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Sample records for carcinoembryonic antigen expression

  1. Antigenic sites in carcinoembryonic antigen.

    PubMed

    Hammarstrom, S; Shively, J E; Paxton, R J; Beatty, B G; Larsson, A; Ghosh, R; Bormer, O; Buchegger, F; Mach, J P; Burtin, P

    1989-09-01

    The epitope reactivities of 52 well-characterized monoclonal antibodies (Mabs) against carcinoembryonic antigen from 11 different research groups were studied using competitive solid-phase immunoassays. About 60% of all possible combinations of Mabs as inhibitors and as the primary binding antibody were investigated. The inhibition data were analyzed by a specially developed computer program "EPITOPES" which measures concordance and discordance in inhibition patterns between Mabs. The analysis showed that 43 of the 52 Mabs (83%) could be classified into one of five essentially noninteracting epitope groups (GOLD 1-5) containing between four and 15 Mabs each. The epitopes recognized by the Mabs belonging to groups 1 to 5 were peptide in nature. With one or two possible exceptions non-classifiable Mabs were either directed against carbohydrate epitopes (4 Mabs) or were inactive in the tests used. Within epitope groups GOLD 1, 4, and 5 two partially overlapping subgroups were distinguished. Mabs with a high degree of carcinoembryonic antigen specificity generally belonged to epitope groups GOLD 1 and 3. PMID:2474375

  2. CD66 carcinoembryonic antigens mediate interactions between Opa-expressing Neisseria gonorrhoeae and human polymorphonuclear phagocytes.

    PubMed

    Gray-Owen, S D; Dehio, C; Haude, A; Grunert, F; Meyer, T F

    1997-06-16

    Colonization of urogenital tissues by the human pathogen Neisseria gonorrhoeae is characteristically associated with purulent exudates of polymorphonuclear phagocytes (PMNs) containing apparently viable bacteria. Distinct variant forms of the phase-variable opacity-associated (Opa) outer membrane proteins mediate the non-opsonized binding and internalization of N. gonorrhoeae by human PMNs. Using overlay assays and an affinity isolation technique, we demonstrate the direct interaction between Opa52-expressing gonococci and members of the human carcinoembryonic antigen (CEA) family which express the CD66 epitope. Gonococci and recombinant Escherichia coli strains synthesizing Opa52 showed specific binding and internalization by transfected HeLa cell lines expressing the CD66 family members BGP (CD66a), NCA (CD66c), CGM1 (CD66d) and CEA (CD66e), but not that expressing CGM6 (CD66b). Bacterial strains expressing either no opacity protein or the epithelial cell invasion-associated Opa50 do not bind these CEA family members. Consistent with their different receptor specificities, Opa52-mediated interactions could be inhibited by polyclonal anti-CEA sera, while Opa50 binding was instead inhibited by heparin. Using confocal laser scanning microscopy, we observed a marked recruitment of CD66 antigen by Opa52-expressing gonococci on both the transfected cell lines and infected PMNs. These data indicate that members of the CEA family constitute the cellular receptors for the interaction with, and internalization of, N. gonorrhoeae. PMID:9218786

  3. Induction of carcinoembryonic antigen expression in a three-dimensional culture system

    NASA Technical Reports Server (NTRS)

    Jessup, J. M.; Brown, D.; Fitzgerald, W.; Ford, R. D.; Nachman, A.; Goodwin, T. J.; Spaulding, G.

    1994-01-01

    MIP-101 is a poorly differentiated human colon carcinoma cell line established from ascites that produces minimal amounts of carcinoembryonic antigen (CEA), a 180 kDa glycoprotein tumor marker, and nonspecific cross-reacting antigen (NCA), a related protein that has 50 and 90 kDa isoforms, in vitro in monolayer culture. MIP-101 produces CEA when implanted into the peritoneum of nude mice but not when implanted into subcutaneous tissue. We tested whether MIP-101 cells may be induced to express CEA when cultured on microcarrier beads in three-dimensional cultures, either in static cultures as non-adherent aggregates or under dynamic conditions in a NASA-designed low shear stress bioreactor. MIP- 101 cells proliferated well under all three conditions and increased CEA and NCA production 3 - 4 fold when grown in three-dimensional cultures compared to MIP-101 cells growing logarithmically in monolayers. These results suggest that three-dimensional growth in vitro simulates tumor function in vivo and that three-dimensional growth by itself may enhance production of molecules that are associated with the metastatic process.

  4. Carcinoembryonic Antigen Expression and Resistance to Radiation and 5-Fluorouracil-Induced Apoptosis and Autophagy.

    PubMed

    Eftekhar, Ebrahim; Jaberie, Hajar; Naghibalhossaini, Fakhraddin

    2016-01-01

    Understanding the mechanism of tumor resistance is critical for cancer therapy. In this study, we investigated the effect of carcinoembryonic antigen (CEA) overexpression on UV-and 5-fluorouracil (5-FU)-induced apoptosis and autophagy in colorectal cancer cells. We used histone deacetylase (HDAC) inhibitor, NaB and DNA demethylating agent, 5-azacytidine (5-AZA) to induce CEA expression in HT29/219 and SW742 colorectal cancer cell lines. MTT assay was used to measure IC50 value of the cells exposed to graded concentrations of 5- FU with either 0.1 mM NaB or 1 μM 5-AZA for 72 h . Using CHO- and SW742-CEA transfectants, we also investigated the effect of CEA expression on UV- and 5-FU-induced apoptosis and autophagy. Treatment of HT29/219 cell line with NaB and 5-AZA increased CEA expression by 29% and 31%, respectively. Compared with control cells, the IC50 value for 5-FU of NaB and 5-AZA-treated cells increased by 40% and 57%, respectively. Treatment of SW742 cells with NaB or 5-AZA increased neither CEA expression nor the IC50 value for 5-FU. In comparison to parental cells, CEA expression also significantly protected transfected cells against UV-induced apoptosis. Decreased proportions of autophagy and apoptosis were also observed in 5-FU treated SW742- and CHO-CEA transfectants. We conclude that CEA expression can effectively protect colorectal cancer cells against radiation and drug-induced apoptosis and autophagy. PMID:27478804

  5. Carcinoembryonic antigen-related cell adhesion molecule 1 is expressed and as a function histotype in ovarian tumors.

    PubMed

    Li, Ning; Yang, Jing-Yan; Wang, Xiao-Ying; Wang, Hai-Tao; Guan, Bing-Xin; Zhou, Cheng-Jun

    2016-02-01

    Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a cell-cell adhesion receptor and is implicated in several cellular functions. It is rarely reported in ovarian tumors. The aim of this study is to determine the expression of CEACAM1 in ovarian tumors, trying to see whether CEACAM1 has different expression patterns as a function of histotype. Antigen expression was examined by immunohistochemistry with mouse anti-human antibody for CEACAM1. Immunohistochemistry was performed using avidin-biotin-diaminobenzide staining. The results were expressed as average score ± SD (0, negative; 8, highest) for each histotype. In ovarian tumors, the benign serous and mucinous cystadenoma negatively or weakly expressed CEACAM1, the malignant epithelial tumors strongly expressed CEACAM1, and there was significant difference between benign epithelial tumor and adenocarcinoma (P < .05). The well-differentiated serous adenocarcinoma expressed CEACAM1 mainly with membrane pattern, and the intermediately and poorly differentiated serous adenocarcinomas expressed CEACAM1 mainly with cytoplasmic pattern (P < .05). In addition, CEACAM1 expression is elevated in solid tumors of ovary but variable as a function of histotype. Compared with membranous expression, the cytoplasmic expression was observed almost in metastatic carcinoma that might decrease the adhesive interactions of the carcinoma cells with the surrounding cells, especially with tumor cells, and this could facilitate the tumor cells to metastasize to distant regions. So, we thought that cytoplasmic CEACAM1 might play an important role in tumor progression, especially in tumor metastasis. PMID:26653024

  6. Regulation by gut commensal bacteria of carcinoembryonic antigen-related cell adhesion molecule expression in the intestinal epithelium.

    PubMed

    Kitamura, Yasuaki; Murata, Yoji; Park, Jung-Ha; Kotani, Takenori; Imada, Shinya; Saito, Yasuyuki; Okazawa, Hideki; Azuma, Takeshi; Matozaki, Takashi

    2015-07-01

    Carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 1 and CEACAM20, immunoglobulin superfamily members, are predominantly expressed in intestinal epithelial cells (IECs) and co-localized at the apical surface of these cells. We here showed that the expression of mouse CEACAM1 and CEACAM20 at both mRNA and protein levels was markedly reduced in IECs of the small intestine by the treatment of mice with antibiotics against Gram-positive bacteria. The expression of both proteins was also decreased in IECs of the small intestine from germ-free mice, compared with that from control specific-pathogen-free mice. Exposure of intestinal organoids to IFN-γ markedly increased the expression of either CEACAM1 or CEACAM20, whereas the exposure to TNF-α increased the expression of the former protein, but not that of the latter. In contrast, the expression of CEACAM20, but not of CEACAM1, in intestinal organoids was markedly increased by exposure to butyrate, a short-chain fatty acid produced by bacterial fermentation in the intestine. Collectively, our results suggest that Gram-positive bacteria promote the mRNA expression of CEACAM1 or CEACAM20 in the small intestine. Inflammatory cytokines or butyrate likely participates in such effects of commensal bacteria. PMID:25908210

  7. Immunomodulatory roles of the carcinoembryonic antigen family of glycoproteins.

    PubMed

    Shao, Ling; Allez, Matthieu; Park, Mee-Sook; Mayer, Lloyd

    2006-08-01

    One of the most remarkable aspects of the immune system is its ability to fashion an immune response most appropriate to the activating stimulus. Although the immune system possesses a number of adaptations to accomplish this, an important theme is local immune regulation by site-specific expression of receptors and ligands. One family of molecules that is gaining attention as modulators of the immune system is the carcinoembryonic antigen cell-adhesion molecule family (CEACAM). Functionally, the carcinoembryonic antigen family can mediate cell-cell contact, host-pathogen interactions, and immune regulation. For example, biliary glycoprotein (CEACAM1) can have direct activity on T cells, leading to the inhibition of helper or cytotoxic T cell function. The expression of carcinoembryonic antigen (CEACAM5) on intestinal epithelial cells is involved in the activation of populations of regulatory CD8(+) T cells, while a distinct subset of regulatory CD8+ T cells is activated by nonspecific cross-reacting antigen (CEACAM6) on placental trophoblasts. Interestingly, the function and phenotype of these cells depend upon the specific member of the carcinoembryonic antigen family expressed, as well as the antigen-presenting molecule with which it associates. Thus, these glycoproteins comprise a family of molecules whose functions can depend on their nature and context. PMID:17057200

  8. Expression of MCRS1 and MCRS2 and their correlation with serum carcinoembryonic antigen in colorectal cancer

    PubMed Central

    Li, Chenguang; Chen, Mingxiao; Zhao, Pingwei; Ayana, Desalegn Admassu; Wang, Lei; Jiang, Yanfang

    2016-01-01

    Cancer-associated genes serve a crucial role in carcinogenesis. The present study aimed to investigate the mRNA expression levels of microspherule protein 1 (MCRS1) and MCRS2 in colorectal cancer (CRC) and their association with clinical variables. The mRNA expression levels of MCRS1 and MCRS2 were assessed by semi-quantitative reverse transcription polymerase chain reaction in the tumor and corresponding non-tumor tissues of 54 newly-diagnosed CRC patients, as well as in the normal colonic mucosa tissue of 19 age/gender-matched healthy controls. Immunofluorescence was also employed to identify the expression of MCRS1 in CRC tissues, while the concentration of serum carcinoembryonic antigen (CEA) was determined by an enzyme-linked immunoassay. The results identified a negative correlation between MCRS1 and MCRS2 expression levels (r=−0.3018, P=0.0266). MCRS1 mRNA expression was significantly increased and MCRS2 mRNA expression was decreased in CRC tissues compared with the levels in the corresponding normal tissues (both P<0.001). An increase in MCRS1 expression and a decrease in MCRS2 expression was detected in advanced stage when compared with early stage CRC patients. Immunofluorescence analysis revealed increased expression of MCRS1 in CRC patients. Furthermore, the expression levels of MCRS1 displayed positive correlation, whilst those of MCRS2 displayed negative correlation, with the serum CEA level in patients with CRC. The results suggest that increased MCRS1 and decreased MCRS2 expression appeared to be involved in the pathogenesis of CRC. The present study provides evidence suggesting that MCRS1 and MCRS2 may identify CRC patients at a risk of disease relapse, and thus, may be potential tools for monitoring disease activity and act as novel diagnostic markers in the treatment of CRC. PMID:27446248

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

    PubMed Central

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

    2015-01-01

    Colorectal cancer is one of the most prevalent cancers in the world. Patients in advanced stages often develop metastases that require chemotherapy and usually show a poor response, have a low survival rate and develop considerable toxicity with adverse symptoms. Gene therapy may act as an adjuvant therapy in attempts to destroy the tumor without affecting normal host tissue. The bacteriophage E gene has demonstrated significant antitumor activity in several cancers, but without any tumor-specific activity. The use of tumor-specific promoters may help to direct the expression of therapeutic genes so they act against specific cancer cells. We used the carcinoembryonic antigen promoter (CEA) to direct E gene expression (pCEA-E) towards colon cancer cells. pCEA-E induced a high cell growth inhibition of human HTC-116 colon adenocarcinoma and mouse MC-38 colon cancer cells in comparison to normal human CCD18co colon cells, which have practically undetectable levels of CEA. In addition, in vivo analyses of mice bearing tumors induced using MC-38 cells showed a significant decrease in tumor volume after pCEA-E treatment and a low level of Ki-67 in relation to untreated tumors. These results suggest that the CEA promoter is an excellent candidate for directing E gene expression specifically toward colon cancer cells. PMID:26053394

  10. Deglycosylation to obtain stable and homogeneous Pichia pastoris-expressed N-A1 domains of carcinoembryonic antigen.

    PubMed

    Sainz-Pastor, Noelia; Tolner, Berend; Huhalov, Alexandra; Kogelberg, Heide; Lee, Yie Chia; Zhu, Delin; Begent, Richard Henry John; Chester, Kerry Ann

    2006-08-15

    Carcinoembryonic antigen (CEA) is a seven domain membrane glycoprotein widely used as a tumour marker for adenocarcinomas and as a target for antibody-directed therapies. Structural models have proposed that the first two domains of CEA (the N terminal and adjoining A1 domains) bind MFE-23, a single chain Fv antibody in experimental clinical use. We aimed to produce recombinant N-A1 to test this hypothesis. The N-A1 domains were expressed as soluble protein with a C-terminal hexahistidine tag (His6-tag) in the yeast Pichia pastoris. His6-tagged N-A1 was captured from the supernatant by batch purification with copper-loaded Streamline Chelating, an immobilised metal affinity chromatography (IMAC) matrix usually utilised in expanded bed techniques. Purified N-A1 was heterogeneous with a molecular weight range from 38 to 188 kDa. Deglycosylation with endoglycosidase H (Endo H) resulted in three discrete molecular weight forms of N-A1, one partially mannosylated, one fully Endo H-digested and one fully Endo H-digested but lacking the His6-tag. These were separated by concanavalin A chromatography followed by HiTrap IMAC. The procedure resulted in single-band-purity, mannose-free N-A1. The binding interaction of MFE-23 to N-A1 was analysed by surface plasmon resonance. The affinity constants retrieved were KD = 4.49 x 10(-9)M for the P. pastoris expressed, native N-A1, and 5.33 x 10(-9) M for the Endo H-treated N-A1. To our knowledge this is the first time that two consecutive domains of CEA have been stably expressed and purified from P. pastoris. This work confirms that the CEA epitope recognised by MFE-23 resides in N-A1. PMID:16678252

  11. Carcinoembryonic antigen in patients with intracranial tumors.

    PubMed

    Suzuki, Y; Tanaka, R

    1980-09-01

    Carcinoembryonic antigen (CEA) in plasma, cerebrospinal fluid (CSF), and tumor cyst fluid obtained from patients with a variety of intracranial tumors was determined by radioimmunoassay Slightly elevated levels of plasma CEA, ranging from 2.6 to 3.8 ng/ml, were noted in six (4%) of 161 patients with primary brain tumors: in three gliomas, two pineal tumors, and one acoustic neurinoma, respectively. On the other hand, 17 (37%) of 46 patients with metastatic brain tumors showed a definite elevation, and most of them had values higher than 5.0 ng/ml. Of 37 patients with primary brain tumors, only one with a pineal germinoma showed a significant elevation of CEA in CSF, whereas eight (44%) of 18 patients with metastatic brain tumors showed high values of CEA in CSF. All six patients with leptomeningeal carcinomatosis showed elevated CEA in CSF. Levels of CEA in tumor cyst fluid were determined in 17 patients with intracranial tumors, including 12 gliomas, two craniopharyngiomas, two metastatic tumors, and one meningioma; elevation of CEA in tumor fluid was noted in two craniopharyngiomas and one metastatic tumor. Sequential determination of CEA of plasma or CSF revealed that the CEA levels were well correlated with the activity of brain tumors. Consequently, the determination of CEA in plasma or CSF is valuable for the differential diagnosis between primary and metastatic brain tumors and for the management of CEA-producing tumors. PMID:7420150

  12. Assaying Carcinoembryonic Antigens by Normalized Saturation Magnetization

    NASA Astrophysics Data System (ADS)

    Huang, Kai-Wen; Chieh, Jen-Jie; Shi, Jin-Cheng; Chiang, Ming-Hsien

    2015-07-01

    Biofunctionalized magnetic nanoparticles (BMNs) that provide unique advantages have been extensively used to develop immunoassay methods. However, these developed magnetic methods have been used only for specific immunoassays and not in studies of magnetic characteristics of materials. In this study, a common vibration sample magnetometer (VSM) was used for the measurement of the hysteresis loop for different carcinoembryonic antigens (CEA) concentrations ( Φ CEA) based on the synthesized BMNs with anti-CEA coating. Additionally, magnetic parameters such as magnetization ( M), remanent magnetization ( M R), saturation magnetization ( M S), and normalized parameters (Δ M R/ M R and Δ M S/ M S) were studied. Here, Δ M R and Δ M s were defined as the difference between any ΦCEA and zero Φ CEA. The parameters M, Δ M R, and Δ M S increased with Φ CEA, and Δ M S showed the largest increase. Magnetic clusters produced by the conjugation of the BMNs to CEAs showed a Δ M S greater than that of BMNs. Furthermore, the relationship between Δ M S/ M S and Φ CEA could be described by a characteristic logistic function, which was appropriate for assaying the amount of CEAs. This analytic Δ M S/ M S and the BMNs used in general magnetic immunoassays can be used for upgrading the functions of the VSM and for studying the magnetic characteristics of materials.

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

    PubMed Central

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

    1990-01-01

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

  14. Expression of human carcinoembryonic antigen-related cell adhesion molecule 6 and alveolar progenitor cells in normal and injured lungs of transgenic mice.

    PubMed

    Lin, Shin-E; Barrette, Anne Marie; Chapin, Cheryl; Gonzales, Linda W; Gonzalez, Robert F; Dobbs, Leland G; Ballard, Philip L

    2015-12-01

    Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is expressed in the epithelium of various primate tissues, including lung airway and alveoli. In human lung, CEACAM6 is developmentally and hormonally regulated, protects surfactant function, has anti-apoptotic activity and is dysregulated in cancers. We hypothesized that alveolar CEACAM6 expression increases in lung injury and promotes cell proliferation during repair. Studies were performed in CEABAC transgenic mice-containing human CEACAM genes. The level of CEACAM6 in adult CEABAC lung was comparable to that in human infants; expression occurred in epithelium of airways and of some alveoli but rarely co-localized with markers of type I or type II cells. Ten days after bleomycin instillation, both the number of CEACAM6(+) cells and immunostaining intensity were elevated in injured lung areas, and there was increased co-localization with type I and II cell markers. To specifically address type II cells, we crossed CEABAC mice with animals expressing EGFP driven by the SP-C promoter. After bleomycin injury, partially flattened, elongated epithelial cells were observed that expressed type I cell markers and were primarily either EGFP(+) or CEACAM6(+). In cell cycle studies, mitosis was greater in CEACAM6(+) non-type II cells versus CEACAM6(+)/EGFP(+) cells. CEACAM6 epithelial expression was also increased after hyperoxic exposure and LPS instillation, suggesting a generalized response to acute lung injuries. We conclude that CEACAM6 expression is comparable in human lung and the CEABAC mouse. CEACAM6 in this model appears to be a marker of a progenitor cell population that contributes to alveolar epithelial cell replenishment after lung injury. PMID:26702074

  15. Nuclear factor-kappa B directs carcinoembryonic antigen-related cellular adhesion molecule 1 receptor expression in Neisseria gonorrhoeae-infected epithelial cells.

    PubMed

    Muenzner, Petra; Billker, Oliver; Meyer, Thomas F; Naumann, Michael

    2002-03-01

    The human-specific pathogen Neisseria gonorrhoeae expresses opacity-associated (Opa) protein adhesins that bind to various members of the carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) family. In this study, we have analyzed the mechanism underlying N. gonorrhoeae-induced CEACAM up-regulation in epithelial cells. Epithelial cells represent the first barrier for the microbial pathogen. We therefore characterized CEACAM expression in primary human ovarian surface epithelial (HOSE) cells and found that CEACAM1-3 (L, S) and CEACAM1-4 (L, S) splice variants mediate an increased Opa(52)-dependent gonoccocal binding to HOSE cells. Up-regulation of these CEACAM molecules in HOSE cells is a direct process that takes place within 2 h postinfection and depends on close contact between microbial pathogen and HOSE cells. N. gonorrhoeae-triggered CEACAM1 up-regulation involves activation of the transcription factor nuclear factor kappaB (NF-kappaB), which translocates as a p50/p65 heterodimer into the nucleus, and an NF-kappaB-specific inhibitory peptide inhibited CEACAM1-receptor up-regulation in N. gonorrhoeae-infected HOSE cells. Bacterial lipopolysaccharides did not induce NF-kappaB and CEACAM up-regulation, which corresponds to our findings that HOSE cells do not express toll-like receptor 4. The ability of N. gonorrhoeae to up-regulate its epithelial receptor CEACAM1 through NF-kappaB suggests an important mechanism allowing efficient bacterial colonization during the initial infection process. PMID:11751883

  16. Novel mouse model for carcinoembryonic antigen-based therapy.

    PubMed

    Chan, Carlos H F; Stanners, Clifford P

    2004-06-01

    Many novel cancer therapies, including immunotherapy and gene therapy, are specifically targeted to tumor-associated molecules, among which carcinoembryonic antigen (CEA) represents a popular example. Discrepancies between preclinical experimental data in animal models and clinical outcome in terms of therapeutic response and toxicity, however, often arise. Preclinical testing can be compromised by the lack of CEA and other closely related human CEA family members in rodents, which lack analogous genes for most human CEA family members. Here, we report the construction of a transgenic mouse with a 187-kb human bacterial artificial chromosome (CEABAC) that contains part of the human CEA family gene cluster including complete human CEA (CEACAM5), CEACAM3, CEACAM6, and CEACAM7 genes. The spatiotemporal expression pattern of these genes in the CEABAC mice was found to be remarkably similar to that of humans. This novel mouse will ensure better assessment than previously utilized models for the preclinical testing of CEA-targeted therapies and perhaps allow the testing of CEACAM6, which is overexpressed in many solid tumors and leukemias, as a therapeutic target. Moreover, expression of CEA family genes in gastrointestinal, breast, hematopoietic, urogenital, and respiratory systems could facilitate other clinical applications, such as the development of therapeutic agents against Neisseria gonorrhoeae infections, which use CEA family members as major receptors. PMID:15194045

  17. Carcinoembryonic antigen continuous epitopes determined by the spot method.

    PubMed

    Solassol, I; Granier, C; Pèlegrin, A

    2001-01-01

    Carcinoembryonic antigen (CEA) is a heavily glycosylated tumor-associated protein with an N-A1-B1-A2-B2-A3-B3 domain structure. Circulating CEA immunoassays are used for monitoring digestive cancer patients, and radiolabeled anti-CEA monoclonal antibodies (MAb) are used for the diagnosis and therapy of CEA-positive tumors. The five major nonoverlapping epitopes (Gold 1-5) have been broadly correlated with the domain organization, but there is no precise localization of the epitopes at the sequence level. In an attempt to identify the peptide sequences corresponding to the five Gold epitopes on the CEA molecule, we prepared a set of 227 overlapping fifteen-mer peptides corresponding to the complete CEA sequence with the SPOT method. Using five high affinity MAbs directed against the five CEA Gold epitopes, we demonstrated that none of these epitopes could be mimicked by a fifteen-mer peptide sequence. However, using rabbit and goat anti-CEA sera, we identified six major continuous antigenic regions. All are included in the Ig-like domains of the CEA: two in the A1 domain (residues 120-134 and 153-164), one each in the A2 (329-337) and A3 domains (508-513), one at the junction between the A3 and B3 domains (553-561) and one in the B3 domain (565-573). A very homologous sequence (common residues VSPRL) was mapped in each of the three A domains. Thus, in terms of occurrence of continuous epitopes, the Ig-like domains A1, A2, A3 and B3 seem to be the most antigenic parts of CEA. These peptide sequences should be good candidates for the future development of site-specific anti-CEA MAbs. PMID:11275797

  18. Electrochemical immunosensor based on hyperbranched structure for carcinoembryonic antigen detection.

    PubMed

    Miao, Jingjing; Wang, Xiaobo; Lu, Liandi; Zhu, Peiyuan; Mao, Chun; Zhao, Haolin; Song, Youchao; Shen, Jian

    2014-08-15

    Sensitive determination of carcinoembryonic antigen (CEA) is very important in clinical research and diagnosis. Herein we report the design and synthesis of a new kind of immunosensor based on the benefits of hyperbranched structure. The hyperbranched polyester was grafted to the surface of indium tin oxides glass (ITO) electrode, and the grafting processes were characterized by attentuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). After CEA and horse radish peroxidase (HRP)-labeled antibody-conjugated AuNPs (HRP-Ab2-AuNPs) bioconjugates were immobilized on the surface of the hyperbranched structure-modified electrode, the optimized conditions of the above electrode were investigated. Moreover, the analytical performance of the proposed immunosensor showed a high sensitivity, a linear range from 0.01 to 80ng/mL with a low detection limit of 2.36pg/mL, and good selectivity for CEA. The designed immunoassay system holds great potential for ultrasensitive electrochemical biosensing of other analytes. PMID:24607616

  19. The Significance of Serum Carcinoembryonic Antigen in Lung Adenocarcinoma

    PubMed Central

    Kim, Jae Jun; Hyun, Kwanyong; Park, Jae Kil; Moon, Seok Whan

    2015-01-01

    Background A raised carcinoembryonic antigen (CEA) may be associated with significant pathology during the postoperative follow-up of lung adenocarcinoma. Methods We reviewed the medical records of 305 patients who underwent surgical resections for primary lung adenocarcinoma at a single institution between April 2006 and February 2013. Results Preoperative CEA levels were significantly associated with age, smoking history, pathologic stage including pT (pathologic tumor stge), pN (pathologic nodal stage) and overall pathological stage, tumor size and differentiation, pathologically positive total lymph node, N1 and N2 lymph node, N2 nodal station (0/1/2=1.83/2.94/7.21 ng/mL, p=0.019), and 5-year disease-free survival (0.591 in group with normal preoperative CEA levels vs. 0.40 in group with high preoperative CEA levels, p=0.001). Preoperative CEA levels were significantly higher than postoperative CEA levels (p<0.001, Wilcoxon signed-rank test). Postoperative CEA level was also significantly associated with disease-free survival (p<0.001). A follow-up serum CEA value of >2.57 ng/mL was found to be the appropriate cutoff value for the prediction of cancer recurrence with sensitivity and specificity of 71.4% and 72.3%, respectively. Twenty percent of patients who had recurrence of disease had a CEA level elevated above this cutoff value prior to radiographic evidence of recurrence. Postoperative CEA, pathologic stage, differentiation, vascular invasion, and neoadjuvant therapy were identified as independent predictors of 5-year disease-free survival in a multivariate analysis. Conclusion The follow-up CEA level can be a useful tool for detecting early recurrence undetected by postoperative imaging studies. The perioperative follow-up CEA levels may be helpful for providing personalized evaluation of lung adenocarcinoma. PMID:26509127

  20. Prognostic significance of carcinoembryonic antigen in colorectal carcinoma. Serum levels before and after resection and before recurrence.

    PubMed

    Chu, D Z; Erickson, C A; Russell, M P; Thompson, C; Lang, N P; Broadwater, R J; Westbrook, K C

    1991-03-01

    The use of carcinoembryonic antigen was evaluated in 425 patients with a mean follow-up of 48 months. The preoperative and postoperative carcinoembryonic antigen levels were predictive of recurrence and survival independent of the tumor stage. In a multivariate regression analysis of age, location, tumor stage, and preoperative and postoperative carcinoembryonic antigen levels, the latter three factors were significant prognostic variables with respect to the adjusted survival. Recurrent disease was found in 42% of patients, excluding patients with stage IV disease. The carcinoembryonic antigen level at recurrence was greater than 5 ng/mL in 79% of the patients and in 89% of the intra-abdominal recurrences. Carcinoembryonic antigen level at recurrence was not predictive of postrecurrence survival except in the subgroup of locoregional disease. The life span in patients with liver and lung metastases was not influenced by carcinoembryonic antigen level at recurrence. Preoperative and postoperative carcinoembryonic antigen levels can indicate a poorer prognostic group of patients with colorectal cancer who may benefit from adjuvant treatment. The carcinoembryonic antigen at recurrence can be used effectively to diagnose intra-abdominal recurrences and project survival after development of local/regional disease. PMID:1998473

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

    PubMed Central

    Wang, Yan; Wang, Mengchun; Li, Yan

    2016-01-01

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

  2. Targeted sonodynamic therapy of cancer using a photosensitizer conjugated with antibody against carcinoembryonic antigen.

    PubMed

    Abe, Hironori; Kuroki, Motomu; Tachibana, Katsuro; Li, Tieli; Awasthi, Aradhana; Ueno, Aruto; Matsumoto, Hisanobu; Imakiire, Takayuki; Yamauchi, Yasushi; Yamada, Hiromi; Ariyoshi, Asami; Kuroki, Masahide

    2002-01-01

    The goal of this study was to develop a strategy for the selective destruction of cancer cells by ultrasonic irradiation in the presence of an antibody-conjugated photosensitizer. To this end, a photoimmunoconjugate (PIC) was prepared between ATX-70, a photosensitizer of a gallium-porphyrin analogue, and F11-39, a high affinity monoclonal antibody (MAb) against carcinoembryonic antigen (CEA), which is often overexpressed in various carcinoma cells. This conjugate, designated F39/ATX-70, retained immunoreactivity against purified CEA and CEA-expressing cells as determined by enzyme-linked immunosorbent assay, flow cytometry and immunofluorescence microscopic analysis. The cytotoxicity of F39/ATX-70 against CEA-expressing human gastric carcinoma cells in vitro was found to be greater than that of ATX-70 when applied in combination with ultrasound irradiation. When in vivo anti-tumor effects in a mouse xenograft model were assessed, intravenous administration of F39/ATX-70 followed by ultrasonic irradiation produced a marked growth inhibition of tumor compared with irradiation alone or irradiation after administration of ATX-70. These results suggest that the PIC between anti-CEA MAb and ATX-70 may have applications in sonodynamic therapy where destruction of CEA-expressing tumor is required. PMID:12168839

  3. Host-related carcinoembryonic antigen cell adhesion molecule 1 promotes metastasis of colorectal cancer.

    PubMed

    Arabzadeh, A; Chan, C; Nouvion, A-L; Breton, V; Benlolo, S; DeMarte, L; Turbide, C; Brodt, P; Ferri, L; Beauchemin, N

    2013-02-14

    Liver metastasis is the predominant cause of colorectal cancer (CRC)-related mortality in developed countries. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a cell adhesion molecule with reduced expression in early phases of CRC development and thus functions as a tumor growth inhibitor. However, CEACAM1 is upregulated in metastatic colon cancer, suggesting a bimodal role in CRC progression. To investigate the role of this protein in the host metastatic environment, Ceacam1(-/-) mice were injected intrasplenically with metastatic MC38 mouse CRC cells. A significant reduction in metastatic burden was observed in Ceacam1(-/-) compared with wild-type (WT) livers. Intravital microscopy showed decreased early survival of MC38 cells in Ceacam1(-/-) endothelial environment. Metastatic cell proliferation within the Ceacam1(-/-) livers was also diminished. Bone marrow-derived cell recruitment, attenuation of immune infiltrates and diminished CCL2, CCL3 and CCL5 chemokine production participated in the reduced Ceacam1(-/-) metastatic phenotype. Transplantations of WT bone marrow (BM) into Ceacam1(-/-) mice fully rescued metastatic development, whereas Ceacam1(-/-) BM transfer into WT mice showed reduced metastatic burden. Chimeric immune cell profiling revealed diminished recruitment of CD11b(+)Gr1(+) myeloid-derived suppressor cells (MDSCs) to Ceacam1(-/-) metastatic livers and adoptive transfer of MDSCs confirmed the involvement of these immune cells in reduction of liver metastasis. CEACAM1 may represent a novel metastatic CRC target for treatment. PMID:22469976

  4. Carcinoembryonic Antigen Cell Adhesion Molecule 1 long isoform modulates malignancy of poorly differentiated colon cancer cells

    PubMed Central

    Arabzadeh, Azadeh; Dupaul-Chicoine, Jeremy; Breton, Valérie; Haftchenary, Sina; Yumeen, Sara; Turbide, Claire; Saleh, Maya; McGregor, Kevin; Greenwood, Celia M T; Akavia, Uri David; Blumberg, Richard S; Gunning, Patrick T; Beauchemin, Nicole

    2015-01-01

    Objective Nearly 20%–29% of patients with colorectal cancer (CRC) succumb to liver or lung metastasis and there is a dire need for novel targets to improve the survival of patients with metastasis. The long isoform of the Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1-L or CC1-L) is a key regulator of immune surveillance in primary CRC, but its role in metastasis remains largely unexplored. We have examined how CC1-L expression impacts on colon cancer liver metastasis. Design Murine MC38 transfected with CC1-L were evaluated in vitro for proliferation, migration and invasion, and for in vivo experimental liver metastasis. Using shRNA silencing or pharmacological inhibition, we delineated the role in liver metastasis of Chemokine (C-C motif) Ligand 2 (CCL2) and Signal Transducer and Activator of Transcription 3 (STAT3) downstream of CC1-L. We further assessed the clinical relevance of these findings in a cohort of patients with CRC. Results MC38-CC1-L-expressing cells exhibited significantly reduced in vivo liver metastasis and displayed decreased CCL2 chemokine secretion and reduced STAT3 activity. Down-modulation of CCL2 expression and pharmacological inhibition of STAT3 activity in MC38 cells led to reduced cell invasion capacity and decreased liver metastasis. The clinical relevance of our findings is illustrated by the fact that high CC1 expression in patients with CRC combined with some inflammation-regulated and STAT3-regulated genes correlate with improved 10-year survival. Conclusions CC1-L regulates inflammation and STAT3 signalling and contributes to the maintenance of a less-invasive CRC metastatic phenotype of poorly differentiated carcinomas. PMID:25666195

  5. Carcinoembryonic antigen promotes colorectal cancer progression by targeting adherens junction complexes

    SciTech Connect

    Bajenova, Olga; Chaika, Nina; Tolkunova, Elena; Davydov-Sinitsyn, Alexander; Gapon, Svetlana; Thomas, Peter; O’Brien, Stephen

    2014-06-10

    Oncomarkers play important roles in the detection and management of human malignancies. Carcinoembryonic antigen (CEA, CEACAM5) and epithelial cadherin (E-cadherin) are considered as independent tumor markers in monitoring metastatic colorectal cancer. They are both expressed by cancer cells and can be detected in the blood serum. We investigated the effect of CEA production by MIP101 colorectal carcinoma cell lines on E-cadherin adherens junction (AJ) protein complexes. No direct interaction between E-cadherin and CEA was detected; however, the functional relationships between E-cadherin and its AJ partners: α-, β- and p120 catenins were impaired. We discovered a novel interaction between CEA and beta-catenin protein in the CEA producing cells. It is shown in the current study that CEA overexpression alters the splicing of p120 catenin and triggers the release of soluble E-cadherin. The influence of CEA production by colorectal cancer cells on the function of E-cadherin junction complexes may explain the link between the elevated levels of CEA and the increase in soluble E-cadherin during the progression of colorectal cancer. - Highlights: • Elevated level of CEA increases the release of soluble E-cadherin during the progression of colorectal cancer. • CEA over-expression alters the binding preferences between E-cadherin and its partners: α-, β- and p120 catenins in adherens junction complexes. • CEA produced by colorectal cancer cells interacts with beta-catenin protein. • CEA over-expression triggers the increase in nuclear beta-catenin. • CEA over-expression alters the splicing of p120 catenin protein.

  6. Differential recognition of members of the carcinoembryonic antigen family by Opa variants of Neisseria gonorrhoeae.

    PubMed Central

    Bos, M P; Grunert, F; Belland, R J

    1997-01-01

    Opacity (Opa) protein variation in Neisseria gonorrhoeae is implicated in the pathogenesis of gonorrhea, possibly by mediating adherence and entry of the bacteria into human tissues. One particular Opa protein mediates adherence to epithelial cells through cell surface proteoglycans. Recently, two other eukaryotic cell receptors for Opa proteins have been reported. These receptors are members of a subgroup of the carcinoembryonic (CEA) gene family that express CD66 antigens. CEA family members vary in their distribution in human tissues. In order to understand whether interactions between Opa and CEA-like molecules play any role in pathogenesis, we must investigate which CEA family members are able to serve as Opa receptors and which Opa proteins recognize CEA-like molecules. We therefore studied HeLa cells that were stably transfected with five different members of the CEA family, i.e., CEA, CEA gene family member 1a (CGM1a), CGM6, nonspecific cross-reacting antigen (NCA), and biliary glycoprotein a (BGPa). We infected these transfectants with all possible 11 Opa variants of gonococcal strain MS11 and determined the numbers of bacteria that were bound and internalized. To account for proteoglycan-mediated adherence, infection assays were also performed in the presence of heparin. Our results show that of the 11 Opa variants of MS11, the same 4 recognized CGM1a and NCA. CGM6, however, was not recognized by any Opa variant of MS11. CEA was recognized by at least 9 of 11 Opa variants, and the BGP transfectants specifically bound and internalized 10 of 11 Opa variants and also bound Opa-negative gonococci. Immunofluorescence experiments showed that clustering of CEA-like molecules occurred upon infection of HeLa transfectants with those Opa variants that interacted specifically with the CEA family member. Together these data show that CEA family members are differentially recognized by gonococcal Opa variants, suggesting that this phenomenon may contribute to cell

  7. Effect of cigarette smoking, pulmonary inflammation, and lung disease on concentrations of carcinoembryonic antigen in serum and secretions.

    PubMed Central

    Stockley, R A; Shaw, J; Whitfield, A G; Whitehead, T P; Clarke, C A; Burnett, D

    1986-01-01

    A specific radioimmunoassay for carcinoembryonic antigen was used to investigate aspects of its measurement in lung disease. The results confirm that serum carcinoembryonic antigen concentrations are higher in healthy smokers and patients with chronic obstructive bronchitis than in healthy non smokers (p less than 0.01). Corticosteroid treatment reduced the concentration in nine patients with bronchitis (p less than 0.05). Other inflammatory lung diseases (bronchiectasis, pneumonia, fibrosing alveolitis) are not associated with a raised serum carcinoembryonic antigen concentration. The sputum concentrations were about 100 times those found in serum and there was a positive correlation (r = 0.611 2p less than 0.01) between the concentrations in sputum and serum in patients with bronchitis. No preferential rise in sputum concentration was found in current smokers or patients with lung carcinoma (n = 16). A higher ratio of carcinoembryonic antigen to albumin concentration (p less than 0.05) was, however, found in lavage fluid obtained from the tumour site than in fluid from "normal" lung in the same patients, suggesting an increase in carcinoembryonic antigen secretion in the vicinity of the tumour. Despite this "local" effect the sputum concentration does not, however, appear to be a useful marker of lung carcinoma and the measurement could not be used as a screening test. PMID:3704962

  8. Nanostructured materials detect epidermal growth factor receptor, neuron specific enolase and carcinoembryonic antigen

    NASA Astrophysics Data System (ADS)

    Stefan-van Staden, Raluca-Ioana; Comnea-Stancu, Ionela Raluca; Surdu-Bob, Carmen Cristina; Badulescu, Marius

    2015-09-01

    New nanostructured materials based on thin films of Cu and Ni deposited on textile material (veil), as well as gold nanostructured microspheres were used for the design of new stochastic sensors. The stochastic sensors were able to detect simultaneously a panel of biomarkers comprising epidermal growth factor receptor, neuron specific enolase, and carcinoembryonic antigen from whole blood samples with high reliabilities - recovery tests higher than 97.00%, with a RSD (%) lower than 0.1%. The stochastic sensors had shown high sensitivities and low determination levels for the detection of the proposed panel of biomarkers making early detection of lung cancer possible by fast screening of whole blood.

  9. Immunophotodiagnosis of colon carcinomas in patients injected with fluoresceinated chimeric antibodies against carcinoembryonic antigen.

    PubMed Central

    Folli, S; Wagnières, G; Pèlegrin, A; Calmes, J M; Braichotte, D; Buchegger, F; Chalandon, Y; Hardman, N; Heusser, C; Givel, J C

    1992-01-01

    Based on previous experiments in nude mice, showing that fluoresceinated monoclonal antibodies against carcinoembryonic antigen localized specifically in human carcinoma xenografts and could be detected by laser-induced fluorescence, we performed a feasibility study to determine whether this immunophotodiagnosis method could be applied in the clinic. Six patients, with known primary colorectal carcinoma, received an i.v. injection of 4.5 or 9 mg of mouse-human chimeric anti-carcinoembryonic antigen monoclonal antibody coupled with 0.10-0.28 mg of fluorescein (molar ratio 1/10 to 1/14). The monoclonal antibody was also labeled with 0.2-0.4 mCi of 125I (1 Ci = 37 GBq). Photodetection of the tumor was done ex vivo on surgically resected tissues for the six patients and in vivo by fluorescence rectosigmoidoscopy for the sixth patient. Upon laser irradiation, clearly detectable heterogeneous green fluorescence from the dye-antibody conjugate was visually observed on all six tumors; almost no such fluorescence was detectable on normal mucosa. The yellowish tissue autofluorescence, which was emitted from both tumor and normal mucosa, could be subtracted by real-time image processing. Radioactivity measurements confirmed the specificity of tumor localization by the conjugate; tissue concentrations of up to 0.059% injected dose per g of tumor and 10 times less (0.006%) per g of normal mucosa were found. The overall results demonstrate the feasibility of tumor immunophotodiagnosis at the clinical level. Images PMID:1518823

  10. Experimental radioimmunotherapy of a xenografted human colonic tumor (GW-39) producing carcinoembryonic antigen

    SciTech Connect

    Goldenberg, D.M.; Gaffar, S.A.; Bennett, S.J.; Beach, J.L.

    1981-11-01

    Experiments were undertaken to evaluate the antitumor effects of 131I-labeled goat antibody immunoglobulin G prepared against carcinoembryonic antigen in hamsters bearing the carcinoembryonic antigen-producing GW-39 human colonic carcinoma. At a single injection of 1 mCi 131I and higher, a marked growth inhibition of GW-39 tumors, as well as a considerable increase in the survival time of the tumor-bearing hamsters, could be achieved. At a dose of 1 mCi, the radioactive affinity-purified antibody appeared to be superior to radioactive normal goat immunoglobulin G in influencing tumor growth and survival time, but no significant difference could be seen at the higher dose of 2 mCi given. Radiobiological calculations indicated that the tumors received, at up to 20 days after therapy, 1325 rads for the specific antibody and only 411 rads for the normal immunoglobulin G preparation. These findings encourage the further evaluation of antibodies to tumor markers for isotopic cancer therapy.

  11. Detection of targeted carcinoembryonic antigens using a micro-fluxgate-based biosensor

    NASA Astrophysics Data System (ADS)

    Lei, Jian; Lei, Chong; Wang, Tao; Yang, Zhen; Zhou, Yong

    2013-11-01

    In this work, a micro-fluxgate-based biosensor was designed for the detection of carcinoembryonic antigen (CEA) labeled by Dynabeads. The sensor with Fe-based amorphous core and three dimension solenoid coils was fabricated by Micro Electro-Mechanical system technology. Sandwich assays are performed using antibody-antigen pair combination of biotin-streptavidin assay on a separated Au film substrate surface with a self-assembled layer. With dc magnetic fields in the range of 560 μT to 875 μT, detection of CEAs with different concentrations was performed and a minimum detectable concentration of 1 pg/ml was achieved. Furthermore, CEA samples with different concentrations can be distinguished.

  12. Surface-enhanced Raman scattering study of carcinoembryonic antigen in serum from patients with colorectal cancers

    NASA Astrophysics Data System (ADS)

    Chen, Gang; Chen, Yanping; Zheng, Xiongwei; He, Cheng; Lu, Jianping; Feng, Shangyuan; Chen, Rong; Zeng, Haisan

    2013-12-01

    In this work, we developed a SERS platform for quantitative detection of carcinoembryonic antigen (CEA) in serum of patients with colorectal cancers. Anti-CEA-functionalized 4-mercaptobenzoic acid-labeled Au/Ag core-shell bimetallic nanoparticles were prepared first and then used to analyze CEA antigen solutions of different concentrations. A calibration curve was established in the range from 5 × 10-3 to 5 × 105 ng/mL. Finally, this new SERS probe was applied for quantitative detection of CEA in serum obtained from 26 colorectal cancer patients according to the calibration curve. The results were in good agreement with that obtained by electrochemical luminescence method, suggesting that SERS immunoassay has high sensitivity and specificity for CEA detection in serum. A detection limit of 5 pg/ml was achieved. This study demonstrated the feasibility and great potential for developing this new technology into a clinical tool for analysis of tumor markers in the blood.

  13. Development of a PMMA Electrochemical Microfluidic Device for Carcinoembryonic Antigen Detection

    NASA Astrophysics Data System (ADS)

    Van Anh, Nguyen; Van Trung, Hoang; Tien, Bui Quang; Binh, Nguyen Hai; Ha, Cao Hong; Le Huy, Nguyen; Loc, Nguyen Thai; Thu, Vu Thi; Lam, Tran Dai

    2016-05-01

    In this study, a poly(methyl methacrylate) (PMMA) microfluidic device fabricated by an inexpensive CO2 laser etching system was developed for detection of carcino-embryonic antigens (CEA). The device was capable of working in continuous mode and was designed with the aid of numerical simulation. The detection of target CEA was based on immuno-assay via magnetic particles and electrochemical sensing. The as-prepared microfluidic can be used to detect CEA at the relatively low concentration of 150 pg mL-1. The device could be reused many times, since the capture and removal of magnetic particles in the assay could be manipulated by an external magnetic field. The proposed approach appears to be suitable for high-throughput and automated analysis of large biomolecules such as tumor markers and pathogens.

  14. Graphene oxide supported rhombic dodecahedral Cu2O nanocrystals for the detection of carcinoembryonic antigen.

    PubMed

    Feng, Taotao; Chen, Xiaoyu; Qiao, Xiuwen; Sun, Zhao; Wang, Haining; Qi, Yu; Hong, Chenglin

    2016-02-01

    In this work, a simple electrochemical immunosensor was developed for the detection of carcinoembryonic antigen (CEA) based on rhombic dodecahedral Cu2O nanocrystals-graphene oxide-gold nanoparticles (rCu2O-GO-AuNPs). GO as the template and surfactant resulting in rCu2O exhibit improved rhombic dodecahedral structure uniformity and excellent electrochemical performance. Moreover, GO was found to be able to effectively improve the long stability of rCu2O on the electrode response. Under optimal conditions, the immunosensor showed a low limit of detection (0.004 ng ml(-1)) and a large linear range (0.01-120 ng ml(-1)). This work presents a potential alternative for the diagnostic applications of GO-supported special morphology materials in biomedicine and biosensors. PMID:26596552

  15. Electrochemical Immunoassay of Carcinoembryonic Antigen Based on A Lead Sulfide Nanoparticle Label

    SciTech Connect

    Wang, Shengfu; Zhang, Xing; Mao, Xun; Zeng, Qingxiang; Xu, Hui; Lin, Yuehe; Chen, Wei; Liu, Guodong

    2008-10-01

    We describe a Lead sulfide nanoparticle (PbS NP) based electrochemical immunoassay to detect a tumor biomarker, carcinoembryonic antigen (CEA). Cubic PbS NPs were prepared and functionalized with thioglycolic acid (TGA), which stabilized the formed NPs and offered carboxyl groups to conjugate with CEA antibodies. PbS NP conjugated with monoclonal CEA antibody was used as a label in an immnorecognition event. After a complete sandwich immunoreaction among the primary CEA antibody (immobilized on the carboxyl-modified magnetic beads), CEA, and the PbS-labeled secondary antibody (PbS-anti-CEA), PbS labels were captured to the magnetic-bead (MB) surface through the antibody-antigen immunocomplex. Electrochemical stripping analysis of the captured PbS was used to quantify the concentration of CEA after an acid-dissolution step. The MBs and the magnetic separation platform were used to integrate a facile antibody immobilization with immunoreactions and the isolation of immunocomplexes from reaction solutions in the immunoassay. The performance of this nanoparticle based electrochemical immunoassay was successfully evaluated with human serum spiked with CEA, indicating that this convenient and sensitive technique offers great promise for rapid, simple, and cost-effective analysis of tumor biomarkers in biological fluids.

  16. Carcinoembryonic antigen in bronchoalveolar lavage fluid in patients with idiopathic pulmonary fibrosis.

    PubMed

    Takahashi, H; Nukiwa, T; Matsuoka, R; Danbara, T; Natori, H; Arai, T; Kira, S

    1985-08-01

    An increased incidence of lung cancer and epithelial metaplasia or hyperplasia which is felt to be as a precursor of cancer, has been reported in patients with idiopathic pulmonary fibrosis (IPF). In this study, carcinoembryonic antigen (CEA) in bronchoalveolar lavage (BAL) fluid was measured in 53 control patients, 31 patients with sarcoidosis, 10 patients with hypersensitivity pneumonitis, 16 patients with primary lung cancer and 26 patients with histologically confirmed IPF. High ratio of CEA to albumin (Alb), exceeding mean + 2SD of nonsmoking control patients, were found in 8 (25%) out of 32 smoking control patients, 4 (44%) out of 9 nonsmoking patients with IPF, 8 (62%) out of 13 smoking patients with IPF, 3 (75%) out of 4 smoking patients with IPF and lung cancer and 13 (81%) out of 16 patients with primary lung cancer, although BAL was performed at the noncancerous parts of the lung in the cases of lung cancer. Furthermore, it was confirmed that CEA increased in BAL fluid in these subjects were different from nonspecific cross-reacting antigen (NCA) which was detectable in the normal lung. Thus we consider that the increase of CEA/Alb ratio in BAL fluid is a possible marker of these early histological disorders in the lung, and also suggests a greater risk of malignant change in the clinical course of IPF. PMID:4068359

  17. [Optical Analysis of the Interaction of Mercaptan Derivatives of Nanogold Particles with Carcinoembryonic Antigen].

    PubMed

    Zeng, Hong-juan; Zhao, Ran-lin; Wang, De-shun; Li, Cai-xia; Liu, Yi-yao

    2016-02-01

    Gold nanoparticles (AuNPs) have been the subject of intense research for use in biomedicine over the past couple of decades. AuNPs, also referred to as colloidal gold, possess some astounding optical and physical properties that have earned them a prime spot among the new promising tools for medical applications. Today, AuNPs are offered to provide the clinical laboratory with more sensitive, faster, and simpler assays, which are also cost-effective. AuNPs can be used to develop point-of- care tests and novel testing strategies such as in drug targeting, disease detection, molecular recognition, and biological labels. The typical structure of AuNPs is spherical nano-sized gold particles, but they can also be composed of a thin gold shell surrounding a dielectric core, such as silica (gold nanoshells). their size range from 0.8 to 250 nm and are characterized by high absorption coefficients. AuNPs have some unique optical properties, such as enhanced absorption and scattering, where the absorption cross-section of AuNPs is 4~5 orders of magnitude greater than that of rhodamine 6G. When AuNPs aggregate, interaction of locally adjacent AuNPs (plasmon-plasmon interaction) shifts their color to blue. Thus, the binding of AuNP-labeled entities to their respective target would lead to aggregation of the nanoparticles and a detectable shift in the optical signal. The strong absorption of AuNPs can also be used in colorimetric detection of analytes by measuring changes in the refractive index of the AuNP's environment caused by adsorption of the target analytes. However, a large number of surface atoms of nanoparticles have huge surplus bonding ability, because of surface effect of gold nanoparticles, result in reuniting and sinking among the nanoparticles which make them unstable. In order to detect traces of carcinoembryonic antigen, one of the tumor targets, a new kind of gold nanoparticle with hyperchormic effect and fluorescence sensitization effect material needs to

  18. Determination of carcinoembryonic antigen and cancer antigen (CA 15-3) in bitches with tumours on mammary gland: preliminary report.

    PubMed

    Valencakova-Agyagosova, A; Frischova, Z; Sevcikova, Z; Hajurka, J; Lepej, J; Szakallova, I; Kredatusova, G; Nagy, V; Ledecky, V

    2014-09-01

    The aim of this work was to determine levels of carcinoembryonic antigen (CEA) and cancer antigen (CA 15-3) in the blood serum of 45 bitches. A modified procedure was used to determine the CEA and CA 15-3 markers with the human kits using the radioimmunoassay method. Samples collected from extirpated tumour of mammary glands were histologically processed and classified as per WHO guidelines. The average age of animals with tumour was 10.00 ± 2.2 years; for healthy bitches average age was 4.2 ± 3.2 years. Values of CEA and CA 15-3 were considered positive, if they exceeded 0.23 ng mL(-1) and 7 IU mL(-1) , respectively. Average levels of CEA in the tumour group were 0.25 ± 0.06 versus 0.20 ± 0.03 in healthy bitches (P = 0.0001). The average CA 15-3 value in bitches with tumour was 8.58 ± 1.27 versus 5.14 ± 1.34 in healthy animals (P < 0.0001). PMID:22947252

  19. Malignant neuroendocrine tumour of the gallbladder with elevated carcinoembryonic antigen: case report and literature review

    PubMed Central

    Furrukh, Muhammad; Qureshi, Asim; Saparamadu, Anna; Kumar, Shiyam

    2013-01-01

    A 58-year-old woman presented to a tertiary care centre with signs and symptoms of acute cholecystitis, cholelithiasis and diagnoses of a high-grade neuroendocrine tumour of the gallbladder primarily with peritoneal and liver metastases. She had a liver abscess secondary to Salmonella and Enterococcus fecalis that was drained and treated with appropriate antibiotics. Interestingly, the serum chromogranin A levels were within normal limits, but carcinoembryonic antigen was elevated, which helped evaluate responses and pick progression. She was treated with 10 cycles of palliative chemotherapy when malignancy associated complications started to recur, that is, cholangitis, worsening pain, cachexia, intestinal obstruction, etc leading to chemotherapy delays. Her disease progressed during these times with rapid deterioration of performance status. She died of septic complications postlaparotomy for intestinal obstruction. Her progression-free survival remained for 8 months with subjective and objective improvements, and her overall survival remained at 13 months. We describe the course of her illness and give a brief review of the literature. PMID:23661652

  20. The role of bile carcinoembryonic antigen in diagnosing bile duct cancer.

    PubMed Central

    Joo, Kwang Ro; Kim, Do Ha; Park, Jong Ho; Bang, Sung-Jo; Shin, Jung Woo; Park, Neung Hwa; Park, Jae Hoo

    2003-01-01

    It is known that the fluids bathing tumors might contain a higher level of the carcinoembryonic antigen (CEA) than those found in the blood. Therefore, we evaluated the role of bile CEA in diagnosing bile duct cancer. One hundred and thirty two patients were prospectively studied. The patients were divided into 3 groups: the bile duct cancer (n=32), pancreatic cancer (n=16), and benign biliary diseases (n=84) groups. Bile samples were obtained on the next day of the biliary drainage procedures. The mean bile CEA level in those with bile duct cancer (120.6 +/- 156.9 ng/mL) was significantly higher than those with pancreatic cancer and benign biliary diseases (32.0 +/- 28.5 ng/mL, 29.3 +/- 56.3 ng/mL). Using the level of 20 ng/mL, the sensitivity and specificity of bile CEA in the diagnosis of bile duct cancer from benign biliary diseases were 65.6% and 66.7%, respectively. Both the bile CEA and total bilirubin level were found to be an independent factor linked to bile duct cancer. This study result suggests that bile CEA level is a useful supplementary test for diagnosing bile duct cancer. PMID:14676443

  1. Pattern recognition of neuron specific enolase and carcinoembryonic antigen in whole blood samples.

    PubMed

    Stefan-van Staden, Raluca-Ioana; Comnea-Stancu, Ionela Raluca; Surdu-Bob, Carmen Cristina; Stanciu-Gavan, Camelia

    2015-02-01

    New tools and methods for pattern recognition of neuron specific enolase (NSE) and carcinoembryonic antigen (CEA) were proposed for the screening of whole blood samples. The new tools were based on stochastic sensors designed using nanoporous gold microspheres, graphite, graphene, diamond paste as well as α-CDs, and 5,10,15,20-tetraphenyl-21H,23H-porphyrin. The best sensor for the assay of CEA was the one based on P/graphite (the limit of determination was 16 fg/ml and sensitivity was 2.32 × 10(7)  s mg(-1)  ml), while for the assay of NSE the, best sensor was the one based on P/graphene (the limit of determination was 7.45 pg/ml and sensitivity was 2.49 × 10(8)  s mg(-1)  ml). The sensor of choice for simultaneous detection of NSE and CEA is the one based on P/graphene because we need high sensitivity and low limit of determination for NSE. To our knowledge, this is the only one screening test for early detection of lung cancer, by identification of NSE and CEA in whole blood samples. PMID:25604868

  2. An Ultrasensitive Chemiluminescence Biosensor for Carcinoembryonic Antigen Based on Autocatalytic Enlargement of Immunogold Nanoprobes

    PubMed Central

    Hao, Minjia; Ma, Zhanfang

    2012-01-01

    A sensitive flow injection chemiluminescence assay for carcinoembryonic antigen (CEA) detection based on signal amplification with gold nanoparticles (NPs) is reported in the present work. The sandwich system of CEA/anti-CEA/goat-anti-mouse IgG functionalized Au nanoparticles was used as the sensing platform. In order to improve detection sensitivity, a further gold enlargement step was developed based on the autocatalytic Au deposition of gold nanoprobes via the reduction of AuCl4− to Au0 on their surface in the presence of NH2OH·HCl. AuCl4−, which is a soluble product of gold nanoprobes, served as an analyte in the CL reaction for the indirect measurement of CEA. Under optimized conditions, the CL intensity of the system was linearly related to the logarithm of CEA concentration in the range of 100 pg·mL−1 to 1,000 ng·mL−1, with a detection limit of 20 pg·mL−1. PMID:23443399

  3. Preoperative carcinoembryonic antigen is related to tumour stage and long-term survival in colorectal cancer.

    PubMed Central

    Chapman, M. A.; Buckley, D.; Henson, D. B.; Armitage, N. C.

    1998-01-01

    Evidence as to the value of preoperative carcinoembryonic antigen (CEA) in guiding treatment for patients with colorectal cancer is conflicting. The aim of this prospective study was to investigate the value of preoperative CEA in predicting tumour factors of proven prognostic value and long-term survival in patients undergoing surgery for colorectal cancer. Preoperative serum CEA, tumour ploidy, stage and grade were ascertained in 277 patients undergoing colorectal cancer surgery. This cohort of patients were followed up for a minimum of 5 years, or until death, in a dedicated colorectal clinic. Patients with an elevated CEA had a 5 year survival of 39%. This increased to 57% if the CEA was normal (P=0.001). The proportion of patients with a raised CEA increased with a more advanced tumour stage (P < 0.000001) and a poorly differentiated tumour grade (P < 0.005). Once stage had been controlled for, CEA was not a predictor of survival. No relationship between tumour ploidy and CEA was found. In conclusion, a raised preoperative serum CEA is likely to be associated with advanced tumour stage and poor long-term survival, compared with patients with a normal value. PMID:9823977

  4. Aptasensor based on tripetalous cadmium sulfide-graphene electrochemiluminescence for the detection of carcinoembryonic antigen.

    PubMed

    Shi, Gui-Fang; Cao, Jun-Tao; Zhang, Jing-Jing; Huang, Ke-Jing; Liu, Yan-Ming; Chen, Yong-Hong; Ren, Shu-Wei

    2014-11-21

    A facile label-free electrochemiluminescence (ECL) aptasensor, based on the ECL of cadmium sulfide-graphene (CdS-GR) nanocomposites with peroxydisulfate as the coreactant, was designed for the detection of carcinoembryonic antigen (CEA). Tripetalous CdS-GR nanocomposites were synthesized through a simple onepot solvothermal method and immobilized on the glassy carbon electrode surface. L-Cystine (L-cys) could largely promote the electron transfer and enhance the ECL intensity. Gold nanoparticles (AuNPs) were assembled onto the L-cys film modified electrode for aptamer immobilization and ECL signal amplification. The aptamer modified with thiol was adsorbed onto the surface of the AuNPs through a Au-S bond. Upon hybridization of the aptamer with the target protein, the sequence could conjugate CEA to form a Y architecture. With CEA as a model analyte, the decreased ECL intensity is proportional to the CEA concentration in the range of 0.01-10.0 ng mL(-1) with a detection limit of 3.8 pg mL(-1) (S/N = 3). The prepared aptasensor was applied to the determination of CEA in human serum samples. The recoveries of CEA in the human serum samples were between 85.0% and 109.5%, and the RSD values were no more than 3.4%. PMID:25209409

  5. Carcinoembryonic antigen-producing adrenal adenoma resected using combined lateral and anterior transperitoneal laparoscopic surgery

    PubMed Central

    Hori, Tomohide; Taniguchi, Kentaro; Kurata, Masashi; Nakamura, Kenji; Kato, Kenji; Ogura, Yoshifumi; Iwasaki, Makoto; Okamoto, Shinya; Yamakado, Koichiro; Yagi, Shintaro; Iida, Taku; Kato, Takuma; Saito, Kanako; Wang, Linan; Kawarada, Yoshifumi; Uemoto, Shinji

    2007-01-01

    A 74-year-old woman presented with symptoms consistent with hyperadrenocorticism and hyperca-techolaminism. She had a cushingoid appearance and her cortisol level was elevated. Her serum dopamine and noradrenalin levels were also elevated. Computed tomography detected a left adrenal mass measuring 3.5 cm × 3.0 cm in diameter. Metaiodobenzylguanidine scintigraphy was negative. Unexpectedly, the serum Serum carcinoembryonic antigen (CEA) level was elevated. Fluorodeoxyglucose positron emission tomography showed increased uptake in the adrenal tumor only, with a maximum standardized uptake value of 2.8. Selective venography and blood sampling revealed that the concentrations of cortisol, catecholamines and CEA were significantly elevated in the vein draining the tumor. A diagnosis of CEA-producing benign adenoma was made. After preoperative management, we performed a combined lateral and anterior transperitoneal laparoscopic adrenectomy. Her vital signs remained stable during surgery. Histopathological examination revealed a benign adenoma. Her cortisol, catecholamine and CEA levels normalized immediately after surgery. We present, to the best of our knowledge, the first case of CEA-producing adrenal adenoma, along with a review of the relevant literature, and discuss our laparoscopic surgery techniques. PMID:18023107

  6. Impact of RAS and BRAF mutations on carcinoembryonic antigen production and pattern of colorectal metastases

    PubMed Central

    Cho, May; Akiba, Chie; Lau, Cecilia; Smith, David; Telatar, Milhan; Afkhami, Michelle; Sentovich, Stephen; Melstrom, Kurt; Fakih, Marwan

    2016-01-01

    AIM: To investigate the impact of RAS and BRAF mutations on the pattern of metastatic disease and carcinoembryonic antigen (CEA) production. METHODS: In this retrospective study, we investigated the impact of RAS and BRAF mutational status on pattern of metastatic disease and CEA production. Only patients presenting with a newly diagnosed metastatic colorectal cancer (CRC) were included. Patients’ characteristics, primary tumor location, site of metastatic disease and CEA at presentation were compared between those with and without RAS and BRAF mutations. RESULTS: Among 174 patients, mutations in KRAS, NRAS and BRAF were detected in 47%, 3% and 6% respectively. RAS mutations (KRAS and NRAS) were more likely to be found in African American patients (87% vs 13%; P value = 0.0158). RAS mutations were associated with a higher likelihood of a normal CEA (< 5 ng/mL) at presentation. BRAF mutations were more likely to occur in females. We were not able to confirm any association between mutational status and site of metastatic disease at initial diagnosis. CONCLUSION: No association was found between RAS and BRAF mutations and sites of metastatic disease at the time of initial diagnosis in our cohort. Patients with RAS mutations were more likely to present with CEA levels < 5 ng/mL. These findings may have clinical implications on surveillance strategies for RAS mutant patients with earlier stages of CRC. PMID:26798444

  7. Crosstalk of carcinoembryonic antigen and transforming growth factor-β via their receptors: comparing human and canine cancer.

    PubMed

    Jensen-Jarolim, Erika; Fazekas, Judit; Singer, Josef; Hofstetter, Gerlinde; Oida, Kumiko; Matsuda, Hiroshi; Tanaka, Akane

    2015-05-01

    There is accumulating evidence that the transforming growth factor beta (TGF-β) and nuclear factor kappa-B (NFκB) pathways are tightly connected and play a key role in malignant transformation in cancer. Immune infiltration by regulatory T- and B-lymphocytes (Tregs, Bregs) has recently gained increased attention for being an important source of TGF-β. There is a plethora of studies examining the pro-tumorigenic functions of carcinoembryonic antigen (CEA), but its receptor CEAR is far less studied. So far, there is a single connecting report that TGF-β also may signal through CEAR. The crosstalk between cancer tissues is further complicated by the expression of CEAR and TGF-β receptors in stromal cells, and implications of TGF-β in epithelial-mesenchymal transition. Furthermore, tumor-infiltrating Tregs and Bregs may directly instruct cancer cells by secreting TGF-β binding to their CEAR. Therefore, both TGF-β and CEA may act synergistically in breast cancer and cause disease progression, and NFκB could be a common crossing point between their signaling. CEAR, TGF-β1-3, TGF-β-R types I-III and NFκB class I and II molecules have an outstanding human-canine sequence identity, and only a canine CEA homolog has not yet been identified. For these reasons, the dog may be a valid translational model patient for investigating the crosstalk of the interconnected CEA and TGF-β networks. PMID:25832000

  8. Simultaneous Detection of α-Fetoprotein and Carcinoembryonic Antigen Based on Si Nanowire Field-Effect Transistors

    PubMed Central

    Zhu, Kuiyu; Zhang, Ye; Li, Zengyao; Zhou, Fan; Feng, Kang; Dou, Huiqiang; Wang, Tong

    2015-01-01

    Primary hepatic carcinoma (PHC) is one of the most common malignancies worldwide, resulting in death within six to 20 months. The survival rate can be improved by effective treatments when diagnosed at an early stage. The α-fetoprotein (AFP) and carcinoembryonic antigen (CEA) have been identified as markers that are expressed at higher levels in PHC patients. In this study, we employed silicon nanowire field-effect transistors (SiNW-FETs) with polydimethylsiloxane (PDMS) microfluidic channels to simultaneously detect AFP and CEA in desalted human serum. Dual-channel PDMS was first utilized for the selective modification of AFP and CEA antibodies on SiNWs, while single-channel PDMS offers faster and more sensitive detection of AFP and CEA in serum. During the SiNW modification process, 0.1% BSA was utilized to minimize nonspecific protein binding from serum. The linear dynamic ranges for the AFP and CEA detection were measured to be 500 fg/mL to 50 ng/mL and 50 fg/mL to 10 ng/mL, respectively. Our work demonstrates the promising potential of fabricated SiNW-FETs as a direct detection kit for multiple tumor markers in serum; therefore, it provides a chance for early stage diagnose and, hence, more effective treatments for PHC patients. PMID:26251912

  9. Simultaneous Detection of α-Fetoprotein and Carcinoembryonic Antigen Based on Si Nanowire Field-Effect Transistors.

    PubMed

    Zhu, Kuiyu; Zhang, Ye; Li, Zengyao; Zhou, Fan; Feng, Kang; Dou, Huiqiang; Wang, Tong

    2015-01-01

    Primary hepatic carcinoma (PHC) is one of the most common malignancies worldwide, resulting in death within six to 20 months. The survival rate can be improved by effective treatments when diagnosed at an early stage. The α-fetoprotein (AFP) and carcinoembryonic antigen (CEA) have been identified as markers that are expressed at higher levels in PHC patients. In this study, we employed silicon nanowire field-effect transistors (SiNW-FETs) with polydimethylsiloxane (PDMS) microfluidic channels to simultaneously detect AFP and CEA in desalted human serum. Dual-channel PDMS was first utilized for the selective modification of AFP and CEA antibodies on SiNWs, while single-channel PDMS offers faster and more sensitive detection of AFP and CEA in serum. During the SiNW modification process, 0.1% BSA was utilized to minimize nonspecific protein binding from serum. The linear dynamic ranges for the AFP and CEA detection were measured to be 500 fg/mL to 50 ng/mL and 50 fg/mL to 10 ng/mL, respectively. Our work demonstrates the promising potential of fabricated SiNW-FETs as a direct detection kit for multiple tumor markers in serum; therefore, it provides a chance for early stage diagnose and, hence, more effective treatments for PHC patients. PMID:26251912

  10. Emerging Role and Targeting of Carcinoembryonic Antigen-related Cell Adhesion Molecule 6 (CEACAM6) in Human Malignancies

    PubMed Central

    Johnson, Benny; Mahadevan, Daruka

    2015-01-01

    Background: Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is a member of the CEA family of cell adhesion proteins that belong to the immunoglobulin superfamily. CEACAM6 is normally expressed on the surface of myeloid (CD66c) and epithelial surfaces. Stiochiomertic expression of members of the CEA family (CEACAM1, 5, 6, 7) on epithelia maintains normal tissue architecture through homo-and hetero-philic interactions. Dysregulated over-expression of CEACAM6 is oncogenic, is associated with anoikis resistance and an invasive phenotype mediated by excessive TGFβ, AKT, FAK and SRC signaling in human malignancies. Methods: Extensive literature review through PubMed was conducted to identify relevant preclinical and clinical research publications regarding CEACAM6 over the last decade and was summarized in this manuscript. Results: CEACAM5 and 6 are over-expressed in nearly 70% of epithelial malignancies including colorectal cancer (CRC), pancreatic ductal adenocarcinoma (PDA), hepatobiliary, gastric, breast, non-small cell lung and head/neck cancers. Importantly, CEACAM6 is a poor prognostic marker in CRC, while its expression correlates with tumor stage, metastasis and post-operative survival in PDA. CEACAM6 appears to be an immune checkpoint suppressor in hematologic malignancies including acute lymphoblastic leukemia and multiple myeloma. Several therapeutic monoclonal antibodies or antibody fragments targeting CEACAM6 have been designed and developed as a targeted therapy for human malignancies. A Llama antibody targeting CEACAM6 is being evaluated in early phase clinical trials. Conclusion: This review focuses on the role of CEACAM6 in the pathogenesis and signaling of the malignant phenotype in solid and hematologic malignancies and highlights its potential as a therapeutic target for anti-cancer therapy.

  11. Baseline carcinoembryonic antigen (CEA) serum levels predict bevacizumab-based treatment response in metastatic colorectal cancer

    PubMed Central

    Prager, Gerald W; Braemswig, Kira H; Martel, Alexandra; Unseld, Matthias; Heinze, Georg; Brodowicz, Thomas; Scheithauer, Werner; Kornek, Gabriela; Zielinski, Christoph C

    2014-01-01

    Carcinoembryonic antigen (CEA) affects tumorigenesis by enhancing tumor cell survival and by inducing tumor angiogenesis. This study aimed to evaluate baseline CEA serum levels to predict bevacizumab-based therapy effect and survival in patients with metastatic colorectal cancer (mCRC). Two hundred and ninety eight mCRC patients receiving chemotherapy plus either bevacizumab or cetuximab were analyzed in a retrospective study. Disease control (DC), progression-free survival (PFS), and overall survival were assessed and related to pretreatment CEA serum levels. Patients with baseline CEA serum levels below the statistical median of 26.8 ng/mL (group I) were compared with patients with higher CEA levels (group II). The cetuximab-based treatment cohort was analyzed for specificity assessment of CEA to predict the anti-vascular endothelial growth factor effect in mCRC. Baseline CEA serum levels inversely correlated with therapeutic response in patients receiving bevacizumab-based treatment (disease control rate, 84% vs 60%), inversely correlated with median PFS leading to a median PFS benefit of 2.1 months for patients in group I when compared with group II, as well as inversely correlated with median overall survival (37.5 months vs 21.4 months). In an independent cohort of 129 patients treated with cetuximab-based therapy, no association of therapeutic response or PFS with CEA serum levels was found. As expected, baseline CEA levels were prognostic for mCRC. These data give first evidence that baseline serum CEA levels might constitute an important predictor for the efficacy of first-line bevacizumab-based therapy in patients with mCRC. Previously, we found that CEA induces angiogenesis independent of VEGF. The data presented here now give first evidence that baseline serum CEA levels in patients might constitute an important predictor for the efficacy of first-line bevacizumab-based therapy for metastatic colorectal cancer. PMID:24850362

  12. Elevated Level of Serum Carcinoembryonic Antigen (CEA) and Search for a Malignancy: A Case Report

    PubMed Central

    Saif, Muhammad W

    2016-01-01

    Carcinoembryonic antigen (CEA) has been shown to be associated with tumor burden in patients with colorectal cancer. However, it is also elevated to a significant degree in a number of other malignant and non-malignant conditions. We report a case of reversible CEA elevation in a patient using lithium for bipolar disorder. A 58-year-old female with a longstanding smoking history and a past medical history of chronic obstructive pulmonary disease (COPD), bipolar illness, hypothyroidism, and obesity was found to have an elevated CEA level of 11.2 ng/ml (normal level <5 ng/ml) in the workup for postmenopausal bleeding. Her history was not positive for malignancy of colorectum, ovaries, thyroid, or breast.  She underwent a large number of imaging and endoscopic studies to evaluate for colorectal, breast, ovarian, and lung cancer; however, it did not reveal any evidence of malignancy. Upon review of her medications, she reported that she had recently started lithium for her bipolar illness. We followed up her CEA level while her dose of lithium was reduced from 450 to 300 mg per day. Her CEA level decreased from 25 mg/dl to 6.1 mg/dl and remained stable over the course of the next eight months. Our case is the first case report that identifies lithium as a potential cause of reversible CEA elevation. The underlying mechanism is yet to be elucidated, but it underscores the importance of investigating the medications as part of the workup. PMID:27446768

  13. Prognostic values of cathepsin B and carcinoembryonic antigen in sera of patients with colorectal cancer.

    PubMed

    Kos, J; Nielsen, H J; Krasovec, M; Christensen, I J; Cimerman, N; Stephens, R W; Brünner, N

    1998-06-01

    The level of cathepsin B (Cat B) was determined in sera obtained preoperatively from 325 patients with colorectal cancer using an ELISA. Control sera from 90 healthy blood donors were analyzed. The levels of Cat B detected included all forms that were present in the sera, i.e., mature enzyme, precursor molecule, and enzyme-inhibitor complexes. The level of Cat B was significantly increased in sera of patients with colorectal cancer. The median level was 10.7 ng/ml versus 2.1 ng/ml in controls (P < 0.0001). A correlation between Cat B serum level and advanced Dukes' stage (P < 0.003) was found, whereas no associations have been found with age, sex, or level of carcinoembryonic antigen (CEA). In survival analysis, the patients with high serum Cat B experienced significantly lower survival probability. At the optimal cutoff value of 9.4 ng/ml, the relative hazard ratio was 1.8 (95% confidence interval, 1.1-2.8; P = 0.016) in the univariate Cox proportional hazards model. The median observation time was 4.4 years (range, 3.2-5.5 years). In multivariate analysis, Dukes' stage was the strongest prognostic variable, followed by age, whereas serum Cat B and CEA were not significant prognostic factors in this model, in accordance with their association with Dukes' stage. When the data for Cat B and CEA were combined, CEA-positive patients were further separated by Cat B into high- and low-risk groups. Patients with high serum levels of both molecules had significantly shorter survival (relative hazard ratio of 2.2; 95% confidence interval, 1.5-3.2; P < 0.0001), as compared with patients with low levels of both molecules. PMID:9626470

  14. Elevated Level of Serum Carcinoembryonic Antigen (CEA) and Search for a Malignancy: A Case Report.

    PubMed

    Asad-Ur-Rahman, Fnu; Saif, Muhammad W

    2016-01-01

    Carcinoembryonic antigen (CEA) has been shown to be associated with tumor burden in patients with colorectal cancer. However, it is also elevated to a significant degree in a number of other malignant and non-malignant conditions. We report a case of reversible CEA elevation in a patient using lithium for bipolar disorder. A 58-year-old female with a longstanding smoking history and a past medical history of chronic obstructive pulmonary disease (COPD), bipolar illness, hypothyroidism, and obesity was found to have an elevated CEA level of 11.2 ng/ml (normal level <5 ng/ml) in the workup for postmenopausal bleeding. Her history was not positive for malignancy of colorectum, ovaries, thyroid, or breast.  She underwent a large number of imaging and endoscopic studies to evaluate for colorectal, breast, ovarian, and lung cancer; however, it did not reveal any evidence of malignancy. Upon review of her medications, she reported that she had recently started lithium for her bipolar illness. We followed up her CEA level while her dose of lithium was reduced from 450 to 300 mg per day. Her CEA level decreased from 25 mg/dl to 6.1 mg/dl and remained stable over the course of the next eight months. Our case is the first case report that identifies lithium as a potential cause of reversible CEA elevation. The underlying mechanism is yet to be elucidated, but it underscores the importance of investigating the medications as part of the workup. PMID:27446768

  15. Inhibition and promotion of tumor growth with adeno-associated virus carcinoembryonic antigen vaccine and Toll-like receptor agonists.

    PubMed

    Triozzi, P L; Aldrich, W; Ponnazhagan, S

    2011-12-01

    Carcinoembryonic antigen (CEA) is a cancer vaccines' target. Several features of recombinant adeno-associated virus (rAAV) are attractive for vaccine applications. Combining other viral vector vaccines with Toll-like receptor (TLR) agonists enhances antitumor immunity. Wild-type and CEA transgenic (Tg) mice were immunized with rAAV-expressing CEA, the TLR9 agonist, oligodinucleotide (ODN)1826 and the TLR7 agonist, imiquimod. Mice were challenged with MC38 colon tumor cells and MC38 cells expressing CEA. rAAV-CEA immunization combined with ODN1826 or imiquimod enhanced CEA-specific T-helper 1 immunity and protected against tumor challenge in wild-type but not in CEA-Tg mice. In contrast, immunization with rAAV-CEA in CEA-Tg mice could abrogate the antitumor effects of ODN1826 and promote tumor growth. Compared to wild-type, CEA-Tg mice were characterized by a greater myeloid suppressor cell and T-helper 2 response to TLR agonists and to syngeneic tumors. Depleting PDCA1(+) plasmacytoid dendritic cells and Gr1(+) myeloid cells increased anti-CEA immune responses in CEA-Tg mice to rAAV-CEA-ODN1826 immunization, whereas depleting CD25(+) T cells did not. There are differences in the response of wild-type and CEA-Tg mice to rAAV-CEA, TLR agonists and syngeneic tumor. In CEA-Tg mice, tumor growth can be promoted with rAAV-CEA and TLR agonists. Dendritic and myeloid cells play a regulatory role. PMID:21869824

  16. Half-antibody functionalized lipid-polymer hybrid nanoparticles for targeted drug delivery to carcinoembryonic antigen presenting pancreatic cancer cells.

    PubMed

    Hu, Che-Ming Jack; Kaushal, Sharmeela; Tran Cao, Hop S; Aryal, Santosh; Sartor, Marta; Esener, Sadik; Bouvet, Michael; Zhang, Liangfang

    2010-06-01

    Current chemotherapy regimens against pancreatic cancer are met with little success as poor tumor vascularization significantly limits the delivery of oncological drugs. High-dose targeted drug delivery, through which a drug delivery vehicle releases a large payload upon tumor localization, is thus a promising alternative strategy against this lethal disease. Herein, we synthesize anti-carcinoembryonic antigen (CEA) half-antibody conjugated lipid-polymer hybrid nanoparticles and characterize their ligand conjugation yields, physicochemical properties, and targeting ability against pancreatic cancer cells. Under the same drug loading, the half-antibody targeted nanoparticles show enhanced cancer killing effect compared to the corresponding nontargeted nanoparticles. PMID:20394436

  17. The carcinoembryonic antigen IgV-like N domain plays a critical role in the implantation of metastatic tumor cells.

    PubMed

    Abdul-Wahid, Aws; Huang, Eric H-B; Cydzik, Marzena; Bolewska-Pedyczak, Eleonora; Gariépy, Jean

    2014-03-01

    The human carcinoembryonic antigen (CEA) is a cell adhesion molecule involved in both homotypic and heterotypic interactions. The aberrant overexpression of CEA on adenocarcinoma cells correlates with their increased metastatic potential. Yet, the mechanism(s) by which its adhesive properties can lead to the implantation of circulating tumor cells and expansion of metastatic foci remains to be established. In this study, we demonstrate that the IgV-like N terminal domain of CEA directly participates in the implantation of cancer cells through its homotypic and heterotypic binding properties. Specifically, we determined that the recombinant N terminal domain of CEA directly binds to fibronectin (Fn) with a dissociation constant in the nanomolar range (K(D) 16 ± 3 nM) and interacts with itself (K(D) 100 ± 17 nM) and more tightly to the IgC-like A(3) domain (K(D) 18 ± 3 nM). Disruption of these molecular associations through the addition of antibodies specific to the CEA N or A(3)B(3) domains, or by adding soluble recombinant forms of the CEA N, A(3) or A(3)B(3) domains or a peptide corresponding to residues 108-115 of CEA resulted in the inhibition of CEA-mediated intercellular aggregation and adherence events in vitro. Finally, pretreating CEA-expressing murine colonic carcinoma cells (MC38.CEA) with rCEA N, A3 or A(3)B(3) modules blocked their implantation and the establishment of tumor foci in vivo. Together, these results suggest a new mechanistic insight into how the CEA IgV-like N domain participates in cellular events that can have a macroscopic impact in terms of cancer progression and metastasis. PMID:24388361

  18. A Sandwich Electrochemical Immunosensor Using Magnetic DNA Nanoprobes for Carcinoembryonic Antigen

    PubMed Central

    Gan, Ning; Jia, Liyong; Zheng, Lei

    2011-01-01

    A novel magnetic nanoparticle-based electrochemical immunoassay of carcinoembryonic antigen (CEA) was designed as a model using CEA antibody-functionalized magnetic beads [DNA/Fe3O4/ZrO2; Fe3O4 (core)/ZrO2 (shell) nano particles (ZMPs)] as immunosensing probes. To design the immunoassay, the CEA antibody and O-phenylenediamine (OPD) were initially immobilized on a chitosan/nano gold composite membrane on a glassy carbon electrode (GCE/CS-nano Au), which was used for CEA recognition. Then, horseradish peroxidase (HRP)-labeled anti-CEA antibodies (HRP-CEA Ab2) were bound to the surface of the synthesized magnetic ZMP nanoparticles as signal tag. Thus, the sandwich-type immune complex could be formed between secondary antibody (Ab2) modified DNA/ZMPs nanochains tagged by HRP and GCE/CS-nano Au. Unlike conventional nanoparticle-based electrochemical immunoassays, the recognition elements of this immunoassay included both electron mediators and enzyme labels, which obviously simplifies the electrochemical measurement process. The sandwich-type immunoassay format was used for online formation of the immunocomplex of CEA captured in the detection cell with an external magnet. The electrochemical signals derived from HRP during the reduction of H2O2 with OPD as electron mediator were measured. The method displayed a high sensitivity for CEA detection in the range of 0.008–200 ng/mL, with a detection limit of 5 pg/mL (estimated at a signal-to-noise ratio of 3). The precision, reproducibility, and stability of the immunoassay were good. The use of the assay was evaluated with clinical serum samples, and the results were in excellent accordance with those obtained using the standard enzyme-linked immunosorbent assay (ELISA) method. Thus, the magnetic nanoparticle-based assay format is a promising approach for clinical applications, and it could be further developed for the detection of other biomarkers in cancer diagnosis. PMID:22174606

  19. Radiocurability by Targeting Tumor Necrosis Factor-{alpha} Using a Bispecific Antibody in Carcinoembryonic Antigen Transgenic Mice

    SciTech Connect

    Larbouret, Christel; Robert, Bruno; Linard, Christine; Teulon, Isabelle; Gourgou, Sophie M.Sc.; Bibeau, Frederic; Martineau, Pierre; Santoro, Lore; Pouget, Jean-Pierre; Pelegrin, Andre; Azria, David

    2007-11-15

    Purpose: Tumor necrosis factor-{alpha} (TNF-{alpha}) enhances radiotherapy (RT) killing of tumor cells in vitro and in vivo. To overcome systemic side effects, we used a bispecific antibody (BsAb) directed against carcinoembryonic antigen (CEA) and TNF-{alpha} to target this cytokine in a CEA-expressing colon carcinoma. We report the evaluation of this strategy in immunocompetent CEA-transgenic mice. Methods and Materials: The murine CEA-transfected colon carcinoma MC-38 was used for all experiments. In vitro, clonogenic assays were performed after RT alone, TNF-{alpha} alone, and RT plus TNF-{alpha}. In vivo, the mice were randomly assigned to treatment groups: control, TNF-{alpha}, BsAb, BsAb plus TNF-{alpha}, RT, RT plus TNF-{alpha}, and RT plus BsAb plus TNF-{alpha}. Measurements of endogenous TNF-{alpha} mRNA levels and evaluation of necrosis (histologic evaluation) were assessed per treatment group. Results: In vitro, combined RT plus TNF-{alpha} resulted in a significant decrease in the survival fraction at 2 Gy compared with RT alone (p < 0.00001). In vivo, we observed a complete response in 5 (50%) of 10, 2 (20%) of 10, 2 (18.2%) of 11, and 0 (0%) of 12 treated mice in the RT plus BsAb plus TNF-{alpha}, RT plus TNF-{alpha}, RT alone, and control groups, respectively. This difference was statistically significant when TNF-{alpha} was targeted with the BsAb (p = 0.03). The addition of exogenous TNF-{alpha} to RT significantly increased the endogenous TNF-{alpha} mRNA level, particularly when TNF-{alpha} was targeted with BsAb (p < 0.01). The percentages of necrotic area were significantly augmented in the RT plus BsAb plus TNF-{alpha} group. Conclusion: These results suggest that targeting TNF-{alpha} with the BsAb provokes RT curability in a CEA-expressing digestive tumor syngenic model and could be considered as a solid rationale for clinical trials.

  20. One year survival rate in patients with carcinoma of the lung treated with split course irradiation: correlation with serum value of carcinoembryonic antigen. [Linear accelerator

    SciTech Connect

    Bolla, M.; Vrousos, C.; Agnus-Delord, C.; Kolodie, H.; Paramelle, B.

    1980-08-01

    Carcinoembryonic antigen (CEA) radio immune assay was performed in 51 patients with lung carcinoma before and at various times after loco-regional radiotherapy. Increase of CEA level correlated with metastasis or progression of the disease; decrease in CEA levels reflected significant regression or stabilization of the disease.

  1. A chitosan-Au-hyperbranched polyester nanoparticles-based antifouling immunosensor for sensitive detection of carcinoembryonic antigen.

    PubMed

    Sun, Chong; Ma, Lie; Qian, Qiuhui; Parmar, Soniya; Zhao, Wenbo; Zhao, Bo; Shen, Jian

    2014-09-01

    Analysts are always interested in finding new functional nanomaterials and devices with good properties for electrochemical sensor applications. In this paper, hyperbranched polyester nanoparticles with carboxylic acid functional groups (HBPE-CA NPs) were synthesized and combined with chitosan wrapped around Au nanoparticles (CS-Au NPs) to prepare a novel and sensitive electrochemical immunosensor by adsorption of carcinoembryonic antibody (anti-CEA) on the (HBPE-CA)/CS-Au NPs modified glass carbon electrode (GCE). Under the optimized conditions, the proposed immunosensor displayed a good amperometric response to carcinoembryonic antigen (CEA). Moreover, based on the antibiofouling properties, the immunosensor could be used for the direct detection of CEA in whole blood, and exhibited a wide detection range (1-10(7) fg mL(-1)), and a low detection limit of 0.251 fg mL(-1) (signal/noise = 3). Control experiments were also carried out by using ascorbic acid (AA), uric acid (UA), human immunoglobulin G (IgG), BSA and glucose in the absence of CEA. The good stability and repeatability of this immunosensor were also proven. Importantly, the results of the detection of clinical whole blood specimens with the proposed immunosensor showed good consistency with the data determined by enzyme-linked immunosorbent assay (ELISA) in serum samples. Furthermore, the developed immunosensor could provide a promising immunoassay strategy for clinical applications, since the values we measured in whole blood directly are likely closer to the real values. PMID:24957417

  2. Sorafenib Decreases Tumor Exposure to an Anti-carcinoembryonic Antigen Monoclonal Antibody in a Mouse Model of Colorectal Cancer.

    PubMed

    Thomas, Veena A; Balthasar, Joseph P

    2016-07-01

    In this investigation, we test the hypothesis that treatment with sorafenib, an anti-angiogenic agent, decreases tumor vascularization and, consequently, hinders the delivery of monoclonal antibodies (mAb) to xenograft tumors. Severe combined immunodeficiency mice bearing carcinoembryonic antigen (CEA) expressing tumor xenografts were divided into control and sorafenib-treated groups. Sorafenib was administered to the latter group at 50 mg/kg IP every 48 h, starting 4 days post-tumor implantation. When tumors attained a size of 200-300 mm(3), mice were evaluated for (a) tumor microvessel density (using immunohistochemical analysis), (b) tumor macromolecular extravasation (using Evans Blue Dye (EBD)), (c) pharmacokinetics of an anti-CEA mAb, T84.66, following an intravenous dose of 10 mg/kg, and (d) intra-tumoral spatial distribution of T84.66 (using autoradiography). Sorafenib treatment resulted in a substantial reduction in tumor growth rate, a visible reduction in tumor microvessel density, and in a 46.4% decrease in EBD extravasation in tumor tissue (p < 0.0455). For control and treated mice, no significant difference was found for the area under the mAb plasma concentration-time curve (AUC(0-7d): 1.67 × 10(3) ± 1.28 × 10(2) vs. 1.76 × 10(3) ± 1.75 × 10(2) nM × day, p = 0.51). However, tumor AUC(0-7d) was reduced by 40.8% in sorafenib-treated mice relative to that observed in control mice (5.61 × 10(2) ± 4.27 × 10(1) vs. 9.48 × 10(2) ± 5.61 × 10(1) nM × day, p < 0.001). Sorafenib therapy was also found to markedly alter mAb tumor spatial distribution. The results collectively suggest that sorafenib treatment causes a significant reduction in mAb delivery to, and distribution within, solid tumors. PMID:27029796

  3. Correlation of disease activity and serum level of carcinoembryonic antigen in acquired idiopathic generalized anhidrosis: A case report.

    PubMed

    Honma, Masaru; Iinuma, Shin; Kanno, Kyoko; Komatsu, Shigetsuna; Minami-Hori, Masako; Ishida-Yamamoto, Akemi

    2015-09-01

    Hypohidrosis and anhidrosis are congenital or acquired conditions which are characterized by inadequate sweating. Acquired idiopathic generalized hypohidrosis/anhidrosis (AIGA) includes idiopathic pure sudomotor failure (IPSF), which has the following distinct features: sudden onset in youth, increased serum immunoglobulin E and responds favorably to systemic corticosteroid. No clinical markers reflecting the disease severity or activity have been established. Here, we report a case of AIGA in a Japanese patient successfully treated with repeated methylprednisolone pulse therapy. In this case, serum carcinoembryonic antigen (CEA) levels increased up to 19.8 ng/mL along with aberrant CEA immunoreactivity of eccrine sweat glands. Interestingly, the serum CEA level normalized as sweating improved with repeated methylprednisolone pulse therapy. Therefore, serum CEA level may serve as a useful clinical marker of hypohidrosis or anhidrosis. PMID:25958966

  4. A novel label-free microfluidic paper-based immunosensor for highly sensitive electrochemical detection of carcinoembryonic antigen.

    PubMed

    Wang, Yang; Xu, Huiren; Luo, Jinping; Liu, Juntao; Wang, Li; Fan, Yan; Yan, Shi; Yang, Yue; Cai, Xinxia

    2016-09-15

    In this work, a highly sensitive label-free paper-based electrochemical immunosensor employing screen-printed working electrode (SPWE) for detection of carcinoembryonic antigen (CEA) was fabricated. In order to raise the detection sensitivity and immobilize anti-CEA, amino functional graphene (NH2-G)/thionine (Thi)/gold nanoparticles (AuNPs) nanocomposites were synthesized and coated on SPWE. The principle of the immunosensor determination was based on the fact that the decreased response currents of Thi were proportional to the concentrations of corresponding antigens due to the formation of antibody-antigen immunocomplex. Experimental results revealed that the immunoassay enabled the determination of standard CEA solutions with linear working ranges of 50pgmL(-1) to 500ngmL(-1), the limit of detections for CEA is 10pgmL(-1) (S/N=3) and its corresponding correlation coefficients were 0.996. Furthermore, the proposed immunosensor could be used for the determination of clinical serum samples. A large number of clinical serum samples were detected and the relative errors between measured values and reference concentrations were calculated. Results showed that this novel paper-based electrochemical immunosensor could provide a new platform for low cost, sensitive, specific, and point-of-care diagnosis in cancer detection. PMID:27132007

  5. The Genome-Wide Analysis of Carcinoembryonic Antigen Signaling by Colorectal Cancer Cells Using RNA Sequencing.

    PubMed

    Bajenova, Olga; Gorbunova, Anna; Evsyukov, Igor; Rayko, Michael; Gapon, Svetlana; Bozhokina, Ekaterina; Shishkin, Alexander; O'Brien, Stephen J

    2016-01-01

    Сarcinoembryonic antigen (CEA, CEACAM5, CD66) is a promoter of metastasis in epithelial cancers that is widely used as a prognostic clinical marker of metastasis. The aim of this study is to identify the network of genes that are associated with CEA-induced colorectal cancer liver metastasis. We compared the genome-wide transcriptomic profiles of CEA positive (MIP101 clone 8) and CEA negative (MIP 101) colorectal cancer cell lines with different metastatic potential in vivo. The CEA-producing cells displayed quantitative changes in the level of expression for 100 genes (over-expressed or down-regulated). They were confirmed by quantitative RT-PCR. The KEGG pathway analysis identified 4 significantly enriched pathways: cytokine-cytokine receptor interaction, MAPK signaling pathway, TGF-beta signaling pathway and pyrimidine metabolism. Our results suggest that CEA production by colorectal cancer cells triggers colorectal cancer progression by inducing the epithelial- mesenchymal transition, increasing tumor cell invasiveness into the surrounding tissues and suppressing stress and apoptotic signaling. The novel gene expression distinctions establish the relationships between the existing cancer markers and implicate new potential biomarkers for colorectal cancer hepatic metastasis. PMID:27583792

  6. Sensitive electrochemical immunoassay of carcinoembryonic antigen with signal dual-amplification using glucose oxidase and an artificial catalase.

    PubMed

    Tang, Juan; Tang, Dianping; Li, Qunfang; Su, Biling; Qiu, Bin; Chen, Guonan

    2011-07-01

    A new dual-amplification strategy of electrochemical signal based on the catalytic recycling of the product was developed for the antigen-antibody interaction by glucose oxidase (GOD)- conjugated gold-silver hollow microspheres (AuAgHSs) coupled with an artificial catalase, Prussian blue nanoparticles (PB), on a graphene-based immunosensing platform. The first signal amplification introduced in this study was based on the labeled GOD on the AuAgHSs toward the catalytic oxidation of glucose. The generated H(2)O(2) was catalytically reduced by the immobilized PB on the graphene nanosheets with the second amplification. With a sandwich-type immunoassay format, carcinoembryonic antigen (CEA) was monitored as a model analyte by using the synthesized AuAgHSs as labels in pH 6.0 phosphate buffer containing 10mM glucose. Under optimal conditions, the electrochemical immunosensor exhibited a wide dynamic range of 0.005-50 ng mL(-1) with a low detection limit (LOD) of 1.0 pg mL(-1) CEA (at 3σ). Both the intra- and inter-assay coefficients of variation (CVs) were lower than 10%. The specificity and stability of the immunosensor were acceptable. In addition, the assay was evaluated for clinical serum specimens, and received a good correlation with those obtained by the referenced electrochemiluminescent (ECL). PMID:21641413

  7. A network signal amplification strategy of ultrasensitive photoelectrochemical immunosensing carcinoembryonic antigen based on CdSe/melamine network as label.

    PubMed

    Li, Jiaojiao; Zhang, Yong; Kuang, Xuan; Wang, Zhiling; Wei, Qin

    2016-11-15

    Taking advantage of CdSe/melamine network as label and Au-TiO2 as substrate, this work developed a novel kind of signal amplification strategy for fabricating photoelectrochemical (PEC) immunoassay. The melamine, a star-shaped triamino molecule, was firstly used for readily capturing CdSe QDs and forming a CdSe/melamine network, which was formed through strong interactions between the carboxyl groups of TGA-stabilized CdSe QDs and the three amino groups of each melamine molecule. In this strategy, the primary antibody (Ab1) was immobilized onto Au-TiO2 substrate, which made the photoelectric conversion efficiency increase significantly. After the formed Ab2-CdSe/melamine network labels were captured onto the electrode surface via the specific antibody-antigen interaction, the photoelectric activity could be further enhanced via the interaction between the Au-TiO2 substrate and CdSe/melamine network. Due to this amplification of PEC signals and the special structure of the label, the fabricated PEC immunosensor was applied for sensitive and specific detection of cancer biomarker carcinoembryonic antigen (CEA), and displayed a wide linear range (0.005-1000ngmL(-1)) and low detection limit (5pgmL(-1)). In addition, the immunosensor was performed with good stability and reproducibility, and the results to analyze human serum samples were satisfactory. PMID:27281106

  8. Nanoporous gold as a solid support for protein immobilization and development of an electrochemical immunoassay for prostate specific antigen and carcinoembryonic antigen

    PubMed Central

    Pandey, Binod; Demchenko, Alexei V.; Stine, Keith J.

    2013-01-01

    Nanoporous gold (NPG) was utilized as a support for immobilizing alkaline phosphatase (ALP) conjugated to monoclonal antibodies against either prostate specific antigen (PSA) or carcinoembryonic antigen (CEA). The antibody-ALP conjugates were coupled to self-assembled monolayers of lipoic acid and used in direct kinetic assays. Using the enzyme substrate p-aminophenylphosphate, the product p-aminophenol was detected by its oxidation near 0.1 V (vs. Ag|AgCl) using square wave voltammetry. The difference in peak current arising from oxidation of p-aminophenol before and after incubation with biomarker increased with biomarker concentration. The response to these two biomarkers was linear up to 10 ng mL-1 for CEA and up to 30 ng mL-1 for PSA. The effect of interference on the PSA assay was studied using bovine serum albumin (BSA) as a model albumin protein. The effect of interference from a serum matrix was examined for the PSA assay using newborn calf serum. A competitive version of the immunoassay using antigen immobilized onto the NPG surface was highly sensitive at lower antigen concentration. Estimates of the surface coverage of the antibody-ALP conjugates on the NPG surface are presented. PMID:23935216

  9. Increased Glucose-induced Secretion of Glucagon-like Peptide-1 in Mice Lacking the Carcinoembryonic Antigen-related Cell Adhesion Molecule 2 (CEACAM2).

    PubMed

    Ghanem, Simona S; Heinrich, Garrett; Lester, Sumona G; Pfeiffer, Verena; Bhattacharya, Sumit; Patel, Payal R; DeAngelis, Anthony M; Dai, Tong; Ramakrishnan, Sadeesh K; Smiley, Zachary N; Jung, Dae Y; Lee, Yongjin; Kitamura, Tadahiro; Ergun, Suleyman; Kulkarni, Rohit N; Kim, Jason K; Giovannucci, David R; Najjar, Sonia M

    2016-01-01

    Carcinoembryonic antigen-related cell adhesion molecule 2 (CEACAM2) regulates food intake as demonstrated by hyperphagia in mice with the Ceacam2 null mutation (Cc2(-/-)). This study investigated whether CEACAM2 also regulates insulin secretion. Ceacam2 deletion caused an increase in β-cell secretory function, as assessed by hyperglycemic clamp analysis, without affecting insulin response. Although CEACAM2 is expressed in pancreatic islets predominantly in non-β-cells, basal plasma levels of insulin, glucagon and somatostatin, islet areas, and glucose-induced insulin secretion in pooled Cc2(-/-) islets were all normal. Consistent with immunofluorescence analysis showing CEACAM2 expression in distal intestinal villi, Cc2(-/-) mice exhibited a higher release of oral glucose-mediated GLP-1, an incretin that potentiates insulin secretion in response to glucose. Compared with wild type, Cc2(-/-) mice also showed a higher insulin excursion during the oral glucose tolerance test. Pretreating with exendin(9-39), a GLP-1 receptor antagonist, suppressed the effect of Ceacam2 deletion on glucose-induced insulin secretion. Moreover, GLP-1 release into the medium of GLUTag enteroendocrine cells was increased with siRNA-mediated Ceacam2 down-regulation in parallel to an increase in Ca(2+) entry through L-type voltage-dependent Ca(2+) channels. Thus, CEACAM2 regulates insulin secretion, at least in part, by a GLP-1-mediated mechanism, independent of confounding metabolic factors. PMID:26586918

  10. In vivo subcellular imaging of tumors in mouse models using a fluorophore-conjugated anti-carcinoembryonic antigen antibody in two-photon excitation microscopy

    PubMed Central

    Koga, Shigehiro; Oshima, Yusuke; Honkura, Naoki; Iimura, Tadahiro; Kameda, Kenji; Sato, Koichi; Yoshida, Motohira; Yamamoto, Yuji; Watanabe, Yuji; Hikita, Atsuhiko; Imamura, Takeshi

    2014-01-01

    Recently, there has been growing interest in applying fluorescence imaging techniques to the study of various disease processes and complex biological phenomena in vivo. To apply these methods to clinical settings, several groups have developed protocols for fluorescence imaging using antibodies against tumor markers conjugated to fluorescent substances. Although these probes have been useful in macroscopic imaging, the specificity and sensitivity of these methods must be improved to enable them to detect micro-lesions in the early phases of cancer, resulting in better treatment outcomes. To establish a sensitive and highly specific imaging method, we used a fluorophore-conjugated anti-carcinoembryonic antigen (CEA) antibody to perform macroscopic and microscopic in vivo imaging of inoculated cancer cells expressing GFP with or without CEA. Macroscopic imaging by fluorescence zoom microscopy revealed that bio-conjugation of Alexa Fluor 594 to the anti-CEA antibody allowed visualization of tumor mass consisting of CEA-expressing human cancer cells, but the background levels were unacceptably high. In contrast, microscopic imaging using a two-photon excitation microscope and the same fluorescent antibody resulted in subcellular-resolution imaging that was more specific and sensitive than conventional imaging using a fluorescence zoom microscope. These results suggest that two-photon excitation microscopy in conjunction with fluorophore-conjugated antibodies could be widely adapted to detection of cancer-specific cell-surface molecules, both in cancer research and in clinical applications. PMID:25117702

  11. The granulocyte receptor carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) directly associates with Vav to promote phagocytosis of human pathogens.

    PubMed

    Schmitter, Tim; Pils, Stefan; Sakk, Vadim; Frank, Ronald; Fischer, Klaus-Dieter; Hauck, Christof R

    2007-03-15

    The human granulocyte-specific receptor carcinoembryonic antigen-related cell adhesion molecule (CEACAM)3 is critically involved in the opsonin-independent recognition of several bacterial pathogens. CEACAM3-mediated phagocytosis depends on the integrity of an ITAM-like sequence within the cytoplasmic domain of CEACAM3 and is characterized by rapid stimulation of the GTPase Rac. By performing a functional screen with CEACAM3-expressing cells, we found that overexpression of a dominant-negative form of the guanine nucleotide exchange factor Vav, but not the dominant-negative versions SWAP70, Dock2, or ELMO1 interfered with CEACAM3-initiated phagocytosis. Moreover, small interfering RNA-mediated silencing of Vav reduced uptake and abrogated the stimulation of Rac in response to bacterial CEACAM3 engagement. In Vav1/Vav2-deficient cells, CEACAM3-mediated internalization was only observed after re-expression of Vav. Vav colocalized with CEACAM3 upon bacterial infection, coimmunoprecipitated in a complex with CEACAM3, and the Vav Src homology 2 domain directly associated with phosphorylated Tyr(230) of CEACAM3. In primary human granulocytes, TAT-mediated transduction of dominant-negative Vav, but not SWAP70, severely impaired the uptake of CEACAM3-binding bacteria. These data support the view that, different from canonical ITAM signaling, the CEACAM3 ITAM-like sequence short-wires bacterial recognition and Rac stimulation via a direct association with Vav to promote rapid phagocytosis and elimination of CEACAM-binding human pathogens. PMID:17339478

  12. The potential of carcinoembryonic antigen, p53, Ki-67 and glutathion Stransferase-π as clinico-histopathological markers for colorectal cancer☆

    PubMed Central

    He, Zhenyu; Shi, Chuanbing; Wen, Hao; Li, Fanglong; Wang, Baolin; Wang, Jie

    2010-01-01

    Objective Colorectal cancer is one of the major contributors to cancer death worldwide. Lack of reliable colorectal cancer markers has hampered the management of these cancer patients. Our main purpose was to study the correlation between histopathological variables of colorectal adenocarcinomas and identify histopathological markers that are of prognostic value in patients with colorectal cancer. Methods In the present study, we examined the expression of carcinoembryonic antigen (CEA), p53, Ki-67 and glutathion Stransferase (GST) -π by using immunohistochemical staining methods in 126 colorectal carcinoma patients and evaluated the lymph node metastasis status in these patients by histopathological examination. Results The positive rates of CEA, p53, Ki-67 and GST-π expression in the colorectal cancer tissue specimens examined were 95.23%, 55.56%, 53.38% and 82.30%, respectively. Expression of p53 and Ki-67 was significantly correlated with the Dukes stages of the tumor, with higher levels of these proteins in Dukes'C and D tumors than those in Dukes' A and B tumors. Furthermore, the expression of p53, GST-π and Ki-67 correlated with prognosis of patients with colorectal cancer. Additionally, the expression of p53 in colorectal cancer was closely related to the expression of Ki-67 and the expression of GST-π was directly correlated with that of p53. Conclusion The expression of CEA, p53, Ki-67 and GST-π was correlated with various clinical features of patients with colorectal cancer. The combined use of these histopathological markers appeared to be a promising tool in predicting the prognosis of patients with this type of cancer. PMID:23554611

  13. Electrochemical immunosensor for carcinoembryonic antigen based on nanosilver-coated magnetic beads and gold-graphene nanolabels.

    PubMed

    Chen, Huafeng; Tang, Dianping; Zhang, Bing; Liu, Bingqian; Cui, Yuling; Chen, Guonan

    2012-03-15

    A novel redox-active magnetic nanostructure was synthesized by using a wet chemical method for high-efficiency electrochemical immunoassay of carcinoembryonic antigen (CEA, as a model analyte). The nanostructures based on the combination of a magnetic nanocore, a layer of electroactive poly(o-phenylenediamine) (PPD), and a silver metallic shell displayed good adsorption properties for the attachment of anti-CEA antibody selective to CEA. The magnetic nanostructure presented good redox behaviors to facilitate and modulate the way it was integrated into a magnetic carbon paste electrode. The assay was based on a sandwich-type immunoassay protocol by using nanogold-patterned graphene oxide nanoscales (AuNP-GO), conjugated with horseradish peroxidase-labeled anti-CEA, as secondary antibodies and biofunctionalized magnetic nanostructures as immunosensing probes. Under optimal conditions, the nanoparticle-based immunocomposites exhibited good electrochemical responses for the determination of CEA, and allowed the detection of CEA at a concentration as low as 1.0 pg mL(-1) at a signal-to-noise ratio of 3. In addition, the magnetic immunosensing had good reproducibility, and acceptable accuracy, and could be successfully applied for the detection of CEA in the clinical serum specimens. Significantly, by controlling the target biomolecules, this assay can be easily extended for use with other immunosensings, and thus represents a versatile design routine. PMID:22365686

  14. Ultrasensitive non-enzymatic immunosensor for carcino-embryonic antigen based on palladium hybrid vanadium pentoxide/multiwalled carbon nanotubes.

    PubMed

    Han, Jian; Jiang, Liping; Li, Faying; Wang, Ping; Liu, Qing; Dong, Yunhui; Li, Yueyun; Wei, Qin

    2016-03-15

    A novel and sensitive sandwich-type non-enzymatic electrochemical immunosensor was fabricated for quantitative monitoring of carcino-embryonic antigen (CEA). Nanocomposite of stannic oxide/reduced graphene oxide was used as substrate material to increase the specific surface area and enhance the conductivity of the glassy carbon electrode. Gold nanoparticles (Au NPs) were introduced to link substrate materials and primary antibodies (Ab1) and accelerate the electron transfer in this system. At the same time, the palladium nanoparticles (Pd NPs)-vanadium pentoxide (V2O5)/multiwalled carbon nanotubes (MWCNTs) were used as the label of secondary antibodies (Ab2). This composite label has shown excellent catalytic activity towards the reduction of H2O2. The nanomaterial-based signal amplification can improve the sensitivity and lower the limit of detection. The proposed immunosensor showed wide linear range from 0.5 pgmL(-1) to 25 ngmL(-1) with limit of detection of 0.17 pgmL(-1). This novel immunosensor was used to analyze serum sample. The results indicated that this immunosensor may find huge potential application for quantitative detection of CEA in the clinical diagnosis. PMID:26562331

  15. Evaluation of pleural CYFRA 21-1 and carcinoembryonic antigen in the diagnosis of malignant pleural effusions.

    PubMed Central

    Salama, G.; Miédougé, M.; Rouzaud, P.; Mauduyt, M. A.; Pujazon, M. C.; Vincent, C.; Carles, P.; Serre, G.

    1998-01-01

    CYFRA 21-1 assay, measuring cytokeratin 19 fragments, was compared with carcinoembryonic antigen (CEA) assay, as an addition to cytological analysis for the diagnosis of malignant effusions. Both markers were determined with commercial enzyme immunoassays in pleural fluid from 196 patients. Cytological analysis and/or pleural biopsy confirmed the malignant origin of the effusion in 99 patients (76 carcinomas, nine pleural mesotheliomas and 14 non-epithelial malignancies). Effusions were confirmed as benign in 97 patients (33 cardiac failures, 39 infectious diseases--including 12 tuberculosis-- and 25 miscellaneous effusions). Both markers were significantly higher in malignant than in benign effusions. All the patients with non-epithelial malignancies presented CYFRA and CEA values lower than the 95% diagnostic specificity thresholds (100 and 6 ng ml(-1) respectively). The diagnostic sensitivity in the group of carcinomas and mesotheliomas was similar for CYFRA (58.8%) and CEA (64.7%). However, CEA had a significantly higher sensitivity in carcinomas (72.4% vs 55.3%), while CYFRA had a clearly higher sensitivity in mesotheliomas (89.9% vs 0%). Interestingly, 12 out of the 16 malignant effusions with a negative cytology were CEA and/or CYFRA positive. Regarding their high diagnostic sensitivity and their complementarity, CEA and CYFRA appear to be very useful for the diagnosis of malignant pleural effusions when cytology is negative. Images Figure 1 PMID:9472646

  16. Nanogold/mesoporous carbon foam-mediated silver enhancement for graphene-enhanced electrochemical immunosensing of carcinoembryonic antigen.

    PubMed

    Lin, Dajie; Wu, Jie; Ju, Huangxian; Yan, Feng

    2014-02-15

    Nanogold functionalized mesoporous carbon foam (Au/MCF) coupling with a signal amplification by C-Au synergistic silver enhancement was designed for sensitive electrochemical immunosensing of biomarker. The Au/MCF was prepared by in situ growth of nanogold on carboxylated MCF and used as a tracing tag to label signal antibody via the inherent interaction between protein and nanogold. The immunosensor was prepared by covalently immobilizing capture antibody on an electrochemically reduced graphene oxide/chitosan film modified glassy carbon electrode. Through a sandwich-type immunoreaction, Au/MCF tags were captured on the immunoconjugates to induce a silver deposition process. The electrochemical stripping signal of the deposited silver was used to monitor the immunoreaction. The Au/MCF-mediated silver enhancement along with the graphene-promoted electron transfer led to high detection sensitivity of carcinoembryonic antigen. Under optimal conditions, the proposed immunoassay method showed wide linear range from 0.05 pg mL(-1) to 1 ng mL(-1) and a detection limit down to 0.024 pg mL(-1). The newly designed amplification strategy holds great potential for ultrasensitive electrochemical biosensing of other analytes. PMID:24041661

  17. Three-dimensional electrochemical immunosensor for sensitive detection of carcinoembryonic antigen based on monolithic and macroporous graphene foam.

    PubMed

    Liu, Jiyang; Wang, Jiao; Wang, Tianshu; Li, Dan; Xi, Fengna; Wang, Jin; Wang, Erkang

    2015-03-15

    A high performance three-dimensional (3D) electrochemical immunosensor was developed for sensitive detection of the tumor biomarker, carcinoembryonic antigen (CEA). Monolithic and macroporous graphene foam grown by chemical vapor deposition (CVD) served as the scaffold of the free-standing 3D electrode. Immuno-recognition interface was fabricated via simple and non-covalent immobilization of antibody using lectin-mediated strategy. Briefly, the well-known lectin macromolecule (concanavalin A, Con A) monolayer was functionalized on 3D graphene (3D-G) using in-situ polymerized polydopamine as the linker. Then the widely used horseradish peroxidase (HRP)-labeled antibody (anti-CEA) in immunoassays was efficiently immobilized to demonstrate the recognition interface via the biospecific affinity of lectin with sugarprotein. The 3D immunosensor is able to detect CEA with a wide linear range (0.1-750.0ngml(-1)), low detection limit (~90pgml(-1) at a signal-to-noise ratio of 3), and short incubation time (30min). Furthermore, this biosensor was used for the detection of the CEA level in real serum samples. PMID:25461170

  18. Combination of circulating tumor cells with serum carcinoembryonic antigen enhances clinical prediction of non-small cell lung cancer.

    PubMed

    Chen, Xi; Wang, Xu; He, Hua; Liu, Ziling; Hu, Ji-Fan; Li, Wei

    2015-01-01

    Circulating tumor cells (CTCs) have emerged as a potential biomarker in the diagnosis, prognosis, treatment, and surveillance of lung cancer. However, CTC detection is not only costly, but its sensitivity is also low, thus limiting its usage and the collection of robust data regarding the significance of CTCs in lung cancer. We aimed to seek clinical variables that enhance the prediction of CTCs in patients with non-small cell lung cancer (NSCLC). Clinical samples and pathological data were collected from 169 NSCLC patients. CTCs were detected by CellSearch and tumor markers were detected using the Luminex xMAP assay. Univariate analyses revealed that histology, tumor stage, tumor size, invasiveness, tumor grade and carcinoembryonic antigen (CEA) were associated with the presence of CTCs. However, the level of CTCs was not associated with the degree of nodal involvement (N) or tumor prognostic markers Ki-67, CA125, CA199, Cyfra21-1, and SCCA. Using logistic regression analysis, we found that the combination of CTCs with tumor marker CEA has a better disease prediction. Advanced stage NSCLC patients with elevated CEA had higher numbers of CTCs. These data suggest a useful prediction model by combining CTCs with serum CEA in NSCLC patients. PMID:25996878

  19. Combination of Circulating Tumor Cells with Serum Carcinoembryonic Antigen Enhances Clinical Prediction of Non-Small Cell Lung Cancer

    PubMed Central

    He, Hua; Liu, Ziling; Hu, Ji-Fan; Li, Wei

    2015-01-01

    Circulating tumor cells (CTCs) have emerged as a potential biomarker in the diagnosis, prognosis, treatment, and surveillance of lung cancer. However, CTC detection is not only costly, but its sensitivity is also low, thus limiting its usage and the collection of robust data regarding the significance of CTCs in lung cancer. We aimed to seek clinical variables that enhance the prediction of CTCs in patients with non-small cell lung cancer (NSCLC). Clinical samples and pathological data were collected from 169 NSCLC patients. CTCs were detected by CellSearch and tumor markers were detected using the Luminex xMAP assay. Univariate analyses revealed that histology, tumor stage, tumor size, invasiveness, tumor grade and carcinoembryonic antigen (CEA) were associated with the presence of CTCs. However, the level of CTCs was not associated with the degree of nodal involvement (N) or tumor prognostic markers Ki-67, CA125, CA199, Cyfra21-1, and SCCA. Using logistic regression analysis, we found that the combination of CTCs with tumor marker CEA has a better disease prediction. Advanced stage NSCLC patients with elevated CEA had higher numbers of CTCs. These data suggest a useful prediction model by combining CTCs with serum CEA in NSCLC patients. PMID:25996878

  20. An immune sandwich assay of carcinoembryonic antigen based on the joint use of upconversion phosphors and magnetic beads.

    PubMed

    Li, Yaohua; Wu, Zhengjun; Liu, Zhihong

    2015-06-21

    We herein report a sensitive and selective immunosensor for carcinoembryonic antigen (CEA) based on the joint use of upconversion phosphors (UCPs) and magnetic beads (MBs). UCPs as the signal probe were designed with a core-shell structure which provided a 40-fold enhancement of the luminescence intensity. Poly(acrylic acid) (PAA)-modified UCPs were covalently conjugated with the anti-CEA antibody (coating), and streptavidin functionalized magnetic beads were combined with another biotin-tagged anti-CEA antibody. With the assistance of a magnet, the as-formed immune sandwich in the presence of CEA can be readily separated from the assay matrix. The immunosensor showed a linear dynamic range for CEA within 0.05-20 ng mL(-1) in a buffered aqueous solution, and 0.1-20 ng mL(-1) in a human serum sample. The sensor was highly specific to CEA. Our results have suggested the potential application of the UCP-MB based immunoassay for CEA in clinical analysis. PMID:25882752

  1. Quantitative real-time detection of carcinoembryonic antigen (CEA) from pancreatic cyst fluid using 3-D surface molecular imprinting.

    PubMed

    Yu, Yingjie; Zhang, Qi; Buscaglia, Jonathan; Chang, Chung-Chueh; Liu, Ying; Yang, Zhenhua; Guo, Yichen; Wang, Yantian; Levon, Kalle; Rafailovich, Miriam

    2016-07-21

    In this study, a sensitive, yet robust, biosensing system with real-time electrochemical readout was developed. The biosensor system was applied to the detection of carcinoembryonic antigen (CEA), which is a common marker for many cancers such as pancreatic, breast, and colon cancer. Real time detection of CEA during a medical procedure can be used to make critical decisions regarding further surgical intervention. CEA was templated on gold surface (RMS roughness ∼3-4 nm) coated with a hydrophilic self-assembled monolayer (SAM) on the working electrode of an open circuit potentiometric network. The subsequent removal of template CEA makes the biosensor capable of CEA detection based on its specific structure and conformation. The molecular imprinting (MI) biosensor was further calibrated using the potentiometric responses in solutions with known CEA concentrations and a detection limit of 0.5 ng ml(-1) was achieved. Potentiometric sensing was then applied to pancreatic cyst fluid samples obtained from 18 patients when the cyst fluid was also evaluated using ELISA in a certified pathology laboratory. Excellent agreement was obtained between the quantitation of CEA obtained by both the ELISA and MI biosensor detection for CEA. A 3-D MI model, using the natural rms roughness of PVD gold layers, is presented to explain the high degree of sensitivity and linearity observed in those experiments. PMID:27193921

  2. Evaluation of primary lung cancer with indium 111 anti-carcinoembryonic antigen (type ZCE-025) monoclonal antibody scintigraphy

    SciTech Connect

    Krishnamurthy, S.; Morris, J.F.; Antonovic, R.; Ahmed, A.; Galey, W.T.; Duncan, C.; Krishnamurthy, G.T. )

    1990-02-01

    A study was undertaken to test whether indium 111 (111In)-labeled anti-carcinoembryonic antigen (CEA) (type ZCE 025) monoclonal intact antibody (MoAb) would concentrate in primary lung cancer enabling its detection and localization by scintigraphy. The scintigraphic results were correlated with chest radiograph, computed tomograph (CT), bronchoscopy, surgical resection, and tumor CEA analysis. Twenty adult male patients with clinical suspicion of primary lung cancer were studied. Each subject was infused with 4 to 5 mCi of 111In anti-CEA ZCE 025 MoAb, and planar and tomographic scintiphotos were obtained on days 3 and 6 or 7 postinfusion. The scintigraphy was true-positive in 12 of 16 patients with primary lung cancer, eight of nine patients with squamous cell carcinoma, and four of seven with adenocarcinoma; it was true-negative in three of four patients with benign lung disease with an overall accuracy of 75%. In seven patients with confirmed primary lung cancer, but with negative bronchoscopic findings, the scintigraphy was true-positive in four. In 11 patients with definitely positive or suspicious malignancy by bronchoscopy the monoclonal scintigraphy was positive in eight. In true-positive cases, the location and size of the lesion by 111In anti-CEA ZCE 025 MoAb imaging correlated well with CT findings and also tumor mass at surgery. Only one of 12 tumors stained positive for CEA had serum CEA levels greater than 10 ng/ml, indicating nonleakage of the tumor antigen into general circulation in early lung cancer. It is concluded that 111In anti-CEA ZCE 025 MoAb planar and tomographic imaging shows potential to serve as a noninvasive diagnostic test in the evaluation of primary lung cancer. The lung lesion is likely to be malignant if it concentrates 111In anti-CEA ZCE 025 MoAb and benign if it does not.

  3. Engulfment of Neisseria gonorrhoeae: revealing distinct processes of bacterial entry by individual carcinoembryonic antigen-related cellular adhesion molecule family receptors.

    PubMed

    McCaw, Shannon E; Liao, Edward H; Gray-Owen, Scott D

    2004-05-01

    Individual Neisseria gonorrhoeae colony opacity-associated (Opa) protein variants can bind up to four different carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) receptors. Most human cells encountered by gonococci express a combination of CEACAM receptors, thereby complicating the elucidation of intracellular signaling pathways triggered by individual receptors. Here, we compare the process of bacterial engulfment by a panel of stably transfected HeLa epithelial cell lines expressing each CEACAM receptor in isolation. CEACAM1 and CEACAM3 each contain proteinaceous transmembrane and cytoplasmic domains; however, the processes of neisserial uptake mediated by these receptors differ with respect to their susceptibilities to both tyrosine kinase inhibitors and the actin microfilament-disrupting agent cytochalasin D. Neisserial uptake mediated by glycosylphosphatidylinositol (GPI)-anchored CEACAM5 and CEACAM6 was not significantly affected by any of a broad spectrum of inhibitors tested. However, cleavage of the GPI anchor by phosphatidylinositol-specific phospholipase C reduced bacterial uptake by HeLa cells expressing CEACAM5, consistent with a single zipper-like mechanism of uptake mediated by this receptor. Regardless of the CEACAM receptor expressed, internalized gonococci were effectively killed by a microtubule-dependent process that required acidification of the bacterium-containing phagosome. Given the phase-variable nature of neisserial Opa proteins, these results indicate that the mechanism of bacterial engulfment and the cellular response to gonococcal infection depend on both the receptor specificities of the neisserial Opa protein variants expressed and the spectrum of CEACAM receptors present on target cells, each of which determines the combination of receptors ultimately engaged. PMID:15102784

  4. Loss of Mammal-specific Tectorial Membrane Component Carcinoembryonic Antigen Cell Adhesion Molecule 16 (CEACAM16) Leads to Hearing Impairment at Low and High Frequencies*

    PubMed Central

    Kammerer, Robert; Rüttiger, Lukas; Riesenberg, Rainer; Schäuble, Constanze; Krupar, Rosemarie; Kamp, Annegret; Sunami, Kishiko; Eisenried, Andreas; Hennenberg, Martin; Grunert, Fritz; Bress, Andreas; Battaglia, Sebastiano; Schrewe, Heinrich; Knipper, Marlies; Schneider, Marlon R.; Zimmermann, Wolfgang

    2012-01-01

    The vertebrate-restricted carcinoembryonic antigen gene family evolves extremely rapidly. Among their widely expressed members, the mammal-specific, secreted CEACAM16 is exceptionally well conserved and specifically expressed in the inner ear. To elucidate a potential auditory function, we inactivated murine Ceacam16 by homologous recombination. In young Ceacam16−/− mice the hearing threshold for frequencies below 10 kHz and above 22 kHz was raised. This hearing impairment progressed with age. A similar phenotype is observed in hearing-impaired members of Family 1070 with non-syndromic autosomal dominant hearing loss (DFNA4) who carry a missense mutation in CEACAM16. CEACAM16 was found in interdental and Deiters cells and was deposited in the tectorial membrane of the cochlea between postnatal days 12 and 15, when hearing starts in mice. In cochlear sections of Ceacam16−/− mice tectorial membranes were significantly more often stretched out as compared with wild-type mice where they were mostly contracted and detached from the outer hair cells. Homotypic cell sorting observed after ectopic cell surface expression of the carboxyl-terminal immunoglobulin variable-like N2 domain of CEACAM16 indicated that CEACAM16 can interact in trans. Furthermore, Western blot analyses of CEACAM16 under reducing and non-reducing conditions demonstrated oligomerization via unpaired cysteines. Taken together, CEACAM16 can probably form higher order structures with other tectorial membrane proteins such as α-tectorin and β-tectorin and influences the physical properties of the tectorial membrane. Evolution of CEACAM16 might have been an important step for the specialization of the mammalian cochlea, allowing hearing over an extended frequency range. PMID:22544735

  5. Leptin Resistance Contributes to Obesity in Mice with Null Mutation of Carcinoembryonic Antigen-related Cell Adhesion Molecule 1.

    PubMed

    Heinrich, Garrett; Russo, Lucia; Castaneda, Tamara R; Pfeiffer, Verena; Ghadieh, Hilda E; Ghanem, Simona S; Wu, Jieshen; Faulkner, Latrice D; Ergün, Süleyman; McInerney, Marcia F; Hill, Jennifer W; Najjar, Sonia M

    2016-05-20

    Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes hepatic insulin clearance. Consistently, mice with null mutation of Ceacam1 (Cc1(-/-)) exhibit impaired insulin clearance with increased lipid production in liver and redistribution to white adipose tissue, leading to visceral obesity at 2 months of age. When the mutation is propagated on the C57/BL6J genetic background, total fat mass rises significantly with age, and glucose intolerance and systemic insulin resistance develop at 6 months of age. This study was carried out to determine the mechanisms underlying the marked increase in total fat mass in 6-month-old mutants. Indirect calorimetry analysis showed that Cc1(-/-) mice develop hyperphagia and a significant reduction in physical activity, in particular in the early hours of the dark cycle, during which energy expenditure is only slightly lower than in wild-type mice. They also exhibit increased triglyceride accumulation in skeletal muscle, due in part to incomplete fatty acid β-oxidation. Mechanistically, hypothalamic leptin signaling is reduced, as demonstrated by blunted STAT3 phosphorylation in coronal sections in response to an intracerebral ventricular injection of leptin. Hypothalamic fatty-acid synthase activity is also elevated in the mutants. Together, the data show that the increase in total fat mass in Cc1(-/-) mice is mainly attributed to hyperphagia and reduced spontaneous physical activity. Although the contribution of the loss of CEACAM1 from anorexigenic proopiomelanocortin neurons in the arcuate nucleus is unclear, leptin resistance and elevated hypothalamic fatty-acid synthase activity could underlie altered energy balance in these mice. PMID:27002145

  6. Correlation of the Serum Level of Carcinoembryonic Antigen and Prolactin with Different Stages of Colorectal Carcinoma According to Dukes' Staging.

    PubMed

    Rahman, M R; Sheikh, S H; Lima, I J; Islam, M R; Faisal, M; Islam, M S; Faruk, M O; Jalal, M T

    2016-01-01

    Carcinoembryonic antigen (CEA) is well established tumor marker for colorectal cancers worldwide. Recent studies show that serum prolactin level is also raised in colorectal cancers. The purpose of the study is to evaluate the correlation of serum CEA and Prolactin with Dukes' staging of colorectal carcinomas. Between January 2013 and June 2013, Serum CEA and Serum Prolactin were measured by radioimmunoassay from 103 patients who were histopathologically diagnosed as colorectal carcinomas. Evaluation of the stages of the colorectal cancers was done on the basis of preoperative investigations and postoperative histopathology and correlated with Preoperative Serum CEA and Serum Prolactin. Results were presented as median value, range and percentage. Male to female ratio was 1.4:1 with median age of 42.26 years (range 17-78 years). Most of the patients in this series presented with carcinoma rectum (42%). Most of the patients (52%) were found in Dukes' stage C and 27% and 15% cases were found as Dukes' stage B and Dukes' stage D respectively. Stage of the disease is directly proportionate to percentage of the patient with high serum prolactin except early stage (Dukes' A-50%, Dukes' B-28.6%, Dukes' C-33.3% & Dukes' D-46.7%). Similarly serum CEA level is directly proportionate to tumor stage (Dukes' A-0%, Dukes' B-32%, Dukes' C-40.7% & Dukes' D-74.7%). A preoperative high serum CEA value suggests advanced disease either locally or with distant metastasis. In contrast preoperative high serum prolactin (hyperprolactinaemia) did not suggest advanced disease as it can be elevated even in early stage of disease. Serum CEA and Serum Prolactin both are valuable tumor markers but serum CEA could not be replaced by serum Prolactin. Serum Prolactin may be a helpful marker in earlier stages of the colorectal cancer. PMID:26931251

  7. Association of pretreatment serum carcinoembryonic antigen levels with chemoradiation-induced downstaging and downsizing of rectal cancer

    PubMed Central

    YEO, SEUNG-GU

    2016-01-01

    The aim of this study was to identify pretreatment clinical parameters associated with preoperative chemoradiotherapy (CRT)-induced downstaging and downsizing of locally advanced rectal cancer (LARC T3–4 or N+). Data from 51 LARC patients, who received preoperative CRT and radical surgery between 2010 and 2013, were retrospectively analyzed. Rectal adenocarcinoma was histologically confirmed in all patients, who ranged in age between 41 and 81 years (median, 64 years). CRT consisted of 50.4 Gy pelvic radiotherapy with concurrent chemotherapy using 5-fluorouracil and leucovorin. After a median interval of 7 weeks post-CRT, the patients underwent total mesorectal excision. Downstaging was defined as the transition from cStage II–III to ypStage 0-I. The longest tumor diameter was measured pre- and post-CRT using computed tomography or magnetic resonance imaging, and based on the surgical specimen, respectively. Downstaging was observed in 16 (31.4%) patients, including 5 (9.8%) with a pathological complete response. The median downsizing rate was 60%. The serum carcinoembryonic antigen (CEA) levels were 0.8–153.9 ng/ml (median, 4.4 ng/ml). The maximum standardized uptake value was 4.7–33.9 (median, 10.8). On univariate analysis, cT stage, tumor size and CEA level were associated with downstaging. On multivariate analysis, only CEA level (≤5 ng/ml) was a significant predictor of downstaging (odds ratio = 16.0; 95% confidence interval: 1.8–146.7; P=0.014). CEA level was the only factor significantly associated with downsizing (>60%) in the univariate analysis. These results demonstrated that pretreatment serum CEA levels are significantly associated with downstaging as well as downsizing of LARC following preoperative CRT. Therefore, this parameter may be useful in personalizing the management of LARC patients. PMID:27073681

  8. A common genetic variant of fucosyltransferase 2 correlates with serum carcinoembryonic antigen levels and affects cancer screening in patients with primary sclerosing cholangitis

    PubMed Central

    Wannhoff, Andreas; Folseraas, Trine; Brune, Maik; Rupp, Christian; Friedrich, Kilian; Knierim, Johannes; Weiss, Karl Heinz; Sauer, Peter; Flechtenmacher, Christa; Schirmacher, Peter; Stremmel, Wolfgang; Hov, Johannes R

    2015-01-01

    Background Primary sclerosing cholangitis (PSC) patients are at increased risk of biliary tract cancer, and carcinoembryonic antigen (CEA) serum levels might be used for screening. Objective To examine cancer screening with CEA in PSC patients and analyse how serum CEA levels are affected by genetic variants of fucosyltransferase (FUT) 2 and 3. Methods In a retrospective cohort analysis we evaluated CEA levels in 226 PSC patients, including 19 with biliary malignancy, and investigated how FUT2 and FUT3 SNPs affected CEA levels. Receiver-operating-characteristic (ROC) analysis was performed and cut-off values were determined based on Youden’s index. A control cohort contained 240 patients, including 28 with biliary malignancy. Results Median CEA concentration was lower in cancer-free patients (1.4 ng/mL) than in cancer patients (2.0 ng/mL, P = 0.014). ROC analysis revealed an area under the curve (AUC) of 0.671, the optimal cut-off was 3.2 ng/mL. The FUT2 variant rs601338 (G428A) correlated with CEA levels, and the effect was most prominent in a subgroup of patients genetically incapable of expressing CA19-9. The AUC improved if ROC analysis was performed separately for wild-type (AUC: 0.731) and homozygous mutant (AUC: 0.816) G428A. The influence of FUT2 on CEA was confirmed in the control cohort. Conclusions CEA is interesting for biliary-malignancy screening in PSC patients, especially in patients who do not express CA19-9. This is the first study to show that the combined use of CEA measurement and FUT genotyping is clinically beneficial and that it might enhance the early detection of biliary malignancy in clinical practice. This approach could also be effective when screening for other common gastrointestinal malignancies. PMID:26966527

  9. Major Protein of Carcinoembryonic Antigen Gene Family - CD66c, A Novel Marker in Colon Carcinoma

    PubMed Central

    Nataraj, Suma M; Prema, Chaitra Linganna; Vimalambike, Manjunath Gubbanna; Shivalingaiah, Sheeladevi Chandakavadi; Sundaram, Shivakumar; Kumar, Anjali Pradeep; Math, Ananda Kuruvatti

    2016-01-01

    Introduction In view of rising trend of the incidence of colorectal carcinoma in the Indian population due to adoption of western lifestyles and behaviours, we investigated the expression of the new emerging stem cell biomarker, CD66c in colorectal carcinoma of Indian origin. Aim To study the expression of CD66c in human colorectal carcinoma and to correlate level of marker expression with tumour staging. Materials and Methods This hospital based prospective study was conducted on 26 colorectal carcinoma patients in the age group of 20 years to 70 years. Surgically resected tumour specimens along with adjacent normal tissue were collected taking necessary precautions, paraffin embedded sections were prepared and used for histological and immunohistochemical analysis of CD66c. Statistical analysis Descriptive statistical measures like mean, standard deviation, percentage was applied. Other inferential statistical tests like Chi-square, Fisher’s-exact test and one-way ANOVA was applied to find out the association of CD66c with different stages. The difference were interpreted as statistically significant when p <0.05. Results CD66c showed differential expression with membrane positivity in normal colorectal epithelial cells and cytoplasmic expression in tumour cells. There was significant correlation between CD66c expression and tumour site (p=0.02) with colon carcinoma showing positive expression compared to the rectal carcinoma. There was no significant correlation between CD66c staining and tumour stage (p=0.947). No significant relationship was observed between CD66c expression and other clinicopathologic variables studied such as sex (p=0.552), age (p=0.713) and tumour grade (p=0.263). Conclusion CD66c can be specifically used for colon carcinoma and may be a novel marker in colon carcinoma stem cell isolation. The quantification of CD66c can be further verified by flow cytometry and RT-PCR. Further studies can be carried out using CD66c alone or in

  10. Measurement of serum carcinoembryonic antigen, carbohydrate antigen 19-9, cytokeratin-19 fragment and matrix metalloproteinase-7 for detecting cholangiocarcinoma: a preliminary case-control study.

    PubMed

    Lumachi, Franco; Lo Re, Giovanni; Tozzoli, Renato; D'Aurizio, Federica; Facomer, Flavio; Chiara, Giordano B; Basso, Stefano M M

    2014-11-01

    Cholangiocarcinoma is a malignant tumor of the liver arising from the bile duct epithelium, accounting for 10-25% of all primary hepatic cancers. The clinical presentation of this tumor is not specific and the diagnosis of early cholangiocarcinoma is difficult, especially in patients with other biliary diseases. Measurement of serum carbohydrate antigen (CA) 19-9 and carcinoembryonic antigen (CEA) are commonly used to monitor response to therapy, but are also useful for confirming the presence of a cholangiocarcinoma. In this setting, other biomarkers have been previously tested, including cytokeratin-19 fragment (CYFRA 21-1) and the matrix metalloproteinase-7 (MMP7). The purpose of this retrospective study was to determine the clinical usefulness of the assay of serum CEA, CA 19-9, CYFRA 21-1 and MMP7, individually and together, as tumor markers for the diagnosis of cholangiocarcinoma. Twenty-four patients (14 men, 10 women, 62.6±8.2 years of age) with histologically-confirmed cholangiocarcinoma (cases) and 25 age- and sex-matched patients with benign liver disease (controls) underwent measurement of these biomarkers. The mean values of all serum markers of patients with cholangiocarcinoma were significantly higher (p<0.01) than that of the controls. No correlation was found between serum tumor markers and total bilirubin, aspartate aminotransferase (AST) and alkaline phosphatase (ALP). The sensitivity, specificity and accuracy were: CEA: 52%, 55%, and 58%; CA 19-9: 74%, 82% and 78%; CYFRA 21-1: 76%, 79% and 78%; MMP7: 78%, 77% and 80%, respectively. The combination of all serum markers afforded 92.0% sensitivity and 96% specificity in detecting cholangiocarcinoma, showing the highest diagnostic accuracy (94%). In conclusion, our preliminary results suggest that the measurement of all four biomarkers together can help in the early detection of cholangiocarcinoma. PMID:25368272

  11. Prognosis Can Be Predicted More Accurately Using Pre- and Postchemoradiotherapy Carcinoembryonic Antigen Levels Compared to Only Prechemoradiotherapy Carcinoembryonic Antigen Level in Locally Advanced Rectal Cancer Patients Who Received Neoadjuvant Chemoradiotherapy

    PubMed Central

    Sung, SooYoon; Son, Seok Hyun; Kay, Chul Seung; Lee, Yoon Suk

    2016-01-01

    Abstract We aimed to evaluate the prognostic value of a change in the carcinoembryonic antigen (CEA) level during neoadjuvant chemoradiotherapy (nCRT) in patients with locally advanced rectal cancer. A total of 110 patients with clinical T3/T4 or node-positive disease underwent nCRT and curative total mesorectal resection from February 2006 to December 2013. Serum CEA level was measured before nCRT, after nCRT, and then again after surgery. A cut-off value for CEA level to predict prognosis was determined using the maximally selected log-rank test. According to the test, patients were classified into 3 groups, based on their CEA levels (Group A: pre-CRT CEA ≤3.2; Group B: pre-CRT CEA level >3.2 and post-CRT CEA ≤2.8; and Group C: pre-CRT CEA >3.2 and post-CRT CEA >2.8). The median follow-up time was 31.1 months. The 3-year disease-free survival (DFS) rates of Group A and Group B were similar, while Group C showed a significantly lower 3-year DFS rate (82.5% vs. 89.5% vs. 55.1%, respectively, P = 0.001). Other clinicopathological factors that showed statistical significance on univariate analysis were pre-CRT CEA, post-CRT CEA, tumor distance from the anal verge, surgery type, downstage, pathologic N stage, margin status and perineural invasion. The CEA group (P = 0.001) and tumor distance from the anal verge (P = 0.044) were significant prognostic factors for DFS on multivariate analysis. Post-CRT CEA level may be a useful prognostic factor in patients whose prognosis cannot be predicted exactly by pre-CRT CEA levels alone in the neoadjuvant treatment era. Combined pre-CRT CEA and post-CRT CEA levels enable us to predict prognosis more accurately and determine treatment and follow-up policies. Further large-scale studies are necessary to validate the prognostic value of CEA levels. PMID:26962798

  12. Coronavirus species specificity: murine coronavirus binds to a mouse-specific epitope on its carcinoembryonic antigen-related receptor glycoprotein.

    PubMed Central

    Compton, S R; Stephensen, C B; Snyder, S W; Weismiller, D G; Holmes, K V

    1992-01-01

    Like most coronaviruses, the coronavirus mouse hepatitis virus (MHV) exhibits strong species specificity, causing natural infection only in mice. MHV-A59 virions use as a receptor a 110- to 120-kDa glycoprotein (MHVR) in the carcinoembryonic antigen (CEA) family of glycoproteins (G. S. Dveksler, M. N. Pensiero, C. B. Cardellichio, R. K. Williams, G. S. Jiang, K. V. Holmes, and C. W. Dieffenbach, J. Virol. 65:6881-6891, 1991; and R. K. Williams, G. S. Jiang, and K. V. Holmes, Proc. Natl. Acad. Sci. USA 88:5533-5536, 1991). The role of virus-receptor interactions in determining the species specificity of MHV-A59 was examined by comparing the binding of virus and antireceptor antibodies to cell lines and intestinal brush border membranes (BBM) from many species. Polyclonal antireceptor antiserum (anti-MHVR) raised by immunization of SJL/J mice with BALB/c BBM recognized MHVR specifically in immunoblots of BALB/c BBM but not in BBM from adult SJL/J mice that are resistant to infection with MHV-A59, indicating a major difference in epitopes between MHVR and its SJL/J homolog which does not bind MHV (7). Anti-MHVR bound to plasma membranes of MHV-susceptible murine cell lines but not to membranes of human, cat, dog, monkey, or hamster cell lines. Cell lines from these species were resistant to MHV-A59 infection, and only the murine cell lines tested were susceptible. Pretreatment of murine fibroblasts with anti-MHVR prevented binding of radiolabeled virions to murine cells and prevented virus infection. Solid-phase virus-binding assays and virus overlay protein blot assays showed that MHV-A59 virions bound to MHVR on intestinal BBM from MHV-susceptible mouse strains but not to proteins on intestinal BBM from humans, cats, dogs, pigs, cows, rabbits, rats, cotton rats, or chickens. In immunoblots of BBM from these species, both polyclonal and monoclonal antireceptor antibodies that block MHV-A59 infection of murine cells recognized only the murine CEA-related glycoprotein

  13. Second antibody clearance of /sup 131/I-labeled anti-carcinoembryonic antigen for improved tumor imaging

    SciTech Connect

    Sharkey, R.M.; Primus, F.J.; Goldenberg, D.M.

    1985-05-01

    The authors have investigated the use of a second antibody (SA) directed against the radiolabeled primary anti-tumor antibody (PA) to enhance the clearance rate of the PA from the circulation and nontarget tissues. Administration of 50 or 250 ..mu..g of anti-goat IgG (SA) hr after the administration of 10 ..mu..g of /sup 131/I-goat anti-carcinoembryonic antigen antibody (PA) to hamsters bearing human colonic tumor xenografts resulted in a 5-fold reduction in the level of circulating PA after 4 hr in comparison to the control group only given /sup 131/I-PA. The percentage of PA in the blood decreased rapidly over 72 hr in animals given 250 ..mu..g of the SA, but at 50 ..mu..g of SA the level of activity in the blood after 24 hr was similar to the control. Tumor accretion was identical after 4 hr, but after 24 hr the animals given 250 ..mu..g of SA had 2-3 fold less PA in the tumor than either the control group or the 50 ..mu..g dose of SA. Tumor/nontumor ratios for all major organs but the spleen improved 6-8 fold within 48 hr after injection of 250 ..mu..g of the SA with tumor/blood ratios as high as 40:1. A SA dose of 50 ..mu..g resulted in a significantly higher tumor/blood ratio after only 4 hr; tumor/nontumor ratios at later times were similar to the control group. Tumors located in the hind legs were visible in all groups by imaging 24 hr after injection of the SA, but only the 250 ..mu..g dose of SA showed a significant reduction in total body activity. These results suggest that the SA approach may be used to reduce the total background radioactivity while maintaining tumor accretion of /sup 131/I-PA to allow for selective tumor imaging.

  14. CEA (Carcinoembryonic Antigen) Test

    MedlinePlus

    ... may also be used as a marker for medullary thyroid cancer and cancers of the rectum, lung , ... ulcerative colitis , rectal polyps , emphysema , and benign breast disease. ^ Back to top Proudly sponsored by ... Learn more ...

  15. A robust electrochemiluminescence immunoassay for carcinoembryonic antigen detection based on a microtiter plate as a bridge and Au@Pd nanorods as a peroxidase mimic.

    PubMed

    Zhang, Yong; Pang, Xuehui; Wu, Dan; Ma, Hongmin; Yan, Zhaoqing; Zhang, Jiatao; Du, Bin; Wei, Qin

    2016-01-01

    The common drawbacks of most traditional electrochemiluminescence (ECL) immunoassays are the strict storage conditions for the ECL electrode and the steric hindrance caused by bovine serum albumin and antigen. The strict storage conditions require that the modified electrode must be stored at 4 °C before measurement, which may cause the degradation of protein molecules and low reproducibility as the time goes by. The steric hindrance can hinder electron transfer between the electrode and the electrochemical active substance unable to transmit proteins on the electrode surface. The current study takes a 96-well microtiter plate (MTP) as a bridge for analyte pre-treatment and Au@Pd nanorods as a peroxidase mimic to assemble a simple and robust ECL immunoassay. Advantages of such assay include not only high sensitivity but also robust detection circumstance. We demonstrated the method by detecting carcinoembryonic antigen from human serum and obtained a good detection limit of 3 fg mL(-1). PMID:26609799

  16. Expression of Carcinoembryonic Cell Adhesion Molecule 6 and Alveolar Epithelial Cell Markers in Lungs of Human Infants with Chronic Lung Disease.

    PubMed

    Gonzales, Linda W; Gonzalez, Robert; Barrette, Anne Marie; Wang, Ping; Dobbs, Leland; Ballard, Philip L

    2015-12-01

    The membrane protein carcinoembryonic antigen cell adhesion molecule (CEACAM6) is expressed in the epithelium of various tissues, participating in innate immune defense, cell proliferation and differentiation, with overexpression in gastrointestinal tract, pancreatic and lung tumors. It is developmentally and hormonally regulated in fetal human lung, with an apparent increased production in preterm infants with respiratory failure. To further examine the expression and cell localization of CEACAM6, we performed immunohistochemical and biochemical studies in lung specimens from infants with and without chronic lung disease. CEACAM6 protein and mRNA were increased ~4-fold in lungs from infants with chronic lung disease as compared with controls. By immunostaining, CEACAM6 expression was markedly increased in the lung parenchyma of infants and children with a variety of chronic lung disorders, localizing to hyperplastic epithelial cells with a ~7-fold elevated proliferative rate by PCNA staining. Some of these cells also co-expressed membrane markers of both type I and type II cells, which is not observed in normal postnatal lung, suggesting they are transitional epithelial cells. We suggest that CEACAM6 is both a marker of lung epithelial progenitor cells and a contributor to the proliferative response after injury due to its anti-apoptotic and cell adhesive properties. PMID:26374831

  17. Relationship between serum carcinoembryonic antigen level and epidermal growth factor receptor mutations with the influence on the prognosis of non-small-cell lung cancer patients

    PubMed Central

    Cai, Zuxun

    2016-01-01

    Objective To investigate the relationship between serum carcinoembryonic antigen (CEA) level and epidermal growth factor receptor (EGFR) gene mutations in non-small-cell lung cancer (NSCLC) patients and to analyze the influence of CEA level on postoperative survival time in lung cancer patients. Methods A total of 296 patients who were treated in Thoracic Surgery Department of Henan Provincial Chest Hospital from September 2011 to September 2013 were recruited. The level of tumor markers, such as CEA, was determined before the surgery, and EGFR gene mutations were detected after surgery. Thereby, the relationship between tumor makers, including CEA, and EGFR mutation and its influence on prognosis could be investigated. Results Among 296 patients, the positive rate of EGFR gene mutation was 37.84% (112/296); the mutation occurred more frequently in nonsmokers, adenocarcinoma patients, women, and patients aged <60 years (P<0.05). Both tumor markers and chemosensitivity indicators were related to the profile of EGFR mutations. Elevated squamous cell carcinoma and Cyfra21-1 as well as positively expressed ERCC1 were more common in patients with wild-type EGFR (P<0.05), whereas increased CEA level was observed more frequently in patients with EGFR gene mutation (P=0.012). The positive rate of EGFR gene mutations was higher as the serum CEA level increased, that is, the positive rate in patients with serum CEA level <5, 5–20, and >20 μg/L was 39.81%, 45.32%, and 65.47%, respectively (P=0.004). Logistic regression analysis showed that CEA level was an independent factor in predicting EGFR gene mutations, and serum CEA level was also an independent factor in affecting the prognosis of NSCLC patients, as the overall 2-year survival rate was 73.86% in elevated CEA group and 86.43% in normal group (P<0.01). Conclusion The prognosis of NSCLC patients receiving resection can be predicted according to serum CEA level, which is associated with EGFR mutations in NSCLC patients

  18. Neutron-capture therapy of human cancer: in vivo results on tumor localization of boron-10-labeled antibodies to carcinoembryonic antigen in the gw-39 tumor model system

    SciTech Connect

    Goldenberg, D.M.; Sharkey, R.M.; Primus, F.J.; Mizusawa, E.; Hawthorne, M.F.

    1984-01-01

    Antibody against carcinoembryonic antigen (CEA) was conjugated with p-(1,2-dicarba-closo-(1-/sup 3/H)dodecaboran(12)-2-yl)benzenediazonium ion by an azo-coupling reaction, resulting in 30 boron atoms per IgG molecule with no loss of antibody protein. Antibody immunoreactivity was not appreciably affected by this conjugation and was stable after incubation in vitro in hamster plasma for 24 hr. The efficacy of the boron-conjugated anti-CEA IgG for localizing selectively in CEA-containing human colonic carcinomas propagated in the hind leg musculature of hamsters was evaluated by labeling the antibodies with /sup 131/I and determining distribution of the radioactivity in vivo. The results show that the boron-conjugated antibodies retain selective localization in the tumors, thus indicating their suitability for transporting boron-10 to tumors for use in neutron-capture therapy of cancer. 17 references, 3 tables, 2 figures.

  19. Imaging with indium111-labeled anticarcinoembryonic antigen monoclonal antibody ZCE-025 of recurrent colorectal or carcinoembryonic antigen-producing cancer in patients with rising serum carcinoembryonic antigen levels and occult metastases

    SciTech Connect

    Patt, Y.Z.; Lamki, L.M.; Shanken, J.; Jessup, J.M.; Charnsangavej, C.; Ajani, J.A.; Levin, B.; Merchant, B.; Halverson, C.; Murray, J.L. )

    1990-07-01

    We tested whether nuclear imaging with indium111 (111In)-labeled murine monoclonal (MoAb) anticarcinoembryonic antigen (anti-CEA) ZCE-025 antibody could detect recurrent disease in patients with a rising serum CEA level but negative findings for computed tomographic (CT) scans of the abdomen and pelvis, chest radiograph, and colonoscopy or barium enema. Twenty patients with a history of completely resected CEA-producing adenocarcinoma and a rising serum CEA level were given an intravenous infusion of 2 mg of 111In-labeled ZCE-025 mixed with 38 mg of unlabeled ZCE-025. Planar and single-photon emission CT (SPECT) scans were acquired at 72 and 144 hours, and in 19 of the 20 patients these were positive. Of those 19, 13 underwent exploratory surgery, and cancer was found in 10, and two had a diagnostic biopsy, which confirmed cancer. Three patients who had negative laparotomies and all four patients who did not undergo surgery or biopsy were followed radiologically. In all seven, cancer was subsequently detected at the sites suggested by the ZCE-025 scan. Thus, tumor was confirmed in all 19 patients with positive scans. Five of 13 patients who were explored benefited from the study and the exploratory laparotomy, as disease was entirely resected in four or was subjected to definitive radiation therapy to the pelvis in the fifth. In two additional patients who were not explored, MoAb imaging resulted in definitive therapy to regionally confined recurrent disease. 111In-labeled anti-CEA MoAb ZCE-025 scanning in patients with rising CEA successfully imaged metastatic colorectal cancer that eluded detection by other methods and affected the care given to some. These results suggest an important role for 111In-labeled ZCE-025 scanning among patients with rising CEA and otherwise occult metastatic cancer.

  20. Natural T-cell response against MHC class I epitopes of epithelial cell adhesion molecule, her-2/neu, and carcinoembryonic antigen in patients with colorectal cancer.

    PubMed

    Nagorsen, D; Keilholz, U; Rivoltini, L; Schmittel, A; Letsch, A; Asemissen, A M; Berger, G; Buhr, H J; Thiel, E; Scheibenbogen, C

    2000-09-01

    The antigens epithelial cell adhesion molecule (Ep-CAM), her-2/neu, and carcinoembryonic antigen (CEA) are potential T-cell targets in antigen-specific vaccination-based cancer therapy. We performed this study to evaluate whether a natural specific T-cell response against these tumor-associated antigens (TAAs) already exists in patients with colorectal carcinoma (CRC). We used the IFN-gamma ELISPOT assay to detect circulating TAA-reactive T cells directly ex vivo in unstimulated peripheral blood mononuclear cells. We analyzed the T-cell response in peripheral blood mononuclear cells of 22 HLA-A2-positive patients with CRC and 8 HLA-A2-positive healthy subjects against 3 HLA A2-restricted peptide epitopes of the TAAs Ep-CAM (GLKAGVIAV), her-2/neu (IISAVVGIL), and CEA (YLSGANLNL). Seven of 22 patients but none of the 8 healthy subjects had T cells specifically secreting IFN-gamma in response to one to three of these antigens (n = 4, Ep-CAM; n = 5, her-2/neu; n = 6, CEA). In three of the seven responding patients, TAA-reactive T cells were further characterized by flow cytometry. In all three patients, the majority of these T cells have a CD3+CD8+IFN-gamma+CD69+CD45RA+ phenotype, resembling activated effector-type T cells. T-cell responses occurred only in patients with metastatic disease (Dukes' stages C and D). The results of this study indicate that natural T-cell responses against TAAs occur in approximately one-half of CRC patients with involvement of lymph nodes or distant metastases, but not in CRC patients with disease confined to the intestinum. PMID:10987297

  1. [Clinical evaluation of the tumor marker CA 19-9 in comparison with carcinoembryonic antigen (CEA) in surgical pre- and postoperative diagnosis].

    PubMed

    Lorenz, M; Happ, J; Hottenrott, C; Maul, F D; Baum, R P; Hör, G; Encke, A

    1986-02-01

    A new tumor marker (CA 19-9) was investigated. CA 19-9 is a tumor-associated antigen which is detected by a monoclonal antibody. CA 19-9 (CIS-Centocor) was compared simultaneously with CEA (carcinoembryonic antigen) in 347 patients. 123 patients with gastrointestinal tumors showed a sensitivity of 31% for CA 19-9 (CEA 49%), combination increased sensitivity to 58%. The highest sensitivity was found in pancreas carcinoma (CA 19-9 75%, CEA 66%, combination 92%); it was lower in gastric, colon, and oesophagus carcinomas. In relapsed colorectal carcinomas sensitivity was 53% (CEA 78%, combination 85%). In cases of relapse, tumor markers may become positive even if they were not detectable before resection of the primary tumor. Specificity for CA 19-9 was 100% (CEA 84%) compared to a group of non-malignant diseases including patients with inflammations and patients with nicotin abuse (n = 102). Because of its high specificity and superior sensitivity to CEA in pancreas carcinomas CA 19-9 should be determined in primary and relapse diagnosis in combination with CEA. PMID:3459133

  2. Assessing Clinical Significance of Serum CA15-3 and Carcinoembryonic Antigen (CEA) Levels in Breast Cancer Patients: A Meta-Analysis.

    PubMed

    Fu, Yijie; Li, Hui

    2016-01-01

    BACKGROUND Breast cancer is the most common malignant cancer in women worldwide. The tumor markers Cancer Antigen 15-3 (CA15-3) and Carcinoembryonic Antigen (CEA) are frequently used for screening and monitoring breast cancer. MATERIAL AND METHODS We conducted a meta-analysis of 13 published case-control studies to assess the associations between serum levels of CA15-3 and CEA with breast cancer susceptibility, including 1179 cases and 493 controls. The analyses were performed on malignant tumor and benign tumor, as well as in different subgroups with respect to the patient ethnicities and clinical tumor stages. RESULTS This systematic review and meta-analysis of association studies shows that serum levels of CA15-3 and CEA are potential biomarkers for breast cancer monitoring. When stratified by clinical stage, we noticed that although malignant tumors in all stages show elevated levels of CA15-3, it is greatly associated with the tumor stage, as it increases as breast tumor stage worsens. CONCLUSIONS This study clarifies the inconsistent conclusions from multiple studies, and provides a precise estimation for clinical utility of 2 important biomarkers, CA15-3 and CEA, in breast cancer monitoring. Thus, our study will shed lights on the prognosis of breast cancer patients. PMID:27596019

  3. Single-step cycle pulse operation of the label-free electrochemiluminescence immunosensor based on branched polypyrrole for carcinoembryonic antigen detection.

    PubMed

    Zhu, Wenjuan; Wang, Qi; Ma, Hongmin; Lv, Xiaohui; Wu, Dan; Sun, Xu; Du, Bin; Wei, Qin

    2016-01-01

    A novel label-free electrochemiluminescence (ECL) immunosensor based on luminol functional-Au NPs@polypyrrole has been developed for the detection of carcinoembryonic antigen (CEA). In this work, polypyrrole prepared by chemical polymerization provided a large surface area to load amounts of gold nanoparticles (Au NPs). Au NPs could not only attach abundant luminol for the enhancement of ECL signal, but also provide a friendly microenvironment for the immobilization of antibodies. Moreover, 1-butylpyridinium tetrafluroborate ([BPy]BF4) were used to disperse luminol functional-Au NPs@polypyrrole nanocomposites, resulting in the film-formation of composites on the electrode, which could improve the stability of immunosensor. In particular, employment of single-step cycle pulse could limit the consecutive reaction between luminol and H2O2 efficiently, thus leading to stable and strong signals. The proposed method presents good ECL response for the detection of CEA allowing a wide linear range from 0.01 pg/mL to 10 ng/mL and a limit of detection as low as 3 fg/mL. The immunosensor would be a promising tool in the early diagnosis of CEA due to its high sensitivity, simplicity and cost-effective. PMID:27091590

  4. Magnetic immunoassay coupled with inductively coupled plasma mass spectrometry for simultaneous quantification of alpha-fetoprotein and carcinoembryonic antigen in human serum

    NASA Astrophysics Data System (ADS)

    Zhang, Xing; Chen, Beibei; He, Man; Zhang, Yiwen; Xiao, Guangyang; Hu, Bin

    2015-04-01

    The absolute quantification of glycoproteins in complex biological samples is a challenge and of great significance. Herein, 4-mercaptophenylboronic acid functionalized magnetic beads were prepared to selectively capture glycoproteins, while antibody conjugated gold and silver nanoparticles were synthesized as element tags to label two different glycoproteins. Based on that, a new approach of magnetic immunoassay-inductively coupled plasma mass spectrometry (ICP-MS) was established for simultaneous quantitative analysis of glycoproteins. Taking biomarkers of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) as two model glycoproteins, experimental parameters involved in the immunoassay procedure were carefully optimized and analytical performance of the proposed method was evaluated. The limits of detection (LODs) for AFP and CEA were 0.086 μg L- 1 and 0.054 μg L- 1 with the relative standard deviations (RSDs, n = 7, c = 5 μg L- 1) of 6.5% and 6.2% for AFP and CEA, respectively. Linear range for both AFP and CEA was 0.2-50 μg L- 1. To validate the applicability of the proposed method, human serum samples were analyzed, and the obtained results were in good agreement with that obtained by the clinical chemiluminescence immunoassay. The developed method exhibited good selectivity and sensitivity for the simultaneous determination of AFP and CEA, and extended the applicability of metal nanoparticle tags based on ICP-MS methodology in multiple glycoprotein quantifications.

  5. Preparation of Au-polydopamine functionalized carbon encapsulated Fe3O4 magnetic nanocomposites and their application for ultrasensitive detection of carcino-embryonic antigen

    PubMed Central

    Ji, Lei; Yan, Tao; Li, Yan; Gao, Jian; Wang, Qi; Hu, Lihua; Wu, Dan; Wei, Qin; Du, Bin

    2016-01-01

    A novel carbon encapsulated Fe3O4 nanoparticles embedded in two-dimensional (2D) porous graphitic carbon nanocomposites (Fe3O4@C@PGC nanocomposites) were synthesized by situ synthesis strategy, which provided a sensor platform owing to a large aspect ratio and porous structure. Polydopamine (PDA) were modified on the surface of Fe3O4@C@PGC nanocomposites through self-polymerization of dopamine, acting as both the reductant and template for one-step synthesis of gold nanoparticles. The prepared Au/PDA/Fe3O4@C@PGC nanocomposites show ferromagnetic features, extremely excellent electron transfer, large specific surface area and excellent dispersing property. These are conducive to the electrochemical signal output and the immobilization of antibody. In this work, a highly label-free sensitive magnetic immunosensor was developed based on Au/PDA/Fe3O4@C@PGC nanocomposites for the detection of carcino-embryonic antigen (CEA). The magnetic glassy carbon electrode was used to fix the Au/PDA/Fe3O4@C@PGC nanocomposites with the help of magnetic force. Under the optimal conditions, the immunosensor exhibited a wide linear range (0.001 ng/mL–20.0 ng/mL), a low detection limit (0.33 pg/mL), good reproducibility, selectivity and acceptable stability. The proposed sensing strategy may provide a potential application in the detection of other cancer biomarkers. PMID:26868035

  6. Single-step cycle pulse operation of the label-free electrochemiluminescence immunosensor based on branched polypyrrole for carcinoembryonic antigen detection

    PubMed Central

    Zhu, Wenjuan; Wang, Qi; Ma, Hongmin; Lv, Xiaohui; Wu, Dan; Sun, Xu; Du, Bin; Wei, Qin

    2016-01-01

    A novel label-free electrochemiluminescence (ECL) immunosensor based on luminol functional-Au NPs@polypyrrole has been developed for the detection of carcinoembryonic antigen (CEA). In this work, polypyrrole prepared by chemical polymerization provided a large surface area to load amounts of gold nanoparticles (Au NPs). Au NPs could not only attach abundant luminol for the enhancement of ECL signal, but also provide a friendly microenvironment for the immobilization of antibodies. Moreover, 1-butylpyridinium tetrafluroborate ([BPy]BF4) were used to disperse luminol functional-Au NPs@polypyrrole nanocomposites, resulting in the film-formation of composites on the electrode, which could improve the stability of immunosensor. In particular, employment of single-step cycle pulse could limit the consecutive reaction between luminol and H2O2 efficiently, thus leading to stable and strong signals. The proposed method presents good ECL response for the detection of CEA allowing a wide linear range from 0.01 pg/mL to 10 ng/mL and a limit of detection as low as 3 fg/mL. The immunosensor would be a promising tool in the early diagnosis of CEA due to its high sensitivity, simplicity and cost-effective. PMID:27091590

  7. A sandwich-type electrochemical immunosensor for carcinoembryonic antigen based on signal amplification strategy of optimized ferrocene functionalized Fe₃O₄@SiO₂ as labels.

    PubMed

    Feng, Taotao; Qiao, Xiuwen; Wang, Haining; Sun, Zhao; Hong, Chenglin

    2016-05-15

    A sandwich-type electrochemical immunosensor was developed for sensitive detection of carcinoembryonic antigen (CEA) by using ferroferric oxide@silica-amino groups (Fe3O4@SiO2-NH2) as carriers and gold nanoparticles-graphene oxide (GO-AuNPs) as platform. The Fe3O4@SiO2-NH2 surface was used as linked reagents for co-immobilization of ferrocenecarboxylic acid (Fc-COOH) and secondary anti-CEA (Ab2) to prepare the signal probe, and it also could hasten the decomposition of hydrogen peroxide (H2O2) to amplify signals. Differential pulse voltammetry (DPV) was successfully used to quantify CEA. Under the optimized conditions, the designed immunosensor shows an excellent analytical performance wide dynamic response range of CEA concentration from 0.001 ng mL(-1) to 80 ng mL(-1) with a relatively low detection limit of 0.0002 ng mL(-1) (S/N=3), and high specificity and good reproducibility. The proposed immunosensor was successfully used to determine CEA in spiked human serum samples. PMID:26686923

  8. Comparison of a chimeric anti-carcinoembryonic antigen antibody conjugated with visible or near-infrared fluorescent dyes for imaging pancreatic cancer in orthotopic nude mouse models

    NASA Astrophysics Data System (ADS)

    Maawy, Ali A.; Hiroshima, Yukihiko; Kaushal, Sharmeela; Luiken, George A.; Hoffman, Robert M.; Bouvet, Michael

    2013-12-01

    The aim of this study was to evaluate a set of visible and near-infrared dyes conjugated to a tumor-specific chimeric antibody for high-resolution tumor imaging in orthotopic models of pancreatic cancer. BxPC-3 human pancreatic cancer was orthotopically implanted into pancreata of nude mice. Mice received a single intravenous injection of a chimeric anti-carcinoembryonic antigen antibody conjugated to one of the following fluorophores: 488-nm group (Alexa Fluor 488 or DyLight 488); 550-nm group (Alexa Fluor 555 or DyLight 550); 650-nm group (Alexa Fluor 660 or DyLight 650), or the 750-nm group (Alexa Fluor 750 or DyLight 755). After 24 h, the Olympus OV100 small-animal imaging system was used for noninvasive and intravital fluorescence imaging of mice. Dyes were compared with respect to depth of imaging, resolution, tumor-to-background ratio (TBR), photobleaching, and hemoglobin quenching. The longer wavelength dyes had increased depth of penetration and ability to detect the smallest tumor deposits and provided the highest TBRs, resistance to hemoglobin quenching, and specificity. The shorter wavelength dyes were more photostable. This study showed unique advantages of each dye for specific cancer imaging in a clinically relevant orthotopic model.

  9. Evaluation of the use of decision-support software in carcino-embryonic antigen (CEA)-based follow-up of patients with colorectal cancer

    PubMed Central

    2012-01-01

    Background The present paper is a first evaluation of the use of "CEAwatch", a clinical support software system for surgeons for the follow-up of colorectal cancer (CRC) patients. This system gathers Carcino-Embryonic Antigen (CEA) values and automatically returns a recommendation based on the latest values. Methods Consecutive patients receiving follow-up care for CRC fulfilling our in- and exclusion criteria were identified to participate in this study. From August 2008, when the software was introduced, patients were asked to undergo the software-supported follow-up. Safety of the follow-up, experiences of working with the software, and technical issues were analyzed. Results 245 patients were identified. The software-supported group contained 184 patients; the control group contained 61 patients. The software was safe in finding the same amount of recurrent disease with fewer outpatient visits, and revealed few technical problems. Clinicians experienced a decrease in follow-up workload of up to 50% with high adherence to the follow-up scheme. Conclusion CEAwatch is an efficient software tool helping clinicians working with large numbers of follow-up patients. The number of outpatient visits can safely be reduced, thus significantly decreasing workload for clinicians. PMID:22390356

  10. Au-F127 strawberry-like nanospheres as an electrochemical interface for sensitive detection of carcinoembryonic antigen in real sample.

    PubMed

    Li, Juan; Xie, Hangqing; Liu, Yuhong; Ren, Hang; Zhao, Wenbo; Huang, Xiaohua

    2015-11-01

    Nanomaterial-based signal-amplification strategies hold a great promise in realizing sensitive biological detection. A simple label-free electrochemical immunosensor for sensitive detection of carcinoembryonic antigen (CEA) was developed by immobilizing anti-CEA antibodies onto the Au-F127 strawberry-like nanospheres modified glassy carbon electrode (Au-F127/GCE). The Au-F127 strawberry-like nanospheres offered a large surface and multifunctional substrate for the effective immobilization of anti-CEA and the existence of Au could accelerate electron transfer and make the electrochemical signal amplified. The Au-F127 nanocomposites and anti-CEA were characterized by transmission electron microscopy (TEM), polycrystalline electron diffraction ring pattern, ultra-violet visible (UV-vis) spectra and attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectra. Electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) were employed to verify the stepwise assembly of the immunosensor and evaluated the analytical performance of the fabricated immunosensor, respectively. The immunosensor showed a wide liner response range between 0.01 and 80 ng mL(-1) with a low detection limit of 0.24 pg mL(-1) at a signal-to-noise (S/N) ratio of 3. Additionally, the proposed method was successfully applied to determine CEA in human serum samples with satisfactory results. PMID:26452840

  11. Label-Free Electrochemiluminescent Immunosensor for Detection of Carcinoembryonic Antigen Based on Nanocomposites of GO/MWCNTs-COOH/Au@CeO₂.

    PubMed

    Pang, Xuehui; Li, Jianxiu; Zhao, Yongbei; Wu, Dan; Zhang, Yong; Du, Bin; Ma, Hongmin; Wei, Qin

    2015-09-01

    A high-sensitivity electrochemiluminescence (ECL) sensor was conducted to detect carcinoembryonic antigen (CEA). Nanocomposites of graphene oxide/carboxylated multiwall carbon nanotubes/gold/cerium oxide nanoparticles (GO/MWCNTs-COOH/Au@CeO2) were used as antibody carriers and sensing platforms to modify on glassy carbon electrodes (GCE). CeO2 nanoparticles were first exploited as an ECL luminescent material and the possible ECL mechanism was proposed in this work. GO/MWCNTs-COOH was used as a loading matrix for CeO2 nanoparticles because of the superior conductivity and large specific surface area. Au nanoparticles were further deposited on this matrix to attach anti-CEA and enhance the sensitivity of immunosensor. The proposed sensing platform showed excellent cathodic ECL performance and sensitive response to CEA. The effects of experimental conditions on the ECL performance were investigated. The proposed immunosensor showed the broad linear range (0.05-100 ng/mL) and the low detection limit (LOD, 0.02 ng/mL, signal-to-noise ratio = 3) according to the selected experimental conditions. The excellent analysis performance for determination of CEA in the human serum samples simplied this immunosensor displayed high sensitivity and excellent repeatability. More importantly, this conducted immunosensor broadens the use scope of CeO2 nanoparticles. PMID:26271682

  12. Increased electrocatalyzed performance through hairpin oligonucleotide aptamer-functionalized gold nanorods labels and graphene-streptavidin nanomatrix: Highly selective and sensitive electrochemical biosensor of carcinoembryonic antigen.

    PubMed

    Wen, Wei; Huang, Jing-Yi; Bao, Ting; Zhou, Jun; Xia, Hong-Xing; Zhang, Xiu-Hua; Wang, Sheng-Fu; Zhao, Yuan-Di

    2016-09-15

    We report a triplex signal amplification strategy for sensitive biosensing of cancer biomarker by taking advantage of hairpin-shaped oligonucleotide-functionalized gold nanorods (HO-GNRs), graphene and the avidin-biotin reation. The strategy expands electrochemical detection of carcinoembryonic antigen (CEA) by using an aptamer as biosensor's recognition element and HO-GNRs as signal enhancer. To construct this biosensor, the GNR was used as a carrier of horseradish peroxidase (HRP) and HO aptamer with a biotin at the 3'-end and a thiol at the 5'-end, which amplified the electrochemical response because of a large molar ratio of HRP to HO. In the presence of target CEA, the binding reactions of CEA with the loop portions of the HOs caused HOs' loop-stem structure opened and exposed the biotins, and then HRP-GNRs-HO conjugates were captured on graphene and streptavidin modified electrodes via the reaction between the exposed biotins and preimmobilized streptavidins. The accumulation of HRP effectively catalyzed the hydrogen peroxide-mediated oxidation of o-phenylenediamine to generate an electrochemical reduction current for CEA detection. Under optimal conditions, the electrochemical biosensor exhibited a wide dynamic range of 5pgmL(-1) and 50ngmL(-1) toward CEA standards with a low detection limit of 1.5pgmL(-1) (signal-to-noise ratio of 3). The proposed biosensor accurately detected CEA concentration in 8 human serum samples from patients with lung diseases, showing excellent correlations with standard chemiluminescence immunoassay. Furthermore, these results of target DNA detection made it abundantly clear that the proposed strategy can also be extended for detection of other relative biomarkers using different functional DNA structures, which shows great prospects in single-nucleotide polymorphisms analysis, biomedical sensing and application for accurate clinical diseases diagnostic. PMID:27111123

  13. Improved tumor localization with increasing dose of indium-111-labeled anti-carcinoembryonic antigen monoclonal antibody ZCE-025 in metastatic colorectal cancer

    SciTech Connect

    Patt, Y.Z.; Lamki, L.M.; Haynie, T.P.; Unger, M.W.; Rosenblum, M.G.; Shirkhoda, A.; Murray, J.L.

    1988-08-01

    Monoclonal antibodies (MoAbs) against carcinoembryonic antigen (CEA) react with human colorectal cancer cells, and when labeled with a gamma-emitting radioisotope, may help to localize known and occult metastatic disease. We tested ZCE-025, a high-affinity immune gamma globulin1 (IgG1) MoAb anti-CEA that does not react with normal granulocyte glycoproteins in a phase I/II trial to determine the reagent's toxicity and its maximum efficacy in detecting metastatic colorectal cancer. Increasing doses of unlabeled ZCE-025 were mixed with 1 mg of Indium-111 (111In)-radiolabeled MoAb and administered intravenously (IV) to 34 patients who had metastatic colorectal cancer. Planar nuclear or single photon emission computed tomographic (SPECT) scans were performed 48 to 72 and 120 to 144 hours later. Total dose of MoAb and scanning sensitivity (number of imaged lesions/number of known lesions) were correlated up to 80 mg. At doses of 2.5 to 20 mg, a mean of 22% of the lesions were imaged; at 40 mg, 77% were imaged (P less than .01). Liver metastases were detected as areas of increased activity (hot) at the 40 mg dose but showed decreased MoAb uptake at lower doses. At the 40 mg dose normal liver parenchymal uptake of the labeled MoAb was lower with respect to blood pool compared with the other doses. At 80 mg, however, sensitivity of detection declined to 21%. One milligram of 111In-labeled ZCE-025 antibody coinfused with 39 mg of unlabeled antibody appeared optimal for detecting metastatic colorectal cancer, particularly in the liver. Although the exact mechanism(s) for this dose effect is currently unknown, a partial blocking effect of unlabeled antibody with a change in MoAb biodistribution may be occurring.

  14. Photoelectrochemical Immunosensor for Detection of Carcinoembryonic Antigen Based on 2D TiO2 Nanosheets and Carboxylated Graphitic Carbon Nitride

    PubMed Central

    Wang, Huan; Wang, Yaoguang; Zhang, Yong; Wang, Qi; Ren, Xiang; Wu, Dan; Wei, Qin

    2016-01-01

    Carcinoembryonic antigen (CEA) was used as the model, an ultrasensitive label-free photoelectrochemical immunosensor was developed using 2D TiO2 nanosheets and carboxylated graphitic carbon nitride (g-C3N4) as photoactive materials and ascorbic acid as an efficient electron donor. 2D TiO2 nanosheets was sythsized by surfactant self-assembly method and proved to have higher photoelectrochemical signals than TiO2 nanoparticles. Firstly, carboxylated g-C3N4 could be attached to 2D TiO2 nanosheets through the bond formed between carboxyl group of carboxylated g-C3N4 and TiO2. And the photocurrent of g-C3N4/TiO2 drastically enhances compared to carboxylated g-C3N4 and TiO2. Then, antibody of CEA was bonded to TiO2 through the dentate bond formed between carboxyl group of anti-CEA and TiO2, leading to the decrease of the photocurrents. As proven by PEC experiments and electrochemical impedance spectroscopy (EIS) analysis, the fabrication process of the immunosensor is successful. Under the optimal conditions, the intensity decreased linearly with CEA concentration in the range of 0.01~10 ng/mL. The detection limit is 2.1 pg/mL. The work provides an effective method for the detection of tumor markers and can be extended for the application in food safety and environmental monitoring analysis. PMID:27263659

  15. In vitro autoradiography of carcinoembryonic antigen in tissue from patients with colorectal cancer using multifunctional antibody TF2 and 67/68Ga-labeled haptens by pretargeting

    PubMed Central

    Hall, Håkan; Velikyan, Irina; Blom, Elisabeth; Ulin, Johan; Monazzam, Azita; Påhlman, Lars; Micke, Patrick; Wanders, Alkwin; McBride, William; Goldenberg, David M.; Långström, Bengt

    2012-01-01

    The carcinoembryonic antigen (CEA) was visualized in vitro in tissue from patients with colorectal cancer with trivalent bispecific antibody TF2 and two hapten molecules, [67/68Ga]Ga-IMP461 and [67/68Ga]Ga-IMP485 by means of pretargeting. Colorectal cancer tissue samples obtained from surgery at Uppsala University Hospital, were frozen fresh and cryosectioned. The two hapten molecules comprising 1,4,7-triazacyclononanetriacetic acid chelate moiety (NOTA) were labeled with 67Ga or 68Ga. The autoradiography was conducted by incubating the tissue samples with the bispecific antibody TF2, followed by washing and incubation with one of the radiolabeled hapten molecules. After washing, drying and exposure to phosphor imager plates, the autoradiograms were analyzed and compared to standard histochemistry (hematoxylin-eosin). Pronounced binding was found in the tissue from colorectal cancer using the bispecific antibody TF2 and either of the haptens [67/68Ga]Ga-IMP461 and [67/68Ga]Ga-IMP485. Distinct binding was also detected in the epithelium of most samples of neighboring tissue, taken at a minimum of 10 cm from the site of the tumor. It is concluded that pretargeting CEA with the bispecific antibody TF2 followed by the addition of 67/68Ga-labeled hapten is extremely sensitive for visualizing this marker for colorectal cancer. This methodology is therefore a very specific complement to other histochemical techniques in the diagnosis of biopsies or in samples taken from surgery. Use of the pretargeting technique in vivo may also be an advance in diagnosing patients with colorectal cancer, either using 67Ga and SPECT or 68Ga and PET. PMID:23133809

  16. The combination of preoperative serum C-reactive protein and carcinoembryonic antigen is a useful prognostic factor in patients with esophageal squamous cell carcinoma: a combined ROC analysis

    PubMed Central

    Huang, Ying; Liu, Jin-Shi; Feng, Ji-Feng

    2015-01-01

    Background The prognostic value of inflammatory index in esophageal cancer (EC) has not been established. In the present study, therefore, we initially evaluated a novel prognostic system, named the COCC (COmbination of C-reactive protein [CRP] and carcinoembryonic antigen [CEA]), for making a prognosis in patients with esophageal squamous cell carcinoma (ESCC). Methods A total of 327 patients with ESCC between January 2006 and December 2008 were included in this retrospective study. The COCC was calculated by combined CRP and CEA according to the logistic equation. The Kaplan–Meier method was used to calculate the cancer-specific survival (CSS), and the difference was assessed by the log-rank test. Cox regression analyses were performed to evaluate the prognostic factors. Results In our study, COCC was defined as CRP +0.71 CEA according to the logistic equation. Receiver operating characteristic curves for CSS prediction were plotted to verify the optimum cutoff points for CRP, CEA, and COCC, which were 9.8 mg/L, 4.2 ng/mL, and 8.0, respectively. Patients with COCC ≤8.0 had a significantly better CSS than patients with COCC >8.0 (53.1% vs 15.3%, P<0.001). Multivariate analysis revealed that COCC was an independent prognostic factor in patients with ESCC (P=0.006). In addition, the area under the curve (AUC) was 0.722 for COCC, 0.645 for CRP, and 0.618 for CEA, indicating that COCC was superior to CRP or CEA for CSS prediction. Conclusion The COCC is an independent prognostic factor in patients with ESCC. We conclude that COCC was superior to CRP or CEA as a more precise prognostic factor in patients with ESCC. PMID:25914550

  17. Mechanistic Control of Carcinoembryonic Antigen-related Cell Adhesion Molecule-1 (CEACAM1) Splice Isoforms by the Heterogeneous Nuclear Ribonuclear Proteins hnRNP L, hnRNP A1, and hnRNP M*

    PubMed Central

    Dery, Kenneth J.; Gaur, Shikha; Gencheva, Marieta; Yen, Yun; Shively, John E.; Gaur, Rajesh K.

    2011-01-01

    Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is expressed in a variety of cell types and is implicated in carcinogenesis. Alternative splicing of CEACAM1 pre-mRNA generates two cytoplasmic domain splice variants characterized by the inclusion (L-isoform) or exclusion (S-isoform) of exon 7. Here we show that the alternative splicing of CEACAM1 pre-mRNA is regulated by novel cis elements residing in exon 7. We report the presence of three exon regulatory elements that lead to the inclusion or exclusion of exon 7 CEACAM1 mRNA in ZR75 breast cancer cells. Heterologous splicing reporter assays demonstrated that the maintenance of authentic alternative splicing mechanisms were independent of the CEACAM1 intron sequence context. We show that forced expression of these exon regulatory elements could alter CEACAM1 splicing in HEK-293 cells. Using RNA affinity chromatography, three members of the heterogeneous nuclear ribonucleoprotein family (hnRNP L, hnRNP A1, and hnRNP M) were identified. RNA immunoprecipitation of hnRNP L and hnRNP A1 revealed a binding motif located central and 3′ to exon 7, respectively. Depletion of hnRNP A1 or L by RNAi in HEK-293 cells promoted exon 7 inclusion, whereas overexpression led to exclusion of the variable exon. By contrast, overexpression of hnRNP M showed exon 7 inclusion and production of CEACAM1-L mRNA. Finally, stress-induced cytoplasmic accumulation of hnRNP A1 in MDA-MB-468 cells dynamically alters the CEACAM1-S:CEACAM1:L ratio in favor of the l-isoform. Thus, we have elucidated the molecular factors that control the mechanism of splice-site recognition in the alternative splicing regulation of CEACAM1. PMID:21398516

  18. Prognostic Value of Pretreatment Carcinoembryonic Antigen After Definitive Radiotherapy With or Without Concurrent Chemotherapy for Squamous Cell Carcinoma of the Uterine Cervix

    SciTech Connect

    Huang, Eng-Yen; Hsu, Hsuan-Chih; Sun, Li-Min; Chanchien, Chan-Chao; Lin, Hao; Chen, Hui-Chun; Tseng, Chih-Wen; Ou, Yu-Che; Chang, Hung-Yao; Fang, Fu-Min; Huang, Yu-Jie; Wang, Chang-Yu; Lu, Hsien-Ming; Tsai, Ching-Chou; and others

    2011-11-15

    Purpose: To evaluate whether pretreatment carcinoembryonic antigen (CEA) levels have a prognostic role in patients after definitive radiotherapy for squamous cell carcinoma (SCC) of the uterine cervix. Methods and Materials: A retrospective study of 550 patients was performed. The SCC antigen (SCC-Ag) and CEA levels were regarded as elevated when they were {>=}2 and {>=}5 ng/mL, respectively. A total of 208 patients underwent concurrent chemoradiotherapy (CCRT). The Kaplan-Meier method was used to calculate the distant metastasis (DM), local failure (LF), disease-free survival (DFS), and overall survival (OS) rates. Multivariate analysis was performed using the Cox proportional hazards model. The hazard ratio (HR) with 95% confidence interval (CI) was evaluated for the risk of a poor prognosis. Results: Compared with the patients with normal CEA/SCC-Ag levels, CEA levels {>=}10 ng/mL but without elevated SCC-Ag levels was an independent factor for LF (HR, 51.81; 95% CI, 11.51-233.23; p < .001), DM (HR, 6.04; 95% CI, 1.58-23.01; p = .008), DFS (HR, 10.17; 95% CI, 3.18-32.56; p < .001), and OS (HR, 5.75; 95% CI, 1.82-18.18; p = .003) after RT alone. However, no significant role for CEA was noted in patients with SCC-Ag levels {>=}2 ng/mL. In patients undergoing CCRT, a CEA level {>=}10 ng/mL was an independent factor for LF (HR, 2.50; 95% CI, 1.01-6.21; p = .047), DM (HR, 3.41; 95% CI, 1.56-7.46; p = .002), DFS (HR, 2.73; 95% CI, 1.39-5.36; p = .003), and OS (HR, 3.93; 95% CI 1.99-7.75; p < .001). A SCC-Ag level of {>=}40 ng/mL was another prognostic factor for DM, DFS, and OS in patients undergoing not only CCRT, but also RT alone. The 5-year OS rate for CCRT patients with CEA <10 ng/mL and {>=}10 ng/mL was 75.3% and 35.8%, respectively (p < .001). CCRT was an independent factor for better OS (HR, 0.69; 95% CI, 0.50-0.97; p = .034). Conclusion: Pretreatment CEA levels in patients with SCC of the uterine cervix provide complementary information for predicting LF, DM

  19. The Role of Biliary Carcinoembryonic Antigen-Related Cellular Adhesion Molecule 6 (CEACAM6) as a Biomarker in Cholangiocarcinoma

    PubMed Central

    Rose, J. Bart; Correa-Gallego, Camilo; Li, Yu; Nelson, James; Alseidi, Adnan; Helton, W. Scott; Allen, Peter J.; D’Angelica, Michael I.; DeMatteo, Ronald P.; Fong, Yuman; Kingham, T. Peter; Kowdley, Kris V.; Jarnagin, William R.; Rocha, Flavio G.

    2016-01-01

    Objective The aim of the present study is to determine if CEACAM6 can be detected in the bile of patients with biliary cancer and can serve as a diagnostic biomarker for cholangiocarcinoma. Summary Background Data Distinguishing bile duct carcinoma from other diagnoses is often difficult using endoscopic or percutaneous techniques. The cell surface protein CEACAM6 is over-expressed in many gastrointestinal cancers and may be selectively elevated in biliary adenocarcinoma. Methods Bile from patients with benign biliary disease and cholangiocarcinoma (hilar, intrahepatic and distal) was collected at the time of index operation. The concentration of CEACAM6 was quantified by sandwich enzyme-linked immunosorbent assay (ELISA) and correlated to pathologic diagnosis. Diagnostic capability of CEACAM6 was evaluated by Wilcoxon rank-sum, linear regression, multiple regression, and receiver operating characteristic (ROC) curve analysis. Results Bile from 83 patients was analyzed: 42 with benign disease and 41 with cholangiocarcinoma. Patients in the benign cohort were younger, predominantly female, and had lower median biliary CEACAM6 levels than patients in the malignant cohort (7.5 ng/ml vs. 40 ng/ml; p = <.001). ROC curve analysis determined CEACAM6 to be a positive predictor cholangiocarcinoma with a CEACAM6 level >14 ng/ml associated with 87.5% sensitivity, 69.1% specificity, and a likelihood ratio of 2.8 (AUC 0.74). Multiple regression analysis suggested elevated alkaline phosphatase and the presence of biliary endoprostheses may influence CEACAM6 levels. Conclusion Biliary CEACAM6 can identify patients with extrahepatic cholangiocarcinoma with a high degree of sensitivity and should be investigated further as a potential screening tool. PMID:26974538

  20. Vertebrate Cells Express Protozoan Antigen after Hybridization

    NASA Astrophysics Data System (ADS)

    Crane, Mark St. J.; Dvorak, James A.

    1980-04-01

    Epimastigotes, the invertebrate host stage of Trypanosoma cruzi, the protozoan parasite causing Chagas' disease in man, were fused with vertebrate cells by using polyethylene glycol. Hybrid cells were selected on the basis of T. cruzi DNA complementation of biochemical deficiencies in the vertebrate cells. Some clones of the hybrid cells expressed T. cruzi-specific antigen. It might be possible to use selected antigens obtained from the hybrids as vaccines for immunodiagnosis or for elucidation of the pathogenesis of Chagas' disease.

  1. Pretreatment carcinoembryonic antigen level is a risk factor for para-aortic lymph node recurrence in addition to squamous cell carcinoma antigen following definitive concurrent chemoradiotherapy for squamous cell carcinoma of the uterine cervix

    PubMed Central

    2012-01-01

    Background To identify pretreatment carcinoembryonic antigen (CEA) levels as a risk factor for para-aortic lymph node (PALN) recurrence following concurrent chemoradiotherapy (CCRT) for cervical cancer. Methods From March 1995 to January 2008, 188 patients with squamous cell carcinoma (SCC) of the uterine cervix were analyzed retrospectively. No patient received PALN irradiation as the initial treatment. CEA and squamous cell carcinoma antigen (SCC-Ag) were measured before and after radiotherapy. PALN recurrence was detected by computer tomography (CT) scans. We analyzed the actuarial rates of PALN recurrence by using Kaplan-Meier curves. Multivariate analyses were carried out with Cox regression models. We stratified the risk groups based on the hazard ratios (HR). Results Both pretreatment CEA levels ≥ 10 ng/mL and SCC-Ag levels < 10 ng/mL (p < 0.001, HR = 8.838), SCC-Ag levels ≥ 40 ng/mL (p < 0.001, HR = 12.551), and SCC-Ag levels of 10-40 ng/mL (p < 0.001, HR = 4.2464) were significant factors for PALN recurrence. The corresponding 5-year PALN recurrence rates were 51.5%, 84.8%, and 27.5%, respectively. The 5-year PALN recurrence rate for patients with both low (< 10 ng/mL) SCC and CEA was only 9.6%. CEA levels ≥ 10 ng/mL or SCC-Ag levels ≥ 10 ng/mL at PALN recurrence were associated with overall survival after an isolated PALN recurrence. Pretreatment CEA levels ≥ 10 ng/mL were also associated with survival after an isolated PALN recurrence. Conclusions Pretreatment CEA ≥ 10 ng/mL is an additional risk factor of PALN relapse following definitive CCRT for SCC of the uterine cervix in patients with pretreatment SCC-Ag levels < 10 ng/mL. More comprehensive examinations before CCRT and intensive follow-up schedules are suggested for early detection and salvage in patients with SCC-Ag or CEA levels ≥ 10 ng/mL. PMID:22289572

  2. Initial clinical experience evaluating Yttrium-90-chimeric T84.66 anticarcinoembryonic antigen antibody and autologous hematopoietic stem cell support in patients with carcinoembryonic antigen-producing metastatic breast cancer.

    PubMed

    Wong, J Y; Somlo, G; Odom-Maryon, T; Williams, L E; Liu, A; Yamauchi, D; Wu, A M; Yazaki, P; Wilczynski, S; Shively, J E; Forman, S; Doroshow, J H; Raubitschek, A A

    1999-10-01

    cT84.66 is a human/murine IgG1 with high affinity and specificity for carcinoembryonic antigen (CEA). An earlier Phase I trial defined the maximum tolerated dose for 90Y-diethylenetriaminepentaacetic acid (DTPA)-cT84.66 at 22 mCi/m2. Dose-limiting toxicities were reversible leukopenia and thrombocytopenia. The purpose of this Phase I trial was to evaluate the feasibility and toxicities of administering higher activities of 90Y-DTPA-cT84.66 with stem cell support in patients with CEA-producing breast cancer. Patients with CEA-producing breast cancer refractory to standard therapies underwent peripheral stem cell collection followed by infusion of 111indium-DTPA-cT84.66. Those patients demonstrating tumor targeting received a single therapy dose of 90Y-DTPA-cT84.66, followed by Ca-DTPA infusion for 72 h posttherapy. Stem cells were reinfused following a divided schedule. To date, seven patients have been accrued to this trial. Each patient received an imaging dose of (111)In-cT84.66. Six patients demonstrated tumor imaging and received a single cycle of 90Y-cT84.66 at 15 mCi/m2 (three patients) and 22.5 mCi/m2 (three patients). One patient did not demonstrate tumor imaging and was not treated. At these administered activities, 90Y-cT84.66 was well tolerated. No dose-limiting toxicities have been observed. All patients demonstrated hematopoietic recovery after stem cell infusion. One patient demonstrated stable disease for 4 months; one patient had stable disease and reduction of bone pain for 3 months; and a third patient experienced >50% reduction of an ovarian metastasis, resolution of malignant pleural effusion, stable pleural metastases, and stable bone scan for 14 months. Preliminary results from this ongoing Phase I trial are promising and demonstrate the feasibility and potential for antitumor effects of stem cell supported 90Y-cT84.66 therapy in patients with CEA-producing breast cancers. PMID:10541368

  3. The pregnancy-specific glycoprotein (PSG) gene cluster on human chromosome 19: Fine structure of the 11 PSG genes and identification of 6 new genes forming a third subgroup within the carcinoembryonic antigen (CEA) family

    SciTech Connect

    Teglund, S.; Fraengsmyr, L.; Hammarstroem, S.

    1994-10-01

    The human pregnancy-specific glycoprotein (PSG) genes belong to the carcinoembryonic antigen (CEA) family, which in turn is a member of the immunoglobulin superfamily. We have analyzed a 700-kb cosmid contig spanning the PSG region on chromosome 19q13.2. The region contains 11 closely related PSG genes organized in tandem with a highly conserved structure and organization. Seven novel genes (CGM12 to CGM18) were found in the PSG region. CGM12 belongs to the CEA subgroup and appears to be a pseudogene. CGM13 to CGM18 forms a third new subgroup within the CEA gene family. The members of this new subgroup show 94-99% identity to each other but only 70-80% to other members of either the CEA or the PSG subgroups. They are composed of exons encoding two IgC-like domains and short hydrophilic carboxyl terminals similar to those of the PSGs. Unlike any of the known CEA family genes, however, they seem to lack the exon for an IgV-like N-terminal domain. 54 refs., 6 figs., 4 tabs.

  4. Developmental expression of autoimmune target antigens during organogenesis.

    PubMed Central

    Akashi, T; Eishi, Y

    1991-01-01

    A common factor existing among autoimmune target antigens was sought in association with their developmental expression during organogenesis. Autoimmunity against a certain organ was experimentally induced in rats by deliberate immunization with whole tissue extract of the respective organ. Histopathological changes in a target organ of the immunized rats were recorded, and tissue specificity of the raised autoantibodies was immunohistologically examined with tissue sections of normal adult rats. These immune sera were also reacted with tissue sections of a target organ in each stage of organogenesis, and the time of first expression of the target antigen was determined for each immune serum. As a result, induced autoantibodies were directed only to a limited number of tissue antigens, such as thyroid follicular antigens [gestation day 17 (17 GD)], salivary ductal antigens (18 GD), anterior pituitary antigens (21 GD), gastric parietal cell antigens (22 GD), neural myelin antigens (2 days after birth), retinal photo-receptor cell antigens (3 days after birth) and testicular germ cell antigens (4 weeks after birth). They were first expressed on the day indicated in parentheses. Comparing with the development of the immune system, which was monitored by demonstrating CD4- and/or CD8-positive cells in the developing thymus and spleen, a common feature of these potential autoimmune target antigens was found to be that they were expressed either in parallel with, or after, but never before, the development of the immune system. This observation might suggest why only a limited number of self antigens can be autoimmune target antigens among the enormously large number of antigen determinants existing in the whole extract of each organ. Images Figure 1 Figure 2 Figure 3 PMID:1769700

  5. Specific tumor labeling enhanced by polyethylene glycol linkage of near infrared dyes conjugated to a chimeric anti-carcinoembryonic antigen antibody in a nude mouse model of human pancreatic cancer

    NASA Astrophysics Data System (ADS)

    Maawy, Ali A.; Hiroshima, Yukihiko; Zhang, Yong; Luiken, George A.; Hoffman, Robert M.; Bouvet, Michael

    2014-10-01

    Labeling of metastatic tumors can aid in their staging and resection of cancer. Near infrared (NIR) dyes have been used in the clinic for tumor labeling. However, there can be a nonspecific uptake of dye by the liver, lungs, and lymph nodes, which hinders detection of metastasis. In order to overcome these problems, we have used two NIR dyes (DyLight 650 and 750) conjugated to a chimeric anti-carcinoembryonic antigen antibody to evaluate how polyethylene glycol linkage (PEGylation) can improve specific tumor labeling in a nude mouse model of human pancreatic cancer. The conjugated PEGylated and non-PEGylated DyLight 650 and 750 dyes were injected intravenously into non-tumor-bearing nude mice. Serum samples were collected at various time points in order to determine serum concentrations and elimination kinetics. Conjugated PEGylated dyes had significantly higher serum dye concentrations than non-PEGylated dyes (p=0.005 for the 650 dyes and p<0.001 for the 750 dyes). Human pancreatic tumors subcutaneously implanted into nude mice were labeled with antibody-dye conjugates and serially imaged. Labeling with conjugated PEGylated dyes resulted in significantly brighter tumors compared to the non-PEGylated dyes (p<0.001 for the 650 dyes; p=0.01 for 750 dyes). PEGylation of the NIR dyes also decreased their accumulation in lymph nodes, liver, and lung. These results demonstrate enhanced selective tumor labeling by PEGylation of dyes conjugated to a tumor-specific antibody, suggesting their future clinical use in fluorescence-guided surgery.

  6. Paper-based colorimetric immunosensor for visual detection of carcinoembryonic antigen based on the high peroxidase-like catalytic performance of ZnFe2O4-multiwalled carbon nanotubes.

    PubMed

    Liu, Weiyan; Yang, Hongmei; Ding, Yanan; Ge, Shenguang; Yu, Jinghua; Yan, Mei; Song, Xianrang

    2014-01-01

    A new paper-based colorimetric immunosensor for the detection of carcinoembryonic antigen (CEA) was developed based on the intrinsic peroxidase activity of ZnFe2O4-multiwalled carbon nanotubes (ZnFe2O4@MWNTs). The immunosensor platform was prepared by depositing chitosan and porous gold onto filter paper and entrapping the primary antibodies (Ab1) onto the layers. Secondary antibodies (Ab2) were assembled on the surface of the functionalized ZnFe2O4@MWNTs. The immunosensor response was quantified as a color change resulting from ZnFe2O4@MWNTs catalyzing the oxidation of 3,3',5,5'-tetramethylbenzidine in the presence of H2O2. The catalytic performance of ZnFe2O4@MWNTs was higher than ZnFe2O4 due to the high electrical conductance of MWNTs, moreover, the electron communications between ZnFe2O4@MWNTs and substrates are electrically "wired". Detection was achieved by measuring the color change when the concentrations of CEA were different. The color change can be quantified with the naked eye but a digitalized picture can also be used to provide more sensitive comparison to a calibrated color scheme. This method was simple for CEA detection with a linear range from 0.005 to 30 ng mL(-1) and a detection limit of 2.6 pg mL(-1). Such an equipment-free immunoassay has great potential in resource-limited environments. PMID:24205509

  7. Randomised Phase I/II trial assessing the safety and efficacy of radiolabelled anti-carcinoembryonic antigen I131 KAb201 antibodies given intra-arterially or intravenously in patients with unresectable pancreatic adenocarcinoma

    PubMed Central

    2009-01-01

    Background Advanced pancreatic cancer has a poor prognosis, and the current standard of care (gemcitabine based chemotherapy) provides a small survival advantage. However the drawback is the accompanying systemic toxicity, which targeted treatments may overcome. This study aimed to evaluate the safety and tolerability of KAb201, an anti-carcinoembryonic antigen monoclonal antibody, labelled with I131 in pancreatic cancer (ISRCTN 16857581). Methods Patients with histological/cytological proven inoperable adenocarcinoma of the head of pancreas were randomised to receive KAb 201 via either the intra-arterial or intravenous delivery route. The dose limiting toxicities within each group were determined. Patients were assessed for safety and efficacy and followed up until death. Results Between February 2003 and July 2005, 25 patients were enrolled. Nineteen patients were randomised, 9 to the intravenous and 10 to the intra-arterial arms. In the intra-arterial arm, dose limiting toxicity was seen in 2/6 (33%) patients at 50 mCi whereas in the intravenous arm, dose limiting toxicity was noted in 1/6 patients at 50 mCi, but did not occur at 75 mCi (0/3). The overall response rate was 6% (1/18). Median overall survival was 5.2 months (95% confidence interval = 3.3 to 9 months), with no significant difference between the intravenous and intra-arterial arms (log rank test p = 0.79). One patient was still alive at the time of this analysis. Conclusion Dose limiting toxicity for KAb201 with I131 by the intra-arterial route was 50 mCi, while dose limiting toxicity was not reached in the intravenous arm. PMID:19243606

  8. Pharmacokinetics of an anti-carcinoembryonic antigen monoclonal antibody conjugated to a bifunctional transition metal carborane complex (venus flytrap cluster) in tumor-bearing mice.

    PubMed

    Beatty, B G; Paxton, R J; Hawthorne, M F; Williams, L E; Rickard-Dickson, K J; Do, T; Shively, J E; Beatty, J D

    1993-08-01

    An anticarcinoembryonic antigen (CEA) monoclonal antibody, T84.66, has been conjugated to a metallocarborane complex (Venus flytrap cluster, VFC) containing 57Co. This radioimmunoconjugate, 57Co-VFC-T84.66, retained > 90% immunoreactivity, was stable in serum (7 days) and demonstrated good localization in LS174T tumor xenografts. Pharmacokinetics of 57Co-VFC-T84.66 in tumor-bearing mice were compared to T84.66 Mab conjugated with either DTPA or its benzylisothiocyanate derivative (BzDTPA) labeled with 111In. Whole-body half-life for VFC-T84.66 was less (t1/2 = 62 hr) than that for either DTPA-T84.66 (t1/2 = 157 hr) or BzDTPA-T84.66 (t1/2 = 167 hr). Blood clearance was similar for all three radioimmunoconjugates (t1/2 = 22 hr). Hepatic uptake of the radiolabel was rapid and remained constant for 7 days for both DTPA radioimmunoconjugates (DTPA radioimmunoconjugate = 13.7 +/- 1.5 %ID/g; BzDTPA radioimmunoconjugate = 10.4 +/- 1.7%ID/g). For VFC, however, liver radioactivity decreased from 19.1 +/- 0.6%ID/g at 1 hr to 0.9 +/- 0.1 %ID/g 7 days postinjection, suggesting a possible role for VFC radioimmunoconjugate in the imaging and therapy of liver metastases. PMID:8326387

  9. Expression of Plasmodium falciparum surface antigens in Escherichia coli.

    PubMed Central

    Ardeshir, F; Flint, J E; Reese, R T

    1985-01-01

    The asexual blood stages of the human malarial parasite Plasmodium falciparum produce many antigens, only some of which are important for protective immunity. Most of the putative protective antigens are believed to be expressed in schizonts and merozoites, the late stages of the asexual cycle. With the aim of cloning and characterizing genes for important parasite antigens, we used late-stage P. falciparum mRNA to construct a library of cDNA sequences inserted in the Escherichia coli expression vector pUC8. Nine thousand clones from the expression library were immunologically screened in situ with serum from Aotus monkeys immune to P. falciparum, and 95 clones expressing parasite antigens were identified. Mice were immunized with lysates from 49 of the bacterial clones that reacted with Aotus sera, and the mouse sera were tested for their reactivity with parasite antigens by indirect immunofluorescence, immunoprecipitation, and immunoblotting assays. Several different P. falciparum antigens were identified by these assays. Indirect immunofluorescence studies of extracellular merozoites showed that three of these antigens appear to be located on the merozoite surface. Thus, we have identified cDNA clones to three different P. falciparum antigens that may be important in protective immunity. Images PMID:3887406

  10. Expression of Treponema pallidum Antigens in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Walfield, Alan M.; Hanff, Philip A.; Lovett, Michael A.

    1982-04-01

    Treponema pallidum DNA was cloned in a bacteriophage. Clones were screened for expression of Treponema pallidum antigens by an in situ radio-immunoassay on nitrocellulose, with the use of subsequent reactions with syphilitic serum and radioiodinated Staphylococcus aureus protein A. One clone, which gave a strong signal, codes for at least seven antigens that react specifically with human antibodies to Treponema pallidum.

  11. A focused immune response targeting the homotypic binding domain of the carcinoembryonic antigen blocks the establishment of tumor foci in vivo.

    PubMed

    Abdul-Wahid, Aws; Huang, Eric H-B; Lu, Huixin; Flanagan, Jean; Mallick, Amirul Islam; Gariépy, Jean

    2012-12-15

    Metastatic forms of cancers remain the main cause of death in cancer patients. In this study, we demonstrate that directing a sustained antibody response towards the homotypic binding function of CEA interferes with the implantation and development of tumor foci in CEA-expressing transgenic (CEA.Tg) mice. Specifically, vaccinating CEA.Tg mice with a recombinant, altered self-form of the CEA Ig V-like N domain led to the production of circulating IgG1 and IgG2a antibodies that inhibited CEA-mediated adhesion of murine carcinoma expressing CEA (MC38.CEA) and mediated antibody-dependent lysis of tumor cells. Moreover, vaccinated CEA.Tg mice were resistant to the development of tumor nodules in the lungs and the peritoneal cavity, suggesting that mounting a focused antibody response to the CEA N domain may represent a simple therapeutic strategy to control the establishment of metastatic foci in cancer patients. PMID:22495743

  12. Diagnostic utility of serum and pleural fluid carcinoembryonic antigen, and cytokeratin 19 fragments in patients with effusion from nonsmall cell lung cancer

    PubMed Central

    Sharma, Sushil Kumar; Bhat, Sanjay; Chandel, Vikas; Sharma, Mayank; Sharma, Pulkit; Gupta, Sakul; Sharma, Sashank; Bhat, Aijaz Ahmed

    2015-01-01

    Aims: To assess the diagnostic value of CEA and CYFRA 21-1 (cytokeratin 19 fragments) in serum and pleural fluid in non small cell lung cancer with malignant pleural effusion (MPE). Settings and Design: Two subsets of patients were recruited with lymphocytic exudative effusion, one subset constituted diagnosed patients of NSCLC with malignant pleural effusion and the other subset of constituted with Tubercular pleural effusion. Materials and Methods: CYFRA 21-1 and CEA levels were measured using Electrochemilumiscence Immunoassay (ECLIA). The test principle used the Sandwich method. For both the tests, results are determined via a calibration curve which is instrument specifically generated by 2 - point calibration and a master curve provided via reagent barcode. Statistical Analysis Used: All data are expressed as means ± SD and percentage. All the parametric variables were analysed by student-t test where as non parametric variables were compared by Mann-Whitney U-test Statistical significance was accepted for P values < 0.05. Software used were SPSS 11.5, and MS excel 2007. In order to compare the performance of the tumor markers, receiver operating characteristic (ROC) curves were constructed and compared with area under the curve (AUC). The threshold for each marker was selected based on the best diagnostic efficacy having achieved equilibrium between sensitivity and specificity. Results: In cases serum CYFRA21-1 levels had mean value of 34.1 ± 29.9 with a range of 1.6-128.3 where as in controls serum CYFRA21-1 levels had mean value of 1.9 ± 1.0 with a range of 0.5–4.7. In cases serum CEA levels had mean value of 24.9 ± 47.3 with a range of 1.0, 267.9 where as in controls serum CEA levels had mean value of 1.9 ± 1.4 with a range of 0.2-6.8. The difference in the means of serum CYFRA 21-l (P = 0.000) and CEA (P = 0.046) were statistically significant. In cases pleural fluid CYFRA21-1 levels had mean value of 160.1 ± 177.1 with a range of 5.4–517

  13. A Novel Malaria Vaccine Candidate Antigen Expressed in Tetrahymena thermophila

    PubMed Central

    Eleni-Muus, Janna; Aldag, Ingo; Samuel, Kay; Creasey, Alison M.; Hartmann, Marcus W. W.; Cavanagh, David R.

    2014-01-01

    Development of effective malaria vaccines is hampered by the problem of producing correctly folded Plasmodium proteins for use as vaccine components. We have investigated the use of a novel ciliate expression system, Tetrahymena thermophila, as a P. falciparum vaccine antigen platform. A synthetic vaccine antigen composed of N-terminal and C-terminal regions of merozoite surface protein-1 (MSP-1) was expressed in Tetrahymena thermophila. The recombinant antigen was secreted into the culture medium and purified by monoclonal antibody (mAb) affinity chromatography. The vaccine was immunogenic in MF1 mice, eliciting high antibody titers against both N- and C-terminal components. Sera from immunized animals reacted strongly with P. falciparum parasites from three antigenically different strains by immunofluorescence assays, confirming that the antibodies produced are able to recognize parasite antigens in their native form. Epitope mapping of serum reactivity with a peptide library derived from all three MSP-1 Block 2 serotypes confirmed that the MSP-1 Block 2 hybrid component of the vaccine had effectively targeted all three serotypes of this polymorphic region of MSP-1. This study has successfully demonstrated the use of Tetrahymena thermophila as a recombinant protein expression platform for the production of malaria vaccine antigens. PMID:24489871

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

    PubMed

    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

  15. Expression of Lewis antigenic determinants in colorectal adenocarcinomas.

    PubMed

    Blasco, E; Torrado, J; Cosme, A; Alvarez, E; Zugasti, A; Gutierrez-Hoyos, A; Arenas, J I

    1989-01-01

    Expression of type 1 and type 2 chain Lewis antigens was studied in 32 rectal adenocarcinoma specimens; the results were correlated with the patients' Lewis phenotype and secretor status. In addition, the pattern of expression of these antigens was analyzed in adjacent and distant normal mucosa. We used an indirect immunofluorescence technique with p-phenylenediamine counterstaining (Oriol technique) and a panel of monoclonal antibodies directed against the different antigenic specificities. Normal distal colonic mucosa only expresses monofucosylated structures (Lea and X) arising from activity of the alpha 1-3,4-fucosyltransferase coded by the Le gene. Rectal adenocarcinomas also show Lea and X, but also reexpress blood group antigens ABH and exhibit difucosylated determinants (Leb and Y). The accumulation of mono- and difucosylated type 2 chain in neoplastic processes, independently of the Le and Se genes, could be due to the enzymes coded by reactivation of the H and X genes. Blood group antigens form a complex signal code, genetically regulated, which intervenes in differentiation, growth and cellular recognition processes, and which may undergo important modifications during malignant transformation. These alterations could be useful in the diagnosis and prognosis of some types of carcinoma. PMID:2476347

  16. Protein Expression Analysis of Melanocyte Differentiation Antigen TRP-2.

    PubMed

    Avogadri, Francesca; Gnjatic, Sacha; Tassello, Jodie; Frosina, Denise; Hanson, Nicole; Laudenbach, Megan; Ritter, Erika; Merghoub, Taha; Busam, Klaus J; Jungbluth, Achim A

    2016-03-01

    Melanocyte differentiation antigens, such as gp100, tyrosinase, and Melan-A and their corresponding antibodies HMB45, T311, and A103, are major diagnostic tools in surgical pathology. Little is known about tyrosinase-related protein 2 (TRP-2, or dopachrome tautomerase/DCT) another melanocyte differentiation antigen, which is an enzymatic component of melanogenesis. We identified a commercial reagent to TRP-2, monoclonal antibody (mAb) C-9 and undertook a comprehensive analysis to assess its specificity and usefulness for surgical pathology. Subsequently, we analyzed panels of normal tissues and tumors. We show that TRP-2 is regularly expressed in melanocytes of the normal skin. In cutaneous nevi, TRP-2 is present in junctional as well as in dermal nevocytes. In malignant tumors, C-9 reactivity is restricted to melanocytic and related lesions and present in 84% and 58% of primary and metastatic melanomas, respectively. Ten primary melanomas of the anorectal mucosa were all positive. Like the other melanocyte differentiation antigens, TRP-2 was absent in 6 desmoplastic melanomas. Also, only 2 of 9 angiomyolipomas were TRP-2 positive. We conclude that mAb C-9 is a valuable reagent for the analysis of TRP-2 expression in archival surgical pathology material. The expression pattern of TRP-2 in melanocytic and related lesions appears to parallel other melanocyte differentiation antigens, although the overall incidence is lower than other antigens, such as Melan-A or gp100. PMID:26894771

  17. 5T4 oncofetal antigen expression in ovarian carcinoma.

    PubMed

    Wrigley, E.; McGown, A.T.; Rennison, J.; Swindell, R.; Crowther, D.; Starzynska, T.; Stern, P.L.

    1995-07-01

    5T4 oncofetal antigen is defined by a monoclonal antibody raised against human placental trophoblast, and recognizes a 72 kD glycoprotein expressed in many different carcinomas but detected only at low levels in some normal epithelia. Analysis of the patterns of expression of 5T4 oncofetal antigen in colorectal carcinomas has indicated a significant association between the presence of the antigen in tumor cells and metastatic spread. The 5T4 antigen expression of 72 epithelial ovarian carcinomas has been investigated by immunohistochemistry; 71% of the carcinomas demonstrated positive 5T4 immunoreactivity in adenocarcinoma cells and/or associated stromal tissue. In order to assess any relationship to prognosis, the 5T4 phenotypes were analyzed with respect to various clinicopathologic features of the tumors and the clinical outcome of the patients assessed by survival and disease-free interval. There was a significant correlation between 5T4 expression and more advanced stage of disease (FIGO stages III and IV) (P < 0.001) and with poorly differentiated tumors (P = 0.036) compared to well or moderately differentiated tumors. Patients with tumors expressing 5T4 were less likely to respond well to adjuvant therapy (P = 0.030) and had a significantly worse outlook in terms of survival (P = 0.033) and disease-free interval (P = 0.033). This significance was not demonstrated as acting independently of FIGO stage and tumor differentiation. PMID:11578488

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

    PubMed Central

    2012-01-01

    Background Fibroadenomas are benign human breast tumors, characterized by proliferation of epithelial and stromal components of the terminal ductal unit. They may grow, regress or remain unchanged, as the hormonal environment of the patient changes. Expression of antigen TF in mucin or mucin-type glycoproteins and of galectin-3 seems to contribute to proliferation and transformations events; their expression has been reported in ductal breast cancer and in aggressive tumors. Findings Lectin histochemistry, immunohistochemistry, and immunofluorescence were used to examine the expression and distribution of antigen TF and galectin-3. We used lectins from Arachis hypogaea, Artocarpus integrifolia, and Amaranthus lecuocarpus to evaluate TF expression and a monoclonal antibody to evaluate galectin-3 expression. We used paraffin-embedded blocks from 10 breast tissues diagnosed with fibroadenoma and as control 10 healthy tissue samples. Histochemical and immunofluorescence analysis showed positive expression of galectin-3 in fibroadenoma tissue, mainly in stroma, weak interaction in ducts was observed; whereas, in healthy tissue samples the staining was also weak in ducts. Lectins from A. leucocarpus and A. integrifolia specificaly recognized ducts in healthy breast samples, whereas the lectin from A. hypogaea recognized ducts and stroma. In fibroadenoma tissue, the lectins from A. integrifolia, A. Hypogaea, and A. leucocarpus recognized mainly ducts. Conclusions Our results suggest that expression of antigen TF and galectin-3 seems to participate in fibroadenoma development. PMID:23265237

  19. Antigenic structures stably expressed by recombinant TGEV-derived vectors.

    PubMed

    Becares, Martina; Sanchez, Carlos M; Sola, Isabel; Enjuanes, Luis; Zuñiga, Sonia

    2014-09-01

    Coronaviruses (CoVs) are positive-stranded RNA viruses with potential as immunization vectors, expressing high levels of heterologous genes and eliciting both secretory and systemic immune responses. Nevertheless, its high recombination rate may result in the loss of the full-length foreign gene, limiting their use as vectors. Transmissible gastroenteritis virus (TGEV) was engineered to express porcine reproductive and respiratory syndrome virus (PRRSV) small protein domains, as a strategy to improve heterologous gene stability. After serial passage in tissue cultures, stable expression of small PRRSV protein antigenic domains was achieved. Therefore, size reduction of the heterologous genes inserted in CoV-derived vectors led to the stable expression of antigenic domains. Immunization of piglets with these TGEV vectors led to partial protection against a challenge with a virulent PRRSV strain, as immunized animals showed reduced clinical signs and lung damage. Further improvement of TGEV-derived vectors will require the engineering of vectors with decreased recombination rate. PMID:25108114

  20. Exosome targeting of tumor antigens expressed by cancer vaccines can improve antigen immunogenicity and therapeutic efficacy.

    PubMed

    Rountree, Ryan B; Mandl, Stefanie J; Nachtwey, James M; Dalpozzo, Katie; Do, Lisa; Lombardo, John R; Schoonmaker, Peter L; Brinkmann, Kay; Dirmeier, Ulrike; Laus, Reiner; Delcayre, Alain

    2011-08-01

    MVA-BN-PRO (BN ImmunoTherapeutics) is a candidate immunotherapy product for the treatment of prostate cancer. It encodes 2 tumor-associated antigens, prostate-specific antigen (PSA), and prostatic acid phosphatase (PAP), and is derived from the highly attenuated modified vaccinia Ankara (MVA) virus stock known as MVA-BN. Past work has shown that the immunogenicity of antigens can be improved by targeting their localization to exosomes, which are small, 50- to 100-nm diameter vesicles secreted by most cell types. Exosome targeting is achieved by fusing the antigen to the C1C2 domain of the lactadherin protein. To test whether exosome targeting would improve the immunogenicity of PSA and PAP, 2 additional versions of MVA-BN-PRO were produced, targeting either PSA (MVA-BN-PSA-C1C2) or PAP (MVA-BN-PAP-C1C2) to exosomes, while leaving the second transgene untargeted. Treatment of mice with MVA-BN-PAP-C1C2 led to a striking increase in the immune response against PAP. Anti-PAP antibody titers developed more rapidly and reached levels that were 10- to 100-fold higher than those for mice treated with MVA-BN-PRO. Furthermore, treatment with MVA-BN-PAP-C1C2 increased the frequency of PAP-specific T cells 5-fold compared with mice treated with MVA-BN-PRO. These improvements translated into a greater frequency of tumor rejection in a PAP-expressing solid tumor model. Likewise, treatment with MVA-BN-PSA-C1C2 increased the antigenicity of PSA compared with treatment with MVA-BN-PRO and resulted in a trend of improved antitumor efficacy in a PSA-expressing tumor model. These experiments confirm that targeting antigen localization to exosomes is a viable approach for improving the therapeutic potential of MVA-BN-PRO in humans. PMID:21670078

  1. Identification of Enterococcus faecalis antigens specifically expressed in vivo

    PubMed Central

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

    2015-01-01

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

  2. HLA antigen expression in enteropathy associated T cell lymphoma.

    PubMed Central

    Ashton-Key, M; Singh, N; Pan, L X; Smith, M E

    1996-01-01

    AIMS: To investigate the occurrence of abnormal patterns of HLA-ABC and HLA-DR expression in enteropathy associated T cell lymphoma and to relate such abnormalities to the Epstein Barr virus (EBV) status of the tumours. METHODS: Eleven enteropathy associated T cell lymphomas were immunostained with HC10 (HLA-ABC heavy chain) and TAL 1B5 (HLA-DR alpha chain) monoclonal antibodies and polyclonal anti-beta 2 microglobulin (beta 2m, the HLA-ABC light chain) antibodies. In situ hybridisation for EBV using EBER probes was performed on all cases. RESULTS: Tumour cells of two of 11 patients were EBER positive. One of these showed partial, and the other, complete loss of beta 2m. HLA-DR expression was undetectable in both patients. Of the remaining nine EBER negative tumours, two were HLA-ABC heavy chain negative or showed only occasional positive cells and five of nine showed partial or complete loss of the HLA-ABC light chain, beta 2m. Seven of the nine cases were either negative for HLA-DR or showed weak expression in a proportion of tumour cells. CONCLUSIONS: These data show that low or absent HLA-ABC and HLA-DR antigen expression occurs commonly in enteropathy associated T cell lymphoma. These abnormal patterns of HLA expression may be associated with escape from immune attack which, in a minority of patients, could be directed against EBV antigens. Images PMID:8813950

  3. Cloning, heterologous expression and antigenicity of a schistosome cercarial protease.

    PubMed

    Price, H P; Doenhoff, M J; Sayers, J R

    1997-05-01

    A gene coding for the 30 kDa Schistosoma mansoni cercarial protease was amplified using the polymerase chain reaction (PCR) from genomic DNA templates. Cloning and sequencing of several independent PCR clones revealed the presence of an intron additional to the one described in the original cloning of the gene. The 3 exons were cloned into expression vectors so that they could be expressed as separate glutathione-S-transferase (GST) translational fusions. Recombinant bacteria carrying these expression plasmids expressed the fusion proteins at high levels. Western blotting of bacterial lysates with sera raised against the native S. mansoni cercarial protease showed that all 3 exons were recognized. Thus we have produced recombinant bacteria capable of providing large amounts of an S. mansoni antigen for immunological studies and evaluation as a candidate vaccine. PMID:9149415

  4. Expression Cloning of Camelid Nanobodies Specific for Xenopus Embryonic Antigens

    PubMed Central

    Itoh, Keiji; Sokol, Sergei Y.

    2014-01-01

    Developmental biology relies heavily on the use of conventional antibodies, but their production and maintenance involves significant effort. Here we use an expression cloning approach to identify variable regions of llama single domain antibodies (known as nanobodies), which recognize specific embryonic antigens. A nanobody cDNA library was prepared from lymphocytes of a llama immunized with Xenopus embryo lysates. Pools of bacterially expressed cDNAs were sib-selected for the ability to produce specific staining patterns in gastrula embryos. Three different nanobodies were isolated: NbP1 and NbP3 stained yolk granules, while the reactivity of NbP7 was predominantly restricted to the cytoplasm and the cortex. The isolated nanobodies recognized specific protein bands in immunoblot analysis. A reverse proteomic approach identified NbP1 target antigen as EP45/Seryp, a serine protease inhibitor. Given the unique stability of nanobodies and the ease of their expression in diverse systems, we propose that nanobody cDNA libraries represent a promising resource for molecular markers for developmental biology. PMID:25285446

  5. Antigenic characterisation of yeast-expressed lyssavirus nucleoproteins.

    PubMed

    Kucinskaite, Indre; Juozapaitis, Mindaugas; Serva, Andrius; Zvirbliene, Aurelija; Johnson, Nicholas; Staniulis, Juozas; Fooks, Anthony R; Müller, Thomas; Sasnauskas, Kestutis; Ulrich, Rainer G

    2007-12-01

    In Europe, three genotypes of the genus Lyssavirus, family Rhabdoviridae, are present, classical rabies virus (RABV, genotype 1), European bat lyssavirus type 1 (EBLV-1, genotype 5) and European bat lyssavirus type 2 (EBLV-2, genotype 6). The entire authentic nucleoprotein (N protein) encoding sequences of RABV (challenge virus standard, CVS, strain), EBLV-1 and EBLV-2 were expressed in yeast Saccharomyces cerevisiae at high level. Purification of recombinant N proteins by caesium chloride gradient centrifugation resulted in yields between 14-17, 25-29 and 18-20 mg/l of induced yeast culture for RABV-CVS, EBLV-1 and EBLV-2, respectively. The purified N proteins were evaluated by negative staining electron microscopy, which revealed the formation of nucleocapsid-like structures. The antigenic conformation of the N proteins was investigated for their reactivity with monoclonal antibodies (mAbs) directed against different lyssaviruses. The reactivity pattern of each mAb was virtually identical between immunofluorescence assay with virus-infected cells, and ELISA and dot blot assay using the corresponding recombinant N proteins. These observations lead us to conclude that yeast-expressed lyssavirus N proteins share antigenic properties with naturally expressed virus protein. These recombinant proteins have the potential for use as components of serological assays for lyssaviruses. PMID:17619134

  6. Plasmodium knowlesi Sporozoite Antigen: Expression by Infectious Recombinant Vaccinia Virus

    NASA Astrophysics Data System (ADS)

    Smith, Geoffrey L.; Godson, G. Nigel; Nussenzweig, Victor; Nussenzweig, Ruth S.; Barnwell, John; Moss, Bernard

    1984-04-01

    The gene coding for the circumsporozoite antigen of the malaria parasite Plasmodium knowlesi was inserted into the vaccinia virus genome under the control of a defined vaccinia virus promoter. Cells infected with the recombinant virus synthesized polypeptides of 53,000 to 56,000 daltons that reacted with monoclonal antibody against the repeating epitope of the malaria protein. Furthermore, rabbits vaccinated with the recombinant virus produced antibodies that bound specifically to sporozoites. These data provide evidence for expression of a cloned malaria gene in mammalian cells and illustrate the potential of vaccinia virus recombinants as live malaria vaccines.

  7. Antigen

    MedlinePlus

    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 ...

  8. Plasmodium falciparum Variant Surface Antigen Expression Patterns during Malaria

    PubMed Central

    2005-01-01

    The variant surface antigens expressed on Plasmodium falciparum–infected erythrocytes are potentially important targets of immunity to malaria and are encoded, at least in part, by a family of var genes, about 60 of which are present within every parasite genome. Here we use semi-conserved regions within short var gene sequence “tags” to make direct comparisons of var gene expression in 12 clinical parasite isolates from Kenyan children. A total of 1,746 var clones were sequenced from genomic and cDNA and assigned to one of six sequence groups using specific sequence features. The results show the following. (1) The relative numbers of genomic clones falling in each of the sequence groups was similar between parasite isolates and corresponded well with the numbers of genes found in the genome of a single, fully sequenced parasite isolate. In contrast, the relative numbers of cDNA clones falling in each group varied considerably between isolates. (2) Expression of sequences belonging to a relatively conserved group was negatively associated with the repertoire of variant surface antigen antibodies carried by the infected child at the time of disease, whereas expression of sequences belonging to another group was associated with the parasite “rosetting” phenotype, a well established virulence determinant. Our results suggest that information on the state of the host–parasite relationship in vivo can be provided by measurements of the differential expression of different var groups, and need only be defined by short stretches of sequence data. PMID:16304608

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

    PubMed

    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

  10. Expression of major histocompatibility antigens in human chronic pancreatitis.

    PubMed

    Jalleh, R P; Gilbertson, J A; Williamson, R C; Slater, S D; Foster, C S

    1993-10-01

    T-lymphocytic infiltration of the exocrine pancreas and liver in patients with chronic pancreatitis has suggested that cell mediated immune mechanisms may play a part in the pathogenesis of this disease. As expression of major histocompatibility (MHC) antigens is a prerequisite for organ specific autoimmunity, the expression of HLA class I (beta 2-microglobulin) and class II (HLA-DR) determinants have been analysed, together with the presence of T-lymphocytes, in 93 patients (64 men and 29 women, mean age 40.6 years) having an operation for chronic pancreatitis. Ethanol (63 patients), recurrent acute pancreatitis (12), congenital lesions (2), and unknown (16) were suggested to be the causes of the disease. Immunohistochemical staining of formalin fixed and paraffin wax embedded tissue sections used conventional immunohistochemical techniques with specific anti-serum samples. No MHC expression was identified in 10 histologically normal pancreatic control specimens or in four cases of chronic pancreatitis secondary to obstruction by neuroendocrine tumours within the head of the pancreas. beta 2-microglobulin expression by pancreatic exocrine epithelial cells was seen in 76 chronic pancreatitis specimens (82%) while HLA-DR was present in 61 (66%). Simultaneous expression of both class I and II determinants was seen in 53 (57%) of cases. MHC determinant expression was not found in 10 cases (11%) of chronic pancreatitis. In the positive specimens, expression was confined to ductal and ductular (interlobular and intralobular) epithelium with no staining of acinar cells. Staining was not related to the suspected cause of the disease or age. T-lymphocytes were more prominent in chronic pancreatitis mean (SEM) (131 (15) cells per high powered field) than controls (5 (1), p < 0.01). Aberrant MHC expression by exocrine pancreatic epithelial cells occurring in the presence of an appreciable T-cell infiltration confirmed that the appropriate cellular conditions were present for

  11. The relationship between MHC antigen expression and metastasis.

    PubMed

    Gopas, J; Rager-Zisman, B; Bar-Eli, M; Hämmerling, G J; Segal, S

    1989-01-01

    much less understood) are involved in the selection of the metastatic cell population. 5. Immunogenicity of tumors is not necessarily determined by high levels of MHC antigen expression; it is also dependent on the level of TSA. Thus, immunoselection mediated by T lymphocytes during metastasis formation could be directed against both MHC and TSA antigens. Therefore, low expression of MHC antigens by metastatic cells as a result of immunoselection is not always observed.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2678949

  12. Expression patterns of the T antigen and the cryptic T antigen in rat fetuses: detection with the lectin amaranthin.

    PubMed

    Sata, T; Zuber, C; Rinderle, S J; Goldstein, I J; Roth, J

    1990-06-01

    The lectin amaranthin, purified from the seeds of Amaranthus caudatus, has been shown to react specifically with the Gal beta 1,3GalNAc-alpha and the NeuAc alpha 2,3Gal beta 1,3GalNAc-alpha sequence which represent the T antigen and the cryptic T antigen, respectively. We report here the development of labeling techniques that apply amaranthin to stain paraffin sections from rat fetuses. Amaranthin staining was inhibited by pre-incubation of lectin-gold complexes with 10 mM Gal beta 1,3GalNAc-alpha-O-benzyl (synthetic T antigen) or 10 mM Gal beta 1,3GalNAc-alpha-O-aminophenylethyl-human serum albumin (T antigen neoglycoprotein), asialoglycophorin, asialofetuin, and asialomucin. The beta-elimination reaction also abolished the lectin staining demonstrating specificity for O-glycosidically linked structures. A comparison with monoclonal anti-T antigen antibody immunostaining demonstrated that amaranthin detects the T antigen and its cryptic form in tissue sections. Application of the galactose oxidase-Schiff sequence abolished amaranthin (and anti-T antibody) binding to the T antigen but not to its cryptic form, and therefore permitted their differentiation in tissue sections. Histochemical evidence was obtained indicating that amaranthin is a more specific anti-T reagent than peanut lectin. Data are presented that show the differential expression of the T antigen and the cryptic T antigen in organs and cells of rat fetuses late in gestation. Therefore, amaranthin can be used for histochemical detection of the T antigen and the cryptic T antigen, and facilitates discrimination between them. PMID:2335739

  13. Preferentially Expressed Antigen of Melanoma Prevents Lung Cancer Metastasis

    PubMed Central

    Sun, Zhengwang; Li, Lei; Lin, Zaijun; Xu, Wei; Han, Shuai; Cao, Wenjiao; Xu, Yunfei; Song, Dianwen; Yang, Xinghai; Xiao, Jianru

    2016-01-01

    Lung cancer is the most common cause of cancer death worldwide. The poor survival rate is largely due to the extensive local invasion and metastasis. However, the mechanisms underlying the invasion and metastasis of lung cancer cells remain largely elusive. In this study, we examined the role of preferentially expressed antigen of melanoma (PRAME) in lung cancer metastasis. Our results show that PRAME is downregulated in lung adenocarcinoma and lung bone metastasis compared with normal human lung. Knockdown of PRAME decreases the expression of E-Cadherin and promotes the proliferation, invasion, and metastasis of lung cancer cells by regulating multiple critical genes, most of which are related to cell migration, including MMP1, CCL2, CTGF, and PLAU. Clinical data analysis reveals that the expression of MMP1 correlates with the clinical features and outcome of lung adenocarcinoma. Taken together, our data demonstrate that PRAME plays a role in preventing the invasion and metastasis of lung adenocarcinoma and novel diagnostic or therapeutic strategies can be developed by targeting PRAME. PMID:27391090

  14. Testis expressed 19 is a novel cancer-testis antigen expressed in bladder cancer.

    PubMed

    Zhong, Jianhua; Chen, Yan; Liao, Xinhui; Li, Jiaqiang; Wang, Han; Wu, Chenglong; Zou, Xiaowen; Yang, Gang; Shi, Jing; Luo, Liya; Liu, Litao; Deng, Jianping; Tang, Aifa

    2016-06-01

    Bladder cancer exhibits high mortality as a result of limited therapeutic options and a high recurrence rate. Accordingly, novel treatments such as immunotherapy have emerged as promising therapeutic modalities to prolong overall patient survival and effect a disease cure, which has renewed enthusiasm for the identification of tumor-specific target antigens. Cancer-testis (CT) antigens are recognized as ideal targets for immunotherapy because of their expression features and high immunogenicity profiles. Here, we investigate the expression pattern of a novel CT antigen, testis-expressed 19 (TEX19), in patients with bladder carcinoma and among multiple human tissues. Six bladder cancer cell lines (T24, UM-UC-3, J82, 5637, SW780, and RT4) were also analyzed for TEX19 expression. Our results reveal that TEX19 expression in normal tissue is restricted to human testis. In addition, TEX19 mRNA expression was detected in 60 % (24/40) bladder cancer samples, whereas 58.20 % (110/189) were positive for TEXT19 protein expression. Compared to low-grade tumors, TEX19 exhibited increased expression in high-grade tumors, from 53.69 to 77.14 %, respectively (P = 0.011). TEX19 was also expressed in all six bladder cancer cell lines. Together, our findings suggest that TEX19 represents a novel CT gene and might play a role in the progression of bladder cancer and that this gene therefore provides a potential target for immunotherapy treatment strategies against bladder cancer. PMID:26695143

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

    PubMed

    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

  16. Protective Antigens Against Glanders Identified by Expression Library Immunization

    PubMed Central

    Whitlock, Gregory C.; Robida, Mark D.; Judy, Barbara M.; Qazi, Omar; Brown, Katherine A.; Deeraksa, Arpaporn; Taylor, Katherine; Massey, Shane; Loskutov, Andrey; Borovkov, Alex Y.; Brown, Kevin; Cano, Jose A.; Magee, D. Mitchell; Torres, Alfredo G.; Estes, D. Mark; Sykes, Kathryn F.

    2011-01-01

    Burkholderia are highly evolved Gram-negative bacteria that primarily infect solipeds but are transmitted to humans by ingestion and cutaneous or aerosol exposures. Heightened concern over human infections of Burkholderia mallei and the very closely related species B. pseudomallei is due to the pathogens’ proven effectiveness as bioweapons, and to the increased potential for natural opportunistic infections in the growing diabetic and immuno-compromised populations. These Burkholderia species are nearly impervious to antibiotic treatments and no vaccine exists. In this study, the genome of the highly virulent B. mallei ATCC23344 strain was examined by expression library immunization for gene-encoded protective antigens. This protocol for genomic-scale functional screening was customized to accommodate the unusually large complexity of Burkholderia, and yielded 12 new putative vaccine candidates. Five of the candidates were individually tested as protein immunogens and three were found to confer significant partial protection against a lethal pulmonary infection in a murine model of disease. Determinations of peripheral blood cytokine and chemokine profiles following individual protein immunizations show that interleukin-2 (IL-2) and IL-4 are elicited by the three confirmed candidates, but unexpectedly interferon-γ and tumor necrosis factor-α are not. We suggest that these pathogen components, discovered using genetic immunization and confirmed in a conventional protein format, will be useful toward the development of a safe and effective glanders vaccine. PMID:22125550

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

    SciTech Connect

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

    2009-03-30

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

  18. HLA Class II Antigen Expression in Colorectal Carcinoma Tumors as a Favorable Prognostic Marker12

    PubMed Central

    Sconocchia, Giuseppe; Eppenberger-Castori, Serenella; Zlobec, Inti; Karamitopoulou, Eva; Arriga, Roberto; Coppola, Andrea; Caratelli, Sara; Spagnoli, Giulio Cesare; Lauro, Davide; Lugli, Alessandro; Han, Junyi; Iezzi, Giandomenica; Ferrone, Cristina; Ferlosio, Amedeo; Tornillo, Luigi; Droeser, Raoul; Rossi, Piero; Attanasio, Antonio; Ferrone, Soldano; Terracciano, Luigi

    2014-01-01

    The goal of this study was to determine the frequency of HLA class II antigen expression in colorectal carcinoma (CRC) tumors, its association with the clinical course of the disease, and the underlying mechanism(s). Two tissue microarrays constructed with 220 and 778 CRC tumors were stained with HLA-DR, DQ, and DP antigen-specific monoclonal antibody LGII-612.14, using the immunoperoxidase staining technique. The immunohistochemical staining results were correlated with the clinical course of the disease. The functional role of HLA class II antigens expressed on CRC cells was analyzed by investigating their in vitro interactions with immune cells. HLA class II antigens were expressed in about 25% of the 220 and 21% of the 778 tumors analyzed with an overall frequency of 23%. HLA class II antigens were detected in 19% of colorectal adenomas. Importantly, the percentage of stained cells and the staining intensity were significantly lower than those detected in CRC tumors. However, HLA class II antigen staining was weakly detected only in 5.4% of 37 normal mucosa tissues. HLA class II antigen expression was associated with a favorable clinical course of the disease. In vitro stimulation with interferon gamma (IFNγ) induced HLA class II antigen expression on two of the four CRC cell lines tested. HLA class II antigen expression on CRC cells triggered interleukin-1β (IL-1β) production by resting monocytes. HLA class II antigen expression in CRC tumors is a favorable prognostic marker. This association may reflect stimulation of IL-1β production by monocytes. PMID:24563618

  19. Expression and immunogenicity of novel subunit enterovirus 71 VP1 antigens.

    PubMed

    Xu, Juan; Wang, Shixia; Gan, Weihua; Zhang, Wenhong; Ju, Liwen; Huang, Zuhu; Lu, Shan

    2012-04-20

    Hand, foot, and mouth disease (HFMD) is a common viral illness in young children. HFMD is caused by viruses belonging to the enterovirus genus of the picornavirus family. Recently, enterovirus 71 (EV71) has emerged as a virulent agent for HFMD with severe clinical outcomes. In the current report, we conducted a pilot antigen engineering study to optimize the expression and immunogenicity of subunit VP1 antigen for the design of EV71 vaccines. DNA immunization was adopted as a simple technical approach to test different designs of VP1 antigens without the need to express VP1 protein in vitro first. Our studies indicated that the expression and immunogenicity of VP1 protein can be improved with alternated VP1 antigen designs. Data presented in the current report revealed novel pathways to optimize the design of VP1 antigen-based EV71 vaccines. PMID:22450314

  20. Production of recombinant botulism antigens: a review of expression systems.

    PubMed

    Moreira, G M S G; Cunha, C E P; Salvarani, F M; Gonçalves, L A; Pires, P S; Conceição, F R; Lobato, F C F

    2014-08-01

    Botulism is a paralytic disease caused by intoxication with neurotoxins produced by Clostridium botulinum. Despite their similar mechanism of action, the botulinum neurotoxins (BoNT) are classified in eight serotypes (A to H). As to veterinary medicine, the impact of this disease is essentially economic, since different species of production animals can be affected, especially by BoNT/C and D. In human health, botulism is feared in a possible biological warfare, what would involve mainly the BoNT/A, B, E and F. In both cases, the most effective way to deal with botulism is through prevention, which involves vaccination. However, the current vaccines against this disease have several drawbacks on their process of production and, besides this, can be dangerous to producers since it requires certain level of biosafety. This way, recombinant vaccines have been shown to be a great alternative for the development of vaccines against both animal and human botulism. All BoNTs have a 50-kDa light chain (LC) and a 100-kDa heavy chain (HC). The latter one presents two domains of 50 kDa, called the N-terminal (HN) and C-terminal (HC) halves. Among these regions, the HC alone seem to confer the proper immune response against intoxication. Since innumerous studies describe the expression of these distinct regions using different systems, strategies, and protocols, it is difficult to define the best option for a viable vaccine production. Thereby, the present review describes the problematic of botulism and discusses the main advances for the viable production of vaccines for both human and veterinary medicine using recombinant antigens. PMID:24930432

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

    SciTech Connect

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

    1987-05-01

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

  2. Virulence of Shigella flexneri Hybrids Expressing Escherichia coli Somatic Antigens

    PubMed Central

    Gemski, P.; Sheahan, D. G.; Washington, O.; Formal, S. B.

    1972-01-01

    The genes controlling either Escherichia coli somatic antigen 8 or 25 were conjugally transferred to virulent Shigella flexneri 2a recipients to determine whether the aquisition of these antigens would affect the virulence of the resulting hybrid. A high proportion of such hybrids were found to be rough and hence were avirulent. Some smooth S. flexneri hybrids which replaced their native group antigens with E. coli factor 25 were still virulent in the animal models employed. All S. flexneri O-8 hybrids were uniformly avirulent. Our finding, that S. flexneri hybrids with the chemically divergent E. coli O-8 repeat unit are avirulent whereas some hybrids with the chemically related O-25 repeat unit retain virulence, suggests that the chemical composition and structure of the O side chain of somatic antigens may represent one determining factor for bacterial penetration of mucosal epithelial cells, the primary step in the pathogenesis of bacillary dysentery. Images PMID:4569915

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

    SciTech Connect

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

    1984-08-01

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

  4. IL-12 release by engineered T cells expressing chimeric antigen receptors can effectively Muster an antigen-independent macrophage response on tumor cells that have shut down tumor antigen expression.

    PubMed

    Chmielewski, Markus; Kopecky, Caroline; Hombach, Andreas A; Abken, Hinrich

    2011-09-01

    During malignant progression cancer cells tend to lose cell surface expression of MHC and other immune antigens, making them invisible to cytotoxic T cells and therefore inaccessible to tumor antigen-directed immunotherapy. Moreover, cancer cell variants that have lost antigen expression frequently contribute to deadly tumor relapses that occur following treatments that had been initially effective. In an effort to destroy antigen-loss cancer cells in tumors, we created a strategy that combines a chimeric antigen receptor (CAR)-redirected T-cell attack with an engineered local release of the cytokine interleukin 12 (IL-12), which recruits and reinforces macrophage function. Cytotoxic T cells were engineered to release inducible IL-12 upon CAR engagement in the tumor lesion, resulting in destruction of antigen-loss cancer cells that would normally escape. Importantly, elimination of the antigen-loss cancer cells was accompanied by an accumulation of activated macrophages that was critical to the antitumor response, because removing the macrophages abolished the response and restoring them reengaged it. Neutralizing TNF-α also abrogated the elimination of antigen-loss cancer cells, implying this proinflammatory factor in the process. Taken together, our results show how IL-12 supplementation by CAR T cells can target otherwise inaccessible tumor lesions, in a manner associated with reduced systemic toxicity, by recruiting and activating innate immune cells for a proinflammatory response. PMID:21742772

  5. Expression of Human Herpesvirus-6 Antigens in Benign and Malignant Lymphoproliferative Diseases

    PubMed Central

    Luppi, Mario; Barozzi, Patrizia; Garber, Richard; Maiorana, Antonio; Bonacorsi, Goretta; Artusi, Tullio; Trovato, Raffaella; Marasca, Roberto; Torelli, Giuseppe

    1998-01-01

    Immunohistochemistry was used to look for the expression of human herpesvirus-6 (HHV-6) antigens in a well characterized series of benign, atypical, and malignant lymphoid lesions, which tested positive for the presence of HHV-6 DNA. A panel of specific antibodies against HHV-6 antigens, characteristic either of the early (p41) or late (p101K, gp106, and gp116) phases of the viral cycle, was applied to the lymphoid tissues from 15 non-Hodgkin’s lymphomas, 14 Hodgkin’s disease cases, 5 angioimmunoblastic lymphadenopathies with dysproteinemia, 14 reactive lymphadenopathies, and 2 cases of sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease). In lymphomatous tissues, the expression of late antigens was documented only in reactive cells, and mainly in plasma cells. Of interest, the expression of the early p41 antigen was detected in the so-called “mummified” Reed-Sternberg cells, in two Hodgkin’s disease cases. In reactive lymphadenopathies, the HHV-6 late antigen-expressing cells were plasma cells, histiocytes, and rare granulocytes distributed in interfollicular areas. In both cases of Rosai-Dorfman disease, the p101K showed an intense staining in follicular dendritic cells of germinal centers, whereas the gp106 exhibited an intense cytoplasmic reaction in the abnormal histiocytes, which represent the histological hallmark of the disease. The expression of HHV-6 antigens is tightly controlled in lymphoid tissues. The lack of HHV-6 antigen expression in neoplastic cells and the limited expression in degenerating Reed-Sternberg cells argue against a major pathogenetic role of the virus in human lymphomagenesis. The detection of a rather unique pattern of viral late antigen expression in Rosai-Dorfman disease suggests a possible pathogenetic involvement of HHV-6 in some cases of this rare lymphoproliferative disorder. PMID:9736030

  6. Effect of Lewis blood group antigen expression on bacterial adherence to COS-1 cells.

    PubMed Central

    Gaffney, R A; Schaeffer, A J; Anderson, B E; Duncan, J L

    1994-01-01

    Epithelial cells from secretor individuals demonstrate decreased bacterial adherence compared with cells from nonsecretors. Lewis blood group antigen expression is one component of the secretor/nonsecretor phenotype and several epidemiologic studies have suggested a link between Lewis blood group antigen phenotype and susceptibility to urinary tract infections. In this study, we examined the possibility that the expression of the difucosylated Lewis blood group determinants, Leb and Ley (associated with the secretor phenotype), made cells less susceptible to Escherichia coli adherence by masking receptors for pili. COS-1 cells, which do not produce Lewis (Lea, Leb, Le(x), and Ley) blood group antigens, were used as target cells for bacterial adherence. The surface blood group antigen expression pattern of the cells was then modified by cotransfection with plasmids containing DNA inserts encoding alpha (1,2)-fucosyltransferase and alpha (1,3)- and alpha (1,4)-fucosyltransferases, resulting in the expression of Leb and Ley. E. coli HB101 expressing various adhesins (type 1, PapJ96, PapIA2, PapAD110, Prs, and S) from recombinant plasmids bound equally well to untransfected cells and transfected cells expressing Lea and Le(x) (nonsecretor phenotype) and Leb and Ley (secretor phenotype) antigens. We conclude that the presence of Leb and Ley antigens on cells from secretors does not alone mask receptors for E. coli pili or hinder bacterial adherence. PMID:8005692

  7. Novel methods for expression of foreign antigens in live vector vaccines

    PubMed Central

    Wang, Jin Yuan; Harley, Regina H.; Galen, James E.

    2013-01-01

    Bacterial live vector vaccines represent a vaccine development strategy that offers exceptional flexibility. In this approach, genes encoding protective antigens of unrelated bacterial, viral or parasitic pathogens are expressed in an attenuated bacterial vaccine strain that delivers these foreign antigens to the immune system, thereby eliciting relevant immune responses. Rather than expressing these antigens using low copy expression plasmids, here we pursue expression of foreign proteins from the live vector chromosome. Our strategy is designed to compensate for the inherent disadvantage of loss of gene dosage (vs. plasmid-based expression) by integrating antigen-encoding gene cassettes into multiple chromosomal sites already inactivated in an attenuated Salmonella enterica serovar Typhi vaccine candidate. We tested expression of a cassette encoding the green fluorescent protein (GFPuv) integrated separately into native guaBA, htrA or clyA chromosomal loci. Using single integrations, we show that expression levels of GFPuv are significantly affected by the site of integration, regardless of the inclusion of additional strong promoters within the incoming cassette. Using cassettes integrated into both guaBA and htrA, we observe cumulative synthesis levels from two integration sites superior to single integrations. Most importantly, we observe that GFPuv expression increases in a growth phase-dependent manner, suggesting that foreign antigen synthesis may be “tuned” to the physiology of the live vaccine. We expect this novel platform expression technology to prove invaluable in the development of a wide variety of multivalent live vector vaccines, capable of expressing multiple antigens from both chromosomal and plasmid-based expression systems within a single strain. PMID:23406777

  8. Expression and immunogenicity of novel subunit enterovirus 71 VP1 antigens

    SciTech Connect

    Xu, Juan; Wang, Shixia; Gan, Weihua; Zhang, Wenhong; Ju, Liwen; Huang, Zuhu; Lu, Shan

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer EV71 is a major emerging infectious disease in many Asian countries. Black-Right-Pointing-Pointer Inactivated EV71 vaccines are in clinical studies but their safety and efficacy are unknown. Black-Right-Pointing-Pointer Developing subunit based EV71 vaccines is significant and novel antigen design is needed. Black-Right-Pointing-Pointer DNA immunization is an efficient tool to test the immunogenicity of VP1 based EV71 vaccines. Black-Right-Pointing-Pointer Multiple VP1 antigens are developed showing immunogenic potential. -- Abstract: Hand, foot, and mouth disease (HFMD) is a common viral illness in young children. HFMD is caused by viruses belonging to the enterovirus genus of the picornavirus family. Recently, enterovirus 71 (EV71) has emerged as a virulent agent for HFMD with severe clinical outcomes. In the current report, we conducted a pilot antigen engineering study to optimize the expression and immunogenicity of subunit VP1 antigen for the design of EV71 vaccines. DNA immunization was adopted as a simple technical approach to test different designs of VP1 antigens without the need to express VP1 protein in vitro first. Our studies indicated that the expression and immunogenicity of VP1 protein can be improved with alternated VP1 antigen designs. Data presented in the current report revealed novel pathways to optimize the design of VP1 antigen-based EV71 vaccines.

  9. Cloning, expression, and antigenic characterization of recombinant protein of Mycoplasma gallisepticum expressed in Escherichia coli.

    PubMed

    Rocha, T S; Tramuta, C; Catania, S; Matucci, A; Giuffrida, M G; Baro, C; Profiti, M; Bertolotti, L; Rosati, S

    2015-04-01

    Mycoplasma gallisepticum (MG) is a member of the most important avian mycoplasmas, causing chronic respiratory disease in chickens and leading to important economic losses in the poultry industry. Recombinant technology represents a strategic approach used to achieve highly reliable and specific diagnostic tests in veterinary diseases control: in particular this aspect is crucial for confirming mycoplasma infection and for maintaining mycoplasma-free breeder flocks. In this study, we identified a component of the pyruvate dehydrogenase dihydrolipoamide acetyltransferase (i.e., E2) protein by 2-dimensional electrophoresis (2-DE), characterized it in immunoblotting assays, and analyzed its recombinant (r-E2) in a rec-ELISA test. For full-length protein expression in Escherichia coli (EC) a point mutation was introduced. A rabbit antiserum produced against r-E2 was tested in a Western Blot using different samples of Mycoplasma species. The results showed the applicability of site-directed mutagenesis, with a good yield of the r-E2 after purification. Also, anti-E2 serum reacted with all the tested MG strains showing no cross reaction with other mycoplasmas. The developed E2 ELISA test was capable of detecting MG antibodies in the sera examined. Those results demonstrate the antigenic stability of the E2 protein which could represent a recombinant antigen with potential diagnostic applications. PMID:25667423

  10. Expression of PCV2 antigen in the ovarian tissues of gilts

    PubMed Central

    TUMMARUK, Padet; PEARODWONG, Pachara

    2015-01-01

    The present study was performed to determine the expression of porcine circovirus type 2 (PCV2) antigen in the ovarian tissue of naturally infected gilts. Ovarian tissues were obtained from 11 culled gilts. The ovarian tissues sections were divided into two groups according to PCV2 DNA detection using PCR. PCV2 antigen was assessed in the paraffin embedded ovarian tissue sections by immunohistochemistry. A total of 2,131 ovarian follicles (i.e., 1,437 primordial, 133 primary, 353 secondary and 208 antral follicles), 66 atretic follicles and 131 corpora lutea were evaluated. It was found that PCV2 antigen was detected in 280 ovarian follicles (i.e., 239 primordial follicles, 12 primary follicles, 10 secondary follicles and 19 antral follicles), 1 atretic follicles and 3 corpora lutea (P<0.05). PCV2 antigen was detected in primordial follicles more often than in secondary follicles, atretic follicles and corpora lutea (P<0.05). The detection of PCV2 antigen was found mainly in oocytes. PCV2 antigen was found in both PCV2 DNA positive and negative ovarian tissues. It can be concluded that PCV2 antigen is expressed in all types of the ovarian follicles and corpora lutea. Further studies should be carried out to determine the influence of PCV2 on porcine ovarian function and oocyte quality. PMID:26522687

  11. Expression of PCV2 antigen in the ovarian tissues of gilts.

    PubMed

    Tummaruk, Padet; Pearodwong, Pachara

    2016-03-01

    The present study was performed to determine the expression of porcine circovirus type 2 (PCV2) antigen in the ovarian tissue of naturally infected gilts. Ovarian tissues were obtained from 11 culled gilts. The ovarian tissues sections were divided into two groups according to PCV2 DNA detection using PCR. PCV2 antigen was assessed in the paraffin embedded ovarian tissue sections by immunohistochemistry. A total of 2,131 ovarian follicles (i.e., 1,437 primordial, 133 primary, 353 secondary and 208 antral follicles), 66 atretic follicles and 131 corpora lutea were evaluated. It was found that PCV2 antigen was detected in 280 ovarian follicles (i.e., 239 primordial follicles, 12 primary follicles, 10 secondary follicles and 19 antral follicles), 1 atretic follicles and 3 corpora lutea (P<0.05). PCV2 antigen was detected in primordial follicles more often than in secondary follicles, atretic follicles and corpora lutea (P<0.05). The detection of PCV2 antigen was found mainly in oocytes. PCV2 antigen was found in both PCV2 DNA positive and negative ovarian tissues. It can be concluded that PCV2 antigen is expressed in all types of the ovarian follicles and corpora lutea. Further studies should be carried out to determine the influence of PCV2 on porcine ovarian function and oocyte quality. PMID:26522687

  12. The effects of Ostertagia occidentalis somatic antigens on ovine TLR2 and TLR4 expression

    PubMed Central

    BORJI, Hassan; HAGHPARAST, Alireza; SOLEIMANI, Nooshinmehr; AZIZZADEH, Mohammad; NAZEMSHIRAZI, Mohammad Hossein

    2015-01-01

    Background: Recognition of helminth-derived pathogen associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), including toll like receptors (TLRs) is the first step towards initiating anti–helminth immune responses. Methods : Using somatic antigens of Ostertagia occidentalis, an important abomasal parasite of ruminants, the expression of ovine TLR2 and TLR4 in peripheral blood mononuclear cells (PBMCs) was analyzed by real-time quatitative reverse-transcription polymerase chain reaction (qRT-PCR). Somatic antigens of O. occidentalis were prepared to stimulate ovine PBMCs in a time and dose dependent manner. Results : A high expression of TLR2 and TLR4 was observed in PBMCs cultured with somatic antigens of the parasites specially when PBMCs were cultured with 100 µg/ml of somatic antigens and incubated for 2h. Up-regulation of TLR2 expression was more pronounced and evident in our study. Conclsusion : Somatic antigens of O. occidentalis have immunostimulatory and dominant role on peripheral immune cells. This study provide for the first time evidence of induction of TLRs in ovine PBMCs by somatic antigen of O. occidentalis PMID:26622306

  13. Antigenic properties and diagnostic potential of puumala virus nucleocapsid protein expressed in insect cells.

    PubMed Central

    Vapalahti, O; Lundkvist, A; Kallio-Kokko, H; Paukku, K; Julkunen, I; Lankinen, H; Vaheri, A

    1996-01-01

    Puumala virus (PUU) is a member of the genus Hantavirus in the family Bunyaviridae and the causative agent of nephropathia epidemica, a European form of hemorrhagic fever with renal syndrome. Sera of nephropathia epidemica patients react specifically with PUU nucleocapsid (N) protein. In order to safely provide large quantities of antigen for diagnostic purposes, PUU Sotkamo strain N protein was expressed by using the baculovirus system in Sf9 insect cells to up to 30 to 50% of the total cellular protein. The recombinant N protein (bac-PUU-N) was solubilized with 6 M urea, dialyzed, and purified by anion-exchange liquid chromatography. In an immunoglobulin M mu-capture assay purified and unpurified bac-PUU-N antigen showed identical results compared with the results of a similar assay based on native PUU antigen grown in Vero E6 cells. An immunoglobulin G monoclonal antibody-capture assay based on unpurified bac-PUU-N also showed results identical to those of an assay with native PUU-N antigen. Moreover, a panel of monoclonal antibodies reactive with eight different epitopes showed identical reactivity patterns with both natural and bac-PUU-N antigen, while two epitopes in PUU-N expressed as a fusion protein in Escherichia coli were not recognized. Puumala hantavirus N protein expressed by the baculovirus system offers a safe and inexpensive source of specific antigen for large-scale diagnostic and seroepidemiological purposes. PMID:8748286

  14. EXPRESSION SYSTEM-DEPENDENT MODULATION OF HIV-1 ENVELOPE GLYCOPROTEIN ANTIGENICITY AND IMMUNOGENICITY

    PubMed Central

    Kong, Leopold; Sheppard, Neil C.; Stewart-Jones, Guillaume B.E.; Robson, Cynthia L.; Chen, Hongying; Xu, Xiaodong; Krashias, George; Bonomelli, Camille; Scanlan, Christopher N.; Kwong, Peter D.; Jeffs, Simon A.; Jones, Ian M.; Sattentau, Quentin J.

    2010-01-01

    Recombinant expression systems differ in the type of glycosylation they impart on expressed antigens such as the Human Immunodeficiency Virus Type-1 (HIV-1) envelope glycoproteins, potentially affecting their biological properties. We performed head-to-head antigenic, immunogenic and molecular profiling of two distantly-related Env surface (gp120) antigens produced in different systems: a) mammalian (293F) cells in the presence of kifunensine which impart only high mannose glycans; b) insect (Spodoptera frugiperda, Sf9) cells, which confer mainly paucimannosidic glycans; c) Sf9 cells recombinant for mammalian glycosylation enzymes (Sf9 Mimic™), which impart high mannose, hybrid and complex glycans without sialic acid; d) 293F cells, which impart high mannose, hybrid and complex glycans with sialic acid. Molecular models revealed a significant difference in gp120 glycan coverage between the Sf9- and wild-type mammalian cell-derived material that is predicted to impact upon ligand binding sites proximal to glycans. Modelling of solvent-exposed surface electrostatic potentials showed that sialic acid imparts a significant negative surface charge that may influence gp120 antigenicity and immunogenicity. Gp120 expressed in systems that do not incorporate sialic acid displayed increased ligand binding to the CD4-binding and CD4–induced sites compared to those expressed in the system that does, and imparted other more subtle differences in antigenicity in a gp120 subtype-specific manner. Non-sialic acid-containing gp120 was significantly more immunogenic than the sialyated version when administered in two different adjuvants, and induced higher titres of antibodies competing for CD4 binding site ligand-gp120 interaction. These findings suggest that non-sialic acid imparting systems yield gp120 immunogens with modified antigenic and immunogenic properties, considerations which should be considered when selecting expression systems for glycosylated antigens to be used

  15. Cloning, Characterization, and Expression of a 200-Kilodalton Diagnostic Antigen of Babesia bigemina†

    PubMed Central

    Tebele, N.; Skilton, R. A.; Katende, J.; Wells, C. W.; Nene, V.; McElwain, T.; Morzaria, S. P.; Musoke, A. J.

    2000-01-01

    Current serological tests for Babesia bigemina use semipurified merozoite antigens derived from infected erythrocytes. One of the major drawbacks of these tests is that antigen quality can vary from batch to batch. Since the quality of the antigen contributes to the sensitivity and specificity of serological tests, the use of standardized recombinant antigens should ensure consistency in assay quality. Previously, a 200-kDa merozoite antigen (p200) was identified as a candidate diagnostic antigen for use in a serological assay for the detection of B. bigemina antibodies in infected cattle. In this study, we have cloned, characterized, and expressed p200. A 3.5-kbp cDNA clone encoding p200 was isolated and shown to be almost full length, lacking approximately 300 bp at the 5′ end. The predicted amino acid sequence shows that p200 consists of a long, highly charged central repeat region of an uninterrupted α helix, indicative of a fibrous protein. Immunoelectron microscopy localized p200 to the merozoite cytoplasm, suggesting that the antigen may be a structural protein involved in forming filament structures within the cytoskeleton. The 3.5-kbp cDNA was expressed in bacteria as a fusion protein with glutathione S-transferase (GST), but the yield was poor. To improve the yield, cDNA fragments encoding antigenic domains of p200 were expressed as fusions with GST. One of these fusion proteins, C1A-GST, is composed of a 7-kDa fragment of the p200 repeat region and contains epitopes that react strongly with sera from cattle experimentally infected with B. bigemina. Recombinant C1A-GST should permit the development of an improved enzyme-linked immunosorbent assay for the detection of antibodies against B. bigemina. PMID:10834983

  16. Antigenic variation in African trypanosomes: the importance of chromosomal and nuclear context in VSG expression control

    PubMed Central

    Glover, Lucy; Hutchinson, Sebastian; Alsford, Sam; McCulloch, Richard; Field, Mark C; Horn, David

    2013-01-01

    African trypanosomes are lethal human and animal parasites that use antigenic variation for evasion of host adaptive immunity. To facilitate antigenic variation, trypanosomes dedicate approximately one third of their nuclear genome, including many minichromosomes, and possibly all sub-telomeres, to variant surface glycoprotein (VSG) genes and associated sequences. Antigenic variation requires transcription of a single VSG by RNA polymerase I (Pol-I), with silencing of other VSGs, and periodic switching of the expressed gene, typically via DNA recombination with duplicative translocation of a new VSG to the active site. Thus, telomeric location, epigenetic controls and monoallelic transcription by Pol-I at an extranucleolar site are prominent features of VSGs and their expression, with telomeres, chromatin structure and nuclear organization all making vitally important contributions to monoallelic VSG expression control and switching. We discuss VSG transcription, recombination and replication control within this chromosomal and sub-nuclear context. PMID:24047558

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

    PubMed Central

    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

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

    PubMed

    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

  19. Humoral immunity to human breast cancer: antigen definition and quantitative analysis of mRNA expression.

    PubMed

    Scanlan, M J; Gout, I; Gordon, C M; Williamson, B; Stockert, E; Gure, A O; Jäger, D; Chen, Y T; Mackay, A; O'Hare, M J; Old, L J

    2001-03-30

    The ability of the immune system to recognize structurally altered, amplified or aberrantly expressed proteins can be used to identify molecules of etiologic relevance to cancer and to define targets for cancer immunotherapy. In the current study, ninety-four distinct antigens reactive with serum IgG from breast cancer patients were identified by immunoscreening breast cancer-derived cDNA expression libraries (SEREX). A serological profile was generated for each antigen on the basis of reactivity with allogeneic sera from normal individuals and cancer patients, and mRNA expression profiles for coding sequences were assembled based upon the tissue distribution of expressed sequence tags, Northern blots and real-time RT-PCR. Forty antigens reacted exclusively with sera from cancer patients. These included well-characterized tumor antigens, e.g. MAGE-3, MAGE-6, NY-ESO-1, Her2neu and p53, as well as newly-defined breast cancer antigens, e.g. kinesin 2, TATA element modulatory factor 1, tumor protein D52 and MAGE D, and novel gene products, e.g. NY-BR-62, NY-BR-75, NY-BR-85, and NY-BR-96. With regard to expression profiles, two of the novel gene products, NY-BR-62 and NY-BR-85, were characterized by a high level of testicular mRNA expression, and were overexpressed in 60% and 90% of breast cancers, respectively. In addition, mRNA encoding tumor protein D52 was overexpressed in 60% of breast cancer specimens, while transcripts encoding SNT-1 signal adaptor protein were downregulated in 70% of these cases. This study adds to the growing list of breast cancer antigens defined by SEREX and to the ultimate objective of identifying the complete repertoire of immunogenic gene products in human cancer (the cancer immunome). PMID:12747765

  20. Direct evidence for cytotoxicity associated with expression of hepatitis delta virus antigen.

    PubMed

    Cole, S M; Gowans, E J; Macnaughton, T B; Hall, P D; Burrell, C J

    1991-05-01

    It has been postulated that hepatocyte injury resulting from infection with hepatitis D virus may be caused by a direct virus cytotoxicity in contrast to immune-mediated injury associated with hepatitis B virus. We have transfected HeLa and HepG2 continuous cell lines with a recombinant plasmid containing the hepatitis D antigen gene under the inducible control of the human metallothionein promoter. The addition of zinc to the cell culture medium then led to the expression of hepatitis D antigen associated with, in the short term, a significant reduction in the rate of RNA but not DNA synthesis and, in the longer term, cell death. The necrotic cells had pyknotic nuclei and shrunken eosinophilic cytoplasm; these necrotic cells resembled the apoptotic bodies seen in hepatitis D virus-related hepatitis. The level of hepatitis D antigen in individual cells that produced these changes was similar to the level of hepatitis D antigen in hepatocytes from a chimpanzee with acute hepatitis D virus infection. We conclude that the expression of hepatitis D antigen resulted in significant cytotoxic changes in these cells, providing strong support for the view that hepatitis D antigen may be specifically cytotoxic to infected hepatocytes in vivo. PMID:1709411

  1. Isolation and characterization of recombinant lambda gt11 bacteriophages expressing four different Mycobacterium intracellulare antigens.

    PubMed Central

    Morris, S L; Rouse, D A; Hussong, D; Chaparas, S D

    1990-01-01

    Four bacteriophages expressing different immunoreactive recombinant Mycobacterium intracellulare antigens were isolated from a lambda gt11 library with monoclonal antibodies to M. intracellulare. These four antibodies reacted with native M. intracellulare proteins of 54, 43, 40/38, and 22 kilodaltons. Southern blot hybridizations with DNA probes prepared from insert fragments of these bacteriophages confirmed the M. intracellulare derivation of the inserts. The physical maps of the immunoreactive phages were deduced by restriction enzyme digestions. The molecular weights of the expressed recombinant antigens were determined by Western (immuno-) blotting. Images PMID:2136733

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

    PubMed

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

    2016-05-01

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

  3. Alkyl Hydroperoxide Reductases C and D Are Major Antigens Constitutively Expressed by Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Olsen, Ingrid; Reitan, Liv J.; Holstad, Gudmund; Wiker, Harald G.

    2000-01-01

    Antigens characteristic for Mycobacterium avium subspecies paratuberculosis were identified by crossed immunoelectrophoresis (CIE) and by absorbing out cross-reactive antigens by using a polyclonal and polyvalent Mycobacterium avium subspecies avium antiserum. Two antigens were present in M. avium subsp. paratuberculosis and not detected in Mycobacterium avium subsp. avium. They were identified as antigens 17 and 20 in a CIE reference system for M. avium subsp. paratuberculosis antigens. Purified antigen 20 was identified as alkyl hydroperoxide reductase C (AhpC) while the N-terminal part of purified antigen 17 showed 80% homology with alkyl hydroperoxide reductase D (AhpD) of Mycobacterium tuberculosis. AhpC had a nonreduced mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular mass of 45 kDa and is probably a homodimer linked with disulfide bridges in its native form. AhpD had a mobility corresponding to 19 kDa. Monospecific rabbit antiserum against AhpC and AhpD reacted with 9 strains of M. avium subsp. paratuberculosis but not with 20 other mycobacterial strains except for a Mycobacterium gordonae strain, against which a weak cross-reactive band was produced. Goats experimentally infected with M. avium subsp. paratuberculosis had strong gamma interferon (IFN-γ) responses toward both AhpC and AhpD, and they also had antibodies against AhpC. The ability of AhpC and AhpD to induce IFN-γ production shows that these proteins potentially could be used in future vaccines or in diagnostic assays. These results further show that AhpC and AhpD are immunologically important proteins which are constitutively and highly expressed in M. avium subsp. paratuberculosis without the bacteria being submitted to oxidative stress and that the specificities of antigens can be a matter of different levels of protein expression in various species as well as distinct structural differences. PMID:10639449

  4. Internalization and re-expression of antigens of human melanoma cells following exposure to monoclonal antibody

    SciTech Connect

    Wang, B.S.; Lumanglas, A.L.; Silva, J.; Ruszala-Mallon, V.; Durr, F.E.

    1987-04-15

    Modulation of the surface membrane of human Sk-Mel-28 melanoma cells by monoclonal antibody (MoAb) 96.5 recognizing p97 determinants was examined using direct radioimmunoassay and indirect fluorescent antibody-staining techniques. It was determined that the majority of /sup 111/In-labeled antibody that remained associated with cells after a 24-hr incubation at 37 degrees C had been internalized because MoAb 96.5 was no longer visible on the cell surface. A second treatment of these cells with the same antibody 24 hr later not only increased the cell-associated radioactivity, reflecting an increase of total antibody bound, but also rendered these cells membrane immunofluorescent again, indicating the re-expression of surface antigens. Autoradiographs of the electrophoretically analyzed membrane components of Sk-Mel-28 cells further demonstrated the appearance of newly synthesized 97-kDa proteins that were immunoprecipitable with MoAb 96.5. Taken together, the present findings suggest that p97 antigens undergo endocytosis in Sk-Mel-28 cells following exposure to MoAb 96.5. However, the same antigens were regenerated and expressed on the cell surface within a period of 24 hr. The re-expression of tumor cell surface antigen following initial internalization of the MoAb-antigen complex may have implications for diagnosis and therapy.

  5. Stable Expression of Lentiviral Antigens by Quality-Controlled Recombinant Mycobacterium bovis BCG Vectors

    PubMed Central

    Hart, Bryan E.; Asrican, Rose; Lim, So-Yon; Sixsmith, Jaimie D.; Lukose, Regy; Souther, Sommer J. R.; Rayasam, Swati D. G.; Saelens, Joseph W.; Chen, Ching-ju; Seay, Sarah A.; Berney-Meyer, Linda; Magtanong, Leslie; Vermeul, Kim; Pajanirassa, Priyadharshini; Jimenez, Amanda E.; Ng, Tony W.; Tobin, David M.; Porcelli, Steven A.; Larsen, Michelle H.; Schmitz, Joern E.; Haynes, Barton F.; Jacobs, William R.; Lee, Sunhee

    2015-01-01

    The well-established safety profile of the tuberculosis vaccine strain, Mycobacterium bovis bacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ΔleuCD transformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freeze-thaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging both in vitro and in vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ΔleuCD vaccine expressing HIV gp120 that retained stable full-length expression after 1024-fold amplification in vitro and following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >1068-fold amplification in vitro and induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ΔleuCD lots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches. PMID:25924766

  6. Stable Expression of Lentiviral Antigens by Quality-Controlled Recombinant Mycobacterium bovis BCG Vectors.

    PubMed

    Hart, Bryan E; Asrican, Rose; Lim, So-Yon; Sixsmith, Jaimie D; Lukose, Regy; Souther, Sommer J R; Rayasam, Swati D G; Saelens, Joseph W; Chen, Ching-Ju; Seay, Sarah A; Berney-Meyer, Linda; Magtanong, Leslie; Vermeul, Kim; Pajanirassa, Priyadharshini; Jimenez, Amanda E; Ng, Tony W; Tobin, David M; Porcelli, Steven A; Larsen, Michelle H; Schmitz, Joern E; Haynes, Barton F; Jacobs, William R; Lee, Sunhee; Frothingham, Richard

    2015-07-01

    The well-established safety profile of the tuberculosis vaccine strain, Mycobacterium bovis bacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ΔleuCD transformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freeze-thaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging both in vitro and in vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ΔleuCD vaccine expressing HIV gp120 that retained stable full-length expression after 10(24)-fold amplification in vitro and following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >10(68)-fold amplification in vitro and induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ΔleuCD lots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches. PMID:25924766

  7. Targeted Surface Expression of an Exogenous Antigen in Stably Transfected Babesia bovis

    PubMed Central

    Laughery, Jacob M.; Knowles, Donald P.; Schneider, David A.; Bastos, Reginaldo G.; McElwain, Terry F.; Suarez, Carlos E.

    2014-01-01

    Babesia bovis is a tick-borne intraerythocytic protozoan responsible for acute disease in cattle which can be controlled by vaccination with attenuated B. bovis strains. Emerging B. bovis transfection technologies may increase the usefulness of these live vaccines. One use of transfected B. bovis parasites may be as a vaccine delivery platform. Previous transfection methods for B. bovis were limited by single expression sites and intracellular expression of transfected antigens. This study describes a novel transfection system in which two exogenous genes are expressed: one for selection and the other for a selected antigen designed to be delivered to the surface of the parasites. The strategy for duplicating the number of transfected genes was based on the use of the putative bidirectional promoter of the B. bovis 1.4 Kb ef-1α intergenic region. The ability of this region to regulate two independent expression sites was demonstrated using a luciferase assay on transiently transfected B. bovis parasites and then incorporated into a stable transfection plasmid to control independent expression of the selectable marker GFP-BSD and another gene of interest. A chimeric gene was synthetized using sequences from the protective B-cell epitopes of Rhipicephalus microplus tick antigen Bm86 along with sequences from the surface exposed B. bovis major surface antigen-1. This chimeric gene was then cloned into the additional expression site of the transfection plasmid. Transfection of the B. bovis Mo7 strain with this plasmid resulted in stable insertion into the ef-1α locus and simultaneous expression of both exogenous genes. Expression of the Bm86 epitopes on the surface of transfected merozoites was demonstrated using immunofluorescence analyses. The ability to independently express multiple genes by the inclusion of a bidirectional promoter and the achievement of surface expression of foreign epitopes advances the potential of transfected B. bovis as a future vaccine

  8. Targeted surface expression of an exogenous antigen in stably transfected Babesia bovis.

    PubMed

    Laughery, Jacob M; Knowles, Donald P; Schneider, David A; Bastos, Reginaldo G; McElwain, Terry F; Suarez, Carlos E

    2014-01-01

    Babesia bovis is a tick-borne intraerythocytic protozoan responsible for acute disease in cattle which can be controlled by vaccination with attenuated B. bovis strains. Emerging B. bovis transfection technologies may increase the usefulness of these live vaccines. One use of transfected B. bovis parasites may be as a vaccine delivery platform. Previous transfection methods for B. bovis were limited by single expression sites and intracellular expression of transfected antigens. This study describes a novel transfection system in which two exogenous genes are expressed: one for selection and the other for a selected antigen designed to be delivered to the surface of the parasites. The strategy for duplicating the number of transfected genes was based on the use of the putative bidirectional promoter of the B. bovis 1.4 Kb ef-1α intergenic region. The ability of this region to regulate two independent expression sites was demonstrated using a luciferase assay on transiently transfected B. bovis parasites and then incorporated into a stable transfection plasmid to control independent expression of the selectable marker GFP-BSD and another gene of interest. A chimeric gene was synthetized using sequences from the protective B-cell epitopes of Rhipicephalus microplus tick antigen Bm86 along with sequences from the surface exposed B. bovis major surface antigen-1. This chimeric gene was then cloned into the additional expression site of the transfection plasmid. Transfection of the B. bovis Mo7 strain with this plasmid resulted in stable insertion into the ef-1α locus and simultaneous expression of both exogenous genes. Expression of the Bm86 epitopes on the surface of transfected merozoites was demonstrated using immunofluorescence analyses. The ability to independently express multiple genes by the inclusion of a bidirectional promoter and the achievement of surface expression of foreign epitopes advances the potential of transfected B. bovis as a future vaccine

  9. Expression of early activation antigen (CD69) during human thymic development.

    PubMed Central

    Jung, L K; Haynes, B F; Nakamura, S; Pahwa, S; Fu, S M

    1990-01-01

    The novel early activation antigen, EA1, has been shown to be induced by mitogens, antigens and the tumour promoter, phorbol myristate acetate (PMA), on human lymphocytes. This antigen has been designated to be CD69. EA1 has also been shown to be expressed on thymocytes without exogenous activation stimuli. In order to characterize further the expression of EA1 on thymocytes, the ontogeny of its expression was studied. EA1 appeared between 7 and 9.5 weeks of gestation, after colonization of the thymic rudiment with CD7+ T cell precursors, but before the onset of compartmentalization of the thymus into cortical and medullary zones. After cortico-medullary differentiation, the majority of medullary thymocytes expressed EA1 while only a fraction of the cortical thymocytes expressed this antigen. In the fetal and post-natal cortex, EA1 expression appeared to cluster in the subcapsular cortex. EA1+ cells were also scattered throughout the inner cortex. By two-colour fluorocytometric analysis of post-natal thymocytes, it was shown that EA1 was expressed on 30 to 65% of thymocytes. EA1 was expressed on CD4+ CD8+ as well as on the more immature CD4- CD8- thymocytes. In contrast to circulating T cells, thymocytes were much less responsive to PMA stimulation for the expression of EA1. Molecular characterization showed that EA1 on thymocytes had the same structure as that of activated peripheral T cells. In addition, thymic EA1 was constitutively phosphorylated. Thus, EA1 expression is acquired early during thymic development after colonization of the thymic rudiment by CD7+ T cell precursors. However, the specific role that EA1 may play in the activation and function of developing thymocytes remains to be determined. Images Fig. 7 Fig. 8 PMID:2204504

  10. Permanent cell line expressing human factor VIII-related antigen established by hybridization.

    PubMed Central

    Edgell, C J; McDonald, C C; Graham, J B

    1983-01-01

    A permanent human cell line, EA . hy 926, has been established that expresses at least one highly differentiated function of vascular endothelium, factor VIII-related antigen. This line was derived by fusing human umbilical vein endothelial cells with the permanent human cell line A549. Hybrid cells that survived in selective medium had more chromosomes than either progenitor cell type and included a marker chromosome from the A549 line. Factor VIII-related antigen can be identified intracellularly in the hybrids by immunofluorescence and accumulates in the culture fluid. Expression of factor VIII-related antigen by these hybrid cells has been maintained for more than 100 cumulative population doublings, including more than 50 passages and three cloning steps. This is evidence that EA . hy 926 represents a permanent line. Images PMID:6407019

  11. Surface antigen expression in chronic lymphocytic leukemia: clustering analysis, interrelationships and effects of chromosomal abnormalities.

    PubMed

    Hulkkonen, J; Vilpo, L; Hurme, M; Vilpo, J

    2002-02-01

    Chronic lymphocytic leukemia (CLL) is a phenotypically distinguishable form of B-lymphoid leukemias. The regularity of surface membrane antigen expression patterns, their interrelationships as well as the effects of the three frequent chromosomal aberrations, ie 11q deletion, 13q deletion and trisomy 12, were investigated in 35 classic CLL cases by flow cytometry. The two-way cluster analysis of 31 individual antigens revealed three expression patterns: (1) most cells in most cases positive (CD5, CD19, CD20, CD23, CD27, CD40, CD45, CD45RA); (2) most cells in most cases negative (CD10, CD14, CD34, CD122, CD154, mIgG); and (3) a mixed pattern with a variable number of positive cases and a variable percentage of positive cells in individual cases (CD11c, CD21, CD22, CD25, CD38, CD45RO, CD79b, CD80, CD95, CD124, CD126, CD130, FMC7, mIgD, mIgkappa, mIglambda, mIgM). The expressions of several antigens were strongly interdependent, even when antigens belonged to entirely different gene families. Such antigen pairs were: CD11c/CD21; CD19/CD45; CD19/CD79b; CD22/CD45RA; CD23/Igkappa; CD25/mIgM; CD27/CD45; CD45/CD79b; CD45RA/Igkappa. In contrast, the expression of some antigens was mutually exclusive, the best examples being CD45RA/CD45RO, CD38/CD80 and CD45RA/CD80. Deletion of chromosome arm 11q attenuated expression of splicing variant CD45RA, but enhanced CD45RO expression. In contrast, cases of trisomy 12 were associated with enhanced CD45RA and attenuated CD45RO expression. Similarly, trisomy 12 was associated with enhanced CD27 and mIgkappa expression. The variable levels of signaling surface membrane antigens, their interactions and interference by genetic aberrations are likely to affect the clinical progression and drug response of CLL. PMID:11840283

  12. The expression of the Hu (paraneoplastic encephalomyelitis/sensory neuronopathy) antigen in human normal and tumor tissues.

    PubMed Central

    Dalmau, J.; Furneaux, H. M.; Cordon-Cardo, C.; Posner, J. B.

    1992-01-01

    Using immunohistochemistry or Western blot analysis, the authors have studied the expression of the Hu antigen (a neuronal protein identified by the serum of patients with small cell lung cancer and paraneoplastic encephalomyelitis/sensory neuronopathy) in normal human tissues and 115 tumors of different histologic types. In normal tissue, the Hu antigen is highly restricted to the nervous system. In lung tumors, the Hu antigen is restricted in its expression to all small cell carcinomas. A few other neuroendocrine-related tumors, especially neuroblastomas (50%), also express the antigen. Images Figure 1 Figure 2 Figure 3 PMID:1415481

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

    PubMed

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

    2016-08-01

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

  14. Differences in the expression of mucus-associated antigens between proximal and distal human colon adenocarcinomas.

    PubMed Central

    Bara, J.; Nardelli, J.; Gadenne, C.; Prade, M.; Burtin, P.

    1984-01-01

    An immunohistological study showed differences in the expression of mucus-associated gastric M1 and intestinal M3 antigens between the proximal (100 cases) and distal (200 cases) colonic adenocarcinomas. Such a regional difference was not observed in the normal colon. A total of 55% and 78% of proximal tumours produced M1 and M3 antigens, respectively (versus 13% and 47% in the distal tumours). The high percentage of M1 positive proximal cancers could be explained by the higher percentage (i) of mucus-producing tumours, such as signet ring cell (6% vs 1%) or mucinous adenocarcinomas (29% vs 11%); and (ii) of M1(+) well-differentiated adenocarcinomas (45% vs 8.5%) and the presence of undifferentiated carcinoma producing M1 antigens (12% vs 0%). These latter carcinomas were found in older patients (mean age 78 years vs 66 years). These results suggest that, on the proximal side, the stem cells were more often engaged in a differentiation process involving the expression of M antigens than were those of the distal side. Moreover, the proximal stem cells more frequently produce a foetal differentiation program showing simultaneous expression of M3 and M1 antigens (in 48% of proximal tumours, vs 11.5% for the distal side). Around 12% of proximal adenocarcinomas (vs 2% of distal tumours) contained stem cells engaged in a cell differentiation program not observed in the normal adult or foetal colon, involving the predominant expression of M1 antigens associated with an undifferential histological pattern. Images Figure 2 PMID:6324842

  15. Induction of cancer testis antigen expression in circulating acute myeloid leukemia blasts following hypomethylating agent monotherapy

    PubMed Central

    Srivastava, Pragya; Paluch, Benjamin E.; Matsuzaki, Junko; James, Smitha R.; Collamat-Lai, Golda; Blagitko-Dorfs, Nadja; Ford, Laurie Ann; Naqash, Rafeh; Lübbert, Michael; Karpf, Adam R.; Nemeth, Michael J.; Griffiths, Elizabeth A.

    2016-01-01

    Cancer testis antigens (CTAs) are promising cancer associated antigens in solid tumors, but in acute myeloid leukemia, dense promoter methylation silences their expression. Leukemia cell lines exposed to HMAs induce expression of CTAs. We hypothesized that AML patients treated with standard of care decitabine (20mg/m2 per day for 10 days) would demonstrate induced expression of CTAs. Peripheral blood blasts serially isolated from AML patients treated with decitabine were evaluated for CTA gene expression and demethylation. Induction of NY-ESO-1 and MAGEA3/A6, were observed following decitabine. Re-expression of NY-ESO-1 and MAGEA3/A6 was associated with both promoter specific and global (LINE-1) hypomethylation. NY-ESO-1 and MAGEA3/A6 mRNA levels were increased irrespective of clinical response, suggesting that these antigens might be applicable even in patients who are not responsive to HMA therapy. Circulating blasts harvested after decitabine demonstrate induced NY-ESO-1 expression sufficient to activate NY-ESO-1 specific CD8+ T-cells. Induction of CTA expression sufficient for recognition by T-cells occurs in AML patients receiving decitabine. Vaccination against NY-ESO-1 in this patient population is feasible. PMID:26883197

  16. RECOMBINANT SIMIAN VARICELLA VIRUSES EXPRESSING RESPIRATORY SYNCYTIAL VIRUS ANTIGENS ARE IMMUNOGENIC

    PubMed Central

    Ward, Toby M.; Traina-Dorge, Vicki; Davis, Kara A.; Gray, Wayne L.

    2013-01-01

    SUMMARY Recombinant simian varicella viruses (rSVVs) were engineered to express respiratory syncytial virus (RSV) antigens. The RSV surface glycoprotein G and second matrix protein M2 (22k) genes were cloned into the SVV genome, and recombinant viruses were characterized in vitro and in vivo. rSVVs were also engineered to express the membrane-anchored or secreted forms of the RSV G protein as well as an RSV G lacking its chemokine mimicry motif (CX3C), which may have different effects on priming the host immune response. The RSV genes were efficiently expressed in rSVV/RSV infected Vero cells as RSV G and M2 transcripts were detected by RT-PCR and RSV antigens were detected by immunofluorescence and immunoblot assays. The rSVVs replicated efficiently in Vero cell culture. Rhesus macaques immunized with rSVV/RSV-G and rSVV/RSV-M2 vaccines produced antibody responses to SVV and RSV antigens. The results demonstrate that recombinant varicella viruses are suitable vectors for expression of RSV antigens and may represent a novel vaccine strategy for immunization against both pathogens. PMID:18272766

  17. ENCYSTATION AND EXPRESSION OF CYST ANTIGENS BY 'GIARDIA LAMBLIA' IN VITRO

    EPA Science Inventory

    The cyst form of Giardia lamblia is responsible for transmission of giardiasis, a major waterborne intestinal disease. These studies demonstrate for the first time expression of cyst antigens and encystation of G. lamblia in vitro by both morphologic and immunologic criteria. The...

  18. Prognostic significance of 5T4 oncofetal antigen expression in colorectal carcinoma.

    PubMed

    Starzynska, T; Marsh, P J; Schofield, P F; Roberts, S A; Myers, K A; Stern, P L

    1994-05-01

    The 5T4 oncofetal antigen is a 72 kDa glycoprotein defined by a monoclonal antibody raised against human placental trophoblast and is expressed in many different carcinomas but detected only at low levels in some normal epithelia. Immunohistochemical analysis of the patterns of expression in colorectal carcinomas has indicated a significant association between the presence of the antigen in tumour cells and metastatic spread. The 5T4 antigen phenotype of 72 colorectal cancers has been compared with the clinical outcome of the patients in order to assess its relationship with prognosis. Forty per cent of tumours were 5T4 positive; the remainder were either unlabelled or exhibited stroma-associated labelling only. There was a significant correlation between 5T4 expression in the malignant cells and unfavourable course of disease (P < 0.001). The 5 year survival with 5T4-positive tumours was 22% compared with 75% for patients with 5T4-negative tumours; median survival was 24 versus > 90 months respectively. Stratified analysis showed that 5T4 antigen tumour positivity was acting independently of each of stage, site of tumour, age or sex. There were significant differences in survival for patients with Dukes' B and C stage carcinomas (P = 0.001 and P = 0.034). The results suggest that in colorectal cancer immunohistochemical assessment of 5T4 expression may be useful in identifying patients at high risk for tumour recurrence and for whom additional treatment strategies might be most appropriate. PMID:8180020

  19. Viral Engineering of Chimeric Antigen Receptor Expression on Murine and Human T Lymphocytes.

    PubMed

    Hammill, Joanne A; Afsahi, Arya; Bramson, Jonathan L; Helsen, Christopher W

    2016-01-01

    The adoptive transfer of a bolus of tumor-specific T lymphocytes into cancer patients is a promising therapeutic strategy. In one approach, tumor specificity is conferred upon T cells via engineering expression of exogenous receptors, such as chimeric antigen receptors (CARs). Here, we describe the generation and production of both murine and human CAR-engineered T lymphocytes using retroviruses. PMID:27581020

  20. Identification of antigenic components of Staphylococcus epidermidis expressed during human infection.

    PubMed

    Pourmand, Mohammad R; Clarke, Simon R; Schuman, Richard F; Mond, James J; Foster, Simon J

    2006-08-01

    A spectrum of in vivo-expressed Staphylococcus epidermidis antigens was identified by probing a bacteriophage lambda library of S. epidermidis genomic DNA with human serum from infected and uninfected individuals. This analysis resulted in identification of 53 antigen-encoding loci. Six antigenic polypeptides were expressed from these loci and purified. These polypeptides were the propeptide, mature amidase, and repeat sequence domains of the major autolysin AtlE, GehD (lipase), and two members of a conserved family of surface proteins (ScaA [AaE] and ScaB). AtlE, ScaA, and ScaB all exhibit human ligand binding capacity. Screening a bank of human serum samples revealed that there were significant increases in the amounts of reactive immunoglobulin G in infected individuals compared to the amounts in healthy individuals for the repeat sequence and mature amidase domains of AtlE, ScaB, and GehD. Vaccination of mice with recombinant antigens stimulated an immune response which in vitro opsonized S. epidermidis. In this study we identified prospective candidate antigens for prophylaxis or immunotherapy to control disease. PMID:16861652

  1. High Expression of Water-Soluble Recombinant Antigenic Domains of Toxoplasma gondii Secretory Organelles

    PubMed Central

    Yang, Zhaoshou; Ahn, Hye-Jin

    2014-01-01

    Recombinant antigenic proteins of Toxoplasma gondii are alternative source of antigens which are easily obtainable for serodiagnosis of toxoplasmosis. In this study, highly antigenic secretory organellar proteins, dense granular GRA2 and GRA3, rhoptrial ROP2, and micronemal MIC2, were analyzed by bioinformatics approach to express as water-soluble forms of antigenic domains. The transmembrane region and disorder tendency of 4 secretory proteins were predicted to clone the genes into pGEX-4T-1 vector. Recombinant plasmids were transformed into BL21 (DE3) pLysS E. coli, and GST fusion proteins were expressed with IPTG. As a result, GST fusion proteins with GRA225-105, GRA339-138, ROP2324-561, and MIC21-284 domains had respectively higher value of IgG avidity. The rGST-GRA225-105 and rGST-GRA339-138 were soluble, while rGST-ROP2324-561 and rGST-MIC21-284 were not. GRA231-71, intrinsically unstructured domain (IUD) of GRA2, was used as a linker to enhance the solubility. The rGST-GRA231-71-ROP2324-561, a chimeric protein, appeared to be soluble. Moreover, rGST-GRA231-71-MIC21-284 was also soluble and had higher IgG avidity comparing to rGST-MIC21-284. These 4 highly expressed and water-soluble recombinant antigenic proteins may be promising candidates to improve the serodiagnosis of toxoplasmosis in addition to the major surface antigen of SAG1. PMID:25246715

  2. High expression of water-soluble recombinant antigenic domains of Toxoplasma gondii secretory organelles.

    PubMed

    Yang, Zhaoshou; Ahn, Hye-Jin; Nam, Ho-Woo

    2014-08-01

    Recombinant antigenic proteins of Toxoplasma gondii are alternative source of antigens which are easily obtainable for serodiagnosis of toxoplasmosis. In this study, highly antigenic secretory organellar proteins, dense granular GRA2 and GRA3, rhoptrial ROP2, and micronemal MIC2, were analyzed by bioinformatics approach to express as water-soluble forms of antigenic domains. The transmembrane region and disorder tendency of 4 secretory proteins were predicted to clone the genes into pGEX-4T-1 vector. Recombinant plasmids were transformed into BL21 (DE3) pLysS E. coli, and GST fusion proteins were expressed with IPTG. As a result, GST fusion proteins with GRA225-105, GRA339-138, ROP2324-561, and MIC21-284 domains had respectively higher value of IgG avidity. The rGST-GRA225-105 and rGST-GRA339-138 were soluble, while rGST-ROP2324-561 and rGST-MIC21-284 were not. GRA231-71, intrinsically unstructured domain (IUD) of GRA2, was used as a linker to enhance the solubility. The rGST-GRA231-71-ROP2324-561, a chimeric protein, appeared to be soluble. Moreover, rGST-GRA231-71-MIC21-284 was also soluble and had higher IgG avidity comparing to rGST-MIC21-284. These 4 highly expressed and water-soluble recombinant antigenic proteins may be promising candidates to improve the serodiagnosis of toxoplasmosis in addition to the major surface antigen of SAG1. PMID:25246715

  3. Expression and self-assembly of recombinant capsid protein from the antigenically distinct Hawaii human calicivirus.

    PubMed Central

    Green, K Y; Kapikian, A Z; Valdesuso, J; Sosnovtsev, S; Treanor, J J; Lew, J F

    1997-01-01

    The Norwalk and Hawaii viruses are antigenically distinct members of the family Caliciviridae and are considered to be important etiologic agents of epidemic gastroenteritis, with most studies focusing on the role of Norwalk virus. To further investigate the importance of Hawaii virus, Hawaii virus-like particles (VLPs) were produced by expression of its capsid protein in the baculovirus system and these VLPs were used as the antigen in an enzyme-linked immunosorbent assay that was efficient in the detection of a serologic response to Hawaii virus. The ready availability of Hawaii VLPs should enable larger-scale epidemiological studies to further elucidate the importance of this agent. PMID:9196224

  4. Attenuated recombinant vaccinia virus expressing oncofetal antigen (tumor-associated antigen) 5T4 induces active therapy of established tumors.

    PubMed

    Mulryan, Kate; Ryan, Matthew G; Myers, Kevin A; Shaw, David; Wang, Who; Kingsman, Susan M; Stern, Peter L; Carroll, Miles W

    2002-10-01

    The human oncofetal antigen 5T4 (h5T4) is a transmembrane glycoprotein overexpressed by a wide spectrum of cancers, including colorectal, ovarian, and gastric, but with a limited normal tissue expression. Such properties make 5T4 an excellent putative target for cancer immunotherapy. The murine homologue of 5T4 (m5T4) has been cloned and characterized, which allows for the evaluation of immune intervention strategies in "self-antigen" in vivo tumor models. We have constructed recombinant vaccinia viruses based on the highly attenuated and modified vaccinia virus ankara (MVA strain), expressing h5T4 (MVA-h5T4), m5T4 (MVA-m5T4), and Escherichia coli LacZ (MVA-LacZ). Immunization of BALB/c and C57BL/6 mice with MVA-h5T4 and MVA-m5T4 constructs induced antibody responses to human and mouse 5T4, respectively. C57BL/6 and BALB/c mice vaccinated with MVA-h5T4 were challenged with syngeneic tumor line transfectants, B16 melanoma, and CT26 colorectal cells that express h5T4. MVA-h5T4-vaccinated mice showed significant tumor retardation compared with mice vaccinated with MVA-LacZ or PBS. In active treatment studies, inoculation with MVA-h5T4 was able to treat established CT26-h5T4 lung tumor and to a lesser extent B16.h5T4 s.c. tumors. Additionally, when C57BL/6 mice vaccinated with MVA-m5T4 were challenged with B16 cells expressing m5T4, resulting growth of the tumors was significantly retarded compared with control animals. Furthermore, mice vaccinated with MVA-m5T4 showed no signs of autoimmune toxicity. These data support the use of MVA-5T4 for tumor immunotherapy. PMID:12481437

  5. Human fibroblasts in idiopathic retroperitoneal fibrosis express HLA-DR antigens.

    PubMed

    Lee, I

    1991-09-01

    Idiopathic retroperitoneal fibrosis (IRF) is a rare human disease characterized by non-neoplastic fibroblastic proliferation associated with chronic inflammatory cells; its pathogenesis is obscure. We undertook an immunohistochemical study for the expression of HLA-DR antigens and other immune-related markers by retroperitoneal proliferating fibroblasts and inflammatory cells from 2 IRF patients. Patterns of immunoreactivity were compared with those expressed by human nodular fasciitis (NF) and granulation tissue. In IRF, most fibroblasts immunostained strongly for HLA-DR antigens, whereas fibroblasts in NF and granulation tissue did, not immunostain at all. The fibroblasts did not immunostain for interleukin 2 receptor, C3b receptor, CD-4, CD-8, or Leu-M1 in any of the tissue studied. Most macrophages and lymphocytes in IRF and NF immunostained Strangly for HLA-DR antigens. In IRF, the CD-4 and CD-8 immunostained T-lymphocytes appeared equally distributed. The expression of HLA-DR antigens by fibroblasts in IRF indicates that this rare disease may indeed be an immune-associated hypersensitivity disorder. PMID:1777134

  6. Precision cancer immunotherapy: optimizing dendritic cell-based strategies to induce tumor antigen-specific T-cell responses against individual patient tumors.

    PubMed

    Osada, Takuya; Nagaoka, Koji; Takahara, Masashi; Yang, Xiao Yi; Liu, Cong-Xiao; Guo, Hongtao; Roy Choudhury, Kingshuk; Hobeika, Amy; Hartman, Zachary; Morse, Michael A; Lyerly, H Kim

    2015-05-01

    Most dendritic cell (DC)-based vaccines have loaded the DC with defined antigens, but loading with autologos tumor-derived antigens would generate DCs that activate personalized tumor-specific T-cell responses. We hypothesized that DC matured with an optimized combination of reagents and loaded with tumor-derived antigens using a clinically feasible electroporation strategy would induce potent antitumor immunity. We first studied the effects on DC maturation and antigen presentation of the addition of picibanil (OK432) to a combination of zoledronic acid, tumor necrosis factor-α, and prostaglandin E2. Using DC matured with the optimized combination, we tested 2 clinically feasible sources of autologous antigen for electroloading, total tumor mRNA or total tumor lysate, to determine which stimulated more potent antigen-specific T cells in vitro and activated more potent antitumor immunity in vivo. The combination of tumor necrosis factor-α/prostaglandin E2/zoledronic acid/OK432 generated DC with high expression of maturation markers and antigen-specific T-cell stimulatory function in vitro. Mature DC electroloaded with tumor-derived mRNA [mRNA electroporated dendritic cell (EPDC)] induced greater expansion of antigen-specific T cells in vitro than DC electroloaded with tumor lysate (lysate EPDC). In a therapeutic model of MC38-carcinoembryonic antigen colon cancer-bearing mice, vaccination with mRNA EPDC induced the most efficient anti-carcinoembryonic antigen cellular immune response, which significantly suppressed tumor growth. In conclusion, mature DC electroloaded with tumor-derived mRNA are a potent cancer vaccine, especially useful when specific tumor antigens for vaccination have not been identified, allowing autologous tumor, and if unavailable, allogeneic cell lines to be used as an unbiased source of antigen. Our data support clinical testing of this strategy. PMID:25839441

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

    PubMed Central

    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

  8. Identification of Preferentially Expressed Antigen of Melanoma as a Potential Tumor Suppressor in Lung Adenocarcinoma

    PubMed Central

    Huang, Quan; Li, Lin; Lin, Zaijun; Xu, Wei; Han, Shuai; Zhao, Chenglong; Li, Lei; Cao, Wenjiao; Yang, Xinghai; Wei, Haifeng; Xiao, Jianru

    2016-01-01

    Background Preferentially expressed antigen of melanoma (PRAME) is known as a tumor-associated antigen that is altered in a variety of malignancies, including lung cancer. However, the role of PRAME in lung cancer remains unclear. Material/Methods We analyzed the expression of PRAME in human lung adenocarcinomas and studied the function of PRAME using small interfering RNA (siRNA)-induced gene knockdown in lung cancer cell lines PC9 and A549. Results We found that PRAME expression is down-regulated in lung adenocarcinomas. Knockdown of PRAME promoted proliferation and suppressed apoptosis of PC9 and A549 cells. Conclusions In line with its roles in controlling cell growth, RPAME regulates multiple critical cell-growth related genes, including IGF1R oncogene. IGF1R up-regulation contributes to increase of cell growth upon the knockdown of PRAME. Taken together, our results suggest that PRAME has inhibitory roles in lung cancer. PMID:27241212

  9. Different serotypes of dengue viruses differently regulate the expression of the host cell antigen processing machinery.

    PubMed

    Gan, Chye Sheng; Yusof, Rohana; Othman, Shatrah

    2015-09-01

    Dengue virus (DV) infection demonstrates an intriguing virus-induced intracellular membrane alteration that results in the augmentation of major histocompatibility complex (MHC) class I-restricted antigen presentation. As oppose to its biological function in attracting CD8(+) T-cells, this phenomenon appears to facilitate the immune evasion. However, the molecular events that attribute to the dysregulation of the antigen presenting mechanism (APM) by DV remain obscure. In this study, we aimed to characterize the host cell APM upon infection with all serotypes of whole DV. Cellular RNA were isolated from infected cells and the gene expressions of LMP2, LMP7, TAP1, TAP2, TAPBP, CALR, CANX, PDIA3, HLA-A and HLA-B were analyzed via quantitative PCR. The profiles of the gene expression were further validated. We showed that all four DV serotypes modulate host APM at the proteasomal level with DV2 showing the most prominent expression profile. PMID:25981524

  10. Two methods for the quantitative analysis of surface antigen expression in acute myeloid leukemia (AML).

    PubMed

    Woźniak, Jolanta

    2004-01-01

    The expression of lineage molecules (CD13 and CD33), c-Kit receptor (CD117), CD34, HLA-DR and adhesion molecule CD49d was assessed in acute myeloid leukemia (AML) blast cells from 32 cases, using direct and indirect quantitative cytometric analysis. High correlation (r=0.8) was found between antigen expression intensity values calculated by direct analysis method (ABC) and by indirect analysis method (RFI). Moreover, the differences in expression intensity of CD13, CD117 and CD34 antigens were found between leukemic and normal myeloblasts. This may be helpful in identification of leukemic cells in the diagnostics of minimal residual disease after treatment in AML patients. PMID:15493582

  11. Lymphocyte expression of a 90 kD brush border antigen.

    PubMed Central

    Van Leer, E H; Moullier, P; Ronco, P; Verroust, P

    1987-01-01

    This study analyses the expression by rat lymphocytes of six renal brush border (BB) antigens defined by a set of monoclonal antibodies and demonstrates that a 90 kD protein (gp 90), synthesized by BB and glomeruli is found on the surface of T and B cells. The other antigens, including the 330 kD protein involved in Heymann's nephritis--a model of epimembranous glomerulonephritis--are not detectable on the surface of lymphocytes. Immunochemical studies indicate that gp 90 and the related protein immunoprecipitated from thymocyte membranes co-migrate in SDS-PAGE. Quantitative binding analysis shows that the number of antigenic sites is in the same order of magnitude on thymocytes, spleen and bone-marrow lymphocytes as well as on two lines of pre-B and pre-T cells, but considerably lower on a highly differentiated helper T cell line. Images Fig. 4 PMID:3301100

  12. Interferon-γ Reduces Melanosomal Antigen Expression and Recognition of Melanoma Cells by Cytotoxic T Cells

    PubMed Central

    Le Poole, I. Caroline; Riker, Adam I.; Quevedo, M. Eugenia; Stennett, Lawrence S.; Wang, Ena; Marincola, Francesco M.; Kast, W. Martin; Robinson, June K.; Nickoloff, Brian J.

    2002-01-01

    In malignant melanoma, tumor-infiltrating lymphocytes are frequently reactive with melanosomal antigens. Achieving complete remissions by peptide therapy is frequently hampered by metastases evading immune recognition. The tumor microenvironment seems to favor reduced expression of target antigens by melanoma cells. Among candidate factors, interferon-γ (IFN-γ) (102 to 103 U/ml) suppressed expression of antigens MART-1, TRP-1, and gp100 by M14 melanoma cells as shown by immunohistology and fluorescence-activated cell sorting analysis, reducing MART-1 expression by >65%. Northern blot analysis revealed that reduced expression was regulated at the transcriptional level, demonstrating a 79% reduction in MART-1 transcript abundance after 32 hours of IFN-γ treatment. To evaluate consequences of IFN-γ exposure for immune recognition, MART-1-responsive T cells were reacted with pretreated HLA-matched melanoma cells. Cytotoxicity was reduced up to 78% by IFN-γ pretreatment, and was restored by addition of MART-1 peptide AAGIGILTV for 2 hours. Examination of melanoma lesions by quantitative reverse transcriptase-polymerase chain reaction revealed up to 188-fold more abundant IFN-γ transcripts when compared to control skin. Laser capture microdissection and immunohistology localized most IFN-γ-producing T cells to the tumor stroma. Reduced MART-1 expression was frequently observed in adjacent tumor cells. Consequently, IFN-γ may enhance inflammatory responses yet hamper effective recognition of melanoma cells. PMID:11839572

  13. Glycoprotein screening in colorectal cancer based on differentially expressed Tn antigen.

    PubMed

    Wei, Hongyun; Cheng, Zongyong; Ouyang, Chunhui; Zhang, Yu; Hu, Yanyan; Chen, Shuijiao; Wang, Chunlian; Lu, Fanggen; Zhang, Jie; Wang, Yongjun; Liu, Xiaowei

    2016-09-01

    Colorectal cancer (CRC) is one of the most common cancers worldwide, and the identification of new biomarkers for CRC is valuable for its diagnosis and treatment. We aimed to screen differentially expressed glycoproteins (especially O-glycoproteins) and to identify diagnostic or therapeutic candidates for colorectal cancer (CRC) based on different Tn antigen expression levels. Fresh cancer tissues and adjacent healthy tissues were obtained from CRC patients and classified into three groups based on their Tn antigen expression: CRC with negative Tn expression (CRC Tn‑), CRC with positive Tn expression (CRC Tn+) and normal control without Tn expression (NC). Protein extractions were separated and identified by iTRAQ technology. Glycoproteins and O-glycoproteins were selected using UniProt and DAVID. Deep bioinformatic analysis, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KO), was used to annotate this O-glycoprotein interaction network. Subsequently, two O‑glycoproteins were verified by western blotting and immunohistochemistry in either LS174T cells or CRC tissues. We found that 330 differentially expressed proteins were identified by iTRAQ between CRC Tn‑ and NC tissues, 317 between CRC Tn+ and NC tissues, and 316 between CRC Tn‑ and Tn+ tissues. Of the 316 proteins, 55 glycoproteins and 19 O‑glycoproteins were identified and analyzed via deep informatics. Namely, different Tn antigen expression levels in CRC led to differential protein expression patterns, especially for glycoproteins and O‑glycoproteins. Decorin and SORBS1, two representative functional O-glycoproteins, were significantly downregulated in the CRC Tn+ tissues compared with the level in the CRC Tn‑ or NC tissues. Based on this deep bioinformatic analysis, Decorin and SORBS1 are hypothesized to be involved in the TGF‑β and PPAR‑γ signaling pathways, respectively. PMID:27432485

  14. Expression and Immune Responses to MAGE Antigens Predict Survival in Epithelial Ovarian Cancer

    PubMed Central

    Daudi, Sayeema; Eng, Kevin H.; Mhawech-Fauceglia, Paulette; Morrison, Carl; Miliotto, Anthony; Beck, Amy; Matsuzaki, Junko; Tsuji, Takemasa; Groman, Adrienne; Gnjatic, Sacha; Spagnoli, Guillo; Lele, Shashikant; Odunsi, Kunle

    2014-01-01

    The MAGE cancer-testis antigens (CTA) are attractive candidates for immunotherapy. The aim of this study was to determine the frequency of expression, humoral immunity and prognostic significance of MAGE CTA in human epithelial ovarian cancer (EOC). mRNA or protein expression frequencies were determined for MAGE-A1, -A3, -A4, -A10 and -C1 (CT7) in tissue samples obtained from 400 patients with EOC. The presence of autologous antibodies against the MAGE antigens was determined from 285 serum samples. The relationships between MAGE expression, humoral immunity to MAGE antigens, and clinico-pathologic characteristics were studied. The individual frequencies of expression were as follows: A1: 15% (42/281), A3: 36% (131/390), A4: 47% (186/399), A10: 52% (204/395), C1: 16% (42/267). Strong concordant expression was noted with MAGE-A1:–A4, MAGE-A1:–C1 and MAGE-A4:–A10 (p<0.0005). Expression of MAGE-A1 or -A10 antigens resulted in poor progression free survival (PFS) (OR 1.44, CI 1.01–2.04, p = 0.044 and OR 1.3, CI 1.03–1.64, p = 0.03, respectively); whereas, MAGE-C1 expression was associated with improved PFS (OR 0.62, CI 0.42–0.92, p = 0.016). The improved PFS observed for MAGE-C1 expression, was diminished by co-expression of MAGE-A1 or -A10. Spontaneous humoral immunity to the MAGE antigens was present in 9% (27/285) of patients, and this predicted poor overall survival (log-rank test p = 0.0137). These findings indicate that MAGE-A1, MAGE-A4, MAGE-A3, and MAGE-A10 are priority attractive targets for polyvalent immunotherapy in ovarian cancer patients. PMID:25101620

  15. Expression of major histocompatibility complex class II antigens in porcine leptospiral nephritis.

    PubMed

    Radaelli, E; Del Piero, F; Aresu, L; Sciarrone, F; Vicari, N; Mattiello, S; Tagliabue, S; Fabbi, M; Scanziani, E

    2009-09-01

    Class II major histocompatibility complex (MHCII) is required for the presentation of antigens to CD4 helper T cells. During nephritis, not only primary antigen presenting cells such as histiocytes and lymphocytes, but also cytokine-stimulated tubular epithelial cells express MHCII. Leptospirosis in fattening pigs is characterized by several degrees of nephritis, from absence of lesions to severe multifocal tubulo-interstitial inflammation. Renal tissue from 20 8-month-old pigs with spontaneous nephritis and 6 control pigs without renal lesions were investigated for leptospirosis by indirect immunohistochemistry (IHC) and polymerase chain reaction (PCR). IHC for MHCII also was performed on renal samples. Serum samples were tested for different serovars of Leptospira interrogans. Control pigs were free of interstitial nephritis and negative for leptospirosis by all tests. In pigs with nephritis, serology was positive for serovar Pomona in 19/20 pigs. In 16 of these 19 pigs, leptospiral renal infection was confirmed by PCR and/or indirect IHC. Nephritic lesions were classified histologically into perivascular lymphocytic (4 pigs), lymphofollicular (6 pigs), lymphohistiocytic (8 pigs), and neutrophilic (2 pigs) pattern. MHCII expression by histiocytes and lymphocytes was observed in all lesions. Prominent MHCII expression in regenerating tubular epithelium was observed in lymphofollicular and lymphohistiocytic nephritis. No tubular colocalization between leptospiral and MHCII antigen was observed. Results suggest that during leptospiral nephritis, MHCII contributes to the intensity of the inflammatory response. Furthermore de novo MHCII expression in regenerating tubules may play a role in the defence mechanism against leptospiral tubular colonization. PMID:19179617

  16. Preferentially Expressed Antigen in Melanoma (PRAME) and the PRAME Family of Leucine-Rich Repeat Proteins.

    PubMed

    Hermes, Nora; Kewitz, Stefanie; Staege, Martin S

    2016-01-01

    Preferentially expressed antigen in melanoma (PRAME) is the best characterized member of the PRAME family of leucine-rich repeat (LRR) proteins. Mammalian genomes contain multiple members of the PRAME family whereas in other vertebrate genomes only one PRAME-like LRR protein was identified. PRAME is a cancer/testis antigen that is expressed at very low levels in normal adult tissues except testis but at high levels in a variety of cancer cells. In contrast to most other cancer/testis antigens, PRAME is expressed not only in solid tumors but also in leukemia cells. Expression of PRAME and other members of the PRAME family is regulated epigenetically. PRAME interacts with varying pathways that might be directly involved in the malignant phenotype of cancer cells. For instance, PRAME is able to dominantly repress retinoic acid signaling in these cells. On the other hand, PRAME-derived peptides can be recognized as epitopes by cytotoxic T cells and PRAME represents an attractive target for immunological treatment strategies. PMID:26694250

  17. Successful vaccination with a polyvalent live vector despite existing immunity to an expressed antigen.

    PubMed

    Flexner, C; Murphy, B R; Rooney, J F; Wohlenberg, C; Yuferov, V; Notkins, A L; Moss, B

    1988-09-15

    A global vaccination strategy must take into account production and delivery costs as well as efficacy and safety. A heat-stable, polyvalent vaccine that requires only one inoculation and induces a high level of humoral and cellular immunity against several diseases is therefore desirable. A new approach is to use live microorganisms such as mycobacteria, enteric bacteria, adenoviruses, herpesviruses and poxviruses as vaccine vectors. A potential limitation of live polyvalent vaccines, however, is existing immunity within the target population not only to the vector, but to any of the expressed antigens. This could restrict replication of the vector, curtail expression of antigens, and reduce the total immune response to the vaccine. Recently acquired immunity to vaccinia virus can severely limit the efficacy of a live recombinant vaccinia-based vaccine, so a strategy involving closely spaced inoculations with the same vector expressing different antigens may present difficulties. We have constructed a recombinant vaccinia virus that expresses surface proteins from two diverse pathogens, influenza A virus haemagglutinin and herpes simplex virus type 1 (HSV-1) glycoprotein D. Mice that had recently recovered from infection with either HSV-1 or influenza A virus could still be effectively immunized with the double recombinant. PMID:2842693

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

    PubMed

    Maeland, Johan A; Radtke, Andreas

    2013-12-27

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

  19. Plant expressed coccidial antigens as potential vaccine candidates in protecting chicken against coccidiosis.

    PubMed

    Sathish, Kota; Sriraman, Rajan; Subramanian, B Mohana; Rao, N Hanumantha; Kasa, Balaji; Donikeni, Jagan; Narasu, M Lakshmi; Srinivasan, V A

    2012-06-22

    Coccidiosis is a disease caused by intracellular parasites belonging to the genus Eimeria. In the present study, we transiently expressed two coccidial antigens EtMIC1 and EtMIC2 as poly histidine-tagged fusion proteins in tobacco. We have evaluated the protective efficacy of plant expressed EtMIC1 as monovalent and as well as bi-valent formulation where EtMIC1 and EtMIC2 were used in combination. The protective efficacy of these formulations was evaluated using homologous challenge in chickens. We observed better serum antibody response, weight gain and reduced oocyst shedding in birds immunized with EtMIC1 and EtMIC2 as bivalent formulation compared to monovalent formulation. However, IFN-γ response was not significant in birds immunized with EtMIC1 compared to the birds immunized with EtMIC2. Our results indicate the potential use of these antigens as vaccine candidates. PMID:22554463

  20. Cloning and expression in Escherichia coli of Mycoplasma gallisepticum antigens recognized by sera from infected chickens.

    PubMed

    Krause, D C; Kleven, S H; Lee, K K

    1990-01-01

    A clone bank of Mycoplasma gallisepticum (MG) strain A5969 DNA was prepared in the expression vector phage lambda gt11. Approximately 75% of the resulting phages were recombinants, based upon the insertional inactivation of the lacZ gene of the vector. Clones were screened immunologically with serum prepared from specific-pathogen-free white leghorn chickens that had been infected with aerosolized MG. Approximately 250 clones, or less than 1% of the recombinant phage, reacted positively to various degrees with the test serum and failed to react with serum from uninfected specific-pathogen-free control chickens. A single clone was chosen at random for comparison with a vector control by western immunoblot, revealing a polypeptide of 140,000 molecular weight in the clone profile but not the control profile that reacted with immune serum. Clones expressing MG antigens recognized during infection may provide an improved means for antigen preparation for serologic diagnosis of mycoplasmosis. PMID:2142422

  1. CD40 antigen is expressed by endothelial cells and tumor cells in Kaposi's sarcoma.

    PubMed Central

    Pammer, J.; Plettenberg, A.; Weninger, W.; Diller, B.; Mildner, M.; Uthman, A.; Issing, W.; Stürzl, M.; Tschachler, E.

    1996-01-01

    The CD40 antigen is a member of the tumor necrosis factor receptor/nerve growth factor receptor superfamily and is involved in cell proliferation, differentiation, and survival. Using different monoclonal antibodies, we found CD40 expression by immunohistochemistry on CD31- and CD34-positive Kaposi's sarcoma spindle cells in all tumors of 18 HIV-1 seropositive and 4 HIV-1 seronegative patients. Western blot analysis of tumor lysates detected a 48- to 50-kd glycoprotein corresponding to the CD40 antigen expressed by B lymphocytes. CD40 expression was also detectable in one of four cultures of spindle cells derived from Kaposi sarcoma tissue. Treatment of the CD40-positive spindle cells but not of the CD40-negative ones with interferon-gamma up-regulated CD40 surface expression. Besides on Kaposi sarcoma tumor cells, CD40 was distinctly present on vascular endothelial cells in areas within and adjacent to the tumors and in benign inflammatory lesions such as granulation tissue of HIV-1-negative patients. In contrast, CD34-negative endothelia of thin walled vessels, most likely lymphatics, were predominantly CD40 negative. Only faint or no CD40 expression was found on endothelial cells in normal skin. We conclude from our data that expression of the CD40 antigen by endothelial cells is up-regulated during tissue inflammation. As signaling through CD40 is able to increase cell survival, expression of CD40 by Kaposi sarcoma tumor cells might play an important role in the pathogenesis of this neoplasm. Images Figure 1 Figure 2 Figure 3 Figure 5 PMID:8623911

  2. Restricted expression of LW antigen on subsets of human B and T lymphocytes.

    PubMed

    Oliveira, O L; Thomas, D B; Lomas, C G; Tippett, P

    1984-01-01

    NIM-M8 is a monoclonal IgM antibody, specific for the LWab antigen as shown by its reaction with red cells of all donors except those lacking LWa, LWb and LWab. Indirect immunofluorescent staining and cell sorter analyses have shown that LWab is present on a subpopulation of human lymphocytes. Cell fractionation studies indicate that subsets of both B and T cells express LWab and it may, therefore, provide a further marker for heterogeneity in these lymphocyte populations. PMID:6443217

  3. Identification, expression, and immunogenicity of Kaposi's sarcoma-associated herpesvirus-encoded small viral capsid antigen.

    PubMed Central

    Lin, S F; Sun, R; Heston, L; Gradoville, L; Shedd, D; Haglund, K; Rigsby, M; Miller, G

    1997-01-01

    We describe a recombinant antigen for use in serologic tests for antibodies to Kaposi's sarcoma (KS)-associated herpesvirus (KSHV). The cDNA for a small viral capsid antigen (sVCA) was identified by immunoscreening of a library prepared from the BC-1 body cavity lymphoma cell line induced into KSHV lytic gene expression by sodium butyrate. The cDNA specified a 170-amino-acid peptide with homology to small viral capsid proteins encoded by the BFRF3 gene of Epstein-Barr virus and the ORF65 gene of herpesvirus saimiri. KSHV sVCA was expressed from a 0.85-kb mRNA present late in lytic KSHV replication in BC-1 cells. This transcript was sensitive to phosphonoacetic acid and phosphonoformic acid, inhibitors of herpesvirus DNA replication. KSHV sVCA expressed in mammalian cells or Escherichia coli or translated in vitro was recognized as an antigen by antisera from KS patients. Rabbit antisera raised to KSHV sVCA expressed in E. coli detected a 22-kDa protein in KSHV-infected human B cells. Overexpressed KSHV sVCA purified from E. coli and used as an antigen in immunoblot screening assay did not cross-react with EBV BFRF3. Antibodies to sVCA were present in 89% of 47 human immunodeficiency virus (HIV)-positive patients with KS, in 20% of 54 HIV-positive patients without KS, but in none of 122 other patients including children born to HIV-seropositive mothers and patients with hemophilia, autoimmune disease, or nasopharyngeal carcinoma. Low-titer antibody was detected in three sera from 28 healthy subjects. Antibodies to recombinant sVCA correlate with KS in high-risk populations. Recombinant sVCA can be used to examine the seroepidemiology of infection with KSHV in the general population. PMID:9060668

  4. Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies

    PubMed Central

    Tang, Jonathan CY; Drokhlyansky, Eugene; Etemad, Behzad; Rudolph, Stephanie; Guo, Binggege; Wang, Sui; Ellis, Emily G; Li, Jonathan Z; Cepko, Constance L

    2016-01-01

    The ability to detect and/or manipulate specific cell populations based upon the presence of intracellular protein epitopes would enable many types of studies and applications. Protein binders such as nanobodies (Nbs) can target untagged proteins (antigens) in the intracellular environment. However, genetically expressed protein binders are stable regardless of antigen expression, complicating their use for applications that require cell-specificity. Here, we created a conditional system in which the stability of an Nb depends upon an antigen of interest. We identified Nb framework mutations that can be used to rapidly create destabilized Nbs. Fusion of destabilized Nbs to various proteins enabled applications in living cells, such as optogenetic control of neural activity in specific cell types in the mouse brain, and detection of HIV-infected human cells by flow cytometry. These approaches are generalizable to other protein binders, and enable the rapid generation of single-polypeptide sensors and effectors active in cells expressing specific intracellular epitopes. DOI: http://dx.doi.org/10.7554/eLife.15312.001 PMID:27205882

  5. Fezf2 Orchestrates a Thymic Program of Self-Antigen Expression for Immune Tolerance.

    PubMed

    Takaba, Hiroyuki; Morishita, Yasuyuki; Tomofuji, Yoshihiko; Danks, Lynett; Nitta, Takeshi; Komatsu, Noriko; Kodama, Tatsuhiko; Takayanagi, Hiroshi

    2015-11-01

    Self-tolerance to immune reactions is established via promiscuous expression of tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs), leading to the elimination of T cells that respond to self-antigens. The transcriptional regulator Aire has been thought to be sufficient for the induction of TRAs, despite some indications that other factors may promote TRA expression in the thymus. Here, we show that the transcription factor Fezf2 directly regulates various TRA genes in mTECs independently of Aire. Mice lacking Fezf2 in mTECs displayed severe autoimmune symptoms, including the production of autoantibodies and inflammatory cell infiltration targeted to peripheral organs. These responses differed from those detected in Aire-deficient mice. Furthermore, Fezf2 expression and Aire expression are regulated by distinct signaling pathways and promote the expression of different classes of proteins. Thus, two independent factors, Fezf2 and Aire, permit the expression of TRAs in the thymus to ensure immune tolerance. PMID:26544942

  6. Expression of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris using the GAP promoter.

    PubMed

    Vassileva, A; Chugh, D A; Swaminathan, S; Khanna, N

    2001-06-01

    High-level expression and efficient assembly of Hepatitis B surface Antigen (HBsAg) particles have been reported in Pichia pastoris by integrating a single copy of the HBsAg gene under the control of the alcohol oxidase (AOX1) promoter. However, the time taken to reach peak product concentration is usually very long ( approximately 240 h). In this paper, we describe the expression of HBsAg in P. pastoris using the recently described glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Unlike the previously described AOX1 promoter based system (in which biomass is generated first followed by methanol-induced antigen production), biomass generation and antigen production occur simultaneously in medium containing glycerol or glucose. Maximal levels of HBsAg expression in case of the single copy AOX1 integrant (attained after 6 days of induction) exceeded the levels of antigen produced by the single copy GAP integrant. However, this was offset by continuous antigen production by the GAP clone. In an attempt to further enhance antigen production levels of the GAP clones, we isolated multicopy Pichia integrants containing up to four copies of the GAP promoter-driven constitutive expression cassette using the Zeocin screening procedure. The data demonstrated a direct correlation between the gene dosage and the levels of HBsAg expressed by the GAP clones. The effect of copy number was additive and the four copy clone resulted in about four-fold higher yield of HBsAg. The majority of HBsAg produced in the constitutive expression system was found to be of particulate form, based on sedimentation behaviour and particle-specific ELISA, suggesting that it has the potential to serve as an effective immunogen. These particles were sensitive to thiol reagents. We also explored the possibility of secreting the GAP expressed HBsAg in P. pastoris. In-frame fusion of the Saccharomyces cerevisiae alpha-factor secretion signal under the constitutive GAP promoter resulted in

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

    PubMed Central

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

    1986-01-01

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

  8. A Phase 1 Study of a Vaccine Targeting Preferentially Expressed Antigen in Melanoma and Prostate-specific Membrane Antigen in Patients With Advanced Solid Tumors

    PubMed Central

    Weber, Jeffrey S.; Vogelzang, Nicholas J.; Ernstoff, Marc S.; Goodman, Oscar B.; Cranmer, Lee D.; Marshall, John L.; Miles, Sabrina; Rosario, Dar; Diamond, David C.; Qiu, Zhiyong; Obrocea, Mihail; Bot, Adrian

    2013-01-01

    Summary Preferentially expressed antigen in melanoma (PRAME) and prostate-specific membrane antigen (PSMA) are tumor-associated antigens implicated in cellular differentiation, genetic stability, and angiogenesis. MKC1106-PP is an immunotherapeutic regimen cotargeting PRAME and PSMA, comprised of a recombinant plasmid (pPRA-PSM encoding fragments derived from both antigens) and 2 peptides (E-PRA and E-PSM derived from PRAME and PSMA, respectively). This multicenter study evaluated MKC1106-PP with a fixed plasmid dose and 2 different peptide doses, administered by intralymph node injection in a prime-boost sequence in human leukocyte antigen-A*0201 and tumor-antigen-positive patients with progressing metastatic solid tumors who had failed standard therapy. Immune monitoring was done by tetramer and enzymatic-linked immune spot analysis. The treatment was well tolerated, with no significant differences in safety, immune response, and clinical outcome relative to peptide doses. Fifteen of 24 evaluable patients showed an immune response, as defined by the expansion of PRAME-specific or PSMA-specific T cells in the blood. There were no partial or complete responses by the Response Evaluation Criteria in Solid Tumors. Seven patients showed stable disease (SD) for 6 months or longer, or prostate specific antigen decline: 4 of 10 with prostate carcinoma, 2 of 2 with renal clear cell carcinoma, and 1 of 10 with metastatic melanoma. In addition, there was an association between the induction and persistence of antigen-specific T cells in blood above baseline levels and disease control, defined as SD for 6 months or longer. These results support further development of MKC1106-PP in specific clinical indications. PMID:21760528

  9. Immunological screening of a glycoprotein antigen expressed by Zajdela ascites hepatoma cells on normal rat tissues and tumour cells.

    PubMed

    Nato, F; Goulut, C; Mirshahi, M; Bourrillon, R

    1991-10-01

    Expression of the glycoprotein MII2 antigen originally identified in Zajdela ascites hepatoma cells was investigated in several normal rat tissues and in more or less differentiated tumours using biochemical and immunological approaches. SDS-polyacrylamide gel electrophoresis followed by fluorography or immunoblotting with an antiserum raised against the purified MII2 antigen revealed that this antigen was absent from normal liver cells. ELISA assays, indirect immunofluorescence and immunoprecipitation experiments using the same antiserum showed that this glycoprotein was not expressed in various normal tissues such as liver, spleen, lung, pancreas, intestine and stomach, but it was unexpectedly detected in kidney and thymic tissues. However, the molecular weight of the antigens immunoprecipitated from kidney and thymus was lower than the one of MII2 (Mr of 60,000 versus 110,000-160,000 for purified MII2). No staining was observed in embryonic rat liver at 10 and 20 days of development. Moreover, this antigen was present on the surface of Morris hepatoma 7777, another rapidly proliferating and poorly differentiated hepatocellular carcinoma. In contrast, this antigen was not detected on the surface of in vitro Zajdela hepatoma cells (ZHC) or of partially differentiated hepatomas (Faza) which have recovered some hepatic functions. In addition, the MII2 antigen was found on the human non-hepatic HT-29 tumour cell line, under its undifferentiated form (HT-29 G+ subline). The possible relationships between the expression of this antigen and both the malignant transformation process and the differentiation process are discussed. PMID:1656518

  10. The expression of desmosomal and corneodesmosomal antigens shows specific variations during the terminal differentiation of epidermis and hair follicle epithelia.

    PubMed

    Mils, V; Vincent, C; Croute, F; Serre, G

    1992-09-01

    Using five monoclonal antibodies (MAb), we studied by indirect immunofluorescence the desmosomes and a junctional structure specific to cornified layers, the corneodesmosome, in normal and plantar epidermis and in the various sheaths of the anagen hair follicle. The monoclonal antibodies DP1&2.2-15, PG5.1, and DG3.10, specific for desmoplakins I/II, plakoglobin, and desmoglein I, respectively, were used to study the desmosome antigens, and G36-19 and G20-21 to study the corneodesmosome antigens. The distribution and sequence of expression of the five antigens allowed the nine epithelial differentiation pathways studied to be merged into four distinct families: non-plantar epidermis, characterized by the absence of desmosome and corneodesmosome antigens in the stratum corneum; the outer root sheath of the hair follicle, which behaves like the viable layers of the epidermis with regard to the desmosome antigens but does not express the corneodesmosome antigens; plantar epidermis and the three components of the inner root sheath in which the corneodesmosome antigens are present up to the desquamating layer; and the three components of the hair shaft, which are characterized by the absence of expression of both the desmosome and the corneodesmosome antigens in its mature portion. PMID:1506670

  11. Interferon-beta downregulates expression of VLA-4 antigen and antagonizes interferon-gamma-induced expression of HLA-DQ on human peripheral blood monocytes.

    PubMed

    Soilu-Hänninen, M; Salmi, A; Salonen, R

    1995-07-01

    We have studied the effect of recombinant human IFN-beta on the basal and IFN-gamma-induced expression of adhesion molecules and class II MHC antigens on human peripheral blood monocytes and on ICAM-1 (intercellular adhesion molecule-1) expression of a human umbilical vein endothelial cell line (EAhy 926). We show that IFN-beta downregulates both basal and IFN-gamma-induced expression of VLA-4 (very late activation antigen-4) antigen on monocytes, but has no effect on the expression of CD11a, CD11b, CD11c, L-selectin, CD18, ICAM-1, beta 1-integrin or CD44 on monocytes or ICAM-1 on EAhy 926 cells. We also show that IFN-beta antagonizes the IFN-gamma-induced expression of HLA-DQ-antigen, but not HLA-DR or HLA-DP antigens on monocyte surface. These findings may partially explain the beneficial effect of IFN-beta in multiple sclerosis, since VLA-4-antigen is critical for leukocyte recruitment into inflamed brain and downregulation of HLA-class II expression diminishes antigen presenting capacity of monocytes. PMID:7642754

  12. Changes in tumor-antigen expression profile as human small-cell lung cancers progress

    PubMed Central

    Ge, Li-Sheng; Hoa, Neil T.; Lambrecht, Nils; Dacosta-Iyer, Maria; Ouyang, Yi; Abolhoda, Amir; Jadus, Martin R.

    2015-01-01

    Objective 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. Methods 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. Results 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. Conclusion Our findings suggested that more immunological targets became available as the tumor responded to chemotherapy and proceeded toward its terminal stages. PMID:26175925

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

    PubMed

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

    2015-02-01

    DNA vaccines require improvement for human use because they are generally weak stimulators of the immune system in humans. The efficacy of DNA vaccines can be improved using a viral replicon as vector to administer antigen of pathogen. In this study, we comprehensively evaluated the conventional non-viral DNA, viral replicon DNA or viral replicon particles (VRP) vaccines encoding different forms of anthrax protective antigen (PA) for specific immunity and protective potency against anthrax. Our current results clearly suggested that these viral replicon DNA or VRP vaccines derived from Semliki Forest virus (SFV) induced stronger PA-specific immune responses than the conventional non-viral DNA vaccines when encoding the same antigen forms, which resulted in potent protection against challenge with the Bacillus anthracis strain A16R. Additionally, the naked PA-expressing SFV replicon DNA or VRP vaccines without the need for high doses or demanding particular delivery regimens elicited robust immune responses and afforded completely protective potencies, which indicated the potential of the SFV replicon as vector of anthrax vaccines for use in clinical application. Therefore, our results suggest that these PA-expressing SFV replicon DNA or VRP vaccines may be suitable as candidate vaccines against anthrax. PMID:25102364

  14. Comparison of Class II HLA antigen expression in normal and carcinomatous human breast cells

    SciTech Connect

    Bernard, D.J.; Maurizis, J.C.; Chassagne, J.; Chollet, P.; Plagne, R.

    1985-03-01

    Class II HLA antigen expression in breast carcinoma and normal breast gland cells was compared using a method more accurate than immunofluorescence. This new method involves labeling membrane proteins with /sup 131/I and the anti-Class II HLA monoclonal antibody with /sup 125/I. The isolation and purification of the doubly labeled (/sup 125/I-/sup 131/I) immune complex was performed by affinity chromatography and chromatofocusing successively. When the specific activity of glycoproteins is known, the amount of glycoprotein which bind specifically to the anti-Class II HLA monoclonal antibody can be deduced. In breast carcinoma cells, 1.5 to 2% of the purified glycoproteins bind specifically to the monoclonal antibody, whereas less than 0.3% of normal breast gland cells binds. In contrast, leukemic cells, of which 80 to 90% possess Class II HLA antigens, 2 to 3% of Class II HLA glycoproteins bind specifically with the anti-Class II HLA monoclonal antibody.

  15. Expression of the common acute lymphoblastic leukemia antigen (CD10) in mesenchymal tumors.

    PubMed Central

    Mechtersheimer, G.; Möller, P.

    1989-01-01

    The expression of the CD10 antigen, formerly designated as common acute lymphoblastic leukemia antigen and recently identified as neutral endopeptidase, was examined immunohistochemically in 26 benign and in 55 malignant mesenchymal tumors. CD10 expression was found in 4 of 4 leiomyomas, 7 of 10 leiomyosarcomas, 1 of 6 rhabdomyosarcomas, 2 of 2 Triton tumors, 1 of 2 aggressive fibromatoses, 1 of 3 fibrosarcomas, 1 of 4 synovial sarcomas, 1 of 1 giant cell tumors of tendon sheath, 4 of 4 malignant fibrous histiocytomas, 3 of 3 Ewing's sarcomas, and 2 of 3 osteosarcomas. Furthermore, CD10 was expressed consistently in the myoepithelial compartment of 12 fibroadenomas and, in 7 of these cases, in a minor stromal cell population, probably of (myo-) fibroblastic origin. Tumors of adipose tissue (4 lipomas, 5 liposarcomas), tumors of autonomic ganglia (2 ganglioneuromas, 1 ganglioneuroblastoma, 2 neuroblastomas), tumors of peripheral nerves with purely schwannian differentiation (7 malignant schwannomas), and tumors of disputed origin were consistently CD10-negative, however, as were single cases of fibroma and chondrosarcoma. These findings indicate that the expression of CD10 is a frequent but not obligatory feature in some mesenchymal tumors. Therefore CD10 is of value in the differential diagnosis of mesenchymal tumors. Images Figure 1 Figure 2 Figure 3 PMID:2541615

  16. Enhanced expression of codon optimized Mycobacterium avium subsp. paratuberculosis antigens in Lactobacillus salivarius

    PubMed Central

    Johnston, Christopher D.; Bannantine, John P.; Govender, Rodney; Endersen, Lorraine; Pletzer, Daniel; Weingart, Helge; Coffey, Aidan; O'Mahony, Jim; Sleator, Roy D.

    2014-01-01

    It is well documented that open reading frames containing high GC content show poor expression in A+T rich hosts. Specifically, G+C-rich codon usage is a limiting factor in heterologous expression of Mycobacterium avium subsp. paratuberculosis (MAP) proteins using Lactobacillus salivarius. However, re-engineering opening reading frames through synonymous substitutions can offset codon bias and greatly enhance MAP protein production in this host. In this report, we demonstrate that codon-usage manipulation of MAP2121c can enhance the heterologous expression of the major membrane protein (MMP), analogous to the form in which it is produced natively by MAP bacilli. When heterologously over-expressed, antigenic determinants were preserved in synthetic MMP proteins as shown by monoclonal antibody mediated ELISA. Moreover, MMP is a membrane protein in MAP, which is also targeted to the cellular surface of recombinant L. salivarius at levels comparable to MAP. Additionally, we previously engineered MAP3733c (encoding MptD) and show herein that MptD displays the tendency to associate with the cytoplasmic membrane boundary under confocal microscopy and the intracellularly accumulated protein selectively adheres to the MptD-specific bacteriophage fMptD. This work demonstrates there is potential for L. salivarius as a viable antigen delivery vehicle for MAP, which may provide an effective mucosal vaccine against Johne's disease. PMID:25237653

  17. The origin of Ia antigen-expressing cells in the rat kidney.

    PubMed Central

    Gurner, A. C.; Smith, J.; Cattell, V.

    1987-01-01

    The lineage of Ia antigen expressing (Ia+) cells that have been detected in the parenchyma and interstitium of the rat kidney has not been defined. The authors have studied the origins of Ia+ cells in chimeric rats using monoclonal antibodies to define cells of bone marrow and parenchymal origin. PVGc RTI rats (recipients) received intravenously 2 X 10(6) bone marrow cells from F1 hybrid PVG RTIc/RTIu rats (donors) 1 day after 1000 rads whole body irradiation. Ia chimerism was monitored in blood and isolated glomeruli by immunofluorescence and in frozen sections by immunoperoxidase, using monoclonal antibodies MRC OX3 (anti-Ia RTIu), MRC OX4 (anti-RTIc and u), and MRC OXI (anti-rat leukocyte common antigen). In normal F1 hybrid kidneys, glomerular cell counts were as follows: OXI+, 7.19 +/- 0.23/gl; OX4+, 3.03 +/- 0.14; OX3+, 2.34 +/- 0.1 (76% detectable expression of RTIu). OXI+, OX4+, and OX3+ cells were codistributed in cells in the interstitium between renal tubules. Proximal tubules were weakly OX4+, OX3+. In chimeric rats 5 days after irradiation, blood leukocytes, and renal OX1+ and OX4+ cells were depleted; OX3+ cells were not detected; by 4 weeks blood leukocytes were restored to normal numbers, and 85% of Ia+ cells were OX3+. By 6 weeks OXI+ and OX4+ cells were restored in glomeruli and interstitium, with increasing expression of OX3+ cells; at 10 weeks 75% of glomerular Ia+ cells were OX3+ (equivalent to detectable level of OX3+ cells in normal F1 hybrids) and OX1+, OX4+, and OX3+ cells appeared in equivalent numbers in the interstitium. Groups of proximal tubules were OX4+ and OX3-. These results in established bone marrow chimeras show that in the normal rat kidney bone marrow derived leukocytes expressing Ia antigen are present in the glomerulus and interstitium. Ia antigen is also expressed on some proximal tubular cells. There is no evidence for endothelial Ia positivity. Images Figure 4 Figure 5 PMID:2437803

  18. Studies on the Thomsen-Friedenreich antigen in human colon with the lectin Amaranthin. Normal and neoplastic epithelium express only cryptic T antigen.

    PubMed

    Sata, T; Roth, J; Zuber, C; Stamm, B; Rinderle, S J; Goldstein, I J; Heitz, P U

    1992-02-01

    The lectin Amaranthin has been shown to be highly specific for the galactose beta 1,3 N-acetylgalactosamine-alpha and sialic acid alpha 2,3 galactose beta 1,3 N-acetylgalactosamine-alpha sequence which represents the Thomsen-Friedenreich (T) antigen and its cryptic form, respectively. Previously, we demonstrated the usefulness of gold-labeled Amaranthin for the histochemical detection of the T antigen and its cryptic form. Application of the galactose oxidase (GO)-Schiff sequence abolished lectin binding to the T antigen but not its cryptic form, and therefore permitted their differentiation. In the present study we have analyzed by light and electron microscopy the distribution and subcellular localization of Amaranthin binding sites in normal, dysplastic and neoplastic colonic epithelium. Furthermore, a monoclonal antibody raised against synthetic galactose bera 1,3 N-acetylgalactosamine-alpha-bovine serum albumin was applied as a reagent for the T antigen. In normal colonic mucosa, two different Amaranthin staining patterns existed: (a) reactivity restricted to the lower portion of the crypts which was principally observed in the left colon, and (b) reactivity along the entire length of the crypts and in the surface epithelium with goblet cell staining in the upper portion of the crypts which was principally observed in the right colon. This Amaranthin staining was resistant to GO-Schiff treatment. No immunostaining with the monoclonal anti-T antigen was observed. Investigation of transitional mucosa, adenocarcinomas of different degrees of differentiation and mucinous carcinomas as well as adenomas with different degrees of dysplasia all revealed positive Amaranthin staining. The lectin staining was resistant to GO-Schiff treatment, and immunolabeling with the monoclonal antibody against the T antigen was absent. These results indicate that only the cryptic form of the T antigen is expressed in normal, dysplastic and neoplastic human colonic epithelium. PMID

  19. Expression of the Major Surface Antigen of Plasmodium knowlesi Sporozoites in Yeast

    NASA Astrophysics Data System (ADS)

    Sharma, Shobhona; Godson, G. Nigel

    1985-05-01

    The circumsporozoite protein, a surface antigen of the sporozoite stage of the monkey malarial parasite Plasmodium knowlesi, was expressed in the yeast Saccharomyces cerevisiae by using an expression vector containing the 5' regulatory region of the yeast alcohol dehydrogenase I gene. It was necessary to eliminate the entire 5' upstream region of the parasite DNA to obtain the expression of this protein. Only the circumsporozoite precursor protein was produced by the yeast transformants, as detected by immunoblotting. About 55 and 20 percent of the circumsporozoite protein produced in yeast was associated with the 25,000g and 150,000g particulate fractions, respectively. The protein could be solubilized in Triton X-100 and was stable in solubilized extracts.

  20. CD80 antigen expression as a predictor of ex vivo chemosensitivity in chronic lymphocytic leukemia.

    PubMed

    Kivekäs, Ilkka; Hulkkonen, Janne; Hurme, Mikko; Vilpo, Leena; Vilpo, Juhani

    2002-05-01

    We investigated the correlation between expression of 31 surface membrane antigens and chemosensitivity of peripheral blood mononuclear cells from 36 patients with CLL. The sensitivity of CLL cells to nine drugs (2'-chlorodeoxyadenosine, cisplatin, chlorambucil, cyclosporin A, doxorubicin, fludarabine, prednisolone, verapamil and vincristine) and two types of irradiation (gamma and UV-irradiation) was determined from dose-response curves of 4-day cultures ex vivo. The results indicated that the CLL cases responding to purine analogs (2'-chlorodeoxyadenosine and fludarabine) can be identified according to CD80 expression: all resistant cases had low or negative CD80 expression. No other correlations were revealed. CD80 may be a surrogate chemosensitivity marker for purine analogs. PMID:11916516

  1. Hepatic expression of the woodchuck hepatitis virus X-antigen during acute and chronic infection and detection of a woodchuck hepatitis virus X-antigen antibody response.

    PubMed

    Jacob, J R; Ascenzi, M A; Roneker, C A; Toshkov, I A; Cote, P J; Gerin, J L; Tennant, B C

    1997-12-01

    The expression and localization of the woodchuck hepatitis virus X-antigen (WHxAg) was examined and compared with other markers of a woodchuck hepatitis virus (WHV) infection using rabbit antisera generated against recombinant WHxAg produced in bacteria. Cellular fractionation studies showed that WHxAg was localized to the soluble and cytoskeletal fractions of the cell when assayed by immunoprecipitation of [35S]-met-cys labeled extracts derived from primary cultures of acute WHV-infected hepatocytes. Immunohistochemical examination of liver from chronic WHV-infected animals showed WHV core antigen (WHcAg) and WHxAg expression in non-neoplastic tissue. The WHxAg was found localized to the cytoplasm of infected cells, similar to WHcAg. WHxAg expression was diminished in the foci of altered hepatocytes and in hepatocellular adenomas but was found in only 1 of 11 hepatocellular carcinomas (HCC). Hepatic biopsies from woodchucks experimentally inoculated with WHV were examined during the acute phase of infection and during convalescence for WHcAg and WHxAg expression by immunohistochemistry. Concurrent expression of WHcAg and WHxAg was observed during the viremic phase of infection. The two antigens exhibited similar localization to the cell cytoplasm, similar distribution within the liver lobule, and similar patterns of clearance during convalescence. An immune response to WHxAg was documented in some woodchucks following acute WHV infection. These studies further define the woodchuck model of HBV infection and should allow for the investigation of the role of hepadnaviral X-antigen expression in the pathogenesis of chronic hepatitis and HCC. PMID:9398005

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

    SciTech Connect

    Habets, W.J.; Sillekens, P.T.G.; Hoet, M.H.; Schalken, J.A.; Roebroek, A.J.M.; Leunissen, J.A.M.; Van de Ven, W.J.M.; Van Venrooij, W.J.

    1987-04-01

    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 lambdaHB''-1 from a phage lambdagt11 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 lambdaHB''-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 lambdaHB''-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 lambdaHB''-1 cDNA insert and subsequent translation resulted in a protein product with the molecular size of the B'' protein. These data demonstrate that clone lambdaHB''-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.

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

    PubMed

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

    2008-07-17

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

  4. Mycoplasma gallisepticum in vivo induced antigens expressed during infection in chickens.

    PubMed

    Ron, Merav; Gorelick-Ashkenazi, Anna; Levisohn, Sharon; Nir-Paz, Ran; Geary, Steven J; Tulman, Edan; Lysnyansky, Inna; Yogev, David

    2015-02-25

    Until now only a few genes encoding virulence factors have been characterized in the avian pathogen Mycoplasma gallisepticum. In order to identify candidate targets associated with infection we applied an immunoscreening technique-in vivo induced antigen technology (IVIAT)-to detect immunogens of M. gallisepticum strain Rlow expressed preferentially during in vivo infection. We identified 13 in vivo-induced (IVI) proteins that correspond to different functional categories including: previously reported putative virulence factors (GapA, PlpA, Hlp3, VlhA 1.07 and VlhA 4.01), transport (PotE, MGA_0241 and 0654), translation (L2, L23, ValS), chaperone (GroEL) and a protein with unknown function (MGA_0042). To validate the in vivo antigenic reactivity, 10 IVI proteins were tested by Western blot analysis using serum samples collected from chickens experimentally (with strain Rlow) and naturally (outbreaks, N=3) infected with M. gallisepticum. All IVI proteins tested were immunogenic. To corroborate these results, we tested expression of IVI genes in chickens experimentally infected with M. gallisepticum Rlow, and in MRC-5 human lung fibroblasts cell culture by using relative real time reverse-transcription PCR (RT-PCR). With the exception of MGA_0338, all six genes tested (MGA_1199, 0042, 0654, 0712, 0928 and 0241) were upregulated at least at one time point during experimental infection (2-4 week post-infection). In contrast, the expression of seven out of eight IVI genes (MGA_1199, 0152, 0338, 0042, 0654, 0712, 0928) were downregulated in MRC-5 cell culture at both 2 and 4h PI; MGA_0241 was upregulated 2h PI. Our data suggest that the identified IVI antigens may have important roles in the pathogenesis of M. gallisepticum infection in vivo. PMID:25575879

  5. PD-1 expression conditions T cell avidity within an antigen-specific repertoire

    PubMed Central

    Simon, Sylvain; Vignard, Virginie; Florenceau, Laetitia; Dreno, B.; Khammari, A.; Lang, F.; Labarriere, N.

    2016-01-01

    ABSTRACT Despite its negative regulatory role on tumor-specific T cells, Programmed cell death 1 (PD-1) is also a marker of activated tumor-infiltrating T cells. In cancer, PD-1 blockade partially reverses T cell dysfunction allowing the amplification of tumor reactive T cells. Here, we investigated the role of PD-1 signaling on effector/memory human T cells specific for shared melanoma antigens, derived from blood. We documented for the first time the existence of melanoma-specific T cell clones unable to express PD-1. This stable feature was due to the persistent methylation of the PDCD1 promoter. These PD-1neg clones were of lower avidity than their PD-1pos counterparts, suggesting that high-affinity-specific T cell clones unable to express PD-1 are not or rarely present in peripheral blood, as they are probably eliminated by negative selection, due to their high reactivity. We also documented the existence of such PD-1neg T cell clones in melanoma tumor-infiltrating lymphocytes (TIL), which also exhibited a lower functional avidity than PD-1pos TIL clones. This clearly shows that PD-1 expression identifies antigen-specific T cell clonotypes of high functional avidity. Finally, we demonstrated that PD-1 blockade during the in vitro selection process of Melan-A-specific T cells favored the amplification of higher avidity T cell clonotypes. This preferential amplification of high-avidity memory T cells upon PD-1 blockade resonates with the expansion of reactive T cells, including neo-antigen-specific T cells observed in anti-PD-1-treated patients. This feature should also be a useful biomarker of clinical efficiency, while providing new insights for adoptive transfer treatments. PMID:26942093

  6. Children with postsurgical capillary leak syndrome can be distinguished by antigen expression on neutrophils and monocytes

    NASA Astrophysics Data System (ADS)

    Tarnok, Attila; Pipek, Michal; Valet, Guenter; Richter, Jacqueline; Hambsch, Joerg; Schneider, Peter

    1999-04-01

    Our initial studies indicate that children who develop post- operative capillary leak syndrome (CLS) following cardiac surgery with cardiopulmonary bypass (CPB) can be distinguished based on their pre-operative level of circulating cytokines an adhesion molecules. We tested flow cytometric analysis of surface antigen expression as a potential assay for risk assessment of CLS. 24th preoperative blood samples were stained with monoclonal antibodies for the adhesion molecules ICAM-1, LFA1, MAC1, (beta) -integrin, activation markers CD25, CD54, CD69, HLA- DR, CD14 or CD4. Cells were measured on a dual-laser flow cytometer calibrated with microbeads. Antigen expression was detected as mean fluorescence intensity. The data indicate, that neutrophils of CLS patients express preoperatively higher levels of LFA1 and monocytes higher levels of HLA-DR and activation markers thus are in a state of activation. This could in combination with surgical trauma and CPB lead to their additional stimulation and migration into sites of inflammation and induce postoperative CLS. It is planned to set up a Flow-Classification program for individual risk assessment. By discriminate analysis over 80 percent of the patients were correctly classified. Our preliminary study indicates that flow cytometry with its low samples requirements and rapid access of the results could be a powerful tool to perform risk assessment prior to pediatric open heart surgery.

  7. Molecular cloning and expression in Pichia pastoris of a hypoallergenic antigen 5.

    PubMed

    Vinzón, Sabrina E; Pirpignani, María L; Nowicki, Cristina; Biscoglio de Jiménez Bonino, Mirtha

    2010-09-01

    Stings by insects from the Hymenoptera order can cause life-threatening allergic reactions and impair life quality. Immunotherapy with venom extracts is the most extensively employed treatment to reduce morbidity and mortality, but purified and safer allergy vaccines are needed. Antigen 5 is an important allergen of vespid venoms. We previously reported that Antigen 5 from Polybia scutellaris (Poly s 5) is likely to be a hypoallergenic variant. On the basis of such findings, this work deals with the recombinant expression and purification of Poly s 5 in Pichia pastoris. In order to overcome non-native glycosylation of the recombinant protein, it was necessary to delete a glycosylation site. On the other hand, different strategies were attempted to obtain a satisfactory yield of the protein; moreover, the influence of the methanol concentration in the expression medium was investigated and found to be crucial. Mass spectrometry, N-terminal sequencing, and IgG-binding inhibition assays were performed. Results allowed us to confirm the immunological equivalence between the recombinant and the natural proteins. In conclusion, a novel protocol for the recombinant expression of Poly s 5 in P. pastoris was designed thus bringing about a high yield of the protein useful for clinical and scientific purposes. PMID:20371379

  8. Antigen recognition and presentation in periapical tissues: a role for TLR expressing cells?

    PubMed

    Desai, S V; Love, R M; Rich, A M; Seymour, G J

    2011-02-01

    Bacteria are the prime cause of periapical diseases and root canal microbiology is a well-researched area of endodontics. Antigen-presenting cells (APCs) are present in periapical lesions of endodontic origin and play a substantial role in recognizing, processing and presenting pathogenic antigens to the adaptive immune system such as an effective and long-lasting immune response is generated against the specific pathogens. Toll-like receptors (TLRs) are germ-line encoded pathogen recognition receptors (PRR) expressed by various APCs which induce their maturation, lead to gene transcription in the nucleus and the production of several pro- and anti-inflammatory cytokines. Thirteen TLRs have been discovered, 10 of which have been identified in humans so far. Preliminary studies of dental pulp tissue have demonstrated various cell types expressing different TLRs in response to commonly encountered microorganisms. However, there is little information available regarding the expression and function of the various TLRs in human periapical lesions. This review discusses the interactions of various APCs in periapical lesions and the possible roles of different TLRs and APCs in pulp/periapical pathogen recognition and presentation to the adaptive immune system in the initiation and sustaining of periapical diseases. PMID:21083574

  9. Induction of embryonic major histocompatibility complex antigen expression by gamma-IFN.

    PubMed

    Warner, C M; Almquist, C D; Toulimat, M H; Xu, Y

    1993-07-01

    Preimplantation mouse embryos were incubated in vitro with mouse recombinant gamma-interferon (IFN). The effect of the gamma-IFN on major histocompatibility complex (MHC) class I antigen expression was tested using an ELISA procedure. It was found that there is a doubling of Db antigens and a tripling of Qa-2 antigens on C57BL/6 mouse embryos cultured from the 8-cell stage for 24 h in the presence of 10(5) units/ml gamma-IFN. The effect of gamma-IFN on the rate of preimplantation embryonic development was tested by culturing 2-cell embryos for 48 h and 8-cell embryos for 24 h in the presence of varying concentrations of gamma-IFN up to 10(6) units/ml. Two methods were used to assess the cell number per embryo after the culture period: incorporation of [3H]thymidine into DNA, and direct counting of nuclei in fixed and stained embryos. Both methods showed that treatment with gamma-IFN increases the rate of development of preimplantation mouse embryos. Since rate of preimplantation embryonic development is genetically controlled by the Ped gene, it is suggested that gamma-IFN has a direct effect on the Ped gene phenotype of preimplantation mouse embryos. PMID:8229991

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

    PubMed Central

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

    2013-01-01

    Summary 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 CART 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

  11. Identification and preliminary characterization of Treponema pallidum protein antigens expressed in Escherichia coli.

    PubMed

    Stamm, L V; Kerner, T C; Bankaitis, V A; Bassford, P J

    1983-08-01

    We have previously described the construction in Escherichia coli K-12 of a hybrid plasmid colony bank of Treponema pallidum (Nichols strain) genomic DNA. By screening a portion of this bank with an in situ immunoassay, we identified six E. coli clones that express T. pallidum antigens. In this study, the recombinant plasmids from each of these clones have been analyzed in E. coli maxicells and have been found to encode a number of proteins that are not of vector pBR322 origin and are, therefore, of treponemal origin. In each case, several of these proteins can be specifically precipitated from solubilized maxicell extracts by high-titer experimental rabbit syphilitic serum. Certain of these proteins are also precipitated by high-titer latent human syphilitic sera (HSS). The T. pallidum DNA inserts in these plasmids range in size from 6.2 to 14 kilobase pairs, and from the restriction patterns of the inserts and the protein profiles generated by each plasmid in maxicells, it is apparent that we have recovered a total of four unique clones from our colony bank. Recombinant plasmids pLVS3 and pLVS5 were of particular interest. Plasmid pLVS3 encodes three major protein antigens with molecular weights of 39,000, 35,000, and 25,000. These three proteins, which were not recognized by pooled normal human sera, were efficiently precipitated by most secondary HSS, latent HSS, and late HSS tested. These proteins were also precipitated, although somewhat inefficiently, by most primary HSS tested. Plasmid pLVS5 encodes a major protein antigen with a molecular weight of 32,000 and several minor protein antigens that, although efficiently precipitated by experimental rabbit syphilitic serum, were generally not recognized by the various HSS tested. Evidence is presented indicating that the protein antigens encoded by plasmids pLVS3 and pLVS5 are specific for pathogenic treponemal species. We have also demonstrated that immunoglobulin G antibodies directed against these protein

  12. Biochemical characterization of the recombinant Boophilus microplus Bm86 antigen expressed by transformed Pichia pastoris cells.

    PubMed

    Montesino, R; Cremata, J; Rodríguez, M; Besada, V; Falcón, V; de la Fuente, J

    1996-02-01

    In the present paper we report the biochemical characteristics of the recombinant tick (Boophilus microplus) gut antigen Bm86 that previously has been cloned, expressed and recovered at high levels in the methylotrophic yeast Pichia pastoris. The results demonstrate that rBm86 had a modification at position 92 (Thr replaced by Ile) and aggregated, forming particles ranging between 17 and 40 nm. The rBm86 was N-glycosylated, having at least two non-glycosylated sequons (Asn-329 and Asn-363) and a ratio of only 0.4/65 (free Cys/total Cys)/mol of protein. PMID:8867893

  13. Human Skin Cells That Express Stage-Specific Embryonic Antigen 3 Associate with Dermal Tissue Regeneration

    PubMed Central

    Vega Crespo, Agustin; Awe, Jason P.; Reijo Pera, Renee

    2012-01-01

    Abstract Stage-specific embryonic antigen 3 (SSEA3) is a glycosphingolipid that has previously been used to identify cells with stem cell-like, multipotent, and pluripotent characteristics. A rare subpopulation of SSEA3-expressing cells exists in the dermis of adult human skin. These SSEA3-expressing cells undergo a significant increase in cell number in response to injury, suggesting a possible role in regeneration. These SSEA3-expressing regeneration-associated (SERA) cells were derived through primary cell culture, purified by fluorescence-activated cell sorting (FACS), and characterized. Longer in vitro culture of the primary skin cells led to lower SSEA3 expression stability after FACS-based purification, suggesting that the current culture conditions may need to be optimized to permit the large-scale expansion of SERA cells. The SERA cells demonstrated a global transcriptional state that was most similar to bone marrow- and fat-derived mesenchymal stem cells (MSCs), and the highest expressing SSEA3-expressing cells co-expressed CD105 (clone 35). However, while a rare population of MSCs was observed in primary human skin cell cultures that could differentiate into adipocytes, osteoblasts, or chondrocytes, SERA cells did not possess this differentiation capacity, suggesting that there are at least two different rare subpopulations in adult human skin primary cultures. The identification, efficient purification, and large-scale expansion of these rare subpopulations (SERA cells and MSCs) from heterogeneous adult human skin primary cell cultures may have applications for future patient-specific cellular therapies. PMID:23514702

  14. The expression of endothelial cell surface antigens by AIDS-associated Kaposi's sarcoma. Evidence for a vascular endothelial cell origin.

    PubMed Central

    Rutgers, J. L.; Wieczorek, R.; Bonetti, F.; Kaplan, K. L.; Posnett, D. N.; Friedman-Kien, A. E.; Knowles, D. M.

    1986-01-01

    The authors investigated 19 cases of Kaposi's sarcoma (KS) obtained from patients with the acquired immune deficiency syndrome (AIDS) for their expression of Factor VIII-related antigen (FVIIIRAg), HLA-DR (Ia) antigens, OKM1, and three distinctive vascular, but not lymphatic, endothelial-cell-associated antigens, E92, OKM5, and HCl. Antigen expression was demonstrated by immunoperoxidase staining of cryostat sections. FVIIIRAg is strongly expressed by the cells lining the vascular spaces (VCs) but is absent, weakly or focally, and variably expressed by the spindle cell (SC) component of KS. The VC component of each KS lesion examined strongly expressed E92, moderately expressed HCl, and weakly expressed OKM5. In contrast, the entire SC component of each KS lesion studied strongly expressed E92 and OKM5 and weakly expressed HCl. Neither the VCs nor the SCs expressed OKM1. These studies provide strong and compelling evidence for the vascular endothelial cell histogenesis of both the vascular and spindle cell components of KS, demonstrate the intertumor and intratumor phenotypic heterogeneity of KS, and suggest that monoclonal antibodies OKM5 and anti-E92 are the best currently available immunohistochemical markers for identifying the spindle cell component of AIDS-associated KS in cryostat sections. Images Figure 1 PMID:3953772

  15. Enhancing Antitumor Efficacy of Chimeric Antigen Receptor T Cells Through Constitutive CD40L Expression

    PubMed Central

    Curran, Kevin J; Seinstra, Beatrijs A; Nikhamin, Yan; Yeh, Raymond; Usachenko, Yelena; van Leeuwen, Dayenne G; Purdon, Terence; Pegram, Hollie J; Brentjens, Renier J

    2015-01-01

    Adoptive cell therapy with genetically modified T cells expressing a chimeric antigen receptor (CAR) is a promising therapy for patients with B-cell acute lymphoblastic leukemia. However, CAR-modified T cells (CAR T cells) have mostly failed in patients with solid tumors or low-grade B-cell malignancies including chronic lymphocytic leukemia with bulky lymph node involvement. Herein, we enhance the antitumor efficacy of CAR T cells through the constitutive expression of CD40 ligand (CD40L, CD154). T cells genetically modified to constitutively express CD40L (CD40L-modified T cells) demonstrated increased proliferation and secretion of proinflammatory TH1 cytokines. Further, CD40L-modified T cells augmented the immunogenicity of CD40+ tumor cells by the upregulated surface expression of costimulatory molecules (CD80 and CD86), adhesion molecules (CD54, CD58, and CD70), human leukocyte antigen (HLA) molecules (Class I and HLA-DR), and the Fas-death receptor (CD95). Additionally, CD40L-modified T cells induced maturation and secretion of the proinflammatory cytokine interleukin-12 by monocyte-derived dendritic cells. Finally, tumor-targeted CD19-specific CAR/CD40L T cells exhibited increased cytotoxicity against CD40+ tumors and extended the survival of tumor-bearing mice in a xenotransplant model of CD19+ systemic lymphoma. This preclinical data supports the clinical application of CAR T cells additionally modified to constitutively express CD40L with anticipated enhanced antitumor efficacy. PMID:25582824

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

    PubMed

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

    2012-06-01

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

  17. Expression and antibody generation of the cancer-testis antigen, BIOT2-S.

    PubMed

    Wang, J-Y; Cao, M; Guo, M-R; Li, S; Yang, X-F; Wang, M; Fang, J; Zhao, J

    2015-01-01

    Biot2-S is a mouse cancer-testis antigen gene that was identified using the cross-reactive serological analysis of recombinant cDNA expression libraries (SEREX) technique in the State Key Laboratory of Biotherapy, West China Hospital, Sichuan University. To express BIOT2-S and generate its antibody for further investigation, the Biot2-S prokaryotic recombinant expression vector Biot2-S/pGEX6P-1 was constructed with Escherichia coli DH5α as a cloning vector, and BIOT2-S was expressed in E. coli Rosetta (DE3). The recombinant BIOT2-S was expressed in the form of an inclusion body and the targeted recombinant BIOT2-S was produced at the level of approximately 25% total bacterial proteins after being induced with optimum conditions (0.2 mM isopropyl-β-D-thiogalactopyranoside for 6 h at 37°C). The target protein was purified by glutathione S-transferase (GST)-trap FF affinity chromatography and detected by western blot. The purified recombinant protein was further confirmed by electrospray ionization quadrupole time-of-flight mass spectrometry after removal of the GST-tags. Then the purified BIOT2-S was used to immunize adult rabbits to generate its antibody. The antibody was purified and its specificity determined. The titer of the antibody was shown to reach 10(4) and the antibody was demonstrated to be able recognize the corresponding protein in the testes of mouse and chicken; the tumor cell lines CT-26 and S180 also reacted with the antibody. This study provides a valuable foundation for further research on the cancer-testis antigen BIOT2-S. PMID:26345800

  18. A novel laboratory technique demonstrating the influences of RHD zygosity and the RhCcEe phenotype on erythrocyte D antigen expression

    PubMed Central

    McGann, Patrick T.; Despotovic, Jenny M.; Howard, Thad A.; Ware, Russell E.

    2015-01-01

    D antigen is the most immunogenic and clinically relevant antigen within the complex Rh blood group system. Variability of D antigen expression was first described decades ago but has rarely been investigated quantitatively, particularly in the context of RHD zygosity along with RhCcEe serological phenotype. With IRB approval, 107 deidentified blood samples were analyzed. Rh phenotypes were determined serologically by saline technique using monoclonal antibodies against D, C, c, E, and e antigens. RHD zygosity was determined using both PCR-restriction fragment length polymorphisms and quantitative real-time PCR techniques. A novel and robust method was developed for quantitation of erythrocyte D antigen sites using calibrated microspheres and flow cytometry, allowing correlation of D antigen density with RHD zygosity and expression of Rh CcEe antigens. Subjects homozygous for RHD expressed nearly twice the number of D antigen sites compared with RHD hemizygotes (33,560 ± 8,222 for DD versus 17,720 ± 4,471 for Dd, P < 0.0001). Expression of c or E antigens was associated with significantly increased erythrocyte D antigen expression, whereas presence of C or e antigens reduced expression. These data and this novel quantitation method will be important for future studies investigating the clinical relevance of D antigen variability. PMID:22121029

  19. Class II major histocompatibility complex antigen expression on peripheral blood monocytes in patients with inflammatory bowel disease.

    PubMed Central

    Gardiner, K R; Crockard, A D; Halliday, M I; Rowlands, B J

    1994-01-01

    Macrophage major histocompatibility complex (MHC) class II antigen expression is associated with defective antigen presentation to T lymphocytes in animals and is predictive of patient outcome after major trauma or sepsis. In this study, class II antigen (HLA-DR and DQ) expression on peripheral blood monocytes was investigated in patients with inflammatory bowel disease in relation to disease activity and outcome. The percentage positivity and fluorescent intensity of expression of HLA-DR and DQ antigens on monocytes were determined in whole blood samples using dual colour immunofluorescence labelling and flow cytometry. Disease activity was assessed using clinical and laboratory indices. There was no significant difference in percentage positivity or fluorescent intensity of class II antigen expression between patients with Crohn's disease, those with ulcerative colitis, and healthy volunteers. The percentage of monocytes displaying HLA-DR positivity was significantly decreased in patients with active ulcerative colitis (active %: 49.5 (5.6); inactive %: 78.9 (6.9); p = 0.01). Data expressed as mean (SEM). In patients requiring surgical resection of diseased bowel, the percentage of monocytes displaying HLA-DR positivity (51.9 (4.0) %) was significantly reduced compared with patients receiving medical treatment alone (81.1 (3.5) %; p < 0.001). Reduced monocyte HLA-DR expression is therefore associated with disease activity and seems to predict outcome in patients with inflammatory bowel disease. PMID:8174990

  20. Genetic Mechanism of Human Neutrophil Antigen 2 Deficiency and Expression Variations

    PubMed Central

    Li, Yunfang; Mair, David C.; Schuller, Randy M.; Li, Ling; Wu, Jianming

    2015-01-01

    Human neutrophil antigen 2 (HNA-2) deficiency is a common phenotype as 3–5% humans do not express HNA-2. HNA-2 is coded by CD177 gene that associates with human myeloproliferative disorders. HNA-2 deficient individuals are prone to produce HNA-2 alloantibodies that cause a number of disorders including transfusion-related acute lung injury and immune neutropenia. In addition, the percentages of HNA-2 positive neutrophils vary significantly among individuals and HNA-2 expression variations play a role in human diseases such as myelodysplastic syndrome, chronic myelogenous leukemia, and gastric cancer. The underlying genetic mechanism of HNA-2 deficiency and expression variations has remained a mystery. In this study, we identified a novel CD177 nonsense single nucleotide polymorphism (SNP 829A>T) that creates a stop codon within the CD177 coding region. We found that all 829TT homozygous individuals were HNA-2 deficient. In addition, the SNP 829A>T genotypes were significantly associated with the percentage of HNA-2 positive neutrophils. Transfection experiments confirmed that HNA-2 expression was absent on cells expressing the CD177 SNP 829T allele. Our data clearly demonstrate that the CD177 SNP 829A>T is the primary genetic determinant for HNA-2 deficiency and expression variations. The mechanistic delineation of HNA-2 genetics will enable the development of genetic tests for diagnosis and prognosis of HNA-2-related human diseases. PMID:26024230

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

    PubMed Central

    Suryanarayana, Nagendra; Vanlalhmuaka; Mankere, Bharti; Verma, Monika; Thavachelvam, Kulanthaivel; Tuteja, Urmil

    2016-01-01

    Bacillus anthracis secretory protein protective antigen (PA) is primary candidate for subunit vaccine against anthrax. Attempts to obtain large quantity of PA from Escherichia coli expression system often result in the formation of insoluble inclusion bodies. Therefore, it is always better to produce recombinant proteins in a soluble form. In the present study, we have obtained biologically active recombinant PA in small scale E. coli shake culture system using three different expression constructs. The PA gene was cloned in expression vectors bearing trc, T5, and T7 promoters and transformed into their respective E. coli hosts. The growth conditions were optimized to obtain maximum expression of PA in soluble form. The expression construct PA-pET32c in DE3-pLysS E. coli host resulted in a maximum production of soluble PA (15 mg L−1) compared to other combinations. Purified PA was subjected to trypsin digestion and binding assay with lethal factor to confirm the protein's functionality. Biological activity was confirmed by cytotoxicity assay on J774.1 cells. Balb/c mice were immunized with PA and the immunogenicity was tested by ELISA and toxin neutralization assay. This study highlights the expression of soluble and biologically active recombinant PA in larger quantity using simpler E. coli production platform. PMID:26966576

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

    PubMed

    Suryanarayana, Nagendra; Vanlalhmuaka; Mankere, Bharti; Verma, Monika; Thavachelvam, Kulanthaivel; Tuteja, Urmil

    2016-01-01

    Bacillus anthracis secretory protein protective antigen (PA) is primary candidate for subunit vaccine against anthrax. Attempts to obtain large quantity of PA from Escherichia coli expression system often result in the formation of insoluble inclusion bodies. Therefore, it is always better to produce recombinant proteins in a soluble form. In the present study, we have obtained biologically active recombinant PA in small scale E. coli shake culture system using three different expression constructs. The PA gene was cloned in expression vectors bearing trc, T5, and T7 promoters and transformed into their respective E. coli hosts. The growth conditions were optimized to obtain maximum expression of PA in soluble form. The expression construct PA-pET32c in DE3-pLysS E. coli host resulted in a maximum production of soluble PA (15 mg L(-1)) compared to other combinations. Purified PA was subjected to trypsin digestion and binding assay with lethal factor to confirm the protein's functionality. Biological activity was confirmed by cytotoxicity assay on J774.1 cells. Balb/c mice were immunized with PA and the immunogenicity was tested by ELISA and toxin neutralization assay. This study highlights the expression of soluble and biologically active recombinant PA in larger quantity using simpler E. coli production platform. PMID:26966576

  3. Cytotoxic T lymphocyte antigen 4 expression in human breast cancer: implications for prognosis.

    PubMed

    Yu, Haiming; Yang, Junlan; Jiao, Shunchang; Li, Ying; Zhang, Wei; Wang, Jiandong

    2015-07-01

    To examine the relationship between cytotoxic T lymphocyte antigen 4 (CTLA-4) expression and breast cancer prognosis, CTLA-4 expression was immunohistochemically detected in paraffin-embedded specimens of primary tumors from 130 patients with breast cancer who had a mean follow-up period of 112 months. CTLA-4 expressed in cytoplasm of breast cancer cells and in cytoplasm and cell membranes of interstitial lymphocytes. Univariate analysis (log-rank) associated higher density of interstitial CTLA-4(+) lymphocytes with longer DFS and OS, but higher tumor CTLA-4 expression with shorter OS. After controlling for age, clinical stage, Scarff-Bloom-Richardson grade, tumor thrombus, ER, PR, HER2 and Ki-67, multivariate analysis (Cox) showed that density of interstitial CTLA-4(+) lymphocytes independently predicted longer DFS (HR 0.315, P = 0.002) and OS (HR 0.313, P = 0.005), whereas tumor CTLA-4 expression independently predicted shorter DFS (HR 2.176, P = 0.029) and OS (HR 2.820, P = 0.007), i.e., patients with high CTLA-4(+) lymphocyte density and CTLA-4(low) tumor cells had the best prognoses. These results indicated that CTLA-4 expression in lymphocytes was associated with better prognosis, but that in tumor cells was associated with worse prognosis. Patients' CTLA-4 profiles might thus be used to predict the benefits and toxicity of CTLA-4 blockade. PMID:25893809

  4. Expression of Cancer Testis Antigens in Colorectal Cancer: New Prognostic and Therapeutic Implications

    PubMed Central

    Czerewaty, Michał; Deskur, Anna; Safranow, Krzysztof; Urasińska, Elżbieta; Starzyńska, Teresa

    2016-01-01

    Background. While cancer/testis antigens (CTAs) are restricted in postnatal tissues to testes and germ line-derived cells, their role in cancer development and the clinical significance of their expression still remain to be better defined. Objective. The aim of this study was to investigate the level of CTA expression in colon samples from patients with colorectal cancer (CRC) in relation to patient clinical status. Methods. Forty-five patients with newly diagnosed colorectal cancer were included in the study. We selected a panel of 18 CTAs that were previously detected in CRC as well as some new gene candidates, and their expression was detected at the mRNA level by employing RQ-PCR. Additionally, we evaluated CTA expression in three colon cancer cell lines (CL-188, HTB-39, and HTB-37) after exposure to the DNA methylation-modifying drug 5-azacytidine. Results. We report that 6 out of 18 (33%) CTAs tested (MAGEA3, OIP5, TTK, PLU1, DKKL1, and FBXO39) were significantly (p < 0.05) overexpressed in tumor tissue compared with healthy colon samples isolated from the same patients. Conclusions. Moreover, we found that MAGEA3, PLU-1, and DKKL expression positively correlated with disease progression, evaluated according to the Dukes staging system. Finally, 5-azacytidine exposure significantly upregulated expression of CTAs on CRC cells, which indicates that this demethylation agent could be employed therapeutically to enhance the immune response against tumor cells.

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

    PubMed

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

    2013-05-20

    Parasite-induced and parasite-regulated larval capsule formation and host immunosuppression are two major characteristics that are unique in Trichinella spp. infections, but the molecule(s) and mechanism(s) that mediate these processes remain largely unknown. Trichinella pseudospiralis and Trichinella spiralis, are obviously different with respect to these two characteristics. A comparative study of these two species, in particular their antigen expression profiles at different developmental stages (the main molecules involved in the cross-talk or interaction between each parasite and its host), may help us better understand the parasite molecules and mechanisms involved. Here, we constructed cDNA libraries from T. pseudospiralis adults (Ad), newborn larvae (NBL) and muscle larvae (ML) mRNA and screened them with pig anti-T. pseudospiralis serum collected 26, 32 and 60 days post-infection (p.i.). The most abundant antigens were found to vary among life-cycle stages. Pyroglutamy peptidase 1-like and 6-phosphogluconolactonase-like genes predominated in the Ad stage and a serine protease (SS2-1-like gene) predominated in NBL similar to that observed in T. spiralis. Muscle larvae expressed proteasome activator complex subunit 3-like and 21 kDa excretory/secretory protein-like genes. This study indicated that parasites of two species may utilise different molecules and mechanisms for larvae capsule formation and host immunosuppression during their infections. Proteins of antigenic genes identified in this study may be also good candidates for diagnosis, treatment or vaccination for T. pseudospiralis infection, and also for the differential diagnosis of two species' infections. PMID:23433603

  6. Expression of blood group antigens in urinary tract tumours: prospective fluorescence study using cryostat sections of fresh frozen tissues.

    PubMed Central

    Thorpe, S J; Abel, P; Henderson, D; Jones, N; Feizi, T

    1986-01-01

    Cryostat sections of fresh frozen tissues were used in a prospective study of blood group H and A antigen fluorescence in 73 transitional cell carcinomas of the bladder. The aim was to evaluate antigen expression without subjecting the tumour tissues to organic solvents that extract blood group active glycolipids. Deletion of the genetically predicted antigen was twice as common in tumours of pT1 or greater stage than those of pTa stage and also twice as common in poorly differentiated than in moderately well differentiated tumours. The considerable heterogeneity and overlap, however, in patterns of reactivity in tumours of various histopathological stages and grades and the effect of secretor status on antigenicity meant that there was no obvious antigenic feature that correlated precisely with invasive stage or differentiation grade. It remains to be determined whether the antigen positive and antigen negative tumours represent different disease entities with differing clinical courses. Our results indicate, however, that studies of the blood group antigens in urinary tract tumours are more likely to be of value in research into biochemical disorders in the neoplastic process than in routine clinical assessment as a guide to treatment. Images Fig 1 Fig 2 Fig 3 PMID:3540013

  7. A microarray method for identifying tumor antigens by screening a tumor cDNA expression library against cancer sera

    PubMed Central

    Whittemore, Kurt; Sykes, Kathryn

    2013-01-01

    The immune system responds to tumor cells. The challenge has been how to effectively use these responses to treat or protect against cancer. Toward the goal of developing a cancer vaccine, we are pursuing methodologies for the discovery and testing of useful antigens. We present an array-based approach for discovering these B cell antigens by directly screening for specific host-sera reactivity to lysates from tumor-derived cDNA expression libraries. Several cancer-specific antigens were identified, and these are currently being validated as potential candidates. PMID:23851590

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

    PubMed

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

    1983-02-01

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

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

    PubMed

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

    2015-05-01

    Streptococcus mutans, which consists of four serotypes, c, e, f, and k, possesses a 190-kDa cell surface protein antigen (PA) for initial tooth adhesion. We used Western blot analysis to determine PA expression in 750 S. mutans isolates from 150 subjects and found a significantly higher prevalence of the isolates with PA expression defects in serotypes f and k compared to serotypes c and e. Moreover, the defect patterns could be classified into three types; no PA expression on whole bacterial cells and in their supernatant samples (Type N1), PA expression mainly seen in supernatant samples (Type N2), and only low expression of PA in the samples of whole bacterial cells (Type W). The underlying reasons for the defects were mutations in the gene encoding PA as well as in the transcriptional processing of this gene for Type N1, defects in the sortase gene for Type N2, and low mRNA expression of PA for Type W. Since cellular hydrophobicity and phagocytosis susceptibility of the PA-defective isolates were significantly lower than those of the normal expression isolates, the potential implication of such defective isolates in systemic diseases involving bacteremia other than dental caries was suggested. Additionally, multilocus sequence typing was utilized to characterize S. mutans clones that represented a proportion of isolates with PA defects of 65-100%. Therefore, we described the molecular basis for variation defects in PA expression of S. mutans. Furthermore, we also emphasized the strong association between PA expression defects and serotypes f and k as well as the clonal relationships among these isolates. PMID:25792295

  10. Mutually Exclusive Expression of Virulence Genes by Malaria Parasites Is Regulated Independently of Antigen Production

    PubMed Central

    Dzikowski, Ron; Frank, Matthias; Deitsch, Kirk

    2006-01-01

    The primary virulence determinant of Plasmodium falciparum malaria parasite–infected cells is a family of heterogeneous surface receptors collectively referred to as PfEMP1. These proteins are encoded by a large, polymorphic gene family called var. The family contains approximately 60 individual genes, which are subject to strict, mutually exclusive expression, with the single expressed var gene determining the antigenic, cytoadherent, and virulence phenotype of the infected cell. The mutually exclusive expression pattern of var genes is imperative for the parasite's ability to evade the host's immune response and is similar to the process of “allelic exclusion” described for mammalian Ig and odorant receptor genes. In mammalian systems, mutually exclusive expression is ensured by negative feedback inhibition mediated by production of a functional protein. To investigate how expression of the var gene family is regulated, we have created transgenic lines of parasites in which expression of individual var loci can be manipulated. Here we show that no such negative feedback system exists in P. falciparum and that this process is dependent solely on the transcriptional regulatory elements immediately adjacent to each gene. Transgenic parasites that are selected to express a var gene in which the PfEMP1 coding region has been replaced by a drug-selectable marker silence all other var genes in the genome, thus effectively knocking out all PfEMP1 expression and indicating that the modified gene is still recognized as a member of the var gene family. Mutually exclusive expression in P. falciparum is therefore regulated exclusively at the level of transcription, and a functional PfEMP1 protein is not necessary for viability or for proper gene regulation in cultured parasites. PMID:16518466

  11. Heat Shock Protein-90 Inhibitors Enhance Antigen Expression on Melanomas and Increase T Cell Recognition of Tumor Cells

    PubMed Central

    Haggerty, Timothy J.; Dunn, Ian S.; Rose, Lenora B.; Newton, Estelle E.; Pandolfi, Franco; Kurnick, James T.

    2014-01-01

    In an effort to enhance antigen-specific T cell recognition of cancer cells, we have examined numerous modulators of antigen-expression. In this report we demonstrate that twelve different Hsp90 inhibitors (iHsp90) share the ability to increase the expression of differentiation antigens and MHC Class I antigens. These iHsp90 are active in several molecular and cellular assays on a series of tumor cell lines, including eleven human melanomas, a murine B16 melanoma, and two human glioma-derived cell lines. Intra-cytoplasmic antibody staining showed that all of the tested iHsp90 increased expression of the melanocyte differentiation antigens Melan-A/MART-1, gp100, and TRP-2, as well as MHC Class I. The gliomas showed enhanced gp100 and MHC staining. Quantitative analysis of mRNA levels showed a parallel increase in message transcription, and a reporter assay shows induction of promoter activity for Melan-A/MART-1 gene. In addition, iHsp90 increased recognition of tumor cells by T cells specific for Melan-A/MART-1. In contrast to direct Hsp90 client proteins, the increased levels of full-length differentiation antigens that result from iHsp90 treatment are most likely the result of transcriptional activation of their encoding genes. In combination, these results suggest that iHsp90 improve recognition of tumor cells by T cells specific for a melanoma-associated antigen as a result of increasing the expressed intracellular antigen pool available for processing and presentation by MHC Class I, along with increased levels of MHC Class I itself. As these Hsp90 inhibitors do not interfere with T cell function, they could have potential for use in immunotherapy of cancer. PMID:25503774

  12. A novel neutralization sensitive and subdominant RAP-1-related antigen (RRA) is expressed by babesia bovis merozoites

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: The Babesia bovis genome encodes a rap-1 related gene denominated RAP-1 related antigen (RRA). In this study, we analyzed the pattern of expression, immunogenicity and functional relevance of RRA. Methods: Phylogenetic analysis was performed using the program Phylip. Expression of rra wa...

  13. Expression of a Chimeric Antigen Receptor in Multiple Leukocyte Lineages in Transgenic Mice.

    PubMed

    Yong, Carmen S M; Westwood, Jennifer A; Schröder, Jan; Papenfuss, Anthony T; von Scheidt, Bianca; Moeller, Maria; Devaud, Christel; Darcy, Phillip K; Kershaw, Michael H

    2015-01-01

    Genetically modified CD8+ T lymphocytes have shown significant anti-tumor effects in the adoptive immunotherapy of cancer, with recent studies highlighting a potential role for a combination of other immune subsets to enhance these results. However, limitations in present genetic modification techniques impose difficulties in our ability to fully explore the potential of various T cell subsets and assess the potential of other leukocytes armed with chimeric antigen receptors (CARs). To address this issue, we generated a transgenic mouse model using a pan-hematopoietic promoter (vav) to drive the expression of a CAR specific for a tumor antigen. Here we present a characterization of the immune cell compartment in two unique vav-CAR transgenic mice models, Founder 9 (F9) and Founder 38 (F38). We demonstrate the vav promoter is indeed capable of driving the expression of a CAR in cells from both myeloid and lymphoid lineage, however the highest level of expression was observed in T lymphocytes from F38 mice. Lymphoid organs in vav-CAR mice were smaller and had reduced cell numbers compared to the wild type (WT) controls. Furthermore, the immune composition of F9 mice differed greatly with a significant reduction in lymphocytes found in the thymus, lymph node and spleen of these mice. To gain insight into the altered immune phenotype of F9 mice, we determined the chromosomal integration site of the transgene in both mouse strains using whole genome sequencing (WGS). We demonstrated that compared to the 7 copies found in F38 mice, F9 mice harbored almost 270 copies. These novel vav-CAR models provide a ready source of CAR expressing myeloid and lymphoid cells and will aid in facilitating future experiments to delineate the role for other leukocytes for adoptive immunotherapy against cancer. PMID:26505904

  14. Expression of a Chimeric Antigen Receptor in Multiple Leukocyte Lineages in Transgenic Mice

    PubMed Central

    Yong, Carmen S. M.; Westwood, Jennifer A.; Schröder, Jan; Papenfuss, Anthony T.; von Scheidt, Bianca; Moeller, Maria; Devaud, Christel

    2015-01-01

    Genetically modified CD8+ T lymphocytes have shown significant anti-tumor effects in the adoptive immunotherapy of cancer, with recent studies highlighting a potential role for a combination of other immune subsets to enhance these results. However, limitations in present genetic modification techniques impose difficulties in our ability to fully explore the potential of various T cell subsets and assess the potential of other leukocytes armed with chimeric antigen receptors (CARs). To address this issue, we generated a transgenic mouse model using a pan-hematopoietic promoter (vav) to drive the expression of a CAR specific for a tumor antigen. Here we present a characterization of the immune cell compartment in two unique vav-CAR transgenic mice models, Founder 9 (F9) and Founder 38 (F38). We demonstrate the vav promoter is indeed capable of driving the expression of a CAR in cells from both myeloid and lymphoid lineage, however the highest level of expression was observed in T lymphocytes from F38 mice. Lymphoid organs in vav-CAR mice were smaller and had reduced cell numbers compared to the wild type (WT) controls. Furthermore, the immune composition of F9 mice differed greatly with a significant reduction in lymphocytes found in the thymus, lymph node and spleen of these mice. To gain insight into the altered immune phenotype of F9 mice, we determined the chromosomal integration site of the transgene in both mouse strains using whole genome sequencing (WGS). We demonstrated that compared to the 7 copies found in F38 mice, F9 mice harbored almost 270 copies. These novel vav-CAR models provide a ready source of CAR expressing myeloid and lymphoid cells and will aid in facilitating future experiments to delineate the role for other leukocytes for adoptive immunotherapy against cancer. PMID:26505904

  15. The B-cell tumor–associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor

    PubMed Central

    Schmitt, Thomas M.; Baskar, Sivasubramanian; Lupo-Stanghellini, Maria Teresa; Nishida, Tetsuya; Yamamoto, Tori N.; Bleakley, Marie; Turtle, Cameron J.; Chang, Wen-Chung; Greisman, Harvey A.; Wood, Brent; Maloney, David G.; Jensen, Michael C.; Rader, Christoph; Riddell, Stanley R.

    2010-01-01

    Monoclonal antibodies and T cells modified to express chimeric antigen receptors specific for B-cell lineage surface molecules such as CD20 exert antitumor activity in B-cell malignancies, but deplete normal B cells. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) was identified as a highly expressed gene in B-cell chronic lymphocytic leukemia (B-CLL), but not normal B cells, suggesting it may serve as a tumor-specific target for therapy. We analyzed ROR1-expression in normal nonhematopoietic and hematopoietic cells including B-cell precursors, and in hematopoietic malignancies. ROR1 has characteristics of an oncofetal gene and is expressed in undifferentiated embryonic stem cells, B-CLL and mantle cell lymphoma, but not in major adult tissues apart from low levels in adipose tissue and at an early stage of B-cell development. We constructed a ROR1-specific chimeric antigen receptor that when expressed in T cells from healthy donors or CLL patients conferred specific recognition of primary B-CLL and mantle cell lymphoma, including rare drug effluxing chemotherapy resistant tumor cells that have been implicated in maintaining the malignancy, but not mature normal B cells. T-cell therapies targeting ROR1 may be effective in B-CLL and other ROR1-positive tumors. However, the expression of ROR1 on some normal tissues suggests the potential for toxi-city to subsets of normal cells. PMID:20702778

  16. Expression of the duffy antigen/receptor for chemokines (DARC) by the inflamed synovial endothelium.

    PubMed

    Patterson, Angela M; Siddall, Hilary; Chamberlain, Giselle; Gardner, Lucy; Middleton, Jim

    2002-05-01

    The expression of chemokine binding sites on the endothelial cells of venules in inflamed synovia was examined and whether the Duffy antigen/receptor for chemokines (DARC) was involved. In situ binding assays were performed to determine the expression of chemokine binding sites from rheumatoid (n = 10) and non-rheumatoid (n = 10) synovia. The expression of DARC protein and mRNA was examined by immunohistochemistry and northern blotting. The involvement of DARC in chemokine binding was studied by incubating sections with blocking antibodies to DARC (Fy3 and 6), to find out if these reduced 125I-IL-8 binding. Binding of radiolabelled chemokines IL-8, RANTES, MCP-1, but not MIP-1alpha, was found on venular endothelial cells in inflamed synovia from both rheumatoid and non-rheumatoid patients. Excess homologous unlabelled chemokine displaced binding and excess unlabelled RANTES could displace radiolabelled IL-8 binding. DARC protein expression was demonstrated on venular endothelial cells in all samples and DARC mRNA could be detected in extracts from synovia. There was downregulation of DARC protein and mRNA in rheumatoid samples. Binding of IL-8 to both rheumatoid and non-rheumatoid synovia was significantly reduced in the presence of anti-DARC Fy3 and Fy6 monoclonal antibodies. These findings show the expression of a multispecific chemokine binding site on the inflamed synovial endothelium, with evidence for involvement of DARC. This suggests a potential role for DARC in the inflammatory processes involved in synovitis. PMID:12081195

  17. Prostate-specific membrane antigen protein expression in tumor tissue and risk of lethal prostate cancer

    PubMed Central

    Kasperzyk, Julie L.; Finn, Stephen P.; Flavin, Richard; Fiorentino, Michelangelo; Lis, Rosina; Hendrickson, Whitney K.; Clinton, Steven K.; Sesso, Howard D.; Giovannucci, Edward L.; Stampfer, Meir J.; Loda, Massimo; Mucci, Lorelei A.

    2013-01-01

    Background Over-expression of prostate-specific membrane antigen (PSMA) in tumor tissue and serum has been linked to increased risk of biochemical recurrence in surgically treated prostate cancer patients, but no studies have assessed its association with disease-specific mortality. Methods We examined whether high PSMA protein expression in prostate tumor tissue was associated with lethal disease, and with tumor biomarkers of progression, among participants of two US-based cohorts (n=902, diagnosed 1983–2004). We used Cox proportional hazards regression to calculate multivariable hazard ratios (HR) and 95% confidence intervals (CI) of lethal prostate cancer, defined as disease-specific death or development of distant metastases (n=95). Partial Spearman rank correlation coefficients were used to correlate PSMA with tumor biomarkers. Results During an average 13 years of follow-up, higher PSMA expression at prostatectomy was significantly associated with lethal prostate cancer (age-adjusted HRQuartile(Q)4vs.Q1=2.42; p-trend<0.01). This association was attenuated and non-significant (multivariable-adjusted HRQ4vs.Q1=1.01; p-trend=0.52) after further adjusting for Gleason score and PSA at diagnosis. High PSMA expression was significantly (p<0.05) correlated with higher Gleason score and PSA at diagnosis, increased tumor angiogenesis, lower vitamin D receptor and androgen receptor expression, and absence of ERG expression. Conclusions High tumor PSMA expression was not an independent predictor of lethal prostate cancer in the current study. PSMA expression likely captures, in part, malignant features of Gleason grade and tumor angiogenesis. Impact PSMA is not a strong candidate biomarker for predicting prostate cancer-specific mortality in surgically treated patients. PMID:24130224

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

    PubMed Central

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

    1989-01-01

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

  19. Tumor-specific up-regulation of the nonclassical class I HLA-G antigen expression in renal carcinoma.

    PubMed

    Ibrahim, E C; Guerra, N; Lacombe, M J; Angevin, E; Chouaib, S; Carosella, E D; Caignard, A; Paul, P

    2001-09-15

    HLA-G is a nonclassical class I antigen mainly expressed at the maternofetal interface during pregnancy where it is thought to down-modulate maternal immune response against the semiallogeneic fetus. Recent studies indicate that ectopic up-regulation of HLA-G expression on melanoma cells may also favor their escape from antitumor immune response. HLA-G expression was here investigated on paraffin-embedded tumor and adjacent normal renal tissues of 18 renal cell carcinoma (RCC) patients. We provide evidence that HLA-G antigen is differentially expressed in carcinoma and normal renal cells and that up-regulation of this antigen in the tumor cells is more frequent than alterations of other MHC class I or class II antigens. We also demonstrated that HLA-G cell surface expression and secretion is maintained in a tumor cell line (DM) established from an HLA-G-positive RCC lesion. Furthermore, we show that type I (alpha and beta) and, in particular, type II (gamma) IFN treatment enhances steady-state mRNA levels and cell surface expression of HLA-G in the DM cell line. As several studies suggest that HLA-G displays various functional features that allow down-modulation of immune response in vitro, we propose that selective in vivo expression of HLA-G may participate in the impairment of antitumor immunity in RCC. PMID:11559559

  20. Generation of Salmonella ghost cells expressing fimbrial antigens of enterotoxigenic Escherichia coli and evaluation of their antigenicity in a murine model

    PubMed Central

    Kim, Chan Song; Hur, Jin; Eo, Seong Kug; Park, Sang-Youel; Lee, John Hwa

    2016-01-01

    Salmonella Typhimurium ghost cells expressing K88ab, K88ac, K99, and FasA fimbriae of enterotoxigenic Escherichia coli (ETEC) in their envelopes were constructed. The genes encoding the fimbriae were individually cloned into an expression plasmid, pMMP81, carrying the asd gene, which was subsequently electroporated into the Δasd S. Typhimurium mutant. Plasmid pJHLP99, carrying the phiX174 lysis gene E, was also subsequently electroporated into the Salmonella mutant. The presence of the individual fimbriae on the ghost cells was examined by Western blot analysis. Forty BALB/c mice were equally divided into 2 groups of 20 mice each. Group A mice were intramuscularly vaccinated with a mixture of the 4 ghost cells expressing the individual fimbriae. The group B mice were inoculated with sterile phosphate-buffered saline as a control. The antigen-specific serum IgG concentrations were significantly higher in group A than in group B from week 2 until week 6 after inoculation. In addition, the antigen-specific IgA concentrations in fecal samples were significantly higher in group A than in group B at week 2 after inoculation. A large difference between the groups in the number of antigen-specific IgA-secreting cells in the small intestine was observed by immunohistochemical study. Also, the splenic lymphocyte proliferative responses were significantly greater in group A than in the control mice. These results suggest that vaccination with our Salmonella ghost cells can induce both humoral and cell-mediated immune responses and that the increased number of antigen-specific IgA-secreting cells in the small intestine may be correlated with the elevated fecal IgA immune response. PMID:26733731

  1. Electroporation Enhances Immunogenicity of a DNA Vaccine Expressing Woodchuck Hepatitis Virus Surface Antigen in Woodchucks▿

    PubMed Central

    Liu, Katherine H.; Ascenzi, Mary A.; Bellezza, Christine A.; Bezuidenhout, Abraham J.; Cote, Paul J.; Gonzalez-Aseguinolaza, Gloria; Hannaman, Drew; Luxembourg, Alain; Evans, Claire F.; Tennant, Bud C.; Menne, Stephan

    2011-01-01

    The development of therapeutic vaccines for chronic hepatitis B virus (HBV) infection has been hampered by host immune tolerance and the generally low magnitude and inconsistent immune responses to conventional vaccines and proposed new delivery methods. Electroporation (EP) for plasmid DNA (pDNA) vaccine delivery has demonstrated the enhanced immunogenicity of HBV antigens in various animal models. In the present study, the efficiency of the EP-based delivery of pDNA expressing various reporter genes first was evaluated in normal woodchucks, and then the immunogenicity of an analog woodchuck hepatitis virus (WHV) surface antigen (WHsAg) pDNA vaccine was studied in this model. The expression of reporter genes was greatly increased when the cellular uptake of pDNA was facilitated by EP. The EP of WHsAg-pDNA resulted in enhanced, dose-dependent antibody and T-cell responses to WHsAg compared to those of the conventional hypodermic needle injection of WHsAg-pDNA. Although subunit WHsAg protein vaccine elicited higher antibody titers than the DNA vaccine delivered with EP, T-cell response rates were comparable. However, in WHsAg-stimulated mononuclear cell cultures, the mRNA expression of CD4 and CD8 leukocyte surface markers and Th1 cytokines was more frequent and was skewed following DNA vaccination compared to that of protein immunization. Thus, the EP-based vaccination of normal woodchucks with pDNA-WHsAg induced a skew in the Th1/Th2 balance toward Th1 immune responses, which may be considered more appropriate for approaches involving therapeutic vaccines to treat chronic HBV infection. PMID:21389124

  2. Gene cloning, expression and immunogenicity of the protective antigen subolesin in Dermacentor silvarum.

    PubMed

    Hu, Yonghong; Zeng, Hua; Zhang, Jincheng; Wang, Duo; Li, Dongming; Zhang, Tiantian; Yang, Shujie; Liu, Jingze

    2014-02-01

    Subolesin (4D8), the ortholog of insect akirins, is a highly conserved protective antigen and thus has the potential for development of a broad-spectrum vaccine against ticks and mosquitoes. To date, no protective antigens have been characterized nor tested as candidate vaccines against Dermacentor silvarum bites and transmission of associated pathogens. In this study, we cloned the open reading frame (ORF) of D. silvarum 4D8 cDNA (Ds4D8), which consisted of 498 bp encoding 165 amino acid residues. The results of sequence alignments and phylogenetic analysis demonstrated that D. silvarum 4D8 (Ds4D8) is highly conserved showing more than 81% identity of amino acid sequences with those of other hard ticks. Additionally, Ds4D8 containing restriction sites was ligated into the pET-32(a+) expression vector and the recombinant plasmid was transformed into Escherichia coli rosetta. The recombinant Ds4D8 (rDs4D8) was induced by isopropyl β-D-thiogalactopyranoside (IPTG) and purified using Ni affinity chromatography. The SDS-PAGE results showed that the molecular weight of rDs4D8 was 40 kDa, which was consistent with the expected molecular mass considering 22 kDa histidine-tagged thioredoxin (TRX) protein from the expression vector. Western blot results showed that rabbit anti-D. silvarum serum recognized the expressed rDs4D8, suggesting an immune response against rDs4D8. These results provided the basis for developing a candidate vaccine against D. silvarum ticks and transmission of associated pathogens. PMID:24623890

  3. Gene Cloning, Expression and Immunogenicity of the Protective Antigen Subolesin in Dermacentor silvarum

    PubMed Central

    Hu, Yonghong; Zeng, Hua; Zhang, Jincheng; Wang, Duo; Li, Dongming; Zhang, Tiantian; Yang, Shujie

    2014-01-01

    Subolesin (4D8), the ortholog of insect akirins, is a highly conserved protective antigen and thus has the potential for development of a broad-spectrum vaccine against ticks and mosquitoes. To date, no protective antigens have been characterized nor tested as candidate vaccines against Dermacentor silvarum bites and transmission of associated pathogens. In this study, we cloned the open reading frame (ORF) of D. silvarum 4D8 cDNA (Ds4D8), which consisted of 498 bp encoding 165 amino acid residues. The results of sequence alignments and phylogenetic analysis demonstrated that D. silvarum 4D8 (Ds4D8) is highly conserved showing more than 81% identity of amino acid sequences with those of other hard ticks. Additionally, Ds4D8 containing restriction sites was ligated into the pET-32(a+) expression vector and the recombinant plasmid was transformed into Escherichia coli rosetta. The recombinant Ds4D8 (rDs4D8) was induced by isopropyl β-D-thiogalactopyranoside (IPTG) and purified using Ni affinity chromatography. The SDS-PAGE results showed that the molecular weight of rDs4D8 was 40 kDa, which was consistent with the expected molecular mass considering 22 kDa histidine-tagged thioredoxin (TRX) protein from the expression vector. Western blot results showed that rabbit anti-D. silvarum serum recognized the expressed rDs4D8, suggesting an immune response against rDs4D8. These results provided the basis for developing a candidate vaccine against D. silvarum ticks and transmission of associated pathogens. PMID:24623890

  4. Effect of particulation on the immunogenic and protective properties of the recombinant Bm86 antigen expressed in Pichia pastoris.

    PubMed

    García-García, J C; Montero, C; Rodríguez, M; Soto, A; Redondo, M; Valdés, M; Méndez, L; de la Fuente, J

    1998-02-01

    The recombinant Bm86 tick antigen expressed in Pichia pastoris is obtained in a highly particulated form, as a distinguish feature of this expression system. This particulated protein, the active principle of the recombinant vaccine Gavac against the cattle tick, have shown high immunogenic and protective properties, probably associated with its own characteristics. To evaluate the effects of particulation on the properties of Bm86, three groups of calves were immunized with particulated or non-particulated recombinant Bm86 and the anti-Bm86 antibody response determined. Animals were challenged with a controlled tick infestation and the protective capacities of both proteins assessed. Humoral immune response and protection in cattle vaccinated with the particulated antigen were higher. These experiments suggested that particulation of the Bm86 expressed in P. pastoris is an important feature for the protective properties of the antigen in vaccine preparations. PMID:9607058

  5. Identification of endothelial antigens relevant to transplant coronary artery disease from a human endothelial cell cDNA expression library.

    PubMed

    Ationu, A

    1998-06-01

    Accelerated transplant coronary artery disease (TxCAD) results in increased expression of antiendothelial antibodies whose target antigens remain largely unidentified. One of these endothelial antigens has been identified as vimentin, a cytoskeletal protein present in cells of the blood vessel walls. In the present study, SDS-PAGE and Western blot analysis of human endothelial cell (EAHy 926) lysates probed with sera from a TxCAD patient were used to confirm immunoreactivity of antiendothelial antibodies towards several endothelial proteins. To further elucidate the identity of these putative antigens, a human endothelial cell (EAHy 926) cDNA expression library was immunoscreened with serum obtained from a TxCAD patient. Two positive cDNA clones were identified by partial nucleotide sequence analysis and GenBank/EMBL database searches for homology as the 85 kDa human CD36 antigen (a cell surface glycoprotein expressed in various cells including epithelial and endothelial cells) and a 50 kDa keratin-like protein (a member of the intermediate filament protein expressed in epithelial cells). These results are the first to demonstrate that human CD36 antigen and a keratin-like protein may be additional target proteins for the anti-endothelial antibodies associated with TxCAD. PMID:9852639

  6. Antigenic validation of recombinant hemagglutinin-neuraminidase protein of Newcastle disease virus expressed in Saccharomyces cerevisiae.

    PubMed

    Khulape, S A; Maity, H K; Pathak, D C; Mohan, C Madhan; Dey, S

    2015-09-01

    The outer membrane glycoprotein, hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) is important for virus infection and subsequent immune response by host, and offers target for development of recombinant antigen-based immunoassays and subunit vaccines. In this study, the expression of HN protein of NDV is attempted in yeast expression system. Yeast offers eukaryotic environment for protein processing and posttranslational modifications like glycosylation, in addition to higher growth rate and easy genetic manipulation. Saccharomyces cerevisiae was found to be better expression system for HN protein than Pichia pastoris as determined by codon usage analysis. The complete coding  sequence of HN gene was amplified with the histidine tag, cloned in pESC-URA under GAL10 promotor and transformed in Saccharomyces cerevisiae. The recombinant HN (rHN) protein was characterized by western blot, showing glycosylation heterogeneity as observed with other eukaryotic expression systems. The recombinant protein was purified by affinity column purification. The protein could be further used as subunit vaccine. PMID:26435147

  7. Expression profile of mucin-associated sialyl-Tn antigen in Chinese patients with different colorectal lesions (adenomas, carcinomas)

    PubMed Central

    Xu, Feng; Fan, Cuizhen; Fan, Shanshan; Liu, Fuquan; Wen, Tao; An, Guangyu; Feng, Guosheng

    2015-01-01

    Background: The sialyl-Tn (sTn) antigen is a mucin-associated carbohydrate antigen expressed by numerous human carcinomas, and is also claimed to be a prognostic factor in colorectal cancer. But the associations between sTn and colorectal cancer remain elusive and controversial. Here, we investigated the expression profile of sTn antigen in a series of human colorectal tissue samples including normal colon, colorectal adenomas, and colorectal carcinomas (CRCs), with an aim to analyzing whether sTn plays a role in the progression and development of Chinese patients with CRCs. Methods: Immunohistochemical staining of sTn antigen was performed in formalin-fixed, paraffin-embedded colonic sections from 4 healthy controls, 44 patients with colorectal adenomas, and 186 patients with primary CRCs. Results: No sTn antigen was detected in normal colonic tissues. There were 41 of 44 patients with colorectal adenomas (93.2%), and 141 of 186 patients with CRCs (75.8%) found to express sTn antigen. The patterns of sTn localization were different in adenomas and carcinomas of colonic tissues. Colorectal adenomas showed predominant supranuclear distribution of sTn antigen, while carcinomas revealed apical membrane, mucin droplet and diffuse cytoplasmic localization. Notably, sTn was significantly associated with the degree of differentiation (P = 0.006) and perineural invasion (P = 0.041) of the tumors, but was independent of age, gender, tumor location, depth of penetration, status of lymph nodes, lymphovascular invasion and TNM stage. Conclusions: These results indicate that sTn may play a role in initiating colorectal carcinogenesis and promoting tumor progression. Determination of sTn expression and localization may assist in evaluating malignant status of colorectal lesions. PMID:26617889

  8. Stress-induced alterations in interferon production and class II histocompatibility antigen expression

    NASA Technical Reports Server (NTRS)

    Sonnenfeld, G.; Cunnick, J. E.; Armfield, A. V.; Wood, P. G.; Rabin, B. S.

    1992-01-01

    Mild electric foot-shock has been shown to be a stressor that can alter immune responses. Male Lewis rats were exposed to one session of 16 5.0-s 1.6-mA foot-shocks. Production of interferon-gamma by splenocytes in response to concanavalin-A was decreased in spleens from the shocked rats compared to control spleens. Spleen cells from rats treated with nadolol, a peripherally acting beta-adrenergic receptor antagonist, and then shocked, showed dose-dependent attenuation of the suppression of interferon-gamma production. This suggests that catecholamines mediate shock-induced suppression of interferon-gamma production. The percentage of splenic mononuclear cells expressing class II histocompatibility (Ia) antigens on their surfaces from spleens of shocked rats was determined by flow cytometry. Significantly decreased class II positive mononuclear cells were present in the spleens of shocked rats in comparison to the spleens of control rats. This may reflect an alteration of cell trafficking or decreased production of class II antigens.

  9. Investigation of expression of 5T4 antigen in cervical cancer.

    PubMed

    Jones, H; Roberts, G; Hole, N; McDicken, I W; Stern, P

    1990-01-01

    A monoclonal antibody detecting amniotrophoblastic antigen 5T4 has shown reactivity against various neoplastic cell lines and tumour specimens but with a relatively restricted normal tissue expression. This antibody has been investigated as a potential indicator of premalignant changes identified as cervical intra-epithelial neoplasia and malignant cervical lesions using immunohistochemistry on frozen tissue biopsies. The basal cells of normal cervical stratified epithelium exhibited faint staining, but a general increase in intensity and extent of specific labeling of this tissue was seen from the first premalignant stage through to carcinoma. In most cases, this was in accordance with the distribution of dysplastic cells, and was accompanied by increased specific staining of the stromal tissue. All invasive squamous carcinomas of the cervix were 5T4 antigen positive. Common inflammatory non-malignant diseases did show a certain degree of epithelial and stromal reactivity. These results, showing 5T4 reactivity with neoplastic and pre-neoplastic lesions, may provide a quantitative basis for its potential use as a tumour marker in the immunochemical detection on immunoassay of cervical cancer. PMID:2404512

  10. Monoclonal antibodies reveal cell-type-specific antigens in the sexually dimorphic olfactory system of Manduca sexta. II. Expression of antigens during postembryonic development.

    PubMed

    Hishinuma, A; Hockfield, S; McKay, R; Hildebrand, J G

    1988-01-01

    Two classes of monoclonal antibodies specific to the olfactory system of Manduca sexta have been isolated: the olfactory-specific antibody (OSA), which specifically recognizes many or all olfactory receptor cells (ORCs) in both males and females, and the male olfactory-specific antibody (MOSA), which stains male-specific receptor cells (principally or exclusively sex-pheromone receptors present only in antennae of males; Hishinuma et al., 1988). In the investigation reported here, we examined the expression of the antigens during postembryonic development in order to correlate the presence of particular antigens with the status of differentiation of the ORCs or with their acquisition of particular functions. As assessed immunocytochemically, the OSA recognizes certain epithelial cells in the antennal imaginal disk of the fifth-instar larva. Later, during the first 70 hr of adult development, when differentiative cell divisions are occurring in the antennal epithelium to generate ORCs and the other cells that make up olfactory sensilla, no cells are stained. Immediately after this period of mitoses, the OSA immunoreactivity reappears exclusively in the ORCs, which begin to elaborate axons as an early event in their differentiation. On immunoblots, the OSA recognizes specific sets of molecules (distinguished on the basis of their apparent molecular weights): 53,000 and 59,000 Da antigens in the disk epithelial cells in the last-instar larva; 53,000, 59,000, and 66,000 Da antigens in the ORCs from 15 to 60% of metamorphic adult development; and 42,000, 59,000, and 66,000 Da antigens in the ORCs from 60 to 100% of adult development. The MOSA also recognizes a subset of the epithelial cells in the antennal disks in male and female larvae.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3339412

  11. Elevated expression of T-bet in mycobacterial antigen-specific CD4(+) T cells from patients with tuberculosis.

    PubMed

    Yang, Bingfen; Zhai, Fei; Jiang, Jing; Wang, Xinjing; Cao, Zhihong; Cheng, Xiaoxing

    2015-01-01

    T-bet is a T-box transcriptional factor that controls the differentiation and effector functions of CD4 T cells. In this study, we studied the role of T-bet in regulating CD4(+) T cell immunity against tuberculosis (TB). T-bet expression in Mycobacterium tuberculosis antigen-specific CD4(+) T cells was significantly higher in patients with active TB than in individuals with latent TB infection (p<0.0001). Comparison of T-bet expression in TCM and TEM subsets showed that CD4(+)T-bet(+)M. tuberculosis antigen-specific CD4(+) T cells had significantly lower frequency of TCM (p=0.003) and higher frequency of TEM (p=0.003) than CD4(+)T-bet(-) cells. The expression of PD-1 in antigen-specific CD4(+) T cells was significantly higher in patients with TB than in individuals with latent TB infection (p=0.006). CD4(+)CD154(+)T-bet(+) T cells had significantly higher expression of PD-1 than CD4(+)CD154(+)T-bet(-) T cells (p=0.0028). It is concluded that T-bet expression might be associated with differentiation into effector memory cells and PD-1 expression in mycobacterial antigen-specific CD4(+) T cells. PMID:26302932

  12. The ganglioside antigen GD2 is surface-expressed in Ewing sarcoma and allows for MHC-independent immune targeting

    PubMed Central

    Kailayangiri, S; Altvater, B; Meltzer, J; Pscherer, S; Luecke, A; Dierkes, C; Titze, U; Leuchte, K; Landmeier, S; Hotfilder, M; Dirksen, U; Hardes, J; Gosheger, G; Juergens, H; Rossig, C

    2012-01-01

    Background: Novel treatment strategies are needed to cure disseminated Ewing sarcoma. Primitive neuroectodermal features and a mesenchymal stem cell origin are both compatible with aberrant expression of the ganglioside antigen GD2 and led us to explore GD2 immune targeting in this cancer. Methods: We investigated GD2 expression in Ewing sarcoma by immunofluorescence staining. We then assessed the antitumour activity of T cells expressing a chimeric antigen receptor specific for GD2 against Ewing sarcoma in vitro and in vivo. Results: Surface GD2 was detected in 10 out of 10 Ewing sarcoma cell lines and 3 out of 3 primary cell cultures. Moreover, diagnostic biopsies from 12 of 14 patients had uniform GD2 expression. T cells specifically modified to express the GD2-specific chimeric receptor 14. G2a-28ζ efficiently interacted with Ewing sarcoma cells, resulting in antigen-specific secretion of cytokines. Moreover, chimeric receptor gene-modified T cells from healthy donors and from a patient exerted potent, GD2-specific cytolytic responses to allogeneic and autologous Ewing sarcoma, including tumour cells grown as multicellular, anchorage-independent spheres. GD2-specific T cells further had activity against Ewing sarcoma xenografts. Conclusion: GD2 surface expression is a characteristic of Ewing sarcomas and provides a suitable target antigen for immunotherapeutic strategies to eradicate micrometastatic cells and prevent relapse in high-risk disease. PMID:22374462

  13. A Recombinant Raccoon Poxvirus Vaccine Expressing both Yersinia pestis F1 and Truncated V Antigens Protects Animals against Lethal Plague

    PubMed Central

    Rocke, Tonie E.; Kingstad-Bakke, Brock; Berlier, Willy; Osorio, Jorge E.

    2014-01-01

    In previous studies, we demonstrated in mice and prairie dogs that simultaneous administration of two recombinant raccoon poxviruses (rRCN) expressing Yersinia pestis antigens (F1 and V307—a truncated version of the V protein) provided superior protection against plague challenge compared to individual single antigen constructs. To reduce costs of vaccine production and facilitate implementation of a sylvatic plague vaccine (SPV) control program for prairie dogs, a dual antigen construct is more desirable. Here we report the construction and characterization of a novel RCN-vectored vaccine that simultaneously expresses both F1 and V307 antigens. This dual antigen vaccine provided similar levels of protection against plague in both mice and prairie dogs as compared to simultaneous administration of the two single antigen constructs and was also shown to protect mice against an F1 negative strain of Y. pestis. The equivalent safety, immunogenicity and efficacy profile of the dual RCN-F1/V307 construct warrants further evaluation in field efficacy studies in sylvatic plague endemic areas. PMID:26344891

  14. A recombinant raccoon poxvirus vaccine expressing both Yersinia pestis F1 and truncated V antigens protects animals against lethal plague.

    USGS Publications Warehouse

    Rocke, Tonie E.; Kingstad-Bakke, B; Berlier, W; Osorio, J.E.

    2014-01-01

    In previous studies, we demonstrated in mice and prairie dogs that simultaneous administration of two recombinant raccoon poxviruses (rRCN) expressing Yersinia pestis antigens (F1 and V307-a truncated version of the V protein) provided superior protection against plague challenge compared to individual single antigen constructs. To reduce costs of vaccine production and facilitate implementation of a sylvatic plague vaccine (SPV) control program for prairie dogs, a dual antigen construct is more desirable. Here we report the construction and characterization of a novel RCN-vectored vaccine that simultaneously expresses both F1 and V307 antigens. This dual antigen vaccine provided similar levels of protection against plague in both mice and prairie dogs as compared to simultaneous administration of the two single antigen constructs and was also shown to protect mice against an F1 negative strain of Y. pestis.. The equivalent safety, immunogenicity and efficacy profile of the dual RCN-F1/V307 construct warrants further evaluation in field efficacy studies in sylvatic plague endemic areas.

  15. A Recombinant Raccoon Poxvirus Vaccine Expressing both Yersinia pestis F1 and Truncated V Antigens Protects Animals against Lethal Plague.

    PubMed

    Rocke, Tonie E; Kingstad-Bakke, Brock; Berlier, Willy; Osorio, Jorge E

    2014-01-01

    In previous studies, we demonstrated in mice and prairie dogs that simultaneous administration of two recombinant raccoon poxviruses (rRCN) expressing Yersinia pestis antigens (F1 and V307-a truncated version of the V protein) provided superior protection against plague challenge compared to individual single antigen constructs. To reduce costs of vaccine production and facilitate implementation of a sylvatic plague vaccine (SPV) control program for prairie dogs, a dual antigen construct is more desirable. Here we report the construction and characterization of a novel RCN-vectored vaccine that simultaneously expresses both F1 and V307 antigens. This dual antigen vaccine provided similar levels of protection against plague in both mice and prairie dogs as compared to simultaneous administration of the two single antigen constructs and was also shown to protect mice against an F1 negative strain of Y. pestis. The equivalent safety, immunogenicity and efficacy profile of the dual RCN-F1/V307 construct warrants further evaluation in field efficacy studies in sylvatic plague endemic areas. PMID:26344891

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

    PubMed

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

    2016-02-01

    Hendra virus (HeV) is an emerging zoonotic paramyxovirus within the genus Henipavirus that has caused severe morbidity and mortality in humans and horses in Australia since 1994. HeV infection of host cells is mediated by the membrane bound attachment (G) and fusion (F) glycoproteins, that are essential for receptor binding and fusion of viral and cellular membranes. The eukaryotic unicellular parasite Leishmania tarentolae has recently been established as a powerful tool to express recombinant proteins with mammalian-like glycosylation patterns, but only few viral proteins have been expressed in this system so far. Here, we describe the purification of a truncated, Strep-tag labelled and soluble version of the HeV attachment protein (sHeV G) expressed in stably transfected L. tarentolae cells. After Strep-tag purification the identity of sHeV G was confirmed by immunoblotting and mass spectrometry. The functional binding of sHeV G to the HeV cell entry receptor ephrin-B2 was confirmed in several binding assays. Generated polyclonal rabbit antiserum against sHeV G reacted with both HeV and Nipah virus (NiV) G proteins in immunofluorescence assay and efficiently neutralised NiV infection, thus further supporting the preserved antigenicity of the purified protein. PMID:26585033

  17. Transcriptional activation by EBV nuclear antigen 1 is essential for the expression of EBV's transforming genes

    PubMed Central

    Altmann, Markus; Pich, Dagmar; Ruiss, Romana; Wang, Jindong; Sugden, Bill; Hammerschmidt, Wolfgang

    2006-01-01

    EBV is a paradigm for human tumor viruses because, although it infects most people benignly, it also can cause a variety of cancers. Both in vivo and in vitro, EBV infects B lymphocytes in G0, induces them to become blasts, and can maintain their proliferation in cell culture or in vivo as tumors. How EBV succeeds in these contrasting cellular environments in expressing its genes that control the host has not been explained. We have genetically dissected the EBV nuclear antigen 1 (EBNA1) gene that is required for replication of the viral genome, to elucidate its possible role in the transcription of viral genes. Strikingly, EBNA1 is essential to drive transcription of EBV's transforming genes after infection of primary B lymphocytes. PMID:16966603

  18. A polysaccharide fraction from medicinal herb Prunella vulgaris downregulates the expression of herpes simplex virus antigen in Vero cells.

    PubMed

    Chiu, Lawrence Chi-Ming; Zhu, Wen; Ooi, Vincent Eng-Choon

    2004-07-01

    Herpes simplex viruses (HSV) are pathogenic. With the emergence of drug-resistant strains of HSV, new antiviral agents, especially those with different modes of action, are urgently needed. Prunella vulgaris L. (Labiatae), a perennial plant commonly found in China and Europe, has long been used as a folk medicine to cure ailments. In this study, a polysaccharide fraction was prepared from Prunella vulgaris (PPV), and its effects on the expressions of HSV-1 and HSV-2 antigens in their host Vero cells were investigated with flow cytometry. The HSV antigen increased time-dependently in the infected cells, and PPV reduced its expression. The effective concentrations of PPV with 50% reductions of the HSV-1 and HSV-2 antigens were 20.6 and 20.1 microg/ml, respectively. The novelty of PPV is that it also reduces the antigen expression of acyclovir-resistant strain of HSV-1. After incubations with 25-100 microg/ml of PPV the HSV antigen-positive cells were reduced by 24.8-92.6%, respectively, showing that this polysaccharide fraction has a different mode of anti-HSV action from acyclovir. Results from this study show that PPV is effective against both the HSV-1 and HSV-2 infections, and flow cytometry offers a quantitative and highly reproducible anti-HSV drug-susceptibility assay. PMID:15182906

  19. Enhanced Expression of Interferon-γ-Induced Antigen-Processing Machinery Components in a Spontaneously Occurring Cancer1

    PubMed Central

    Cerruti, Fulvia; Martano, Marina; Petterino, Claudio; Bollo, Enrico; Morello, Emanuela; Bruno, Renato; Buracco, Paolo; Cascio, Paolo

    2007-01-01

    In human tumors, changes in the surface expression and/or function of major histocompatibility complex (MHC) class I antigens are frequently found and may provide malignant cells with a mechanism to escape control of the immune system. This altered human lymphocyte antigen (HLA) class I phenotype can be caused by either structural alterations or dysregulation of genes encoding subunits of HLA class I antigens and/or components of the MHC class I antigen-processing machinery (APM). Herein we analyze the expression of several proteins involved in the generation of MHC class I epitopes in feline injection site sarcoma, a spontaneously occurring tumor in cats that is an informativemodel for the study of tumor biology in other species, including humans. Eighteen surgically removed primary fibrosarcoma lesions were analyzed, and an enhanced expression of two catalytic subunits of immunoproteasomes, PA28 and leucine aminopeptidase, was found in tumors compared to matched normal tissues. As a functional counterpart of these changes in protein levels, proteasomal activities were increased in tissue extracts from fibrosarcomas. Taken together, these results suggest that alterations in the APM system may account for reduced processing of selected tumor antigens and may potentially provide neoplastic fibroblasts with a mechanism for escape from T-cell recognition and destruction. PMID:18030364

  20. Immunogenicity of recombinant BCGs expressing predicted antigenic epitopes of bovine viral diarrhea virus E2 gene.

    PubMed

    Liu, Dongxu; Lu, Huijun; Shi, Kun; Su, Fengyan; Li, Jianming; Du, Rui

    2014-10-01

    To develop a vaccine to prevent diseases caused by Mycobacterium tuberculosis and bovine viral diarrhea virus (BVDV) simultaneously, recombinant Bacillus Calmette-Guerin (rBCG) vaccines expressing different regions of the BVDV E2 gene were constructed. Using DNASTAR 6.0 software, potential antigenic epitopes were predicted, and six regions were chosen to generate recombinant plasmids with the pMV361 vector (pMV361-E2-1, pMV361-E2-2, pMV361-E2-3, pMV361-E2-4, pMV361-E2-5 and pMV361-E2-6, respectively). The recombinant plasmids were transformed into BCG, and protein expression was thermally induced at 45 °C. Mice were immunized with 5 × 10(6) CFU/200 µL of each rBCG strain. Compared with other groups, BVDV E2 specific antibody titers were higher in mice immunized with rBCG-E2-6. Ratios and numbers of CD4+, CD8+ and IL-12 expressing spleen lymphocytes of the rBCG-E2-6 group also were higher than those of other groups. Thus, the rBCG-E2-6 vaccine showed the highest immunogenicity of all groups based on the humoral and cellular responses to vaccination. PMID:25135492

  1. A new Epstein-Barr virus transactivator, R, induces expression of a cytoplasmic early antigen.

    PubMed Central

    Hardwick, J M; Lieberman, P M; Hayward, S D

    1988-01-01

    Several Epstein-Barr virus (EBV) early promoters respond to a new EBV transactivator encoded by BRLF1, designated R. Transactivation was measured in chloramphenicol acetyltransferase assays on Raji, BHK, and Vero cells that were cotransfected with the transactivator and target promoters linked to the cat gene. The divergent promoter of BamHI-H was particularly responsive to R transactivation. This large promoter region consists of a leftward TATA box for the NotI repeat gene (BHLF1) and a probable rightward TATA box for the EA-R gene (BHRF1) separated by 940 base pairs of unusual sequence complexity. Sequences within this divergent promoter region appear to confer inducibility by EBV transactivators R and Z (BZLF1). The Z transactivator stimulated expression in both the leftward and rightward directions, and R stimulated expression primarily in the rightward direction, but the MS transactivator (BMLF1) had no activity in either direction. The adenovirus E3 promoter also responded to the R transactivator, but several other herpesvirus and human promoters were nonresponsive. When the divergent promoter was linked to the EA-R gene as it is in the EBV genome, the R and Z transactivators also induced the expression of EA-R in cotransfected cells. This cytoplasmic early antigen is encoded by BHRF1 and may be anchored in intracellular membranes by a carboxy-terminal transmembrane region. Images PMID:2836611

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

    PubMed

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

    2013-01-01

    Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) is being evaluated in multiple clinical trials. Our current approach to adoptive immunotherapy is based on a second generation CAR (designated CD19RCD28) that signals through a CD28 and CD3-ζ endodomain. T cells are electroporated with DNA plasmids from the Sleeping Beauty (SB) transposon/transposase system to express this CAR. Stable integrants of genetically modified T cells can then be retrieved when co-cultured with designer artificial antigen presenting cells (aAPC) in the presence of interleukin (IL)-2 and 21. Here, we reveal how the platform technologies of SB-mediated transposition and CAR-dependent propagation on aAPC were adapted for human application. Indeed, we have initiated clinical trials in patients with high-risk B-lineage malignancies undergoing autologous and allogeneic hematopoietic stem-cell transplantation (HSCT). We describe the process to manufacture clinical grade CD19-specific T cells derived from healthy donors. Three validation runs were completed in compliance with current good manufacturing practice for Phase I/II trials demonstrating that by 28 days of co-culture on γ-irradiated aAPC ∼10(10) T cells were produced of which >95% expressed CAR. These genetically modified and propagated T cells met all quality control testing and release criteria in support of infusion. PMID:23741305

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

    PubMed Central

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

    2013-01-01

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

  4. Differential expression of T cell antigens in normal peripheral blood lymphocytes: a quantitative analysis by flow cytometry.

    PubMed Central

    Ginaldi, L; Farahat, N; Matutes, E; De Martinis, M; Morilla, R; Catovsky, D

    1996-01-01

    AIMS: To obtain reference values of the level of expression of T cell antigens on normal lymphocyte subsets in order to disclose differences which could reflect their function or maturation stages, or both. METHODS: Peripheral blood from 15 healthy donors was processed by flow cytometry with triple colour analysis. For each sample phycoerythrin (PE) conjugated CD2, CD4, CD5, CD8, and CD56 monoclonal antibodies were combined with Cy5-R-phycoerythrin (TC) conjugated CD3 and fluorescein isothiocyanate (FITC) conjugated CD7; CD2- and CD7-PE were also combined with CD3-TC and CD4-FITC. Standard microbeads with different capacities to bind mouse immunoglobulins were used to convert the mean fluorescence intensity (MFI) values of the lymphocyte subsets identified by multiparametric flow cytometry into the number of antigen molecules per cell, measured as antibody binding capacity (ABC). RESULTS: CD4+ (helper/inducer) T cells exhibit a higher CD3 antigen expression compared with CD8+ (suppressor/ cytotoxic) T lymphocytes. Within the CD4+ T cells, the CD4+CD7- subset expressed a lower level of CD3 compared with CD4+CD7+ and CD8+CD7+ cells, and higher CD2 and CD5 expression than the main CD3+CD7+ subset. Major differences in antigen expression were also detected between CD3+ T cells and CD3-CD56+ natural killer (NK) cells: NK cells exhibited higher levels of CD7 and CD56 and lower levels of CD2 and CD5 than T cells. Significantly lower CD5 expression was also detected in the small CD5+ B lymphocyte subset compared with T cells. CONCLUSIONS: Quantitative flow cytometry with triple colour analysis may be used to detect antigen modulations in disease states and to increase the accuracy of diagnosis by comparison with findings in normal counterparts. Images PMID:8813949

  5. Expression and T cell recognition of hybrid antigens with amino-terminal domains encoded by Qa-2 region of major histocompatibility complex and carboxyl termini of transplantation antigens.

    PubMed

    Stroynowski, I; Forman, J; Goodenow, R S; Schiffer, S G; McMillan, M; Sharrow, S O; Sachs, D H; Hood, L

    1985-05-01

    Coding potential of the Q6 gene from the Qa-2a region of BALB/c Crgl mice was analyzed by a combination of hybrid class I gene construction and DNA-mediated gene transfer. Recombinant genes were created by exon shuffling of the 5' coding region of the Q6 gene and the 3' coding region of a gene encoding a transplantation antigen (Kd, Dd, or Ld), or the inverse. Some of these hybrid class I genes were expressed in the transfected mouse fibroblasts (L cells). The hybrid class I molecules encoded by the 5' end of the Q6 gene and the 3' end of the Ld gene precipitated as 45,000 mol wt molecules associated with beta 2-microglobulin. The expression of the hybrid proteins indicates that 926 basepairs of the 5' flanking region upstream of the structural Q6 gene contain a promoter that functions as a transcription initiation site in L cells. The 3' portion of the Q6 gene appears to be responsible for the lack of cell surface expression of the intact Q6 and the hybrid Ld/Q6 genes in mouse fibroblasts. Accordingly, this portion of the Q6 class I gene may play a regulatory role in tissue-specific expression. Serological analyses of hybrid Q6 proteins suggested that Q6 may be a structural gene for CR (H-2 crossreactive) antigen found normally on subpopulations of lymphocytes. If this identification is correct, Q6 gene will define a new category of class I genes encoding approximately 40,000 mol wt molecules and carrying a characteristic truncated cytoplasmic tail. Analysis of L cells transfected with Q6 hybrid genes demonstrated also that the cytotoxic T cells specific for Qa-2a region-coded antigens recognize the amino-terminal alpha 1-alpha 2 domain of Q6 fusion products. This recognition can be blocked by anti-Qa-2a alloantiserum and monoclonal antibodies reactive with the alpha 3-beta 2-microglobulin portion of the Q6 hybrids. We propose that the structural requirements for the anti-Qa-2a cytotoxic T lymphocyte-specific epitopes on target molecules are the same as for anti

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

    PubMed Central

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

    2015-01-01

    Burkholderia pseudomallei and Burkholderia mallei are the etiologic agents of melioidosis and glanders, respectively. These bacteria are highly infectious via the respiratory route and can cause severe and often fatal diseases in humans and animals. Both species are considered potential agents of biological warfare; they are classified as category B priority pathogens. Currently there are no human or veterinary vaccines available against these pathogens. Consequently efforts are directed towards the development of an efficacious and safe vaccine. Lipopolysaccharide (LPS) is an immunodominant antigen and potent stimulator of host immune responses. B. mallei express LPS that is structurally similar to that expressed by B. pseudomallei, suggesting the possibility of constructing a single protective vaccine against melioidosis and glanders. Previous studies of others have shown that antibodies against B. mallei or B. pseudomallei LPS partially protect mice against subsequent lethal virulent Burkholderia challenge. In this study, we evaluated the protective efficacy of recombinant Salmonella enterica serovar Typhimurium SL3261 expressing B. mallei O antigen against lethal intranasal infection with Burkholderia thailandensis, a surrogate for biothreat Burkholderia spp. in a murine model that mimics melioidosis and glanders. All vaccine-immunized mice developed a specific antibody response to B. mallei and B. pseudomallei O antigen and to B. thailandensis and were significantly protected against challenge with a lethal dose of B. thailandensis. These results suggest that live-attenuated SL3261 expressing B. mallei O antigen is a promising platform for developing a safe and effective vaccine. PMID:26148026

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

    PubMed

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

    2016-01-01

    To obtain adhesive and safe lactic acid bacteria (LAB) strains for expressing heterologous antigens, we screened LAB inhabitants in intestine of Tibetan chickens by analyzing their adhesion and safety properties and the selected LAB was engineered to express heterologous antigen (UTEpi C-A) based on chromosomal integration strategy. We demonstrated that a new Lactobacillu salivarius TCMM17 strain is strongly adhesive to chicken intestinal epithelial cells, contains no endogenous plasmids, is susceptible to tested antimicrobials, and shows no toxicities. In order to examine the potential of TCMM17 strain as heterogenous antigen delivering vehicle, we introduced a UTEpi C-A expression cassette in its chromosome by constructing a non-replicative plasmid (pORI280-UUTEpi C-AD). The recombinant TCMM17 strain (∆TCMM17) stably was found to keep the gene cassette through 50 generations, and successfully displayed EpiC encoded by the cassette on its surface. This work provides a universal platform for development of novel oral vaccines and expression of further antigens of avian pathogens. PMID:26618736

  8. Roles of lymphatic endothelial cells expressing peripheral tissue antigens in CD4 T-cell tolerance induction.

    PubMed

    Rouhani, Sherin J; Eccles, Jacob D; Riccardi, Priscila; Peske, J David; Tewalt, Eric F; Cohen, Jarish N; Liblau, Roland; Mäkinen, Taija; Engelhard, Victor H

    2015-01-01

    Lymphatic endothelial cells (LECs) directly express peripheral tissue antigens and induce CD8 T-cell deletional tolerance. LECs express MHC-II molecules, suggesting they might also tolerize CD4 T cells. We demonstrate that when β-galactosidase (β-gal) is expressed in LECs, β-gal-specific CD8 T cells undergo deletion via the PD-1/PD-L1 and LAG-3/MHC-II pathways. In contrast, LECs do not present endogenous β-gal in the context of MHC-II molecules to β-gal-specific CD4 T cells. Lack of presentation is independent of antigen localization, as membrane-bound haemagglutinin and I-Eα are also not presented by MHC-II molecules. LECs express invariant chain and cathepsin L, but not H2-M, suggesting that they cannot load endogenous antigenic peptides onto MHC-II molecules. Importantly, LECs transfer β-gal to dendritic cells, which subsequently present it to induce CD4 T-cell anergy. Therefore, LECs serve as an antigen reservoir for CD4 T-cell tolerance, and MHC-II molecules on LECs are used to induce CD8 T-cell tolerance via LAG-3. PMID:25857745

  9. Roles of lymphatic endothelial cells expressing peripheral tissue antigens in CD4 T-cell tolerance induction

    PubMed Central

    Rouhani, Sherin J.; Eccles, Jacob D.; Riccardi, Priscila; Peske, J. David; Tewalt, Eric F.; Cohen, Jarish N.; Liblau, Roland; Mäkinen, Taija; Engelhard, Victor H.

    2015-01-01

    Lymphatic endothelial cells (LECs) directly express peripheral tissue antigens and induce CD8 T-cell deletional tolerance. LECs express MHC-II molecules, suggesting they might also tolerize CD4 T cells. We demonstrate that when β-galactosidase (β-gal) is expressed in LECs, β-gal-specific CD8 T cells undergo deletion via the PD-1/PD-L1 and LAG-3/MHC-II pathways. In contrast, LECs do not present endogenous β-gal in the context of MHC-II molecules to β-gal-specific CD4 T cells. Lack of presentation is independent of antigen localization, as membrane-bound haemagglutinin and I-Eα are also not presented by MHC-II molecules. LECs express invariant chain and cathepsin L, but not H2-M, suggesting that they cannot load endogenous antigenic peptides onto MHC-II molecules. Importantly, LECs transfer β-gal to dendritic cells, which subsequently present it to induce CD4 T-cell anergy. Therefore, LECs serve as an antigen reservoir for CD4 T-cell tolerance, and MHC-II molecules on LECs are used to induce CD8 T-cell tolerance via LAG-3. PMID:25857745

  10. High-level expression of recombinant dengue viral NS-1 protein and its potential use as a diagnostic antigen.

    PubMed

    Huang, J L; Huang, J H; Shyu, R H; Teng, C W; Lin, Y L; Kuo, M D; Yao, C W; Shaio, M F

    2001-11-01

    The prevalence of NS1 Ab response in patients with dengue viral infection and the potential of using recombinant NS1 protein as a diagnostic antigen for dengue viral infection were investigated. In this study, the full-length and C-terminal half of NS1 proteins (rNS1, rNS1-C) were highly expressed (10-30 mg/l) and further purified and refolded. The good antigenicity of the full-length rNS1 protein was confirmed by interaction with 19 dengue NS1-specific monoclonal antibodies (MAbs) in ELISA; however, the antigenicity of rNS1-C was relatively lower. The full-length rNS1 antigen also differentiated reliably between sera from dengue virus-infected patients and sera from normal controls. When rNS1 was used as an antigen to detect human anti-NS1 IgM and IgG Ab, the anti-NS1 Ab response was found in 15 of 17 patients (88%) with primary dengue infection and all 16 patients (100%) with secondary dengue infection. These results indicated that using the full-length rNS1 whose antigenicity is restored as ELISA antigen, a high anti-NS1 antibody prevalence could be detected in patients with either primary or secondary dengue infection. This finding suggested that the anti-NS1 antibody appeared not only in secondary and severe dengue virus infection and might not correlate the pathogenesis of dengue hemorrhagic fever. The study also verified that our purified rNS1 protein showed similar immunological properties as native dengue viral proteins. Genetic engineering production of recombinant NS1 antigen could provide a safe and valuable resource for dengue virus serodiagnosis. PMID:11596093

  11. A mouse model for chronic lymphocytic leukemia based on expression of the SV40 large T antigen.

    PubMed

    ter Brugge, Petra J; Ta, Van B T; de Bruijn, Marjolein J W; Keijzers, Guido; Maas, Alex; van Gent, Dik C; Hendriks, Rudi W

    2009-07-01

    The simian virus 40 (SV40) T antigen is a potent oncogene able to transform many cell types and has been implicated in leukemia and lymphoma. In this report, we have achieved sporadic SV40 T-antigen expression in mature B cells in mice, by insertion of a SV40 T antigen gene in opposite transcriptional orientation in the immunoglobulin (Ig) heavy (H) chain locus between the D and J(H) segments. SV40 T-antigen expression appeared to result from retention of the targeted germline allele and concomitant antisense transcription of SV40 large T in mature B cells, leading to chronic lymphocytic leukemia (CLL). Although B-cell development was unperturbed in young mice, aging mice showed accumulation of a monoclonal B-cell population in which the targeted IgH allele was in germline configuration and the wild-type IgH allele had a productive V(D)J recombination. These leukemic B cells were IgD(low)CD5(+) and manifested nonrandom usage of V, D, and J segments. V(H) regions were either unmutated, with preferential usage of the VH11 family, or manifested extensive somatic hypermutation. Our findings provide an animal model for B-CLL and show that pathways activated by SV40 T antigen play important roles in the pathogenesis of B-CLL. PMID:19332766

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

    PubMed

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

    2016-02-29

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of food-borne illness around the world and can have significant health implications in humans, poultry and other animals. Flagellin (FliC) is the primary component of bacterial flagella. It has been shown that the FliC of S. Enteritidis is a significant antigenic structure and can elicit strong humoral responses against S. Enteritidis infection in chickens. Here, we constructed a FliC antigen library using a yeast surface expression system. Yeast cells expressing FliC peptide antigens were labeled with chicken sera against S. Enteritidis and sorted using FACS. The analyses of FliC peptides revealed that the FliC linear antigenicity in chickens resided on three domains which were able to elicit strong humoral responses in vivo. Animal experiments further revealed that the antibodies elicited by these antigenic domains were able to significantly inhibit the invasion of S. Enteritidis into the liver and spleen of chickens. These findings will facilitate our better understanding of the humoral responses elicited by FliC in chickens upon infection by S. Enteritidis. PMID:26854340

  13. Mesenchymal stem cells expressing neural antigens instruct a neurogenic cell fate on neural stem cells.

    PubMed

    Croft, Adam P; Przyborski, Stefan A

    2009-04-01

    The neurogenic response to injury in the postnatal brain is limited and insufficient for restoration of function. Recent evidence suggests that transplantation of mesenchymal stem cells (MSCs) into the injured brain is associated with improved functional recovery, mediated in part through amplification in the endogenous neurogenic response to injury. In the current study we investigate the interactions between bone marrow-derived MSCs and embryonic neural stem cells (NSCs) plus their differentiated progeny using an in vitro co-culture system. Two populations of MSCs were used, MSCs induced to express neural antigens (nestin+, Tuj-1+, GFAP+) and neural antigen negative MSCs. Following co-culture of induced MSCs with differentiating NSC/progenitor cells a significant increase in Tuj-1+ neurons was detected compared to co-cultures of non-induced MSCs in which an increase in astrocyte (GFAP+) differentiation was observed. The effect was mediated by soluble interactions between the two cell populations and was independent of any effect on cell death and proliferation. Induced and non-induced MSCs also promoted the survival of Tuj-1+ cell progeny in long-term cultures and both promoted axonal growth, an effect also seen in differentiating neuroblastoma cells. Therefore, MSCs provide instructive signals that are able to direct the differentiation of NSCs and promote axonal development in neuronal progeny. The data indicates that the nature of MSC derived signals is dependent not only on their microenvironment but on the developmental status of the MSCs. Pre-manipulation of MSCs prior to transplantation in vivo may be an effective means of enhancing the endogenous neurogenic response to injury. PMID:19159625

  14. Abnormal cell surface antigen expression in individuals with variant CD45 splicing and histiocytosis.

    PubMed

    Boxall, Sally; McCormick, James; Beverley, Peter; Strobel, Stephan; De Filippi, Paola; Dawes, Ritu; Klersy, Catherine; Clementi, Rita; De Juli, Emanuella; Ferster, Aline; Wallace, Diana; Aricò, Maurizio; Danesino, Cezare; Tchilian, Elma

    2004-03-01

    Hemophagocytic lymphohistiocytosis (HLH) and Langerhans cell histiocytosis (LCH) are members of a group of rare heterogenous disorders, the histiocytoses, characterized by uncontrolled accumulation of pleomorphic infiltrates of leukocytes. The etiology of these diseases is mainly unknown. CD45 is a hemopoietic cell specific tyrosine phosphatase essential for antigen receptor mediated signaling in lymphocytes and different patterns of CD45 splicing are associated with distinct functions. Recently a polymorphism (C77G) in exon 4 of CD45 causing abnormal CD45 splicing and a point mutation affecting CD45 dimerization were implicated in multiple sclerosis in humans and lymphoproliferation and autoimmunity in mice respectively. Here we show that two patients with HLH exhibited abnormal CD45 splicing caused by the C77G variant allele, while a further 21 HLH patients have normal CD45. We have also examined 62 LCH patients and found three to have the C77G mutation. Peripheral blood thymus-derived (T) CD8(+) cells from normal individuals carrying the C77G mutation show a significant decrease in the proportion of cells expressing L-selectin and increased frequency of cells with LFA-1(hi) expression. It remains to be established whether C77G is a contributing factor in these histiocytic disorders. PMID:14630980

  15. The use of directed evolution to create a stable and immunogenic recombinant BCG expressing a modified HIV-1 Gag antigen.

    PubMed

    Chapman, Rosamund; Bourn, William R; Shephard, Enid; Stutz, Helen; Douglass, Nicola; Mgwebi, Thandi; Meyers, Ann; Chin'ombe, Nyasha; Williamson, Anna-Lise

    2014-01-01

    Numerous features make Mycobacterium bovis BCG an attractive vaccine vector for HIV. It has a good safety profile, it elicits long-lasting cellular immune responses and in addition manufacturing costs are affordable. Despite these advantages it is often difficult to express viral antigens in BCG, which results in genetic instability and low immunogenicity. The aim of this study was to generate stable recombinant BCG (rBCG) that express high levels of HIV antigens, by modification of the HIV genes. A directed evolution process was applied to recombinant mycobacteria that expressed HIV-1 Gag fused to the green fluorescent protein (GFP). Higher growth rates and increased GFP expression were selected for. Through this process a modified Gag antigen was selected. Recombinant BCG that expressed the modified Gag (BCG[pWB106] and BCG[pWB206]) were more stable, produced higher levels of antigen and grew faster than those that expressed the unmodified Gag (BCG[pWB105]). The recombinant BCG that expressed the modified HIV-1 Gag induced 2 to 3 fold higher levels of Gag-specific CD4 T cells than those expressing the unmodified Gag (BCG[pWB105]). Mice primed with 10(7) CFU BCG[pWB206] and then boosted with MVA-Gag developed Gag-specific CD8 T cells with a frequency of 1343±17 SFU/10(6) splenocytes, 16 fold greater than the response induced with MVA-Gag alone. Levels of Gag-specific CD4 T cells were approximately 5 fold higher in mice primed with BCG[pWB206] and boosted with MVA-Gag than in those receiving the MVA-Gag boost alone. In addition mice vaccinated with BCG[pWB206] were protected from a surrogate vaccinia virus challenge. PMID:25061753

  16. Rat adenocarcinoma 13762 expresses tumor rejection antigens but tumor-bearing animals exhibit tumor-specific immunosuppression.

    PubMed

    Frey, A B; Appleman, L J

    1993-11-01

    Rat adenocarcinoma 13762 was adapted to continuous growth in culture and used in a variety of experiments to investigate the immune response to inoculation of animals with replication-defective tumor cells. The results demonstrate that 13762 cells express tumor-specific tumor rejection antigens that elicit protective immunity to tumorigenic challenge. By several criteria there is no apparent humoral component of the anti-tumor immunity; however, anti-tumor immunity is characterized by nylon-wool nonadherent spleen T cells. Anti-tumor T cells demonstrate tumoricidal activity in local adoptive transfer assays and are not found in spleens of naive animals or animals immunized against either nontumorigenic Rat 1 cells or a syngeneic fibrosarcoma. Despite the expression of tumor rejection antigens 13762 tumor, and the demonstrable ability of injection of irradiated tumor to induce anti-tumor immunity, tumors elicited in unimmunized syngeneic animals grow progressively. The reasons for growth of antigenic tumor are unknown but are shown not to be due to defective antigen expression in 13762 tumor since, in addition to being able to elicit T cell immune response in immunized animals, 13762 tumor expresses MHC Class I molecules and can be a target for allogeneic T cell recognition in vitro. These data suggest that in tumor-bearing animals an effective anti-tumor immune response is either not initiated or down-regulated. Since animals bearing 13762 tumors can be immunized against an unrelated syngeneic sarcoma, can produce humoral responses to several protein antigens, and can produce delayed type hypersensitivity response against dinitrofluorobenzene, the immune response to 13762-induced tumors appears specifically suppressed. In support of this contention, 13762 cells express high levels of transforming growth factor beta 1 in vitro which is postulated to impact upon the nascent anti-tumor immune response. PMID:8403560

  17. Subsets of Human Dendritic Cell Precursors Express Different Toll-like Receptors and Respond to Different Microbial Antigens

    PubMed Central

    Kadowaki, Norimitsu; Ho, Stephen; Antonenko, Svetlana; de Waal Malefyt, Rene; Kastelein, Robert A.; Bazan, Fernando; Liu, Yong-Jun

    2001-01-01

    Toll-like receptors (TLRs) are ancient microbial pattern recognition receptors highly conserved from Drosophila to humans. To investigate if subsets of human dendritic cell precursors (pre-DC), including monocytes (pre-DC1), plasmacytoid DC precursors (pre-DC2), and CD11c+ immature DCs (imDCs) are developed to recognize different microbes or microbial antigens, we studied their TLR expression and responses to microbial antigens. We demonstrate that whereas monocytes preferentially express TLR 1, 2, 4, 5, and 8, plasmacytoid pre-DC strongly express TLR 7 and 9. In accordance with these TLR expression profiles, monocytes respond to the known microbial ligands for TLR2 (peptidoglycan [PGN], lipoteichoic acid) and TLR4 (lipopolysaccharide), by producing tumor necrosis factor (TNF)-α and interleukin (IL)-6. In contrast, plasmacytoid pre-DCs only respond to the microbial TLR9-ligand, CpG-ODNs (oligodeoxynucleotides [ODNs] containing unmethylated CpG motifs), by producing IFN-α. CD11c+ imDCs preferentially express TLR 1, 2, and 3 and respond to TLR 2-ligand PGN by producing large amounts of TNF-α, and to viral double-stranded RNA-like molecule poly I:C, by producing IFN-α and IL-12. The expression of distinct sets of TLRs and the corresponding difference in reactivity to microbial molecules among subsets of pre-DCs and imDCs support the concept that they have developed through distinct evolutionary pathways to recognize different microbial antigens. PMID:11561001

  18. Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens.

    PubMed

    Kadowaki, N; Ho, S; Antonenko, S; Malefyt, R W; Kastelein, R A; Bazan, F; Liu, Y J

    2001-09-17

    Toll-like receptors (TLRs) are ancient microbial pattern recognition receptors highly conserved from Drosophila to humans. To investigate if subsets of human dendritic cell precursors (pre-DC), including monocytes (pre-DC1), plasmacytoid DC precursors (pre-DC2), and CD11c(+) immature DCs (imDCs) are developed to recognize different microbes or microbial antigens, we studied their TLR expression and responses to microbial antigens. We demonstrate that whereas monocytes preferentially express TLR 1, 2, 4, 5, and 8, plasmacytoid pre-DC strongly express TLR 7 and 9. In accordance with these TLR expression profiles, monocytes respond to the known microbial ligands for TLR2 (peptidoglycan [PGN], lipoteichoic acid) and TLR4 (lipopolysaccharide), by producing tumor necrosis factor (TNF)-alpha and interleukin (IL)-6. In contrast, plasmacytoid pre-DCs only respond to the microbial TLR9-ligand, CpG-ODNs (oligodeoxynucleotides [ODNs] containing unmethylated CpG motifs), by producing IFN-alpha. CD11c(+) imDCs preferentially express TLR 1, 2, and 3 and respond to TLR 2-ligand PGN by producing large amounts of TNF-alpha, and to viral double-stranded RNA-like molecule poly I:C, by producing IFN-alpha and IL-12. The expression of distinct sets of TLRs and the corresponding difference in reactivity to microbial molecules among subsets of pre-DCs and imDCs support the concept that they have developed through distinct evolutionary pathways to recognize different microbial antigens. PMID:11561001

  19. Binding to histo-blood group antigen-expressing bacteria protects human norovirus from acute heat stress

    PubMed Central

    Li, Dan; Breiman, Adrien; le Pendu, Jacques; Uyttendaele, Mieke

    2015-01-01

    This study aims to investigate if histo-blood group antigen (HBGA) expressing bacteria have any protective role on human norovirus (NoV) from acute heat stress. Eleven bacterial strains were included, belonging to Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Clostridium difficile, Bifidobacterium adolescentis, and B. longum. HBGA expression of the bacteria as well as binding of human NoV virus-like particles (VLPs, GI.1, and GII.4 strains) to the bacteria were detected by flow cytometry. NoV VLPs pre-incubated with HBGA expressing or non-HBGA expressing bacteria were heated and detected by both direct ELISA and porcine gastric mucin-binding assay. The NoV-binding abilities of the bacteria correlated well with their HBGA expression profiles. Two HBGA expressing E. coli (LMG8223 and LFMFP861, both GI.1 and GII.4 binders) and one non-HBGA expressing E. coli (ATCC8739, neither GI.1 nor GII.4 binder) were selected for the heat treatment test with NoV VLPs. Compared with the same cell numbers of non-HBGA expressing E. coli, the presence of HBGA-expressing E. coli could always maintain higher antigen integrity, as well as mucin-binding ability of NoV VLPs of both GI.1 and GII.4 after heat-treatment at 90°C for 2 min. These results indicate that HBGA-expressing bacteria may protect NoVs during the food processing treatments, thereby facilitating their transmission. PMID:26191052

  20. Influenza virus infection elicits protective antibodies and T cells specific for host cell antigens also expressed as tumor-associated antigens: a new view of cancer immunosurveillance.

    PubMed

    Iheagwara, Uzoma K; Beatty, Pamela L; Van, Phu T; Ross, Ted M; Minden, Jonathan S; Finn, Olivera J

    2014-03-01

    Most tumor-associated antigens (TAA) are self-molecules that are abnormally expressed in cancer cells and become targets of antitumor immune responses. Antibodies and T cells specific for some TAAs have been found in healthy individuals and are associated with lowered lifetime risk for developing cancer. Lower risk for cancer has also been associated with a history of febrile viral diseases. We hypothesized that virus infections could lead to transient expression of abnormal forms of self-molecules, some of which are TAAs; facilitated by the adjuvant effects of infection and inflammation, these molecules could elicit specific antibodies, T cells, and lasting immune memory simultaneously with immunity against viral antigens. Such infection-induced immune memory for TAA would be expected to provide life-long immune surveillance of cancer. Using influenza virus infection in mice as a model system, we tested this hypothesis and demonstrated that influenza-experienced mice control 3LL mouse lung tumor challenge better than infection-naive control mice. Using 2D-difference gel electrophoresis and mass spectrometry, we identified numerous molecules, some of which are known TAAs, on the 3LL tumor cells recognized by antibodies elicited by two successive influenza infections. We studied in detail immune responses against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H4, HSP90, malate dehydrogenase 2, and annexin A2, all of which were overexpressed in influenza-infected lungs and in tumor cells. Finally, we show that immune responses generated through vaccination against peptides derived from these antigens correlated with improved tumor control. PMID:24778322

  1. Cloning of cDNA of major antigen of foot and mouth disease virus and expression in E. coli

    NASA Astrophysics Data System (ADS)

    Küpper, Hans; Keller, Walter; Kurz, Christina; Forss, Sonja; Schaller, Heinz

    1981-02-01

    Double-stranded DNA copies of the single-stranded genomic RNA of foot and mouth disease virus have been cloned into the Escherichia coli plasmid pBR322. A restriction map of the viral genome was established and aligned with the biochemical map of foot and mouth disease virus. The coding sequence for structural protein VP1, the major antigen of the virus, was identified and inserted into a plasmid vector where the expression of this sequence is under control of the phage λ PL promoter. In an appropriate host the synthesis of antigenic polypeptide can be demonstrated by radioimmunoassay.

  2. Mycobacterium bovis BCG priming induces a strong potentiation of the antibody response induced by recombinant BCG expressing a foreign antigen.

    PubMed Central

    Gheorghiu, M; Lagranderie, M R; Gicquel, B M; Leclerc, C D

    1994-01-01

    Several recent studies have demonstrated that strong cellular or humoral immune responses can be induced against foreign antigens expressed by recombinant Mycobacterium bovis BCG. It has therefore been suggested that BCG could represent one of the best candidate vectors for live recombinant vaccines. However, a large percentage of the human population has been immunized by BCG, and this priming could modify the immune response to future recombinant BCG vaccines. In the present study, we have therefore compared the immune responses induced in naive and BCG-primed mice by two recombinant BCG vaccines expressing either beta-galactosidase or human immunodeficiency virus type 1 Nef antigens. Our results demonstrated that BCG priming limits the growth of recombinant BCG in mouse spleen or lymph nodes. This reduction in BCG growth was associated with decreased proliferative responses against Nef or beta-galactosidase antigens. This suppression, however, never exceeded 50%. Interestingly, in contrast to these reduced T-cell responses, BCG-primed mice developed high levels of anti-beta-galactosidase antibodies after immunization with recombinant BCG expressing this antigen. This stimulation of antibody responses was not due to polyclonal stimulation or to a nonspecific adjuvant effect of BCG. The isotypic patterns of anti-beta-galactosidase antibody responses induced by the recombinant BCG were similar in naive and BCG-primed mice. These results indicate that priming with BCG will not be a limitation for the use of recombinant BCG vaccines in humans. PMID:7927686

  3. Constitutive expression of major histocompatibility complex class II antigens in pulmonary epithelium and endothelium varies among different species.

    PubMed

    Houser, Stuart L; Benjamin, Louis C; Wain, John C; Madsen, Joren C; Allan, James S

    2004-02-27

    We have observed high constitutive levels of class II antigen expression on porcine and human coronary endothelium, but not on the endothelium of rats and mice. This study examines whether a similar interspecies difference exists in the expression of class II molecules on pulmonary epithelium and endothelium. Lung tissues from naïve human, porcine, and rodent sources were stained with the monoclonal antibody ISCR3 and examined by light microscopy. Immunoperoxidase staining of class II molecules was observed on human and porcine pulmonary epithelium and endothelium, but was absent in rats and mice. By using an antibody with cross-species reactivity, we demonstrated that naïve swine pulmonary epithelium and endothelium, unlike those of rodent species, express basal levels of class II antigens in a manner similar to that observed in human lung tissue. These interspecies differences may explain experimental differences observed between murine and large-animal constructs. PMID:15084944

  4. Expression of cutaneous lymphocyte-associated antigen by CD8+ T cells specific for a skin-tropic virus

    PubMed Central

    Koelle, David M.; Liu, Zhi; McClurkan, Christopher M.; Topp, Max S.; Riddell, Stanley R.; Pamer, Eric G.; Johnson, Andrew S.; Wald, Anna; Corey, Lawrence

    2002-01-01

    Virus-specific CD8+ T cells traffic to infected tissues to promote clearance of infection. We used herpes simplex virus type 2 (HSV-2) as a model system to investigate CD8+ T cell trafficking to the skin in humans. Using human leukocyte antigen (HLA) class I tetramers, we observed that HSV-specific CD8+ T cells in the peripheral blood expressed high levels of cutaneous lymphocyte-associated antigen (CLA). In contrast, CD8+ T cells specific for non–skin-tropic herpesviruses lacked CLA expression. CLA-positive HSV-2–specific CD8+ T cells had the characteristics of central memory cells, expressing CCR7, CD62L, and CD28, and they proliferated briskly in response to antigen. CLA is related to a functional E-selectin ligand, and both E-selectin and CLA-positive cells were detected in HSV-2–infected skin. HSV-2–specific T cells adhered to cells transfected with E-selectin. A higher proportion of HSV-specific CD8+ T cells recovered from herpes lesions express CLA compared with blood, consistent with a role for CLA in skin homing. To our knowledge, this is the first report of expression of tissue-specific adhesion-associated molecules by virus-specific CD8+ T cells. The evaluation of vaccines for skin and mucosal pathogens should include study of the induction of appropriate tissue-specific homing molecules. PMID:12189248

  5. Chronic Hepatitis B Virus Infection: The Relation between Hepatitis B Antigen Expression, Telomere Length, Senescence, Inflammation and Fibrosis

    PubMed Central

    Tachtatzis, Phaedra M.; Marshall, Aileen; Aravinthan, Aloysius; Verma, Suman; Penrhyn-Lowe, Sue; Mela, Marianna; Scarpini, Cinzia; Davies, Susan E.; Coleman, Nicholas; Alexander, Graeme J. M.

    2015-01-01

    Background Chronic Hepatitis B virus (HBV) infection can lead to the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma. We hypothesized that HBV might accelerate hepatocyte ageing and investigated the effect of HBV on hepatocyte cell cycle state and biological age. We also investigated the relation between inflammation, fibrosis and cell cycle phase. Methods Liver samples from patients with chronic HBV (n = 91), normal liver (n = 55) and regenerating liver (n = 15) were studied. Immunohistochemistry for cell cycle phase markers and HBV antigens was used to determine host cell cycle phase. Hepatocyte-specific telomere length was evaluated by quantitative fluorescent in-situ hybridization (Q-FISH) in conjunction with hepatocyte nuclear area and HBV antigen expression. The effects of induced cell cycle arrest and induced cellular senescence on HBV production were assessed in vitro. Results 13.7% hepatocytes in chronic HBV had entered cell cycle, but expression of markers for S, G2 and M phase was low compared with regenerating liver. Hepatocyte p21 expression was increased (10.9%) in chronic HBV and correlated with liver fibrosis. Mean telomere length was reduced in chronic HBV compared to normal. However, within HBV-affected livers, hepatocytes expressing HBV antigens had longer telomeres. Telomere length declined and hepatocyte nuclear size increased as HBV core antigen (HBcAg) expression shifted from the nucleus to cytoplasm. Nuclear co-expression of HBcAg and p21 was not observed. Cell cycle arrest induced in vitro was associated with increased HBV production, in contrast to 
in vitro induction of cellular senescence, which had no effect. Conclusion Chronic HBV infection was associated with hepatocyte G1 cell cycle arrest and accelerated hepatocyte ageing, implying that HBV induced cellular senescence. However, HBV replication was confined to biologically younger hepatocytes. Changes in the cellular location of HBcAg may be related to the

  6. Characterization of MAX.3 antigen, a glycoprotein expressed on mature macrophages, dendritic cells and blood platelets: identity with CD84.

    PubMed Central

    Krause, S W; Rehli, M; Heinz, S; Ebner, R; Andreesen, R

    2000-01-01

    MAX.3 is a monoclonal antibody that preferentially reacts with mature macrophages (MAC), monocyte-derived dendritic cells, megakaryocytes and platelets. In this study, we describe the characterization, purification and identification of the MAX.3 antigen. Immunoprecipitation and SDS/PAGE revealed different molecular masses of MAX.3 antigen in MAC (60-90 kDa) and platelets (58-64 kDa), whereas a similar size (45 kDa) was observed in both cell types after digestion with N-glycosidase F. Lectin affinity and sequential treatment with different glycosidases suggests complex type glycosylation of MAX.3 antigen in MAC and hybrid type glycosylation in platelets. Amino acid sequencing led to the identification of a corresponding cDNA clone and showed its identity to the sequence of the CD84 antigen, a member of the CD2 family of cell surface molecules. MAX.3/CD84 was further studied by immunohistochemistry and a variable expression was found on tissue MAC, confirming this antigen to be mainly a marker for MAC in situ. PMID:10698700

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

    SciTech Connect

    Bujak, Emil; Pretto, Francesca; Ritz, Danilo; Gualandi, Laura; Wulhfard, Sarah; Neri, Dario

    2014-09-10

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

  8. [Construction and expression of a eukaryotic vector co-expressing immunodominant antigens of CFP10, ESAT6, Ag85A and Ag85B of Mycobacterium tuberculosis].

    PubMed

    Li, Wu; Deng, Guangcun; Liu, Xiaoming; Wang, Yujiong

    2014-02-01

    CFP10, ESAT6, Antigen 85A (Ag85A) and antigen 85B (Ag85B) are the key immunodominant antigens of Mycobacterium tuberculosis. In order to construct a eukaryotic vector able to co-express the four genes in one vector, we amplified the target gene fragments encoding the CFP10, ESAT6, Ag85A and Ag85B antigens and inserted them into the multicloning site of the shuttle plasmid vector pcDNA3.1 (+), of which the CFP10 and ESAT6 encoding genes were in frame fused with a linker encoding (Gly4Ser)3 residue, before the fused gene was inserted downstream of CMV promoter with a bovine growth hormone poly A(BGH pA) sequence at the 3'-end; Ag85A and Ag85B encoding genes were fused with a separation of internal ribosome entry site (IRES) sequence before the fused gene cassette was inserted downstream of RSV promoter with a BGH pA sequence at the 3'-end. The final plasmid containing all four genes was confirmed by sequence analysis and designated as pcDNA-CFP10-ESAT6-Ag85A-Ag85B (pcDNA-CEAB). In order to verify the ability of this construct to express target proteins, we then transfected the recombinant plasmid into Human embryonic kidney (HEK) 293T cells and harvested the cell lysates, and the cell lysates were then separated by SDS-PAGE and subjected to Western blot analysis 48 h after transfection. All four of the target proteins were detected in the cell lysates against the respective specific antibodies, suggesting that we have successfully constructed a eukaryotic vector co-expressing the four immunodominant antigens of Mycobacterium tuberculosis, which lay a foundation for the further study of the immunogenicity and protective activity of the four antigens. PMID:24941747

  9. Efficacy of Recombinant Canine Distemper Virus Expressing Leishmania Antigen against Leishmania Challenge in Dogs.

    PubMed

    Miura, Ryuichi; Kooriyama, Takanori; Yoneda, Misako; Takenaka, Akiko; Doki, Miho; Goto, Yasuyuki; Sanjoba, Chizu; Endo, Yasuyuki; Fujiyuki, Tomoko; Sugai, Akihiro; Tsukiyama-Kohara, Kyoko; Matsumoto, Yoshitsugu; Sato, Hiroki; Kai, Chieko

    2015-01-01

    Canine distemper virus (CDV) vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV-LACK, rCDV-TSA, and rCDV-LmSTI1, respectively). Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears) with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV-LACK showed markedly smaller nodules without ulceration. Although the rCDV-TSA- and rCDV-LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV-LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs. PMID:26162094

  10. Efficacy of Recombinant Canine Distemper Virus Expressing Leishmania Antigen against Leishmania Challenge in Dogs

    PubMed Central

    Yoneda, Misako; Takenaka, Akiko; Doki, Miho; Goto, Yasuyuki; Sanjoba, Chizu; Endo, Yasuyuki; Fujiyuki, Tomoko; Sugai, Akihiro; Tsukiyama-Kohara, Kyoko; Matsumoto, Yoshitsugu; Sato, Hiroki; Kai, Chieko

    2015-01-01

    Canine distemper virus (CDV) vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV–LACK, rCDV–TSA, and rCDV–LmSTI1, respectively). Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears) with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV–LACK showed markedly smaller nodules without ulceration. Although the rCDV–TSA- and rCDV–LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV–LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs. PMID:26162094

  11. Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms.

    PubMed

    Ford, Nicole R; Hecht, Karen A; Hu, DeHong; Orr, Galya; Xiong, Yijia; Squier, Thomas C; Rorrer, Gregory L; Roesijadi, Guritno

    2016-03-18

    The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins comprising either enhanced green fluorescent protein (EGFP) or single chain antibodies engineered with a tetracysteine tagging sequence. Of interest were the site-specific binding of (1) the fluorescent biarsenical probe AsCy3 and AsCy3e to the tetracysteine tagged fusion proteins and (2) high and low molecular mass antigens, the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT), to biosilica-immobilized single chain antibodies. Analysis of biarsenical probe binding using fluorescence and structured illumination microscopy indicated differential colocalization with EGFP in nascent and mature biosilica, supporting the use of either EGFP or bound AsCy3 and AsCy3e in studying biosilica maturation. Large increases in the lifetime of a fluorescent analogue of TNT upon binding single chain antibodies provided a robust signal capable of discriminating binding to immobilized antibodies in the transformed frustule from nonspecific binding to the biosilica matrix. In conclusion, our results demonstrate an ability to engineer diatoms to create antibody-functionalized mesoporous silica able to selectively bind chemical and biological agents for the development of sensing platforms. PMID:26746113

  12. Redirecting T-Cell Specificity to EGFR Using mRNA to Self-limit Expression of Chimeric Antigen Receptor.

    PubMed

    Caruso, Hillary G; Torikai, Hiroki; Zhang, Ling; Maiti, Sourindra; Dai, Jianliang; Do, Kim-Anh; Singh, Harjeet; Huls, Helen; Lee, Dean A; Champlin, Richard E; Heimberger, Amy B; Cooper, Laurence J N

    2016-06-01

    Potential for on-target, but off-tissue toxicity limits therapeutic application of genetically modified T cells constitutively expressing chimeric antigen receptors (CARs) from tumor-associated antigens expressed in normal tissue, such as epidermal growth factor receptor (EGFR). Curtailing expression of CAR through modification of T cells by in vitro-transcribed mRNA species is one strategy to mitigate such toxicity. We evaluated expression of an EGFR-specific CAR coded from introduced mRNA in human T cells numerically expanded ex vivo to clinically significant numbers through coculture with activating and propagating cells (AaPC) derived from K562 preloaded with anti-CD3 antibody. The density of AaPC could be adjusted to affect phenotype of T cells such that reduced ratio of AaPC resulted in higher proportion of CD8 and central memory T cells that were more conducive to electrotransfer of mRNA than T cells expanded with high ratios of AaPC. RNA-modified CAR T cells produced less cytokine, but demonstrated similar cytolytic capacity as DNA-modified CAR T cells in response to EGFR-expressing glioblastoma cells. Expression of CAR by mRNA transfer was transient and accelerated by stimulation with cytokine and antigen. Loss of CAR abrogated T-cell function in response to tumor and normal cells expressing EGFR. We describe a clinically applicable method to propagate and modify T cells to transiently express EGFR-specific CAR to target EGFR-expressing tumor cells that may be used to limit on-target, off-tissue toxicity to normal tissue. PMID:27163741

  13. Immunization with recombinantly expressed glycan antigens from Schistosoma mansoni induces glycan-specific antibodies against the parasite

    PubMed Central

    Prasanphanich, Nina Salinger; Luyai, Anthony E; Song, Xuezheng; Heimburg-Molinaro, Jamie; Mandalasi, Msano; Mickum, Megan; Smith, David F; Nyame, A Kwame; Cummings, Richard D

    2014-01-01

    Schistosomiasis caused by infection with parasitic helminths of Schistosoma spp. is a major global health problem due to inadequate treatment and lack of a vaccine. The immune response to schistosomes includes glycan antigens, which could be valuable diagnostic markers and vaccine targets. However, no precedent exists for how to design vaccines targeting eukaryotic glycoconjugates. The di- and tri-saccharide motifs LacdiNAc (GalNAcβ1,4GlcNAc; LDN) and fucosylated LacdiNAc (GalNAcβ1,4(Fucα1-3)GlcNAc; LDNF) are the basis for several important schistosome glycan antigens. They occur in monomeric form or as repeating units (poly-LDNF) and as part of a variety of different glycoconjugates. Because chemical synthesis and conjugation of such antigens is exceedingly difficult, we sought to develop a recombinant expression system for parasite glycans. We hypothesized that presentation of parasite glycans on the cell surface would induce glycan-specific antibodies. We generated Chinese hamster ovary (CHO) Lec8 cell lines expressing poly-LDN (L8-GT) and poly-LDNF (L8-GTFT) abundantly on their membrane glycoproteins. Sera from Schistosoma mansoni-infected mice were highly cross-reactive with the cells and with cell-surface N-glycans. Immunizing mice with L8-GT and L8-GTFT cells induced glycan-specific antibodies. The L8-GTFT cells induced a sustained booster response, with antibodies that bound to S. mansoni lysates and recapitulated the exquisite specificity of the anti-parasite response for particular presentations of LDNF antigen. In summary, this recombinant expression system promotes successful generation of antibodies to the glycans of S. mansoni, and it can be adapted to study the role of glycan antigens and anti-glycan immune responses in many other infections and pathologies. PMID:24727440

  14. CTLA-4 expression on antigen-specific cells but not IL-10 secretion is required for oral tolerance.

    PubMed

    Fowler, Sanna; Powrie, Fiona

    2002-10-01

    CD4(+) T cells play a vital role in mediating the tolerance induced at mucosal sites following exposure to non-pathogenic stimuli, and further understanding of the precise mechanisms by which these cells prevent aberrant responses is required. We have developed a model using transfer of DO11.10 TCR-transgenic bone marrow into irradiated recipients in which it has been possible to track antigen-specific CD4(+) cells in mesenteric lymph nodes (mLN), Peyer's patches (PP) and lamina propria following primary exposure to antigen. Using this model we have demonstrated initial activation in all three gut-associated lymphoid tissue compartments characterized by increases in the frequency of transgenic cells expressing CD69 and CD25. These cells subsequently enter a state of hyporesponsiveness both locally in the mLN and PP and in the periphery following feeding and challenge. Investigating the role of CTLA-4 either using anti-CTLA-4 mAb or by generating chimeras using DO11.10xCTLA-4(-/-) mice as donors we have clearly shown that antigen-specific cells require the expression of this regulatory molecule for oral tolerance. In contrast, oral tolerance was intact in chimeras generated using DO11.10xIL-10(-/-) cells, indicating that secretion of this cytokine by antigen-specific cells is not required. PMID:12355454

  15. Systemic Combination Virotherapy for Melanoma with Tumor Antigen-Expressing Vesicular Stomatitis Virus and Adoptive T-cell Transfer

    PubMed Central

    Rommelfanger, Diana M.; Wongthida, Phonphimon; Diaz, Rosa M.; Kaluza, Karen M.; Thompson, Jill M.; Kottke, Timothy J.; Vile, Richard G.

    2013-01-01

    Oncolytic virotherapy offers the potential to treat tumors both as a single agent and in combination with traditional modalities such as chemotherapy and radiotherapy. Here we describe an effective, fully systemic treatment regimen, which combines virotherapy, acting essentially as an adjuvant immunotherapy, with adoptive cell transfer (ACT). The combination of ACT with systemic administration of a vesicular stomatitis virus (VSV) engineered to express the endogenous melanocyte antigen glycoprotein 100 (gp100) resulted in regression of established melanomas and generation of antitumor immunity. Tumor response was associated with in vivo T-cell persistence and activation as well as treatment-related vitiligo. However, in a proportion of treated mice, initial tumor regressions were followed by recurrences. Therapy was further enhanced by targeting an additional tumor antigen with the VSV-antigen + ACT combination strategy, leading to sustained response in 100% of mice. Together, our findings suggest that systemic virotherapy combined with antigen-expressing VSV could be used to support and enhance clinical immunotherapy protocols with adoptive T-cell transfer, which are already used in the clinic. PMID:22836753

  16. Expression and morphology of enterotoxigenic Escherichia coli surface antigen CS31A in E. coli K12 and Vibrio cholerae.

    PubMed

    Kuehni-Boghenbor, Kathrin; Jordi, Helene A; Frey, Joachim; Vilei, Edy M; Favre, Didier; Stoffel, Michael H

    2012-06-01

    Enterotoxigenic Escherichia coli (ETEC) is known as a worldwide cause of diarrheal disease. The pathogenesis involves the attachment of the microorganisms to the mucosa and the production of enterotoxins. Surface expression of CS31A fimbriae was assessed by Western blots, dot blots, immunofluorescence, and electron microscopy using negative staining and immunogold labeling. These investigations revealed significant differences in both the morphology of the wild-type and recombinant strains and the antigen exposure of CS31A in the wild-type and recombinant strains. In the wild-type ETEC strain, expression of CS31A was subject to phase variation. The recombinant E. coli strain produced CS31A but was prone to epitope shedding. In Vibrio cholerae vaccine strain CVD 103-HgR, the recombinant CS31A antigen was expressed but was only found intracellularly. Thus, E. coli strains seem to lend themselves better to the development of recombinant vaccines expressing ETEC-specific antigens at the cell's surface than strains from other orders or genera such as V. cholerae. PMID:22607531

  17. Expression of H type 1 antigen of ABO histo-blood group in normal colon and aberrant expressions of H type 2 and H type 3/4 antigens in colon cancer.

    PubMed

    Fujitani, N; Liu, Y; Toda, S; Shirouzu, K; Okamura, T; Kimura, H

    2000-05-01

    We have immunohistochemically examined the distribution of the H antigens of type 1, type 2 and type 3/4 chains of the ABO(H) histo-blood group system in human normal colon and in colon cancer using three monoclonal antibodies specific for each of the H type 1/2, H type 2, and the H type 3/4 chain. We unexpectedly found that mucosa of the normal colon from secretors but not that from nonsecretors expressed only H type 1 and did not express H type 2 or H type 3/4. The H type 1 was expressed in goblet cells. Positive goblet cells expressing H type 1 were decreased in number progressively from the proximal colon to the rectum. In tumors, 4 (57%) of 7 cancer tissues of the proximal colon from secretors expressed no H type 1, whereas all 8 cancer tissues of the distal colon from secretors expressed H type 1. The aberrant expressions of H type 2 and H type 3/4 (47 and 67%, respectively) were found in cancer tissues from both the proximal and the distal colon. Tumors from nonsecretors did not express any H antigens. Our results suggested that the expression of H type 1 in the normal colon and the aberrant expressions of H type 2 and H type 3/4 in colon cancer tissues were regulated by FUT2-encoded Se type alpha(1,2)fucosyltransferase. However, UEA-I-positive substance(s) rather than H type 2 were uniquely expressed throughout the normal colon and in colon cancers from both secretors and nonsecretors. PMID:11261842

  18. Effect of oestradiol and pathogen-associated molecular patterns on class II-mediated antigen presentation and immunomodulatory molecule expression in the mouse female reproductive tract

    PubMed Central

    Ochiel, Daniel O; Rossoll, Richard M; Schaefer, Todd M; Wira, Charles R

    2012-01-01

    Cells of the female reproductive tract (FRT) can present antigen to naive and memory T cells. However, the effects of oestrogen, known to modulate immune responses, on antigen presentation in the FRT remain undefined. In the present study, DO11.10 T-cell antigen receptor transgenic mice specific for the class II MHC-restricted ovalbumin (OVA) 323–339 peptide were used to study the effects of oestradiol and pathogen-associated molecular patterns on antigen presentation in the FRT. We report here that oestradiol inhibited antigen presentation of OVA by uterine epithelial cells, uterine stromal cells and vaginal cells to OVA-specific memory T cells. When ovariectomized animals were treated with oestradiol for 1 or 3 days, antigen presentation was decreased by 20–80%. In contrast, incubation with PAMP increased antigen presentation by epithelial cells (Pam3Cys), stromal cells (peptidoglycan, Pam3Cys) and vaginal cells (Pam3Cys). In contrast, CpG inhibited both stromal and vaginal cell antigen presentation. Analysis of mRNA expression by reverse transcription PCR indicated that oestradiol inhibited CD40, CD80 and class II in the uterus and CD40, CD86 and class II in the vagina. Expression in isolated uterine and vaginal cells paralleled that seen in whole tissues. In contrast, oestradiol increased polymeric immunoglobulin receptor mRNA expression in the uterus and decreased it in the vagina. These results indicate that antigen-presenting cells in the uterus and vagina are responsive to oestradiol, which inhibits antigen presentation and co-stimulatory molecule expression. Further, these findings suggest that antigen-presenting cells in the uterus and vagina respond to selected Toll-like receptor agonists with altered antigen presentation. PMID:22043860

  19. Embryonic expression of murine 5T4 oncofoetal antigen is associated with morphogenetic events at implantation and in developing epithelia.

    PubMed

    Barrow, Katie M; Ward, Christopher M; Rutter, Jennifer; Ali, Sumia; Stern, Peter L

    2005-08-01

    Overexpression of 5T4 oncofoetal antigen, an early marker of ES cell differentiation, in vitro increases cellular motility and decreases adhesion, properties relevant to development and cancer. Embryonic expression of m5T4 antigen is first detected on trophectoderm at implantation and is restricted to extra-embryonic tissues to embryonic day (E) 11.5. In the embryo, significant m5T4 expression is detected at E12.5 in hindbrain roofplate and in various epithelia derived from all germ layers. In keratin 14-expressing epithelia, there is a congruent 5T4 expression pattern with many of these cells being Ki-67 positive. In brain, expression is observed in roofplate, ependymal layers, choroid plexus, and subventricular zones of lateral ventricles at E14.5. By E17.5, expression is decreased in the subventricular zone with further restriction to choroid plexus in adult brain. Our data demonstrate a limited 5T4 expression profile during embryogenesis associated with actively cycling, undifferentiated epithelial progenitor cells that may contribute to their migration. PMID:15977177

  20. The preferentially expressed antigen in melanoma (PRAME) inhibits myeloid differentiation in normal hematopoietic and leukemic progenitor cells

    PubMed Central

    Guthrie, Katherine A.; Cummings, Carrie L.; Sabo, Kathleen; Wood, Brent L.; Gooley, Ted; Yang, Taimei; Epping, Mirjam T.; Shou, Yaping; Pogosova-Agadjanyan, Era; Ladne, Paula; Stirewalt, Derek L.; Abkowitz, Janis L.; Radich, Jerald P.

    2009-01-01

    The preferentially expressed antigen in melanoma (PRAME) is expressed in several hematologic malignancies, but either is not expressed or is expressed at only low levels in normal hematopoietic cells, making it a target for cancer therapy. PRAME is a tumor-associated antigen and has been described as a corepressor of retinoic acid signaling in solid tumor cells, but its function in hematopoietic cells is unknown. PRAME mRNA expression increased with chronic myeloid leukemia (CML) disease progression and its detection in late chronic-phase CML patients before tyrosine kinase inhibitor therapy was associated with poorer therapeutic responses and ABL tyrosine kinase domain point mutations. In leukemia cell lines, PRAME protein expression inhibited granulocytic differentiation only in cell lines that differentiate along this lineage after all-trans retinoic acid (ATRA) exposure. Forced PRAME expression in normal hematopoietic progenitors, however, inhibited myeloid differentiation both in the presence and absence of ATRA, and this phenotype was reversed when PRAME was silenced in primary CML progenitors. These observations suggest that PRAME inhibits myeloid differentiation in certain myeloid leukemias, and that its function in these cells is lineage and phenotype dependent. Lastly, these observations suggest that PRAME is a target for both prognostic and therapeutic applications. PMID:19625708

  1. Surface membrane antigen expression changes induced in vitro by exogenous growth factors in chronic lymphocytic leukemia cells.

    PubMed

    Vilpo, J; Hulkkonen, J; Hurme, M; Vilpo, L

    2002-09-01

    The factors determining the growth and survival of cells in B chronic lymphocytic leukemia (CLL) have remained poorly understood. We investigated the effects of optimal mitogen combinations (OMCs) on the expression of 26 surface membrane antigens among 33 CLL patients. The seven OMCs used were selected after pre-testing 14 combinations of (1) S. aureus Cowan I (SAC), (2) interleukin-2 (IL-2), (3) tumor necrosis factor alpha (TNF-alpha) and (4) 12-O-tetradecanoylphorbol 13-acetate (TPA; also known as phorbol 12-myristate 13-acetate or PMA). In flow cytometry we revealed that OMCs induced statistically highly significant upregulation of the expression of CD5, CD11c, CD19, CD22, CD23, CD25, CD38, CD40, CD45, CD45RO, CD95, CD126, CD130 and FMC7, and downregulation of CD20 and CD124 expression. Interestingly, the expression of CD27, CD45RA, CD79b, CD80, CD122 and that of the immunoglobulin gene superfamily members CD21, Ig-kappa, Ig-lambda, Ig-delta and Ig-micro were not significantly affected under similar conditions. The expression of several antigens was co-regulated, suggesting common regulatory pathways. These antigens include CD11c/CD5, CD11c/CD22, CD11c/CD126, CD11c/FMC7 as well as CD27/CD45, CD27/CD45RA and CD27/CD79b. Upregulation of surface antigen expression, induced by OMCs, should be applicable in antibody therapy in vitro and in vivo, and in negative stem cell selection for autotransplantation. Furthermore, the current strategy to enhance cell surface antigen expression may be a versatile tool to raise humoral and cell-mediated host defense against CLL cells. Upregulation of proteins mediating positive growth signals (eg CD25, CD40) and negative signals or apoptosis (eg CD95) may be used to sensitize cells to chemotherapy and programmed cell death. PMID:12200683

  2. Human platelet antigen genotyping and expression of CD109 (human platelet antigen 15) mRNA in various human cell types.

    PubMed

    Hwang, Sang Mee; Kim, Mi Jung; Chang, Ho Eun; Hong, Yun Ji; Kim, Taek Soo; Song, Eun Young; Park, Kyoung Un; Song, Junghan; Han, Kyou-Sup

    2013-01-01

    CD109 gene encodes a glycosylphosphatidylinositol-linked glycoprotein found in a subset of platelets and endothelial cell, and human platelet antigen (HPA) 15 is found on CD109. We evaluated the HPA genotype and/or the CD109 mRNA expression on two peripheral blood stem cells (PBSC), two peripheral bloods (PB), 12 granulocyte products, natural killer (NK)-92, B-lymphocyte (CO88BV59-1), K-562 leukemia cell line, human embryonic stem cell (hESC), and human fibroblasts (HF). HPA genotyping was performed by SNaPshot assay and CD109 mRNA expression was evaluated by real-time PCR with SYBR green and melting curve analysis. Genotype HPA-15a/-15a was found in PBSC#1 and two granulocyte products, and HPA-15a/-15b was found in PBSC#2, eight granulocyte products, NK-92, K-562, hESC, and HF, and HPA-15b/-15b was found in two granulocyte products. CD109 mRNA expression was highly increased in HF and increased in CD34+ and CD34- PBSCs and some granulocyte products, compared to the PB. However, the increase of expression level varied among the PBSC and granulocyte products. The CD109 mRNA expression of NK-92, K-562, hESC, and CO 88BV59-1 was not detected. HPA genotype was evaluated in various cells and the expression of CD109, which contains HPA 15, was different among cell lines and high in HF and PBSCs. PMID:23509816

  3. Preferentially Expressed Antigen of Melanoma (PRAME) and Wilms’ Tumor 1 (WT 1) Genes Expression in Childhood Acute Lymphoblastic Leukemia, Prognostic Role and Correlation with Survival

    PubMed Central

    Khateeb, Engy El; Morgan, Dalia

    2014-01-01

    BACKGROUND: Acute lymphocytic leukemia (ALL) is the most common hematologic malignancy in children. In young children it is also largely curable, with more than 90% of afflicted children achieving long-term remission. PRAME (Preferentially expressed antigen of melanoma) gene belongs to Group 3 class I HLA-restricted widely expressed antigens in which genes encoding widely expressed tumor antigens have been detected in many normal tissues as well as in histologically different types of tumors with no preferential expression on a certain type of cancer. It has been found to be expressed in a variety of cancer cells as leukemia & lymphoma. PRAME monitoring can be useful for detection of minimal residual disease and subsequent relapses particularly those leukemias in which specific tumor markers are unavailable. Wilms’ tumor1 (WT1) gene was identified as a gene that plays an important role in normal kidney development and inactivation of its function was shown to result in the development of Wilms’ tumors in paediatric patients. Disruption of WT1 function has been implicated in the formation of many different tumor types. AIM: to study how PRAME & WT 1 genes expression patterns influence cancer susceptibility & prognosis. PATIENTS & METHODS: 50 patients with denovo childhood acute lymphoblastic leukemia, as well as 50 age and sex matched apparently healthy volunteers were genotyped for PRAME and WT1 genes expression by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: PRAME gene was expressed in 34 of the patients (68%) and WT1 gene was expressed in 26 of the patients (52%). Expression of both genes was significantly higher compared to controls (P < 0.0001). Analysis of relapse free survival among our patients revealed that patients expressing PRAME gene or WT1 gene had better relapse free survival (p value=0.02 and 0.01 respectively). Relapse free survival increased significantly among patients coexpressing PRAME and WT 1(p value =0

  4. The relative roles of MHC and non-MHC antigens in bone marrow transplantation in rats. Graft acceptance and antigenic expression on donor red blood cells.

    PubMed

    Pinto, M; Gill, T J; Kunz, H W; Dixon-McCarthy, B D

    1983-06-01

    In order to investigate the influence of MHC and non-MHC genes in bone marrow transplantation, various combinations of congenic and inbred strains of rats were used as donors and recipients. A standard regimen of busulfan and cyclophosphamide treatment was used to condition the recipients. The resultant survival patterns of the animals indicated that: (1) a difference across the entire RT1 (MHC) complex is sufficient for the induction of fatal graft-versus-host disease (GVHD) in 100% of the engrafted animals; and (2) the blood group antigens RT2 and RT3, which are controlled by non-MHC genes, do not cause bone marrow graft rejection or GVHD. There were sequential changes of expression in surface alloantigens on the red cells in different donor-recipient combinations without other hematologic changes in the busulfan-cyclophosphamide conditioned bone marrow chimeras. PMID:6346598

  5. Immune response profile elicited by the model antigen ovalbumin expressed in fusion with the bacterial OprI lipoprotein.

    PubMed

    Basto, Afonso P; Badenes, Marina; Almeida, Sílvia C P; Martins, Carlos; Duarte, António; Santos, Dulce M; Leitão, Alexandre

    2015-03-01

    The use of immunogenic formulations targeting pattern recognition receptors towards modulation of immune responses is a promising strategy to develop better vaccines against infectious and malignant diseases. Molecules targeting TLR2 offer interesting properties that are relevant for vaccine development, including the possibility to covalently attach the lipidic ligands to the antigens. However, the type of immune response elicited by these formulations is still controversial. In this work, we used the model antigen ovalbumin (OVA) expressed in fusion with the bacterial lipoprotein OprI in order to characterize the immunomodulatory properties of this TLR ligand. Murine bone marrow-derived dendritic cells stimulated with OprI-OVA fusion lipoprotein produced high levels of the pro-inflammatory cytokines TNF-α and IL-6 and also IL-10, IL-12(p70) and IL-27, while TGF-β and IL-23 were not detected. Using OT-II and OT-I mice, an enhancement of MHC class II and class I antigen presentation was observed for the OVA antigen in fusion with OprI. Mice immunized by intraperitoneal route with this fusion lipoprotein in prime-boost protocols developed strong specific antibody responses including IgG1, IgG2c, IgG2b, IgG3 and IgE. These results, together with data obtained by restimulation of splenocytes from the immunized mice, point to an immune response profile that does not correspond to a strict Th1 or Th2 polarization. Finally, in a challenge experiment using a melanoma syngeneic mouse model (B16-OVA), prophylactic inoculation with OprI fused with the unrelated antigen eGFP increased the tumor growth, while the fusion with the tumor-associated antigen OVA delayed the tumor growth and increased mice survival. PMID:25467796

  6. Expression and Cellular Immunogenicity of a Transgenic Antigen Driven by Endogenous Poxviral Early Promoters at Their Authentic Loci in MVA

    PubMed Central

    Orubu, Toritse; Alharbi, Naif Khalaf; Lambe, Teresa; Gilbert, Sarah C.; Cottingham, Matthew G.

    2012-01-01

    CD8+ T cell responses to vaccinia virus are directed almost exclusively against early gene products. The attenuated strain modified vaccinia virus Ankara (MVA) is under evaluation in clinical trials of new vaccines designed to elicit cellular immune responses against pathogens including Plasmodium spp., M. tuberculosis and HIV-1. All of these recombinant MVAs (rMVA) utilize the well-established method of linking the gene of interest to a cloned poxviral promoter prior to insertion into the viral genome at a suitable locus by homologous recombination in infected cells. Using BAC recombineering, we show that potent early promoters that drive expression of non-functional or non-essential MVA open reading frames (ORFs) can be harnessed for immunogenic expression of recombinant antigen. Precise replacement of the MVA orthologs of C11R, F11L, A44L and B8R with a model antigen positioned to use the same translation initiation codon allowed early transgene expression similar to or slightly greater than that achieved by the commonly-used p7.5 or short synthetic promoters. The frequency of antigen-specific CD8+ T cells induced in mice by single shot or adenovirus-prime, rMVA-boost vaccination were similarly equal or marginally enhanced using endogenous promoters at their authentic genomic loci compared to the traditional constructs. The enhancement in immunogenicity observed using the C11R or F11L promoters compared with p7.5 was similar to that obtained with the mH5 promoter compared with p7.5. Furthermore, the growth rates of the viruses were unimpaired and the insertions were genetically stable. Insertion of a transgenic ORF in place of a viral ORF by BAC recombineering can thus provide not only a potent promoter, but also, concomitantly, a suitable insertion site, potentially facilitating development of MVA vaccines expressing multiple recombinant antigens. PMID:22761956

  7. Expression of Leu M1 antigen on a monoclonal B cell line established from a patient with rheumatoid arthritis.

    PubMed

    Takei, M; Kang, H; Tomura, K; Ikeda, E; Karasaki, M; Nakauchi, H; Okumura, K; Sawada, S

    1989-11-01

    The purpose of this study is to show that anti-Leu M1 antibody (anti-CD15), which has different staining characteristics in lymphoid and non-lymphoid cells, reacted against the surface antigen of a defined monoclonal B cell line. This antibody recognizes the sugar moiety, lacto-N-fucopentaose (LNF-III), which is linked to the cell membrane protein in several kinds of cells, but not in B cells. However, a human monoclonal B-cell line (TKS-1) which was established from the peripheral blood of a patient with rheumatoid arthritis, expressed the Leu M1 antigen spontaneously. The analysis of surface markers using a fluorescence-activated cell sorter (FACS) has revealed that the surface markers of TKS-1 were anti-mu, delta, kappa, HLA-DR, DQ, Leu 12 (CD19) and Leu M1 (CD15). TKS-1 cells were not reactive with any of the following antibodies: anti-OK M1 (CD11b), Leu M2, Leu M3 (CD14), Leu M4, Leu 1 (CD5), Leu 2 (CD8), Leu 3 (CD4), Leu 4 (CD3), Leu 7 and Leu 11 (CD16). In addition, TKS-1 was positive to Epstein-Barr nuclear antigen, weakly positive to non-specific esterase without staining inhibition by NaF, and negative to peroxidase. TKS-1 cells produced IgM in the culture supernatant and have kappa-light chain rearrangement in its DNA. As shown in other studies, distribution of Leu M1 is very wide. This antigen is not a specific immunodiagnostic marker to distinguish the cell type. We conclude that it is possible to express Leu M1 antigen on the membrane of a B-cell lineage cell. PMID:2575080

  8. Cloning, sequence analysis, and expression of the large subunit of the human lymphocyte activation antigen 4F2

    SciTech Connect

    Lumadue, J.A.; Glick, A.B.; Ruddle, F.H.

    1987-12-01

    Among the earliest expressed antigens on the surface of activated human lymphocytes is the surface antigen 4F2. The authors have used DNA-mediated gene transfer and fluorescence-activated cell sorting to obtain cell lines that contain the gene encoding the large subunit of the human 4F2 antigen in a mouse L-cell background. Human DNAs cloned from these cell lines were subsequently used as hybridization probes to isolate a full-length cDNA clone expressing 4F2. Sequence analysis of the coding region has revealed an amino acid sequence of 529 residues. Hydrophobicity plotting has predicted a probable structure for the protein that includes an external carboxyl terminus, an internal leader sequence, a single hydrophobic transmembrane domain, and two possible membrane-associated domains. The 4F2 cDNA detects a single 1.8-kilobase mRNA in T-cell and B-cell lines. RNA gel blot analysis of RNA derived from quiescent and serum-stimulated Swiss 3T3 fibroblasts reveals a cell-cycle modulation of 4F2 gene expression: the mRNA is present in quiescent fibroblasts but increases 8-fold 24-36 hr after stimulation, at the time of maximal DNA synthesis.

  9. Cell-Free Expression and In Situ Immobilization of Parasite Proteins from Clonorchis sinensis for Rapid Identification of Antigenic Candidates

    PubMed Central

    Ju, Jung Won; Kim, Ho-Cheol; Shin, Hyun-Il; Kim, Yu Jung; Kim, Dong-Myung

    2015-01-01

    Progress towards genetic sequencing of human parasites has provided the groundwork for a post-genomic approach to develop novel antigens for the diagnosis and treatment of parasite infections. To fully utilize the genomic data, however, high-throughput methodologies are required for functional analysis of the proteins encoded in the genomic sequences. In this study, we investigated cell-free expression and in situ immobilization of parasite proteins as a novel platform for the discovery of antigenic proteins. PCR-amplified parasite DNA was immobilized on microbeads that were also functionalized to capture synthesized proteins. When the microbeads were incubated in a reaction mixture for cell-free synthesis, proteins expressed from the microbead-immobilized DNA were instantly immobilized on the same microbeads, providing a physical linkage between the genetic information and encoded proteins. This approach of in situ expression and isolation enables streamlined recovery and analysis of cell-free synthesized proteins and also allows facile identification of the genes coding antigenic proteins through direct PCR of the microbead-bound DNA. PMID:26599101

  10. Single cell tuning of Myc expression by antigen receptor signal strength and interleukin-2 in T lymphocytes

    PubMed Central

    Preston, Gavin C; Sinclair, Linda V; Kaskar, Aneesa; Hukelmann, Jens L; Navarro, Maria N; Ferrero, Isabel; MacDonald, H Robson; Cowling, Victoria H; Cantrell, Doreen A

    2015-01-01

    Myc controls the metabolic reprogramming that supports effector T cell differentiation. The expression of Myc is regulated by the T cell antigen receptor (TCR) and pro-inflammatory cytokines such as interleukin-2 (IL-2). We now show that the TCR is a digital switch for Myc mRNA and protein expression that allows the strength of the antigen stimulus to determine the frequency of T cells that express Myc. IL-2 signalling strength also directs Myc expression but in an analogue process that fine-tunes Myc quantity in individual cells via post-transcriptional control of Myc protein. Fine-tuning Myc matters and is possible as Myc protein has a very short half-life in T cells due to its constant phosphorylation by glycogen synthase kinase 3 (GSK3) and subsequent proteasomal degradation. We show that Myc only accumulates in T cells exhibiting high levels of amino acid uptake allowing T cells to match Myc expression to biosynthetic demands. The combination of digital and analogue processes allows tight control of Myc expression at the population and single cell level during immune responses. PMID:26136212

  11. Cancer testis antigens and NY-BR-1 expression in primary breast cancer: prognostic and therapeutic implications

    PubMed Central

    2013-01-01

    Background Cancer–testis antigens (CTA) comprise a family of proteins, which are physiologically expressed in adult human tissues solely in testicular germ cells and occasionally placenta. However, CTA expression has been reported in various malignancies. CTAs have been identified by their ability to elicit autologous cellular and or serological immune responses, and are considered potential targets for cancer immunotherapy. The breast differentiation antigen NY-BR-1, expressed specifically in normal and malignant breast tissue, has also immunogenic properties. Here we evaluated the expression patterns of CTAs and NY-BR-1 in breast cancer in correlation to clinico-pathological parameters in order to determine their possible impact as prognostic factors. Methods The reactivity pattern of various mAbs (6C1, MA454, M3H67, 57B, E978, GAGE #26 and NY-BR-1 #5) were assessed by immunohistochemistry in a tissue micro array series of 210 randomly selected primary invasive breast cancers in order to study the diversity of different CTAs (e.g. MAGE-A, NY-ESO-1, GAGE) and NY-BR-1. These expression data were correlated to clinico-pathological parameters and outcome data including disease-free and overall survival. Results Expression of at least one CTA was detectable in the cytoplasm of tumor cells in 37.2% of the cases. NY-BR-1 expression was found in 46.6% of tumors, respectively. Overall, CTA expression seemed to be linked to adverse prognosis and M3H67 immunoreactivity specifically was significantly correlated to shorter overall and disease-free survival (p=0.000 and 0.024, respectively). Conclusions Our findings suggest that M3H67 immunoreactivity could serve as potential prognostic marker in primary breast cancer patients. The exclusive expression of CTAs in tumor tissues as well as the frequent expression of NY-BR-1 could define new targets for specific breast cancer therapies. PMID:23731661

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

    PubMed

    Zudaire, E; Simpson, S J; Illa, I; Montuenga, L M

    2004-06-01

    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 PCNA cellular index (PCNA-I) was obtained. Measurements of the DNA content of the cells have also been carried out by means of microdensitometry of Feulgen-stained, thick sections of midgut. A comparison between the PCNA nuclear level and the DNA content was performed. The PCNA levels were significantly different among the cells of the five regions studied: caeca, anterior ventricle, medial ventricle, posterior ventricle and ampullae of the Malpighian tubules. We have studied in more detail the region with highest PCNA-I, i.e. the caeca. The quality and the quantity of food eaten under ad libitum conditions were highly correlated with both the PCNA and DNA levels in the caeca cells. Locusts fed a diet with a close to optimal P:C content (P 21%, C 21%) showed the highest PCNA and DNA content. In locusts fed a food that also contained a 1:1 ratio of P to C but was diluted three-fold by addition of indigestible cellulose (P 7%, C 7%), a compensatory increase in consumption was critical to maintaining PCNA levels. Our measurements also showed that the nuclear DNA content of the mature and differentiated epithelial cells was several-fold higher than the levels in the undifferentiated stem cells of the regenerative nests. These results, combined with the low number of mitotic figures found in the regenerative nests of the caeca and the marked variation in PCNA levels among groups, suggest that some type of DNA endoreduplication process may be taking place. Our data also indicate that

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

    PubMed Central

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

    2015-01-01

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

  14. Enriched HLA-DQ3 phenotype and decreased class I major histocompatibility complex antigen expression in recurrent respiratory papillomatosis.

    PubMed Central

    Bonagura, V R; Siegal, F P; Abramson, A L; Santiago-Schwarz, F; O'Reilly, M E; Shah, K; Drake, D; Steinberg, B M

    1994-01-01

    Respiratory papillomas, caused by human papillomaviruses, are benign tumors that recur following removal. We evaluated immune function and major histocompatibility complex (MHC) phenotype and expression in these patients. MHC-independent immune function appeared normal. The frequency of peripheral blood MHC class II phenotypes was highly enriched for DQ3 and DR11, one split of DR5. Class I MHC antigen expression on papilloma tissue was markedly reduced. Together, these phenomena may facilitate papillomavirus evasion of the cellular immune response. Images PMID:7496977

  15. Live attenuated rubella vectors expressing SIV and HIV vaccine antigens replicate and elicit durable immune responses in rhesus macaques

    PubMed Central

    2013-01-01

    Background Live attenuated viruses are among our most potent and effective vaccines. For human immunodeficiency virus, however, a live attenuated strain could present substantial safety concerns. We have used the live attenuated rubella vaccine strain RA27/3 as a vector to express SIV and HIV vaccine antigens because its safety and immunogenicity have been demonstrated in millions of children. One dose protects for life against rubella infection. In previous studies, rubella vectors replicated to high titers in cell culture while stably expressing SIV and HIV antigens. Their viability in vivo, however, as well as immunogenicity and antibody persistence, were unknown. Results This paper reports the first successful trial of rubella vectors in rhesus macaques, in combination with DNA vaccines in a prime and boost strategy. The vectors grew robustly in vivo, and the protein inserts were highly immunogenic. Antibody titers elicited by the SIV Gag vector were greater than or equal to those elicited by natural SIV infection. The antibodies were long lasting, and they were boosted by a second dose of replication-competent rubella vectors given six months later, indicating the induction of memory B cells. Conclusions Rubella vectors can serve as a vaccine platform for safe delivery and expression of SIV and HIV antigens. By presenting these antigens in the context of an acute infection, at a high level and for a prolonged duration, these vectors can stimulate a strong and persistent immune response, including maturation of memory B cells. Rhesus macaques will provide an ideal animal model for demonstrating immunogenicity of novel vectors and protection against SIV or SHIV challenge. PMID:24041113

  16. Rates of MAGE-A3 and PRAME expressing tumors in FFPE tissue specimens from bladder cancer patients: potential targets for antigen-specific cancer immunotherapeutics

    PubMed Central

    Lerut, Evelyne; Van Poppel, Hendrik; Joniau, Steven; Gruselle, Olivier; Coche, Thierry; Therasse, Patrick

    2015-01-01

    Introduction: Antigen-specific active immunotherapy is an investigational therapeutic approach of potential interest for bladder cancer regardless of disease stage. Clinical development of antigen-specific immunotherapeutics against bladder cancer must be preceded by assessment of the expression of relevant genes in bladder tumors. The objectives of this study (NCT01706185) were to assess the rate of expression of the MAGE-A3 and PRAME genes in bladder tumors and to investigate the feasibility of using formalin-fixed paraffin-embedded (FFPE) tumor tissues for testing. Materials and methods: Archived FFPE bladder tumor specimens (any stage) were tested for mRNA expression of MAGE-A3 and PRAME using antigen-specific quantitative reverse transcription polymerase chain reaction assays. Data on patients and tumor characteristics were obtained from hospital records to investigate these characteristics’ possible association with the antigen expression. Results: Over 92% of the 156 tumors examined gave valid antigen test results. Of the tumors with a valid test, 46.5% were MAGE-A3-positive, 32.2% were PRAME-positive and 59.7% positive for at least one of them. Exploratory analyses of possible associations between antigen expression and patient or tumor characteristics did not identify clear associations between antigen expression and any of the variables investigated. Conclusions: Assessment of tumor antigen mRNA expression by using FFPE bladder tissues was feasible. The rates of MAGE-A3-positive and PRAME-positive tumors indicate that both antigens may be interesting targets for immunotherapeutics against bladder cancer. PMID:26464715

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

    PubMed Central

    Rooney, Barrie; Piening, Turid; Büscher, Philippe; Rogé, Stijn; Smales, C. Mark

    2015-01-01

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

  18. Mucosal Blood Group Antigen Expression Profiles and HIV Infections: A Study among Female Sex Workers in Kenya

    PubMed Central

    Chanzu, Nadia Musimbi; Mwanda, Walter; Oyugi, Julius; Anzala, Omu

    2015-01-01

    Background The ABO blood group antigens are carbohydrate moieties expressed on human red blood cells however; these antigens can also be expressed on some other cells particularly the surface of epithelial cells and may be found in mucosal secretions. In many human populations 80% secrete ABO antigens (termed ‘secretors’) while 20% do not (termed ‘non-secretors’). Furthermore, there are disease conditions that are associated with secretor status. Objective To investigate correlations between secretor status and HIV infection among female sex workers in Nairobi, Kenya. Methodology This cross-sectional study recruited 280 female sex workers aged 18–65 years from the Pumwani Majengo cohort, Kenya. Blood typing was determined by serological techniques using monoclonal antibodies to the ABO blood group antigens. Secretor phenotyping was determined using anti-H specific lectins specific to salivary, vaginal and cervical blood group H antigen using the agglutination inhibition technique and correlated to individual HIV sero-status. Participants were additionally screened for Bacterial vaginosis, Neisseria gonorrhoea and Trichomonas vaginalis. Results Out of the 280 participants, 212 (75.7%) were secretors and 68 (24.3%) were non-secretors. The incidence of all infections: HIV, Bacterial vaginosis, Neisseria gonorrhoea and Trichomonas vaginalis was higher among secretors compared to non-secretors. However, this difference was only statistically significant for HIV infection incidence rates: HIV infected secretors (83.7%) versus HIV un-infected secretors (71.8%) (p = 0.029) Based on ABO phenotype stratification, the incidence of HIV infection was higher among blood group A secretors (26/52 = 50%), in comparison to B (12/39 = 33.3%: p = 0.066), AB (3/9 = 33.3%: p = 0.355), and O secretors (36/112 = 32.1%: p = 0.028). Conclusion This is the first report to document the variable expression of the ABH blood group antigens profiling secretor and non-secretor phenotypes

  19. Tissue-specific expression of Le(Y) antigen in high endothelial venules of human lymphoid tissues.

    PubMed

    Tanegashima, A; Ushiyama, I; Nishi, K; Yamamoto, H; Fukunaga, T

    1999-12-01

    In this study, we demonstrated that the anti-Le(Y) antibody (BM-1) especially reacted with high endothelial venules (HEVs) in peripheral lymph nodes of blood group O individuals. The Le(Y) expression on HEVs showed a unique tissue-specific pattern, i.e., a large amount of the Le(Y) expression in peripheral lymph nodes and no or small amounts in mesenteric lymph node. Statistical analysis showed that there was the significant difference between the percentage of Le(Y)-positive HEVs in peripheral lymph nodes and mesenteric lymph nodes. No expression of Le(Y) was observed in vessels of Payer's patch, thymus, spleen and other non-lymphoid organs. In blood group A or B individuals, the reactivity between HEVs and anti-Le(Y) antibody increased after enzyme digestion with alpha-N-acetylgalactosaminidase or alpha-galactosidase. These findings show that the expression of difucosylated blood group ABH antigens are especially expressed on HEVs in peripheral lymph nodes. Furthermore, the tissue-specific pattern suggests that these antigens may be related to intercellular adhesion between lymphocytes and HEVs. PMID:11133021

  20. Spleen-Dependent Regulation of Antigenic Variation in Malaria Parasites: Plasmodium knowlesi SICAvar Expression Profiles in Splenic and Asplenic Hosts

    PubMed Central

    Lapp, Stacey A.; Korir-Morrison, Cindy; Jiang, Jianlin; Bai, Yaohui; Corredor, Vladimir; Galinski, Mary R.

    2013-01-01

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

  1. Assessment by electron-microscopy of recombinant Vibrio cholerae and Salmonella vaccine strains expressing enterotoxigenic Escherichia coli-specific surface antigens.

    PubMed

    Ziethlow, V; Favre, D; Viret, J-F; Frey, J; Stoffel, M H

    2008-03-01

    Diarrhoea caused by enterotoxigenic Escherichia coli (ETEC) requires adhesion of microorganisms to enterocytes. Hence, a promising approach to immunoprophylaxis is to elicit antibodies against colonisation factor antigens (CFAs). Genes encoding the most prevalent ETEC-specific surface antigens were cloned into Vibrio cholerae and Salmonella vaccine strains. Expression of surface antigens was assessed by electron-microscopy. Whereas negative staining was effective in revealing CFA/I and CS3, but not CS6, immunolabelling allowed identification of all surface antigens examined. The V. cholerae vaccine strain CVD103 did not express ETEC-specific colonisation factors, whereas CVD103-HgR expressed CS3 only. However, expression of both CFA/I and CS3 was demonstrated in Salmonella Ty21a. PMID:18093230

  2. Isolation and characterization of recombinant lambda gt11 bacteriophages expressing eight different mycobacterial antigens of potential immunological relevance.

    PubMed Central

    Andersen, A B; Worsaae, A; Chaparas, S D

    1988-01-01

    A genomic lambda gt11 DNA library of Mycobacterium tuberculosis was screened for expression of mycobacterial protein antigens with murine monoclonal antibodies. The reactivity patterns of the monoclonal antibodies ranged from those showing a limited interspecies reactivity to antibodies widely cross-reactive among different mycobacterial species. Twelve recombinant bacteriophages were isolated, containing eight mycobacterial genes (paa, pab, pac, pad, paeA, paeB, pafA, and pafB) encoding protein antigens. Physical maps of the phages were generated and the products of the recombinant genes were analyzed by immunoblotting techniques. PaeA and PaeB are distinct proteins but were shown to share an epitope. A similar condition was observed between PafA and PafB. Among the phages isolated, two groups expressed epitopes specific for M. tuberculosis and Mycobacterium bovis BCG. One group of phages produced an antigenic determinant which is found in M. tuberculosis and Mycobacterium marinum but not in M. bovis BCG. Images PMID:2451643

  3. Expression of E-cadherin, beta-catenin and Ki-67 antigen and their reciprocal relationships in mammary adenocarcinomas in bitches.

    PubMed

    Nowak, Marcin; Madej, Janusz A; Dziegiel, Piotr

    2007-01-01

    In progression of tumours, resulting from, i.e., release of cells from the parental tumour and development of metastases, expression of cell adhesion molecules (CAM) plays a significant role. CAM, including E-cadherin and the linked to it beta-catenin, determine the extent of adhesion between normal and neoplastically altered cells. Moreover, the unbound form of beta-catenin in a cell nucleus may affect the rate of cell proliferation This study aimed at demonstrating intensity and localisation of E-cadherin and beta-catenin expression as related to expression of the proliferation-associated antigen, Ki-67 in mammary adenocarcinomas of bitches. The study was performed on 35 cases of the above mentioned tumours. On paraffin sections immunohistochemical reactions were performed using monoclonal antibodies directed against E-cadherin, beta-catenin and Ki-67 antigen. In the studies a membranous expression of E-cadherin, a cytoplasmic-nuclear expression of beta-catenin and nuclear expression of Ki-67 antigen were demonstrated. Statistical calculations using Spearman's test demonstrated a pronounced positive correlation between expression of beta-catenin and Ki-67 antigen and absence of correlation between expression of E-cadherin and Ki-67 antigen. No correlation could be detected between expression intensities of E-cadherin and beta-catenin. PMID:17951173

  4. CD4+ T Cells and Toll-Like Receptors Recognize Salmonella Antigens Expressed in Bacterial Surface Organelles

    PubMed Central

    Bergman, Molly A.; Cummings, Lisa A.; Barrett, Sara L. Rassoulian; Smith, Kelly D.; Lara, J. Cano; Aderem, Alan; Cookson, Brad T.

    2005-01-01

    A better understanding of immunity to infection is revealed from the characteristics of microbial ligands recognized by host immune responses. Murine infection with the intracellular bacterium Salmonella generates CD4+ T cells that specifically recognize Salmonella proteins expressed in bacterial surface organelles such as flagella and membrane vesicles. These natural Salmonella antigens are also ligands for Toll-like receptors (TLRs) or avidly associated with TLR ligands such as lipopolysaccharide (LPS). PhoP/PhoQ, a regulon controlling Salmonella virulence and remodeling of LPS to resist innate immunity, coordinately represses production of surface-exposed antigens recognized by CD4+ T cells and TLRs. These data suggest that genetically coordinated surface modifications may provide a growth advantage for Salmonella in host tissues by limiting both innate and adaptive immune recognition. PMID:15731032

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

    PubMed

    Ozawa, Keiya

    2014-03-01

    Adoptive T-cell therapy using chimeric antigen receptor (CAR) technology is a novel approach to cancer immuno-gene therapy. CARs are hybrid proteins consisting of target-antigen-specific single-chain antibody fragment fused to intracellular T-cell activation domains (CD28 or CD137/CD3 zeta receptor). CAR-expressing engineered T lymphocytes can directly recognize and kill tumor cells in an HLA independent manner. In the United States, promising results have been obtained in the clinical trials of adoptive immuno-gene therapy using CD19-CAR-T lymphocytes for the treatment of refractory B-cell malignancies, including chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). In this review article, CD19-CAR-T gene therapy for refractory B-cell non-Hodgkin lymphoma is discussed. PMID:24724418

  6. Surface antigen expression and correlation with variable heavy-chain gene mutation status in chronic lymphocytic leukemia.

    PubMed

    Vilpo, Juhani; Tobin, Gerard; Hulkkonen, Janne; Hurme, Mikko; Thunberg, Ulf; Sundström, Christer; Vilpo, Leena; Rosenquist, Richard

    2003-01-01

    Recent studies have demonstrated that B-cell chronic lymphocytic leukemia (CLL) consists of two clinical entities with either somatically hypermutated (M-CLL) or unmutated (UM-CLL) immunoglobulin variable heavy-chain (VH) regions. In view of the fact that the cellular biology of these two subsets of disease is currently unexplored, we performed an extensive analysis of the surface antigen expression and correlated this with the VH gene mutation status in a cohort of 32 CLL patients. Using polymerase chain reaction amplification and nucleotide sequencing, the VH genes were shown to be mutated in 10 cases (31%) and unmutated in 22 (69%). The expression of 27 surface membrane antigens in peripheral blood leukemic cells was analyzed by flow cytometry, measuring both the percentage of positive cells as well as the geometric mean fluorescence intensity (GMF). Most of the surface membrane antigens (CD5, CD11c, CD19, CD20, CD21, CD22, CD23, CD25, CD40, CD45, VD79b, CD80, CD95, CD122, CD124, CD126, CD130, CD154, IgM, and IgD) showed a similar expression pattern in both UM-CLL and M-CLL patients. The similarity of M-CLL and UM-CLL, as demonstrated here for the first time with many protein markers, indicates a considerably homogeneous phenotype in both subsets. Furthermore, CD27 was strongly expressed in all cases, which may suggest a memory cell phenotype for both M-CLL and UM-CLL. More positive cells in the UM-CLL group were observed regarding CD38, but CD38 was not a good predictor of VH gene mutation status. Seventy percent of the M-CLL cases, but only 36% of UM-CLL cases, were Ig-lambda+. The most striking differential expression, however, was observed in the two slicing variants of the common leukocyte antigen CD45, namely CD45RO and CD45RA. CD45RO expression was significantly associated with M-CLL, whereas the GMF intensity of CD45RA tended to be associated with UM-CLL. The role of these CD45 splicing variants in the pathogenesis of CLL deserves further investigation

  7. Expression of chimeric antigen receptors in natural killer cells with a regulatory-compliant non-viral method

    PubMed Central

    Li, Linhong; Liu, Linda N; Feller, Stephanie; Allen, Cornell; Shivakumar, Rama; Fratantoni, Joseph; Wolfraim, Lawrence A.; Fujisaki, Hiroyuki; Campana, Dario; Chopas, Nicholas; Dzekunov, Sergey; Peshwa, Madhusudan

    2009-01-01

    Natural killer1 cells hold promise for cancer therapy. NK cytotoxicity can be enhanced by expression of chimeric antigen receptors that re-direct specificity toward target cells by engaging cell surface molecules expressed on target cells. We developed a regulatory-compliant, scalable non-viral approach to engineer NK cells to be target-specific based on transfection of mRNA encoding chimeric receptors. Transfection of eGFP mRNA into ex vivo expanded NK cells (N=5) or purified unstimulated NK cells from peripheral blood (N=4) resulted in good cell viability with eGFP expression in 85% ± 6% and 86% ± 4%, 24 hours after transfection, respectively. An mRNA encoding a receptor directed against CD19 (anti-CD19-BB-z) was also transfected into NK cells efficiently. Ex vivo expanded and purified unstimulated NK cells expressing anti-CD19-BB-z exhibited enhanced cytotoxicity against CD19+ target cells resulting in ≥80% lysis of acute lymphoblastic leukemia and B-lineage chronic lymphocytic leukemia cells at effector target ratios lower than 10:1. The target-specific cytotoxicity for anti-CD19-BB-z mRNA-transfected NK cells was observed as early as 3 hours after transfection and persisted for up to 3 days. The method described here should facilitate the clinical development of NK-based antigen-targeted immunotherapy for cancer. PMID:19745843

  8. Targeting of folate receptor β on acute myeloid leukemia blasts with chimeric antigen receptor-expressing T cells.

    PubMed

    Lynn, Rachel C; Poussin, Mathilde; Kalota, Anna; Feng, Yang; Low, Philip S; Dimitrov, Dimiter S; Powell, Daniel J

    2015-05-28

    T cells expressing a chimeric antigen receptor (CAR) can produce dramatic results in lymphocytic leukemia patients; however, therapeutic strategies for myeloid leukemia remain limited. Folate receptor β (FRβ) is a myeloid-lineage antigen expressed on 70% of acute myeloid leukemia (AML) patient samples. Here, we describe the development and evaluation of the first CARs specific for human FRβ (m909) in vitro and in vivo. m909 CAR T cells exhibited selective activation and lytic function against engineered C30-FRβ as well as endogenous FRβ(+) AML cell lines in vitro. In mouse models of human AML, m909 CAR T cells mediated the regression of engrafted FRβ(+) THP1 AML in vivo. In addition, we demonstrated that treatment of AML with all-trans retinoic acid (ATRA) enhanced FRβ expression, resulting in improved immune recognition by m909 CAR T cells. Because many cell surface markers are shared between AML blasts and healthy hematopoietic stem and progenitor cells (HSCs), we evaluated FRβ expression and recognition of HSCs by CAR T cells. m909 CAR T cells were not toxic against healthy human CD34(+) HSCs in vitro. Our results indicate that FRβ is a promising target for CAR T-cell therapy of AML, which may be augmented by combination with ATRA. PMID:25887778

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

    PubMed

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

    2015-10-01

    Porcine pleuropneumonia is an infectious disease caused by Actinobacillus pleuropneumoniae. The identification of A. pleuropneumoniae genes, specially expressed in vivo, is a useful tool to reveal the mechanism of infection. IVIAT was used in this work to identify antigens expressed in vivo during A. pleuropneumoniae infection, using sera from individuals with chronic porcine pleuropneumonia. Sequencing of DNA inserts from positive clones showed 11 open reading frames with high homology to A. pleuropneumoniae genes. Based on sequence analysis, proteins encoded by these genes were involved in metabolism, replication, transcription regulation, and signal transduction. Moreover, three function-unknown proteins were also indentified in this work. Expression analysis using quantitative real-time PCR showed that most of the genes tested were up-regulated in vivo relative to their expression levels in vitro. IVI (in vivoinduced) genes that were amplified by PCR in different A. pleuropneumoniae strains showed that these genes could be detected in almost all of the strains. It is demonstrated that the identified IVI antigen may have important roles in the infection of A. pleuropneumoniae. PMID:26059519

  10. Targeting of folate receptor β on acute myeloid leukemia blasts with chimeric antigen receptor–expressing T cells

    PubMed Central

    Lynn, Rachel C.; Poussin, Mathilde; Kalota, Anna; Feng, Yang; Low, Philip S.; Dimitrov, Dimiter S.

    2015-01-01

    T cells expressing a chimeric antigen receptor (CAR) can produce dramatic results in lymphocytic leukemia patients; however, therapeutic strategies for myeloid leukemia remain limited. Folate receptor β (FRβ) is a myeloid-lineage antigen expressed on 70% of acute myeloid leukemia (AML) patient samples. Here, we describe the development and evaluation of the first CARs specific for human FRβ (m909) in vitro and in vivo. m909 CAR T cells exhibited selective activation and lytic function against engineered C30-FRβ as well as endogenous FRβ+ AML cell lines in vitro. In mouse models of human AML, m909 CAR T cells mediated the regression of engrafted FRβ+ THP1 AML in vivo. In addition, we demonstrated that treatment of AML with all-trans retinoic acid (ATRA) enhanced FRβ expression, resulting in improved immune recognition by m909 CAR T cells. Because many cell surface markers are shared between AML blasts and healthy hematopoietic stem and progenitor cells (HSCs), we evaluated FRβ expression and recognition of HSCs by CAR T cells. m909 CAR T cells were not toxic against healthy human CD34+ HSCs in vitro. Our results indicate that FRβ is a promising target for CAR T-cell therapy of AML, which may be augmented by combination with ATRA. PMID:25887778

  11. The piggyBac Transposon-Mediated Expression of SV40 T Antigen Efficiently Immortalizes Mouse Embryonic Fibroblasts (MEFs)

    PubMed Central

    Cui, Jing; Zhang, Hongmei; Chen, Xiang; Li, Ruidong; Wu, Ningning; Chen, Xian; Wen, Sheng; Zhang, Junhui; Yin, Liangjun; Deng, Fang; Liao, Zhan; Zhang, Zhonglin; Zhang, Qian; Yan, Zhengjian; Liu, Wei; Ye, Jixing; Deng, Youlin; Wang, Zhongliang; Qiao, Min; Luu, Hue H.; Haydon, Rex C.; Shi, Lewis L.; Liang, Houjie; He, Tong-Chuan

    2014-01-01

    Mouse embryonic fibroblasts (MEFs) are mesenchymal stem cell (MSC)-like multipotent progenitor cells and can undergo self-renewal and differentiate into to multiple lineages, including bone, cartilage and adipose. Primary MEFs have limited life span in culture, which thus hampers MEFs’ basic research and translational applications. To overcome this challenge, we investigate if piggyBac transposon-mediated expression of SV40 T antigen can effectively immortalize mouse MEFs and that the immortalized MEFs can maintain long-term cell proliferation without compromising their multipotency. Using the piggyBac vector MPH86 which expresses SV40 T antigen flanked with flippase (FLP) recognition target (FRT) sites, we demonstrate that mouse embryonic fibroblasts (MEFs) can be efficiently immortalized. The immortalized MEFs (piMEFs) exhibit an enhanced proliferative activity and maintain long-term cell proliferation, which can be reversed by FLP recombinase. The piMEFs express most MEF markers and retain multipotency as they can differentiate into osteogenic, chondrogenic and adipogenic lineages upon BMP9 stimulation in vitro. Stem cell implantation studies indicate that piMEFs can form bone, cartilage and adipose tissues upon BMP9 stimulation, whereas FLP-mediated removal of SV40 T antigen diminishes the ability of piMEFs to differentiate into these lineages, possibly due to the reduced expansion of progenitor populations. Our results demonstrate that piggyBac transposon-mediated expression of SV40 T can effectively immortalize MEFs and that the reversibly immortalized piMEFs not only maintain long-term cell proliferation but also retain their multipotency. Thus, the high transposition efficiency and the potential footprint-free natures may render piggyBac transposition an effective and safe strategy to immortalize progenitor cells isolated from limited tissue supplies, which is essential for basic and translational studies. PMID:24845466

  12. Whole Pichia pastoris Yeast Expressing Measles Virus Nucleoprotein as a Production and Delivery System to Multimerize Plasmodium Antigens

    PubMed Central

    Jacob, Daria; Ruffie, Claude; Dubois, Myriam; Combredet, Chantal; Amino, Rogerio; Formaglio, Pauline; Gorgette, Olivier; Pehau-Arnaudet, Gérard; Guery, Charline; Puijalon, Odile; Barale, Jean-Christophe; Ménard, Robert; Tangy, Frédéric; Sala, Monica

    2014-01-01

    Yeasts are largely used as bioreactors for vaccine production. Usually, antigens are produced in yeast then purified and mixed with adjuvants before immunization. However, the purification costs and the safety concerns recently raised by the use of new adjuvants argue for alternative strategies. To this end, the use of whole yeast as both production and delivery system appears attractive. Here, we evaluated Pichia pastoris yeast as an alternative vaccine production and delivery system for the circumsporozoite protein (CS) of Plasmodium, the etiologic agent of malaria. The CS protein from Plasmodium berghei (Pb) was selected given the availability of the stringent C57Bl/6 mouse model of infection by Pb sporozoites, allowing the evaluation of vaccine efficacy in vivo. PbCS was multimerized by fusion to the measles virus (MV) nucleoprotein (N) known to auto-assemble in yeast in large-size ribonucleoprotein rods (RNPs). Expressed in P. pastoris, the N-PbCS protein generated highly multimeric and heterogenic RNPs bearing PbCS on their surface. Electron microscopy and immunofluorescence analyses revealed the shape of these RNPs and their localization in peripheral cytoplasmic inclusions. Subcutaneous immunization of C57Bl/6 mice with heat-inactivated whole P. pastoris expressing N-PbCS RNPs provided significant reduction of parasitemia after intradermal challenge with a high dose of parasites. Thus, in the absence of accessory adjuvants, a very low amount of PbCS expressed in whole yeast significantly decreased clinical damages associated with Pb infection in a highly stringent challenge model, providing a proof of concept of the intrinsic adjuvancy of this vaccine strategy. In addition to PbCS multimerization, the N protein contributed by itself to parasitemia delay and long-term mice survival. In the future, mixtures of whole recombinant yeasts expressing relevant Plasmodium antigens would provide a multivalent formulation applicable for antigen combination screening

  13. Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems.

    PubMed

    Gottschamel, Johanna; Lössl, Andreas; Ruf, Stephanie; Wang, Yanliang; Skaugen, Morten; Bock, Ralph; Clarke, Jihong Liu

    2016-07-01

    Dengue fever is a disease in many parts of the tropics and subtropics and about half the world's population is at risk of infection according to the World Health Organization. Dengue is caused by any of the four related dengue virus serotypes DEN-1, -2, -3 and -4, which are transmitted to people by Aedes aegypti mosquitoes. Currently there is only one vaccine (Dengvaxia(®)) available (limited to a few countries) on the market since 2015 after half a century's intensive efforts. Affordable and accessible vaccines against dengue are hence still urgently needed. The dengue envelop protein domain III (EDIII), which is capable of eliciting serotype-specific neutralizing antibodies, has become the focus for subunit vaccine development. To contribute to the development of an accessible and affordable dengue vaccine, in the current study we have used plant-based vaccine production systems to generate a dengue subunit vaccine candidate in tobacco. Chloroplast genome engineering was applied to express serotype-specific recombinant EDIII proteins in tobacco chloroplasts using both constitutive and ethanol-inducible expression systems. Expression of a tetravalent antigen fusion construct combining EDIII polypeptides from all four serotypes was also attempted. Transplastomic EDIII-expressing tobacco lines were obtained and homoplasmy was verified by Southern blot analysis. Northern blot analyses showed expression of EDIII antigen-encoding genes. EDIII protein accumulation levels varied for the different recombinant EDIII proteins and the different expression systems, and reached between 0.8 and 1.6 % of total cellular protein. Our study demonstrates the suitability of the chloroplast compartment as a production site for an EDIII-based vaccine candidate against dengue fever and presents a Gateway(®) plastid transformation vector for inducible transgene expression. PMID:27116001

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

    NASA Astrophysics Data System (ADS)

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

    1983-04-01

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

  15. Advances in alfalfa mosaic virus-mediated expression of anthrax antigen in planta

    SciTech Connect

    Brodzik, R.; Bandurska, K.; Deka, D.; Golovkin, M.; Koprowski, H. . E-mail: h_koprowski@jefferson.edu

    2005-12-16

    Plant viruses show great potential for production of pharmaceuticals in plants. Such viruses can harbor a small antigenic peptide(s) as a part of their coat proteins (CP) and elicit an antigen-specific immune response. Here, we report the high yield and consistency in production of recombinant alfalfa mosaic virus (AlMV) particles for specific presentation of the small loop 15 amino acid epitope from domain-4 of the Bacillus anthracis protective antigen (PA-D4s). The epitope was inserted immediately after the first 25 N-terminal amino acids of AlMV CP to retain genome activation and binding of CP to viral RNAs. Recombinant AlMV particles were efficiently produced in tobacco, easily purified for immunological analysis, and exhibited extended stability and systemic proliferation in planta. Intraperitional injections of mice with recombinant plant virus particles harboring the PA-D4s epitope elicited a distinct immune response. Western blotting and ELISA analysis showed that sera from immunized mice recognized both native PA antigen and the AlMV CP.

  16. Activated rat T cells synthesize and express functional major histocompatibility class II antigens.

    PubMed Central

    Broeren, C P; Wauben, M H; Lucassen, M A; Van Meurs, M; Van Kooten, P J; Boog, C J; Claassen, E; Van Eden, W

    1995-01-01

    In the present report, we studied the presence and functional significance of major histocompatibility complex (MHC) class II antigen on rat T cells. Most rat T-cell lines cultured in vitro were found to be MHC class II+. Also, these T-cell lines were shown to synthesize MHC class II molecules. Immunohistochemical and flow cytometric double stainings for T-cell receptor (TCR) and MHC class II showed that in vivo as well a large proportion of T cells was MHC class II+. The immunohistochemical staining of spleen sections enabled us to characterize the MHC class II+ and MHC class II- T cells. It was shown that resting T cells in vivo were MHC class II-. In contrast, activated T cells, as determined by their localization in the marginal zone of the spleen, proved to be MHC class II+. Finally, T-cell clones were found to be able to present peptidic antigens, but could only poorly present more complex exogenous antigens, probably due to inefficient uptake of such antigens. These features would endow activated rat T cells with the capacity to present cell-specific self-proteins, such as TCR, to regulatory CD4+ MHC class II-restricted T cells, as was described by our group elsewhere. Images Figure 2 Figure 5 PMID:7750994

  17. Advances in alfalfa mosaic virus-mediated expression of anthrax antigen in planta.

    PubMed

    Brodzik, R; Bandurska, K; Deka, D; Golovkin, M; Koprowski, H

    2005-12-16

    Plant viruses show great potential for production of pharmaceuticals in plants. Such viruses can harbor a small antigenic peptide(s) as a part of their coat proteins (CP) and elicit an antigen-specific immune response. Here, we report the high yield and consistency in production of recombinant alfalfa mosaic virus (AlMV) particles for specific presentation of the small loop 15 amino acid epitope from domain-4 of the Bacillus anthracis protective antigen (PA-D4s). The epitope was inserted immediately after the first 25 N-terminal amino acids of AlMV CP to retain genome activation and binding of CP to viral RNAs. Recombinant AlMV particles were efficiently produced in tobacco, easily purified for immunological analysis, and exhibited extended stability and systemic proliferation in planta. Intraperitional injections of mice with recombinant plant virus particles harboring the PA-D4s epitope elicited a distinct immune response. Western blotting and ELISA analysis showed that sera from immunized mice recognized both native PA antigen and the AlMV CP. PMID:16236249

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

    PubMed

    Joergensen, Louise; Bengtsson, Dominique C; Bengtsson, Anja; Ronander, Elena; Berger, Sanne S; Turner, Louise; Dalgaard, Michael B; Cham, Gerald K K; Victor, Michala E; Lavstsen, Thomas; Theander, Thor G; Arnot, David E; Jensen, Anja T R

    2010-01-01

    The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens play a major role in cytoadhesion of infected erythrocytes (IE), antigenic variation, and immunity to malaria. The current consensus on control of variant surface antigen expression is that only one PfEMP1 encoded by one var gene is expressed per cell at a time. We measured var mRNA transcript levels by real-time Q-PCR, analysed var gene transcripts by single-cell FISH and directly compared these with PfEMP1 antigen surface expression and cytoadhesion in three different antibody-selected P. falciparum 3D7 sub-lines using live confocal microscopy, flow cytometry and in vitro adhesion assays. We found that one selected parasite sub-line simultaneously expressed two different var genes as surface antigens, on single IE. Importantly, and of physiological relevance to adhesion and malaria pathogenesis, this parasite sub-line was found to bind both CD31/PECAM1 and CD54/ICAM1 and to adhere twice as efficiently to human endothelial cells, compared to infected cells having only one PfEMP1 variant on the surface. These new results on PfEMP1 antigen expression indicate that a re-evaluation of the molecular mechanisms involved in P. falciparum adhesion and of the accepted paradigm of absolutely mutually exclusive var gene transcription is required. PMID:20824088

  19. Interaction of serologically defined colon cancer antigen-3 with Arf6 and its predominant expression in the mouse testis.

    PubMed

    Sakagami, Hiroyuki; Hara, Yoshinobu; Fukaya, Masahiro

    2016-09-01

    ADP ribosylation factor 6 (Arf6) is a small GTPase that regulates endosomal trafficking and actin cytoskeleton remodeling. Here, we identified the serologically defined colon antigen-3 (SDCCAG3) as an Arf6-interacting protein by yeast two-hybrid screening with a constitutively active Arf6 mutant. SDCCAG3 interacts specifically with Arf6 among the Arf family members through its 101  C-terminal amino acids. SDCCAG3 is expressed most intensely in the testis at the mRNA and protein levels. In the testis, SDCCAG3 is expressed in spermatocytes and spermatids. We also show that full-length SDCCAG3, but not a mutant lacking the ability to interact with Arf6, is recruited to the midbody during cytokinesis when expressed exogenously in HeLa cells. These findings suggest that SDCCAG3 might function in endosomal trafficking downstream of Arf6. PMID:27373827

  20. Identification of genes expressed during Toxoplasma gondii infection by in vivo-induced antigen technology (IVIAT) with positive porcine sera.

    PubMed

    Tao, Qing; Xiao, Jiang; Wang, Yifan; Fang, Kun; Li, Nizhi; Hu, Min; Zhou, Yanqin; Zhao, Junlong

    2014-08-01

    Infection of pigs with Toxoplasma gondii is a common source of human toxoplasmosis and causes serious economic losses. In vivo-induced antigen technology (IVIAT) is an effective immunological technique to identify the antigens that a pathogen specifically expressed during infection. To discover the genes that are important in T. gondii infection of pigs, we employed IVIAT using sera from infected pigs. Fourteen antigens were identified including microneme protein 11 (MIC11), dense granule protein 5 (GRA5), 18 kDa cyclophilin (C-18), serine proteinase inhibitor (PI), calmodulin (CaM), leucine-rich repeat protein ( LRRP), D-3-phosphoglycerate dehydrogenase (D3PD), elongation factor 1-gamma (EF1), and 6 hypothetical proteins. The increased transcription levels of 5 (MIC11, GRA5, C-18, PI, and CaM) of the 14 molecules identified by IVIAT were confirmed by real-time PCR. The full length or partial proteins encoded by these 5 genes were expressed in Escherichia coli , and their immunogenicity was confirmed by Western blot analysis with positive porcine sera. Further functional studies were conducted with CaM. Suppression of CaM expression by RNA interference decreased T. gondii tachyzoites cell attachment, invasion, and egress but did not influence their replication. The proteins identified in this study are predicted to be involved in cell invasion, ion-protein binding, protein folding, biosynthesis, and metabolism. The results of the functional analysis support the hypothesis that CaM contributes to parasite pathogenesis during infection. These results may have significant implications for the discovery of candidate molecules for the development of potential therapies and preventive measures against toxoplasmosis in pigs. PMID:24646180

  1. Enhanced Protective Efficacy of Nonpathogenic Recombinant Leishmania tarentolae Expressing Cysteine Proteinases Combined with a Sand Fly Salivary Antigen

    PubMed Central

    Taheri, Tahereh; Taslimi, Yasaman; Doustdari, Fatemeh; Seyed, Negar; Torkashvand, Fatemeh; Meneses, Claudio; Papadopoulou, Barbara; Kamhawi, Shaden; Valenzuela, Jesus G.; Rafati, Sima

    2014-01-01

    Background Novel vaccination approaches are needed to prevent leishmaniasis. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The nonpathogenic to humans lizard protozoan parasite, Leishmania (L) tarentolae, has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models. Correspondingly, pre-exposure to sand fly saliva or immunization with a salivary protein has been shown to protect mice against cutaneous leishmaniasis. Methodology/Principal Findings Here, we tested the efficacy of a novel combination of established protective parasite antigens expressed by L. tarentolae together with a sand fly salivary antigen as a vaccine strategy against L. major infection. The immunogenicity and protective efficacy of different DNA/Live and Live/Live prime-boost vaccination modalities with live recombinant L. tarentolae stably expressing cysteine proteinases (type I and II, CPA/CPB) and PpSP15, an immunogenic salivary protein from Phlebotomus papatasi, a natural vector of L. major, were tested both in susceptible BALB/c and resistant C57BL/6 mice. Both humoral and cellular immune responses were assessed before challenge and at 3 and 10 weeks after Leishmania infection. In both strains of mice, the strongest protective effect was observed when priming with PpSP15 DNA and boosting with PpSP15 DNA and live recombinant L. tarentolae stably expressing cysteine proteinase genes. Conclusion/Significance The present study is the first to use a combination of recombinant L. tarentolae with a sand fly salivary antigen (PpSP15) and represents a novel promising vaccination approach against leishmaniasis. PMID:24675711

  2. Immunoglobulin (Ig) D in Labeo rohita is widely expressed and differentially modulated in viral, bacterial and parasitic antigenic challenges.

    PubMed

    Basu, Madhubanti; Lenka, Saswati S; Paichha, Mahismita; Swain, Banikalyan; Patel, Bhakti; Banerjee, Rajanya; Jayasankar, Pallipuram; Das, Surajit; Samanta, Mrinal

    2016-10-15

    Immunoglobulins (Igs) play critical roles in protecting host against diverse pathogenic invasion and diseases. Among all Ig isotypes, IgD is the most recently-evolved and enigmatic molecule detected in all vertebrates species except birds. In South-East Asia, Labeo rohita (rohu) is the leading candidate fish species for freshwater aquaculture, and this article describes about IgD gene expression in rohu following viral, bacterial and parasitic antigenic challenges. The partial cDNA (761bp) of Labeo rohita-IgD (LrIgD) was cloned and submitted in the GenBank with the accession no KT883581. Phylogenetically, LrIgD was closely related to grass carp IgD. Analysis of LrIgD gene expression in juveniles by quantitative real-time PCR (qRT-PCR) assay revealed gradual increase in IgD expression with the advancement of time. In the healthy rohu fingerlings, LrIgD expression occurred predominantly in kidney followed by liver and spleen. In response to rhabdoviral antigenic stimulation, LrIgD expression was significantly enhanced in all tested tissues. In bacterial (Aeromonas hydrophila) infection, transcripts of LrIgD increased more dramatically in liver followed by kidney and gill. In parasitic (Argulus) infection, most significant expression of IgD was noted in the skin, followed by kidney, liver, spleen and gill. These results collectively suggest the key role of IgD in the immune response of rohu during viral, bacterial and parasitic infections. PMID:27590429

  3. Suppressed CD31 Expression in Sarcoma-180 Tumors after Injection with Toxoplasma gondii Lysate Antigen in BALB/c Mice

    PubMed Central

    Pyo, Kyoung-Ho; Jung, Bong-Kwang; Chai, Jong-Yil

    2010-01-01

    The anti-tumorigenic effects of Toxoplasma gondii (RH) antigens were studied in a murine sarcoma-180 tumor model. To determine the anti-tumor effects, the reduction in tumor size and expression of CD31 (an angiogenesis marker in the tumor tissue) were examined after injection of BALB/c mice with T. gondii lysate antigen (TLA) or formalin-fixed, proliferation-inhibited, T. gondii tachyzoites. Tumors were successfully produced by an intradermal injection of sarcoma-180 cells with plain Matrigel in the mid-backs of mice. After injection with TLA or formalin-fixed T. gondii tachyzoites, the increase in tumor size and weight nearly stopped while tumor growth continued in control mice that were injected with PBS. CD31 expression in TLA-treated or formalin-fixed T. gondii-injected mice was lower than the control mice. Accordingly, the present study shows that the treatment of mice with formalin-fixed T. gondii or TLA in the murine sarcoma-180 tumor model results in a decrease of both tumor size and CD31 expression. PMID:20585536

  4. Prokaryotic expression, purification and antigenicity analysis of African swine fever virus pK205R protein.

    PubMed

    Wu, Xulong; Xiao, Lu; Peng, Bin; Wang, Yin; Yang, Zexiao; Yao, Xueping; Hu, Ling; Lin, Xingyu

    2016-01-01

    African swine fever is an acute, febrile and highly virulent porcine disease causing serious economic losses worldwide. The pK205R protein of the African swine fever virus (ASFV) is largely expressed in the early stages of infection, which has given the K205R gene extensive attention. In this study, the ASFV K205R was cloned and expressed in Escherichia coli BL21 (DE3). Expression of histidine-tagged pK205R with a molecular mass of 44 kDa was determined by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Optimisation of culture conditions allowed induction of the recombinant protein with 0.4 mM Isopropyl β-D-thiogalactoside (IPTG) at 37°C for 2 h. The protein existed in cellular supernatant and was purified using a Ni-NTA resin column. The purified protein was used to immunize rabbits four times to enable the production of polyclonal antibodies, and the antiserum titre was detected by ELISA. The results showed that the purified pK205R can react with ASFV positive serum specifically by Western blotting. The pK205R had high antigenicity, which indicated that pK205R could be used as an antigen for detection of ASFV-specific antibodies in ELISA testing, and the recombinant protein could contribute to further research of the action and structure of pK205R. PMID:27096786

  5. Induction of plasmacytomas secreting antigen-specific monoclonal antibodies with a retrovirus expressing v-abl and c-myc.

    PubMed Central

    Weissinger, E M; Mischak, H; Largaespada, D A; Kaehler, D A; Mitchell, T; Smith-Gill, S J; Risser, R; Mushinski, J F

    1991-01-01

    ABL-MYC, a recombinant murine retrovirus that expresses v-abl and c-myc, rapidly induces transplantable mono- or oligoclonal plasmacytomas in BALB/c mice. To determine if the targets for transformation of this retrovirus are antigen-committed B lymphocytes and to explore this system as an alternative technique for producing antigen-specific monoclonal antibodies, plasmacytomas were induced in mice that had been immunized with two different types of immunogens, hen egg white lysozyme and sheep red blood cells. The majority of these plasmacytomas secreted immunogen-specific antibodies. Plasmacytomas induced in unimmunized mice did not react with hen egg white lysozyme or sheep red blood cells. The specific antibodies were comparable in concentration, specificity, and affinity to monoclonal antibodies obtained with conventional hybridoma technology, but, in addition to IgGs and IgMs, they included specific IgA antibodies, which are rare among splenic-derived hybridomas. Our results demonstrate that a principal target for ABL-MYC is an antigen-committed B lymphocyte. In addition this procedure provides an alternative method for the production of monoclonal antibodies, without a requirement for hetero-caryon formation by cell fusion techniques. Images PMID:1924333

  6. Development of an ELISA based on the baculovirus-expressed capsid protein of porcine circovirus type 2 as antigen.

    PubMed

    Liu, Changming; Ihara, Takeshi; Nunoya, Tetsuo; Ueda, Susumu

    2004-03-01

    The genome of porcine circovirus type 2 (PCV2) contains two major open reading frames, which have been shown to encode the virus capsid and replication-associated proteins. The capsid protein is a major structural protein of the virus; it can be a suitable target antigen for detecting PCV2-specific antibodies to monitor PCV2 infection. To produce the antigen, the capsid protein coding sequence was cloned into a baculovirus transfer vector, and a recombinant capsid (rC) protein of PCV2 was expressed as a combined fusion protein in frame with a C-terminal peptide of six histidines. The affinity-purified rC protein was used as coating antigen to develop an ELISA for detecting the virus-specific antibodies in swine sera. The rC protein-based ELISA (rcELISA) was evaluated by examining a panel of 49 PCV2-positive and 49 PCV2-negative swine sera. In comparative experiments of immunoperoxidase monolayer assay (IPMA) using 102 field sera, there was 89.2% coincidence between data obtained by the rcELISA and IPMA. The rcELISA achieved 88.5% specificity and 89.4% sensitivity for detection of PCV2 antibody in the field sera. The assay showed no cross-reactivity with antibodies to PCV type 1, porcine reproductive and respiratory syndrome virus and porcine parvovirus. The results suggest that the rcELISA is suitable for routine serodiagnosis and epidemiological surveys of PCV2-associated diseases. PMID:15107550

  7. Expression of senescent antigen on erythrocytes infected with a knobby variant of the human malaria parasite Plasmodium falciparum

    SciTech Connect

    Winograd, E.; Greenan, J.R.T.; Sherman, I.W.

    1987-04-01

    Erythrocytes infected with a knobby variant of Plasmodium falciparum selectively bind IgG autoantibodies in normal human serum. Quantification of membrane-bound IgG, by use of /sup 125/I-labeled protein A, revealed that erythrocytes infected with the knobby variant bound 30 times more protein A than did noninfected erythrocytes; infection with a knobless variant resulted in less than a 2-fold difference compared with noninfected erythrocytes. IgG binding to knobby erythrocytes appeared to be related to parasite development, since binding of /sup 125/I-labeled protein A to cells bearing young trophozoites (less than 20 hr after parasite invasion) was similar to binding to uninfected erythrocytes. By immunoelectron microscopy, the membrane-bound IgG on erythrocytes infected with the knobby variant was found to be preferentially associated with the protuberances (knobs) of the plasma membrane. The removal of aged or senescent erythrocytes from the peripheral circulation is reported to involve the binding of specific antibodies to an antigen (senescent antigen) related to the major erythrocyte membrane protein band 3. Since affinity-purified autoantibodies against band 3 specifically bound to the plasma membrane of erythrocytes infected with the knobby variant of P. falciparum, it is clear that the malaria parasite induces expression of senescent antigen.

  8. Expression of major histocompatibility complex class I and class II antigens in human Schwann cell cultures and effects of infection with Mycobacterium leprae.

    PubMed

    Samuel, N M; Mirsky, R; Grange, J M; Jessen, K R

    1987-06-01

    Recent experiments on rats have raised the possibility that Schwann cells can present antigens to T lymphocytes. We have investigated whether this mechanism might be relevant in leprosy by determining under what conditions human Schwann cells express class I and class II antigens, and whether infection with Mycobacterium leprae affects this expression. The distribution of these antigens was examined on human Schwann cells in dissociated cell cultures derived from human fetal peripheral nerves. We find that both Schwann cells and fibroblastic cells in these cultures normally express class I antigens but not class II antigens. When Schwann cells are infected with live Mycobacterium leprae for 48 h, 73% of Schwann cells phagocytose the bacteria. Mycobacterium leprae prevents 3H-thymidine incorporation into cultured human Schwann cells, but does not affect class I expression in these cells. Treatment of normal and Mycobacterium leprae infected cultures with gamma-interferon for 72 h induces class II expression on most Schwann cells but not on the majority of fibroblastic cells. The fact that human Schwann cells infected with Mycobacterium leprae can be induced by gamma-interferon to express class II antigens suggests that they may be able to present Mycobacterium leprae antigens to T lymphocytes and thus initiate immune responses against the bacteria. We suggest that a failure of this response, such as that seen within nerve trunks in lepromatous leprosy, is caused by deficient class II expression on Schwann cells. This deficiency in class II expression, in turn, may be caused by the reduced gamma-interferon production characteristic of lepromatous leprosy. PMID:3115648

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

    PubMed

    Bachmann, Anna; Petter, Michaela; Tilly, Ann-Kathrin; Biller, Laura; Uliczka, Karin A; Duffy, Michael F; Tannich, Egbert; Bruchhaus, Iris

    2012-01-01

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

  10. Small-lymphoid cells and myeloid antigen expression in a patient with IgG myeloma: A case report

    PubMed Central

    JIANG, PENGJUN; XIA, WEN; SUN, XUEMEI; DAI, XINGBIN; LI, LIN

    2016-01-01

    Multiple myeloma is defined as a malignant proliferation of a single clone of plasma cells resulting in monoclonal immunoglobulin production. Due to the number of plasma cell morphological variants, difficulty is often faced during morphological diagnosis. The current study describes the case of a 49-year-old woman presenting with atypical plasma cell morphology detected by a bone marrow examination. Flow cytometric immunophenotyping determined the nature of the neoplastic cells as monoclonal myeloma cells with myeloid antigen expression. Serum electrophoresis with immunofixation and subsequent clinical findings confirmed this diagnosis. Therefore, the immunophenotyping of plasma cells in myelomas may be useful for the diagnosis of cases with atypical plasma cell morphology. PMID:26998140

  11. Local expression of matrix metalloproteinases, cathepsins, and their inhibitors during the development of murine antigen-induced arthritis

    PubMed Central

    Schurigt, Uta; Stopfel, Nadine; Hückel, Marion; Pfirschke, Christina; Wiederanders, Bernd; Bräuer, Rolf

    2005-01-01

    Cartilage and bone degradation, observed in human rheumatoid arthritis (RA), are caused by aberrant expression of proteinases, resulting in an imbalance of these degrading enzymes and their inhibitors. However, the role of the individual proteinases in the pathogenesis of degradation is not yet completely understood. Murine antigen-induced arthritis (AIA) is a well-established animal model of RA. We investigated the time profiles of expression of matrix metalloproteinase (MMP), cathepsins, tissue inhibitors of matrix metalloproteinases (TIMP) and cystatins in AIA. For primary screening, we revealed the expression profile with Affymetrix oligonucleotide chips. Real-time polymerase chain reaction (PCR) analyses were performed for the validation of array results, for tests of more RNA samples and for the completion of the time profile. For the analyses at the protein level, we used an MMP fluorescence activity assay and zymography. By a combination of oligonucleotide chips, real-time PCR and zymography, we showed differential expressions of several MMPs, cathepsins and proteinase inhibitors in the course of AIA. The strongest dysregulation was observed on days 1 and 3 in the acute phase. Proteoglycan loss analysed by safranin O staining was also strongest on days 1 and 3. Expression of most of the proteinases followed the expression of pro-inflammatory cytokines. TIMP-3 showed an expression profile similar to that of anti-inflammatory interleukin-4. The present study indicates that MMPs and cathepsins are important in AIA and contribute to the degradation of cartilage and bone. PMID:15642138

  12. Modulation of Cellular and Viral Gene Expression by the Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus

    PubMed Central

    Renne, Rolf; Barry, Chris; Dittmer, Dirk; Compitello, Nicole; Brown, Patrick O.; Ganem, Don

    2001-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV-8), is the likely etiological agent of Kaposi's sarcoma and primary effusion lymphoma. Common to these malignancies is that tumor cells are latently infected with KSHV. Viral gene expression is limited to a few genes, one of which is the latency-associated nuclear antigen (LANA), the product of ORF73. Examination of the primary sequence of LANA reveals some structural features reminiscent of transcription factors, leading us to hypothesize that LANA may regulate viral and cellular transcription during latency. In reporter gene-based transient transfection assays, we found that LANA can have either positive or negative effects on gene expression. While expression of a reporter gene from several synthetic promoters was increased in the presence of LANA, expression from the human immunodeficiency virus (HIV) long terminal repeat (LTR)—and from NF-κB-dependent reporter genes—was reduced by LANA expression. In addition, the promoter of KSHV ORF73 itself is activated up to 5.5-fold by LANA. This autoregulation may be important in tumorigenesis, because two other genes (v-cyclin and v-FLIP) with likely roles in cell growth and survival are also controlled by this element. To identify cellular genes influenced by LANA, we employed cDNA array-based expression profiling. Six known genes (and nine expressed sequence tags) were found to be upregulated in LANA-expressing cell lines. One of these, Staf-50, is known to inhibit expression from the HIV LTR; most of the other known genes are interferon inducible, although the interferon genes themselves were not induced by LANA. These data demonstrate that LANA expression has effects on cellular and viral gene expression. We suggest that, whether direct or indirect in origin, these effects may play important roles in the pathobiology of KSHV infection. PMID:11119614

  13. Oral Administration of Lactococcus lactis Expressing Synthetic Genes of Myelin Antigens in Decreasing Experimental Autoimmune Encephalomyelitis in Rats

    PubMed Central

    Kasarello, Kaja; Kwiatkowska-Patzer, Barbara; Lipkowski, Andrzej W.; Bardowski, Jacek K.; Szczepankowska, Agnieszka K.

    2015-01-01

    Background Multiple sclerosis is a human autoimmunological disease that causes neurodegeneration. One of the potential ways to stop its development is induction of oral tolerance, whose effect lies in decreasing immune response to the fed antigen. It was shown in animal models that administration of specific epitopes of the three main myelin proteins – myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), and proteolipid protein (PLP) – results in induction of oral tolerance and suppression of disease symptoms. Use of bacterial cells to produce and deliver antigens to gut mucosa seems to be an attractive method for oral tolerance induction in treatment of diseases with autoimmune background. Material/Methods Synthetic genes of MOG35-55, MBP85-97, and PLP139-151 myelin epitopes were generated and cloned in Lactococcus lactis under a CcpA-regulated promoter. The tolerogenic effect of bacterial preparations was tested on experimental autoimmune encephalomyelitis, which is the animal model of MS. EAE was induced in rats by intradermal injection of guinea pig spinal cord homogenate into hind paws. Results Rats were administered preparations containing whole-cell lysates of L. lactis producing myelin antigens using different feeding schemes. Our study demonstrates that 20-fold, but not 4-fold, intragastric administration of autoantigen-expressing L. lactis cells under specific conditions reduces the clinical symptoms of EAE in rats. Conclusions The present study evaluated the use of myelin antigens produced in L. lactis in inhibiting the onset of experimental autoimmune encephalomyelitis in rats. Obtained results indicate that application of such recombinant cells can be an attractive method of oral tolerance induction. PMID:26026273

  14. Expression of tissue polypeptide antigen (TPA) in fetal and adult liver: changes in liver disease.

    PubMed Central

    Burt, A D; Stewart, J A; Aitchison, M; MacSween, R N

    1987-01-01

    The distribution of tissue polypeptide antigen (40 kD molecular weight) in normal adult and fetal liver, and in liver disease was investigated and compared with the distribution of low and high molecular weight cytokeratins. In normal liver tissue polypeptide antigen was found only in bile duct epithelium; this distribution is similar to that of high molecular weight cytokeratin, but differs from that of low molecular weight cytokeratins. In liver disease it was found in areas of ductular transformation; in Mallory's bodies; and in alcoholic liver disease and primary biliary cirrhosis in some hepatocytes that did not contain Mallory's bodies. Images Fig 1 Fig 2 Fig 3 Fig 4 Fig 5 Fig 6 PMID:2442199

  15. Production and characterization of an orally immunogenic Plasmodium antigen in plants using a virus-based expression system.

    PubMed

    Webster, Diane E; Wang, Lina; Mulcair, Mark; Ma, Charles; Santi, Luca; Mason, Hugh S; Wesselingh, Steve L; Coppel, Ross L

    2009-12-01

    Increasing numbers of plant-made vaccines and pharmaceuticals are entering the late stage of product development and commercialization. Despite the theoretical benefits of such production, expression of parasite antigens in plants, particularly those from Plasmodium, the causative parasites for malaria, have achieved only limited success. We have previously shown that stable transformation of tobacco plants with a plant-codon optimized form of the Plasmodium yoelii merozoite surface protein 4/5 (PyMSP4/5) gene resulted in PyMSP4/5 expression of up to approximately 0.25% of total soluble protein. In this report, we describe the rapid expression of PyMSP4/5 in Nicotiana benthamiana leaves using the deconstructed tobacco mosaic virus-based magnICON expression system. PyMSP4/5 yields of up to 10% TSP or 1-2 mg/g of fresh weight were consistently achieved. Characterization of the recombinant plant-made PyMSP4/5 indicates that it is structurally similar to PyMSP4/5 expressed by Escherichia coli. It is notable that the plant-made PyMSP4/5 protein retained its immunogenicity following long-term storage at ambient temperature within freeze-dried leaves. With assistance from a mucosal adjuvant the PyMSP4/5-containing leaves induced PyMSP4/5-specific antibodies when delivered orally to naïve mice or mice primed by a DNA vaccine. This study provides evidence that immunogenic Plasmodium antigens can be produced in large quantities in plants using the magnICON viral vector system. PMID:19781007

  16. Surface expression patterns of defined glycan antigens change during Schistosoma mansoni cercarial transformation and development of schistosomula.

    PubMed

    Smit, Cornelis H; Homann, Arne; van Hensbergen, Vincent P; Schramm, Gabriele; Haas, Helmut; van Diepen, Angela; Hokke, Cornelis H

    2015-12-01

    During the complex lifecycle of Schistosoma mansoni, a large variety of glycans is expressed. To many of these glycans, antibodies are induced by the infected host and some might be targets for vaccines or diagnostic tests. Spatial changes in glycan expression during schistosome development are largely unexplored. To study the surface-exposed glycans during the important initial stages of infection, we analyzed the binding of a panel of anti-glycan monoclonal antibodies (mAbs) to cercariae and schistosomula up to 72 h after transformation by immunofluorescence microscopy. The mAb specificity toward their natural targets was studied using a microarray containing a wide range of schistosomal N-glycans, O-glycans and glycosphingolipid glycans. With the exception of GalNAcβ1-4(Fucα1-3)GlcNAc (LDN-F), mono- and multifucosylated GalNAcβ1-4GlcNAc (LDN)-motifs were exposed at the surface of all developmental stages studied. Multifucosylated LDN-motifs were present on cercarial glycocalyx-derived O-glycans as well as cercarial glycolipids. In contrast, the Galβ1-4(Fucα1-3)GlcNAc (Lewis X) and LDN-F-motifs, also expressed on cercarial glycolipids, and in addition on a range of cercarial N- and O-glycans, became surface expressed only after transformation of cercariae to schistosomula. In line with the documented shedding of the O-glycan-rich cercarial glycocalyx after transformation these observations suggest that surface accessible multifucosylated LDN-motifs are mostly expressed by O-glycans in cercariae, but principally by glycosphingolipids in schistosomula. We hypothesize that these temporal changes in surface exposure of glycan antigens are relevant to the interaction with the host during the initial stages of infection with schistosomes and discuss the potential of these glycan antigens as intervention targets. PMID:26347524

  17. Novel Protective Antigens Expressed by Trypanosoma cruzi Amastigotes Provide Immunity to Mice Highly Susceptible to Chagas' Disease▿

    PubMed Central

    Silveira, Eduardo L. V.; Claser, Carla; Haolla, Filipe A. B.; Zanella, Luiz G.; Rodrigues, Mauricio M.

    2008-01-01

    Earlier studies have demonstrated in A/Sn mice highly susceptible to Chagas' disease protective immunity against lethal Trypanosoma cruzi infection elicited by vaccination with an open reading frame (ORF) expressed by amastigotes. In our experiments, we used this mouse model to search for other amastigote-expressed ORFs with a similar property. Fourteen ORFs previously determined to be expressed in this developmental stage were individually inserted into a eukaryotic expression vector containing a nucleotide sequence that encoded a mammalian secretory signal peptide. Immunization with 13 of the 14 ORFs induced specific antibodies which recognized the amastigotes. Three of those immune sera also reacted with trypomastigotes and epimastigotes. After a lethal challenge with Y strain trypomastigotes, the vast majority of plasmid-injected mice succumbed to infection. In some cases, a significant delay in mortality was observed. Only two of these ORFs provided protective immunity against the otherwise lethal infection caused by trypomastigotes of the Y or Colombia strain. These ORFs encode members of the trans-sialidase family of surface antigens related to the previously described protective antigen amastigote surface protein 2 (ASP-2). Nevertheless, at the level of antibody recognition, no cross-reactivity was observed between the ORFs and the previously described ASP-2 from the Y strain. In immunofluorescence analyses, we observed the presence of epitopes related to both proteins expressed by amastigotes of seven different strains. In conclusion, our approach allowed us to successfully identify two novel protective ORFs which we consider interesting for future studies on the immune response to Chagas' disease. PMID:18579696

  18. Novel protective antigens expressed by Trypanosoma cruzi amastigotes provide immunity to mice highly susceptible to Chagas' disease.

    PubMed

    Silveira, Eduardo L V; Claser, Carla; Haolla, Filipe A B; Zanella, Luiz G; Rodrigues, Mauricio M

    2008-08-01

    Earlier studies have demonstrated in A/Sn mice highly susceptible to Chagas' disease protective immunity against lethal Trypanosoma cruzi infection elicited by vaccination with an open reading frame (ORF) expressed by amastigotes. In our experiments, we used this mouse model to search for other amastigote-expressed ORFs with a similar property. Fourteen ORFs previously determined to be expressed in this developmental stage were individually inserted into a eukaryotic expression vector containing a nucleotide sequence that encoded a mammalian secretory signal peptide. Immunization with 13 of the 14 ORFs induced specific antibodies which recognized the amastigotes. Three of those immune sera also reacted with trypomastigotes and epimastigotes. After a lethal challenge with Y strain trypomastigotes, the vast majority of plasmid-injected mice succumbed to infection. In some cases, a significant delay in mortality was observed. Only two of these ORFs provided protective immunity against the otherwise lethal infection caused by trypomastigotes of the Y or Colombia strain. These ORFs encode members of the trans-sialidase family of surface antigens related to the previously described protective antigen amastigote surface protein 2 (ASP-2). Nevertheless, at the level of antibody recognition, no cross-reactivity was observed between the ORFs and the previously described ASP-2 from the Y strain. In immunofluorescence analyses, we observed the presence of epitopes related to both proteins expressed by amastigotes of seven different strains. In conclusion, our approach allowed us to successfully identify two novel protective ORFs which we consider interesting for future studies on the immune response to Chagas' disease. PMID:18579696

  19. Epithelium Expressing the E7 Oncoprotein of HPV16 Attracts Immune-Modulatory Dendritic Cells to the Skin and Suppresses Their Antigen-Processing Capacity

    PubMed Central

    Chandra, Janin; Miao, Yan; Romoff, Natasha; Frazer, Ian H.

    2016-01-01

    Antigen presenting cells (APCs) in skin can promote either antigen-specific effector functions or antigen tolerance, and thus determine clearance or persistence of cutaneous viral infections. Human papillomavirus (HPV) infections can persist in squamous epithelium in immunocompetent individuals, and some persisting HPV infections, particularly with HPV16, promote malignant epithelial transformation. Here, we investigate whether local expression of the HPV16 protein most associated with malignant transformation, HPV16-E7, affects the phenotype and function of APC subsets in the skin. We demonstrate an expanded population of Langerhans cells in HPV16-E7 transgenic skin with distinct cell surface markers which express immune-modulatory enzymes and cytokines not expressed by cells from non transgenic skin. Furthermore, HPV16-E7 transgene expression in keratinocytes attracts new APC subsets to the epidermis. In vivo migration and transport of antigen to the draining lymph node by these APCs is markedly enhanced in HPV16-E7 expressing skin, whereas antigen-processing, as measured by proteolytic cleavage of DQ-OVA and activation of T cells in vivo by APCs, is significantly impaired. These data suggest that local expression of HPV16-E7 in keratinocytes can contribute to persisting infection with this oncogenic virus, by altering the phenotype and function of local APCs. PMID:27031095

  20. Pectinesterase Inhibitor from Jelly Fig (Ficus awkeotsang Makino) Achene Inhibits Surface Antigen Expression by Human Hepatitis B Virus.

    PubMed

    Huang, Yu-Chuen; Jiang, Chii-Ming; Chen, Yu-Jen; Chen, Yu-Yawn

    2013-01-01

    Pectinesterase inhibitor (PEI) isolated from jelly fig (Ficus awkeotsang Makino) is an edible component of a popular drink consumed in Asia. Hepatitis B virus (HBV) infection is prevalent in Asia, and current treatments for HBV infection need improvement. This study aimed to evaluate the effect of PEI on the surface antigen expression by HBV (HBsAg). Human hepatoma cell lines Hep3B and Huh7 served as in vitro models for assessing the cytotoxicity and HBsAg expression. A culture of primary hepatocytes cultured from mice served as the normal counterpart. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. HBsAg expression was evaluated by measuring HBsAg secretion into the culture medium using an enzyme-linked immunosorbent assay. The results showed that PEI did not affect the viability of the human hepatoma cell lines or primary mouse hepatocytes. PEI inhibited the expression of HBsAg in hepatoma cell lines harboring endogenous (Hep3B) and integrated (Huh7) HBV genomes in a concentration- and time-dependent manner, thus implicating a universal activity against HBV gene expression. In conclusion, it suggests that PEI from jelly fig inhibits the expression of human HBsAg in host cells without toxic effects on normal primary hepatocytes. PMID:24302965

  1. Purification and refolding of anti-T-antigen single chain antibodies (scFvs) expressed in Escherichia coli as inclusion bodies.

    PubMed

    Yuasa, Noriyuki; Koyama, Tsubasa; Fujita-Yamaguchi, Yoko

    2014-02-01

    T-antigen (Galβ1-3GalNAcα-1-Ser/Thr) is an oncofetal antigen that is commonly expressed as a carbohydrate determinant in many adenocarcinomas. Since it is associated with tumor progression and metastasis, production of recombinant antibodies specific for T-antigen could lead to the development of cancer diagnostics and therapeutics. Previously, we isolated and characterized 11 anti-T-antigen phage clones from a phage library displaying human single-chain antibodies (scFvs) and purified one scFv protein, 1G11. More recently, we purified and characterized 1E8 scFv protein using a Drosophila S2 expression system. In the current study, four anti-T-antigen scFv genes belonging to Groups 1-4 were purified from inclusion bodies expressed in Escherichia coli cells. Inclusion bodies isolated from E. coli cells were denatured in 3.5 M Gdn-HCl. Solubilized His-tagged scFv proteins were purified using Ni(2+)-Sepharose column chromatography in the presence of 3.5 M Gdn-HCl. Purified scFv proteins were refolded according to a previously published method of step-wise dialysis. Two anti-T-antigen scFv proteins, 1E6 and 1E8 that belong to Groups 1 and 2, respectively, were produced in sufficient amounts, thus allowing further characterization of their binding activity with T-antigen. Specificity and affinity constants determined using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR), respectively, provided evidence that both 1E8 and 1E6 scFv proteins are T-antigen specific and suggested that 1E8 scFv protein has a higher affinity for T-antigen than 1E6 scFv protein. PMID:24647109

  2. Novel recombinant BCG expressing perfringolysin O and the over-expression of key immunodominant antigens; pre-clinical characterization, safety and protection against challenge with Mycobacterium tuberculosis.

    PubMed

    Sun, Ronggai; Skeiky, Yasir A W; Izzo, Angelo; Dheenadhayalan, Veerabadran; Imam, Zakaria; Penn, Erica; Stagliano, Katherine; Haddock, Scott; Mueller, Stefanie; Fulkerson, John; Scanga, Charles; Grover, Ajay; Derrick, Steven C; Morris, Sheldon; Hone, David M; Horwitz, Marcus A; Kaufmann, Stefan H E; Sadoff, Jerald C

    2009-07-16

    Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), has infected approximately two billion individuals worldwide with approximately 9.2 million new cases and 1.6 million deaths annually. Current efforts are focused on making better BCG priming vaccines designed to induce a comprehensive and balanced immunity followed by booster(s) targeting a specific set of relevant antigens in common with the BCG prime. We describe the generation and immunological characterization of recombinant BCG strains with properties associated with lysis of the endosome compartment and over-expression of key Mtb antigens. The endosome lysis strain, a derivative of BCG SSI-1331 (BCG(1331)) expresses a mutant form of perfringolysin O (PfoA(G137Q)), a cytolysin normally secreted by Clostridium perfringens. Integration of the PfoA(G137Q) gene into the BCG genome was accomplished using an allelic exchange plasmid to replace ureC with pfoA(G137Q) under the control of the Ag85B promoter. The resultant BCG construct, designated AERAS-401 (BCG(1331) DeltaureC::OmegapfoA(G137Q)) secreted biologically active Pfo, was well tolerated with a good safety profile in immunocompromised SCID mice. A second rBCG strain, designated AFRO-1, was generated by incorporating an expression plasmid encoding three mycobacterial antigens, Ag85A, Ag85B and TB10.4, into AERAS-401. Compared to the parental BCG strain, vaccination of mice and guinea pigs with AFRO-1 resulted in enhanced immune responses. Mice vaccinated with AFRO-1 and challenged with the hypervirulent Mtb strain HN878 also survived longer than mice vaccinated with the parental BCG. Thus, we have generated improved rBCG vaccine candidates that address many of the shortcomings of the currently licensed BCG vaccine strains. PMID:19500523

  3. A foundation for universal T-cell based immunotherapy: T cells engineered to express a CD19-specific chimeric-antigen-receptor and eliminate expression of endogenous TCR

    PubMed Central

    Torikai, Hiroki; Reik, Andreas; Liu, Pei-Qi; Zhou, Yuanyue; Zhang, Ling; Maiti, Sourindra; Huls, Helen; Miller, Jeffrey C.; Kebriaei, Partow; Rabinovitch, Brian; Lee, Dean A.; Champlin, Richard E.; Bonini, Chiara; Naldini, Luigi; Rebar, Edward J.; Gregory, Philip D.; Holmes, Michael C.

    2012-01-01

    Clinical-grade T cells are genetically modified ex vivo to express a chimeric antigen receptor (CAR) to redirect specificity to a tumor associated antigen (TAA) thereby conferring antitumor activity in vivo. T cells expressing a CD19-specific CAR recognize B-cell malignancies in multiple recipients independent of major histocompatibility complex (MHC) because the specificity domains are cloned from the variable chains of a CD19 monoclonal antibody. We now report a major step toward eliminating the need to generate patient-specific T cells by generating universal allogeneic TAA-specific T cells from one donor that might be administered to multiple recipients. This was achieved by genetically editing CD19-specific CAR+ T cells to eliminate expression of the endogenous αβ T-cell receptor (TCR) to prevent a graft-versus-host response without compromising CAR-dependent effector functions. Genetically modified T cells were generated using the Sleeping Beauty system to stably introduce the CD19-specific CAR with subsequent permanent deletion of α or β TCR chains with designer zinc finger nucleases. We show that these engineered T cells display the expected property of having redirected specificity for CD19 without responding to TCR stimulation. CAR+TCRneg T cells of this type may potentially have efficacy as an off-the-shelf therapy for investigational treatment of B-lineage malignancies. PMID:22535661

  4. Decreased expression of human class II antigens on monocytes from patients with acquired immune deficiency syndrome. Increased expression with interferon-gamma.

    PubMed Central

    Heagy, W; Kelley, V E; Strom, T B; Mayer, K; Shapiro, H M; Mandel, R; Finberg, R

    1984-01-01

    The expression of HLA-DR (a class II histocompatibility antigen) on monocytes isolated from the peripheral blood of normal individuals and patients with acquired immune deficiency syndrome (AIDS) was investigated by the use of dual fluorescent staining and cytofluorometry. In animal models the absence of class II positive monocytes is linked to a failure of T cells to respond to antigens. We now report that patients with AIDS have a paucity of HLA-DR+ monocytes. The percentage of HLA-DR+ monocytes among eight normal individuals ranged from 49.3 to 95.0%+, and only one individual had less than 50% HLA-DR+ monocytes. HLA-DR expression on monocytes from homosexual male patients with lymphadenopathy was similar to that of normal subjects (range, 58.0 to 97.4%+). In contrast, seven of nine patients with AIDS had less than 50% HLA-DR+ monocytes (range, 13.4 to 78.8%+). The in vitro incubation of monocytes from AIDS patients with cloned human interferon-gamma resulted in an increase of the expression of HLA-DR to near normal levels. PMID:6439741

  5. Recombinant Pvs48/45 Antigen Expressed in E. coli Generates Antibodies that Block Malaria Transmission in Anopheles albimanus Mosquitoes

    PubMed Central

    Arévalo-Herrera, Myriam; Vallejo, Andrés F.; Rubiano, Kelly; Solarte, Yezid; Marin, Catherin; Castellanos, Angélica; Céspedes, Nora; Herrera, Sócrates

    2015-01-01

    Transmission of malaria parasites from humans to Anopheles mosquitoes can be inhibited by specific antibodies elicited during malaria infection, which target surface Plasmodium gametocyte/gamete proteins. Some of these proteins may have potential for vaccine development. Pvs48/45 is a P. vivax gametocyte surface antigen orthologous to Pfs48/45, which may play a role during parasite fertilization and thus has potential for transmission blocking (TB) activity. Here we describe the expression of a recombinant Pvs48/45 protein expressed in Escherichia coli as a ∼60kDa construct which we tested for antigenicity using human sera and for its immunogenicity and transmission blocking activity of specific anti-mouse and anti-monkey Pvs48/45 antibodies. The protein reacted with sera of individuals from malaria-endemic areas and in addition induced specific IgG antibody responses in BALB/c mice and Aotus l. griseimembra monkeys. Sera from both immunized animal species recognized native P. vivax protein in Western blot (WB) and immunofluorescence assays. Moreover, sera from immunized mice and monkeys produced significant inhibition of parasite transmission to An. Albimanus mosquitoes as shown by membrane feeding assays. Results indicate the presence of reactive epitopes in the Pvs48/45 recombinant product that induce antibodies with TB activity. Further testing of this protein is ongoing to determine its vaccine potential. PMID:25775466

  6. Antigenic properties and diagnostic potential of baculovirus-expressed infectious bursal disease virus proteins VPX and VP3.

    PubMed

    Martínez-Torrecuadrada, J L; Lázaro, B; Rodriguez, J F; Casal, J I

    2000-07-01

    The routine technique for detecting antibodies specific to infectious bursal disease virus (IBDV) is a serological evaluation by enzyme-linked immunosorbent assay (ELISA) with preparations of whole virions as the antigens. To avoid using complete virus in the standard technique, we have developed two new antigens through the expression of the VPX and VP3 genes in insect cells. VPX and especially VP3 were expressed at high levels in insect cells and simple to purify. The immunogenicity of both proteins was similar to that of the native virus. VPX was able to elicit neutralizing antibodies but VP3 was not. Purified VPX and VP3 were tested in an indirect ELISA with more than 300 chicken sera. There was an excellent correlation between the results of the ELISA using VPX and those of the two commercial kits. VP3 did not perform as well as VPX, and the linear correlation was significantly lower. A comparison with the standard reference technique, seroneutralization, showed that the indirect ELISA was more sensitive. Therefore, VPX-based ELISA is a good alternative to conventional ELISAs that use whole virions. PMID:10882666

  7. Characterization of Tula virus antigenic determinants defined by monoclonal antibodies raised against baculovirus-expressed nucleocapsid protein.

    PubMed

    Lundkvist, A; Vapalahti, O; Plyusnin, A; Sjölander, K B; Niklasson, B; Vaheri, A

    1996-11-01

    Tula virus was recently discovered by RT-PCR in lung samples from European common voles (Microtus arvalis and M. rossiaemeridionalis). Since virus isolation attempts had been unsuccessful, no antigen was available for analysis or for use in immunoassays. To circumvent this, complete Tula virus nucleocapsid protein (bac-TUL-N) was expressed in recombinant baculovirus. Rodent antibody end-point titers to bac-TUL-N and to truncated N fragments indicated that the NH2-terminal region is the major antigenic target and revealed a high cross-reactivity to Puumala virus N. Immunizations with crude bac-TUL-N preparations evoked high antibody responses to native hantavirus N in Balb/c mice and six monoclonal antibodies (Mabs) were generated. Epitope mapping of the Mabs, based on a competitive assay, reactivities to truncated recombinant N fragments, and reactivity patterns to different hantavirus strains, identified five recognition sites on Tula virus N. One epitope, which was identified as specific for Tula virus, was located in a region of N which is highly variable among the hantaviruses (aa 226-293), and four epitopes were mapped to the NH2-terminal region of the protein (aa 1-61). One epitope was expressed only in Tula and Prospect Hill viruses, one epitope in Tula, Prospect Hill, Khabarovsk, and Sin Nombre viruses, while two epitopes were conserved in all examined hantaviruses carried by rodents within the subfamily Arvicolinae of the Muridae family. PMID:8896239

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

    SciTech Connect

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

    2005-09-09

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

  9. Sialyl-Tn antigen expression occurs early during human mammary carcinogenesis and is associated with high nuclear grade and aneuploidy.

    PubMed

    Cho, S H; Sahin, A; Hortobagyi, G N; Hittelman, W N; Dhingra, K

    1994-12-15

    Sialyl-Tn (STn) antigen represents an aberrant glycosylation product of cell surface mucin in adenocarcinomas. We studied its expression in 40 breast carcinomas (35 of which included in situ carcinomas) by performing immunostaining with B72.3 monoclonal antibody. STn expression was observed in 50% of cases and was equally frequent in in situ and in invasive carcinomas. Positive STn staining significantly correlated with high nuclear grade (P = 0.001), aneuploidy (P < 0.001) and high S-phase fraction (P = 0.02). No correlation was observed between STn staining and age, menopausal status, presence of invasive component, or hormone receptor positivity. STn staining may provide an objective marker of dedifferentiation of breast tumors and should be investigated further for its prognostic value in breast cancers and as a biomarker of malignant transformation of breast epithelium. PMID:7987817

  10. Expression of Cpgp40/15 in Toxoplasma gondii: a surrogate system for the study of Cryptosporidium glycoprotein antigens.

    PubMed

    O'Connor, R M; Kim, K; Khan, F; Ward, H D

    2003-10-01

    Cryptosporidium parvum is a waterborne enteric coccidian that causes diarrheal disease in a wide range of hosts. Development of successful therapies is hampered by the inability to culture the parasite and the lack of a transfection system for genetic manipulation. The glycoprotein products of the Cpgp40/15 gene, gp40 and gp15, are involved in C. parvum sporozoite attachment to and invasion of host cells and, as such, may be good targets for anticryptosporidial therapies. However, the function of these antigens appears to be dependent on the presence of multiple O-linked alpha-N-acetylgalactosamine (alpha-GalNAc) determinants. A eukaryotic expression system that would produce proteins bearing glycosylation patterns similar to those found on the native C. parvum glycoproteins would greatly facilitate the molecular and functional characterization of these antigens. As a unique approach to this problem, the Cpgp40/15 gene was transiently expressed in Toxoplasma gondii, and the expressed recombinant glycoproteins were characterized. Antisera to gp40 and gp15 reacted with the surface membranes of tachyzoites expressing the Cpgp40/15 construct, and this reactivity colocalized with that of antiserum to the T. gondii surface protein SAG1. Surface membrane localization was dependent on the presence of the glycophosphatidylinositol anchor attachment site present in the gp15 coding sequence. The presence of terminal O-linked alpha-GalNAc determinants on the T. gondii recombinant gp40 was confirmed by reactivity with Helix pomatia lectin and the monoclonal antibody 4E9, which recognizes alpha-GalNAc residues, and digestion with alpha-N-acetylgalactosaminidase. In addition to appropriate localization and glycosylation, T. gondii apparently processes the gp40/15 precursor into the gp40 and gp15 component glycopolypeptides, albeit inefficiently. These results suggest that a surrogate system using T. gondii for the study of Cryptosporidium biology may be useful. PMID:14500524

  11. Expression of the Escherichia coli K5 capsular antigen: immunoelectron microscopic and biochemical studies with recombinant E. coli.

    PubMed Central

    Kröncke, K D; Boulnois, G; Roberts, I; Bitter-Suermann, D; Golecki, J R; Jann, B; Jann, K

    1990-01-01

    The capsular K5 polysaccharide, a representative of group II capsular antigens of Escherichia coli, has been cloned previously, and three gene regions responsible for polymerization and surface expression have been defined (I. S. Roberts, R. Mountford, R. Hodge, K. B. Jann, and G. J. Boulnois, J. Bacteriol. 170:1305-1310, 1988). In this report, we describe the immunoelectron microscopic analysis of recombinant bacteria expressing the K5 antigen and of mutants defective in either region 1 or region 3 gene functions, as well as the biochemical analysis of the K5 capsular polysaccharide. Whereas the K5 clone expressed the K5 polysaccharide as a well-developed capsule in about 25% of its population, no capsule was observed in whole mount preparations and ultrathin sections of the expression mutants. Immunogold labeling of sections from the region 3 mutant revealed the capsular K5 polysaccharide in the cytoplasm. With the region 1 mutant, the capsular polysaccharide appeared associated with the cell membrane, and, unlike the region 3 mutant polysaccharide, the capsular polysaccharide could be detected in the periplasm after plasmolysis of the bacteria. Polysaccharides were isolated from the homogenized mutants with cetyltrimethylammonium bromide. The polysaccharide from the region 1 mutant had the same size as that isolated from the capsule of the original K5 clone, and both polysaccharides were substituted with phosphatidic acid. The polysaccharide from the region 3 mutant was smaller and was not substituted with phosphatidic acid. These results prompt us to postulate that gene region 3 products are involved in the translocation of the capsular polysaccharide across the cytoplasmic membrane and that region 1 directs the transport of the lipid-substituted capsular polysaccharide through the periplasm and across the outer membrane. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 FIG. 6 PMID:2404935

  12. Ethnic differences in the expression of blood group antigens in the salivary gland secretory cells from German and Japanese non-secretor individuals.

    PubMed

    Tanegashima, A; Nishi, K; Fukunaga, T; Rand, S; Brinkmann, B

    1996-08-01

    Type 1 ABO blood group antigens (peripheral core structure: Gal beta 1-3GlcNAc beta 1-R) are expressed mainly in endodermally-derived tissues, but are not synthesized in mesodermally-derived tissues. In the former tissues, H type 1 antigen is generated largely by alpha-2-L-fucosyltransferase encoded by secretor (Se) gene and acting on the terminal galactose of the type 1 precursor chain. This theory has been generally accepted, and it seems that the expression of ABO blood group antigens is absent, or expressed at a low level, in these tissues from non-secretor individuals. In this immunohistochemical study on the secretory cells of salivary glands, we found ethnic difference between German and Japanese non-secretor individuals in the expression of blood group antigens: i.e. the expression of the type 1 blood group antigens is present in these cells from Japanese non-secretor individuals but absent from German. A possible explanation is that another alpha-2-L-fucosyltransferase, independent of the secretor gene, is present in Japanese non-secretor individuals. PMID:8872110

  13. Constitutive and cytokine-induced expression of human leukocyte antigens and cell adhesion molecules by human myotubes.

    PubMed Central

    Michaelis, D.; Goebels, N.; Hohlfeld, R.

    1993-01-01

    Understanding the immunobiology of muscle is relevant to muscular autoimmune diseases and to gene therapies based on myoblast transfer. We have investigated the constitutive and cytokine-induced intra- and extracellular expression of histocompatibility human leukocyte antigens (HLA) and cell adhesion molecules by multinucleated human myotubes using immunofluorescence microscopy. Myotubes constitutively expressed HLA class I but not HLA class II. Exposure to interferon-gamma, but not tumor necrosis factor-alpha, induced HLA-DR in the cytoplasm and on the surface membrane of approximately 40 to 95% of cultured myotubes. Surface expression was strongest in perinuclear membrane areas, and cytoplasmic expression was strongest at branching points and at the tips of myotubes. HLA-DP and HLA-DQ were not expressed in detectable amounts. Both interferon-gamma and tumor necrosis factor-alpha induced the intercellular adhesion molecule-1 (CD54) in the cytoplasm and on the surface of nearly all myotubes. The distribution of intercellular adhesion molecule-1 and HLA-DR was similar but not identical in double-positive myotubes. The leukocyte function-associated (LFA) adhesion molecules LFA-1 (CD11a/CD18), LFA-2 (CD2), and LFA-3 (CD58) could not be detected in the cytoplasm or on the surface. Our results indicate that cytokine-induced myotubes can participate in immune interactions with T lymphocytes. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8214008

  14. Topoisomerase inhibitors modulate gene expression of B-cell translocation gene 2 and prostate specific antigen in prostate carcinoma cells.

    PubMed

    Chiang, Kun-Chun; Tsui, Ke-Hung; Chung, Li-Chuan; Yeh, Chun-Nan; Chang, Phei-Lang; Chen, Wen-Tsung; Juang, Horng-Heng

    2014-01-01

    Camptothecin (CPT) and doxorubicin (DOX) have been demonstrated to have potent anti-tumor activity. The B-cell translocation gene 2 (BTG2) is involved in the regulation of cell cycle progression. We evaluated the molecular mechanisms of CPT and DOX on cell proliferation and the expressions of BTG2 and prostate specific antigen (PSA) in prostate carcinoma cells. Our results indicated that CPT or DOX treatments induced Go/G1 cell cycle arrest in LNCaP cells and apoptosis at higher dosage. Immunoblot and transient gene expression assay indicated that CPT or DOX treatments induced p53 and BTG2 gene expression, with the later effect dependent on the p53 response element within BTG2 promoter area since mutation of the p53 response element from GGGAAAGTCC to GGAGTCC or from GGCAGAGCCC to GGCACC by site-directed mutagenesis abolished the stimulation of CPT or DOX on the BTG2 promoter activity, which is also supported by our results that cotreatments of pifithrin-α, an inhibitor of p53 dependent transcriptional activation, blocked the induction of CPT or DOX on BTG2 gene expression. CPT or DOX also downregulated the protein expressions of androgen receptor (AR) and PSA. Transient gene expression assays suggested that CPT or DOX's attenuation of PSA promoter activity is dependent on both the androgen and p53 response elements within of the PSA promoter. Our results indicated that CPT and DOX attenuate cell proliferation via upregulation of BTG2 gene expression through the p53-dependent pathway. The CPT and DOX block the PSA gene expression by upregulation of p53 activity and downregulation of androgen receptor activity. PMID:24586533

  15. Topoisomerase Inhibitors Modulate Gene Expression of B-Cell Translocation Gene 2 and Prostate Specific Antigen in Prostate Carcinoma Cells

    PubMed Central

    Chung, Li-Chuan; Yeh, Chun-Nan; Chang, Phei-Lang; Chen, Wen-Tsung; Juang, Horng-Heng

    2014-01-01

    Camptothecin (CPT) and doxorubicin (DOX) have been demonstrated to have potent anti-tumor activity. The B-cell translocation gene 2 (BTG2) is involved in the regulation of cell cycle progression. We evaluated the molecular mechanisms of CPT and DOX on cell proliferation and the expressions of BTG2 and prostate specific antigen (PSA) in prostate carcinoma cells. Our results indicated that CPT or DOX treatments induced Go/G1 cell cycle arrest in LNCaP cells and apoptosis at higher dosage. Immunoblot and transient gene expression assay indicated that CPT or DOX treatments induced p53 and BTG2 gene expression, with the later effect dependent on the p53 response element within BTG2 promoter area since mutation of the p53 response element from GGGAAAGTCC to GGAGTCC or from GGCAGAGCCC to GGCACC by site-directed mutagenesis abolished the stimulation of CPT or DOX on the BTG2 promoter activity, which is also supported by our results that cotreatments of pifithrin-α, an inhibitor of p53 dependent transcriptional activation, blocked the induction of CPT or DOX on BTG2 gene expression. CPT or DOX also downregulated the protein expressions of androgen receptor (AR) and PSA. Transient gene expression assays suggested that CPT or DOX’s attenuation of PSA promoter activity is dependent on both the androgen and p53 response elements within of the PSA promoter. Our results indicated that CPT and DOX attenuate cell proliferation via upregulation of BTG2 gene expression through the p53-dependent pathway. The CPT and DOX block the PSA gene expression by upregulation of p53 activity and downregulation of androgen receptor activity. PMID:24586533

  16. Oral immunization with hepatitis B surface antigen expressed in transgenic plants

    PubMed Central

    Kong, Qingxian; Richter, Liz; Yang, Yu Fang; Arntzen, Charles J.; Mason, Hugh S.; Thanavala, Yasmin

    2001-01-01

    Oral immunogenicity of recombinant hepatitis B surface antigen (HBsAg) derived from yeast (purified product) or in transgenic potatoes (uncooked unprocessed sample) was compared. An oral adjuvant, cholera toxin, was used to increase immune responses. Transgenic plant material containing HBsAg was the superior means of both inducing a primary immune response and priming the mice to respond to a subsequent parenteral injection of HBsAg. Electron microscopy of transgenic plant samples revealed evidence that the HBsAg accumulated intracellularly; we conclude that natural bioencapsulation of the antigen may provide protection from degradation in the digestive tract until plant cell degradation occurs near an immune effector site in the gut. The correlate of protection from hepatitis B virus infection is serum antibody titers induced by vaccination; the protective level in humans is 10 milliunits/ml or greater. Mice fed HBsAg-transgenic potatoes produced HBsAg-specific serum antibodies that exceeded the protective level and, on parenteral boosting, generated a strong long-lasting secondary antibody response. We have also shown the effectiveness of oral delivery by using a parenteral prime-oral boost immunization schedule. The demonstrated success of oral immunization for hepatitis B virus with an “edible vaccine” provides a strategy for contributing a means to achieve global immunization for hepatitis B prevention and eradication. PMID:11553782

  17. Cyclin D1 as a universally expressed mantle cell lymphoma-associated tumor antigen for immunotherapy.

    PubMed

    Wang, M; Sun, L; Qian, J; Han, X; Zhang, L; Lin, P; Cai, Z; Yi, Q

    2009-07-01

    Mantle cell lymphoma (MCL) accounts for 5-10% of all non-Hodgkin lymphomas and has the worst prognosis among all lymphomas. The hallmark of MCL is a t(11;14) translocation that results in overexpression of cyclin D1 by tumor cells of virtually all patients. In this study, we examined whether cyclin D1 could be an effective tumor-associated antigen for immunotherapy. We identified cyclin D1 peptides for HLA-A(*)0201 and generated peptide-specific CD8(+) T-cell lines from HLA-A(*)0201(+) blood donors and MCL patients. These cell lines proliferated in response to cyclin D1 peptide-pulsed stimulatory cells. Moreover, the T cells efficiently lysed peptide-pulsed but not unpulsed T2 cells and autologous dendritic cells; cyclin D1(+) and HLA-A(*)0201(+) human MCL lines MINO, SP53, Jeko-1 and Granta 519; and more importantly, HLA-A(*)0201(+) primary lymphoma cells from MCL patients. No killing was observed with HLA-A(*)0201(-) primary lymphoma cells or HLA-A(*)0201(+) normal blood cells, including B cells. These results indicate that these T cells are potent cytotoxic T cells and recognize cyclin D1 peptides naturally presented by patient lymphoma cells in the context of HLA-A(*)0201 molecules. Taken together, our work identifies cyclin D1 as a potentially important antigen for immunotherapy of MCL. PMID:19225534

  18. Comparison of altered expression of histocompatibility antigens with altered immune function in murine spleen cells treated with ultraviolet radiation and/or TPA

    SciTech Connect

    Pretell, J.O.; Cone, R.E.

    1985-02-01

    Previous studies in our laboratory demonstrated that several treatments that inhibited the ability of cells to stimulate the mixed lymphocyte reaction (MLR) also blocked the shedding of histocompatibility antigens and Ia antigens from murine spleen cells. In the present studies, one of these treatments, ultraviolet radiation (UV), was shown to cause an initial loss in the density of H-2K, IA, and IE antigens prior to the block in shedding observed after culture of these cells. Further analysis revealed that the UV-induced loss of antigens could be prevented by the presence of colchicine during irradiation. Biosynthetic analyses revealed the IA antigen synthesis was also inhibited in the UV-irradiated cells. Examination of the effects of a second agent, 12-0-tetradecanoylphorbol-13-acetate (TPA) on the turnover of histocompatibility antigens revealed that the biosynthesis and shedding of these antigens were accelerated by this agent. However, addition of TPA to UV-irradiated cells did not result in a reversal of the UV-induced block in biosynthesis of IA antigens. Results of immune function assays correlated with the biochemical studies: UV-irradiation inhibited the generation of the MLR, but TPA enhanced this reaction, and addition of TPA to mixed lymphocyte cultures with UV-irradiated stimulators did not reverse the UV-induced inhibition. These results suggest that, although the turnover of histocompatibility antigens may be affected by TPA and UV in an antagonistic fashion, additional factors other than the expression of histocompatibility antigens are operating in the inhibition of stimulation of an MLR by UV radiation or its enhancement by TPA.

  19. Intramolecular trimerization, a novel strategy for making multispecific antibodies with controlled orientation of the antigen binding domains.

    PubMed

    Alvarez-Cienfuegos, Ana; Nuñez-Prado, Natalia; Compte, Marta; Cuesta, Angel M; Blanco-Toribio, Ana; Harwood, Seandean Lykke; Villate, Maider; Merino, Nekane; Bonet, Jaume; Navarro, Rocio; Muñoz-Briones, Clara; Sørensen, Karen Marie Juul; Mølgaard, Kasper; Oliva, Baldo; Sanz, Laura; Blanco, Francisco J; Alvarez-Vallina, Luis

    2016-01-01

    Here, we describe a new strategy that allows the rapid and efficient engineering of mono and multispecific trivalent antibodies. By fusing single-domain antibodies from camelid heavy-chain-only immunoglobulins (VHHs) to the N-terminus of a human collagen XVIII trimerization domain (TIE(XVIII)) we produced monospecific trimerbodies that were efficiently secreted as soluble functional proteins by mammalian cells. The purified VHH-TIE(XVIII) trimerbodies were trimeric in solution and exhibited excellent antigen binding capacity. Furthermore, by connecting with two additional glycine-serine-based linkers three VHH-TIE(XVIII) modules on a single polypeptide chain, we present an approach for the rational design of multispecific tandem trimerbodies with defined stoichiometry and controlled orientation. Using this technology we report here the construction and characterization of a tandem VHH-based trimerbody capable of simultaneously binding to three different antigens: carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR) and green fluorescence protein (GFP). Multispecific tandem VHH-based trimerbodies were well expressed in mammalian cells, had good biophysical properties and were capable of simultaneously binding their targeted antigens. Importantly, these antibodies were very effective in inhibiting the proliferation of human epidermoid carcinoma A431 cells. Multispecific VHH-based trimerbodies are therefore ideal candidates for future applications in various therapeutic areas. PMID:27345490

  20. Intramolecular trimerization, a novel strategy for making multispecific antibodies with controlled orientation of the antigen binding domains

    PubMed Central

    Alvarez-Cienfuegos, Ana; Nuñez-Prado, Natalia; Compte, Marta; Cuesta, Angel M.; Blanco-Toribio, Ana; Harwood, Seandean Lykke; Villate, Maider; Merino, Nekane; Bonet, Jaume; Navarro, Rocio; Muñoz-Briones, Clara; Sørensen, Karen Marie Juul; Mølgaard, Kasper; Oliva, Baldo; Sanz, Laura; Blanco, Francisco J.; Alvarez-Vallina, Luis

    2016-01-01

    Here, we describe a new strategy that allows the rapid and efficient engineering of mono and multispecific trivalent antibodies. By fusing single-domain antibodies from camelid heavy-chain-only immunoglobulins (VHHs) to the N-terminus of a human collagen XVIII trimerization domain (TIEXVIII) we produced monospecific trimerbodies that were efficiently secreted as soluble functional proteins by mammalian cells. The purified VHH-TIEXVIII trimerbodies were trimeric in solution and exhibited excellent antigen binding capacity. Furthermore, by connecting with two additional glycine-serine-based linkers three VHH-TIEXVIII modules on a single polypeptide chain, we present an approach for the rational design of multispecific tandem trimerbodies with defined stoichiometry and controlled orientation. Using this technology we report here the construction and characterization of a tandem VHH-based trimerbody capable of simultaneously binding to three different antigens: carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR) and green fluorescence protein (GFP). Multispecific tandem VHH-based trimerbodies were well expressed in mammalian cells, had good biophysical properties and were capable of simultaneously binding their targeted antigens. Importantly, these antibodies were very effective in inhibiting the proliferation of human epidermoid carcinoma A431 cells. Multispecific VHH-based trimerbodies are therefore ideal candidates for future applications in various therapeutic areas. PMID:27345490

  1. Immunotoxicity of aflatoxin B1: Impairment of the cell-mediated response to vaccine antigen and modulation of cytokine expression

    SciTech Connect

    Meissonnier, Guylaine M.; Pinton, Philippe; Laffitte, Joelle; Cossalter, Anne-Marie; Gong, Yun Yun; Wild, Christopher P.; Bertin, Gerard; Galtier, Pierre; Oswald, Isabelle P.

    2008-09-01

    Aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus flavus or A. parasiticus, is a frequent contaminant of food and feed. This toxin is hepatotoxic and immunotoxic. The present study analyzed in pigs the influence of AFB1 on humoral and cellular responses, and investigated whether the immunomodulation observed is produced through interference with cytokine expression. For 28 days, pigs were fed a control diet or a diet contaminated with 385, 867 or 1807 {mu}g pure AFB1/kg feed. At days 4 and 15, pigs were vaccinated with ovalbumin. AFB1 exposure, confirmed by an observed dose-response in blood aflatoxin-albumin adduct, had no major effect on humoral immunity as measured by plasma concentrations of total IgA, IgG and IgM and of anti-ovalbumin IgG. Toxin exposure did not impair the mitogenic response of lymphocytes but delayed and decreased their specific proliferation in response to the vaccine antigen, suggesting impaired lymphocyte activation in pigs exposed to AFB1. The expression level of pro-inflammatory (TNF-{alpha}, IL-1{beta}, IL-6, IFN-{gamma}) and regulatory (IL-10) cytokines was assessed by real-time PCR in spleen. A significant up-regulation of all 5 cytokines was observed in spleen from pigs exposed to the highest dose of AFB1. In pigs exposed to the medium dose, IL-6 expression was increased and a trend towards increased IFN-{gamma} and IL-10 was observed. In addition we demonstrate that IL-6 impaired in vitro the antigenic- but not the mitogenic-induced proliferation of lymphocytes from control pigs vaccinated with ovalbumin. These results indicate that AFB1 dietary exposure decreases cell-mediated immunity while inducing an inflammatory response. These impairments in the immune response could participate in failure of vaccination protocols and increased susceptibility to infections described in pigs exposed to AFB1.

  2. Expression of Bacillus anthracis protective antigen in transgenic chloroplasts of tobacco, a non-food/feed crop

    PubMed Central

    Watson, Jennifer; Koya, Vijay; Leppla, Stephen H.; Daniell, Henry

    2012-01-01

    The Centers for Disease Control (CDC) lists Bacillus anthracis as a category A agent and estimates the cost of an anthrax attack to exceed US$ 26 billion per 100,000 exposed individuals. Concerns regarding anthrax vaccine purity, a requirement for multiple injections, and a limited supply of the protective antigen (PA), underscore the urgent need for an improved vaccine. Therefore, the 83 kDa immunogenic Bacillus anthracis protective antigen was expressed in transgenic tobacco chloroplasts. The PA gene (pag) was cloned into a chloroplast vector along with the psbA regulatory signals to enhance translation. Chloroplast integration of the transgenes was confirmed by PCR and Southern blot analyses. Crude plant extracts contained up to 2.5 mg full length PA/g of fresh leaf tissue and this showed exceptional stability for several months in stored leaves or crude extracts. Maximum levels of expression were observed in mature leaves under continuous illumination. Co-expression of the ORF2 chaperonin from Bacillus thuringiensis did not increase PA accumulation or induce folding into cuboidal crystals in transgenic chloroplasts. Trypsin, chymotrypsin and furin proteolytic cleavage sites present in PA were protected in transgenic chloroplasts because only full length PA 83 was observed without any degradation products. Both CHAPS and SDS detergents extracted PA with equal efficiency and PA was observed in the soluble fraction. Chloroplast-derived PA was functionally active in lysing mouse macrophages when combined with lethal factor (LF). Crude leaf extracts contained up to 25 μg functional PA/ml. With an average yield of 172 mg of PA per plant using an experimental transgenic cultivar grown in a greenhouse, 400 million doses of vaccine (free of contaminants) could be produced per acre, a yield that could be further enhanced 18-fold using a commercial cultivar in the field. PMID:15474731

  3. Expression of the cluster 1 antigen (neural cell adhesion molecule) in neuroectodermal tumours.

    PubMed Central

    Patel, K.; Frost, G.; Kiely, F.; Phimister, E.; Coakham, H. B.; Kemshead, J. T.

    1991-01-01

    In this study, we have investigated the expression of the neural cell adhesion molecule (NCAM) in the human brain, primary brain tumours and neuroblastoma. Adult brain was found to express discrete isoforms of 180, 170, 140 and 120 kDa, which on neuraminidase treatment resolved into bands of 180, 170, 140, 120 and 95 kDa. Primary brain tumours such as Schwannoma and medulloblastoma expressed embryonic NCAM characterised by a high level of glycosylation, whereas other tumours, e.g. astrocytoma, meningioma, glioma and oligodendroglioma expressed adult NCAM. Post-neuraminidase treatment, differential expression of the 180, 170, 140, 120 and 95 kDa isoforms were noted in these various tumour types. On the other hand, neuroblastoma cell lines were found to express only embryonic NCAM, which after neuraminidase treatment resulted in differential presence of only 180, 140 and 120 kDa proteins. Images Figure 1 Figure 2 PMID:2039710

  4. Expression of a highly antigenic and native-like folded extracellular domain of the human α1 subunit of muscle nicotinic acetylcholine receptor, suitable for use in antigen specific therapies for Myasthenia Gravis.

    PubMed

    Niarchos, Athanasios; Zouridakis, Marios; Douris, Vassilis; Georgostathi, Assimina; Kalamida, Dimitra; Sotiriadis, Alexandros; Poulas, Konstantinos; Iatrou, Kostas; Tzartos, Socrates J

    2013-01-01

    We describe the expression of the extracellular domain of the human α1 nicotinic acetylcholine receptor (nAChR) in lepidopteran insect cells (i-α1-ECD) and its suitability for use in antigen-specific therapies for Myasthenia Gravis (MG). Compared to the previously expressed protein in P. pastoris (y-α1-ECD), i-α1-ECD had a 2-fold increased expression yield, bound anti-nAChR monoclonal antibodies and autoantibodies from MG patients two to several-fold more efficiently and resulted in a secondary structure closer to that of the crystal structure of mouse α1-ECD. Our results indicate that i-α1-ECD is an improved protein for use in antigen-specific MG therapeutic strategies. PMID:24376846

  5. Dexamethasone modulates TCR zeta chain expression and antigen receptor-mediated early signaling events in human T lymphocytes.

    PubMed

    Nambiar, M P; Enyedy, E J; Fisher, C U; Warke, V G; Juang, Y T; Tsokos, G C

    2001-02-25

    Dexamethasone is a potent anti-inflammatory and immunosupressive agent that has complex, yet incompletely defined, effects on the immune response. Here, we explored the effect of dexamethasone on the expression of TCR zeta chain and TCR/CD3-induced early signaling events in human T lymphocytes. Immunoblotting studies using TCR zeta chain specific mAb showed a dose-dependent biphasic effect of dexamethasone on TCR zeta chain expression, that is, it was increased when cells were incubated with 10 nM, whereas the expression was decreased when incubated with 100 nM dexamethasone. The dose-dependent biphasic effect of dexamethsone on the TCR zeta chain expression was also revealed by FACS analysis of permeabilized cells. Time course studies showed that upregulation of the TCR zeta chain at 10 nM dexamethasone reached maximum levels at 24 h and remained elevated up to 48 h. Other subunits of the TCR/CD3 complex were minimally affected under these conditions. The increased expression of the TCR zeta chain following treatment with 10 nM dexamethasone correlated with increased anti-CD3 antibody-induced tyrosine phosphorylation of the TCR zeta chain and downstream signaling intermediate ZAP-70 and PLC gamma with faster kinetics. Similarly, the induction of TCR zeta chain expression at 10 nM dexamethasone correlated with increased and more sustained TCR/CD3-mediated [Ca(2+)](i) response. Reporter gene assays using TCR zeta chain promoter-driven luciferase gene constructs in Jurkat cells showed that treatment with 10 nM dexamethasone increased TCR zeta chain promoter activity and that the region between -160 and +58 was responsible for the observed effect. These results suggest that dexamethasone primarily acts at the transcriptional level and differentially modulates TCR zeta chain expression and antigen receptor-mediated early signaling events in human peripheral T lymphocytes. PMID:11277620

  6. ELEVATED EXPRESSION OF CANCER-ASSOCIATED PROLIFERATING CELL NUCLEAR ANTIGEN IN HIGH-GRADE PROSTATIC INTRAEPITHELIAL NEOPLASIA AND PROSTATE CANCER

    PubMed Central

    Wang, Xiaoyan; Hickey, Robert J.; Malkas, Linda H.; Koch, Michael O.; Li, Lang; Zhang, Shaobo; Sandusky, George E.; Grignon, David J; Eble, John N.; Cheng, Liang

    2011-01-01

    Background Proliferating-cell nuclear antigen (PCNA) plays an important role in DNA replication and repair. The expression and potential utility of this marker in prostatic neoplasia is uncertain. With the development of this new caPCNA selective antibody, we explored the potential utility of this marker in prostate cancer. Methods Using a traditional primary Fab2′ rabbit anti-caPCNA antibody-HRP conjugated secondary anti-Fab2′ antibody format, the expression of the caPCNA was analyzed in prostate tissue from 89 radical prostatectomy specimens. The caPCNA expression was correlated with clinicopathologic characteristics. Results The fraction of cells staining positively with caPCNA antibody in prostatic adenocarcinoma (mean, 23%) was significantly higher than that in benign prostatic epithelium (mean, 2%; p < 0.001) or high-grade prostatic intraepithelial neoplasia (PIN) (mean, 6%; p < 0.05). Moreover, the intensity of caPCNA expression in prostatic adenocarcinoma (mean, 2.9) was significantly higher than that in benign prostatic tissue (mean, 0.7; p < 0.001) or high-grade PIN (mean, 2.0; p < 0.001). Benign prostatic epithelium showed only minimal or negative reactivity. There was significant correlation between the percentage of caPCNA expression and primary Gleason grade (p = 0.01), and with Gleason score (p = 0.02). Adenocarcinomas with positive vascular invasion had a significantly higher percentage of cells staining with caPCNA antibody (p < 0.0001) and a higher intensity of caPCNA expression (p = 0.04). Conclusions Our data indicate that increased expression of the cancer-associated isoform of PCNA is common in prostatic adenocarcinoma and its precursor and may be a useful biomarker. PMID:21031434

  7. Transcriptional Targeting of Mature Dendritic Cells with Adenoviral Vectors via a Modular Promoter System for Antigen Expression and Functional Manipulation

    PubMed Central

    Deinzer, Andrea

    2016-01-01

    To specifically target dendritic cells (DCs) to simultaneously express different therapeutic transgenes for inducing immune responses against tumors, we used a combined promoter system of adenoviral vectors. We selected a 216 bp short Hsp70B′ core promoter induced by a mutated, constitutively active heat shock factor (mHSF) 1 to drive strong gene expression of therapeutic transgenes MelanA, BclxL, and IL-12p70 in HeLa cells, as well as in mature DCs (mDCs). As this involves overexpressing mHSF1, we first evaluated the resulting effects on DCs regarding upregulation of heat shock proteins and maturation markers, toxicity, cytokine profile, and capacity to induce antigen-specific CD8+ T cells. Second, we generated the two-vector-based “modular promoter” system, where one vector contains the mHSF1 under the control of the human CD83 promoter, which is specifically active only in DCs and after maturation. mHSF1, in turn, activates the Hsp70B′ core promotor-driven expression of transgenes MelanA and IL-12p70 in the DC-like cell line XS52 and in human mature and hence immunogenic DCs, but not in tolerogenic immature DCs. These in vitro experiments provide the basis for an in vivo targeting of mature DCs for the expression of multiple transgenes. Therefore, this modular promoter system represents a promising tool for future DC-based immunotherapies in vivo. PMID:27446966

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

    PubMed

    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. Construction of Recombinant Baculoviruses Expressing Infectious Bursal Disease Virus Main Protective Antigen and Their Immune Effects on Chickens.

    PubMed

    Ge, Jingping; An, Qi; Song, Shanshan; Gao, Dongni; Ping, Wenxiang

    2015-01-01

    In order to overcome the limitations of conventional vaccines for infectious bursal disease virus (IBDV), we constructed recombinant dual expression system baculoviruses with VP2 and VP2/4/3, the main protective antigens of IBDV. We compared the immune effects of the baculoviruses in avian cells and detected their control effects on chickens with infectious bursal disease. We used Western blot analysis to measure VP2 protein and VP2/4/3 polyprotein expression in avian cells infected using the Bac-to-Bac baculovirus expression system. The recombinant baculoviruses were used to vaccinate specific pathogen-free chickens, which produced specific protective antibodies and strong cellular immune responses. The results of the virus challenge experiment revealed that the protective efficiency of VP2 and VP2/4/3 virus vaccines were 95.8% and 100%, respectively, both of which were higher than the vaccine group (87.5%), and significantly higher than the control group (50%). The results demonstrated that the immune effect of BV-S-ITRs-VP2/4/3 was superior to that of BV-S-ITRs-VP2. Compared with traditional attenuated vaccine and genetically engineered live vector vaccine, the dual expression viral vector vaccine has good bio-safety. The results of this study provide a foundation for the further development of poultry vaccines, in addition to providing a useful reference for developing non-replicating live vaccines against other viral diseases. PMID:26167907

  10. Eradication of B-ALL using chimeric antigen receptor-expressing T cells targeting the TSLPR oncoprotein.

    PubMed

    Qin, Haiying; Cho, Monica; Haso, Waleed; Zhang, Ling; Tasian, Sarah K; Oo, Htoo Zarni; Negri, Gian Luca; Lin, Yongshun; Zou, Jizhong; Mallon, Barbara S; Maude, Shannon; Teachey, David T; Barrett, David M; Orentas, Rimas J; Daugaard, Mads; Sorensen, Poul H B; Grupp, Stephan A; Fry, Terry J

    2015-07-30

    Adoptive transfer of T cells genetically modified to express chimeric antigen receptors (CARs) targeting the CD19 B cell-associated protein have demonstrated potent activity against relapsed/refractory B-lineage acute lymphoblastic leukemia (B-ALL). Not all patients respond, and CD19-negative relapses have been observed. Overexpression of the thymic stromal lymphopoietin receptor (TSLPR; encoded by CRLF2) occurs in a subset of adults and children with B-ALL and confers a high risk of relapse. Recent data suggest the TSLPR signaling axis is functionally important, suggesting that TSLPR would be an ideal immunotherapeutic target. We constructed short and long CARs targeting TSLPR and tested efficacy against CRLF2-overexpressing B-ALL. Both CARs demonstrated activity in vitro, but only short TSLPR CAR T cells mediated leukemia regression. In vivo activity of the short CAR was also associated with long-term persistence of CAR-expressing T cells. Short TSLPR CAR treatment of mice engrafted with a TSLPR-expressing ALL cell line induced leukemia cytotoxicity with efficacy comparable with that of CD19 CAR T cells. Short TSLPR CAR T cells also eradicated leukemia in 4 xenograft models of human CRLF2-overexpressing ALL. Finally, TSLPR has limited surface expression on normal tissues. TSLPR-targeted CAR T cells thus represent a potent oncoprotein-targeted immunotherapy for high-risk ALL. PMID:26041741

  11. Transcriptional Targeting of Mature Dendritic Cells with Adenoviral Vectors via a Modular Promoter System for Antigen Expression and Functional Manipulation.

    PubMed

    Knippertz, Ilka; Deinzer, Andrea; Dörrie, Jan; Schaft, Niels; Nettelbeck, Dirk M; Steinkasserer, Alexander

    2016-01-01

    To specifically target dendritic cells (DCs) to simultaneously express different therapeutic transgenes for inducing immune responses against tumors, we used a combined promoter system of adenoviral vectors. We selected a 216 bp short Hsp70B' core promoter induced by a mutated, constitutively active heat shock factor (mHSF) 1 to drive strong gene expression of therapeutic transgenes MelanA, BclxL, and IL-12p70 in HeLa cells, as well as in mature DCs (mDCs). As this involves overexpressing mHSF1, we first evaluated the resulting effects on DCs regarding upregulation of heat shock proteins and maturation markers, toxicity, cytokine profile, and capacity to induce antigen-specific CD8(+) T cells. Second, we generated the two-vector-based "modular promoter" system, where one vector contains the mHSF1 under the control of the human CD83 promoter, which is specifically active only in DCs and after maturation. mHSF1, in turn, activates the Hsp70B' core promotor-driven expression of transgenes MelanA and IL-12p70 in the DC-like cell line XS52 and in human mature and hence immunogenic DCs, but not in tolerogenic immature DCs. These in vitro experiments provide the basis for an in vivo targeting of mature DCs for the expression of multiple transgenes. Therefore, this modular promoter system represents a promising tool for future DC-based immunotherapies in vivo. PMID:27446966

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

    SciTech Connect

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

    1988-01-15

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

  13. Construction of Recombinant Baculoviruses Expressing Infectious Bursal Disease Virus Main Protective Antigen and Their Immune Effects on Chickens

    PubMed Central

    Song, Shanshan; Gao, Dongni; Ping, Wenxiang

    2015-01-01

    In order to overcome the limitations of conventional vaccines for infectious bursal disease virus (IBDV), we constructed recombinant dual expression system baculoviruses with VP2 and VP2/4/3, the main protective antigens of IBDV. We compared the immune effects of the baculoviruses in avian cells and detected their control effects on chickens with infectious bursal disease. We used Western blot analysis to measure VP2 protein and VP2/4/3 polyprotein expression in avian cells infected using the Bac-to-Bac baculovirus expression system. The recombinant baculoviruses were used to vaccinate specific pathogen-free chickens, which produced specific protective antibodies and strong cellular immune responses. The results of the virus challenge experiment revealed that the protective efficiency of VP2 and VP2/4/3 virus vaccines were 95.8% and 100%, respectively, both of which were higher than the vaccine group (87.5%), and significantly higher than the control group (50%). The results demonstrated that the immune effect of BV-S-ITRs-VP2/4/3 was superior to that of BV-S-ITRs-VP2. Compared with traditional attenuated vaccine and genetically engineered live vector vaccine, the dual expression viral vector vaccine has good bio-safety. The results of this study provide a foundation for the further development of poultry vaccines, in addition to providing a useful reference for developing non-replicating live vaccines against other viral diseases. PMID:26167907

  14. Targeted expression of SV40 T antigen in the hair follicle of transgenic mice produces an aberrant hair phenotype.

    PubMed

    Keough, R; Powell, B; Rogers, G

    1995-03-01

    Directed expression of SV40 large T antigen (TAg) in transgenic mice can induce tissue-specific tumorigenesis and useful cell lines exhibiting differentiated characteristics can be established from resultant tumor cells. In an attempt to produce an immortalised mouse hair follicle cortical cell line for the study of hair keratin gene control, SV40 TAg expression was targeted to the hair follicles of transgenic mice using a sheep hair gene promoter. Expression of SV40 TAg in the follicle cortex disrupted normal fiber ultrastructure, producing a marked phenotypic effect. Affected hairs were wavy or severely kinked (depending on the severity of the phenotype) producing an appearance ranging from a ruffled coat to a stubble covering the back of the mouse. The transgenic hairs appeared to be weakened at the base of the fibers, leading to premature hair-loss and a thinner pelage, or regions of temporary nudity. No follicle tumors or neoplasia were apparent and immortalisation of cortical cells could not be established in culture. In situ hybridisation studies in the hair follicle using histone H3 as a cell proliferation marker suggested that cell proliferation had ceased prior to commencement of K2.10-TAg expression and was not re-established in the differentiating cortical cells. Hence, TAg was unable to induce cell immortalisation at that stage of cortical cell differentiation. However, transgenic mice developed various other abnormalities including vertebral abnormalities and bladder, liver and intestinal tumors, which resulted in reduced life expectancy. PMID:7542671

  15. Eradication of B-ALL using chimeric antigen receptor–expressing T cells targeting the TSLPR oncoprotein

    PubMed Central

    Qin, Haiying; Cho, Monica; Haso, Waleed; Zhang, Ling; Tasian, Sarah K.; Oo, Htoo Zarni; Negri, Gian Luca; Lin, Yongshun; Zou, Jizhong; Mallon, Barbara S.; Maude, Shannon; Teachey, David T.; Barrett, David M.; Orentas, Rimas J.; Daugaard, Mads; Sorensen, Poul H. B.; Grupp, Stephan A.

    2015-01-01

    Adoptive transfer of T cells genetically modified to express chimeric antigen receptors (CARs) targeting the CD19 B cell–associated protein have demonstrated potent activity against relapsed/refractory B-lineage acute lymphoblastic leukemia (B-ALL). Not all patients respond, and CD19-negative relapses have been observed. Overexpression of the thymic stromal lymphopoietin receptor (TSLPR; encoded by CRLF2) occurs in a subset of adults and children with B-ALL and confers a high risk of relapse. Recent data suggest the TSLPR signaling axis is functionally important, suggesting that TSLPR would be an ideal immunotherapeutic target. We constructed short and long CARs targeting TSLPR and tested efficacy against CRLF2-overexpressing B-ALL. Both CARs demonstrated activity in vitro, but only short TSLPR CAR T cells mediated leukemia regression. In vivo activity of the short CAR was also associated with long-term persistence of CAR-expressing T cells. Short TSLPR CAR treatment of mice engrafted with a TSLPR-expressing ALL cell line induced leukemia cytotoxicity with efficacy comparable with that of CD19 CAR T cells. Short TSLPR CAR T cells also eradicated leukemia in 4 xenograft models of human CRLF2-overexpressing ALL. Finally, TSLPR has limited surface expression on normal tissues. TSLPR-targeted CAR T cells thus represent a potent oncoprotein-targeted immunotherapy for high-risk ALL. PMID:26041741

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

    PubMed Central

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

    2008-01-01

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

  17. Identification of antigenic targets of paraproteins by expression cloning does not support a causal role of chronic antigenic stimulation in the pathogenesis of multiple myeloma and MGUS.

    PubMed

    Preuss, Klaus-Dieter; Held, Gerhard; Kubuschok, Boris; Hung, Chun-Zhu; Malatsidze, Natalia; Wagner, Mathias; Pfreundschuh, Michael

    2007-07-15

    Antigenic targets of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) paraproteins have been suggested to play an important role as growth stimulators in the pathogenesis of these neoplasms. To identify such targets, we screened cDNA libraries from human testis, lung and breast cancer, bovine and porcine muscle and wheat germ for reactivity with paraproteins in the sera from 115 patients with MGUS and MM. Of >6 x 10(8) paraprotein-antigen interactions screened, an IgA paraprotein from a female patient bound to sperm-specific cylicin-2, and 3 IgG paraproteins bound to tripeptidyl-peptidase-II (TPP-2), insulin-like growth-factor binding-protein-2 (IGFBP-2) and porcine kinesin. Specificity was confirmed by reverse Western blots using recombinant antigens. The broad spectrum of auto-, allo- and heteroantigens as targets of human paraproteins in patients without signs of chronic antigenic stimulation renders a causal role of the antigenic stimulus in the pathogenesis of MGUS and MM unlikely. PMID:17373665

  18. Antibodies generated against conserved antigens expressed by bacteria and allergen-bearing fungi suppress airway disease.

    PubMed

    Kin, Nicholas W; Stefanov, Emily K; Dizon, Brian L P; Kearney, John F

    2012-09-01

    There has been a sharp rise in allergic asthma and asthma-related deaths in the developed world, in contrast to many childhood illnesses that have been reduced or eliminated. The hygiene hypothesis proposes that excessively sanitary conditions early in life result in autoimmune and allergic phenomena because of a failure of the immune system to receive proper microbial stimulation during development. We demonstrate that Abs generated against conserved bacterial polysaccharides are reactive with and dampen the immune response against chitin and Aspergillus fumigatus. A reduction in Ag uptake, cell influx, cell activation, and cytokine production occurred in the presence of anti-polysaccharide Abs, resulting in a striking decrease in the severity of allergic airway disease in mice. Overall, our results suggest that Ag exposure--derived from environmental sources, self-antigens, or vaccination--during the neonatal period has dramatic effects on the adult Ab response and modifies the development of allergic airway disease. PMID:22837487

  19. Cloning and expression of an antigenic domain of a major surface protein (Nc-p43) of Neospora caninum.

    PubMed

    Lima, Manoel S da C; Andreotti, Renato; Caetano, Alexandre R; Paiva, Fernando; Matos, Maria de Fátima C

    2007-01-01

    Neospora caninum is an obligate intracellular protozoan that can infect domestic and wild canids, as well as ruminants and equines. It was described in 1988 as causing neuromuscular alterations and death in dogs. Recently, N. caninum has been the focus of considerable attention for its large impact on the dairy industry, given the economic losses related to breeding failures and to a decrease in productivity. ELISA diagnosis of neosporosis has not been widely used in Brazil, mostly because of the assay's cost, and thus the distribution of the disease in the country is not well known. In order to evaluate its ability to react with sera from infected animals from the state of Mato Grosso do Sul, an antigenic determinant domain of a major surface protein (Nc-p43) was produced. The antigenic domain, located in the distal 2/3 region of the C-terminus, was amplified by polymerase chain reaction. The DNA fragments were cloned into pet100/D-TOPO vectors. The recombinant plasmids were transformed into Escherichia coli of the BL21 Star (DE3) strain and induced to express the fused fragment of Nc-p43 as a 29-kDa protein that, when assayed with bovine Neospora-positive serum from a regional sample, was sensitive for identification by immunoblotting. This Nc-p43 fragment may be of use in additional studies targeted at diagnosing N. caninum infection and at evaluating the immunoprotection conferred by the protein fragment to animal hosts. PMID:17706005

  20. The cattle tick antigen, Bm95, expressed in Pichia pastoris contains short chains of N- and O-glycans.

    PubMed

    González, Luis J; Cremata, José A; Guanche, Yazmín; Ramos, Yassel; Triguero, Ada; Cabrera, Gleysin; Montesino, Raquel; Huerta, Vivian; Pons, Tirso; Boué, Oscar; Farnós, Omar; Rodríguez, Manuel

    2004-12-15

    Bm95 is an antigen isolated from Boophilus microplus strains with low susceptibility to antibodies developed in cattle vaccinated with the recombinant Bm86 antigen (Gavac, HeberBiotec S.A., Cuba). It is a Bm86-like surface protein, which by similarity contains seven EGF-like domains and a lipid-binding GPI-anchor site at the C-terminal region. The primary structure of the recombinant (rBm95) protein expressed in Pichia pastoris was completely verified by LC/MS. The four potential glycosylation sites (Asn 122, 163, 329, and 363) are glycosylated partially with short N-glycans, from Man(5)GlcNAc(2) to Man(9)GlcNAc(2) of which, Man(8-9)GlcNAc(2) were the most abundant. O-Glycopeptides are distributed mostly towards the protein N-terminus. While the first N-glycosylated site (Asn(122)) is located between EGF-like domains 2 and 3, where the O-glycopeptides were found, two other N-glycosylated sites (Asn(329) and Asn(363)) are located between EGF-like domains 5 and 6, a region devoid of O-glycosylated Ser or Thr. PMID:15542059

  1. Expression of immunoglobulin receptors with distinctive features indicating antigen selection by marginal zone B cells from human spleen.

    PubMed

    Colombo, Monica; Cutrona, Giovanna; Reverberi, Daniele; Bruno, Silvia; Ghiotto, Fabio; Tenca, Claudya; Stamatopoulos, Kostas; Hadzidimitriou, Anastasia; Ceccarelli, Jenny; Salvi, Sandra; Boccardo, Simona; Calevo, Maria Grazia; De Santanna, Amleto; Truini, Mauro; Fais, Franco; Ferrarini, Manlio

    2013-01-01

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