of various research fields, ranging from ecology, evolution, physiology and toxicology to aquaculture. In the past decades fish has attracted considerable attention for functional genomics, cancer biology and developmental genetics, in particular nuclear transfer for understanding of cytoplasmic-nuclear relationship. This special issue reports on recent progress made in fish stem cells and nuclear transfer.

  9. Antigen presenting cells in the skin of a patient with hair loss and systemic lupus erythematosus

    Directory of Open Access Journals (Sweden)

    Ana Maria Abreu Velez

    2009-09-01

    Full Text Available Context: Hair loss is one of the most striking clinical features of active systemic lupus erythematosus (SLE, however, very few studies have investigated the immunological features of this process. Case report: We describe a 33 years old female who presented with scalp hair loss and arthralgias. Physical examination revealed erythematous plaques on the nose and scalp, with bitemporal hair loss. Scalp biopsies revealed epidermal hyperkeratosis, with a mild interface infiltrate of lymphocytes and histiocytes and a superficial and deep, perivascular and periadnexal infiltrate of mostly CD4 positive cells. Antibodies to HAM 56, CD68, CD1a, S-100, mast cell tryptase and c-kit/CD117 were strongly positive around the hair follicles, and in the adjacent sebaceous glands. Conclusion: We present the first report showing a significant presence of several antigen presenting cells around the hair follicular units in a patient with alopecia in active SLE. Today, antigen presenting cells and dendritic cells (DC are modeled as the master regulators of human immunity. One aspect that has become clearly appreciated is the great diversity of DC subtypes, each with considerable functional differences. Thus, we suggest that APC and DCs are equipped with Pattern Recognition Receptors (PRRs to some hair follicular unit antigens; that these innate sensors recognize conserved molecular patterns on self- tissue, and play a significant role in the pathophysiology of alopecia in SLE patients.

  10. Antigen presenting cells in the skin of a patient with hair loss and systemic lupus erythematosus

    Directory of Open Access Journals (Sweden)

    Ana Maria Abreu Velez

    2009-01-01

    Full Text Available Context: Hair loss is one of the most striking clinical features of active systemic lupus erythematosus (SLE, however, very few studies have investigated the immunological features of this process. Case report: We describe a 33 years old female who presented with scalp hair loss and arthralgias. Physical examination revealed erythematous plaques on the nose and scalp, with bitemporal hair loss. Scalp biopsies revealed epidermal hyperkeratosis, with a mild interface infiltrate of lymphocytes and histiocytes and a superficial and deep, perivascular and periadnexal infiltrate of mostly CD4 positive cells. Antibodies to HAM 56, CD68, CD1a, S-100, mast cell tryptase and c-kit/CD117 were strongly positive around the hair follicles, and in the adjacent sebaceous glands. Conclusion : We present the first report showing a significant presence of several antigen presenting cells around the hair follicular units in a patient with alopecia in active SLE. Today, antigen presenting cells and dendritic cells (DC are modeled as the master regulators of human immunity. One aspect that has become clearly appreciated is the great diversity of DC subtypes, each with considerable functional differences. Thus, we suggest that APC and DCs are equipped with Pattern Recognition Receptors (PRRs to some hair follicular unit antigens; that these innate sensors recognize conserved molecular patterns on self- tissue, and play a significant role in the pathophysiology of alopecia in SLE patients

  11. Immunological analysis of cell-associated antigens of Bacillus anthracis.

    OpenAIRE

    Ezzell, J W; Abshire, T. G.

    1988-01-01

    Sera from Hartley guinea pigs vaccinated with a veterinary live spore anthrax vaccine were compared with sera from guinea pigs vaccinated with the human anthrax vaccine, which consists of aluminum hydroxide-adsorbed culture proteins of Bacillus anthracis V770-NP-1R. Sera from animals vaccinated with the spore vaccine recognized two major B. anthracis vegetative cell-associated proteins that were either not recognized or poorly recognized by sera from animals that received the human vaccine. T...

  12. Complement-dependent transport of antigen into B cell follicles

    DEFF Research Database (Denmark)

    Gonzalez, Santiago F.; Lukacs-Kornek, Veronika; Kuligowski, Michael P.;

    2010-01-01

    Since the original proposal by Fearon and Locksley (Fearon and Locksley. 1996. Science 272: 50-53) that the complement system linked innate and adaptive immunity, there has been a rapid expansion of studies on this topic. With the advance of intravital imaging, a number of recent papers revealed ...... opsonization of influenza and uptake by macrophages, and the capture of virus by dendritic cells residing in the medullary compartment of peripheral lymph nodes....

  13. Cell-mediated immune response of synovial fluid lymphocytes to ureaplasma antigen in Reiter's syndrome

    Directory of Open Access Journals (Sweden)

    Pavlica Ljiljana

    2003-01-01

    Full Text Available INTRODUCTION Reiter's syndrome (RS is an seronegative arthritis that occurs after urogenital or enteric infection which in addition with occular and/or mucocutaneous manifestations presents complete form of disease. According to previous understanding arthritis in the RS is the reactive one, which means that it is impossible to isolate its causative agent. However, there are the more and more authors suggesting that arthritis in the urogenital form of disease is caused by the infective agent in the affected joint. This suggestion is based on numerous studies on the presence of Chlmaydia trachomatis and Ureaplasma urealyticum in the inflamed joint by using new diagnostic methods in molecular biology published in the recent literature [1-3]. Besides, numerous studies of the humoral and cell-mediated immune response to "triggering" bacteria in the affected joint have supported previous suggestions [4-7]. Aim of the study was to determine whether synovial fluid T-cells specifically recognize the "triggering" bacteria presumably responsible for the Reiter's syndrome. METHOD The 3H-thymidine uptake procedure for measuring lymphocyte responses was applied to lymphocytes derived concurrently from synovial fluid (SF and from peripheral blood (PB [8]. Ureaplasma antigen and mitogen PHA stimulated lymphocytes in 24 RS patients (24 PB samples, 9 SF samples and the results were compared with those found in 10 patients with rheumatoid arthritis (RA (10 PB samples, 5 SF samples. Preparation of ureaplasma antigen. Ureaplasma was cultured on cell-free liquid medium [9]. Sample of 8 ml was heat-inactivated for 15 minutes at 601C and permanently stirred with magnetic mixer. The sample was centrifuged at 2000 x g for 40 minutes and than deposits carefully carried to other sterile glass tubes (Corex and recentrifuged at 9000 x g for 30 minutes. The deposit was washed 3 times in sterile 0.9% NaCl, and final sediment was resuspended in 1.2 ml sterile 0.9% Na

  14. A 125I-protein A-binding assay detecting antibodies to cell surface antigens

    International Nuclear Information System (INIS)

    A 125I-protein A-binding assay detecting antibodies to cell surface antigens on human blood cells was developed and evaluated using sera from multitransfused nonleukemic patients sensitized against HLA antigens. The binding assay was found to be reproducible and more sensitive than conventional HLA testing. Seven patients with acute myelogenous leukemia and two patients with acute lymphoblastic leukemia successfully treated by chemotherapy were than investigated. Sera from seven of the patients studied in partial or complete remission demonstrated significant binding to autochthonous leukemic cells obtained from bone marrow or peripheral blood. In two cases sera taken during the leukemic stage demonstrated the most pronounced binding to the patients' own leukemic cells. Sera from four patients with demonstrable significant binding to autochthonous leukemic cells failed to bind to autochthonous remission cells when both types of target cells were tested in parallel. Differences in serum concentrations of IgG, IgA, and IgM were not the cause of the demonstrated increased binding of leukemic sera to autochthonous target cells. We propose that the 125I-protein A-binding assay presented in this paper detects antibodies reacting selectively with acute leukemia cells. (orig.)

  15. Sensitivity and specificity of tritiated thymidine incorporation and ELISPOT assays in identifying antigen specific T cell immune responses

    OpenAIRE

    MacLeod Beth; Slota Meredith; dela Rosa Corazon; Goodell Vivian; Disis Mary L

    2007-01-01

    Abstract Background Standardization of cell-based immunologic monitoring is becoming increasingly important as methods for measuring cellular immunity become more complex. We assessed the ability of two commonly used cell-based assays, tritiated thymidine incorporation (proliferation) and IFN-gamma ELISPOT, to predict T cell responses to HER-2/neu, tetanus toxoid (tt), and cytomegalovirus (CMV) antigens. These antigens were determined to be low (HER-2/neu), moderate (tt), and robustly (CMV) i...

  16. Kinetics of T cell-activation molecules in response to Mycobacterium tuberculosis antigens

    Directory of Open Access Journals (Sweden)

    Antas Paulo RZ

    2002-01-01

    Full Text Available The phenotypic features acquired subsequent to antigen-specific stimulation in vitro were evaluated by means of the kinetic expressions of CD69 and CD25 activation molecules on T lymphocytes and assayed by flow cytometry in response to PPD, Ag85B, and ferritin in PPD-positive healthy control individuals. In response to PHA, CD69 staining on both CD4+ and CD8+ T cells became initially marked after 4 h, peaked at 24 h, and quickly decreased after 120 h. For CD25, a latter expression was detected around 8 h, having increased after 96 h. As expected, the response rate to the mycobacterial antigens was much lower than that to the mitogen. Positive staining was high after 96 h for CD25 and after 24 h for CD69. CD69 expression was significantly enhanced (p < 0.05 on CD8+ as compared to CD4+ T cells. High levels were also found between 96-120 h. Regarding Ag85B, CD25+ cells were mostly CD4+ instead of CD8+ T cells. Moreover, in response to ferritin, a lower CD25 expression was noted. The present data will allow further characterization of the immune response to new mycobacterial-specific antigens and their evaluation for possible inclusion in developing new diagnostic techniques for tuberculosis as well in a new vaccine to prevent the disease.

  17. Relationship between Fc receptors, antigen-binding sites on T and B cells, and H-2 complex-associated determinants.

    Science.gov (United States)

    Basten, A; Miller, J F; Abraham, R

    1975-03-01

    The relationship between H-2 complex-associated determinants, Fc receptors, and specific antigen-recognition sites on T and B cells was examined by binding and functional assays. The Fc receptor was detected by radiolabeled immune complexes or aggregated human IgG. Both these reagents selectively bound to B cells, not to T cells. When spleen cells, from mice primed to several antigens, were exposed to highly substituted radioactive aggregates, their capacity to transfer both a direct and indirect plaque-forming cell response to these antigens was abrogated. Addition of B cells, but not of T cells, restored responsiveness. Complexed Ig binding to Fc receptors was prevented by pretreatment of mixed lymphoid cell populations with antisera directed against membrane components on the same cell (e.g., H-2) and on other cells (e.g., theta). The lack of specificity of inhibition was thought to be due to the formation on cell surfaces of antigen-antibody complexes which would then attach to the Fc receptor during the incubation precedure. Specific blockade of the Fc receptor during the incubation procedure. Specific blockade of the Fc receptor however occurred when B cells were pretreated with the Fab fragments of anti-H-2 antibody. This was demonstrated autoradiographically and by inhibition of aggregate-induced suicide. The blocking activity of ante-H-2 Fab was removed by absorption with spleen cells from thymectomized irradiated mice but not with thymus cells of appropriate specificity. This suggested that the antibodies involved had specificity for determinants on the B-cell membrane distinct from those coded by the K or D end of the H-2 complex, and either absent from, or poorly represented on, thymus cells. Specific antigen-induced suicide of B cells was achieved simply by incubating the cells with radioactive antigen in the cold. T-cell suicide on the other hand required that the 125I-labeled antigen be presented to the T cells at 37 degrees-C on the surface of

  18. A Novel Chimeric Antigen Receptor Against Prostate Stem Cell Antigen Mediates Tumor Destruction in a Humanized Mouse Model of Pancreatic Cancer

    Science.gov (United States)

    Lagisetty, Kiran H.; Tran, Eric; Zheng, Zhili; Gattinoni, Luca; Yu, Zhiya; Burns, William R.; Miermont, Anne M.; Teper, Yaroslav; Rudloff, Udo; Restifo, Nicholas P.; Feldman, Steven A.; Rosenberg, Steven A.; Morgan, Richard A.

    2014-01-01

    Abstract Despite advances in the understanding of its molecular pathophysiology, pancreatic cancer remains largely incurable, highlighting the need for novel therapies. We developed a chimeric antigen receptor (CAR) specific for prostate stem cell antigen (PSCA), a glycoprotein that is overexpressed in pancreatic cancer starting at early stages of malignant transformation. To optimize the CAR design, we used antigen-recognition domains derived from mouse or human antibodies, and intracellular signaling domains containing one or two T cell costimulatory elements, in addition to CD3zeta. Comparing multiple constructs established that the CAR based on human monoclonal antibody Ha1-4.117 had the greatest reactivity in vitro. To further analyze this CAR, we developed a human pancreatic cancer xenograft model and adoptively transferred CAR-engineered T cells into animals with established tumors. CAR-engineered human lymphocytes induced significant antitumor activity, and unlike what has been described for other CARs, a second-generation CAR (containing CD28 cosignaling domain) induced a more potent antitumor effect than a third-generation CAR (containing CD28 and 41BB cosignaling domains). While our results provide evidence to support PSCA as a target antigen for CAR-based immunotherapy of pancreatic cancer, the expression of PSCA on selected normal tissues could be a source of limiting toxicity. PMID:24694017

  19. A novel system of artificial antigen-presenting cells efficiently stimulates Flu peptide-specific cytotoxic T cells in vitro

    International Nuclear Information System (INIS)

    Highlights: → Adoptive immunotherapy depends on relevant numbers of cytolytic T lymphocytes. → An ideal artificial APCs system was successfully prepared in vivo. → Controlled release of IL-2 leads to much more T-cell expansion. → This system is better than general cellular APCs on T-cell expansion. -- Abstract: Therapeutic numbers of antigen-specific cytotoxic T lymphocytes (CTLs) are key effectors in successful adoptive immunotherapy. However, efficient and reproducible methods to meet the qualification remain poor. To address this issue, we designed the artificial antigen-presenting cell (aAPC) system based on poly(lactic-co-glycolic acid) (PLGA). A modified emulsion method was used for the preparation of PLGA particles encapsulating interleukin-2 (IL-2). Biotinylated molecular ligands for recognition and co-stimulation of T cells were attached to the particle surface through the binding of avidin-biotin. These formed the aAPC system. The function of aAPCs in the proliferation of specific CTLs against human Flu antigen was detected by enzyme-linked immunospot assay (ELISPOT) and MTT staining methods. Finally, we successfully prepared this suitable aAPC system. The results show that IL-2 is released from aAPCs in a sustained manner over 30 days. This dramatically improves the stimulatory capacity of this system as compared to the effect of exogenous addition of cytokine. In addition, our aAPCs promote the proliferation of Flu antigen-specific CTLs more effectively than the autologous cellular APCs. Here, this aAPC platform is proved to be suitable for expansion of human antigen-specific T cells.

  20. A novel system of artificial antigen-presenting cells efficiently stimulates Flu peptide-specific cytotoxic T cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Han, Hui [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Peng, Ji-Run, E-mail: pengjr@medmail.com.cn [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Chen, Peng-Cheng; Gong, Lei [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Qiao, Shi-Shi [Department of Hepatobiliary Surgery, The First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052 (China); Wang, Wen-Zhen; Cui, Zhu-Qingqing; Yu, Xin; Wei, Yu-Hua [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Leng, Xi-Sheng, E-mail: lengxs2003@yahoo.com.cn [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China)

    2011-08-05

    Highlights: {yields} Adoptive immunotherapy depends on relevant numbers of cytolytic T lymphocytes. {yields} An ideal artificial APCs system was successfully prepared in vivo. {yields} Controlled release of IL-2 leads to much more T-cell expansion. {yields} This system is better than general cellular APCs on T-cell expansion. -- Abstract: Therapeutic numbers of antigen-specific cytotoxic T lymphocytes (CTLs) are key effectors in successful adoptive immunotherapy. However, efficient and reproducible methods to meet the qualification remain poor. To address this issue, we designed the artificial antigen-presenting cell (aAPC) system based on poly(lactic-co-glycolic acid) (PLGA). A modified emulsion method was used for the preparation of PLGA particles encapsulating interleukin-2 (IL-2). Biotinylated molecular ligands for recognition and co-stimulation of T cells were attached to the particle surface through the binding of avidin-biotin. These formed the aAPC system. The function of aAPCs in the proliferation of specific CTLs against human Flu antigen was detected by enzyme-linked immunospot assay (ELISPOT) and MTT staining methods. Finally, we successfully prepared this suitable aAPC system. The results show that IL-2 is released from aAPCs in a sustained manner over 30 days. This dramatically improves the stimulatory capacity of this system as compared to the effect of exogenous addition of cytokine. In addition, our aAPCs promote the proliferation of Flu antigen-specific CTLs more effectively than the autologous cellular APCs. Here, this aAPC platform is proved to be suitable for expansion of human antigen-specific T cells.

  1. 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.

  2. Tumor associated antigen specific T-cell populations identified in ex vivo expanded TIL cultures

    DEFF Research Database (Denmark)

    Junker, Niels; Kvistborg, Pia; Køllgaard, Tania;

    2012-01-01

    Ex vivo expanded tumor infiltrating lymphocytes (TILs) from malignant melanoma (MM) and head & neck squamous cell carcinoma (HNSCC) share a similar oligoclonal composition of T effector memory cells, with HLA class I restricted lysis of tumor cell lines. In this study we show that ex vivo expanded...... TILs from MM and HNSCC demonstrate a heterogeneous composition in frequency and magnitude of tumor associated antigen specific populations by Elispot IFN¿ quantitation. TILs from MM and HNSCC shared reactivity towards NY ESO-1, cyclin B1 and Bcl-x derived peptides. Additionally we show that dominating...

  3. Expression of myeloid differentiation antigens on normal and malignant myeloid cells.

    OpenAIRE

    Griffin, J D; Ritz, J; Nadler, L M; Schlossman, S F

    1981-01-01

    A series of monoclonal antibodies have been characterized that define four surface antigens (MY3, MY4, MY7, and MY8) of human myeloid cells. They were derived from a fusion of the NS-1 plasmacytoma cell line with splenocytes from a mouse immunized with human acute myelomonocytic leukemia cells. MY3 and MY4 are expressed by normal monocytes and by greater than 90% of patients with acute monocytic leukemia or acute myelomonocytic leukemia, but are detected much less often on other types of myel...

  4. Identification of a Coccidioides immitis antigen 2 domain that expresses B-cell-reactive epitopes.

    OpenAIRE

    Zhu, Y; Tryon, V; Magee, D M; Cox, R. A.

    1997-01-01

    Antigen 2 (Ag2), a major immunoreactive component of Coccidioides immitis mycelium- and spherule-phase cell walls, was recently cloned in our laboratory and was shown to elicit T-cell responses in Coccidioides-immune mice. In this investigation, we evaluated recombinant Ag2 (rAg2) and PCR-generated Ag2 truncations for expression of B-cell-reactive epitopes in enzyme-linked immunosorbent and immunoblot assays with sera from patients with active coccidioidomycosis, a hyperimmune goat anti-Ag2 s...

  5. Making Better Chimeric Antigen Receptors for Adoptive T-cell Therapy.

    Science.gov (United States)

    Maus, Marcela V; June, Carl H

    2016-04-15

    Chimeric antigen receptors (CAR) are engineered fusion proteins constructed from antigen recognition, signaling, and costimulatory domains that can be expressed in cytotoxic T cells with the purpose of reprograming the T cells to specifically target tumor cells. CAR T-cell therapy uses gene transfer technology to reprogram a patient's own T cells to stably express CARs, thereby combining the specificity of an antibody with the potent cytotoxic and memory functions of a T cell. In early-phase clinical trials, CAR T cells targeting CD19 have resulted in sustained complete responses within a population of otherwise refractory patients with B-cell malignancies and, more specifically, have shown complete response rates of approximately 90% in patients with relapsed or refractory acute lymphoblastic leukemia. Given this clinical efficacy, preclinical development of CAR T-cell therapy for a number of cancer indications has been actively investigated, and the future of the CAR T-cell field is extensive and dynamic. Several approaches to increase the feasibility and safety of CAR T cells are currently being explored, including investigation into the mechanisms regulating the persistence of CAR T cells. In addition, numerous early-phase clinical trials are now investigating CAR T-cell therapy beyond targeting CD19, especially in solid tumors. Trials investigating combinations of CAR T cells with immune checkpoint blockade therapies are now beginning and results are eagerly awaited. This review evaluates several of the ongoing and future directions of CAR T-cell therapy.Clin Cancer Res; 22(8); 1875-84. ©2016 AACR SEE ALL ARTICLES IN THIS CCR FOCUS SECTION, "OPPORTUNITIES AND CHALLENGES IN CANCER IMMUNOTHERAPY". PMID:27084741

  6. ImmunoChip study implicates antigen presentation to T cells in narcolepsy.

    Directory of Open Access Journals (Sweden)

    Juliette Faraco

    Full Text Available Recent advances in the identification of susceptibility genes and environmental exposures provide broad support for a post-infectious autoimmune basis for narcolepsy/hypocretin (orexin deficiency. We genotyped loci associated with other autoimmune and inflammatory diseases in 1,886 individuals with hypocretin-deficient narcolepsy and 10,421 controls, all of European ancestry, using a custom genotyping array (ImmunoChip. Three loci located outside the Human Leukocyte Antigen (HLA region on chromosome 6 were significantly associated with disease risk. In addition to a strong signal in the T cell receptor alpha (TRA@, variants in two additional narcolepsy loci, Cathepsin H (CTSH and Tumor necrosis factor (ligand superfamily member 4 (TNFSF4, also called OX40L, attained genome-wide significance. These findings underline the importance of antigen presentation by HLA Class II to T cells in the pathophysiology of this autoimmune disease.

  7. Antigen presenting cells costimulatory signaling during pre-implantation pregnancy 

    Directory of Open Access Journals (Sweden)

    Anna Sławek

    2012-09-01

    Full Text Available  Success of pregnancy depends on many factors. Three phenomena inducing immune tolerance against semi-allogeneic conceptus may play a crucial role in the pre-implantation period of pregnancy: influence of sex hormones in sex cycle, presence of oocyte or embryo and the presence of semen in the female reproductive tract. On the other hand dendritic cells are the most effective antigen-presenting cells in regulation of immune phenomena and also are considered as potent participants in inducing immune tolerance in the pregnancy. They communicate with T cells in cell contact-dependent manner or via cytokines. During cell-cell contacts, costimulatory molecules play a key role and their expression is often dependent on cytokines milieu. Both costimulatory molecules and cytokines influence generation of T regulatory cells. Interactions of these molecules are closely related. In this paper we would like to pay attention to the importance of antigen presenting cells costimulatory potency in immune regulation during a pre-implantation period of pregnancy.

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

    International Nuclear Information System (INIS)

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

  9. Performance-enhancing drugs: design and production of redirected chimeric antigen receptor (CAR) T cells.

    Science.gov (United States)

    Levine, B L

    2015-03-01

    Performance enhancement of the immune system can now be generated through ex vivo gene modification of T cells in order to redirect native specificity to target tumor antigens. This approach combines the specificity of antibody therapy, the expanded response of cellular therapy and the memory activity of vaccine therapy. Recent clinical trials of chimeric antigen receptor (CAR) T cells directed toward CD19 as a stand-alone therapy have shown sustained complete responses in patients with acute lymphoblastic leukemia and chronic lymphocytic leukemia. As these drug products are individually derived from a patient's own cells, a different manufacturing approach is required for this kind of personalized therapy compared with conventional drugs. Key steps in the CAR T-cell manufacturing process include the selection and activation of isolated T cells, transduction of T cells to express CARs, ex vivo expansion of modified T cells and cryopreservation in infusible media. In this review, the steps involved in isolating, genetically modifying and scaling-out the CAR T cells for use in a clinical setting are described in the context of in-process and release testing and regulatory standards. PMID:25675873

  10. Targeted delivery of lipid antigen to macrophages via the CD169/sialoadhesin endocytic pathway induces robust invariant natural killer T cell activation

    OpenAIRE

    Kawasaki, Norihito; Vela, Jose Luis; Nycholat, Corwin M.; Rademacher, Christoph; Khurana, Archana; van Rooijen, Nico; Crocker, Paul R.; Kronenberg, Mitchell; Paulson, James C.

    2013-01-01

    Invariant natural killer T (iNKT) cells induce a protective immune response triggered by foreign glycolipid antigens bound to CD1d on antigen-presenting cells (APCs). A limitation of using glycolipid antigens to stimulate immune responses in human patients has been the inability to target them to the most effective APCs. Recent studies have implicated phagocytic CD169+ macrophages as major APCs in lymph nodes for priming iNKT cells in mice immunized with glycolipid antigen in particulate form...

  11. Calnexin induces expansion of antigen-specific CD4(+) T cells that confer immunity to fungal ascomycetes via conserved epitopes.

    Science.gov (United States)

    Wüthrich, Marcel; Brandhorst, Tristan T; Sullivan, Thomas D; Filutowicz, Hanna; Sterkel, Alana; Stewart, Douglas; Li, Mengyi; Lerksuthirat, Tassanee; LeBert, Vanessa; Shen, Zu Ting; Ostroff, Gary; Deepe, George S; Hung, Chiung Yu; Cole, Garry; Walter, Jennifer A; Jenkins, Marc K; Klein, Bruce

    2015-04-01

    Fungal infections remain a threat due to the lack of broad-spectrum fungal vaccines and protective antigens. Recent studies showed that attenuated Blastomyces dermatitidis confers protection via T cell recognition of an unknown but conserved antigen. Using transgenic CD4(+) T cells recognizing this antigen, we identify an amino acid determinant within the chaperone calnexin that is conserved across diverse fungal ascomycetes. Calnexin, typically an ER protein, also localizes to the surface of yeast, hyphae, and spores. T cell epitope mapping unveiled a 13-residue sequence conserved across Ascomycota. Infection with divergent ascomycetes, including dimorphic fungi, opportunistic molds, and the agent causing white nose syndrome in bats, induces expansion of calnexin-specific CD4(+) T cells. Vaccine delivery of calnexin in glucan particles induces fungal antigen-specific CD4(+) T cell expansion and resistance to lethal challenge with multiple fungal pathogens. Thus, the immunogenicity and conservation of calnexin make this fungal protein a promising vaccine target. PMID:25800545

  12. Hsp70 enhances presentation of FMDV antigen to bovine CD4+ T cells in vitro

    OpenAIRE

    McLaughlin, Kerry; Seago, Julian; Robinson, Lucy; Kelly, Charles; Charleston, Bryan

    2010-01-01

    International audience Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious acute vesicular disease affecting cloven-hoofed animals, including cattle, sheep and pigs. The current vaccine induces a rapid humoral response, but the duration of the protective antibody response is variable, possibly associated with a variable specific CD4+ T cell response. We investigated the use of heat shock protein 70 (Hsp70) as a molecular chaperone to target viral antigen to th...

  13. Identification of two germ-tube-specific cell wall antigens of Candida albicans.

    OpenAIRE

    Ponton, J; J. M. Jones

    1986-01-01

    Outer cell wall layers of intact yeast- and mycelial-phase Candida albicans B311 were extracted with dithiothreitol. Antisera against mycelial-phase organisms were absorbed with yeast-phase organisms or yeast-phase extract and used to stain Western blots of sodium dodecyl sulfate-polyacrylamide gels loaded with yeast- and mycelial-phase extracts. Autoradiography of gels loaded with extracts from organisms surface labeled with 125I was used to detect surface antigens containing proteins. Antig...

  14. Red Cell Alloimmunization to Rhesus Antigen Among Pregnant Women Attending a Tertiary Care Hospital in Oman

    OpenAIRE

    Tamima Al-Dughaishi; Yusra Al Harrasi; Maymoona Al-Duhli; Ikhlass Al-Rubkhi; Nihal Al-Riyami; Al-Riyami, Arwa Z.; Pathare, Anil V.; Vaidyanathan Gowri

    2016-01-01

    Objectives: The detection of maternal alloimmunization against red cell antigens is vital in the management of hemolytic disease of the fetus and newborn. We sought to measure the presence of allosensitization to Rhesus D (RhD) antibodies in antenatal women attending a tertiary care hospital and assess the fetal outcome in sensitized women. Methods: We conducted a retrospective review of pregnant Omani women who registered at the Sultan Qaboos University Hospital between June 2011 and Ju...

  15. A Novel Laser Vaccine Adjuvant Increases the Motility of Antigen Presenting Cells

    OpenAIRE

    Farinelli, Bill; Doukas, Apostolos; Gelfand, Jeffrey Alan; Anderson, Richard Rox; Mei X. Wu; Chen, Xinyuan; Kim, Pilhan; Yun, Seok-Hyun

    2010-01-01

    Background Development of a potent vaccine adjuvant without introduction of any side effects remains an unmet challenge in the field of the vaccine research. Methodology/Principal Findings We found that laser at a specific setting increased the motility of antigen presenting cells (APCs) and immune responses, with few local or systemic side effects. This laser vaccine adjuvant (LVA) effect was induced by brief illumination of a small area of the skin or muscle with a nondestructive...

  16. Improved Activation toward Primary Colorectal Cancer Cells by Antigen-Specific Targeting Autologous Cytokine-Induced Killer Cells

    Directory of Open Access Journals (Sweden)

    Claudia Schlimper

    2012-01-01

    Full Text Available Adoptive therapy of malignant diseases with cytokine-induced killer (CIK cells showed promise in a number of trials; the activation of CIK cells from cancer patients towards their autologous cancer cells still needs to be improved. Here, we generated CIK cells ex vivo from blood lymphocytes of colorectal cancer patients and engineered those cells with a chimeric antigen receptor (CAR with an antibody-defined specificity for carcinoembryonic antigen (CEA. CIK cells thereby gained a new specificity as defined by the CAR and showed increase in activation towards CEA+ colon carcinoma cells, but less in presence of CEA− cells, indicated by increased secretion of proinflammatory cytokines. Redirected CIK activation was superior by CAR-mediated CD28-CD3ζ than CD3ζ signaling only. CAR-engineered CIK cells from colon carcinoma patients showed improved activation against their autologous, primary carcinoma cells from biopsies resulting in more efficient tumour cell lysis. We assume that adoptive therapy with CAR-modified CIK cells shows improved selectivity in targeting autologous tumour lesions.

  17. Association of serum Epstein-Barr nuclear antigen-1 antibodies and intrathecal immunoglobulin synthesis in early multiple sclerosis.

    Science.gov (United States)

    Pfuhl, Catherina; Oechtering, Johanna; Rasche, Ludwig; Gieß, René M; Behrens, Janina R; Wakonig, Katharina; Freitag, Erik; Pache, Florence C; Otto, Carolin; Hofmann, Jörg; Eberspächer, Bettina; Bellmann-Strobl, Judith; Paul, Friedemann; Ruprecht, Klemens

    2015-08-15

    Multiple sclerosis (MS) is associated with Epstein-Barr virus (EBV) infection. A characteristic feature of MS is an intrathecal synthesis of immunoglobulin (Ig)G. In 90 patients with clinically isolated syndromes/early relapsing-remitting MS, serum antibodies to Epstein-Barr nuclear antigen-1, but not to EBV viral capsid antigen, rubella, or varicella zoster virus, were higher (p=0.03) in those with than those without a calculated intrathecal IgG synthesis >0% and correlated with the percentage (r=0.27, p=0.009) and concentration (r=0.27, p=0.012) of intrathecally produced IgG. These findings suggest a link between EBV infection and the events leading to intrathecal IgG synthesis in patients with MS. PMID:26198934

  18. CD4+ T cell-mediated presentation of non-infectious HIV-1virion antigens to HIV-specific CD8+ T cells

    Institute of Scientific and Technical Information of China (English)

    XU Jian-qing; Franco Lori; Julianna Lisziewicz

    2006-01-01

    Background The mechanism of chronic immune activation and impairment of HIV-specific immune responses during chronic infection is not fully understood. However, it is known that high immune activation leads to more rapid progression to AIDS. We hypothesize that CD4+ T cell-mediated viral antigen presentation contributes to this pathologic immune activation in HIV-infected individuals.Methods HIV-specific T cells, responding to noninfectious HIV-1 virions as antigen, were measured by flow cytometric assays. These experimental conditions reflect the in vivo condition where noninfectious HIV-1 represents more than 99% of the antigens.Results CD4+ T cells purified from HIV-infected individuals were capable of cross presenting exogenous noninfectious HIV-1 virions to HIV-1-specific CD8+ T cells. Cross presentation required the entry of HIV-1 to CD4+ T cells and antigen translocation from endoplasmic reticulum to the Golgi complex. Blocking CD4+mediated activation of HIV-specific CD8+ T cells and redirecting the viral antigens to antigen presenting cells improved HIV-specific T cell responses.Conclusions One possible cause of chronic immune activation and impairment of HIV-1 specific T cell responses is represented by HIV-1 harboring CD4+ T cells cross presenting HIV-1 antigen to activate CD8+ T cells. This new mechanism provides the first evidence that cross presentation of noninfectious HIV-1. Virions play a role in the immunopathogenesis of HIV-1 infection.

  19. Plasmodium berghei: immunosuppression of the cell-mediated immune response induced by nonviable antigenic preparations

    International Nuclear Information System (INIS)

    In this work, plasmodial antigens were examined for their ability to suppress the cellular immune response during lethal Plasmodium berghei infection. Splenic enlargement and the number and function of white spleen cells were assessed after injection of normal mice with irradiated parasitized erythrocytes (IPE) or with parasitized erythrocytes (PE) membranes. Both IPE and PE membranes caused splenomegaly and an increase in the number of splenic white cells with concurrent alteration of the relative proportions of T cells and macrophages. The percentage of T lymphocytes was fractionally diminished, but there was a marked increase in Lyt 2.2 positive (suppressor and cytotoxic) T subsets and in the number of splenic macrophage precursors. The pathological enlargement of the spleen was induced by various plasma membrane-derived antigens containing both proteins and carbohydrates. Splenocytes of mice injected with liposomes containing deoxycholate-treated PE or PE fractions showed both diminished interleukin 2 production and a decreased response to mitogen. It appears that some of the changes in the cellular immune response during P. berghei infection are a consequence of the massive provision of a wide spectrum of antigens, capable of suppressing the immune response. Thus, it may be appropriate to evaluate the possible negative effect of parasite epitopes that are candidates for vaccine

  20. Dynamic visualization of dendritic cell-antigen interactions in the skin following transcutaneous immunization.

    Directory of Open Access Journals (Sweden)

    Teerawan Rattanapak

    Full Text Available Delivery of vaccines into the skin provides many advantages over traditional parenteral vaccination and is a promising approach due to the abundance of antigen presenting cells (APC residing in the skin including Langerhans cells (LC and dermal dendritic cells (DDC. However, the main obstacle for transcutaneous immunization (TCI is the effective delivery of the vaccine through the stratum corneum (SC barrier to the APC in the deeper skin layers. This study therefore utilized microneedles (MN and a lipid-based colloidal delivery system (cubosomes as a synergistic approach for the delivery of vaccines to APC in the skin. The process of vaccine uptake and recruitment by specific types of skin APC was investigated in real-time over 4 hours in B6.Cg-Tg (Itgax-EYFP 1 Mnz/J mice by two-photon microscopy. Incorporation of the vaccine into a particulate delivery system and the use of MN preferentially increased vaccine antigen uptake by a highly motile subpopulation of skin APC known as CD207⁺ DC. No uptake of antigen or any response to immunisation by LC could be detected.

  1. Plasmodium berghei: immunosuppression of the cell-mediated immune response induced by nonviable antigenic preparations

    Energy Technology Data Exchange (ETDEWEB)

    Gross, A.; Frankenburg, S.

    1989-01-01

    In this work, plasmodial antigens were examined for their ability to suppress the cellular immune response during lethal Plasmodium berghei infection. Splenic enlargement and the number and function of white spleen cells were assessed after injection of normal mice with irradiated parasitized erythrocytes (IPE) or with parasitized erythrocytes (PE) membranes. Both IPE and PE membranes caused splenomegaly and an increase in the number of splenic white cells with concurrent alteration of the relative proportions of T cells and macrophages. The percentage of T lymphocytes was fractionally diminished, but there was a marked increase in Lyt 2.2 positive (suppressor and cytotoxic) T subsets and in the number of splenic macrophage precursors. The pathological enlargement of the spleen was induced by various plasma membrane-derived antigens containing both proteins and carbohydrates. Splenocytes of mice injected with liposomes containing deoxycholate-treated PE or PE fractions showed both diminished interleukin 2 production and a decreased response to mitogen. It appears that some of the changes in the cellular immune response during P. berghei infection are a consequence of the massive provision of a wide spectrum of antigens, capable of suppressing the immune response. Thus, it may be appropriate to evaluate the possible negative effect of parasite epitopes that are candidates for vaccine.

  2. Scaffolded Antigens in Yeast Cell Particle Vaccines Provide Protection against Systemic Polyoma Virus Infection.

    Science.gov (United States)

    Tipper, Donald J; Szomolanyi-Tsuda, Eva

    2016-01-01

    Background. U65, a self-aggregating peptide scaffold, traps fused protein antigens in yeast cells. Conversion to Yeast Cell Particle (YCP) vaccines by partial removal of surface mannoproteins exposes β-glucan, mediating efficient uptake by antigen-presenting cells (APCs). YCP vaccines are inexpensive, capable of rapid large-scale production and have potential for both parenteral and oral use. Results. YCP processing by alkaline hydrolysis exposes up to 20% of the glucan but converts scaffolded antigen and internal yeast proteins into a common aggregate, preventing selective yeast protein removal. For U65-green fluorescent protein (GFP) or U65-Apolipoprotein A1 (ApoA1) subcutaneous vaccines, maximal IgG responses in mice required 10% glucan exposure. IgG responses to yeast proteins were 5-fold lower. Proteolytic mannoprotein removal produced YCPs with only 6% glucan exposure, insufficiently porous for selective removal of even native yeast proteins. Vaccine efficacy was reduced 10-fold. Current YCP formulations, therefore, are not suitable for human use but have considerable potential for use in feed animal vaccines. Significantly, a YCP vaccine expressing a GFP fusion to VP1, the murine polyoma virus major capsid protein, after either oral or subcutaneous administration, protected mice against an intraperitoneal polyoma virus challenge, reducing viral DNA levels in spleen and liver by >98%. PMID:27213160

  3. Scaffolded Antigens in Yeast Cell Particle Vaccines Provide Protection against Systemic Polyoma Virus Infection

    Directory of Open Access Journals (Sweden)

    Donald J. Tipper

    2016-01-01

    Full Text Available Background. U65, a self-aggregating peptide scaffold, traps fused protein antigens in yeast cells. Conversion to Yeast Cell Particle (YCP vaccines by partial removal of surface mannoproteins exposes β-glucan, mediating efficient uptake by antigen-presenting cells (APCs. YCP vaccines are inexpensive, capable of rapid large-scale production and have potential for both parenteral and oral use. Results. YCP processing by alkaline hydrolysis exposes up to 20% of the glucan but converts scaffolded antigen and internal yeast proteins into a common aggregate, preventing selective yeast protein removal. For U65-green fluorescent protein (GFP or U65-Apolipoprotein A1 (ApoA1 subcutaneous vaccines, maximal IgG responses in mice required 10% glucan exposure. IgG responses to yeast proteins were 5-fold lower. Proteolytic mannoprotein removal produced YCPs with only 6% glucan exposure, insufficiently porous for selective removal of even native yeast proteins. Vaccine efficacy was reduced 10-fold. Current YCP formulations, therefore, are not suitable for human use but have considerable potential for use in feed animal vaccines. Significantly, a YCP vaccine expressing a GFP fusion to VP1, the murine polyoma virus major capsid protein, after either oral or subcutaneous administration, protected mice against an intraperitoneal polyoma virus challenge, reducing viral DNA levels in spleen and liver by >98%.

  4. Scaffolded Antigens in Yeast Cell Particle Vaccines Provide Protection against Systemic Polyoma Virus Infection

    Science.gov (United States)

    Tipper, Donald J.; Szomolanyi-Tsuda, Eva

    2016-01-01

    Background. U65, a self-aggregating peptide scaffold, traps fused protein antigens in yeast cells. Conversion to Yeast Cell Particle (YCP) vaccines by partial removal of surface mannoproteins exposes β-glucan, mediating efficient uptake by antigen-presenting cells (APCs). YCP vaccines are inexpensive, capable of rapid large-scale production and have potential for both parenteral and oral use. Results. YCP processing by alkaline hydrolysis exposes up to 20% of the glucan but converts scaffolded antigen and internal yeast proteins into a common aggregate, preventing selective yeast protein removal. For U65-green fluorescent protein (GFP) or U65-Apolipoprotein A1 (ApoA1) subcutaneous vaccines, maximal IgG responses in mice required 10% glucan exposure. IgG responses to yeast proteins were 5-fold lower. Proteolytic mannoprotein removal produced YCPs with only 6% glucan exposure, insufficiently porous for selective removal of even native yeast proteins. Vaccine efficacy was reduced 10-fold. Current YCP formulations, therefore, are not suitable for human use but have considerable potential for use in feed animal vaccines. Significantly, a YCP vaccine expressing a GFP fusion to VP1, the murine polyoma virus major capsid protein, after either oral or subcutaneous administration, protected mice against an intraperitoneal polyoma virus challenge, reducing viral DNA levels in spleen and liver by >98%.

  5. Changes in tumor-antigen expression profile as human small-cell lung cancers progress

    International Nuclear Information System (INIS)

    Our group has previously observed that in patients with small-cell lung cancers (SCLCs), the expression of a tumor antigen, glioma big potassium (gBK) ion channel, is higher at the time of death than when the cancer is first treated by surgical resection. This study aimed to determine whether this dichotomy was common in other potential lung tumor antigens by examining the same patient samples using our more extensive profile analysis of tumor-antigen precursor protein (TAPP). We then tested the hypothesis that therapeutic intervention may inadvertently cause this increased gBK production. SCLC samples (eight surgical resections and three autopsy samples) and three control lungs were examined by quantitative real-time polymerase chain reaction for 42 potential TAPPs that represent potential T-cell-mediated immunological targets. Twenty-two TAPP mRNAs displayed the same profile as gBK, i.e., more mRNAs were expressed at autopsy than in their surgical counterparts. B-cyclin and mouse double minute 2, human homolog of P53-binding protein were elevated in both autopsy and surgical specimens above the normal-lung controls. When HTB119 cells were incubated with doxorubicin, gBK was strongly induced, as confirmed by intracellular flow cytometry with a gBK-specific antibody. Our findings suggested that more immunological targets became available as the tumor responded to chemotherapy and proceeded toward its terminal stages

  6. Identifying Patient-Specific Epstein-Barr Nuclear Antigen-1 Genetic Variation and Potential Autoreactive Targets Relevant to Multiple Sclerosis Pathogenesis.

    Directory of Open Access Journals (Sweden)

    Monika Tschochner

    Full Text Available Epstein-Barr virus (EBV infection represents a major environmental risk factor for multiple sclerosis (MS, with evidence of selective expansion of Epstein-Barr Nuclear Antigen-1 (EBNA1-specific CD4+ T cells that cross-recognize MS-associated myelin antigens in MS patients. HLA-DRB1*15-restricted antigen presentation also appears to determine susceptibility given its role as a dominant risk allele. In this study, we have utilised standard and next-generation sequencing techniques to investigate EBNA-1 sequence variation and its relationship to HLA-DR15 binding affinity, as well as examining potential cross-reactive immune targets within the central nervous system proteome.Sanger sequencing was performed on DNA isolated from peripheral blood samples from 73 Western Australian MS cases, without requirement for primary culture, with additional FLX 454 Roche sequencing in 23 samples to identify low-frequency variants. Patient-derived viral sequences were used to predict HLA-DRB1*1501 epitopes (NetMHCII, NetMHCIIpan and candidates were evaluated for cross recognition with human brain proteins.EBNA-1 sequence variation was limited, with no evidence of multiple viral strains and only low levels of variation identified by FLX technology (8.3% nucleotide positions at a 1% cut-off. In silico epitope mapping revealed two known HLA-DRB1*1501-restricted epitopes ('AEG': aa 481-496 and 'MVF': aa 562-577, and two putative epitopes between positions 502-543. We identified potential cross-reactive targets involving a number of major myelin antigens including experimentally confirmed HLA-DRB1*15-restricted epitopes as well as novel candidate antigens within myelin and paranodal assembly proteins that may be relevant to MS pathogenesis.This study demonstrates the feasibility of obtaining autologous EBNA-1 sequences directly from buffy coat samples, and confirms divergence of these sequences from standard laboratory strains. This approach has identified a number of

  7. Overnight resting of PBMC changes functional signatures of antigen specific T- cell responses: impact for immune monitoring within clinical trials.

    Directory of Open Access Journals (Sweden)

    Sarah Kutscher

    Full Text Available Polyfunctional CD4 or CD8 T cells are proposed to represent a correlate of immune control for persistent viruses as well as for vaccine mediated protection against infection. A well-suited methodology to study complex functional phenotypes of antiviral T cells is the combined staining of intracellular cytokines and phenotypic marker expression using polychromatic flow cytometry. In this study we analyzed the effect of an overnight resting period at 37 °C on the quantity and functionality of HIV-1, EBV, CMV, HBV and HCV specific CD4 and CD8 T-cell responses in a cohort of 21 individuals. We quantified total antigen specific T cells by multimer staining and used 10-color intracellular cytokine staining (ICS to determine IFNγ, TNFα, IL2 and MIP1β production. After an overnight resting significantly higher numbers of functionally active T cells were detectable by ICS for all tested antigen specificities, whereas the total number of antigen specific T cells determined by multimer staining remained unchanged. Overnight resting shifted the quality of T-cell responses towards polyfunctionality and increased antigen sensitivity of T cells. Our data suggest that the observed effect is mediated by T cells rather than by antigen presenting cells. We conclude that overnight resting of PBMC prior to ex vivo analysis of antiviral T-cell responses represents an efficient method to increase sensitivity of ICS-based methods and has a prominent impact on the functional phenotype of T cells.

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

    Science.gov (United States)

    Parker, Jennifer Janell

    Radiation therapy is a widely used cancer treatment that has the potential to influence anti-tumor immune responses. Both myeloablative and non-myeloablative radiation are often used as part of preparatory regimens for hematopoetic stem cell transplantation, in combination with other chemotherapy or immuno-modulatory (e.g. Anti-thymocyte globulin (ATG)) therapies for both cytotoxic and immune modulatory purposes. However, the mechanisms responsible for the effect of radiation on antigen presenting cell (APC) responsiveness and radioresistance are poorly understood. The first studies described in this thesis were designed to identify and characterize early radiation-induced signaling changes in antigen presenting cells and to determine the effects of these signaling changes on APC receptor expression and function. The NFkappaB pathway in antigen presenting cells was chosen for study because it is activated by radiation in a wide range of other cell types and plays a vital role in the maintenance and regulation of the immune system. The effects of therapeutically relevant doses radiation (2 and 20 Gy) were compared at various timepoints in the human monocytic cell line (U937) using phospho-flow cytometry staining methods and cytometric analysis. These studies demonstrated that radiation-induced changes in the phosphorylation state of NFkappaB family members that were p53 independent. However, these changes were dependent upon activation of ATM in response to single or double-stranded breaks in DNA, as shown in experiments using an inhibitor of ATM and ATM siRNA knockdown U937 cells. In addition, studies examining the effect of radiation on co-stimulatory receptors with and without inhibition of the NFkappaB pathway via phospho-flow cytometry revealed that radiation-induced phosphorylation of NEMO promoted the activation and functional maturation of U937 cells. Furthermore, functional studies using both phospho-flow cytometry and/or mixed lymphocyte reactions to

  9. Current status and regulatory perspective of chimeric antigen receptor-modified T cell therapeutics.

    Science.gov (United States)

    Kim, Mi-Gyeong; Kim, Dongyoon; Suh, Soo-Kyung; Park, Zewon; Choi, Min Joung; Oh, Yu-Kyoung

    2016-04-01

    Chimeric antigen receptor-modified T cells (CAR-T) have emerged as a new modality for cancer immunotherapy due to their potent efficacy against terminal cancers. CAR-Ts are reported to exert higher efficacy than monoclonal antibodies and antibody-drug conjugates, and act via mechanisms distinct from T cell receptor-engineered T cells. These cells are constructed by transducing genes encoding fusion proteins of cancer antigen-recognizing single-chain Fv linked to intracellular signaling domains of T cell receptors. CAR-Ts are classified as first-, second- and third-generation, depending on the intracellular signaling domain number of T cell receptors. This review covers the current status of CAR-T research, including basic proof-of-concept investigations at the cell and animal levels. Currently ongoing clinical trials of CAR-T worldwide are additionally discussed. Owing to the lack of existing approved products, several unresolved concerns remain with regard to safety, efficacy and manufacturing of CAR-T, as well as quality control issues. In particular, the cytokine release syndrome is the major side-effect impeding the successful development of CAR-T in clinical trials. Here, we have addressed the challenges and regulatory perspectives of CAR-T therapy. PMID:26895243

  10. 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.

  11. Cinnamon extract suppresses experimental colitis through modulation of antigen-presenting cells

    Institute of Scientific and Technical Information of China (English)

    Ho-Keun Kwon; Zee Yong Park; Sin-Hyeog Im; Ji-Sun Hwang; Choong-Gu Lee; Jae-Seon So; Anupama Sahoo; Chang-Rok Im; Won Kyung Jeon; Byoung Seob Ko; Sung Haeng Lee

    2011-01-01

    AIM:To investigate the anti-inflammatory effects of cinnamon extract and elucidate its mechanisms for targeting the function of antigen presenting cells. METHODS:Cinnamon extract was used to treat murine macrophage cell line (Raw 264.7),mouse primary antigen-presenting cells (APCs,MHCII+) and CD11c+ dendritic cells to analyze the effects of cinnamon extract on APC function.The mechanisms of action of cinnamon extract on APCs were investigated by analyzing cytokine production,and expression of MHC antigens and co-stimulatory molecules by quantitative real-time PCR and flow cytometry.In addition,the effect of cinnamon extract on antigen presentation capacity and APC-dependent T-cell differentiation were analyzed by [H3]-thymidine incorporation and cytokine analysis,respectively. To confirm the anti-inflammatory effects of cinnamon extract in vivo ,cinnamon or PBS was orally administered to mice for 20 d followed by induction of experimental colitis with 2,4,6 trinitrobenzenesulfonic acid.The protective effects of cinnamon extract against experimental colitis were measured by checking clinical symptoms,histological analysis and cytokine expression profiles in inflamed tissue. RESULTS:Treatment with cinnamon extract inhibited maturation of MHCII+ APCs or CD11c+ dendritic cells (DCs) by suppressing expression of co-stimulatory molecules (B7.1,B7.2,ICOS-L),MHCII and cyclooxygenase (COX)-2.Cinnamon extract induced regulatory DCs (rDCs) that produce low levels of pro-inflammatory cytokines [interleukin (IL)-1β,IL-6,IL-12,interferon (IFN)-γ and tumor necrosis factor (TNF)-α] while expressing high levels of immunoregulatory cytokines (IL-10 and transforming growth factor-β).In addition, rDCs generated by cinnamon extract inhibited APC-dependent T-cell proliferation,and converted CD4+ T cells into IL-10high CD4+ T cells.Furthermore,oral administration of cinnamon extract inhibited development and progression of intestinal colitis by inhibiting expression of COX-2 and pro

  12. Generation of competent bone marrow-derived antigen presenting cells from the deer mouse (Peromyscus maniculatus

    Directory of Open Access Journals (Sweden)

    Farrell Regina M

    2004-09-01

    Full Text Available Abstract Background Human infections with Sin Nombre virus (SNV and related New World hantaviruses often lead to hantavirus cardiopulmonary syndrome (HCPS, a sometimes fatal illness. Lungs of patients who die from HCPS exhibit cytokine-producing mononuclear infiltrates and pronounced pulmonary inflammation. Deer mice (Peromyscus maniculatus are the principal natural hosts of SNV, in which the virus establishes life-long persistence without conspicuous pathology. Little is known about the mechanisms SNV employs to evade the immune response of deer mice, and experimental examination of this question has been difficult because of a lack of methodologies for examining such responses during infection. One such deficiency is our inability to characterize T cell responses because susceptible syngeneic deer mice are not available. Results To solve this problem, we have developed an in vitro method of expanding and generating competent antigen presenting cells (APC from deer mouse bone marrow using commercially-available house mouse (Mus musculus granulocyte-macrophage colony stimulating factor. These cells are capable of processing and presenting soluble protein to antigen-specific autologous helper T cells in vitro. Inclusion of antigen-specific deer mouse antibody augments T cell stimulation, presumably through Fc receptor-mediated endocytosis. Conclusions The use of these APC has allowed us to dramatically expand deer mouse helper T cells in culture and should permit extensive characterization of T cell epitopes. Considering the evolutionary divergence between deer mice and house mice, it is probable that this method will be useful to other investigators using unconventional models of rodent-borne diseases.

  13. Circulating HIV-Specific Interleukin-21(+)CD4(+) T Cells Represent Peripheral Tfh Cells with Antigen-Dependent Helper Functions.

    Science.gov (United States)

    Schultz, Bruce T; Teigler, Jeffrey E; Pissani, Franco; Oster, Alexander F; Kranias, Gregory; Alter, Galit; Marovich, Mary; Eller, Michael A; Dittmer, Ulf; Robb, Merlin L; Kim, Jerome H; Michael, Nelson L; Bolton, Diane; Streeck, Hendrik

    2016-01-19

    A central effort in HIV vaccine development is to generate protective broadly neutralizing antibodies, a process dependent on T follicular helper (Tfh) cells. The feasibility of using peripheral blood counterparts of lymph node Tfh cells to assess the immune response and the influence of viral and vaccine antigens on their helper functions remain obscure. We assessed circulating HIV-specific IL-21(+)CD4(+) T cells and showed transcriptional and phenotypic similarities to lymphoid Tfh cells, and hence representing peripheral Tfh (pTfh) cells. pTfh cells were functionally active and B cell helper quality differed depending on antigen specificity. Furthermore, we found higher frequency of pTfh cells in peripheral blood mononuclear cell specimens from the ALVAC+AIDSVAX (RV144) HIV vaccine trial associated with protective antibody responses compared to the non-protective DNA+Ad5 vaccine trial. Together, we identify IL-21(+)CD4(+) T cells as pTfh cells, implicating them as key populations in the generation of vaccine-evoked antibody responses. PMID:26795249

  14. Rapid and sustained CD4(+) T-cell-independent immunity from adenovirus-encoded vaccine antigens

    DEFF Research Database (Denmark)

    Holst, Peter J; Bartholdy, Christina; Buus, Anette Stryhn;

    2007-01-01

    absence of CD4(+) T-cell help were sustained in the long term and able to expand and control a secondary challenge with LCMV. Our results demonstrate that modifications to the antigen used in adenovirus vaccines may be used to improve the induced T-cell response. Such a strategy for CD4(+) T-cell...... elicited with an adenovirus-encoded minimal epitope covalently linked to beta(2)-microglobulin. We demonstrate that the beta(2)-microglobulin-linked epitope induced an accelerated and augmented CD8(+) T-cell response. Furthermore, the immunity conferred by vaccination with beta(2)-microglobulin......-linked lymphocytic choriomeningitis virus (LCMV)-derived epitopes was long-lived and protective. Notably, in contrast to full-length protein, the response elicited with the beta(2)-microglobulin-linked LCMV-derived epitope was CD4(+) T-cell independent. Furthermore, virus-specific CD8(+) T cells primed in the...

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    Cancer-germline antigens are promising targets for cancer immunotherapy, but whether such therapies will also eliminate the primary tumor stem cell population remains undetermined. We previously showed that long-term cultures of telomerized adult human bone marrow mesenchymal stem cells can...... modulators, makes them promising targets for immunotherapeutic approaches to cancer treatment....... spontaneously evolve into tumor-initiating, mesenchymal stem cells (hMSC-TERT20), which have characteristics of clinical sarcoma cells. In this study, we used the hMSC-TERT20 tumor stem cell model to investigate the potential of cancer-germline antigens to serve as tumor stem cell targets. We found that...

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

    Science.gov (United States)

    Gjerstorff, Morten; Burns, Jorge S; Nielsen, Ole; Kassem, Moustapha; Ditzel, Henrik

    2009-07-01

    Cancer-germline antigens are promising targets for cancer immunotherapy, but whether such therapies will also eliminate the primary tumor stem cell population remains undetermined. We previously showed that long-term cultures of telomerized adult human bone marrow mesenchymal stem cells can spontaneously evolve into tumor-initiating, mesenchymal stem cells (hMSC-TERT20), which have characteristics of clinical sarcoma cells. In this study, we used the hMSC-TERT20 tumor stem cell model to investigate the potential of cancer-germline antigens to serve as tumor stem cell targets. We found that tumorigenic transformation of hMSC-TERT20 cells induced the expression of members of several cancer-germline antigen gene families (ie, GAGE, MAGE-A, and XAGE-1), with promoter hypomethylation and histone acetylation of the corresponding genes. Both in vitro cultures and tumor xenografts derived from tumorigenic hMSC-TERT20 single cell subclones exhibited heterogeneous expression of both GAGE and MAGE-A proteins, and similar patterns of expression were observed in clinical sarcomas. Importantly, histone deacetylase and DNA methyltransferase inhibitors were able to induce more ubiquitous expression levels of cancer-germline antigens in hMSC-TERT20 cells, while their expression levels in primary human mesenchymal stem cells remained unaffected. The expression pattern of cancer-germline antigens in tumorigenic mesenchymal stem cells and sarcomas, plus their susceptibility to enhancement by epigenetic modulators, makes them promising targets for immunotherapeutic approaches to cancer treatment. PMID:19498007

  17. Replication of Chinese hamster embryo cells transformed by temperature-sensitive T-antigen mutants of simian virus 40.

    OpenAIRE

    Robinson, C C; Swartzendruber, D E; Lehman, J M

    1980-01-01

    Chinese hamster embryo cells transformed by simian virus 40 temperature-sensitive T-antigen mutants replicated when confluent at 40.5 degrees C, regardless of the selection method, selection temperature, or virus strain used.

  18. Identification of a novel SEREX antigen family, ECSA, in esophageal squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Murakami Akihiro

    2011-06-01

    Full Text Available Abstract Background Diagnosis of esophageal squamous cell carcinoma (SCC may improve with early diagnosis. Currently it is difficult to diagnose SCC in the early stage because there is a limited number of tumor markers available. Results Fifty-two esophageal SCC SEREX antigens were identified by SEREX (serological identification of antigens by recombinant cDNA expression cloning using a cDNA phage library and sera of patients with esophageal SCC. Sequence analysis revealed that three of these antigens were similar in amino acid sequences, and they were designated as ECSA (esophageal carcinoma SEREX antigen-1, -2 and -3. The ECSA family was also similar to an EST clone, hepatocellular carcinoma-associated antigen 25a (HCA25a. Serum antibody levels to ECSA-1, -2 and -3 were significantly higher in patients with esophageal SCC than in healthy donors. Based on the conserved amino acid sequences, three peptides were synthesized and used for enzyme-linked immunosorbent assays (ELISA. The serum antibody levels against one of these peptides were significantly higher in patients with esophageal SCC. This peptide sequence was also conserved in FAM119A, GOSR1 and BBS5, suggesting that these are also ECSA family members. Reverse transcription followed by quantitative PCR analysis showed that the mRNA expression levels of ECSA-1, -2 and -3 and FAM119A but not of HCA25a, GOSR1 and BBS5 were frequently elevated in esophageal SCC tissues. Conclusions We have identified a new gene family designated ECSA. Serum antibodies against the conserved domain of the ECSA family may be a promising tumor marker for esophageal SCC.

  19. Migratory dendritic cells transfer antigen to a lymph node-resident dendritic cell population for efficient CTL priming.

    Science.gov (United States)

    Allan, Rhys S; Waithman, Jason; Bedoui, Sammy; Jones, Claerwen M; Villadangos, Jose A; Zhan, Yifan; Lew, Andrew M; Shortman, Ken; Heath, William R; Carbone, Francis R

    2006-07-01

    Skin dendritic cells (DCs) are thought to act as key initiators of local T cell immunity. Here we show that after skin infection with herpes simplex virus (HSV), cytotoxic T lymphocyte (CTL) activation required MHC class I-restricted presentation by nonmigratory CD8(+) DCs rather than skin-derived DCs. Despite a lack of direct presentation by migratory DCs, blocking their egress from infected skin substantially inhibited class I-restricted presentation and HSV-specific CTL responses. These results support the argument for initial transport of antigen by migrating DCs, followed by its transfer to the lymphoid-resident DCs for presentation and CTL priming. Given that relatively robust CTL responses were seen with small numbers of skin-emigrant DCs, we propose that this inter-DC antigen transfer functions to amplify presentation across a larger network of lymphoid-resident DCs for efficient T cell activation. PMID:16860764

  20. Amiloride enhances antigen specific CTL by faciliting HBV DNA vaccine entry into cells.

    Directory of Open Access Journals (Sweden)

    Shuang Geng

    Full Text Available The induction of relatively weak immunity by DNA vaccines in humans can be largely attributed to the low efficiency of transduction of somatic cells. Although formulation with liposomes has been shown to enhance DNA transduction of cultured cells, little, if any, effect is observed on the transduction of somatic tissues and cells. To improve the rate of transduction, DNA vaccine delivery by gene gun and the recently developed electroporation techniques have been employed. We report here that to circumvent requirement for such equipment, amiloride, a drug that is prescribed for hypertension treatment, can accelerate plasmid entry into antigen presenting cells (APCs both in vitro and in vivo. The combination induced APCs more dramatically in both maturation and cytokine secretion. Amiloride enhanced development of full CD8 cytolytic function including induction of high levels of antigen specific CTL and expression of IFN-γ+perforin+granzymeB+ in CD8+ T cells. Thus, amiloride is a facilitator for DNA transduction into host cells which in turn enhances the efficiency of the immune responses.

  1. Role of the H-2 complex in the induction of T cell tolerance to self minor histocompatibility antigens

    OpenAIRE

    1983-01-01

    The present study has utilized cytotoxic T lymphocyte (CTL) responses specific for minor histocompatibility (minor H) antigens as an experimental approach to determining whether recognition of self MHC determinants is involved in the induction of T cell tolerance to self antigens. It was observed that C3H.SW splenic T cells from C3H.SW leads to B10 X B10.BR radiation bone marrow chimeras contained CTL precursors (pCTL) reactive against self C3H minor H antigens + H-2k but were tolerant to sel...

  2. Antigen availability determines CD8⁺ T cell-dendritic cell interaction kinetics and memory fate decisions.

    Science.gov (United States)

    Henrickson, Sarah E; Perro, Mario; Loughhead, Scott M; Senman, Balimkiz; Stutte, Susanne; Quigley, Michael; Alexe, Gabriela; Iannacone, Matteo; Flynn, Michael P; Omid, Shaida; Jesneck, Jonathan L; Imam, Sabrina; Mempel, Thorsten R; Mazo, Irina B; Haining, W Nicholas; von Andrian, Ulrich H

    2013-09-19

    T cells are activated by antigen (Ag)-bearing dendritic cells (DCs) in lymph nodes in three phases. The duration of the initial phase of transient, serial DC-T cell interactions is inversely correlated with Ag dose. The second phase, characterized by stable DC-T cell contacts, is believed to be necessary for full-fledged T cell activation. Here we have shown that this is not the case. CD8⁺ T cells interacting with DCs presenting low-dose, short-lived Ag did not transition to phase 2, whereas higher Ag dose yielded phase 2 transition. Both antigenic constellations promoted T cell proliferation and effector differentiation but yielded different transcriptome signatures at 12 hr and 24 hr. T cells that experienced phase 2 developed long-lived memory, whereas conditions without stable contacts yielded immunological amnesia. Thus, T cells make fate decisions within hours after Ag exposure, resulting in long-term memory or abortive effector responses, correlating with T cell-DCs interaction kinetics. PMID:24054328

  3. Seoul virus suppresses NF-κB-mediated inflammatory responses of antigen presenting cells from Norway rats

    OpenAIRE

    Au, Rebecca Y.; Jedlicka, Anne E.; Li, Wei; Pekosz, Andrew; Klein, Sabra L.

    2010-01-01

    Hantavirus infection reduces antiviral defenses, increases regulatory responses, and causes persistent infection in rodent hosts. To address whether hantaviruses alter the maturation and functional activity of antigen presenting cells (APCs), rat bone marrow-derived dendritic cells (BMDCs) and macrophages (BMDMs) were generated and infected with Seoul virus (SEOV) or stimulated with TLR ligands. SEOV infected both DCs and macrophages, but copies of viral RNA, viral antigen, and infectious vir...

  4. Tritium (3H) radiolabeling of protein A and antibody to high specific activity: Application to cell surface antigen radioimmunoassays

    International Nuclear Information System (INIS)

    Staphylococcal protein A and several different immunoglobulins have been radiolabeled to high specific activities (> 106 cpm/μg) by reductive methylation with tritiated (3H) sodium borohydride. The proteins retain excellent functional and antigenic properties. The utility of these reagents in a variety of assays for cell surface antigens is illustrated. The results indicate that this radiolabeling procedure may become the method of choice for many cell surface and solution immunoassays. (Auth.)

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

    OpenAIRE

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

    2013-01-01

    T cells can be redirected to overcome tolerance to cancer by engineering with integrating vectors to express a chimeric antigen receptor (CAR). In preclinical models, we have previously demonstrated that transfection of T cells with messenger RNA (mRNA) coding for a CAR is an alternative strategy that has antitumor efficacy and the potential to evaluate the on-target off-tumor toxicity of new CAR targets safely due to transient mRNA CAR expression. Here, we report the safety observed in four ...

  6. Tumorigenic activity of Merkel cell polyomavirus T antigens expressed in the stratified epithelium of mice

    OpenAIRE

    Spurgeon, Megan E.; Cheng, Jingwei; Bronson, Roderick T.; Lambert, Paul F.; DeCaprio, James A.

    2015-01-01

    Merkel cell polyomavirus (MCPyV) is frequently associated with Merkel cell carcinoma (MCC), a highly aggressive neuroendocrine skin cancer. Most MCC tumors contain integrated copies of the viral genome with persistent expression of the MCPyV large T (LT) and small T (ST) antigen. MCPyV isolated from MCC typically contain wild type ST but truncated forms of LT that retain the N-terminus but delete the C-terminus and render LT incapable of supporting virus replication. To determine the oncogeni...

  7. Activation/Inhibition of mast cells by supra-optimal antigen concentrations

    OpenAIRE

    Huber, Michael

    2013-01-01

    Mast cells (MCs) are tissue resident cells of hemopoietic origin and are critically involved in allergic diseases. MCs bind IgE by means of their high-affinity receptor for IgE (FcεRI). The FcεRI belongs to a family of multi-chain immune recognition receptors and is activated by cross-linking in response to multivalent antigens (Ags)/allergens. Activation of the FcεRI results in immediate release of preformed granular substances (e.g. histamine, heparin, and proteases), generation of arachido...

  8. Engineering a prostate-specific membrane antigen-activated tumor endothelial cell prodrug for cancer therapy

    DEFF Research Database (Denmark)

    Denmeade, Samuel R; Mhaka, Annastasiah M; Rosen, D Marc;

    2012-01-01

    Heterogeneous expression of drug target proteins within tumor sites is a major mechanism of resistance to anticancer therapies. We describe a strategy to selectively inhibit, within tumor sites, the function of a critical intracellular protein, the sarcoplasmic/endoplasmic reticulum calcium...... adenosine triphosphatase (SERCA) pump, whose proper function is required by all cell types for viability. To achieve targeted inhibition, we took advantage of the unique expression of the carboxypeptidase prostate-specific membrane antigen (PSMA) by tumor endothelial cells within the microenvironment of...

  9. A novel strategy to improve antigen presentation for active immunotherapy in cancer. Fusion of the human papillomavirus type 16 E7 antigen to a cell penetrating peptide

    International Nuclear Information System (INIS)

    Facilitating the delivery of exogenous antigens to antigen-presenting cells, ensuing processing and presentation via the major histocompatibility complex class I and induction of an effective immune response are fundamental for an effective therapeutic cancer vaccine. In this regard, we propose the use of cell-penetrating peptides fused to a tumor antigen. To demonstrate this concept we designed a fusion protein comprising a novel cell-penetrating and immunostimulatory peptide corresponding to residues 32 to 51 of the Limulus anti-lipopolysaccharide factor protein (LALF32-51) linked to human papillomavirus 16 E7 antigen (LALF32-51-E7). In this work, we demonstrated that the immunization with LALF32-51-E7 using the TC-1 mouse model induces a potent and long-lasting anti-tumor response supported on an effective E7-specific CD8+T-cell response. The finding that therapeutic immunization with LALF32-51 or E7 alone, or an admixture of LALF32-51 and E7, does not induce significant tumor reduction indicates that covalent linkage between LALF32-51 and E7 is required for the anti-tumor effect. These results support the use of this novel cell-penetrating peptide as an efficient means for delivering therapeutic targets into cellular compartments with the induction of a cytotoxic CD8+T lymphocyte immune response. This approach is promissory for the treatment of tumors associated with the human papillomavirus 16, which is responsible for the 50% of cervical cancer cases worldwide and other malignancies. Furthermore, protein-based vaccines can circumvent the major histocompatibility complex specificity limitation associated with peptide vaccines providing a greater extent in their application

  10. Pre-clinical evaluation of CD38 chimeric antigen receptor engineered T cells for the treatment of multiple myeloma.

    Science.gov (United States)

    Drent, Esther; Groen, Richard W J; Noort, Willy A; Themeli, Maria; Lammerts van Bueren, Jeroen J; Parren, Paul W H I; Kuball, Jürgen; Sebestyen, Zsolt; Yuan, Huipin; de Bruijn, Joost; van de Donk, Niels W C J; Martens, Anton C M; Lokhorst, Henk M; Mutis, Tuna

    2016-05-01

    Adoptive transfer of chimeric antigen receptor-transduced T cells is a promising strategy for cancer immunotherapy. The CD38 molecule, with its high expression on multiple myeloma cells, appears a suitable target for antibody therapy. Prompted by this, we used three different CD38 antibody sequences to generate second-generation retroviral CD38-chimeric antigen receptor constructs with which we transduced T cells from healthy donors and multiple myeloma patients. We then evaluated the preclinical efficacy and safety of the transduced T cells. Irrespective of the donor and antibody sequence, CD38-chimeric antigen receptor-transduced T cells proliferated, produced inflammatory cytokines and effectively lysed malignant cell lines and primary malignant cells from patients with acute myeloid leukemia and multi-drug resistant multiple myeloma in a cell-dose, and CD38-dependent manner, despite becoming CD38-negative during culture. CD38-chimeric antigen receptor-transduced T cells also displayed significant anti-tumor effects in a xenotransplant model, in which multiple myeloma tumors were grown in a human bone marrow-like microenvironment. CD38-chimeric antigen receptor-transduced T cells also appeared to lyse the CD38(+) fractions of CD34(+) hematopoietic progenitor cells, monocytes, natural killer cells, and to a lesser extent T and B cells but did not inhibit the outgrowth of progenitor cells into various myeloid lineages and, furthermore, were effectively controllable with a caspase-9-based suicide gene. These results signify the potential importance of CD38-chimeric antigen receptor-transduced T cells as therapeutic tools for CD38(+) malignancies and warrant further efforts to diminish the undesired effects of this immunotherapy using appropriate strategies. PMID:26858358

  11. CD80 and CD86 Differentially Regulate Mechanical Interactions of T-Cells with Antigen-Presenting Dendritic Cells and B-Cells

    OpenAIRE

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

    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 ...

  12. Interleukin mRNA changes in mast cells stimulated by TSL-1 antigens

    Directory of Open Access Journals (Sweden)

    Arizmendi N.

    2001-06-01

    Full Text Available In this work we analyzed by RT-PCR, the mRNA changes for IL-4, IL-10, TNF and IFN ( induced by TSL-1 antigens in a rat mast cell line (HRMC with mucosal characteristics. The data obtained showed an increase of 65 and 52 % in mRNA expression for IL-4 and TNF respectively and a decrease of 59 and 55 % in mRNAs for IFNγ and IL-10. Our results suggest that TSL-1 antigens induce the release from MC of regulatory molecules, such as IL-4 by an IgE independent mechanism. Our data also provides important information related to the ability of MC to participate not only in the effector phase against the infectious agents, but also in the orchestration of the immune response by the host against parasites.

  13. Further Exploration of the Dendritic Cell Algorithm: Antigen Multiplier and Time Windows

    CERN Document Server

    Gu, Feng; Aickelin, Uwe

    2010-01-01

    As an immune-inspired algorithm, the Dendritic Cell Algorithm (DCA), produces promising performances in the field of anomaly detection. This paper presents the application of the DCA to a standard data set, the KDD 99 data set. The results of different implementation versions of the DXA, including the antigen multiplier and moving time windows are reported. The real-valued Negative Selection Algorithm (NSA) using constant-sized detectors and the C4.5 decision tree algorithm are used, to conduct a baseline comparison. The results suggest that the DCA is applicable to KDD 99 data set, and the antigen multiplier and moving time windows have the same effect on the DCA for this particular data set. The real-valued NSA with constant-sized detectors is not applicable to the data set, and the C4.5 decision tree algorithm provides a benchmark of the classification performance for this data set.

  14. In Vitro Expression of Hepatitis C Virus Non-structure 5 Antigen in the HepG2 Cell Line

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To establish a cell line as a model system for HCV infection and propagation in vitro, a human HepG2 cell line was incubated with a HCV RNA positive serum. The sABC immunological techniques and gold-labeled colloid electron microscopy method were employed to examine the viral proteins in those ceils. The HCV non-structure 5 antigen was first detected in the HepG2 cells 72 h after incubation. The antigen was continuously observed in the cytoplasm as well on the membrane of the HepG2 cells even after 1, 2, 3 and 4 weeks after incubation. The observation of HCV non-struc ture 5 antigen continuously expressed in the HepG2 cells strongly indicates that the cells may have been infected by HCV virus. Therefore, the HepG2 cell line may serve as a potential host for establishment of HCV infection and propagation in vitro.

  15. Role of very late antigen-1 in T-cell-mediated immunity to systemic viral infection

    DEFF Research Database (Denmark)

    Ørding Kauffmann, Susanne; Thomsen, Allan Randrup; Christensen, Jan Pravsgaard

    2006-01-01

    The T-cell response to lymphocytic choriomeningitis virus was studied in mice lacking very late antigen-1 (VLA-1). The generation of virus-specific effector T cells was unimpaired in VLA-1(-/-) mice. In the memory phase, VLA-1 deficiency did not influence the number of memory CD8(+) T cells or th...... current findings indicate that the expression of VLA-1 is not pivotal for T-cell-mediated antiviral immunity to a systemic infection....... their distribution between lymphoid and nonlymphoid organs. Regarding a functional role of VLA-1, we found that intracerebral infection of both VLA-1(-/-) and wild-type (wt) mice resulted in lethal T-cell-mediated meningitis, and quantitative and qualitative analyses of the cellular exudate did not...

  16. Antigen-specific T8+ human clone of cells with a nonspecific augmenting function on the T4 cell-B cell helper interaction

    International Nuclear Information System (INIS)

    The authors isolated a T8+ T3+ Ia+ clone of cells from the peripheral blood mononuclear cells of a healthy subject. The clone was expanded and maintained with autologous feed cells, interleukin 2, and a streptococcal antigen. The T8+ clone of cells responded specifically to the streptococcal antigen, in the absence of accessory cells,and released a soluble factor. Both the cloned cells and the corresponding soluble factor expressed augmenting helper but not suppressor activity. The augmenting helper activity for B cell antibody synthesis was demonstrable only in the presence of autologous T 4 cells. Radioimmunoassay was used to measure antibodies. Although stimulation of the T8+ cloned cells was antigen-specific, the resulting soluble factor elicited nonspecific antibody synthesis in the presence of T4 and B cells. The T8+ cloned cell-derived factor was adsorbed by B cells but not by T4 cells. Preliminary studies suggest that the factor has the properties of a B cell growth factor. They suggest that the T8+ population consists of functionally heterogeneous cell subsets, some that have suppressor function and others that augment the T4+ helper-inducer activity in B cell antibody synthesis

  17. Prolonged antigen presentation by immune complex-binding dendritic cells programs the proliferative capacity of memory CD8 T cells.

    Science.gov (United States)

    León, Beatriz; Ballesteros-Tato, André; Randall, Troy D; Lund, Frances E

    2014-07-28

    The commitment of naive CD8 T cells to effector or memory cell fates can occur after a single day of antigenic stimulation even though virus-derived antigens (Ags) are still presented by DCs long after acute infection is resolved. However, the effects of extended Ag presentation on CD8 T cells are undefined and the mechanisms that regulate prolonged Ag presentation are unknown. We showed that the sustained presentation of two different epitopes from influenza virus by DCs prevented the premature contraction of the primary virus-specific CD8 T cell response. Although prolonged Ag presentation did not alter the number of memory CD8 T cells that developed, it was essential for programming the capacity of these cells to proliferate, produce cytokines, and protect the host after secondary challenge. Importantly, prolonged Ag presentation by DCs was dependent on virus-specific, isotype-switched antibodies (Abs) that facilitated the capture and cross-presentation of viral Ags by FcγR-expressing DCs. Collectively, our results demonstrate that B cells and Abs can regulate the quality and functionality of a subset of antiviral CD8 T cell memory responses and do so by promoting sustained Ag presentation by DCs during the contraction phase of the primary T cell response. PMID:25002751

  18. Production of antigen-specific suppressive T cell factor by radiation leukemia virus-transformed suppressor T cells

    International Nuclear Information System (INIS)

    Hen egg-white lysozyme-specific suppressor T cells induced in C57BL/6 mice have been selected by sequential passage over plates coated with goat anti-mouse Ig and HEL. These suppressor T cells, 80% I-J+, were infected in vitro with radiation leukemia virus and injected intravenously into sublethally irradiated syngeneic recipients. After 4 to 6 months, 6 out of 20 injected mice developed thymic lymphomas, which were maintained by transplantation into histocompatible hosts and subsequently established as permanent cell lines. Cells of these six thymomas were screened for the presence of Thy 1.2, Lyt 1, Lyt 2, I-J/sup b/, and Ig cell surface antigens by direct or indirect immunofluorescence. One tumor was found to express the expected phenotype of suppressor T cells. High-speed supernatants of extracts obtained from L4 cells were able to induce HEL-specific suppression in a T cell proliferative assay, demonstrating the presence of an antigen-specific suppressive T cell factor

  19. Evidence for a new segregant series of B cell antigens that are encoded in the HLA-D region and that stimulate secondary allogenic proliferative and cytotoxic responses

    OpenAIRE

    1980-01-01

    Five new histocompatibility antigens, designated secondary B cell or (SB) antigens, have been identified by secondary allogeneic proliferative and cytotoxic responses. The reagents used to define the SB antigents are lymphocytes primed between donors matched for all known HLA antigens. The SB antigens stimulate weak primary allogeneic proliferative responses (a mean relative response of 8%) but strong secondary proliferative responses. Strong secondary cell-mediated cytotoxicity is generated ...

  20. Identification of Makorin 1 as a novel SEREX antigen of esophageal squamous cell carcinoma

    International Nuclear Information System (INIS)

    Esophageal squamous cell carcinoma (SCC) represents one of the most malignant tumors. To improve the poor prognosis, it is necessary to diagnose esophageal SCC at early stages using new tumor markers. SEREX (serological identification of antigens by recombinant cDNA expression cloning) is suitable for large-scale screening of tumor antigens and has been applied for various types of human tumors. Tumor markers of esophageal squamous cell carcinoma (SCC) were screened by SEREX method. The presence of serum anti-makorin 1 (MKRN1) antibodies (s-MKRN1-Abs) was examined by Western blotting using bacterially expressed MKRN1 protein. The expression levels of MKRN1 mRNA in tissues were examined by RT-PCR. The biological activity of MKRN1 was examined by transfection of ras-NIH3T3 mouse fibroblasts with MKRN1 cDNA. Major ubiquitinated proteins in MKRN1-transfected cells were identified by immunoprecipitation with anti-ubiquitin antibody followed by mass spectrometry. MKRN1 was identified as a novel SEREX antigen of esophageal SCC. Although a total of 18 (25%) of 73 patients with esophageal SCC had s-MKRN1-Abs, none of the 43 healthy donors had a detectable level of s-MKRN1-Abs. There was no correlation between the presence of s-MKRN1-Abs and clinicopathological variables other than histological grading. Well-differentiated tumors were associated significantly with the presence of s-MKRN1-Abs in the patients. The mRNA levels of MKRN1 were frequently higher in esophageal SCC tissues than in the peripheral normal esophageal mucosa. Stable transfection of ras-NIH3T3 cells with MKRN1 cDNA induced prominent morphological changes such as enlargement of the cell body and spreading. Ubiquitination of 80- and 82-kDa proteins were clearly observed in MKRN1-transfected cells but not in the parental cells, which were identified as L-FILIP (filamin A interacting protein 1). MKRN1 is a novel SEREX antigen of esophageal SCC, and s-NKRN1-Abs can be a candidate of diagnostic markers of

  1. Expanded polyfunctional T cell response to mycobacterial antigens in TB disease and contraction post-treatment.

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    James M Young

    Full Text Available BACKGROUND: T cells producing multiple factors have been shown to be required for protection from disease progression in HIV but we have recently shown this not to be the case in TB. Subjects with active disease had a greater proportion of polyfunctional cells responding to ESAT-6/CFP-10 stimulation than their infected but non-diseased household contacts (HHC. We therefore wanted to assess this profile in subjects who had successfully completed standard TB chemotherapy. METHODS: We performed a cross-sectional study using PBMC from TB cases (pre- and post-treatment and HHC. Samples were stimulated overnight with TB antigens (ESAT-6/CFP-10 and PPD and their CD4+ and CD8+ T cells were assessed for production of CD107a, IFN-gamma, IL-2 and TNF-alpha and the complexity of the responses was determined using SPICE and PESTLE software. RESULTS AND CONCLUSIONS: We found that an increase in complexity (i.e., production of more than 1 factor simultaneously of the T cell profile was associated with TB disease and that this was significantly reduced following TB treatment. This implies that T cells are able to respond adequately to TB antigens with active disease (at least initially but the ability of this response to protect the host from disease progression is hampered, presumably due to immune evasion strategies by the bacteria. These findings have implications for the development of new diagnostics and vaccine strategies.

  2. Ly6C(+) monocyte efferocytosis and cross-presentation of cell-associated antigens.

    Science.gov (United States)

    Larson, S R; Atif, S M; Gibbings, S L; Thomas, S M; Prabagar, M G; Danhorn, T; Leach, S M; Henson, P M; Jakubzick, C V

    2016-06-01

    Recently it was shown that circulating Ly6C(+) monocytes traffic from tissue to the draining lymph nodes (LNs) with minimal alteration in their overall phenotype. Furthermore, in the steady state, Ly6C(+) monocytes are as abundant as classical dendritic cells (DCs) within the draining LNs, and even more abundant during inflammation. However, little is known about the functional roles of constitutively trafficking Ly6C(+) monocytes. In this study we investigated whether Ly6C(+) monocytes can efferocytose (acquire dying cells) and cross-present cell-associated antigen, a functional property particularly attributed to Batf3(+) DCs. We demonstrated that Ly6C(+) monocytes intrinsically efferocytose and cross-present cell-associated antigen to CD8(+) T cells. In addition, efferocytosis was enhanced upon direct activation of the Ly6C(+) monocytes through its corresponding TLRs, TLR4 and TLR7. However, only ligation of TLR7, and not TLR4, enhanced cross-presentation by Ly6C(+) monocytes. Overall, this study outlines two functional roles, among others, that Ly6C(+) monocytes have during an adaptive immune response. PMID:26990659

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

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    Adiba Isa

    Full Text Available 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 a 9-42 fold increase of all six HLA-A,-B,-C gene transcripts. Interestingly, prior to stimulation, gene transcripts for all but two alleles were present in similar amounts suggesting that post-transcriptional mechanisms regulate the constitutive expression of HLA-A,-B, and -C. Locus-restricted expression of HLA-A, -B and -C challenges our current understanding of the function of these molecules as regulators of CD8(+ T-cell and NK-cell function and should lead to further inquiries into their expression on other cell types.

  4. 核糖核苷酸还原酶M1、细胞核增殖抗原在基底细胞样乳腺癌组织的表达及意义%Expression and clinical significance of ribonucleotide reductase M1 and proliferation cell nuclear antigen in bassal-like carcinoma of breast in young female patient

    Institute of Scientific and Technical Information of China (English)

    祁旦巳; 周伶俐; 高宝辉; 张旭彤

    2015-01-01

    Objective To study the role of ribonucleotide reductase M 1 (RRM 1) and proliferation cell nuclear antigen (Ki-67) in the pathogenesis mechanism of bassal-like breast carcinoma (BLBC) in young female patient.Methods The clinical data of 52 young female patients (≤35 years old) treated in the Second Affiliated Hospital of Wenzhou Medical University, from June 2009 to May 2014 were analyzed.Of these patients, 22 cases of BLBC and 30 cases of non-BLBC were picked out based on immunohistochemisy, the proteins of RRM1 and Ki-67 were detected in BLBC and non-BLBC cases.Results The positive expression rate of RRM1 in BLBC organization was significantly lower than the non-BLBC group (22.7% vs.86.7% , P < 0.05);The positive expression rate of Ki-67 in BLBC organization was significantly higher than the non-BLBC group (95.5% vs.66.7%, P < 0.05).Conclusion The expression of RRM1 and Ki-67 in BLBC organizations with non-BLBC showed a negative correlation has important significance in prognostic judgment of breast cancer And maybe BLBC for sensitive to chemotherapy drugs gemcitabine.%目的 探讨核糖核苷酸还原酶M1(RRM1)及细胞核增殖抗原(Ki-67)在青年女性患者基底细胞样乳腺癌(BLBC)及非基底细胞样乳腺癌中的表达.方法 收集青年乳腺癌患者(年龄≤35岁)的石蜡标本,根据免疫组织化学结果选出基底细胞样乳腺癌22例,并随机选择非基底细胞样乳腺癌30例,通过免疫组织化学链霉菌抗生物素蛋白-过氧化物酶(SP)法检测RRM1及Ki-67在肿瘤中的表达.结果 RRM1在BLBC组织中的表达阳性率为22.7% (5/22),显著低于非BLBC组织的86.7% (26/30,P<0.05);Ki-67在BLBC组织中表达阳性率为95.5% (21/22),显著高于非BLBC组织的66.7% (20/30,P<0.05).结论 RRM1与Ki-67在BLBC组织与非BLBC组织中的表达呈负相关,检测RRM1与Ki-67有助于判断乳腺癌的预后,并且BLBC可能对代谢类化疗药物吉西他滨敏感.

  5. 增殖细胞核抗原、人类N-myc下游调节基因1在肝细胞性肝癌中的表达及临床意义%Expression of proliferating cell nuclear antigen and human N-myc downstream regulated gene 1 and their significances in hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    李永明; 范文艳; 高建芝; 许娜; 崔鑫华; 徐振平

    2013-01-01

    Objective: To study the expression of proliferating cell nuclear antigen (PCNA) and N-myc downstream regulated gene 1 (NDRG1) in human primary hepatocellular carcinoma, and to confer the biological behavior and the clinical significance in the hepatocellular carcinoma. Methods: Documented specimens were selected, containing 58 cases of primary liver cancer, 34 cases of cirrhosis and 15 cases of normal tissues. H-E staining was performed to observe the morphology, and immunohistochemistry SABC method to study the expression of PCNA and NDRG1 in primary liver cancer. Results: The expression rate of PCNA in primary liver cancer was significantly higher than that of liver cirrhosis tissues and normal liver tissues. The expression of PCNA in hepatocellular carcinoma was not related to the patient gender, age, HbsAg-positive, AFP level, independent of location and diameter of the tumor. NDRG1 expression was gradually decreased in the normal liver tissues, cirrhesis liver tissues and primary liver tissues; There were no significant difference between the cirrhosis group and the normal liver tissue group; The expression of NDRG1 in hepatocellular carcimoma was not related to the patient gender, age, HbsAg-positive, AFP level, in dependent of location and diameter of the tumor. The expression of NDRG1 in HCC was negatively correlated with PCNA in this experiment. Conclusion: PCNA and NDRG1 in hepatocellular carcinoma play an important role in their development processes. Joint detection contributes to the early cancer detection, early diagnosis and early treatment.%目的:研究增殖细胞核抗原(PCNA)及人类N-myc下游调节基因1(NDRG1)在人肝细胞性肝癌中的表达情况,探讨其与肝癌生物学行为的关系及临床意义.方法:选择有存档的原发性肝癌标本58例,肝硬化34例,正常肝组织标本15例,用H-E染色观察组织形态,用免疫组织化学SABC检测PCNA和NDRG1的表达.结果:肝癌组织中PCNA表达明显高于肝硬化组

  6. The Functional Response of B Cells to Antigenic Stimulation: A Preliminary Report of Latent Tuberculosis

    Science.gov (United States)

    du Plessis, Willem J.; Kleynhans, Léanie; du Plessis, Nelita; Stanley, Kim; Malherbe, Stephanus T.; Maasdorp, Elizna; Ronacher, Katharina; Chegou, Novel N.; Walzl, Gerhard; Loxton, Andre G.

    2016-01-01

    Mycobacterium tuberculosis (M.tb) remains a successful pathogen, causing tuberculosis disease numbers to constantly increase. Although great progress has been made in delineating the disease, the host-pathogen interaction is incompletely described. B cells have shown to function as both effectors and regulators of immunity via non-humoral methods in both innate and adaptive immune settings. Here we assessed specific B cell functional interaction following stimulation with a broad range of antigens within the LTBI milieu. Our results indicate that B cells readily produce pro- and anti-inflammatory cytokines (including IL-1β, IL-10, IL-17, IL-21 and TNF-α) in response to stimulation. TLR4 and TLR9 based stimulations achieved the greatest secreted cytokine-production response and BCG stimulation displayed a clear preference for inducing IL-1β production. We also show that the cytokines produced by B cells are implicated strongly in cell-mediated communication and that plasma (memory) B cells (CD19+CD27+CD138+) is the subset with the greatest contribution to cytokine production. Collectively our data provides insight into B cell responses, where they are implicated in and quantifies responses from specific B cell phenotypes. These findings warrant further functional B cell research with a focus on specific B cell phenotypes under conditions of active TB disease to further our knowledge about the contribution of various cell subsets which could have implications for future vaccine development or refined B cell orientated treatment in the health setting. PMID:27050308

  7. Genome-wide analysis of host-chromosome binding sites for Epstein-Barr Virus Nuclear Antigen 1 (EBNA1

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    Wang Pu

    2010-10-01

    Full Text Available Abstract The Epstein-Barr Virus (EBV Nuclear Antigen 1 (EBNA1 protein is required for the establishment of EBV latent infection in proliferating B-lymphocytes. EBNA1 is a multifunctional DNA-binding protein that stimulates DNA replication at the viral origin of plasmid replication (OriP, regulates transcription of viral and cellular genes, and tethers the viral episome to the cellular chromosome. EBNA1 also provides a survival function to B-lymphocytes, potentially through its ability to alter cellular gene expression. To better understand these various functions of EBNA1, we performed a genome-wide analysis of the viral and cellular DNA sites associated with EBNA1 protein in a latently infected Burkitt lymphoma B-cell line. Chromatin-immunoprecipitation (ChIP combined with massively parallel deep-sequencing (ChIP-Seq was used to identify cellular sites bound by EBNA1. Sites identified by ChIP-Seq were validated by conventional real-time PCR, and ChIP-Seq provided quantitative, high-resolution detection of the known EBNA1 binding sites on the EBV genome at OriP and Qp. We identified at least one cluster of unusually high-affinity EBNA1 binding sites on chromosome 11, between the divergent FAM55 D and FAM55B genes. A consensus for all cellular EBNA1 binding sites is distinct from those derived from the known viral binding sites, suggesting that some of these sites are indirectly bound by EBNA1. EBNA1 also bound close to the transcriptional start sites of a large number of cellular genes, including HDAC3, CDC7, and MAP3K1, which we show are positively regulated by EBNA1. EBNA1 binding sites were enriched in some repetitive elements, especially LINE 1 retrotransposons, and had weak correlations with histone modifications and ORC binding. We conclude that EBNA1 can interact with a large number of cellular genes and chromosomal loci in latently infected cells, but that these sites are likely to represent a complex ensemble of direct and indirect EBNA

  8. Regulation of T cell response to leishmania antigens by determinants of histocompatibility leukocyte class I and II molecules

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    Bacellar O.

    1998-01-01

    Full Text Available It has been shown that HLA class I molecules play a significant role in the regulation of the proliferation of T cells activated by mitogens and antigens. We evaluated the ability of mAb to a framework determinant of HLA class I molecules to regulate T cell proliferation and interferon gamma (IFN-g production against leishmania, PPD, C. albicans and tetanus toxoid antigens in patients with tegumentary leishmaniasis and healthy subjects. The anti-major histocompatibility complex (MHC mAb (W6/32 suppressed lymphocyte proliferation by 90% in cultures stimulated with aCD3, but the suppression was variable in cultures stimulated with leishmania antigen. This suppression ranged from 30-67% and was observed only in 5 of 11 patients. IFN-g production against leishmania antigen was also suppressed by anti-HLA class I mAb. In 3 patients IFN-g levels were suppressed by more than 60%, while in the other 2 cultures IFN-g levels were 36 and 10% lower than controls. The suppression by HLA class I mAb to the proliferative response in leishmaniasis patients and in healthy controls varied with the antigens and the patients or donors tested. To determine whether the suppression is directed at antigen presenting cells (APCs or at the responding T cells, experiments with antigen-primed non-adherent cells, separately incubated with W6/32, were performed. Suppression of proliferation was only observed when the W6/32 mAb was added in the presence of T cells. These data provide evidence that a mAb directed at HLA class I framework determinants can suppress proliferation and cytokine secretion in response to several antigens.

  9. Molecular tracking of antigen-specific T cell clones in neurological immune-mediated disorders

    Science.gov (United States)

    Muraro, Paolo A.; Wandinger, Klaus-Peter; Bielekova, Bibiana; Gran, Bruno; Marques, Adriana; Utz, Ursula; McFarland, Henry F.; Jacobson, Steve; Martin, Roland

    2016-01-01

    Summary T cells recognizing self or microbial antigens may trigger or reactivate immune-mediated diseases. Monitoring the frequency of specific T cell clonotypes to assess a possible link with the course of disease has been a difficult task with currently available technology. Our goal was to track individual candidate pathogenic T cell clones, selected on the basis of previous extensive studies from patients with immune-mediated disorders of the CNS, including multiple sclerosis, HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/ TSP) and chronic Lyme neuroborreliosis. We developed and applied a highly specific and sensitive technique to track single CD4+ and CD8+ T cell clones through the detection and quantification of T cell receptor (TCR) α or β chain complementarity-determining region 3 transcripts by real-time reverse transcriptase (RT)-PCR. We examined the frequency of the candidate pathogenic T cell clones in the peripheral blood and CSF during the course of neurological disease. Using this approach, we detected variations of clonal frequencies that appeared to be related to clinical course, significant enrichment in the CSF, or both. By integrating clono-type tracking with direct visualization of antigen-specific staining, we showed that a single T cell clone contributed substantially to the overall recognition of the viral peptide/MHC complex in a patient with HAM/ TSP. T cell clonotype tracking is a powerful new technology enabling further elucidation of the dynamics of expansion of autoreactive or pathogen-specific T cells that mediate pathological or protective immune responses in neurological disorders. PMID:12477694

  10. Application of Adoptive T-Cell Therapy Using Tumor Antigen-Specific T-Cell Receptor Gene Transfer for the Treatment of Human Leukemia

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    Toshiki Ochi

    2010-01-01

    Full Text Available The last decade has seen great strides in the field of cancer immunotherapy, especially the treatment of melanoma. Beginning with the identification of cancer antigens, followed by the clinical application of anti-cancer peptide vaccination, it has now been proven that adoptive T-cell therapy (ACT using cancer antigen-specific T cells is the most effective option. Despite the apparent clinical efficacy of ACT, the timely preparation of a sufficient number of cancer antigen-specific T cells for each patient has been recognized as its biggest limitation. Currently, therefore, attention is being focused on ACT with engineered T cells produced using cancer antigen-specific T-cell receptor (TCR gene transfer. With regard to human leukemia, ACT using engineered T cells bearing the leukemia antigen-specific TCR gene still remains in its infancy. However, several reports have provided preclinical data on TCR gene transfer using Wilms' tumor gene product 1 (WT1, and also preclinical and clinical data on TCR gene transfer involving minor histocompatibility antigen, both of which have been suggested to provide additional clinical benefit. In this review, we examine the current status of anti-leukemia ACT with engineered T cells carrying the leukemia antigen-specific TCR gene, and discuss the existing barriers to progress in this area.

  11. Expression of HSV-1 ICP0 Antigen Peptide in Prokaryotic Cells and Preparation of Specific Antibody

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    As an immediate-early protein of herpes simplex virus, infected-cell polypeptide 0 (ICP0) exhibits complicated interactions with host cells, and its regulatory function on gene expression is of great importance. Since the ICP0 encoding sequence contains many rare codons which are absent in E.coli, and ICP0 is highly unstable in prokaryotic cells, expression of entire ICP0 in prokaryotic cells has never been reported. In order to further investigate the function of ICP0, a recombinant plasmid was constructed by subcloning a cDNA fragment encoding an amino-terminal of 105 residues of the ICP0 protein into pGEX-5x-1 vector. The resulting GST-105 fusion antigen peptide was expressed with high efficiency in E.coli. Antibodies prepared after the immunization of mice with purified fusion protein can recognize not only the denatured ICP0 protein, but also the native ICP0 protein with normal biological conformation.

  12. Intravacuolar Membranes Regulate CD8 T Cell Recognition of Membrane-Bound Toxoplasma gondii Protective Antigen

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    Jodie Lopez

    2015-12-01

    Full Text Available Apicomplexa parasites such as Toxoplasma gondii target effectors to and across the boundary of their parasitophorous vacuole (PV, resulting in host cell subversion and potential presentation by MHC class I molecules for CD8 T cell recognition. The host-parasite interface comprises the PV limiting membrane and a highly curved, membranous intravacuolar network (IVN of uncertain function. Here, using a cell-free minimal system, we dissect how membrane tubules are shaped by the parasite effectors GRA2 and GRA6. We show that membrane association regulates access of the GRA6 protective antigen to the MHC I pathway in infected cells. Although insertion of GRA6 in the PV membrane is key for immunogenicity, association of GRA6 with the IVN limits presentation and curtails GRA6-specific CD8 responses in mice. Thus, membrane deformations of the PV regulate access of antigens to the MHC class I pathway, and the IVN may play a role in immune modulation.

  13. An antigen-mediated selection system for mammalian cells that produce glycosylated single-chain Fv

    International Nuclear Information System (INIS)

    Selection and production of specific antibodies are limiting the development of high-throughput immunoassays such as antibody chips. In this study, we propose an antigen-mediated selection of antibody producers (ASAP) system in mammalian cells. As a model system, transgenes encoding anti-fluorescein ScFv fused to cytokine receptors were introduced to IL-3-dependent cell lines. Addition of fluorescein-conjugated BSA induced growth signal through the ScFv/receptor chimeras, leading to selective expansion of the transduced cells. Cre recombinase was then used to excise the receptor gene flanked by two loxP recognition sites in the introns, resulting in secretion of his-myc-tagged ScFv to the culture medium. When the first loxP site was used in the exon as a linker between ScFv and receptor, enhanced antigen-mediated cell proliferation and production of unexpectedly glycosylated ScFv were achieved. ASAP is the first mammalian selection/production system of recombinant human ScFvs, without need for subcloning and with the advantage of glycosylated product

  14. Phenotypic studies of natural killer cell subsets in human transporter associated with antigen processing deficiency.

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    Jacques Zimmer

    Full Text Available Peripheral blood natural killer (NK cells from patients with transporter associated with antigen processing (TAP deficiency are hyporesponsive. The mechanism of this defect is unknown, but the phenotype of TAP-deficient NK cells is almost normal. However, we noticed a high percentage of CD56(bright cells among total NK cells from two patients. We further investigated TAP-deficient NK cells in these patients and compared them to NK cells from two other TAP-deficient patients with no clinical symptoms and to individuals with chronic inflammatory diseases other than TAP deficiency (chronic lung diseases or vasculitis. Peripheral blood mononuclear cells isolated from venous blood were stained with fluorochrome-conjugated antibodies and the phenotype of NK cells was analyzed by flow cytometry. In addition, (51Chromium release assays were performed to assess the cytotoxic activity of NK cells. In the symptomatic patients, CD56(bright NK cells represented 28% and 45%, respectively, of all NK cells (higher than in healthy donors. The patients also displayed a higher percentage of CD56(dimCD16(- NK cells than controls. Interestingly, this unusual NK cell subtype distribution was not found in the two asymptomatic TAP-deficient cases, but was instead present in several of the other patients. Over-expression of the inhibitory receptor CD94/NKG2A by TAP-deficient NK cells was confirmed and extended to the inhibitory receptor ILT2 (CD85j. These inhibitory receptors were not involved in regulating the cytotoxicity of TAP-deficient NK cells. We conclude that expansion of the CD56(bright NK cell subtype in peripheral blood is not a hallmark of TAP deficiency, but can be found in other diseases as well. This might reflect a reaction of the immune system to pathologic conditions. It could be interesting to investigate the relative distribution of NK cell subsets in various respiratory and autoimmune diseases.

  15. Detection and Characterization of Autoantibodies to Neuronal Cell-Surface Antigens in the Central Nervous System.

    Science.gov (United States)

    van Coevorden-Hameete, Marleen H; Titulaer, Maarten J; Schreurs, Marco W J; de Graaff, Esther; Sillevis Smitt, Peter A E; Hoogenraad, Casper C

    2016-01-01

    Autoimmune encephalitis (AIE) is a group of disorders in which autoantibodies directed at antigens located on the plasma membrane of neurons induce severe neurological symptoms. In contrast to classical paraneoplastic disorders, AIE patients respond well to immunotherapy. The detection of neuronal surface autoantibodies in patients' serum or CSF therefore has serious consequences for the patients' treatment and follow-up and requires the availability of sensitive and specific diagnostic tests. This mini-review provides a guideline for both diagnostic and research laboratories that work on the detection of known surface autoantibodies and/or the identification of novel surface antigens. We discuss the strengths and pitfalls of different techniques for anti-neuronal antibody detection: (1) Immunohistochemistry (IHC) and immunofluorescence on rat/primate brain sections; (2) Immunocytochemistry (ICC) of living cultured hippocampal neurons; and (3) Cell Based Assay (CBA). In addition, we discuss the use of immunoprecipitation and mass spectrometry analysis for the detection of novel neuronal surface antigens, which is a crucial step in further disease classification and the development of novel CBAs. PMID:27303263

  16. Detection and Characterization of Autoantibodies to Neuronal Cell-Surface Antigens in the Central Nervous System

    Directory of Open Access Journals (Sweden)

    Marleen eVan Coevorden-Hameete

    2016-05-01

    Full Text Available Autoimmune encephalitis (AIE is a group of disorders in which autoantibodies directed at antigens located on the plasma membrane of neurons induce severe neurological symptoms. In contrast to classical paraneoplastic disorders, AIE patients respond well to immunotherapy. The detection of neuronal surface autoantibodies in patients’ serum or CSF therefore has serious consequences for the patients’ treatment and follow-up and requires the availability of sensitive and specific diagnostic tests. This mini-review provides a guideline for both diagnostic and research laboratories that work on the detection of known surface autoantibodies and/or the identification of novel surface antigens. We discuss the strengths and pitfalls of different techniques for anti-neuronal antibody detection: 1 Immunohistochemistry and immunofluorescence on rat/ primate brain sections, 2 Immunocytochemistry of living cultured hippocampal neurons, 3 Cell Based Assay (CBA. In addition, we discuss the use of immunoprecipitation and mass spectrometry analysis for the detection of novel neuronal surface antigens, which is a crucial step in further disease classification and the development of novel CBAs.

  17. NKT cell stimulation with glycolipid antigen in vivo: co-stimulation-dependent expansion, Bim-dependent contraction, and hypo-responsiveness to further antigenic challenge1

    Science.gov (United States)

    Kyparissoudis, Konstantinos; Pellicci, Daniel G.; Zhan, Yifan; Lew, Andrew M.; Bouillet, Philippe; Strasser, Andreas; Smyth, Mark J.; Godfrey, Dale I.

    2005-01-01

    Activation of NKT cells using the glycolipid α-galactosylceramide (α-GalCer4) has availed many investigations into their immunoregulatory and therapeutic potential. However, it remains unclear how NKT cells respond to stimulation in vivo, which co-stimulatory pathways are important, and what factors (eg. antigen availability and activation-induced cell death) limit their response. We have explored these questions in the context of anin vivo model of NKT cell dynamics spanning activation, population expansion and subsequent contraction. Neither the B7/CD28 nor the CD40/CD40-L co-stimulatory pathways were necessary for cytokine production by activated NKT cells, either early (2 hours) or late (3 days) following initial stimulation, but both pathways were necessary for normal proliferative expansion of NKT cells in vivo. The pro-apoptotic Bcl-2 family member Bim was necessary for normal contraction of the NKT cell population between days 3-9 after stimulation, suggesting the pool size is regulated by apoptotic cell death in a manner similar to that of conventional T cells. Antigen availability was not the limiting factor for NKT cell expansion in vivo, and a second injection of α-GalCer induced a very blunted response, whereby cytokine production was reduced and further expansion did not occur. This appeared to be a form of anergy that was intrinsic to the NKT cells and not associated with up-regulation of inhibitory NK cell receptors such as NKG2A or Ly49 family members. Furthermore, NKT cells from mice pre-challenged with α-GalCer in vivoshowed little cytokine production and reduced proliferation in vitro. In summary, this study significantly enhances our understanding of how NKT cells respond to α-GalCer in vivo, revealing that the full primary response depends on costimulation via the CD28 and CD40 pathways, with subsequent Bim-dependent contraction. After contraction, the NKT cells are hypo-responsive to further antigenic induced expansion. PMID:16116198

  18. Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells.

    Science.gov (United States)

    Frigault, Matthew J; Lee, Jihyun; Basil, Maria Ciocca; Carpenito, Carmine; Motohashi, Shinichiro; Scholler, John; Kawalekar, Omkar U; Guedan, Sonia; McGettigan, Shannon E; Posey, Avery D; Ang, Sonny; Cooper, Laurence J N; Platt, Jesse M; Johnson, F Brad; Paulos, Chrystal M; Zhao, Yangbing; Kalos, Michael; Milone, Michael C; June, Carl H

    2015-04-01

    This study compared second-generation chimeric antigen receptors (CAR) encoding signaling domains composed of CD28, ICOS, and 4-1BB (TNFRSF9). Here, we report that certain CARs endow T cells with the ability to undergo long-term autonomous proliferation. Transduction of primary human T cells with lentiviral vectors encoding some of the CARs resulted in sustained proliferation for up to 3 months following a single stimulation through the T-cell receptor (TCR). Sustained numeric expansion was independent of cognate antigen and did not require the addition of exogenous cytokines or feeder cells after a single stimulation of the TCR and CD28. Results from gene array and functional assays linked sustained cytokine secretion and expression of T-bet (TBX21), EOMES, and GATA-3 to the effect. Sustained expression of the endogenous IL2 locus has not been reported in primary T cells. Sustained proliferation was dependent on CAR structure and high expression, the latter of which was necessary but not sufficient. The mechanism involves constitutive signaling through NF-κB, AKT, ERK, and NFAT. The propagated CAR T cells retained a diverse TCR repertoire, and cellular transformation was not observed. The CARs with a constitutive growth phenotype displayed inferior antitumor effects and engraftment in vivo. Therefore, the design of CARs that have a nonconstitutive growth phenotype may be a strategy to improve efficacy and engraftment of CAR T cells. The identification of CARs that confer constitutive or nonconstitutive growth patterns may explain observations that CAR T cells have differential survival patterns in clinical trials. PMID:25600436

  19. Engineered nanomaterials cause cytotoxicity and activation on mouse antigen presenting cells

    International Nuclear Information System (INIS)

    Nanomaterials improve everyday products but their safety for human health is poorly known. In this study we explored immunological effects of five different nanomaterials on antigen presenting cells (APC) in vitro. Nanomaterials studied were rutile titanium dioxide (TiO2), amorphous silica-coated rutile titanium dioxide (TiO2-silica), zinc oxide (ZnO), single-walled carbon nanotubes (SWCNT) and multi-walled carbon nanotubes (MWCNT). APCs included mouse macrophages (RAW 264.7 cell line) and murine bone marrow-derived dendritic cells (bmDC). All studied particles were cytotoxic to bmDCs, and ZnO, TiO2 and TiO2-silica-induced dose-dependently cell death also in macrophages. ZnO had the most drastic immunological effects leading to high expression of proinflammatory cytokine, IL-1β, and enhanced production of neutrophil chemoattractant CXCL-9 on both cell types. TiO2 and TiO2-silica stimulated the expression of IL-6, MIP-1α and TNF-α in macrophages, and increased their maturation, antigen presentation and co-stimulation activity. In contrast, SWCNT or MWCNT did not seem to have any significant immunological effects on the cell types studied suggesting that APCs might not be the target cells for carbon nanotubes. Due to diverse effects on different nanomaterials on immune cells we suggest that each new nanomaterial should be extensively studied in vitro and in vivo for risk assessment before their use in final products.

  20. Effective induction of naive and recall T-cell responses by targeting antigen to human dendritic cells via a humanized anti-DC-SIGN antibody.

    NARCIS (Netherlands)

    Tacken, P.J.; Vries, I.J.M. de; Gijzen, K.; Joosten, B.H.G.M.; Wu, D.; Rother, R.P.; Faas, S.J.; Punt, C.J.A.; Torensma, R.; Adema, G.J.; Figdor, C.G.

    2005-01-01

    Current dendritic cell (DC)-based vaccines are based on ex vivo-generated autologous DCs loaded with antigen prior to readministration into patients. A more direct and less laborious strategy is to target antigens to DCs in vivo via specific surface receptors. Therefore, we developed a humanized ant

  1. Identification of the hair cell soma-1 antigen, HCS-1, as otoferlin.

    Science.gov (United States)

    Goodyear, Richard J; Legan, P Kevin; Christiansen, Jeffrey R; Xia, Bei; Korchagina, Julia; Gale, Jonathan E; Warchol, Mark E; Corwin, Jeffrey T; Richardson, Guy P

    2010-12-01

    Hair cells, the mechanosensitive receptor cells of the inner ear, are critical for our senses of hearing and balance. The small number of these receptor cells in the inner ear has impeded the identification and characterization of proteins important for hair cell function. The binding specificity of monoclonal antibodies provides a means for identifying hair cell-specific proteins and isolating them for further study. We have generated a monoclonal antibody, termed hair cell soma-1 (HCS-1), which specifically immunolabels hair cells in at least five vertebrate classes, including sharks and rays, bony fish, amphibians, birds, and mammals. We used HCS-1 to immunoprecipitate the cognate antigen and identified it as otoferlin, a member of the ferlin protein family. Mutations in otoferlin underlie DFNB9, a recessive, nonsyndromic form of prelingual deafness characterized as an auditory neuropathy. Using immunocytochemistry, we find that otoferlin is associated with the entire basolateral membrane of the hair cells and with vesicular structures distributed throughout most of the hair cell cytoplasm. Biochemical assays indicate that otoferlin is tightly associated with membranes, as it is not solubilized by alterations in calcium or salt concentrations. HCS-1 immunolabeling does not co-localize with ribeye, a constituent of synaptic ribbons, suggesting that otoferlin may, in addition to its proposed function in synaptic vesicle release, play additional roles in hair cells. PMID:20809368

  2. Common antigens of streptococcal and non-streptococcal oral bacteria: immunochemical studies of extracellular and cell-wall-associated antigens from Streptococcus sanguis, Streptococcus mutans, Lactobacillus salivarius, and Actinomyces viscosus.

    Science.gov (United States)

    Schöller, M; Klein, J P; Frank, R M

    1981-01-01

    Soluble extracellular antigens (ESA) were prepared from the culture supernatant of exponential growing cells of Streptococcus sanguis OMZ 9 by a combination of ammonium sulfate precipitation and chromatography on a Bio-Gel P6 column. Soluble cell wall antigens (WEA) were obtained from the bacterial pellet by extraction with 1 M phosphate buffer (pH 6). Antisera against whole cells of S. sanguis and S. mutans of different serotypes, 10% trichloroacetic extracts of bacterial cell walls, dextran, ESA, and WEA were prepared by injecting the different antigens several times in rabbits. ESA and WEA were prepared from a representative strain of Bratthall's seven serological groups, Lactobacillus salivarius, and Actinomyces viscosus. All sera showed various agglutinin titers against heat-killed cells, and titers were generally higher with homologous cells. The comparison of the different antigens using agar gel diffusion and immunoelectrophoresis showed the presence of extracellular common antigens in both ESA and WEA between the different strains. Absorption of anti-ESA sera with WEA, and anti-WEA sera with ESA, showed the existence of a specific antigen common to all bacteria in each fraction. Enzymatic treatment of the antigen before immunodiffusion demonstrated the protein nature of the two antigens present in ESA and WEA. Images PMID:6783541

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

    Science.gov (United States)

    Hinrichs, Christian S

    2016-04-01

    Adoptive transfer of T cells genetically engineered to express a tumor-targeting chimeric antigen receptor (CAR) or T-cell receptor (TCR) can mediate cancer regression in some patients. CARs are synthetic single-chain proteins that use antibody domains to target cell surface antigens. TCRs are natural heterodimeric proteins that can target intracellular antigens through recognition of peptides bound to human leukocyte antigens. CARs have shown promise in B-cell malignancies and TCRs in melanoma, but neither approach has achieved clear success in an epithelial cancer. Treatment of epithelial cancers may be particularly challenging because of a paucity of target antigens expressed by carcinomas and not by important healthy tissues. In addition, epithelial cancers may be protected by inhibitory ligands and soluble factors in the tumor microenvironment. One strategy to overcome these negative regulators is to modulate expression of T-cell genes to enhance intrinsic T-cell function. Programmable nucleases, which can suppress inhibitory genes, and inducible gene expression systems, which can enhance stimulatory genes, are entering clinical testing. Other work is delineating whether control of genes for immune checkpoint receptors (e.g.,PDCD1, CTLA4) and cytokine and TCR signaling regulators (e.g.,CBLB, CISH, IL12, IL15) can increase the antitumor activity of therapeutic T cells.Clin Cancer Res; 22(7); 1559-64. ©2016 AACR. PMID:27037253

  4. The cancer-germline antigen SSX2 causes cell cycle arrest and DNA damage in cancer cells

    DEFF Research Database (Denmark)

    Greve, Katrine Buch Vidén; Lindgreen, Jonas; Terp, Mikkel Green;

    2011-01-01

    increase in the number of gamma-H2AX ‘DNA damage foci’, indicating replicative stress, which may lead to genomic instability. As the p53 tumor suppressor is an inducer of G1 arrest after DNA damage and often deregulated in cancer cells, we investigated if the growth reduction due to SSX2 expression was p53...... dependent. The growth reduction was similar in isogenic colon cancer cells with and without p53, indicating that SSX2 is able to inhibit the growth of cancer cells, even in absence of functional p53. Our results show that SSX2 acts as an inhibitor of cancer cell proliferation, possibly through replicative......The SSX family of cancer and germline antigens is mainly expressed in the germ cells of healthy individuals as well as wide range of cancers and is therefore potential targets for immunotherapy. However, little is known about the role of SSX proteins in tumorigenesis and normal cell function. Here...

  5. Antigen and Memory CD8 T Cells: Were They Both Right?

    Directory of Open Access Journals (Sweden)

    Epelman Slava

    2007-06-01

    Full Text Available Picture yourself as a researcher in immunology. To begin your project, you ask a question: Do CD8 T cells require antigen to maintain a memory response? This question is of prime importance to numerous medical fields. In chronologic order, you digest the literature, but unfortunately, you hit a major stumbling block in the 1990s. The crux of the problem is that which so often happens in science: two well-recognized, capable groups emerge with diametrically opposed conclusions, leaving you pondering which set of wellcontrolled data to believe. Fortunately, years later, a surprising group of articles sheds light on this mystery and subtly reconciles these two positions.

  6. Tracking antigen-specific CD8+ T cells in the rat using MHC class I multimers.

    OpenAIRE

    Duplan, Valérie; Suberbielle, Elsa; Napper, Catherine,; Joly, Etienne; Saoudi, Abdelhadi; Gonzalez-Dunia, Daniel

    2007-01-01

    Studies of the quantitative and qualitative aspects of anti-microbial, anti-tumoral or autoreactive immune responses have been greatly facilitated by the possibility to stain antigen-specific CD8(+) T cells using fluorescently labeled multimeric major histocompatibility complex (MHC) class I/peptide complexes. So far, this technology has been developed for human and mouse, but not yet in the rat. Here, we describe the generation of the first rat MHC multimer. We produced a rat RT1(l) Pro5 MHC...

  7. Production of Cloned Korean Native Pig by Somatic Cell Nuclear Transfer

    OpenAIRE

    Hwang, In-Sul; Kwon, Dae-Jin; Oh, Keun Bong; Ock, Sun-A; Chung, Hak-Jae; Cho, In-Cheol; Lee, Jeong-Woong; Im, Gi-Sun; Hwang, Seongsoo

    2015-01-01

    The Korean native pig (KNP) have been considered as animal models for animal biotechnology research because of their relatively small body size and their presumably highly inbred status due to the closed breeding program. However, little is reported about the use of KNP for animal biotechnology researches. This study was performed to establish the somatic cell nuclear transfer (SCNT) protocol for the production of swine leukocyte antigens (SLA) homotype-defined SCNT KNP. The ear fibroblast ce...

  8. Polyethylene glycol-coated graphene oxide attenuates antigen-specific IgE production and enhanced antigen-induced T-cell reactivity in ovalbumin-sensitized BALB/c mice

    Directory of Open Access Journals (Sweden)

    Wu HY

    2014-09-01

    Full Text Available Hsin-Ying Wu,1,* Kun-Ju Lin,2,* Ping-Yen Wang,1 Chi-Wen Lin,3 Hong-Wei Yang,3 Chen-Chi M Ma,3 Yu-Jen Lu,4 Tong-Rong Jan1 1Department and Graduate Institute of Veterinary Medicine, School of Veterinary Medicine, National Taiwan University, Taipei, Taiwan; 2Animal Molecular Imaging Center and Department of Nuclear Medicine, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan; 3Department of Chemical Engineering, National Tsing Hua University, Hsin-Chu, Taiwan; 4Department of Neurosurgery, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan *These authors contributed equally to this work Background: Graphene oxide (GO is a promising nanomaterial for potential application in the versatile field of biomedicine. Graphene-based nanomaterials have been reported to modulate the functionality of immune cells in culture and to induce pulmonary inflammation in mice. Evidence pertaining to the interaction between graphene-based nanomaterials and the immune system in vivo remains scarce. The present study investigated the effect of polyethylene glycol-coated GO (PEG-GO on antigen-specific immunity in vivo. Methods: BALB/c mice were intravenously administered with a single dose of PEG-GO (0.5 or 1 mg/kg 1 hour before ovalbumin (OVA sensitization, and antigen-specific antibody production and splenocyte reactivity were measured 7 days later. Results: Exposure to PEG-GO significantly attenuated the serum level of OVA-specific immunoglobulin E. The production of interferon-γ and interleukin-4 by splenocytes restimulated with OVA in culture was enhanced by treatment with PEG-GO. In addition, PEG-GO augmented the metabolic activity of splenocytes restimulated with OVA but not with the T-cell mitogen concanavalin A. Conclusion: Collectively, these results demonstrate that systemic exposure to PEG-GO modulates several aspects of antigen-specific immune responses, including the serum production of immunoglobulin E and T-cell functionality. Keywords: graphene oxide, T-cell

  9. Increased generation of Foxp3(+) regulatory T cells by manipulating antigen presentation in the thymus.

    Science.gov (United States)

    Lin, Jiqiang; Yang, Lu; Silva, Hernandez Moura; Trzeciak, Alissa; Choi, Yongwon; Schwab, Susan R; Dustin, Michael L; Lafaille, Juan J

    2016-01-01

    Regulatory T-cell (Treg) selection in the thymus is essential to prevent autoimmune diseases. Although important rules for Treg selection have been established, there is controversy regarding the degree of self-reactivity displayed by T-cell receptors expressed by Treg cells. In this study we have developed a model of autoimmune skin inflammation, to determine key parameters in the generation of skin-reactive Treg cells in the thymus (tTreg). tTreg development is predominantly AIRE dependent, with an AIRE-independent component. Without the knowledge of antigen recognized by skin-reactive Treg cells, we are able to enhance skin-specific tTreg cell generation using three approaches. First, we increase medullary thymic epithelial cells by using mice lacking osteoprotegerin or by adding TRANCE (RANKL, Tnfsf11). Second, we inject intrathymically peripheral dendritic cells from skin-draining sites. Finally, we inject skin tissue lysates intrathymically. These findings have implications for enhancing the generation of organ-specific Treg cells in autoimmune diseases. PMID:26923114

  10. Antigen-primed helper T cell function in CBA/N mice is radiosensitive

    International Nuclear Information System (INIS)

    CBA/N mice have an X-linked immunodeficiency that includes a deficient humoral response to sheep red blood cells (SRBC). In order to study the cellular mechanisms of this deficiency we have examined helper T cell function to SRBC in an adoptive transfer system by using 2 different sources of helper T cells. When thymocytes were used as the source of helper T cell precursors in an adoptive transfer system, CBA/N thymocytes were as effective as CBA/Ca thymocytes in inducing CBA/Ca bone marrow cells to develop into both direct and indirect anti-SRBC plaque-forming cells (PFC). However, when SRBC-primed, irradiated recipient mice were used as the source of helper T cells, primed and irradiated CBA/N recipiets developed significantly fewer direct and indirect anti-SRBC PFC than similarly treated CBA/CA recipients when reconstituted with CBA/Ca bone marrow cells and challenged with SRBC. We conclude that antigen-primed helper T cell function in CBA/N mice is radiosensitive. Possible reasons for this are evaluated and discussed

  11. Cell-mediated immune responses to a cloned Plasmodium falciparum antigen

    Energy Technology Data Exchange (ETDEWEB)

    Rollwagen, F.M.; Pacheco, N.D.; Wistar, R. Jr.

    1986-03-05

    A peptide fragment of the Plasmodium falciparum (P.f.) circumsporozoite protein (CSP) containing 32 repeats of the immunodominant tetrapeptide ASN-ALA-ASN-PRO (R32tet32) is currently being evaluated as a vaccine in man. This R32tet32 peptide, prepared by recombinant DNA technology from a cloned P.f. gene fragment, has been examined for its ability to stimulate T-cell proliferation in experimental animals. Groups of mice were injected with either R32tet32 emulsified in Freund's complete adjuvant (CFA), or live, or frozen-thawed P.f. sporozoites. Lymphocytes from such mice were cocultured with varying doses of R32tet32 or irrelevant antigen. Proliferation was assessed by /sup 3/H-thymidine uptake; serum antibody was analyzed by ELISA. A proliferative response was found in mice immunized with R32tet32+CFA as early as day 7 post-injection, and was persistent through at least day 23. No proliferation in response to R32tet32 was observed in lymphocytes taken from mice injected with live or frozen-thawed sporozoites. All three immunogens induced both IgM and IgG antibody to R32tet32. They conclude that exposure to live or frozen-thawed P.f. sporozoites alone is sufficient to generate T-cell helper activity for subsequent antibody production, but that antigen+CFA was necessary to generate significant T-cell proliferative activity.

  12. Cell-mediated immune responses to a cloned Plasmodium falciparum antigen

    International Nuclear Information System (INIS)

    A peptide fragment of the Plasmodium falciparum (P.f.) circumsporozoite protein (CSP) containing 32 repeats of the immunodominant tetrapeptide ASN-ALA-ASN-PRO (R32tet32) is currently being evaluated as a vaccine in man. This R32tet32 peptide, prepared by recombinant DNA technology from a cloned P.f. gene fragment, has been examined for its ability to stimulate T-cell proliferation in experimental animals. Groups of mice were injected with either R32tet32 emulsified in Freund's complete adjuvant (CFA), or live, or frozen-thawed P.f. sporozoites. Lymphocytes from such mice were cocultured with varying doses of R32tet32 or irrelevant antigen. Proliferation was assessed by 3H-thymidine uptake; serum antibody was analyzed by ELISA. A proliferative response was found in mice immunized with R32tet32+CFA as early as day 7 post-injection, and was persistent through at least day 23. No proliferation in response to R32tet32 was observed in lymphocytes taken from mice injected with live or frozen-thawed sporozoites. All three immunogens induced both IgM and IgG antibody to R32tet32. They conclude that exposure to live or frozen-thawed P.f. sporozoites alone is sufficient to generate T-cell helper activity for subsequent antibody production, but that antigen+CFA was necessary to generate significant T-cell proliferative activity

  13. Localization of human immunodeficiency virus antigens in infected cells by scanning/transmission-immunogold techniques

    International Nuclear Information System (INIS)

    An application of high resolution scanning/transmission electron microscopy (STEM) and gold-labelling techniques for the rapid detection of human immunodeficiency virus (HIV) in infected cells has been developed. Experimental in vitro studies for detecting two HIV structural proteins, gp41 and p17, were performed following an indirect labeling procedure that uses monoclonal anti-p17 and anti-gp41 antibodies as primary antibodies and 40 nm gold-linked goat antimouse IgG as secondary antibodies. The cells were then studied by STEM in the scanning mode. Unambiguous localization of the viral antigens was possible by combining the three-dimensional image provided by the secondary electron image and the atomic number-dependent backscattered electron image for the identification of the gold marker. This technique combines both the morphological information and the rapid procedures of scanning electron microscopy with the precise and sensitive antigen detection provided by the use of STEM and immunological methods. The preliminary results of its application to the study of peripheral blood mononuclear cells from four anti-HIV-seropositive patients showing the presence of specific labeling in all of them suggest that it might prove useful for early detection of HIV infection before seroconversion, as well as for quantitative studies

  14. The selective adherence of lymphoblasts to antigenic cell monolayers. A method for determining the specificity of lymphocytes proliferating in response to histocompatibility antigens

    International Nuclear Information System (INIS)

    The specificity and intensity of the immune response of rat lymph nodes draining a skin allograft were examined by exploiting a monolayer of donor-type thoracic duct lymphocytes as an immunoabsorbent. Stable monolayers were produced by attaching lymphocytes from different strains of rat to Petri dishes pretreated with poly-L-lysine. The responding lymph node cells were labelled in vitro with [3H]thymidine, incubated on the monolayer and mechanically separated into non-adherent and adherent fractions. The radioactivity associated with the adherent fraction was 7-8 times greater when the monolayer displayed the immunizing major histocompatibility antigens than when syngeneic or 'third party' monolayers were used. The non-specific adherence to syngeneic monolayers was low and consistent. Immunization to minor histocompatibility antigens may also be studied by this method. (Auth.)

  15. Epstein-Barr virus nuclear antigen 3C targets p53 and modulates its transcriptional and apoptotic activities

    International Nuclear Information System (INIS)

    The p53 tumor suppressor gene is one of the most commonly mutated genes in human cancers and the corresponding encoded protein induces apoptosis or cell-cycle arrest at the G1/S checkpoint in response to DNA damage. To date, previous studies have shown that antigens encoded by human tumor viruses such as SV40 large T antigen, adenovirus E1A and HPV E6 interact with p53 and disrupt its functional activity. In a similar fashion, we now show that EBNA3C, one of the EBV latent antigens essential for the B-cell immortalization in vitro, interacts directly with p53. Additionally, we mapped the interaction of EBNA3C with p53 to the C-terminal DNA-binding and the tetramerization domain of p53, and the region of EBNA3C responsible for binding to p53 was mapped to the N-terminal domain of EBNA3C (residues 130-190), previously shown to interact with a number of important cell-cycle components, specifically SCFSkp2, cyclin A, and cMyc. Furthermore, we demonstrate that EBNA3C substantially represses the transcriptional activity of p53 in luciferase based reporter assays, and rescues apoptosis induced by ectopic p53 expression in SAOS-2 (p53-/-) cells. Interestingly, we also show that the DNA-binding ability of p53 is diminished in the presence of EBNA3C. Thus, the interaction between the p53 and EBNA3C provides new insights into the mechanism(s) by which the EBNA3C oncoprotein can alter cellular gene expression in EBV associated human cancers.

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

    Directory of Open Access Journals (Sweden)

    Hiroshi Fujiwara

    2014-12-01

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

  17. An Enhanced ELISPOT Assay for Sensitive Detection of Antigen-Specific T Cell Responses to Borrelia burgdorferi

    OpenAIRE

    Kellermann, Gottfried H.; Lehmann, Paul V; Diana R. Roen; Chenggang Jin

    2013-01-01

    Lyme Borreliosis is an infectious disease caused by the spirochete Borrelia burgdorferi that is transmitted through the bite of infected ticks. Both B cell-mediated humoral immunity and T cell immunity develop during natural Borrelia infection. However, compared with humoral immunity, the T cell response to Borrelia infection has not been well elucidated. In this study, a novel T cell-based assay was developed and validated for the sensitive detection of antigen-specific T cell response to B....

  18. Thyroperoxidase, an auto-antigen with a mosaic structure made of nuclear and mitochondrial gene modules.

    OpenAIRE

    Libert, F; Ruel, J.; Ludgate, M; Swillens, S; Alexander, N.; Vassart, G.; Dinsart, C

    1987-01-01

    A lambda gt11 cDNA library was constructed from a normal human thyroid and screened with a rabbit anti-porcine thyroperoxidase antibody. A series of thyroperoxidase (TPO) clones were obtained which allowed determination of the complete primary structure of the protein. The library was also screened with serum from a patient with Hashimoto's thyroiditis, an autoimmune disease characterized by the presence in the serum of high titers of autoantibodies directed against the 'microsomal antigen' (...

  19. Infection of SCID mice with Mycobacterium leprae and control with antigen-activated "immune" human peripheral blood mononuclear cells.

    Science.gov (United States)

    Converse, P J; Haines, V L; Wondimu, A; Craig, L E; Meyers, W M

    1995-03-01

    The SCID (severe combined immunodeficient) mouse lacks both B and T cells and tolerates injected mononuclear cells from humans, the principal hosts of Mycobacterium leprae. A SCID mouse model of leprosy could be useful to investigate potential vaccine strategies using human cells in a context in which the growth of the organism is monitored. Initial experiments determined that SCID mice are more susceptible than normal mice to infection and dissemination of M. leprae. Cells from humans, either BCG vaccinated or from countries where leprosy is endemic, were stimulated in vitro with a number of mycobacterial antigens--whole M. leprae, M. leprae cell walls, purified protein derivative of M. tuberculosis, and Mycobacterium bovis BCG--and tested for proliferation and production of interleukin-6, tumor necrosis factor alpha, and gamma interferon. Cell walls were the most efficient and consistent in inducing all of these activities. In vitro-activated human cells retain function better after injection into SCID mice than nonactivated cells. To test the ability of cells to affect the growth of M. leprae in the footpads of SCID mice, cells from a known responder to mycobacterial antigens and from a nonresponder were activated by M. leprae cell wall antigens. The cells were harvested and coinjected with fresh M. leprae into the right hind footpads of SCID mice. After 3 months, there was no growth of M. leprae in the footpads of mice coinjected with cells from the mycobacterial antigen responder, while growth was uninhibited in mice receiving cells from the nonresponder. Future experiments will determine requirements for antigen specificity in inhibiting M. leprae multiplication. PMID:7868226

  20. Kinetics of antigen specific and non-specific polyclonal B-cell responses during lethal Plasmodium yoelii malaria

    Directory of Open Access Journals (Sweden)

    Laurence Rolland

    1992-06-01

    Full Text Available In order to study the kinetics and composition of the polyclonal B-cell activation associated to malaria infection, antigen-specific and non-specific B-cell responses were evaluated in the spleens of mice infected with Plasmodium yoelii 17 XL or injected with lysed erythrocytes or plasma from P. yoelii infected mice or with P. falciparum culture supernatants. Spleen/body weigth ratio, numbers of nucleated spleen cells and Immunoglobulin-containing and Immunoglobulin-secreting cells increased progressively during the course of infection,in parallel to the parasitemia. A different pattern of kinetics was observed when anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell plaque forming cells response were studied: maximum values were observed at early stages of infection, whereas the number of total Immunoglobulin-containing and Immunoglobulin-secreting cells were not yet altered. Conversely, at the end of infection, when these latter values reached their maximum, the anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell specific responses were normal or even infranormal. In mice injected with Plasmodium-derived material, a higher increase in antigen-specific PFC was observed, as compared to the increase of Immunoglobulin-containing and Immunoglobulin-secreting cell numbers. This suggested a "preferential" (antigen-plus mitogen-induced stimulation of antigen-specific cells rather than a generalized non-specific (mitogen-induced triggering of B-lymphocytes. On the basis of these and previous results, it is suggested that polyclonal B-cell activation that takes place during the course of infection appears as a result of successive waves of antigen-specific B-cell activation.

  1. How squalene GLAdly helps generate antigen-specific T cells via antigen-carrying neutrophils and IL-18

    OpenAIRE

    Kedl, Justin D.; Kedl, Ross M.

    2015-01-01

    The mechanisms by which squalene, which in oil-and-water emulsions has been shown to be an excellent formulation for TLR agonists, enhances the magnitude and quality of adaptive immune responses are not thoroughly defined. In this issue of the European Journal of Immunology [Eur. J. Immunol. 2015. 45: XXXX-XXXX], Desbien et al. show that a squalene/TLR4-based adjuvant augments antigen-specific Th1 responses in vaccinated mice through a caspase/IL-18-dependent mechanism. This commentary will d...

  2. Improved method for linear B-cell epitope prediction using antigen's primary sequence.

    Directory of Open Access Journals (Sweden)

    Harinder Singh

    Full Text Available One of the major challenges in designing a peptide-based vaccine is the identification of antigenic regions in an antigen that can stimulate B-cell's response, also called B-cell epitopes. In the past, several methods have been developed for the prediction of conformational and linear (or continuous B-cell epitopes. However, the existing methods for predicting linear B-cell epitopes are far from perfection. In this study, an attempt has been made to develop an improved method for predicting linear B-cell epitopes. We have retrieved experimentally validated B-cell epitopes as well as non B-cell epitopes from Immune Epitope Database and derived two types of datasets called Lbtope_Variable and Lbtope_Fixed length datasets. The Lbtope_Variable dataset contains 14876 B-cell epitope and 23321 non-epitopes of variable length where as Lbtope_Fixed length dataset contains 12063 B-cell epitopes and 20589 non-epitopes of fixed length. We also evaluated the performance of models on above datasets after removing highly identical peptides from the datasets. In addition, we have derived third dataset Lbtope_Confirm having 1042 epitopes and 1795 non-epitopes where each epitope or non-epitope has been experimentally validated in at least two studies. A number of models have been developed to discriminate epitopes and non-epitopes using different machine-learning techniques like Support Vector Machine, and K-Nearest Neighbor. We achieved accuracy from ∼54% to 86% using diverse s features like binary profile, dipeptide composition, AAP (amino acid pair profile. In this study, for the first time experimentally validated non B-cell epitopes have been used for developing method for predicting linear B-cell epitopes. In previous studies, random peptides have been used as non B-cell epitopes. In order to provide service to scientific community, a web server LBtope has been developed for predicting and designing B-cell epitopes (http://crdd.osdd.net/raghava/lbtope/.

  3. Epstein–Barr virus nuclear antigen 3C interact with p73: Interplay between a viral oncoprotein and cellular tumor suppressor

    International Nuclear Information System (INIS)

    The p73 protein has structural and functional homology with the tumor suppressor p53, which plays an important role in cell cycle regulation, apoptosis, and DNA repair. The p73 locus encodes both a tumor suppressor (TAp73) and a putative oncogene (ΔNp73). p73 May play a significant role in p53-deficient lymphomas infected with Epstein–Barr virus (EBV). EBV produces an asymptomatic infection in the majority of the global population, but it is associated with several human B-cell malignancies. The EBV-encoded Epstein–Barr virus nuclear antigen 3C (EBNA3C) is thought to disrupt the cell cycle checkpoint by interacting directly with p53 family proteins. Doxorubicin, a commonly used chemotherapeutic agent, induces apoptosis through p53 and p73 signaling such that the lowΔNp73 level promotes the p73-mediated intrinsic pathway of apoptosis. In this report, we investigated the mechanism by which EBV infection counters p73α-induced apoptosis through EBNA3C. - Highlights: • EBV-encoded EBNA3C suppresses doxorubicin-induced apoptosis in B-cell lymphomas. • EBNA3C binds to p73 to suppress its apoptotic effect. • EBNA3C maintains latency by regulating downstream mitochondrial pathways

  4. Epstein–Barr virus nuclear antigen 3C interact with p73: Interplay between a viral oncoprotein and cellular tumor suppressor

    Energy Technology Data Exchange (ETDEWEB)

    Sahu, Sushil Kumar; Mohanty, Suchitra; Kumar, Amit [Division of Infectious Disease Biology, Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar 751023 (India); Kundu, Chanakya N. [School of Biotechnology, KIIT University, Bhubaneswar (India); Verma, Subhash C. [Department of Microbiology and Immunology, University of Nevada, School of Medicine, Reno, NV 89557 (United States); Choudhuri, Tathagata, E-mail: tatha@ils.res.in [Division of Infectious Disease Biology, Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar 751023 (India); Department of Biotechnology, Siksha Bhavana, Visva Bharati, Santiniketan, Bolpur (India)

    2014-01-05

    The p73 protein has structural and functional homology with the tumor suppressor p53, which plays an important role in cell cycle regulation, apoptosis, and DNA repair. The p73 locus encodes both a tumor suppressor (TAp73) and a putative oncogene (ΔNp73). p73 May play a significant role in p53-deficient lymphomas infected with Epstein–Barr virus (EBV). EBV produces an asymptomatic infection in the majority of the global population, but it is associated with several human B-cell malignancies. The EBV-encoded Epstein–Barr virus nuclear antigen 3C (EBNA3C) is thought to disrupt the cell cycle checkpoint by interacting directly with p53 family proteins. Doxorubicin, a commonly used chemotherapeutic agent, induces apoptosis through p53 and p73 signaling such that the lowΔNp73 level promotes the p73-mediated intrinsic pathway of apoptosis. In this report, we investigated the mechanism by which EBV infection counters p73α-induced apoptosis through EBNA3C. - Highlights: • EBV-encoded EBNA3C suppresses doxorubicin-induced apoptosis in B-cell lymphomas. • EBNA3C binds to p73 to suppress its apoptotic effect. • EBNA3C maintains latency by regulating downstream mitochondrial pathways.

  5. A Fusion Protein between Streptavidin and the Endogenous TLR4 Ligand EDA Targets Biotinylated Antigens to Dendritic Cells and Induces T Cell Responses In Vivo

    Directory of Open Access Journals (Sweden)

    Laura Arribillaga

    2013-01-01

    Full Text Available The development of tools for efficient targeting of antigens to antigen presenting cells is of great importance for vaccine development. We have previously shown that fusion proteins containing antigens fused to the extra domain A from fibronectin (EDA, an endogenous TLR4 ligand, which targets antigens to TLR4-expressing dendritic cells (DC, are highly immunogenic. To facilitate the procedure of joining EDA to any antigen of choice, we have prepared the fusion protein EDAvidin by linking EDA to the N terminus of streptavidin, allowing its conjugation with biotinylated antigens. We found that EDAvidin, as streptavidin, forms tetramers and binds biotin or biotinylated proteins with a Kd ~ 2.6 × 10−14 mol/L. EDAvidin favours the uptake of biotinylated green fluorescent protein by DC. Moreover, EDAvidin retains the proinflammatory properties of EDA, inducing NF-κβ by TLR4-expressing cells, as well as the production of TNF-α by the human monocyte cell line THP1 and IL-12 by DC. More importantly, immunization of mice with EDAvidin conjugated with the biotinylated nonstructural NS3 protein from hepatitis C virus induces a strong anti-NS3 T cell immune response. These results open a new way to use the EDA-based delivery tool to target any antigen of choice to DC for vaccination against infectious diseases and cancer.

  6. A fusion protein between streptavidin and the endogenous TLR4 ligand EDA targets biotinylated antigens to dendritic cells and induces T cell responses in vivo.

    Science.gov (United States)

    Arribillaga, Laura; Durantez, Maika; Lozano, Teresa; Rudilla, Francesc; Rehberger, Federico; Casares, Noelia; Villanueva, Lorea; Martinez, Marta; Gorraiz, Marta; Borrás-Cuesta, Francisco; Sarobe, Pablo; Prieto, Jesús; Lasarte, Juan José

    2013-01-01

    The development of tools for efficient targeting of antigens to antigen presenting cells is of great importance for vaccine development. We have previously shown that fusion proteins containing antigens fused to the extra domain A from fibronectin (EDA), an endogenous TLR4 ligand, which targets antigens to TLR4-expressing dendritic cells (DC), are highly immunogenic. To facilitate the procedure of joining EDA to any antigen of choice, we have prepared the fusion protein EDAvidin by linking EDA to the N terminus of streptavidin, allowing its conjugation with biotinylated antigens. We found that EDAvidin, as streptavidin, forms tetramers and binds biotin or biotinylated proteins with a Kd ~ 2.6 × 10(-14) mol/L. EDAvidin favours the uptake of biotinylated green fluorescent protein by DC. Moreover, EDAvidin retains the proinflammatory properties of EDA, inducing NF- κβ by TLR4-expressing cells, as well as the production of TNF- α by the human monocyte cell line THP1 and IL-12 by DC. More importantly, immunization of mice with EDAvidin conjugated with the biotinylated nonstructural NS3 protein from hepatitis C virus induces a strong anti-NS3 T cell immune response. These results open a new way to use the EDA-based delivery tool to target any antigen of choice to DC for vaccination against infectious diseases and cancer. PMID:24093105

  7. IDENTIFICATION OF ACROSOME AS THE MAIN ANTIGEN OF THE SPERM CELLS PROVOKING AUTOANTIBODIES IN VASECTOMIZED IRANIAN MEN

    Directory of Open Access Journals (Sweden)

    M R Nowroozi

    2008-12-01

    Full Text Available "nVasectomy is one of the extensively used methods of contraception in family planning programs. Antisperm antibodies (ASA develop after vasectomy which can result in auto-immune male infertility. The precise sperm antigens involved in the autoimmune response are still poorly defined, therefore we determined the circulating ASA and identified relevant sperm antigens based on localization of binding sites of ASA to sperm cell antigens, using a rapid, inexpensive and clinically relevant assay in vasectomized men. Results showed that 2.5% of men had ASA at the time of vasectomy, whereas 53.5% of the study population subsequently developed ASA. The numbers of men with circulating ASA increased significantly for the first three months after vasectomy. These antibodies were distinguishable into three groups based on their bindings to different sites of sperm cell antigens including against acrosome and tail in 67.56% and 10.8%, respectively; 21.6% of subjects had antibody to the other parts of the sperm cell antigens. The results of this study are discussed in terms of an autoimmune response against sperm antigens and development of ASA.

  8. Cytomegalovirus-infected cells express Leu-M1 antigen. A potential source of diagnostic error.

    OpenAIRE

    Rushin, J. M.; Riordan, G. P.; Heaton, R. B.; Sharpe, R. W.; Cotelingam, J. D.; Jaffe, E S

    1990-01-01

    The authors examined cytomegalovirus (CMV)-infected tissues and Hodgkin's Disease (HD) cases with immunohistochemical assays for Leu-M1 and CMV. The cytologic characteristics were correlated with immunostaining patterns. Cytomegalovirus-infected cells in lymph node, lung, and esophagus sections showed Cowdry type A inclusions, and many had granular cytoplasmic inclusions. All infected cells showed nuclear staining with an anti-CMV antibody. Leu-M1 reacted with CMV-infected cells in cytoplasmi...

  9. Cell cycle-dependent expression of Ki-67 antigen in human melanoma cells subjected to irradiation and/or hyperthermia

    International Nuclear Information System (INIS)

    The proliferation of human melanoma cells in vitro during the first 3 days after irradiation and/or hyperthermia was followed by two-parameter flow cytometry combining cell cycle analysis on the basis of DNA content with Ki-67 antibody labeling. It was found that cells arrested or delayed in the S and G2 phases of the cell cycle were Ki-67-positive in spite of the antigen's very short half-life. Thus Ki-67 staining failed to reflect those changes in cell proliferation which typically occur in the course of a fractionated radiotherapy as well as those expected in the case of hyperthermia or a combined treatment. 24 refs., 3 figs., 1 tab

  10. Chimeric Antigen Receptor-Modified T Cells for Solid Tumors: Challenges and Prospects

    Directory of Open Access Journals (Sweden)

    Yelei Guo

    2016-01-01

    Full Text Available Recent studies have highlighted the successes of chimeric antigen receptor-modified T- (CART- cell-based therapy for B-cell malignancies, and early phase clinical trials have been launched in recent years. The few published clinical studies of CART cells in solid tumors have addressed safety and feasibility, but the clinical outcome data are limited. Although antitumor effects were confirmed in vitro and in animal models, CART-cell-based therapy still faces several challenges when directed towards solid tumors, and it has been difficult to achieve the desired outcomes in clinical practice. Many studies have struggled to improve the clinical responses to and benefits of CART-cell treatment of solid tumors. In this review, the status quo of CART cells and their clinical applications for solid tumors will be summarized first. Importantly, we will suggest improvements that could increase the therapeutic effectiveness of CART cells for solid tumors and their future clinical applications. These interventions will make treatment with CART cells an effective and routine therapy for solid tumors.

  11. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia.

    Science.gov (United States)

    Kalos, Michael; Levine, Bruce L; Porter, David L; Katz, Sharyn; Grupp, Stephan A; Bagg, Adam; June, Carl H

    2011-08-10

    Tumor immunotherapy with T lymphocytes, which can recognize and destroy malignant cells, has been limited by the ability to isolate and expand T cells restricted to tumor-associated antigens. Chimeric antigen receptors (CARs) composed of antibody binding domains connected to domains that activate T cells could overcome tolerance by allowing T cells to respond to cell surface antigens; however, to date, lymphocytes engineered to express CARs have demonstrated minimal in vivo expansion and antitumor effects in clinical trials. We report that CAR T cells that target CD19 and contain a costimulatory domain from CD137 and the T cell receptor ζ chain have potent non-cross-resistant clinical activity after infusion in three of three patients treated with advanced chronic lymphocytic leukemia (CLL). The engineered T cells expanded >1000-fold in vivo, trafficked to bone marrow, and continued to express functional CARs at high levels for at least 6 months. Evidence for on-target toxicity included B cell aplasia as well as decreased numbers of plasma cells and hypogammaglobulinemia. On average, each infused CAR-expressing T cell was calculated to eradicate at least 1000 CLL cells. Furthermore, a CD19-specific immune response was demonstrated in the blood and bone marrow, accompanied by complete remission, in two of three patients. Moreover, a portion of these cells persisted as memory CAR(+) T cells and retained anti-CD19 effector functionality, indicating the potential of this major histocompatibility complex-independent approach for the effective treatment of B cell malignancies. PMID:21832238

  12. Bifidobacteria Enhance Antigen Sampling and Processing by Dendritic Cells in Pediatric Inflammatory Bowel Disease.

    Science.gov (United States)

    Strisciuglio, Caterina; Miele, Erasmo; Giugliano, Francesca P; Vitale, Serena; Andreozzi, Marialuisa; Vitale, Alessandra; Catania, Maria R; Staiano, Annamaria; Troncone, Riccardo; Gianfrani, Carmen

    2015-07-01

    Bifidobacteria have been reported to reduce inflammation and contribute to intestinal homeostasis. However, the interaction between these bacteria and the gut immune system remains largely unknown. Because of the central role played by dendritic cells (DCs) in immune responses, we examined in vitro the effects of a Bifidobacteria mixture (probiotic) on DC functionality from children with inflammatory bowel disease. DCs obtained from peripheral blood monocytes of patients with Crohn's disease (CD), ulcerative colitis, and noninflammatory bowel disease controls (HC) were incubated with fluorochrome-conjugated particles of Escherichia coli or DQ-Ovalbumin (DQ-OVA) after a pretreatment with the probiotic, to evaluate DC phenotype, antigen sampling and processing. Moreover, cell supernatants were collected to measure tumor necrosis factor alpha, interferon gamma, interleukin 17, and interleukin 10 production by enzyme-linked immunosorbent assay. DCs from CD children showed a higher bacteria particles uptake and DQ-OVA processing after incubation with the probiotic; in contrast, DC from both ulcerative colitis and HC showed no significant changes. Moreover, a marked tumor necrosis factor alpha release was observed in DC from CD after exposure to E. coli particles, whereas the probiotic did not affect the production of this proinflammatory cytokine. In conclusion, the Bifidobacteria significantly improved the antigen uptake and processing by DCs from patients with CD, which are known to present an impaired autophagic functionality, whereas, in DCs from ulcerative colitis and HC, no prominent effect of probiotic mixture was observed. This improvement of antigen sampling and processing could partially solve the impairment of intestinal innate immunity and reduce uncontrolled microorganism growth in the intestine of children with inflammatory bowel disease. PMID:25895109

  13. Nitric Oxide Limits the Expansion of Antigen-Specific T Cells in Mice Infected with the Microfilariae of Brugia pahangi

    Science.gov (United States)

    O'Connor, Richard A.; Devaney, Eileen

    2002-01-01

    Infection of BALB/c mice with the microfilariae (Mf) of the filarial nematode Brugia pahangi results in an antigen-specific proliferative defect that is induced by high levels of NO. Using carboxyfluorescein diacetate succinimydl ester and cell surface labeling, it was possible to identify a population of antigen-specific T cells from Mf-infected BALB/c mice that expressed particularly high levels of CD4 (CD4hi). These cells proliferated in culture only when inducible NO synthase was inhibited and accounted for almost all of the antigen-specific proliferative response under those conditions. CD4hi cells also expressed high levels of CD44, consistent with their status as activated T cells. A similar population of CD4hi cells was observed in cultures from Mf-infected gamma interferon receptor knockout (IFN-γR−/−) mice. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling staining revealed that the CD4+ T cells from Mf-infected wild-type mice were preferentially susceptible to apoptosis compared to CD4+ T cells from IFN-γR−/− mice. These studies suggest that the expansion of antigen-specific T cells in Mf-infected mice is limited by NO. PMID:12379675

  14. B cells pulsed with Helicobacter pylori antigen efficiently activate memory CD8+ T cells from H. pylori-infected individuals.

    Science.gov (United States)

    Azem, Josef; Svennerholm, Ann-Mari; Lundin, B Samuel

    2006-01-01

    Helicobacter pylori infection causes chronic gastritis that may progress to peptic ulcers or gastric adenocarcinoma and thereby cause major world-wide health problems. Previous studies have shown that CD4+ T cells are important in the immune response to H. pylori in humans, but the role of CD8+ T cells is less clear. In order to study the CD8+ T cell response to H. pylori in greater detail, we have evaluated efficient conditions for activation of CD8+ T cells in vitro. We show that H. pylori-reactive CD8+ T cells can be activated most efficiently by B cells or dendritic cells pulsed with H. pylori antigens. We further show that the majority of CD8+ T cells in H. pylori-infected gastric mucosa are memory cells, and that memory CD8+ T cells sorted from peripheral blood of H. pylori-infected individuals respond 15-fold more to H. pylori urease compared to memory cells from uninfected subjects. We conclude that CD8+ T cells do participate in the immune response to H. pylori, and this may have implications for the development of more severe disease outcomes in H. pylori-infected subjects. PMID:16324887

  15. Amplification of rabies virus-induced stimulation of human T-cell lines and clones by antigen-specific antibodies.

    OpenAIRE

    Celis, E; Wiktor, T J; Dietzschold, B.; Koprowski, H

    1985-01-01

    The effect of antigen-specific antibodies on the response of human T-cell lines and clones to rabies virus was studied. Plasmas from rabies-immune vaccine recipients, but not those from nonimmune individuals, enhanced the proliferative response of rabies-reactive T cells to whole inactivated virus or to the purified glycoprotein and nucleocapsid from the rabies virion. Rabies-immune plasma also increased the antigen-induced production of gamma interferon by the rabies-specific T-cell lines. E...

  16. Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD19 can eradicate lymphoma and normal B cells

    OpenAIRE

    Kochenderfer, James N.; Yu, Zhiya; Frasheri, Dorina; Restifo, Nicholas P; Rosenberg, Steven A.

    2010-01-01

    Adoptive T-cell therapy with anti-CD19 chimeric antigen receptor (CAR)–expressing T cells is a new approach for treating advanced B-cell malignancies. To evaluate anti-CD19–CAR-transduced T cells in a murine model of adoptive T-cell therapy, we developed a CAR that specifically recognized murine CD19. We used T cells that were retrovirally transduced with this CAR to treat mice bearing a syngeneic lymphoma that naturally expressed the self-antigen murine CD19. One infusion of anti-CD19–CAR-tr...

  17. Lipopolysaccharide O-antigen prevents phagocytosis of Vibrio anguillarum by rainbow trout (Oncorhynchus mykiss skin epithelial cells.

    Directory of Open Access Journals (Sweden)

    Kristoffer Lindell

    Full Text Available Colonization of host tissues is a first step taken by many pathogens during the initial stages of infection. Despite the impact of bacterial disease on wild and farmed fish, only a few direct studies have characterized bacterial factors required for colonization of fish tissues. In this study, using live-cell and confocal microscopy, rainbow trout skin epithelial cells, the main structural component of the skin epidermis, were demonstrated to phagocytize bacteria. Mutant analyses showed that the fish pathogen Vibrio anguillarum required the lipopolysaccharide O-antigen to evade phagocytosis and that O-antigen transport required the putative wzm-wzt-wbhA operon, which encodes two ABC polysaccharide transporter proteins and a methyltransferase. Pretreatment of the epithelial cells with mannose prevented phagocytosis of V. anguillarum suggesting that a mannose receptor is involved in the uptake process. In addition, the O-antigen transport mutants could not colonize the skin but they did colonize the intestines of rainbow trout. The O-antigen polysaccharides were also shown to aid resistance to the antimicrobial factors, lysozyme and polymyxin B. In summary, rainbow trout skin epithelial cells play a role in the fish innate immunity by clearing bacteria from the skin epidermis. In defense, V. anguillarum utilizes O-antigen polysaccharides to evade phagocytosis by the epithelial cells allowing it to colonize rapidly fish skin tissues.

  18. Antigen presentation by small intestinal epithelial cells uniquely enhances IFN-γ secretion from CD4+ intestinal intraepithelial lymphocytes

    International Nuclear Information System (INIS)

    Highlights: •Small intestinal epithelial cells (sIECs). •sIECs are able to induce antigen specific proliferation of CD4+ IELs. •sIECs induce markedly enhanced IFN-γ secretion by CD4+ IELs. •Induction of enhanced IFN-γ secretion by sIECs is uniquely observed in CD4+ IELs. -- Abstract: Small intestinal epithelial cells (sIECs) express major histocompatibility complex class II molecules even in a normal condition, and are known to function as antigen presenting cells (APCs) at least in vitro. These findings raised the possibility that sIECs play an important role in inducing immune responses against luminal antigens, especially those of intestinal intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs). We herein showed that antigenic stimulation with sIECs induced markedly greater secretion of interferon-gamma (IFN-γ) by CD4+ IELs, but not interleukin (IL)-4, IL-10 and IL-17 although the proliferative response was prominently lower than that with T cell-depleted splenic APCs. In contrast, no enhanced IFN-γ secretion by CD4+ LPLs and primed splenic CD4+ T cells was observed when stimulated with sIECs. Taken together, these results suggest that sIECs uniquely activate CD4+ IELs and induce remarkable IFN-γ secretion upon antigenic stimulation in vivo

  19. Antigen presentation by small intestinal epithelial cells uniquely enhances IFN-γ secretion from CD4{sup +} intestinal intraepithelial lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Hatano, Ryo; Yamada, Kiyoshi; Iwamoto, Taku; Maeda, Nana; Emoto, Tetsuro; Shimizu, Makoto; Totsuka, Mamoru, E-mail: atotuka@mail.ecc.u-tokyo.ac.jp

    2013-06-14

    Highlights: •Small intestinal epithelial cells (sIECs). •sIECs are able to induce antigen specific proliferation of CD4{sup +} IELs. •sIECs induce markedly enhanced IFN-γ secretion by CD4{sup +} IELs. •Induction of enhanced IFN-γ secretion by sIECs is uniquely observed in CD4{sup +} IELs. -- Abstract: Small intestinal epithelial cells (sIECs) express major histocompatibility complex class II molecules even in a normal condition, and are known to function as antigen presenting cells (APCs) at least in vitro. These findings raised the possibility that sIECs play an important role in inducing immune responses against luminal antigens, especially those of intestinal intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs). We herein showed that antigenic stimulation with sIECs induced markedly greater secretion of interferon-gamma (IFN-γ) by CD4{sup +} IELs, but not interleukin (IL)-4, IL-10 and IL-17 although the proliferative response was prominently lower than that with T cell-depleted splenic APCs. In contrast, no enhanced IFN-γ secretion by CD4{sup +} LPLs and primed splenic CD4{sup +} T cells was observed when stimulated with sIECs. Taken together, these results suggest that sIECs uniquely activate CD4{sup +} IELs and induce remarkable IFN-γ secretion upon antigenic stimulation in vivo.

  20. Paper-based assay for red blood cell antigen typing by the indirect antiglobulin test.

    Science.gov (United States)

    Yeow, Natasha; McLiesh, Heather; Guan, Liyun; Shen, Wei; Garnier, Gil

    2016-07-01

    A rapid and simple paper-based elution assay for red blood cell antigen typing by the indirect antiglobulin test (IAT) was established. This allows to type blood using IgG antibodies for the important blood groups in which IgM antibodies do not exist. Red blood cells incubated with IgG anti-D were washed with saline and spotted onto the paper assay pre-treated with anti-IgG. The blood spot was eluted with an elution buffer solution in a chromatography tank. Positive samples were identified by the agglutinated and fixed red blood cells on the original spotting area, while red blood cells from negative samples completely eluted away from the spot of origin. Optimum concentrations for both anti-IgG and anti-D were identified to eliminate the washing step after the incubation phase. Based on the no-washing procedure, the critical variables were investigated to establish the optimal conditions for the paper-based assay. Two hundred ten donor blood samples were tested in optimal conditions for the paper test with anti-D and anti-Kell. Positive and negative samples were clearly distinguished. This assay opens up new applications of the IAT on paper including antibody detection and blood donor-recipient crossmatching and extends its uses into non-blood typing applications with IgG antibody-based diagnostics. Graphical abstract A rapid and simple paper-based assay for red blood cell antigen typing by the indirect antiglobulin test. PMID:27185543