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Sample records for surface antigen levels

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

    PubMed Central

    Li, Sai; Rissanen, Ilona; Zeltina, Antra; Hepojoki, Jussi; Raghwani, Jayna; Harlos, Karl; Pybus, Oliver G.; Huiskonen, Juha T.; Bowden, Thomas A.

    2016-01-01

    Summary Hantaviruses, a geographically diverse group of zoonotic pathogens, initiate cell infection through the concerted action of Gn and Gc viral surface glycoproteins. Here, we describe the high-resolution crystal structure of the antigenic ectodomain of Gn from Puumala hantavirus (PUUV), a causative agent of hemorrhagic fever with renal syndrome. Fitting of PUUV Gn into an electron cryomicroscopy reconstruction of intact Gn-Gc spike complexes from the closely related but non-pathogenic Tula hantavirus localized Gn tetramers to the membrane-distal surface of the virion. The accuracy of the fitting was corroborated by epitope mapping and genetic analysis of available PUUV sequences. Interestingly, Gn exhibits greater non-synonymous sequence diversity than the less accessible Gc, supporting a role of the host humoral immune response in exerting selective pressure on the virus surface. The fold of PUUV Gn is likely to be widely conserved across hantaviruses. PMID:27117403

  2. Kinetics of Hepatitis B Surface Antigen Level in Chronic Hepatitis B Patients who Achieved Hepatitis B Surface Antigen Loss during Pegylated Interferon Alpha-2a Treatment

    PubMed Central

    Li, Ming-Hui; Zhang, Lu; Qu, Xiao-Jing; Lu, Yao; Shen, Ge; Wu, Shu-Ling; Chang, Min; Liu, Ru-Yu; Hu, Lei-Ping; Li, Zhen-Zhen; Hua, Wen-Hao; Song, Shu-Jing; Xie, Yao

    2017-01-01

    Background: Hepatitis B surface antigen (HBsAg) loss/seroconversion is considered to be the ideal endpoint of antiviral therapy and the ultimate treatment goal in chronic hepatitis B (CHB). This study aimed to assess the patterns of HBsAg kinetics in CHB patients who achieved HBsAg loss during the treatment of pegylated interferon (PEG-IFN) α-2a. Methods: A total of 150 patients were enrolled, composing of 83 hepatitis B envelope antigen (HBeAg)-positive and 67 HBeAg-negative patients. Patients were treated with PEG-IFN α-2a180 μg/week until HBsAg loss/seroconversion was achieved, which occurred within 96 weeks. Serum hepatitis B virus deoxyribonucleic acid and serological indicators (HBsAg, anti-HBs, HBeAg, and anti-HBe) were determined before and every 3 months during PEG-IFN α-2a treatment. Biochemical markers and peripheral blood neutrophil and platelet counts were tested every 1–3 months. Results: Baseline HBsAg levels were 2.5 ± 1.3 log IU/ml, and decreased rapidly at 12 and 24 weeks by 48.3% and 88.3%, respectively. The mean time to HBsAg loss was 54.2 ± 30.4 weeks, though most patients needed extended treatment and 30.0% of HBsAg loss occurred during 72–96 weeks. Baseline HBsAg levels were significantly higher in HBeAg-positive patients (2.9 ± 1.1 log IU/ml) compared with HBeAg-negative patients (2.0 ± 1.3 log IU/ml; t = 4.733, P < 0.001), but the HBsAg kinetics were similar. Patients who achieved HBsAg loss within 48 weeks had significantly lower baseline HBsAg levels and had more rapid decline of HBsAg at 12 weeks compared to patients who needed extended treatment to achieve HBsAg loss. Conclusions: Patients with lower baseline HBsAg levels and more rapid decline during early treatment with PEG-IFN are more likely to achieve HBsAg loss during 96 weeks of treatment, and extended therapy longer than 48 weeks may be required to achieve HBsAg loss. PMID:28229987

  3. First evaluation of the serum level of anti-hepatitis B surface antigen after vaccination in Libya.

    PubMed

    Madour, A; Alkout, A; Vanin, S

    2013-12-01

    The hepatitis B virus (HBV) vaccination schedule in Libya follows international recommendations (1st dose at birth, 2nd after 1 month and 3rd after 6 months). This research aimed to evaluate the long-term protection of the HBV immunization programme in Tripoli and to determine the best age to administer booster doses. Serum levels of hepatitis B surface antigen were determined in 277 randomly selected children aged 1-12 years. The response to HBV vaccine in 1-3-year-olds was 93.2%, but this declined with age and at 7-9 years after initial vaccination only 53.1% of children had protective titres (> or = 10 mIU/mL). No significant differences between males and females in antibody persistence or response to vaccine were observed. We recommend continuing the HBV vaccination programme and that a booster dose be given to 6-year-old children to ensure maximum protection during the period of school entry and beyond.

  4. Levels of antibodies to Plasmodium falciparum sporozoite surface antigens reflect malaria transmission rates and are persistent in the absence of reinfection.

    PubMed Central

    Druilhe, P; Pradier, O; Marc, J P; Miltgen, F; Mazier, D; Parent, G

    1986-01-01

    Antibodies reacting with Plasmodium falciparum sporozoite surface antigens were measured by an immunofluorescence assay using wet preparations of sporozoites attached to poly-L-lysine-treated glass slides, a procedure which was found to be more specific than one using glutaraldehyde-treated and dried preparations. Subjects recovering from a first attack were found to be negative. In two African villages which differed in the level at which mosquitoes transmit the disease (1 and 100 infective bites per year and per individual), both the prevalence by age group and the levels of anti-sporozoite antibodies differed markedly, as follows. In the low-transmission area, these antibodies were not detected in subjects aged 2 to 10 years; thereafter, prevalence increased gradually with the age of the subject and reached 90% in subjects aged 50 to 80 years. In the high-transmission area, all of the subjects studied, including the younger ones, were positive. Anti-sporozoite antibody levels were independent of the levels of antibodies directed against blood stages. On average, the mean antibody titers were equal to 1/16 in the first village and 1/1,650 in the second one. These results suggest that stage-specific antibodies reflect the cumulative number of sporozoites inoculated in humans by mosquitoes and may therefore have useful epidemiological applications. In addition, the presence of stage-specific antibodies in the sera of African adults collected at different times after departure from the endemic area indicates that they may last for several years. During the course of this study, we observed a heterogeneity of immunofluorescence labeling in parasite populations prepared from mosquito salivary glands. This raises the question of possible qualitative or quantitative antigenic differences or both between one sporozoite and the other. PMID:3525412

  5. Dose-response association between hepatitis B surface antigen levels and liver cancer risk in Chinese men and women.

    PubMed

    Yang, Yang; Gao, Jing; Li, Hong-Lan; Zheng, Wei; Yang, Gong; Zhang, Wei; Ma, Xiao; Tan, Yu-Ting; Rothman, Nathaniel; Gao, Yu-Tang; Chow, Wong-Ho; Shu, Xiao-Ou; Xiang, Yong-Bing

    2016-07-15

    We aimed at evaluating the risk of liver cancer in different levels of HBsAg among Chinese men and women. We carried out a nested case-control study including 363 cases and 3,511 controls in two population-based cohorts in Shanghai. Plasma samples collected at enrollment were quantified for HBsAg levels using the Architect QT assay. Conditional logistic regression was performed to estimate the odds ratios (ORs) and 95% confidence intervals (95% CIs) for liver cancer, with adjustment for potential confounders. HBsAg was detected in 6.29% of control subjects overall (7.02% in men and 4.98% in women). HBsAg levels were positively associated with liver cancer risk in a dose-response manner (ptrend  < 0.001). Such association showed a significant gender disparity. With increasing levels of HBsAg, liver cancer risks rose more steeply in men than in women. In men, the adjusted ORs increased from 7.27 (95% CI: 3.49-15.15) at the lowest detectable level of HBsAg (5-9 IU/ml) to 7.16 (95% CI: 3.21-15.96), 34.30 (95% CI: 16.94-69.44), and 47.33 (95% CI: 23.50-95.34) at the highest level of HBsAg (≥1,000 IU/ml) compared to those negative for HBsAg. The corresponding ORs were much lower for women, from 1.37 (95% CI: 0.25-7.47), 3.81 (95% CI: 1.09-13.28), 7.36 (95% CI: 2.41-22.46) and 16.86 (95% CI: 7.24-39.27), respectively. HBsAg quantification has potential to distinguish individuals at different risks of liver cancer. Men with the lowest detectable level of HBsAg should still pay attention to their liver cancer risks, but those with a higher level may be given a higher priority in future liver cancer surveillance program.

  6. 21 CFR 660.40 - Hepatitis B Surface Antigen.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Hepatitis B Surface Antigen. 660.40 Section 660.40...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.40 Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this...

  7. 21 CFR 660.40 - Hepatitis B Surface Antigen.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Hepatitis B Surface Antigen. 660.40 Section 660.40...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.40 Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this...

  8. 21 CFR 660.40 - Hepatitis B Surface Antigen.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Hepatitis B Surface Antigen. 660.40 Section 660.40...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.40 Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this...

  9. 21 CFR 660.40 - Hepatitis B Surface Antigen.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Hepatitis B Surface Antigen. 660.40 Section 660.40...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.40 Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this...

  10. 21 CFR 660.40 - Hepatitis B Surface Antigen.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  11. K99 surface antigen of Escherichia coli: antigenic characterization.

    PubMed Central

    Isaacson, R E

    1978-01-01

    K99 prepared by acid precipitation hemagglutinated guinea pig erythrocytes, whereas K99 prepared by chromatography on diethylaminoethyl-Sephadex did not. K99 purified by either procedure hemagglutinated horse erythrocytes. K99 prepared by acid precipitation contained a second antigen not presnet in the K99 prepared by chromatography on diethylaminoethyl-Sephadex. This antigen could be detected by immunoprecipitation with some, but not all, sera prepared against K99-positive Escherichia coli strains. It was assumed that this second antigen is not K99 and is responsible for the guinea pig erythrocyte hemagglutination reaction. Furthermore, the second antigen has an isoelectric point of 4.2, which has been reported by Morris and co-workers to be the isoelectric point of K99. Images PMID:83300

  12. Physiological response of Pichia pastoris GS115 to methanol-induced high level production of the Hepatitis B surface antigen: catabolic adaptation, stress responses, and autophagic processes

    PubMed Central

    2012-01-01

    Background Pichia pastoris is an established eukaryotic host for the production of recombinant proteins. Most often, protein production is under the control of the strong methanol-inducible aox1 promoter. However, detailed information about the physiological alterations in P. pastoris accompanying the shift from growth on glycerol to methanol-induced protein production under industrial relevant conditions is missing. Here, we provide an analysis of the physiological response of P. pastoris GS115 to methanol-induced high-level production of the Hepatitis B virus surface antigen (HBsAg). High product titers and the retention of the protein in the endoplasmic reticulum (ER) are supposedly of major impact on the host physiology. For a more detailed understanding of the cellular response to methanol-induced HBsAg production, the time-dependent changes in the yeast proteome and ultrastructural cell morphology were analyzed during the production process. Results The shift from growth on glycerol to growth and HBsAg production on methanol was accompanied by a drastic change in the yeast proteome. In particular, enzymes from the methanol dissimilation pathway started to dominate the proteome while enzymes from the methanol assimilation pathway, e.g. the transketolase DAS1, increased only moderately. The majority of methanol was metabolized via the energy generating dissimilatory pathway leading to a corresponding increase in mitochondrial size and numbers. The methanol-metabolism related generation of reactive oxygen species induced a pronounced oxidative stress response (e.g. strong increase of the peroxiredoxin PMP20). Moreover, the accumulation of HBsAg in the ER resulted in the induction of the unfolded protein response (e.g. strong increase of the ER-resident disulfide isomerase, PDI) and the ER associated degradation (ERAD) pathway (e.g. increase of two cytosolic chaperones and members of the AAA ATPase superfamily) indicating that potential degradation of HBsAg could

  13. Cell Surface Expression Level Variation between Two Common Human Leukocyte Antigen Alleles, HLA-A2 and HLA-B8, Is Dependent on the Structure of the C Terminal Part of the Alpha 2 and the Alpha 3 Domains

    PubMed Central

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

    2015-01-01

    Constitutive cell surface expression of Human Leukocyte Antigen (HLA) class I antigens vary extremely from tissue to tissue and individual antigens may differ widely in expression levels. Down-regulation of class I expression is a known immune evasive mechanism used by cancer cells and viruses. Moreover, recent observations suggest that even minor differences in expression levels may influence the course of viral infections and the frequency of complications to stem cell transplantation. We have shown that some human multipotent stem cells have high expression of HLA-A while HLA-B is only weakly expressed, and demonstrate here that this is also the case for the human embryonic kidney cell line HEK293T. Using quantitative flow cytometry and quantitative polymerase chain reaction we found expression levels of endogenous HLA-A3 (median 71,204 molecules per cell) 9.2-fold higher than the expression of-B7 (P = 0.002). Transfection experiments with full-length HLA-A2 and -B8 encoding plasmids confirmed this (54,031 molecules per cell vs. 2,466, respectively, P = 0.001) independently of transcript levels suggesting a post-transcriptional regulation. Using chimeric constructs we found that the cytoplasmic tail and the transmembrane region had no impact on the differential cell surface expression. In contrast, ~65% of the difference could be mapped to the six C-terminal amino acids of the alpha 2 domain and the alpha 3 domain (amino acids 176–284), i.e. amino acids not previously shown to be of importance for differential expression levels of HLA class I molecules. We suggest that the differential cell surface expression of two common HLA-A and–B alleles is regulated by a post-translational mechanism that may involve hitherto unrecognized molecules. PMID:26258424

  14. Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of Hepatitis B surface antigen

    PubMed Central

    Gurramkonda, Chandrasekhar; Adnan, Ahmad; Gäbel, Thomas; Lünsdorf, Heinrich; Ross, Anton; Nemani, Satish Kumar; Swaminathan, Sathyamangalam; Khanna, Navin; Rinas, Ursula

    2009-01-01

    Background Hepatitis B is a serious global public health concern. Though a safe and efficacious recombinant vaccine is available, its use in several resource-poor countries is limited by cost. We have investigated the production of Hepatitis B virus surface antigen (HBsAg) using the yeast Pichia pastoris GS115 by inserting the HBsAg gene into the alcohol oxidase 1 locus. Results Large-scale production was optimized by developing a simple fed-batch process leading to enhanced product titers. Cells were first grown rapidly to high-cell density in a batch process using a simple defined medium with low salt and high glycerol concentrations. Induction of recombinant product synthesis was carried out using rather drastic conditions, namely through the addition of methanol to a final concentration of 6 g L-1. This methanol concentration was kept constant for the remainder of the cultivation through continuous methanol feeding based on the on-line signal of a flame ionization detector employed as methanol analyzer in the off-gas stream. Using this robust feeding protocol, maximum concentrations of ~7 grams HBsAg per liter culture broth were obtained. The amount of soluble HBsAg, competent for assembly into characteristic virus-like particles (VLPs), an attribute critical to its immunogenicity and efficacy as a hepatitis B vaccine, reached 2.3 grams per liter of culture broth. Conclusion In comparison to the highest yields reported so far, our simple cultivation process resulted in an ~7 fold enhancement in total HBsAg production with more than 30% of soluble protein competent for assembly into VLPs. This work opens up the possibility of significantly reducing the cost of vaccine production with implications for expanding hepatitis B vaccination in resource-poor countries. PMID:19208244

  15. Serum immunoglobulin levels in Australia antigen positive and Australia antigen negative hepatitis

    PubMed Central

    Peters, C. J.; Johnson, K. M.

    1972-01-01

    Ig levels were determined by radial immunodiffusion in uncomplicated cases of acute hepatitis with or without Australia antigenaemia. Initial sera from Australia antigen negative cases showed a striking elevation in IgM levels when compared to Australia antigen positive cases (6·5 versus 1·9 mg/ml). None of twenty-four Australia antigen positive cases exceeded 3 mg/ml IgM, and only 3/58 Australia antigen negative cases exhibited values below 3 mg/ml. Intial sera from Australia antigen positive and Australia antigen negative subjects did not differ in concentration of IgG, IgA, or IgD. Serial determinations of IgG revealed a transient fall in patients with Australia antigen positive hepatitis, and a rise in Australia antigen negative cases. Asymptomatic, Australia antigen positive, Guaymi Indian subjects were compared to matched Australia antigen negative controls from the same indigenous group and no differences in the concentration of IgG, IgM, IgA or IgD were found, although elevations of IgG and IgM were common in both groups. No evidence of abnormal proteins was found when sera were tested by cellulose acetate electrophoresis or by immunoelectrophoresis versus immunoglobulin-specific antisera. Ultracentrifugal analysis failed to detect `7S' IgM. PMID:4625396

  16. Hepatitis B surface antigen in blood donors: further observations.

    PubMed

    Szmuness, W; Hirsch, R L; Prince, A M; Levine, R W; Harley, E J; Ikram, H

    1975-02-01

    A survey of 128,000 volunteer blood donors from the Greater New York metropolitan area revealed that first-time male donors were positive for hepatitis B surface antigen (HBsAg) 2.5 times more frequently than were first-time female donors; Negroes and Mongols were positive four to 20 times more frequently than Caucasians. The ratio of ad to ay seemed to be higher in non-Caucasian antigen carriers than in Caucasian carriers. Among both Caucasians and non-Caucasians the rate of positivity declined after the age of 50. An excess prevalence of HBsAg was observed in donors with the lowest level of education and in those with the highest level. HBsAg was detectable nine times less frequently among repeat donors than among first-time donors (0.2 vs.1.90 per 1,000). Detection of HBsAg was unrelated to ABO-Rh blood groups. Several mechanisms for these wide variations of antigen detection are possible.

  17. Expression of hepatitis B surface antigen in transgenic banana plants.

    PubMed

    Kumar, G B Sunil; Ganapathi, T R; Revathi, C J; Srinivas, L; Bapat, V A

    2005-10-01

    Embryogenic cells of bananan cv. Rasthali (AAB) have been transformed with the 's' gene of hepatitis B surface antigen (HBsAg) using Agrobacterium mediated transformation. Four different expression cassettes (pHBS, pHER, pEFEHBS and pEFEHER) were utilized to optimize the expression of HBsAg in banana. The transgenic nature of the plants and expression of the antigen was confirmed by PCR, Southern hybridization and reverse transcription (RT)-PCR. The expression levels of the antigen in the plants grown under in vitro conditions as well as the green house hardened plants were estimated by ELISA for all the four constructs. Maximum expression level of 38 ng/g F.W. of leaves was noted in plants transformed with pEFEHBS grown under in vitro conditions, whereas pHER transformed plants grown in the green house showed the maximum expression level of 19.92 ng/g F.W. of leaves. Higher monoclonal antibody binding of 67.87% of the antigen was observed when it was expressed with a C-terminal ER retention signal. The buoyant density in CsCl of HBsAg derived from transgenic banana leaves was determined and found to be 1.146 g/ml. HBsAg obtained from transgenic banana plants is similar to human serum derived one in buoyant density properties. The transgenic plants were grown up to maturity in the green house and the expression of HBsAg in the fruits was confirmed by RT-PCR. These transgenic plants were multiplied under in vitro using floral apex cultures. Attempts were also made to enhance the expression of HBsAg in the leaves of transgenic banana plants by wounding and/or treatment with plant growth regulators. This is the first report on the expression of HBsAg in transgenic banana fruits.

  18. Genetic and antigenic diversity of the surface protective antigen proteins of Erysipelothrix rhusiopathiae.

    PubMed

    To, Ho; Nagai, Shinya

    2007-07-01

    The surface protective antigen (Spa) protein of Erysipelothrix rhusiopathiae has been shown to be highly immunogenic and is a potential candidate for a new vaccine against erysipelas. In this study, we cloned and sequenced spa genes from all E. rhusiopathiae serovar reference strains as well as from a serovar 18 strain which was not classified as any species in the genus Erysipelothrix. Sequence analysis revealed that the Spa proteins could be classified into three molecular species, including SpaA, which was previously found in serovars 1a and 2, and the newly designated SpaB and SpaC proteins. The SpaA protein is produced by E. rhusiopathiae serovars 1a, 1b, 2, 5, 8, 9, 12, 15, 16, 17, and N, the SpaB protein is produced by E. rhusiopathiae serovars 4, 6, 11, 19, and 21, and the SpaC protein is produced only by serovar 18. The amino acid sequence similarity was high among members of each Spa type (96 to 99%) but low between different Spa types ( approximately 60%). The greatest diversity in Spa proteins was found in the N-terminal half of the molecule (50 to 57% similarity), which was shown to be involved in immunoprotection. Coinciding with this, immunoblot analysis revealed that rabbit antisera specific to each Spa reacted strongly with the homologous Spa protein but weakly with heterologous Spa proteins. A mouse cross-protection study showed that the three recombinant Spa (rSpa) proteins elicited complete protection against challenge with homologous strains but that the level of protection against challenge with heterologous strains varied depending on the rSpa protein used for immunization. Our study is the first to demonstrate sequence and antigenic diversity in Spa proteins and to indicate that rSpaC may be the most promising antigen for use as a vaccine component because of its broad cross-protectiveness.

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

    PubMed

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

    2006-01-01

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

  20. 21 CFR 660.1 - Antibody to Hepatitis B Surface Antigen.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Antibody to Hepatitis B Surface Antigen. 660.1... Hepatitis B Surface Antigen § 660.1 Antibody to Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product shall be Antibody to Hepatitis B Surface Antigen. The product...

  1. 21 CFR 660.1 - Antibody to Hepatitis B Surface Antigen.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Antibody to Hepatitis B Surface Antigen. 660.1... Hepatitis B Surface Antigen § 660.1 Antibody to Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product shall be Antibody to Hepatitis B Surface Antigen. The product...

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

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Antibody to Hepatitis B Surface Antigen. 660.1... Hepatitis B Surface Antigen § 660.1 Antibody to Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product shall be Antibody to Hepatitis B Surface Antigen. The product...

  3. 21 CFR 660.1 - Antibody to Hepatitis B Surface Antigen.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Antibody to Hepatitis B Surface Antigen. 660.1... Hepatitis B Surface Antigen § 660.1 Antibody to Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product shall be Antibody to Hepatitis B Surface Antigen. The product...

  4. 21 CFR 660.1 - Antibody to Hepatitis B Surface Antigen.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  5. The CD24 surface antigen in neural development and disease.

    PubMed

    Gilliam, Daniel T; Menon, Vishal; Bretz, Niko P; Pruszak, Jan

    2017-03-01

    A cell's surface molecular signature enables its reciprocal interactions with the associated microenvironments in development, tissue homeostasis and pathological processes. The CD24 surface antigen (heat-stable antigen, nectadrin; small cell lung cancer antigen cluster-4) represents a prime example of a neural surface molecule that has long been known, but whose diverse molecular functions in intercellular communication we have only begun to unravel. Here, we briefly summarize the molecular fundamentals of CD24 structure and provide a comprehensive review of CD24 expression and functional studies in mammalian neural developmental systems and disease models (rodent, human). Striving for an integrated view of the intracellular signaling processes involved, we discuss the most pertinent routes of CD24-mediated signaling pathways and functional networks in neurobiology (neural migration, neurite extension, neurogenesis) and pathology (tumorigenesis, multiple sclerosis).

  6. Prevalence of hepatitis B surface antigen, hepatitis B e antigen and antibody, and antigen subtypes in atomic bomb survivors

    SciTech Connect

    Neriishi, K.; Kodama, K.; Akiba, S. |

    1995-11-01

    On the basis of previous studies showing an association between hepatitis B surface antigen (HBsAg) positivity and radiation exposure in atomic bomb (A-bomb) survivors, we investigated further the active state of hepatitis B virus (HBV) infection by incorporating tests of hepatitis B e antigen (HBeAg) and hepatitis B e antibody (anti-HBe) and HBsAg subtypes into our biennial health examinations. Among 6548 A-bomb survivors for whom HBsAg was assayed between July 1979 and July 1981, 129 persons were HBsAg positive. HBeAg and anti-HBe were measured in 104 of these persons and subtypes of HBsAg in 98 persons. Among those exposed to radiation (average liver dose 0.58 Sv), the odds ratio of HBsAg positivity tended to increase with radiation dose (P for trend = 0.024). The P values for association between the prevalence of HB e antigen and radiation dose were 0.094 and 0.17, respectively. The HB antigen subtype adr was predominant over other subtypes in both Hiroshima and Nagasaki, but the distribution of subtypes did not seem to differ in relation to radiation dose. These results suggested that A-bomb survivors remain in active state of HBV infection and that the mechanism(s) of seroconversion may be impaired. 29 refs., 6 tabs.

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

    PubMed

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

    2016-07-01

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

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

    NASA Astrophysics Data System (ADS)

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

    1980-02-01

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

  9. Prevalence of Serologic Hepatitis B Markers in Blood Donors From Puebla, Mexico: The Association of Relatively High Levels of Anti-Core Antibodies With the Detection of Surface Antigen and Genomic DNA

    PubMed Central

    Sosa-Jurado, Francisca; Hilda Rosas-Murrieta, Nora; Guzman-Flores, Belinda; Perez Zempoaltecalt, Cintia; Patricia Sanchez Torres, Ana; Ramirez Rosete, Leticia; Bernal-Soto, Maribel; Marquez-Dominguez, Luis; Melendez-Mena, Daniel; Angel Mendoza Torres, Miguel; Teresa Lopez Delgado, Maria; Reyes-Leyva, Julio; Vallejo-Ruiz, Veronica; Santos-Lopez, Gerardo

    2016-01-01

    Background The hepatitis B virus (HBV) causes chronic hepatitis, hepatic cirrhosis, and hepatocellular carcinoma. Surface antigen (HBsAg) detection is a definitive test that can confirm HBV infection, while the presence of antibodies against the core protein (anti-HBc) suggests either a previous or ongoing infection or occult hepatitis B infection (OBI). Objectives The aim of the present study was to determine the prevalence of anti-HBc and HBsAg in blood donors. Further, the study aimed to estimate the anti-HBc level at which HBV DNA is detected in putative OBI cases, as well as to search for mutations in the “a” determinant associated with the non-detection of HBsAg in serum. Patients and Methods We conducted a cross-sectional study from 2003–2009. The study included 120,552 blood donors from the state of Puebla, Mexico. Different commercial systems based on microparticles (enzymatic (MEIA) or chemiluminescent (CMIA)) were used to determine the HBsAg and anti-HBc levels. For the detection of HBV DNA, a nested polymerase chain reaction (nested PCR) was used and the genotypes were determined using Sanger sequencing. Results Of the 120,552 blood donors, 1437 (1.19%, 95% CI: 1.12 - 1.26) were reactive to anti-HBc, while 82 (0.066%, 95% CI: 0.053 - 0.079) were reactive to HBsAg. Some 156 plasma samples collected in 2009 from anti-HBc-positive/HBsAg-negative blood donors were submitted for HBV DNA detection in a search for probable OBI. Viral DNA was detected in 27/156 (17.3%, 95% CI: 11.5 - 23.1). Our results show an association between HBV DNA or HBsAg and anti-HBc S/CO levels ≥ 4.0. All DNA samples were identified as genotype H and some “a” determinant mutations were identified, although none corresponded to mutations previously reported to hinder the detection of HBsAg by commercial immunoassays. Conclusions We observed that as the anti-HBc levels increase, there is a higher prevalence of the viral protein HBsAg in blood donors. Samples testing positive

  10. CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

    PubMed Central

    Alonso-Camino, Vanesa; Sánchez-Martín, David; Compte, Marta; Nuñez-Prado, Natalia; Diaz, Rosa M; Vile, Richard; Alvarez-Vallina, Luis

    2013-01-01

    A human single-chain variable fragment (scFv) antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs). The repertoire was fused to a first-generation T cell receptor ζ (TCRζ)-based chimeric antigen receptor (CAR). We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2) bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR) and the selection context (cell synapse), which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells. PMID:23695536

  11. Differences in carcinoembryonic antigen levels between colon and rectal cancer.

    PubMed

    Ding, Yunlong; Xuan, Weibo; Chen, Chunlin; Chen, Zhe; Yang, Ziyi; Zuo, Yunfei; Ren, Shuangyi

    2014-07-01

    The aim of the present study was to investigate the levels of the serum tumor biomarker carcinoembryonic antigen (CEA) in patients with carcinoma of the colon and rectum in different clinical stages. Colorectal cancer (CRC) is one of the most commonly diagnosed types of cancer worldwide and previous studies have reported rapidly updated therapeutic regimes. While the majority of studies focus on CRC as a single entity, certain studies distinguish colon cancer (CC) from rectal cancer (RC), as there is a hypothesis stating that CC and RC are two naturally different entities. CEA is reported to be an important tumor-associated antigen overexpressed in CRC, which is routinely detected as a significant indicator of CRC. Our study aimed to identify potential differences in the expression of CEA between CC and RC, which may, to some degree, reflect the natural differences between the two. We investigated 240 CRC cases between July, 2010 and December, 2012 from The First and Second Affiliated Hospitals of Dalian Medical University, including 117 CC and 123 RC patients with tumors classified by Duke's staging as A-D. The serum CEA level was measured preoperatively by radioimmunoassays as a routinely used auxiliary indicator. The expression of CEA differed between CC and RC, with the former exhibiting variation among the four stages, whereas no variation was observed in RC. In addition, there were differences between CC and RC regarding the CEA level in stage C and D. Furthermore, the CEA level in stage C of CC was significantly lower compared to that in any other stage. In conclusion, the intrinsic distribution of the CEA level between CC and RC suggests that CC and RC may be two naturally different entities; the significantly low CEA level in stage C of CC indicates that stage C may be crucial in the evolution of CC.

  12. Surface proteins, ERAD and antigenic variation in Trypanosoma brucei.

    PubMed

    Tiengwe, Calvin; Muratore, Katherine A; Bangs, James D

    2016-11-01

    Variant surface glycoprotein (VSG) is central to antigenic variation in African trypanosomes. Although much prior work documents that VSG is efficiently synthesized and exported to the cell surface, it was recently claimed that 2-3 fold more is synthesized than required, the excess being eliminated by ER-Associated Degradation (ERAD) (Field et al., ). We now reinvestigate VSG turnover and find no evidence for rapid degradation, consistent with a model whereby VSG synthesis is precisely regulated to match requirements for a functional surface coat on each daughter cell. However, using a mutated version of the ESAG7 subunit of the transferrin receptor (E7:Ty) we confirm functional ERAD in trypanosomes. E7:Ty fails to assemble into transferrin receptors and accumulates in the ER, consistent with retention of misfolded protein, and its turnover is selectively rescued by the proteasomal inhibitor MG132. We also show that ER accumulation of E7:Ty does not induce an unfolded protein response. These data, along with the presence of ERAD orthologues in the Trypanosoma brucei genome, confirm ERAD in trypanosomes. We discuss scenarios in which ERAD could be critical to bloodstream parasites, and how these may have contributed to the evolution of antigenic variation in trypanosomes.

  13. Hepatitis B surface antigen in blood donors. An epidemiologic study.

    PubMed

    Jayaprakash, P A; Shanmugam, J; Hariprasad, D

    1983-01-01

    Of 8085 volunteer donors attending the blood bank at SCTIMST screened for hepatitis B surface antigen (HBsAg) carrier state by counterimmunoelectrophoresis, 103 (1.27%) were HBsAg positive. The personal data of donors showed a higher rate of HBsAg among men than women and in the age group of 21 to 30 years than in the other age groups. A significantly higher rate was noted among donors belonging to the lower socioeconomic group (p less than 0.05).

  14. Complex regulation of transcription from the hepatitis B virus major surface antigen promoter in human hepatoma cell lines.

    PubMed Central

    Raney, A K; Milich, D R; McLachlan, A

    1991-01-01

    A detailed mutational analysis of the regulatory DNA sequence elements that control expression of the hepatitis B virus major surface antigen gene was performed in the human hepatoma cell lines HepG2.1 and Huh7, using transient transfection assays. Seven regions (A to G) of the major surface antigen promoter located within 200 nucleotides of the RNA initiation site have been identified which influence the level of transcription from this promoter. The three distal regions (A to C), located between -188 and -68, appear to possess a level of redundancy in their ability to influence the transcriptional activity from the major surface antigen promoter. The simultaneous deletion of regions A, B, and C resulted in an approximately fourfold reduction in transcription from the major surface antigen promoter. Region D, located between -67 and -49, is an essential element of the major surface antigen promoter. The three proximal regions (E to G) are located within 45 nucleotides of the major transcription initiation site. Region E prevents the negative influence of region F and can compensate for the effect of mutation of region G on transcription from the major surface antigen promoter. Region G can compensate for the effect of the loss of a functional region E sequence on the transcriptional activity of the major surface antigen promoter only in the absence of a functional region F sequence. These results imply that the level of expression of the major surface antigen gene is controlled by the complex interplay between a minimum of six transcription factors which activate and one transcription factor which represses transcription from this gene. PMID:1651407

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

    PubMed

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

    1999-08-01

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

  16. Surface Polysaccharide Mutants Reveal that Absence of O Antigen Reduces Biofilm Formation of Actinobacillus pleuropneumoniae

    PubMed Central

    Hathroubi, S.; Hancock, M. A.; Langford, P. R.; Tremblay, Y. D. N.; Labrie, J.

    2015-01-01

    Actinobacillus pleuropneumoniae is a Gram-negative bacterium belonging to the Pasteurellaceae family and the causative agent of porcine pleuropneumonia, a highly contagious lung disease causing important economic losses. Surface polysaccharides, including lipopolysaccharides (LPS) and capsular polysaccharides (CPS), are implicated in the adhesion and virulence of A. pleuropneumoniae, but their role in biofilm formation is still unclear. In this study, we investigated the requirement for these surface polysaccharides in biofilm formation by A. pleuropneumoniae serotype 1. Well-characterized mutants were used: an O-antigen LPS mutant, a truncated core LPS mutant with an intact O antigen, a capsule mutant, and a poly-N-acetylglucosamine (PGA) mutant. We compared the amount of biofilm produced by the parental strain and the isogenic mutants using static and dynamic systems. Compared to the findings for the biofilm of the parental or other strains, the biofilm of the O antigen and the PGA mutants was dramatically reduced, and it had less cell-associated PGA. Real-time PCR analyses revealed a significant reduction in the level of pgaA, cpxR, and cpxA mRNA in the biofilm cells of the O-antigen mutant compared to that in the biofilm cells of the parental strain. Specific binding between PGA and LPS was consistently detected by surface plasmon resonance, but the lack of O antigen did not abolish these interactions. In conclusion, the absence of the O antigen reduces the ability of A. pleuropneumoniae to form a biofilm, and this is associated with the reduced expression and production of PGA. PMID:26483403

  17. Brewing spoilage Lactobacilli detected using monoclonal antibodies to bacterial surface antigens.

    PubMed

    Whiting, M S; Gares, S L; Ingledew, W M; Ziola, B

    1999-01-01

    A panel of thirteen monoclonal antibodies (Mabs) was assembled that reacts with surface antigens on eight of eleven Lactobacillus brewing spoilage organisms, including one or more of L. brevis, L. buchneri, L. casei-alactosus, L. plantarum, or unspeciated isolate(s). Immunoblotting was done to identify the antigens involved in Mab binding. Antigen stability in situ was tested by protease treatment and by surface antigen extraction of washed bacteria. Protease susceptibility of extracted surface antigens was also examined. In most cases, Lactobacillus surface antigens detected by the Mabs appear to be noncovalently bound proteins readily altered or removed from the bacterium by various environmental conditions. This research identifies brewing conditions that need to be tested to ascertain whether bacterial surface antigen-reactive Mabs can be used for the rapid, sensitive, and specific detection of Lactobacillus brewing spoilage organisms.

  18. Appearance and Distribution of Neuronal Cell Surface and Synaptic Vesicle Antigens in the Developing Rat Superior Cervical Ganglion1

    PubMed Central

    Greif, Karen F.; Reichardt, Louis F.

    2009-01-01

    Monoclonal antibodies directed against a neuronal cell surface heparan sulfate proteoglycan and against a synaptic vesicle protein were used to study the postnatal development of ganglionic neurons and synapses in the rat superior cervical ganglion. Antigen levels in developing ganglia were quantitated by radioimmune assays. Localization of antigens in adult and developing ganglia was carried out using peroxidase-antiperoxidase immunocytochemistry at the light microscopic level. Ultrastructural staining patterns in adult ganglia also were studied. The time course of antigen increases parallels those in previous reports on the accumulation of neurotransmitter enzymes within the ganglion. Both synaptic and surface antigens increase postnatally, with the most rapid changes occurring during the 2nd week. Antibodies stain adult tissue in patterns consistent with the expected distribution of antigens: antibodies directed against synaptic vesicles stain synaptic terminals and cell cytoplasm and those directed against surface proteoglycan stain the plasma membranes of neuronal cell bodies and processes. Variable staining of the cell cytoplasm also is observed. No apparent changes in antigen distribution are observed with the light microscope during development. Variations in the time course of the development of antigens associated with different portions of the proteoglycan molecule suggest that the intracellular processing of this molecule may vary during development. PMID:6212651

  19. Characterization of antigen association with accessory cells: specific removal of processed antigens from the cell surface by phospholipases.

    PubMed Central

    Falo, L D; Haber, S I; Herrmann, S; Benacerraf, B; Rock, K L

    1987-01-01

    To characterize the basis for the cell surface association of processed antigen with the antigen-presenting cell (APC) we analyzed its sensitivity to enzymatic digestion. Antigen-exposed APC that are treated with phospholipase and then immediately fixed lose their ability to stimulate antigen-plus-Ia-specific T-T hybridomas. This effect is seen with highly purified phospholipase A2 and phospholipase C. In addition it is observed with three distinct antigens--ovalbumin, bovine insulin, and poly(LGlu56LLys35LPhe9) [(GluLysPhe)n]. The effect of phospholipases is highly specific. Identically treated APC are equivalent to controls in their ability to stimulate alloreactive hybridomas specific for precisely the same Ia molecule that is corecognized by antigen-plus-Ia-specific hybrids. Furthermore, the antigen-presenting function of enzyme-treated, fixed APC can be reconstituted by the addition of exogenous in vitro processed or "processing independent" antigens. In parallel studies 125I-labeled avidin was shown to specifically bind to APC that were previously exposed and allowed to process biotin-insulin. Biotin-insulin-exposed APC that are pretreated with phospholipase bind significantly less 125I-labeled avidin than do untreated, exposed APC. Identical enzyme treatment does not reduce the binding of avidin to a biotinylated antibody already bound to class II major histocompatibility complex molecules of APC. At least some of the biotin-insulin surface sites are immunologically relevant, because the presentation of processed biotin-insulin by fixed APC is blocked by avidin. This effect is specific. Avidin binding to biotin-insulin-exposed APC does not inhibit allospecific stimulation nor the presentation of unconjugated insulin. These studies demonstrate that phospholipase effectively removes processed cell surface antigen. PMID:3467371

  20. Antibodies to Hepatitis B Surface Antigen Potentiate the Response of Human T Lymphocyte Clones to the Same Antigen

    NASA Astrophysics Data System (ADS)

    Celis, Esteban; Chang, Tse Wen

    1984-04-01

    Human T-helper lymphocyte clones specific for hepatitis B virus surface antigen (HBsAg) proliferate on stimulation with HBsAg in vitro. Antibodies specific for HBsAg, but no other antibodies, augment this proliferative response. In the presence of antibodies to HBsAg, the maximum response could be achieved at HBsAg concentrations that were 1 percent of those required in the absence of the antibodies. These findings suggest that antigen-specific antibodies exert regulatory controls on T cells that recognize the same antigens.

  1. Urinary prostate specific antigen levels after radical prostatectomy.

    PubMed

    Takayama, T K; Vessella, R L; Brawer, M K; True, L D; Noteboom, J; Lange, P H

    1994-01-01

    It was recently demonstrated that urinary prostate specific antigen (PSA) is discordant with serum PSA in many patients after radical prostatectomy. This observation led to the speculation that elevated urinary PSA in the face of undetectable serum PSA may indicate early disease recurrence. We measured urinary PSA levels in 30 patients who had undergone radical prostatectomy for prostate carcinoma and 7 patients who had undergone cystoprostatectomy for bladder cancer. PSA levels of randomly collected urine samples ranged from 0.00 to 22.9 ng./ml. and 0.01 to 8.37 ng./ml., respectively. There was no correlation among urinary and serum PSA levels, pathological stage or type of operation. In 14 patients who had undergone radical prostatectomy and who had measurable levels of urinary PSA voided specimens were divided into initial stream and end stream voided samples. The PSA levels in the end stream voided samples were significantly less than the initial stream sample in 12 of the 14 patients. In men who had undergone radical prostatectomy urethral swab samples were analyzed for PSA. Of 26 patients 24 had detectable levels of urethral swab PSA (range 0.01 to 39.04 ng./ml., median 0.93 ng./ml.). Urethral swab PSA levels did not correlate with serum PSA values or pathological stage of disease. Of 7 patients who had defunctionalized urethras after radical cystoprostatectomy 5 had significantly elevated PSA in the urethral wash or swab samples (range 4.3 to 24.5 ng./ml.). Immunohistochemical analysis of urethrectomy specimens demonstrated positive staining for PSA in 3 of 4 specimens. We conclude that the major source of urinary PSA following total prostatectomy is the urethra itself rather than residual prostate tissue. Measuring serial urinary PSA appears to have limited value in monitoring patients after radical prostatectomy. Whether this urethral PSA can ever contaminate the serum levels of PSA after radical prostatectomy is currently under investigation.

  2. Microchip device with 64-site electrode array for multiplexed immunoassay of cell surface antigens based on electrochemiluminescence resonance energy transfer.

    PubMed

    Wu, Mei-Sheng; Shi, Hai-Wei; He, Li-Jing; Xu, Jing-Juan; Chen, Hong-Yuan

    2012-05-01

    This paper describes a novel on-chip microarray platform based on an electrochemiluminescence resonance energy transfer (ECL-RET) strategy for rapid assay of cancer cell surface biomarkers. This platform consists of 64 antigen-decorated CdS nanorod spots with the diameter of 1.0 cm uniformly distributed on 16 indium tin oxide (ITO) strips, which is coated with a multichannel decorated polydimethylsiloxane (PDMS) slice to realize multiplexed determination of antigens. To shorten the immune reaction time in the microchannels and simplify the device, magnetic stirring and four-channel universal serial bus (USB) ports for plug-and-play were used. When Ru(bpy)(3)(2+) labeled antibodies were selectively captured by the corresponding antigens on the CdS nanorod spot array, ECL-RET from the CdS nanorod (donor) by cathodic emission in the presence of K(2)S(2)O(8) to Ru(bpy)(3)(2+) (acceptor) occurred. With signal amplification of Ru(bpy)(3)(2+) and competitive immunoassay, carcinoembryonic antigen (CEA), α-fetoprotein (AFP), and prostate specific antigen (PSA) as models were detected on this microfluidic device via recording the increased ECL-RET signals on electrode surfaces. Furthermore, this multiplexed competitive immunoassay was successfully used for detecting cancer cell surface antigens via the specific antibody-cell interactions and cell counting via cell surface receptors and antigens on the CdS nanorod surface. This platform provides a rapid and simple but sensitive approach with microliter-level sample volume and holds great promise for multiplexed detection of antigens and antigen-specific cells.

  3. A Monoclonal Antibody Specific to Surface Antigen on Candida krusei

    PubMed Central

    Robert, Raymond; Faure, Odile; Carloti, Arnaud; Lebeau, Bernadette; Bernard, Christian; Marot-Leblond, Agnès; Grillot, Renée; Senet, Jean-Marcel

    1998-01-01

    A monoclonal antibody (MAb; MAb 6B3) which reacts specifically with a cell wall antigen found in all strains or isolates of Candida krusei was developed. MAb 6B3 was extensively tested by immunofluorescence assay for cross-reaction with many Candida, Cryptococcus, Saccharomyces, Trichosporon, and Rhodotorula species and was found to react only with the species C. krusei. The specific epitope is expressed on the surface of fungal cells and appears to reside on a protein moiety. Taking into account the increasing importance of fluconazole-resistant strains in nosocomial fungal infections, the very high degree of specificity of this MAb for C. krusei could be useful for the routine detection of C. krusei in culture or in tissue samples. PMID:9455893

  4. Stable isotope labeling of oligosaccharide cell surface antigens

    SciTech Connect

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

    1998-12-31

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

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

    PubMed

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

    2009-08-01

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

  6. In vitro antigen-induced antibody responses to hepatitis B surface antigen in man. Kinetic and cellular requirements.

    PubMed Central

    Cupps, T R; Gerin, J L; Purcell, R H; Goldsmith, P K; Fauci, A S

    1984-01-01

    In this report we define the parameters of the human immune response after immunization with hepatitis B vaccine. 2 wk after booster immunization, there is significant spontaneous secretion of antibody to hepatitis B surface antigen (anti-HBs IgG), which is not further augmented by stimulation with antigen or pokeweed mitogen (PWM). By 4 wk there is little spontaneous secretion of specific antibody, and low doses of antigen or PWM produce significant increases in the amount of anti-HBs IgG produced. By 8 wk the peripheral blood mononuclear cells are refractory to stimulation by antigen, but anti-HBs IgG is produced in response to PWM. 0.5 yr or more after the last immunization, some individuals will manifest an antigen-induced specific antibody response. This induction of anti-HBs IgG by hepatitis B surface antigen (HBsAg) is monocyte- and T cell-dependent. Note that there is a dichotomy in the T cell response to HBsAg. The specific antibody response is clearly T cell dependent, but no in vitro T cell proliferative response to HBsAG could be demonstrated in the immunized individuals. Although the precise reason for the absent proliferative response to HBsAg despite well-established humoral immunity has not been determined, there was no evidence to suggest nonspecific suppression by HBsAg or the presence of an adherent suppressor cell population. The ability to evaluate antigen-induced, antigen-specific responses to HBsAg will be useful in defining the unique interaction between the human immune response and this clinically important viral agent. PMID:6332826

  7. Bacterial surface antigens defined by monoclonal antibodies: the methanogens

    SciTech Connect

    Conway de Macario, E.; Macario, A.J.L.; Magarinos, M.C.; Jovell, R.J.; Kandler, O.

    1982-01-01

    The methanogens (MB) are unique microbes of great evolutionary interest with applications in biotechnology-bioengineerings and are important in digestive processes. Their cell-wall composition is distinctively different from that of Eubacteria, e.g. the Methanobacteriaceae possess the peptidoglycan pseudomurein rather than murein. The range of cell-wall compositions among MB and their evolutionary and functional significance is not well known. The authors undertook a systematic study of the MB's surface structure using monoclonal antibodies through the following steps: (1) generation of hybridomas that produce antibody to several MB from 3 of their 4 families; (2) development of immunoenzymatic assays for MB's antigens and antibodies; (3) determination of the fine specificity of monoclonal antibodies by inhibition-blocking tests using cell-wall extracts and compounds of known structure; thus a set of monoclonal probes of predetermined specificity was assembled; and (4) resolution of surface determinants of MB representative of the Methanobacteriaceae using the monoclonal probes. Specific markers of MB strains were characterized. Two epitopes were identified within the pseudomurein molecule.

  8. Functionalized Magnetic Nanoparticles for the Detection and Quantitative Analysis of Cell Surface Antigen

    PubMed Central

    Shahbazi-Gahrouei, Daryoush; Abdolahi, Mohammad; Zarkesh-Esfahani, Sayyed Hamid; Laurent, Sophie; Sermeus, Corine; Gruettner, Cordula

    2013-01-01

    Cell surface antigens as biomarkers offer tremendous potential for early diagnosis, prognosis, and therapeutic response in a variety of diseases such as cancers. In this research, a simple, rapid, accurate, inexpensive, and easily available in vitro assay based on magnetic nanoparticles and magnetic cell separation principle was applied to identify and quantitatively analyze the cell surface antigen expression in the case of prostate cancer cells. Comparing the capability of the assay with flow cytometry as a gold standard method showed similar results. The results showed that the antigen-specific magnetic cell separation with antibody-coated magnetic nanoparticles has high potential for quantitative cell surface antigen detection and analysis. PMID:23484112

  9. Investigation of a Novel Hepatitis B Virus Surface Antigen (HBsAg) Escape Mutant Affecting Immunogenicity

    PubMed Central

    Hossain, Md. Golzar; Ueda, Keiji

    2017-01-01

    Mutation in the hepatitis B virus surface antigen (HBsAg) may affect the efficiency of diagnostic immunoassays or success of vaccinations using HBsAg. Thus, antigenicity and immunogenicity analyses of the mutated HBsAg are necessary to develop novel diagnostic tools and efficient vaccinations. Here, the in vitro antigenicity of three wild-type HBsAg open reading frames (ORFs) (adr4, W1S [subtype adr] and W3S [subtype adr]) isolated from clinically infected patients and nineteen synthesized single/double/multiple amino acid-substituted mutants were tested with commercial ELISA kits. Immunofluorescence staining of transfected cells and Western blot analysis confirmed that these ORFs were expressed at comparable levels in HEK-293 cells. W1S and adr4 were clearly detected, whereas W3S could not be detected. Using the same commercial immunoassay kit, we found that the single mutants, K120P and D123T, were marginally reactive, whereas W3S-aW1S and the double mutant, K120P/D123T, exhibited antigenicity roughly equivalent to the wild-type wako1S. On the other hand, the single mutants of W1S, P120K and T123D, significantly impaired the reactivity, while W1S-aW3S and the double mutant of W1S, P120K/T123D, resulted in a complete loss of antigenicity. In addition, ELISA revealed reduced HBs antigenicity of two mutants, W1S N146G and W1S Q129R/G145R. These commercial ELISA-based antigenic reactivities of HBsAg were also strongly correlated with the predicted Ai alterations of affected amino acids due to the specific mutation. In conclusion, this study showed for the first time that lysine (K120) and aspartate (D123) simultaneously affected HBsAg antigenicity, leading to diagnostic failure. These findings will improve diagnostic assays and vaccine development. PMID:28045894

  10. Two distinct antigen systems in human B lymphocytes: identification of cell surface and intracellular antigens using monoclonal antibodies.

    PubMed Central

    Ishii, Y; Takami, T; Yuasa, H; Takei, T; Kikuchi, K

    1984-01-01

    Two distinct antigen systems (L26 and L27) specifically expressed in human B lymphocytes were identified using TB2-2B3 (2B3) and T3-5B3 (5B3) monoclonal antibodies, respectively. Whereas L26 antigen defined by 2B3 were rarely expressed on the surface of B cells but abundant in the cytoplasm, 127 antigens detected by 5B3 was clearly expressed on the cell surface. These two antigens appeared to be restricted in their expression to B cells, as they were found in most B cells but not other cell types including thymocytes, T cells, monocytes and granulocytes. Functional studies demonstrated that L27 was more easily lost from B cells after activation with pokeweed mitogen than was L26. Likewise, plasma cell myeloma, as well as normal plasma cells, was devoid of both L26 and L27, whereas immunoblastic sarcoma of B cell type expressed L26 but not L27. These two antigens co-existed in the same B cell lines including Epstein-Barr virus transformed B cell lines, B cell type acute lymphatic leukaemia (B-ALL) cell line, Burkitt's lymphoma cell lines and myeloma cell lines, but pre-B and common ALL cell lines were entirely negative for both L26 and L27. Immunoprecipitation studies showed that L26 consisted of at least two polypeptide chains with molecular weights of 30K and 33K daltons, which were clearly distinct from HLA-DR antigens. The antigen L27 is presently under study. Images Fig. 2 Fig. 3 PMID:6332692

  11. Bioencapsulation of the hepatitis B surface antigen and its use as an effective oral immunogen.

    PubMed

    Hayden, Celine A; Streatfield, Stephen J; Lamphear, Barry J; Fake, Gina M; Keener, Todd K; Walker, John H; Clements, John D; Turner, Debra D; Tizard, Ian R; Howard, John A

    2012-04-19

    Hepatitis B remains a major global health problem despite the availability of a safe and effective vaccine. Segments of the population lack access to or respond poorly to the parenteral vaccine, perpetuating the infection-transmission cycle. A low cost, orally delivered vaccine has the potential to alleviate many of these problems. Here we describe the expression of a bioencapsulated hepatitis B surface antigen (HBsAg) in maize and its immunogenicity, demonstrating for the first time a commercially feasible oral subunit vaccine production system for a major disease. This work surmounts previous barriers to plant-produced vaccines by expressing HBsAg at much higher levels and retaining antigen immunogenicity post-processing: factors which facilitated a robust immune response in mice without the need for an adjuvant. This method provides a practical solution to the delivery of a low-cost, stable oral vaccine.

  12. Clinical significance of hepatitis B surface antigen mutants

    PubMed Central

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

    2015-01-01

    Hepatitis B virus (HBV) infection is a major public health problem in many countries, with nearly 300 million people worldwide carrying HBV chronic infection and over 1 million deaths per year due to cirrhosis and liver cancer. Several hepatitis B surface antigen (HBsAg) mutations have been described, most frequently due to a single amino acid substitution and seldom to a nucleotide deletion. The majority of mutations are located in the S region, but they have also been found in the pre-S1 and pre-S2 regions. Single amino acid substitutions in the major hydrophilic region of HBsAg, called the “a” determinant, have been associated with immune escape and the consequent failure of HBV vaccination and HBsAg detection, whereas deletions in the pre-S1 or pre-S2 regions have been associated with the development of hepatocellular carcinoma. This review article will focus on the HBsAg mutants and their biological and clinical implications. PMID:26644816

  13. Construction and Screening of Antigen Targeted Immune Yeast Surface Display Antibody Libraries

    SciTech Connect

    Miller, Keith D.; Pefaur, Noah B.; Baird, Cheryl L.

    2008-07-01

    These protocols describe a yeast surface display-based process for the rapid selection of antibodies from immunized mice, eliminating the need for creating and screening hybridoma fusions. A yeast surface display library of single-chain antibody fragments (scFvs) is created from antigen-binding B cells from the splenocytes of immunized mice. The antigen targeted library is then screened for antigen specific scFv by magnetic activated cell sorting (MACS) and fluorescence activated cell sorting (FACS). Library construction and screening can be accomplished in as little as 2 weeks resulting in a panel of scFvs specific for the target antigen.

  14. Construction and Screening of Antigen Targeted Immune Yeast Surface Display Antibody Libraries

    SciTech Connect

    Miller, Keith D.; Pefaur, Noah B.; Baird, Cheryl L.

    2009-08-02

    These protocols describe a yeast surface display-based process for the rapid selection of antibodies from immunized mice, eliminating the need for creating and screening hybridoma fusions. A yeast surface display library of single-chain antibody fragments (scFvs) is created from antigen-binding B cells from the splenocytes of immunized mice. The antigen targeted library is then screened for antigen specific scFv by magneticactivated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Library construction and screening can be accomplished in as little as 2 weeks, resulting in a panel of scFvs specific for the target antigen.

  15. Combined flow cytometric analysis of surface and intracellular antigens reveals surface molecule markers of human neuropoiesis.

    PubMed

    Turaç, Gizem; Hindley, Christopher J; Thomas, Ria; Davis, Jason A; Deleidi, Michela; Gasser, Thomas; Karaöz, Erdal; Pruszak, Jan

    2013-01-01

    Surface molecule profiles undergo dynamic changes in physiology and pathology, serve as markers of cellular state and phenotype and can be exploited for cell selection strategies and diagnostics. The isolation of well-defined cell subsets is needed for in vivo and in vitro applications in stem cell biology. In this technical report, we present an approach for defining a subset of interest in a mixed cell population by flow cytometric detection of intracellular antigens. We have developed a fully validated protocol that enables the co-detection of cluster of differentiation (CD) surface antigens on fixed, permeabilized neural cell populations defined by intracellular staining. Determining the degree of co-expression of surface marker candidates with intracellular target population markers (nestin, MAP2, doublecortin, TUJ1) on neuroblastoma cell lines (SH-SY5Y, BE(2)-M17) yielded a combinatorial CD49f(-)/CD200(high) surface marker panel. Its application in fluorescence-activated cell sorting (FACS) generated enriched neuronal cultures from differentiated cell suspensions derived from human induced pluripotent stem cells. Our data underlines the feasibility of using the described co-labeling protocol and co-expression analysis for quantitative assays in mammalian neurobiology and for screening approaches to identify much needed surface markers in stem cell biology.

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

    SciTech Connect

    Tomavo, S.; Schwarz, R.T.; Dubremetz, J.F. )

    1989-10-01

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

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

    PubMed Central

    Tomavo, S; Schwarz, R T; Dubremetz, J F

    1989-01-01

    The four major surface antigens of Toxoplasma gondii tachyzoites (P43, P35, P30, and P22) were made water soluble by phosphatidylinositol-specific phospholipase C (PI-PLC). These antigens were biosynthetically labeled with 3H-fatty acids, [3H]ethanolamine, and [3H]carbohydrates. Treatment of 3H-fatty-acid-labeled parasite lysates with PI-PLC removed the radioactive label from these antigens. A cross-reacting determinant was exposed on these antigens after PI-PLC treatment. Images PMID:2531282

  18. Induction of surface antigen CD69 expression in T-lymphocytes following exposure to actinomycin D.

    PubMed

    Morgan, C D; Greene, J F; Measel, J W

    1999-10-01

    The expression of surface antigen CD69 in immune response cells is typically associated with the early stage(s) of cell activation, with maximal expression levels within 4 h of appropriate antigenic or mitogenic stimulation, and maintenance of these high expression levels for 18-24 h. The expression profiles of CD69 in human peripheral blood mononuclear cells (PBMC) cultured with actinomycin D prior to mitogenic stimulation were evaluated by direct immunofluorescence using flow cytometry. Pretreatment of PBMC suspensions with low, non-toxic levels of actinomycin D stimulated CD3+ T-lymphocytes to express CD69 in a concentration-dependent manner. Furthermore, CD4+ T-lymphocytes were the primary cells responding in this fashion. Secondary mitogenic stimulation following antibiotic treatment potentiated cellular CD69 expression in these assays. CD69 expression was profoundly suppressed with in vitro actinomycin D concentrations >/=1-2 microg/ml, presumably by interference with cellular transcription/translation mechanisms. Parallel thymidine incorporation assays indicated that actinomycin D effectively inhibited thymidine uptake in a concentration-dependent manner, with complete inhibition at >/=0.1 microg/ml. The evaluation of cell cycling dynamics following antibiotic treatment, with and without secondary mitogen stimulation, indicated no substantial changes in DNA synthesis over controls. The diversity of these responses suggests that expression of CD69 may not solely reflect mitogenic activation status but may, under some conditions, result from induced cellular stress.

  19. Antibodies to dendritic neuronal surface antigens in opsoclonus myoclonus ataxia syndrome.

    PubMed

    Panzer, Jessica A; Anand, Ronan; Dalmau, Josep; Lynch, David R

    2015-09-15

    Opsoclonus myoclonus ataxia syndrome (OMAS) is an autoimmune disorder characterized by rapid, random, conjugate eye movements (opsoclonus), myoclonus, and ataxia. Given these symptoms, autoantibodies targeting the cerebellum or brainstem could mediate the disease or be markers of autoimmunity. In a subset of patients with OMAS, we identified such autoantibodies, which bind to non-synaptic puncta on the surface of live cultured cerebellar and brainstem neuronal dendrites. These findings implicate autoimmunity to a neuronal surface antigen in the pathophysiology of OMAS. Identification of the targeted antigen(s) could elucidate the mechanisms underlying OMAS and provide a biomarker for diagnosis and response to therapy.

  20. Expression and immunological characterisation of Eimeria tenella glycosylphosphatidylinositol-anchored surface antigen-5

    NASA Astrophysics Data System (ADS)

    Ho, Sue-Kim; Nathan, Sheila; Wan, Kiew-Lian

    2016-11-01

    Eimeria tenella is the most pathogenic of the Eimeria species that infect chickens and causes huge economic losses to the poultry industry. The glycosylphosphatidylinositol-anchored surface antigen-5 (SAG5) found on the surface of the parasite has been shown to activate the chicken's immune system. In this study, recombinant SAG5 was expressed, purified and used to investigate the immune-inducing characteristics of the molecule. Chickens were immunized with purified recombinant SAG5 and sera were subjected to Enzyme-linked Immunosorbant Assay (ELISA). Results indicated that specific antibodies against rSAG5 were produced, with IgG detected at a higher level compared to IgA and IgM. Information on the immunological responses elicited by SAG5 provides essential knowledge that will contribute towards the effort to develop more effective strategies against coccidiosis.

  1. Gold Nanoparticle Based Activatable Probe for Sensing Ultra-Low Levels of Prostate Specific Antigen

    PubMed Central

    Liu, Dingbin; Huang, Xinglu; Wang, Zhantong; Jin, Albert; Sun, Xiaolian; Zhu, Lei; Wang, Fu; Ma, Ying; Niu, Gang; HightWalker, Angela R.; Chen, Xiaoyuan

    2013-01-01

    It is still in high demand to develop extremely sensitive and accurate clinical tools for biomarkers of interest for early diagnosis and monitoring of diseases. In this report, we present a highly sensitive and compatible gold nanoparticle (AuNP)-based fluorescence activatable probe for sensing ultra-low levels of prostate-specific antigen (PSA) in patient serum samples. The limit of detection of the newly-developed probe for PSA was pushed down to 0.032 pg/mL, which is more than two orders of magnitude lower than that of the conventional fluorescence probe. The ultrahigh sensitivity of this probe was attributed to the high loading efficiency of the dyes on AuNP surfaces and high fluorescence quenching unquenching abilities of the dye-AuNP pairs. The efficiency and robustness of this probe was investigated in patient serum samples, demonstrating the great potential of this probe in real-world applications. PMID:23683064

  2. Oral immunization with hepatitis B surface antigen expressed in transgenic plants.

    PubMed

    Kong, Q; Richter, L; Yang, Y F; Arntzen, C J; Mason, H S; Thanavala, Y

    2001-09-25

    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.

  3. Localized surface antigens of guinea pig sperm migrate to new regions prior to fertilization

    PubMed Central

    1984-01-01

    We have previously defined distinct localizations of antigens on the surface of the guinea pig sperm using monoclonal antibodies. In the present study we have demonstrated that these antigen localizations are dynamic and can be altered during changes in the functional state of the sperm. Before the sperm is capable of fertilizing the egg, it must undergo capacitation and an exocytic event, the acrosome reaction. Prior to capacitation, the antigen recognized by the monoclonal antibody, PT-1, was restricted to the posterior tail region (principle piece and end piece). After incubation in capacitating media at 37 degrees C for 1 h, 100% of the sperm population showed migration of the PT-1 antigen onto the anterior tail. This redistribution of surface antigen resulted from a migration of the surface molecules originally present on the posterior tail. It did not occur in the presence of metabolic poisons or when tail-beating was prevented. It was temperature-dependent, and did not require exogenous Ca2+. Since the PT- 1 antigen is freely diffusing on the posterior tail before migration, the mechanism of redistribution could involve the alteration of a presumptive membrane barrier. In addition, we observed the redistribution of a second surface antigen after the acrosome reaction. The antigen recognized by the monoclonal antibody, PH-20, was localized exclusively in the posterior head region of acrosome-intact sperm. Within 7-10 min of induction of the acrosome reaction with Ca2+ and A23187, 90-100% of the acrosome-reacted sperm population no longer demonstrated binding of the PH-20 antibody on the posterior head, but showed binding instead on the inner acrosomal membrane. This redistribution of the PH-20 antigen also resulted from the migration of pre-existing surface molecules, but did not appear to require energy. The migration of PH-20 antigen was a selective process; other antigens localized to the posterior head region did not leave the posterior head after the

  4. Inhibition of immune opsonin-independent phagocytosis by antibody to a pulmonary macrophage cell surface antigen

    SciTech Connect

    Parod, R.J.; Godleski, J.J.; Brain J.D.

    1986-03-15

    Unlike other hamster phagoycytes, hamster pulmonary macrophages (PM) avidly ingest albumin-coated latex particles in the absence of serum. They also possess a highly specific cell surface antigen. To evaluate the relationship between these two characteristics, PM were incubated with mouse monoclonal antibody directed against the PM antigen. After unbound antibody was removed, the amount of bound antibody and the phagocytic capability of PM were measured by flow cytometry and fluorescence microscopy. Maximum antibody binding produced a 25% inhibition of ingestion. Particle attachment was not affected. This effect was antigen specific, since neither a nonspecific mouse myeloma protein of the same subclass nor a mouse antibody that bound to another hamster surface antigen had any effect on binding or ingestion. If antigen-specific F(ab')/sub 2/ fragments were introduced both before and during the period of phagocytosis, the inhibition of particle ingestion approached 100%. Particle binding increased at low F(ab')/sub 2/ concentrations but declined at higher concentrations. Because calcium may play a role in the ingestion process, the effect of antibody on /sup 45/Ca uptake was evaluated. It was observed that antigen-specific F(ab')/sub 2/ fragments stimulated /sup 45/Ca uptake, whereas control antibodies did not. These results suggest that the antigen reacting with the anti-hamster PM monoclonal antibody is involved in immune opsonin-independent phagocytosis and that calcium participates in this phagocytic process.

  5. Anaplasma platys Immunoblot Test Using Major Surface Antigens.

    PubMed

    Lai, Tzung-Huei; Parraga, Maria E; Alvarez, Elizabeth; Rikihisa, Yasuko

    2016-09-01

    Anaplasma platys is an uncultivable tick-borne obligatory intracellular bacterium, which is known to infect platelets of dogs. A. platys causes infectious canine cyclic thrombocytopenia in subtropical and tropical regions throughout the world. Several cases of human infection with A. platys infection have also been reported. However, seroprevalence of A. platys exposure and infection has not been determined in most of the regions, in part, due to lack of a simple and reliable assay method. Furthermore, A. platys antigens recognized by dogs are unknown. We previously sequenced gene encoding A. platys major outer membrane proteins P44 and Omp-1X. In the present study, we obtained purified recombinant A. platys P44 and Omp-1X proteins, and using them as antigens in immunoblotting examined seroreactivity in dogs. Of 34 specimens from Venezuela where A. platys infection was previously reported, 25 specimens (73.5%) reacted to rAplP44 and/or rAplOMP-1X. Neither Anaplasma phagocytophilum-seropositive (N = 10) nor A. phagocytophilum-seronegative canine specimens (N = 10) from the geographic regions where A. platys infection has never been reported, reacted rAplP44 or rAplOMP-1X. The result indicates a high A. platys seroprevalence rate in tested dogs from Venezuela and suggests that the immunoblot analysis based on recombinant A. platys major outer membrane proteins can provide a simple and defined tool to enlighten the prevalence of A. platys infection.

  6. Factors important in the extraction, stability and in vitro assembly of the hepatitis B surface antigen derived from recombinant plant systems.

    PubMed

    Smith, Mark L; Keegan, Mark E; Mason, Hugh S; Shuler, Michael L

    2002-01-01

    The expression of vaccine antigens in edible plant material together with their delivery by the oral route constitutes a powerful paradigm, with the potential to dramatically reduce the cost of vaccine production and administration, in addition to improving distribution and patient compliance. These products will be subject to many of the same regulations applied to current injectable vaccines, so reliable methods to quantify antigen and ensure stability in crude plant extracts are required. As a model system the hepatitis B surface antigen (HBsAg) was expressed in soybean and tobacco cell cultures. This complex antigen consists of membrane-associated small surface antigen proteins (p24(s)), disulfide cross-linked to yield dimers and higher multimers. Although the total p24(s) extracted from plant cells was relatively unaffected by detergent concentration, the quantification of antigenically reactive product depended strongly on the ratio of detergent to cell concentration. Furthermore, 1-20% w/v sodium ascorbate improved the measured levels of monoclonal-reactive antigen 4- to 12-fold. Detergent also influenced antigen stability in cell lysates stored at 4 degrees C; under optimum conditions stability was maintained for at least 1 month, whereas excess detergent rendered the antigen susceptible to proteolytic degradation. This proteolysis could be counteracted by the addition of skim milk or its protein component, which stabilized antigenically reactive p24(s) for up to 2 months. The immunologically relevant epitopes of HBsAg are critically dependent on disulfide bonding. By altering the sodium ascorbate concentration or buffer pH the proportion of HBsAg displaying the monoclonal reactive epitopes was increased between 8- and 20-fold. In addition, under certain conditions the dimerized p24(s) could be converted to oligomeric aggregates, resembling the form of the serum-derived antigen. These simple in vitro manipulations, compatible with the goal of a minimally

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

    SciTech Connect

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

    1985-09-01

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

  8. Ultrasensitive detection of HIV-1 p24 antigen using nanofunctionalized surfaces in a capacitive immunosensor.

    PubMed

    Teeparuksapun, Kosin; Hedström, Martin; Wong, Eric Y; Tang, Shixing; Hewlett, Indira K; Mattiasson, Bo

    2010-10-15

    The HIV-1 capsid protein, p24 antigen, is of considerable diagnostic interest because following HIV exposure it is detectable several days earlier than host-generated HIV antibodies (which are the target of almost all current tests used in the field) and can be used to design very sensitive assays without the need for PCR. Here, we present an ultrasensitive capacitive immunosensor that is capable of detecting subattogram per milliliter concentrations of p24 antigen, which to our knowledge is the lowest level of detection ever reported. Dilution studies using p24-spiked human plasma samples indicate that the immunosensor is robust against the interfering effects of a complex biological matrix. Moreover, the capacitive immunosensor assay is rapid (<20 min), label-free, and generates data in real-time, with a portable format in development. Additional optimization of the capture agents and/or surface chemistries may further improve performance, highlighting the potential of this platform to serve as a diagnostic tool for early detection of HIV in field settings.

  9. Assembly of viral particles in Xenopus oocytes: pre-surface-antigens regulate secretion of the hepatitis B viral surface envelope particle.

    PubMed Central

    Standring, D N; Ou, J H; Rutter, W J

    1986-01-01

    Infection with hepatitis B virus (HBV) is associated with the production of a viral envelope particle that contains membrane lipids, surface antigen (S), and two presurface-antigens (pre-S) comprised of the entire S moiety with approximately 55 (pre-S2) and 174 (pre-S1) additional NH2-terminal amino acids. We show here that Xenopus oocytes injected with synthetic S mRNA assemble and secrete characteristic 22-nm viral envelope particles. In contrast, pre-S1 and pre-S2 antigens are synthesized but not secreted. By coinjecting mRNAs, we found that synthesis of high levels of pre-S proteins specifically inhibits S antigen secretion. On the other hand, high levels of S synthesis can drive the secretion of small amounts of either pre-S antigen. These observations are consistent with a model for viral envelope assembly in which both S and pre-S proteins are incorporated into a multimeric particle, presumably via interactions between the S protein domains, while the pre-S amino-terminal moieties regulate the secretion of this structure. Our results indicate that Xenopus oocytes will provide a powerful system for studying the morphogenesis of simple structures of viral or cellular origin. Images PMID:3467308

  10. The Effect of Superparamagnetic Iron Oxide Nanoparticle Surface Charge on Antigen Cross-Presentation.

    PubMed

    Mou, Yongbin; Xing, Yun; Ren, Hongyan; Cui, Zhihua; Zhang, Yu; Yu, Guangjie; Urba, Walter J; Hu, Qingang; Hu, Hongming

    2017-12-01

    Magnetic nanoparticles (NPs) of superparamagnetic iron oxide (SPIO) have been explored for different kinds of applications in biomedicine, mechanics, and information. Here, we explored the synthetic SPIO NPs as an adjuvant on antigen cross-presentation ability by enhancing the intracellular delivery of antigens into antigen presenting cells (APCs). Particles with different chemical modifications and surface charges were used to study the mechanism of action of antigen delivery. Specifically, two types of magnetic NPs, γFe2O3/APTS (3-aminopropyltrimethoxysilane) NPs and γFe2O3/DMSA (meso-2, 3-Dimercaptosuccinic acid) NPs, with the same crystal structure, magnetic properties, and size distribution were prepared. Then, the promotion of T-cell activation via dendritic cells (DCs) was compared among different charged antigen coated NPs. Moreover, the activation of the autophagy, cytosolic delivery of the antigens, and antigen degradation mediated by the proteasome and lysosome were measured. Our results indicated that positive charged γFe2O3/APTS NPs, but not negative charged γFe2O3/DMSA NPs, enhanced the cross-presentation ability of DCs. Increased cross-presentation ability induced by γFe2O3/APTS NPs was associated with increased cytosolic antigen delivery. On the contrary, γFe2O3/DMSA NPs was associated with rapid autophagy. Overall, our results suggest that antigen delivered in cytoplasm induced by positive charged particles is beneficial for antigen cross-presentation and T-cell activation. NPs modified with different chemistries exhibit diverse biological properties and differ greatly in their adjuvant potentials. Thus, it should be carefully considered many different effects of NPs to design effective and safe adjuvants.

  11. The Effect of Superparamagnetic Iron Oxide Nanoparticle Surface Charge on Antigen Cross-Presentation

    NASA Astrophysics Data System (ADS)

    Mou, Yongbin; Xing, Yun; Ren, Hongyan; Cui, Zhihua; Zhang, Yu; Yu, Guangjie; Urba, Walter J.; Hu, Qingang; Hu, Hongming

    2017-01-01

    Magnetic nanoparticles (NPs) of superparamagnetic iron oxide (SPIO) have been explored for different kinds of applications in biomedicine, mechanics, and information. Here, we explored the synthetic SPIO NPs as an adjuvant on antigen cross-presentation ability by enhancing the intracellular delivery of antigens into antigen presenting cells (APCs). Particles with different chemical modifications and surface charges were used to study the mechanism of action of antigen delivery. Specifically, two types of magnetic NPs, γFe2O3/APTS (3-aminopropyltrimethoxysilane) NPs and γFe2O3/DMSA (meso-2, 3-Dimercaptosuccinic acid) NPs, with the same crystal structure, magnetic properties, and size distribution were prepared. Then, the promotion of T-cell activation via dendritic cells (DCs) was compared among different charged antigen coated NPs. Moreover, the activation of the autophagy, cytosolic delivery of the antigens, and antigen degradation mediated by the proteasome and lysosome were measured. Our results indicated that positive charged γFe2O3/APTS NPs, but not negative charged γFe2O3/DMSA NPs, enhanced the cross-presentation ability of DCs. Increased cross-presentation ability induced by γFe2O3/APTS NPs was associated with increased cytosolic antigen delivery. On the contrary, γFe2O3/DMSA NPs was associated with rapid autophagy. Overall, our results suggest that antigen delivered in cytoplasm induced by positive charged particles is beneficial for antigen cross-presentation and T-cell activation. NPs modified with different chemistries exhibit diverse biological properties and differ greatly in their adjuvant potentials. Thus, it should be carefully considered many different effects of NPs to design effective and safe adjuvants.

  12. Expression of the hepatitis B surface S and preS2 antigens in tubers of Solanum tuberosum.

    PubMed

    Joung, Y H; Youm, J W; Jeon, J H; Lee, B C; Ryu, C J; Hong, H J; Kim, H C; Joung, H; Kim, H S

    2004-07-01

    In an attempt to develop an edible vaccine, we transformed a recombinant hepatitis B virus (HBV) gene encoding the middle protein of HBV that contains the surface S and preS2 antigen into potato by Agrobacterium-mediated transformation. The HBV gene was under control of either the CaMV 35S promoter, the double 35S promoter with the AlMV 5' non-translated leader sequence, or the tuber-specific patatin promoter. HBV mRNA levels were higher with the 35S promoter than with the double 35S and patatin promoters; however, the levels of the S and preS2 antigen in the transformed tubers were higher with the patatin promoter than with the CaMV 35S and double promoters. The levels of preS2 antigen produced are the highest reported to date. Transgenic potato tubers were fed to mice, and the mice showed an immune response against the HBV S antigen.

  13. Escherichia coli strains expressing H12 antigens demonstrate an increased ability to attach to abiotic surfaces as compared with E. coli strains expressing H7 antigens.

    PubMed

    Goulter, Rebecca M; Taran, Elena; Gentle, Ian R; Gobius, Kari S; Dykes, Gary A

    2014-07-01

    The role of Escherichia coli H antigens in hydrophobicity and attachment to glass, Teflon and stainless steel (SS) surfaces was investigated through construction of fliC knockout mutants in E. coli O157:H7, O1:H7 and O157:H12. Loss of FliC(H12) in E. coli O157:H12 decreased attachment to glass, Teflon and stainless steel surfaces (p<0.05). Complementing E. coli O157:H12 ΔfliC(H12) with cloned wildtype (wt) fliC(H12) restored attachment to wt levels. The loss of FliCH7 in E. coli O157:H7 and O1:H7 did not always alter attachment (p>0.05), but complementation with cloned fliC(H12), as opposed to cloned fliCH7, significantly increased attachment for both strains compared with wt counterparts (p<0.05). Hydrophobicity determined using bacterial adherence to hydrocarbons and contact angle measurements differed with fliC expression but was not correlated to the attachment to materials included in this study. Purified FliC was used to functionalise silicone nitride atomic force microscopy probes, which were used to measure adhesion forces between FliC and substrates. Although no significant difference in adhesion force was observed between FliC(H12) and FliCH7 probes, differences in force curves suggest different mechanism of attachment for FliC(H12) compared with FliCH7. These results indicate that E. coli strains expressing flagellar H12 antigens have an increased ability to attach to certain abiotic surfaces compared with E. coli strains expressing H7 antigens.

  14. Surface plasmon resonance and NMR analyses of anti Tn-antigen MLS128 monoclonal antibody binding to two or three consecutive Tn-antigen clusters.

    PubMed

    Matsumoto-Takasaki, Ayano; Hanashima, Shinya; Aoki, Ami; Yuasa, Noriyuki; Ogawa, Haruhiko; Sato, Reiko; Kawakami, Hiroko; Mizuno, Mamoru; Nakada, Hiroshi; Yamaguchi, Yoshiki; Fujita-Yamaguchi, Yoko

    2012-03-01

    Tn-antigens are tumour-associated carbohydrate antigens that are involved in metastatic processes and are associated with a poor prognosis. MLS128 monoclonal antibody recognizes the structures of two or three consecutive Tn-antigens (Tn2 or Tn3). Since MLS128 treatment inhibits colon and breast cancer cell growth [Morita, N., Yajima, Y., Asanuma, H., Nakada, H., and Fujita-Yamaguchi, Y. (2009) Inhibition of cancer cell growth by anti-Tn monoclonal antibody MLS128. Biosci. Trends 3, 32-37.], understanding the interaction between MLS128 and Tn-clusters may allow us to the development of novel cancer therapeutics. Although MLS128 was previously reported to have specificity for Tn3 rather than Tn2, similar levels of Tn2/Tn3 binding were unexpectedly observed at 37°C. Thus, thermodynamic analyses were performed via surface plasmon resonance (SPR) using synthetic Tn2- and Tn3-peptides at 10, 15, 20, 25 and 30°C. SPR results revealed that MLS128's association constants for both antigens were highly temperature dependent. Below 25°C MLS128's association constant for Tn3-peptide was clearly higher than that for Tn2-peptide. At 30°C, however, the association constant for Tn2-peptide was higher than that for Tn3-peptide. This reversal of affinity is due to the sharp increase in K(d) for Tn3. These results were confirmed by NMR, which directly measured MLS128-Tn binding in solution. This study suggested that thermodynamic control plays a critical role in the interaction between MLS128/Tn2 and MLS128/Tn3.

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

    PubMed

    Ito, A; Onitake, K

    1987-06-01

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

  16. Continuous flow magnetic cell fractionation based on antigen expression level.

    PubMed

    Schneider, Thomas; Moore, Lee R; Jing, Ying; Haam, Seungjoo; Williams, P Stephen; Fleischman, Aaron J; Roy, Shuvo; Chalmers, Jeffrey J; Zborowski, Maciej

    2006-07-31

    Cell separation is important in medical and biological research and plays an increasingly important role in clinical therapy and diagnostics, such as rare cancer cell detection in blood. The immunomagnetic labeling of cells with antibodies conjugated to magnetic nanospheres gives rise to a proportional relationship between the number of magnetic nanospheres attached to the cell and the cell surface marker number. This enables the potential fractionation of cell populations by magnetophoretic mobility (MM). We exploit this feature with our apparatus, the Dipole Magnet Flow Fractionator (DMFF), which consists of an isodynamic magnetic field, an orthogonally-oriented thin ribbon of cell suspension in continuous sheath flow, and ten outlet flows. From a sample containing a 1:1 mixture of immunomagnetically labeled (label+) and unlabeled (label-) cells, we achieved an increase in enrichment of the label+ cell fraction with increasing outlet numbers in the direction of the magnetic field gradient (up to 10-fold). The total recovery of the ten outlet fractions was 90.0+/-7.7%. The mean MM of label+ cells increased with increasing outlet number by up to a factor of 2.3. The postulated proportionality between the number of attached magnetic beads and the number of cell surface markers was validated by comparison of MM measured by cell tracking velocimetry (CTV) with cell florescence intensity measured by flow cytometry.

  17. Research progress on surface antigen 1 (SAG1) of Toxoplasma gondii

    PubMed Central

    2014-01-01

    Toxoplasma gondii is an obligate intracellular parasitic protozoan that has a wide host range and causes a zoonotic parasitosis called toxoplasmosis. This infection causes significant morbidity, costs for care and loss of productivity and suffering. The most effective measures to minimize this parasite’s harm to patients are prompt diagnosis and treatment and preventing infection. A parasite surface antigen, SAG1, is considered an important antigen for the development of effective diagnostic tests or subunit vaccines. This review covers several aspects of this antigen, including its gene structure, contribution to host invasion, mechanisms of the immune responses and its applications for diagnosis and vaccine development. This significant progress on this antigen provides foundations for further development of more effective and precise approaches to diagnose toxoplasmosis in the clinic, and also have important implications for exploring novel measures to control toxoplasmosis in the near future. PMID:24726014

  18. Strains of Sarcocystis neurona exhibit differences in their surface antigens, including the absence of the major surface antigen SnSAG1.

    PubMed

    Howe, Daniel K; Gaji, Rajshekhar Y; Marsh, Antoinette E; Patil, Bhagyashree A; Saville, William J; Lindsay, David S; Dubey, J P; Granstrom, David E

    2008-05-01

    A gene family of surface antigens is expressed by merozoites of Sarcocystis neurona, the primary cause of equine protozoal myeloencephalitis (EPM). These surface proteins, designated SnSAGs, are immunodominant and therefore excellent candidates for development of EPM diagnostics or vaccines. Prior work had identified an EPM isolate lacking the major surface antigen SnSAG1, thus suggesting there may be some diversity in the SnSAGs expressed by different S. neurona isolates. Therefore, a bioinformatic, molecular and immunological study was conducted to assess conservation of the SnSAGs. Examination of an expressed sequence tag (EST) database revealed several notable SnSAG polymorphisms. In particular, the EST information implied that the EPM strain SN4 lacked the major surface antigen SnSAG1. The absence of this surface antigen from the SN4 strain was confirmed by both Western blot and Southern blot. To evaluate SnSAG polymorphisms in the S. neurona population, 14 strains were examined by Western blots using monospecific polyclonal antibodies against the four described SnSAGs. The results of these analyses demonstrated that SnSAG2, SnSAG3, and SnSAG4 are present in all 14 S. neurona strains tested, although some variance in SnSAG4 was observed. Importantly, SnSAG1 was not detected in seven of the strains, which included isolates from four cases of EPM and a case of fatal meningoencephalitis in a sea otter. Genetic analyses by PCR using gene-specific primers confirmed the absence of the SnSAG1 locus in six of these seven strains. Collectively, the data indicated that there is heterogeneity in the surface antigen composition of different S. neurona isolates, which is an important consideration for development of serological tests and prospective vaccines for EPM. Furthermore, the diversity reported herein likely extends to other phenotypes, such as strain virulence, and may have implications for the phylogeny of the various Sarcocystis spp. that undergo sexual stages

  19. Ultra-sensitive immunosensor for detection of hepatitis B surface antigen using multi-functionalized gold nanoparticles.

    PubMed

    Shourian, M; Ghourchian, H; Boutorabi, M

    2015-10-01

    The signal amplification for analytical purposes has considerable potential in detecting trace levels of analytes for clinical, security or environmental applications. In the present report a strategy based on a sandwich type immunoassay system was designed for the detection of hepatitis B surface antigen which exploits the specific affinity interaction between streptavidin and biotin recognition systems. The method involves the specific coupling of multi-functionalized gold nanoparticles (bearing biotin and luminol molecules) to the streptavidin modified by secondary antibody. The chemiluminescent signal is produced by the gold nanoparticles in the presence of HAuCl4 as catalyst and hydrogen peroxide as oxidant. The immunosensor was able to detect hepatitis B surface antigen in the linear concentration range from 1.7 to 1920 pg mL(-1) and the detection limit of 0.358 pg mL(-1), at signal/noise = 3.

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

    PubMed Central

    Liston, Sean D.; Ovchinnikova, Olga G.

    2016-01-01

    Polysaccharide capsules are surface structures that are critical for the virulence of many Gram-negative pathogenic bacteria. Salmonella enterica serovar Typhi is the etiological agent of typhoid fever. It produces a capsular polysaccharide known as “Vi antigen,” which is composed of nonstoichiometrically O-acetylated α-1,4-linked N-acetylgalactosaminuronic acid residues. This glycan is a component of currently available vaccines. The genetic locus for Vi antigen production is also present in soil bacteria belonging to the genus Achromobacter. Vi antigen assembly follows a widespread general strategy with a characteristic glycan export step involving an ATP-binding cassette transporter. However, Vi antigen producers lack the enzymes that build the conserved terminal glycolipid characterizing other capsules using this method. Achromobacter species possess a Vi antigen-specific depolymerase enzyme missing in S. enterica Typhi, and we exploited this enzyme to isolate acylated Vi antigen termini. Mass spectrometry analysis revealed a reducing terminal N-acetylhexosamine residue modified with two β-hydroxyl acyl chains. This terminal structure resembles one half of lipid A, the hydrophobic portion of bacterial lipopolysaccharides. The VexE protein encoded in the Vi antigen biosynthesis locus shares similarity with LpxL, an acyltransferase from lipid A biosynthesis. In the absence of VexE, Vi antigen is produced, but its physical properties are altered, its export is impaired, and a Vi capsule structure is not assembled on the cell surface. The structure of the lipidated terminus dictates a unique assembly mechanism and has potential implications in pathogenesis and vaccine production. PMID:27226298

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

    PubMed

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

    2016-06-14

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

  2. Accessibility of gonococcal and meningococcal surface antigens: immunogold labeling for quantitative electron microscopy.

    PubMed Central

    Pâques, M; Teppema, J S; Beuvery, E C; Abdillahi, H; Poolman, J T; Verkleij, A J

    1989-01-01

    The parallel application of two electron microscopic immunogold labeling procedures was used to assess the surface exposure and accessibility of gonococcal and meningococcal surface antigens. Monoclonal antibodies were used as markers for the surface antigens, i.e., outer membrane proteins and lipooligosaccharides. To evaluate the labeling densities obtained after incubation of whole bacteria in suspension or ultrathin cryosections of bacteria, a method of electron microscopic quantitation was developed. Incubation of whole bacterial suspensions with monoclonal antibodies and protein A-gold resulted in specific labeling of the bacterial surfaces. However, the labeling densities varied largely in each cell. By contrast, cryosections showed uniform heavy labeling densities at the surface of the outer membranes of all cells. Apparently, by sectioning the cells the antigen-masking barrier could be evaded, and steric hindrance was no longer restrictive. Thus, a better estimate of both the presence and the surface exposure, i.e., the accessibility of antigens, could be made. Such information is essential for us to better understand host-bacterial interactions and to develop new vaccines. Images PMID:2492264

  3. Sarcocystis neurona merozoites express a family of immunogenic surface antigens that are orthologues of the Toxoplasma gondii surface antigens (SAGs) and SAG-related sequences.

    PubMed

    Howe, Daniel K; Gaji, Rajshekhar Y; Mroz-Barrett, Meaghan; Gubbels, Marc-Jan; Striepen, Boris; Stamper, Shelby

    2005-02-01

    Sarcocystis neurona is a member of the Apicomplexa that causes myelitis and encephalitis in horses but normally cycles between the opossum and small mammals. Analysis of an S. neurona expressed sequence tag (EST) database revealed four paralogous proteins that exhibit clear homology to the family of surface antigens (SAGs) and SAG-related sequences of Toxoplasma gondii. The primary peptide sequences of the S. neurona proteins are consistent with the two-domain structure that has been described for the T. gondii SAGs, and each was predicted to have an amino-terminal signal peptide and a carboxyl-terminal glycolipid anchor addition site, suggesting surface localization. All four proteins were confirmed to be membrane associated and displayed on the surface of S. neurona merozoites. Due to their surface localization and homology to T. gondii surface antigens, these S. neurona proteins were designated SnSAG1, SnSAG2, SnSAG3, and SnSAG4. Consistent with their homology, the SnSAGs elicited a robust immune response in infected and immunized animals, and their conserved structure further suggests that the SnSAGs similarly serve as adhesins for attachment to host cells. Whether the S. neurona SAG family is as extensive as the T. gondii SAG family remains unresolved, but it is probable that additional SnSAGs will be revealed as more S. neurona ESTs are generated. The existence of an SnSAG family in S. neurona indicates that expression of multiple related surface antigens is not unique to the ubiquitous organism T. gondii. Instead, the SAG gene family is a common trait that presumably has an essential, conserved function(s).

  4. Serological responses to Cryptosporidium antigens among users of surface- vs. ground-water sources.

    PubMed Central

    Frost, F. J.; Kunde, T. R.; Muller, T. B.; Craun, G. F.; Katz, L. M.; Hibbard, A. J.; Calderon, R. L.

    2003-01-01

    Cryptosporidium oocysts are commonly detected in surface-derived drinking water. However, the public health significance of these findings is unclear. This study compared serological responses to two Cryptosporidium antigen groups for blood donors and college students using chlorinated and filtered river water vs. ground-water sources. The surface water received agricultural and domestic sewage discharges upstream. Participants from the surface-water city had a higher relative prevalence (RP) of a serological response to the 15/17-kDa antigen group (72.3 vs. 52.4%, RP = 1.36, P < 0.001) and to the 27-kDa antigen group (82.6 vs. 72.5%, RP = 1.14, P < 0.02). Multivariate logistic regression analysis found that the people with a shorter duration of residence or drinking bottled water also had a lower seropositivity for each marker. Use of private wells was associated with a higher prevalence of response to the 15/17-kDa markers. Seroconversion to the 15/17-kDa antigen group was more common in the residents of the city using surface water. These findings are consistent with an increased risk of Cryptosporidium infection for users of surface-derived drinking water compared with users of municipal ground-water-derived drinking water. Users of private well water may also have an increased risk of infection. PMID:14959781

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

    PubMed

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

    1964-12-01

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

  6. Surface plasmon field-enhanced fluorescence spectroscopy studies of the interaction between an antibody and its surface-coupled antigen.

    PubMed

    Yu, Fang; Yao, Danfeng; Knoll, Wolfgang

    2003-06-01

    Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) uses the greatly enhanced electromagnetic field of a surface plasmon mode for the excitation of surface-confined fluorophores. The ability to simultaneously monitor the interfacial refractive index changes and the fluorescence signals in real time offers a huge potential for applications of SPFS in surface immunoreaction detection. In this study, gold surfaces were functionalized by mixed self-assembled monolayers exposing an antigen (biotin) at a density that was varied over a wide range. Specific antibody-antigen interactions were observed for anti-biotin antibody solutions passing over the surfaces with a rather high flow speed driven by a home-built liquid-handling system. First, the use of the fluorophores Cy5 and Alexa Fluor 647 in SFPS-based immunoassays was investigated. It was found that Cy5 exhibits strong self-quenching, which makes it rather unsuitable for quantitative measurements. For the in situ measurement of the binding kinetics, an angular "detuning" effect was confirmed to negatively interfere with the fluorescence signal in cases where large SPR signals were detected. An in-depth comparison between the SPR and the fluorescence signal reveals that the fluorescence yield of the dyes depends strongly on the separation distance from the gold surface. And finally, we stress the ability of SPFS to detect binding to surfaces containing extremely diluted antigen density, where the SPR signal failed to follow.

  7. Demonstration of common antigens on cell surface of Clostridium chauvoei and C. septicum by indirect-immunofluorescence assay.

    PubMed

    Hamaoka, T; Terakado, N

    1994-04-01

    The common antigens between Clostridium chauvoei and C. septicum were examined by indirect-immunofluorescence assay (IFA). Antisera to formalized cells of C. chauvoei and C. septicum strains and to EDTA-soluble antigens of these strains were used. The antisera to formalized cells, which have reacted only with homologous antigens in agglutination tests, reacted not only with homologous antigens but also with heterologous antigens in IFA. The antisera to EDTA-soluble antigens, which have shown no reactivities in somatic agglutination tests, reached with both homologous and heterologous antigens in IFA. These results indicate that these species possess common antigens, which are undetected by agglutination tests, on the cell surface and a some of them are solubilized by EDTA-treatment.

  8. Immunogenic Properties of Lactobacillus plantarum Producing Surface-Displayed Mycobacterium tuberculosis Antigens.

    PubMed

    Kuczkowska, Katarzyna; Kleiveland, Charlotte R; Minic, Rajna; Moen, Lars F; Øverland, Lise; Tjåland, Rannei; Carlsen, Harald; Lea, Tor; Mathiesen, Geir; Eijsink, Vincent G H

    2017-01-15

    Tuberculosis (TB) remains among the most deadly diseases in the world. The only available vaccine against tuberculosis is the bacille Calmette-Guérin (BCG) vaccine, which does not ensure full protection in adults. There is a global urgency for the development of an effective vaccine for preventing disease transmission, and it requires novel approaches. We are exploring the use of lactic acid bacteria (LAB) as a vector for antigen delivery to mucosal sites. Here, we demonstrate the successful expression and surface display of a Mycobacterium tuberculosis fusion antigen (comprising Ag85B and ESAT-6, referred to as AgE6) on Lactobacillus plantarum The AgE6 fusion antigen was targeted to the bacterial surface using two different anchors, a lipoprotein anchor directing the protein to the cell membrane and a covalent cell wall anchor. AgE6-producing L. plantarum strains using each of the two anchors induced antigen-specific proliferative responses in lymphocytes purified from TB-positive donors. Similarly, both strains induced immune responses in mice after nasal or oral immunization. The impact of the anchoring strategies was reflected in dissimilarities in the immune responses generated by the two L. plantarum strains in vivo The present study comprises an initial step toward the development of L. plantarum as a vector for M. tuberculosis antigen delivery.

  9. 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

    2010-09-02

    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.

  10. Surface Co-Expression of Two Different PfEMP1 Antigens on Single Plasmodium falciparum-Infected Erythrocytes Facilitates Binding to ICAM1 and PECAM1

    PubMed Central

    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

  11. 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.

  12. Prime-boost immunisation against tropical theileriosis with two parasite surface antigens: evidence for protection and antigen synergy.

    PubMed

    Gharbi, Mohamed; Darghouth, Mohamed Aziz; Weir, William; Katzer, Frank; Boulter, Nicky; Adamson, Rachel; Gilbert, Sarah C; Jongejan, Frans; Westbroek, Irene; Hall, Roger; Tait, Andrew; Shiels, Brian

    2011-09-02

    Current methods for control of tropical theileriosis in cattle suffer from several disadvantages that could be circumvented by development of an effective sub-unit vaccine. Previous work has utilised two major surface antigens (SPAG-1 and Tams1) and conventional adjuvants to provide partial protection against parasite challenge. In this study we have delivered these antigens using the prime-boost system and analysed whether a combination regime can enhance protection against lethal challenge. Delivery of the boost as recombinant protein or expressed from a recombinant MVA vector was also assessed. The results confirmed that immunisation with Tams1 alone could reduce the severity of several disease parameters compared to non-immunised controls and these effects were more marked when recombinant protein was used for boosting compared to MVA delivery. A similar outcome was obtained by immunisation with SPAG-1 alone. Significantly, delivery of SPAG-1 and Tams1 as a cocktail showed enhanced protection. This was manifest by significant improvement in a large range of clinical and parasitological parameters and, most dramatically, by the survival and recovery of 50% of the immunised animals compared to 0% of the controls. Analysis of the antibody response post-challenge showed that while there was a strong response to Tams1, no response to SPAG-1 was detected. In contrast, lymphoproliferation assays showed a significant enhancement of response at day 7 post-challenge in calves of the SPAG-1 group but a dramatic decrease of the proliferation activity in all three groups receiving Tams1. We conclude that immunisation with a cocktail of SPAG-1 and Tams1 generates a synergistic protective response that significantly improves the efficacy of recombinant vaccination against tropical theileriosis. Potential effector mechanisms that could mediate this response are discussed.

  13. Immunoglobulin levels of congenitally athymic rats immunized with thymus-dependent-independent antigens.

    PubMed Central

    Taubman, M A; Ebersole, J L; Smith, D J

    1986-01-01

    The levels of immunoglobulins (Ig) in the serum and saliva of nude (rnu/rnu), congenitally athymic rats and control (rnu/+) rats were studied after injection in the vicinity of the salivary glands with several different T-dependent or T-independent dinitrophenylated antigens. Serum IgG levels were higher in rnu/+ rats than rnu/rnu rats after both primary and secondary immunizations. WHile serum IgM levels were higher in rnu/+ rats after primary immunization, secondary immunization with the most thymic-dependent antigens elicited higher IgM levels in rnu/rnu rats. After primary immunization, serum IgA levels in rnu/rnu rats were significantly lower than rnu/+ levels but no differences between the groups were noted after secondary immunization. Primary immunization with these antigens also demonstrated a markedly reduced salivary IgA producing capability by the athymic rats. Importantly, no such differences in serum or salivary IgA were previously found in the absence of immunization. Thus, immunization accentuated differences in Ig level and isotype distribution between rnu/rnu and rnu/+ rats. PMID:3486816

  14. Expression of cell surface antigens on mast cells: mast cell phenotyping.

    PubMed

    Hauswirth, Alexander W; Florian, Stefan; Schernthaner, Gerit-Holger; Krauth, Maria-Theresa; Sonneck, Karoline; Sperr, Wolfgang R; Valent, Peter

    2006-01-01

    During the past few decades, a number of functionally important cell surface antigens have been detected on human mast cells (MCs). These antigens include the stem cell factor receptor (SCFR/CD117), the high-affinity immunoglobulin E receptor, adhesion molecules, and activation-linked membrane determinants. Several of these antigens (CD2, CD25, CD35, CD88, CD203c) appear to be upregulated on MCs in patients with systemic mastocytosis and therefore are used as diagnostic markers. Quantitative measurement of these markers on MCs is thus of diagnostic value and is usually performed by multicolor-based flow cytometry techniques utilizing a PE- or APC-labeled antibody against CD117 for MCs detection. This chapter gives an overview about the methods of staining of MC in various tissues with special reference to novel diagnostic markers applied in patients with suspected systemic mastocytosis.

  15. Demonstration of a surface antigen of Clostridium tyrobutyricum by use of immunoblotting with a monoclonal antibody.

    PubMed

    Gueguen, F; Robreau, G; Talbot, F; Malcoste, R

    1990-01-01

    A monoclonal antibody, prepared against whole cells of Clostridium tyrobutyricum, recognized a surface antigen extracted by heat treatment or by hot phenol-water treatment. This antigen, after analysis by polyacrylamide gel electrophoresis and immunoblotting, has been shown to present a regularly-spaced ladder pattern similar to those shown by the lipopolysaccharide of many gram-negative bacteria. The proteinase K has been shown to have no effect on the recognition of this epitope by the monoclonal antibody. On the contrary, the inhibition of the antigen reactivity to the monoclonal antibody after a mild periodate oxidation suggests the involvement of a carbohydrate moiety in the epitope. Moreover, the SDS-PAGE analysis of phenol-water extracts has shown an additional compound, detected by silver staining but not recognized by the monoclonal antibody.

  16. [Quantification of the hepatitis B surface antigen in the characterization and follow-up].

    PubMed

    Rodríguez, Manuel; González-Diéguez, María Luisa

    2014-07-01

    The recent availability of commercial techniques for the quantification of the hepatitis B surface antigen (HBsAg) has revived interest in this antigen. In recent years, the antigen's potential as a biomarker of the natural history of the disease and its response to antiviral treatment has been assessed. HBsAg serum values reflect the transcriptional activity of cccDNA; reading these values could therefore complement the reading of hepatitis B virus (HBV) DNA in the categorization of the various phases of chronic HBV infection. During its natural history, HBsAg values progressively decrease from the immune-tolerant phase to the inactive carrier phase. For patients who are HBeAg-negative, the combined reading of HBV DNA and HBsAg can be useful for differentiating inactive carriers from patients with chronic HBeAg-negative hepatitis, for stratifying the risk of developing hepatocarcinoma and for predicting HBsAg clearance.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-01-01

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

  19. Expression and functional properties of the Streptococcus intermedius surface protein antigen I/II.

    PubMed

    Petersen, F C; Pasco, S; Ogier, J; Klein, J P; Assev, S; Scheie, A A

    2001-07-01

    Streptococcus intermedius is associated with deep-seated purulent infections. In this study, we investigated expression and functional activities of antigen I/II in S. intermedius. The S. intermedius antigen I/II appeared to be cell surface associated, with a molecular mass of approximately 160 kDa. Northern blotting indicated that the S. intermedius NCTC 11324 antigen I/II gene was transcribed as a monocistronic message. Maximum expression was seen during the early exponential phase. Insertional inactivation of the antigen I/II gene resulted in reduced hydrophobicity during early exponential phase, whereas no effect was detected during mid- and late exponential phases. Binding to human fibronectin and laminin was reduced in the isogenic mutant, whereas binding to human collagen types I and IV and to rat collagen type I was not significant for either the wild type or the mutant. Compared to the wild type, the capacity of the isogenic mutant to induce interleukin 8 (IL-8) release by THP-1 monocytic cells was significantly reduced. The results indicate that the S. intermedius antigen I/II is involved in adhesion to human receptors and in IL-8 induction.

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

    PubMed

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

    2015-01-01

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

  1. Prostate-Specific Antigen Velocity Before and After Elimination of Factors That Can Confound the Prostate-Specific Antigen Level

    SciTech Connect

    Park, Jessica J.; Chen, Ming-Hui; Loffredo, Marian; D'Amico, Anthony V.

    2012-03-01

    Purpose: Prostate-specific antigen (PSA) velocity, like PSA level, can be confounded. In this study, we estimated the impact that confounding factors could have on correctly identifying a patient with a PSA velocity >2 ng/ml/y. Methods and Materials: Between 2006 and 2010, a total of 50 men with newly diagnosed PC comprised the study cohort. We calculated and compared the false-positive and false-negative PSA velocity >2 ng/ml/y rates for all men and those with low-risk disease using two approaches to calculate PSA velocity. First, we used PSA values obtained within 18 months of diagnosis; second, we used values within 18 months of diagnosis, substituting the prebiopsy PSA for a repeat, nonconfounded PSA that was obtained using the same assay and without confounders. Results: Using PSA levels pre-biopsy, 46% of all men had a PSA velocity >2 ng/ml/y; whereas this value declined to 32% when substituting the last prebiopsy PSA for a repeat, nonconfounded PSA using the same assay and without confounders. The false-positive rate for PSA velocity >2 ng/ml/y was 43% as compared with a false-negative rate of PSA velocity >2 ng/ml/y of 11% (p = 0.0008) in the overall cohort. These respective values in the low-risk subgroup were 60% and 16.7% (p = 0.09). Conclusion: This study provides evidence to explain the discordance in cancer-specific outcomes among groups investigating the prognostic significance of PSA velocity >2 ng/ml/y, and highlights the importance of patient education on potential confounders of the PSA test before obtaining PSA levels.

  2. Modulation of an adhesion-related surface antigen on equine neutrophils by bacterial lipopolysaccharide and antiinflammatory drugs.

    PubMed

    Bochsler, P N; Slauson, D O; Neilsen, N R

    1990-10-01

    . These data suggest that equine PMN express a functional adhesion molecule similar to those found on human PMN and that LPS may enhance the expression of this surface antigen. Expression of this adhesion-related surface antigen on equine PMN does not correlate well with levels of drug-induced diminished adhesion of PMN to endothelium in vitro.

  3. A mimotope of pre-S2 region of surface antigen of viral hepatitis B screened by phage display.

    PubMed

    Zhang, W Y; Wan, Y; Li, D G; Tang, Y; Zhou, W

    2001-09-01

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

  4. Generalized immunological recognition of the major merozoite surface antigen (gp195) of Plasmodium falciparum

    SciTech Connect

    Chang, S.P.; Hui, G.S.N.; Kato, A.; Siddiqui, W.A. )

    1989-08-01

    The antibody response to the Plasmodium falciparum major merozoite surface antigen (gp195) of congenic mouse strains differing in H-2 haplotype has been examined. All seven strains of mice were capable of producing gp195-specific antibodies. Generalized immune recognition of gp195 by mice of diverse H-2 haplotypes distinguished gp195 from the P. falciparum circumsporozoite protein and the 230-kDa and 48/45-kDa gamete surface antigens. However, the H-2 genetic locus appeared to influence the specificity of gp105-specific antibodies. Immunoblot patterns of mouse sera with parasite antigens revealed a complex pattern of reactivity with terminal and intermediate processing fragments of gp195. The majority of immunoblot bands observed were similar for all of the mouse strains; however, there were several strains that additionally recognized a few unique fragments or displayed more intense reactivities with specific processing fragments. These results suggest that while individuals of diverse major histocompatibility complex makeup are capable of recognizing the gp195 antigen, the recognition of specific gp195 B-cell and T-cell epitopes may be under control of the major histocompatibility complex.

  5. Flow cytometry protocols for surface and intracellular antigen analyses of neural cell types.

    PubMed

    Menon, Vishal; Thomas, Ria; Ghale, Arun R; Reinhard, Christina; Pruszak, Jan

    2014-12-18

    Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of protocols for flow cytometric analysis of the cluster of differentiation (CD) surface antigens and intracellular antigens in neural cell types. Our step-by-step description of the methodological procedures include: the harvesting of neural in vitro cultures, an optional carboxyfluorescein succinimidyl ester (CFSE)-labeling step, followed by surface antigen staining with conjugated CD antibodies (e.g., CD24, CD54), and subsequent intracellar antigen detection via primary/secondary antibodies or fluorescently labeled Fab fragments (Zenon labeling). The video demonstrates the most critical steps. Moreover, principles of experimental planning, the inclusion of critical controls, and fundamentals of flow cytometric analysis (identification of target population and exclusion of debris; gating strategy; compensation for spectral overlap) are briefly explained in order to enable neurobiologists with limited prior knowledge or specific training in flow cytometry to assess its utility and to better exploit this powerful methodology.

  6. Functional variation of the antigen I/II surface protein in Streptococcus mutans and Streptococcus intermedius.

    PubMed

    Petersen, F C; Assev, S; van der Mei, H C; Busscher, H J; Scheie, A A

    2002-01-01

    Although Streptococcus intermedius and Streptococcus mutans are regarded as members of the commensal microflora of the body, S. intermedius is often associated with deep-seated purulent infections, whereas S. mutans is frequently associated with dental caries. In this study, we investigated the roles of the S. mutans and S. intermedius antigen I/II proteins in adhesion and modulation of cell surface characteristics. By using isogenic mutants, we show that the antigen I/II in S. mutans, but not in S. intermedius, was involved in adhesion to a salivary film under flowing conditions, as well as in binding to rat collagen type I. Binding to human fibronectin was a common function associated with the S. mutans and S. intermedius antigen I/II. Adhesion of S. mutans or S. intermedius to human collagen types I or IV was negligible. Hydrophobicity, as measured by water contact angles, and zeta potentials were unaltered in the S. intermedius mutant. The S. mutans isogenic mutants, on the other hand, exhibited more positive zeta potentials at physiological pH values than did the wild type. The results indicate common and species-specific roles for the antigen I/II in mediating the attachment of S. mutans and S. intermedius to host components and in determining cell surface properties.

  7. Antigenicity of UV radiation-induced murine tumors correlates positively with the level of adenosine deaminase activity.

    PubMed

    Aukerman, S L; Fidler, I J

    1987-01-01

    The specific activities of adenosine deaminase (ADA) in 16 murine tumor cell lines derived from seven UV light-induced neoplasms (melanoma and fibrosarcoma) were determined. In each case, the specific activity of ADA correlated positively with the antigenicity of the tumor cells. Highly antigenic cell lines that regress upon introduction into syngeneic hosts had on average 4- to 6-fold higher ADA specific activities than cell lines of low antigenicity that grow progressively in syngeneic hosts. The antigenic differences are probably not related to intracellular cAMP levels, as the level of cAMP differed only 2-fold between the two groups of cell lines.

  8. HLA class I is most tightly linked to levels of tapasin compared with other antigen-processing proteins in glioblastoma

    PubMed Central

    Thuring, Camilla; Follin, Elna; Geironson, Linda; Freyhult, Eva; Junghans, Victoria; Harndahl, Mikkel; Buus, Søren; Paulsson, Kajsa M

    2015-01-01

    Background: Tumour cells can evade the immune system by dysregulation of human leukocyte antigens (HLA-I). Low quantity and/or altered quality of HLA-I cell surface expression is the result of either HLA-I alterations or dysregulations of proteins of the antigen-processing machinery (APM). Tapasin is an APM protein dedicated to the maturation of HLA-I and dysregulation of tapasin has been linked to higher malignancy in several different tumours. Methods: We studied the expression of APM components and HLA-I, as well as HLA-I tapasin-dependency profiles in glioblastoma tissues and corresponding cell lines. Results: Tapasin displayed the strongest correlation to HLA-I heavy chain but also clustered with β2-microglobulin, transporter associated with antigen processing (TAP) and LMP. Moreover, tapasin also correlated to survival of glioblastoma patients. Some APM components, for example, TAP1/TAP2 and LMP2/LMP7, showed variable but coordinated expression, whereas ERAP1/ERAP2 displayed an imbalanced expression pattern. Furthermore, analysis of HLA-I profiles revealed variable tapasin dependence of HLA-I allomorphs in glioblastoma patients. Conclusions: Expression of APM proteins is highly variable between glioblastomas. Tapasin stands out as the APM component strongest correlated to HLA-I expression and we proved that HLA-I profiles in glioblastoma patients include tapasin-dependent allomorphs. The level of tapasin was also correlated with patient survival time. Our results support the need for individualisation of immunotherapy protocols. PMID:26313662

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

    PubMed

    Uchida, Tetsuya; Taneichi, Maiko

    2014-01-01

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

  10. Detection and Characterization of Autoantibodies to Neuronal Cell-Surface Antigens in the Central Nervous System

    PubMed Central

    van Coevorden-Hameete, Marleen H.; Titulaer, Maarten J.; Schreurs, Marco W. J.; de Graaff, Esther; Sillevis Smitt, Peter A. E.; Hoogenraad, Casper C.

    2016-01-01

    Autoimmune encephalitis (AIE) is a group of disorders in which autoantibodies directed at antigens located on the plasma membrane of neurons induce severe neurological symptoms. In contrast to classical paraneoplastic disorders, AIE patients respond well to immunotherapy. The detection of neuronal surface autoantibodies in patients’ serum or CSF therefore has serious consequences for the patients’ treatment and follow-up and requires the availability of sensitive and specific diagnostic tests. This mini-review provides a guideline for both diagnostic and research laboratories that work on the detection of known surface autoantibodies and/or the identification of novel surface antigens. We discuss the strengths and pitfalls of different techniques for anti-neuronal antibody detection: (1) Immunohistochemistry (IHC) and immunofluorescence on rat/primate brain sections; (2) Immunocytochemistry (ICC) of living cultured hippocampal neurons; and (3) Cell Based Assay (CBA). In addition, we discuss the use of immunoprecipitation and mass spectrometry analysis for the detection of novel neuronal surface antigens, which is a crucial step in further disease classification and the development of novel CBAs. PMID:27303263

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

    NASA Astrophysics Data System (ADS)

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

    2007-04-01

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

  12. Chemical Characterization of a New Surface Antigenic Polysaccharide from a Mutant of Staphylococcus aureus

    PubMed Central

    Wu, Teresa C. M.; Park, James T.

    1971-01-01

    A mutant of Staphylococcus aureus H was isolated by virtue of its inability to agglutinate with antibodies against teichoic acid of S. aureus. Immunological studies revealed that the mutant, S. aureus T, possessed a new surface antigen in addition to having the antigenic determinant of the wild-type strain, the ribitol teichoic acid. The presence of this additional surface component rendered strain T resistant to staphylococcal typing phages, presumably by masking the phage-receptor sites. The polymer was separated from teichoic acid by chromatography on diethylaminoethyl cellulose and was shown to be composed of two amino sugars, N-acetyl-d-fucosamine and N-acetyl-d-mannosamin uronic acid. Images PMID:5001874

  13. Cell surface antigens of human trophoblast: definition of an apparently unique system with a monoclonal antibody.

    PubMed Central

    Mueller, U W; Hawes, C S; Jones, W R

    1986-01-01

    An epitope with apparent specificity for the surface of human syncytiotrophoblast was defined by a murine monoclonal antibody, FDO46B (IgG1, kappa). The epitope was predominantly expressed during the first trimester of pregnancy. Binding was detected on frozen tissue sections and on cultured trophoblast by the immunoperoxidase technique. It was also detected on the surface of a small percentage (less than 10%) of cultured choriocarcinoma cells (JEG-3). A panel of human tissues was negative, as were normal and malignant human lymphocytes. The antigen bearing the FDO46B epitope was still expressed by trophoblast after culture in the presence of tunicamycin, indicating that it is possibly protein in nature. This antigen may have potential utility as a target for a contraceptive vaccine. Images Figure 1 Figure 2 Figure 3 PMID:2428734

  14. Impact of positive hepatitis B surface antigen on the outcome of IVF treatment.

    PubMed

    Lee, Vivian Chi Yan; Ng, Ernest Hung Yu; Yeung, William Shu Biu; Ho, Pak Chung

    2010-11-01

    There has been controversy about the effect of hepatitis B virus (HBV) infection on pregnancy outcome after IVF treatment. A total of 1676 couples undergoing their first IVF cycle were included in this study. The prevalence of HBV infection in the female partners of the subfertile population seeking treatment with assisted reproductive technology was 7.8-9.6% during the study period. The ongoing pregnancy rate was not significantly different between couples with HBV-seropositive wives and seronegative ones (26.7% versus 30.2%). The ongoing pregnancy rate and the live-birth rate of couples with both partners being HBV surface antigen positive was not significantly different from couples with discordant HBV serostatus and those couples with both partners being HBV surface antigen negative (23% versus 29% versus 30%, respectively; 23% versus 27% versus 27%, respectively). The percentage of normal sperm morphology in HBV-seropositive husbands was significantly lower than that of seronegative counterparts (5.0% versus 10.0%, P=0.009). In conclusion, there was no adverse effect of HBV infection on the assisted reproduction outcomes. This study was the first report on the live-birth rate of hepatitis B (HBV) seropositive couples, revealing the effect on the pregnancy outcome of IVF. The prevalence of HBV infection in the female partners of subfertile population seeking treatment with ART was 7.8-9.6%. Both the ongoing pregnancy rate and live-birth rate were not significantly different among couples with both partners being positive for HBV surface antigen, couples with discordant HBV serostatus and those couples with both partners being negative for HBV surface antigen. The percentage of normal sperm morphology in HBV-seropositive husbands was significantly lower than that of seronegative counterparts.

  15. Fast, antigen-saving multiplex immunoassay to determine levels and avidity of mouse serum antibodies to pertussis, diphtheria, and tetanus antigens.

    PubMed

    Stenger, Rachel M; Smits, Mieke; Kuipers, Betsy; Kessen, Sabine F M; Boog, Claire J P; van Els, Cécile A C M

    2011-04-01

    To enhance preclinical evaluation of serological immune responses to the individual diphtheria, tetanus, and pertussis (DTP) components of DTP combination vaccines, a fast hexavalent bead-based method was developed. This multiplex immunoassay (MIA) can simultaneously determine levels of specific mouse serum IgG antibodies to P antigens P.69 pertactin (P.69 Prn), filamentous hemagglutinin (FHA), pertussis toxin (Ptx), and combined fimbria type 2 and 3 antigens (Fim2/3) and to diphtheria toxin (Dtx) and tetanus toxin (TT) in a single well. The mouse DTP MIA was shown to be specific and sensitive and to correlate with the six single in-house enzyme-linked immunosorbent assays (ELISAs) for all antigens. Moreover, the MIA was expanded to include avidity measurements of DTP antigens in a multivalent manner. The sensitivities of the mouse DTP avidity MIA per antigen were comparable to those of the six individual in-house avidity ELISAs, and good correlations between IgG concentrations obtained by both methods for all antigens tested were shown. The regular and avidity mouse DTP MIAs were reproducible, with good intra- and interassay coefficients of variability (CV) for all antigens. Finally, the usefulness of the assay was demonstrated in a longitudinal study of the development and avidity maturation of specific IgG antibodies in mice having received different DTP vaccines. We conclude that the hexaplex mouse DTP MIA is a specific, sensitive, and high-throughput alternative for ELISA to investigate the quantity and quality of serological responses to DTP antigens in preclinical vaccine studies.

  16. Age-dependent B cell Autoimmunity to a Myelin Surface Antigen in Pediatric Multiple Sclerosis

    PubMed Central

    McLaughlin, Katherine A.; Chitnis, Tanuja; Newcombe, Jia; Franz, Bettina; Kennedy, Julia; McArdel, Shannon; Kuhle, Jens; Kappos, Ludwig; Rostasy, Kevin; Pohl, Daniela; Gagne, Donald; Ness, Jayne M.; Tenembaum, Silvia; O'Connor, Kevin C.; Viglietta, Vissia; Wong, Susan J.; Tavakoli, Norma P.; de Seze, Jerome; Khoury, Samia J.; Bar-Or, Amit; Hafler, David A.; Banwell, Brenda; Wucherpfennig, Kai W.

    2009-01-01

    Multiple sclerosis (MS) typically manifests in early to mid adulthood, but there is increasing recognition of pediatric-onset MS, aided by improvements in imaging techniques. The immunological mechanisms of disease are largely unexplored in pediatric-onset MS, in part because studies have historically focused on adult-onset disease. We investigated autoantibodies to myelin surface antigens in a large cohort of pediatric MS cases by flow cytometric labeling of transfectants that expressed different myelin proteins. While antibodies to native myelin oligodendrocyte glycoprotein (MOG) were uncommon among adult-onset patients, a subset of pediatric patients had serum antibodies that brightly labeled the MOG transfectant. Antibodies to two other myelin surface antigens were largely absent. Affinity purification of MOG antibodies as well as competition of binding with soluble MOG documented their binding specificity. The prevalence of such autoantibodies was highest among patients with a very early onset of MS: 38.7% of patients less than 10 years of age at disease onset had MOG antibodies, compared to 14.7% of patients in the 10–18 year age group. B cell autoimmunity to this myelin surface antigen is therefore most common in patients with a very early onset of MS. PMID:19687098

  17. Variable expression of activation-linked surface antigens on human mast cells in health and disease.

    PubMed

    Valent, P; Schernthaner, G H; Sperr, W R; Fritsch, G; Agis, H; Willheim, M; Bühring, H J; Orfao, A; Escribano, L

    2001-02-01

    Mast cells (MC) are multipotent effector cells of the immune system. They contain an array of biologically active mediator substances in their granules. MC also express a number of functionally important cell surface antigens, including stem cell factor receptor (SCFR=kit=CD117), high affinity IgER (FcepsilonRI), or CSaR (CD88). Respective ligands can induce or promote degranulation, migration, or cytokine production. Other integral surface molecules can mediate adhesion or cell aggregation. Recent data suggest that a number of critical molecules are variably expressed on the surface of human MC. In fact, depending on the environment (organ), stage of cell maturation, type of disease, and other factors, MC express variable amounts of activation-linked antigens (CD25, CD63, CD69, CD88), cell recognition molecules (CD2, CD11, CD18, CD50, CD54), or cytokine receptors. At present, however, little is known about the mechanisms and regulation of expression of such antigens. The present article gives an overview of MC phenotypes in health and disease, and attempts to provide explanations for the phenotypic variability of MC.

  18. Enzyme-linked immunosorbent assays for detection of equine antibodies specific to Sarcocystis neurona surface antigens.

    PubMed

    Hoane, Jessica S; Morrow, Jennifer K; Saville, William J; Dubey, J P; Granstrom, David E; Howe, Daniel K

    2005-09-01

    Sarcocystis neurona is the primary causative agent of equine protozoal myeloencephalitis (EPM), a common neurologic disease of horses in the Americas. We have developed a set of enzyme-linked immunosorbent assays (ELISAs) based on the four major surface antigens of S. neurona (SnSAGs) to analyze the equine antibody response to S. neurona. The SnSAG ELISAs were optimized and standardized with a sample set of 36 equine sera that had been characterized by Western blotting against total S. neurona parasite antigen, the current gold standard for S. neurona serology. The recombinant SnSAG2 (rSnSAG2) ELISA showed the highest sensitivity and specificity at 95.5% and 92.9%, respectively. In contrast, only 68.2% sensitivity and 71.4% specificity were achieved with the rSnSAG1 ELISA, indicating that this antigen may not be a reliable serological marker for analyzing antibodies against S. neurona in horses. Importantly, the ELISA antigens did not show cross-reactivity with antisera to Sarcocystis fayeri or Neospora hughesi, two other equine parasites. The accuracy and reliability exhibited by the SnSAG ELISAs suggest that these assays will be valuable tools for examining the equine immune response against S. neurona infection, which may help in understanding the pathobiology of this accidental parasite-host interaction. Moreover, with modification and further investigation, the SnSAG ELISAs have potential for use as immunodiagnostic tests to aid in the identification of horses affected by EPM.

  19. Detection of antibodies against hepatitis B virus surface antigen and hepatitis C virus core antigen in plasma with a waveguide-mode sensor.

    PubMed

    Shimizu, Takenori; Tanaka, Torahiko; Uno, Shigeyuki; Ashiba, Hiroki; Fujimaki, Makoto; Tanaka, Mutsuo; Awazu, Koichi; Makishima, Makoto

    2017-02-09

    In large-scale disasters, such as huge significant earthquakes, on-site examination for blood typing and infectious disease screening will be very helpful to save lives of victims who need surgical treatment and/or blood transfusion. However, physical damage, such as building collapse, electric power failure and traffic blockage, disrupts the capacity of the medical system. Portable diagnostic devices are useful in such cases of emergency. In this study, we evaluated a waveguide-mode sensor for detection of anti-hepatitis virus antibodies. First, we examined whether we can detect antigen-antibody interaction on a sensor chip immobilized hepatitis B virus surface (HBs) antigen and hepatitis C virus (HCV) core antigen using monoclonal mouse antibodies for HBs antigen and HCV core antigen. We obtained significant changes in the reflectance spectra, which indicate specific antigen-antibody interaction for anti-HBs antibody and anti-HCV antibody. Next, we examined the effect of horseradish peroxidase-conjugated secondary antibody using aminoethyl carbazole as the peroxidase substrate and found that the colorimetric reaction increases detection sensitivity for anti-HBs antibody more than 300 times. Finally, we successfully detected anti-HBs antibody in human blood samples with an enhancing method using a peroxidase reaction. Thus, a portable device utilizing a waveguide-mode sensor may be applied to on-site blood testing in emergency settings.

  20. Prevalence of hepatitis B surface antigen among women receiving prenatal care at the Palm Beach County Health Department.

    PubMed

    Malecki, J M; Guarin, O; Hulbert, A; Brumback, C L

    1986-03-01

    Pregnant women receiving prenatal care at the maternity clinics of the Palm Beach County Health Department were tested for hepatitis B surface antigen. Routine screening of pregnant women for the antigen is discussed. The results of this study indicate the need for routine screening of our medically indigent pregnant population.

  1. [Clinical studies of sixteen cases with pulmonary cryptococcosis mainly with respect to serum level of cryptococcal antigen].

    PubMed

    Dohtsu, Yasumasa; Ishimatsu, Yuji; Takatani, Hiroshi; Minami, Kazunori; Inoue, Keiji; Kohara, Norihiro; Yanagihara, Katsunori; Higashiyama, Yasuhito; Miyazaki, Yoshitsugu; Hirakata, Yoichi; Kohno, Shigeru

    2005-09-01

    Clinical studies of sixteen cases with pulmonary cryptococcosis, during the past six years between 1998 and 2004, were peformed mainly with respect to serum cryptococcal antigen titer. Serum cryptococcal antigen was positive in twelve of 16 cases, the other three cases were diagnosed by VATS, the other one by positive culture of cryptococcus in BALF. In these twelve cases, the serum cryptococcal antigen titer was continuously tested after treatment. The serum cryptococcal antigen titer decreased from half to 6 months after treatment. And the cryptococcal Ag changed to negative in six of the 12 cases by antifungal agents from 5 to 19 months. But four cases whose pneumonia was severe tended to have a high titer level of cryptococcal antigen and were positive for a long period. In the Chest CT of four pulmonary cryptococcosis case with negative cryptococcal antigen, all of the maximum nodule size was less than or equal to 15mm in diameter.

  2. A strategy of antigen incorporation into exosomes: comparing cross-presentation levels of antigens delivered by engineered exosomes and by lentiviral virus-like particles.

    PubMed

    Lattanzi, Laura; Federico, Maurizio

    2012-11-26

    Among strategies aimed at developing new nanoparticle-based vaccines, exosomes hold much promise. They are nanovesicles released by basically all eukaryotic cell types originating from intraluminal vesicles which accumulate in multivesicular bodies. Exosomes have immunogenic properties whose strength correlates with the amounts of associated antigens. Engineering antigens to target them in exosomes represents the last frontier in terms of nanoparticle-based vaccines. Here we report a new method to incorporate protein antigens in exosomes relying on the unique properties of a mutant of the HIV-1 Nef protein, Nef(mut). This is a biologically inactive mutant we found incorporating into exosomes at high levels also when fused at its C-terminus with foreign proteins. We compared both biochemical and antigenic properties of Nef(mut) exosomes with those of previously characterized Nef(mut) -based lentiviral virus-like particles (VLPs). We found that exosomes incorporate Nef(mut) and fusion protein derivatives with similar efficiency of VLPs. When an envelope fusion protein was associated with both exosomes and VLPs to favor cross-presentation of associated antigens, Nef(mut) and its derivatives incorporated in exosomes were cross-presented at levels at least similar to what observed when the antigens were delivered by engineered VLPs. This occurred despite exosomes entered target cells with an apparent lower efficiency than VLPs. The unique properties of HIV-1 Nef(mut) in terms of exosome incorporation efficiency, carrier of foreign antigens, and lack of anti-cellular effects open the way toward the development of a flexible, safe, cost-effective exosome-based CD8(+) T cell vaccine platform.

  3. "Mycoplasmal antigen modulation," a novel surface variation suggested for a lipoprotein specifically localized on Mycoplasma mobile.

    PubMed

    Wu, Heng Ning; Kawaguchi, Chie; Nakane, Daisuke; Miyata, Makoto

    2012-05-01

    Mycoplasma mobile, a pathogen of freshwater fish, glides easily across surfaces, colonizes on the fish gill, and causes necrosis. The cell surface is differentiated into three parts: the head, neck, and body. Mobile variable surface proteins (Mvsps) localizing at each of these parts may be involved in surface variation including phase variation and antigenic variation, although no proof exists. In this study, we examined this possibility by focusing on MvspI, the largest Mvsp. Immunofluorescence microscopy showed that MvspI is expressed on the surfaces of all cells. When anti-MvspI antibody was added at concentrations over 0.8 nM, MvspI was observed to decrease over time. After 72 h of cultivation with the antibody, the fluorescence intensity and amount of MvspI decreased up to 13 and 39%, respectively, compared to those of cells grown without antibody. These changes were reversed by the removal of the antibody. Such effects were not observed when another antibody targeting other Mvsps was used, suggesting that the decrease is specific to the relationship between MvspI and the antibody. Cell growth was also inhibited by the antibody, but the decrease in MvspI could not be explained by the selective growth of MvspI-negative variants or by the inhibition of growth with other conditions. The decrease in MvspI caused by the antibody binding may suggest a novel type of surface variation, designated here as "mycoplasmal antigen modulation."

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

    PubMed

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

    2011-12-01

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

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

    PubMed Central

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

    2016-01-01

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

  6. Levels of Schistosoma mansoni Circulating Antigen in Chronic Hepatitis C Patients with Different Stages of Liver Fibrosis.

    PubMed

    Attallah, Abdelfattah M; El-Far, Mohamed; Omran, Mohamed M; Farid, Khaled; Attallah, Ahmed A; Abd-Elaziz, Dalal; El-Bendary, Mohamed S; El-Dosoky, Ibrahim; Ismail, Hisham

    2016-01-01

    The goal of this study was to determine the levels of S. mansoni antigen in different liver fibrosis stages with chronic hepatitis C (CHC) Egyptian patients. A total of 174 CHC patients showing HCV-NS4 antigen and HCV- RNA in their sera were included. S. mansoni antigen was detected in serum using Western blot and ELISA. The levels of interferon-γ (IFN- γ) were determined using ELISA. The 50 kDa S. mansoni antigen discriminated patients infected with S. mansoni from healthy individuals with 0.93 area under curve (AUC), 92% sensitivity, and 97% specificity. The level of S. mansoni antigen (μg/ml) was significantly (P < 0.0001) increased with the progression of liver fibrosis stages (26.9 ± 17.5 in F1, 42.1 ± 25.2 in F2, 49.8 ± 30.3 in F3 and 62.2 ± 26.3 μg/mL in F4 liver cirrhosis), 26.9 ± 17.59 in significant fibrosis (F2-F4); 51.2 ± 27.9 in advanced fibrosis (F3-F4). A significant correlation (r = 0.506; P < 0.0001) was shown between the levels of the S. mansoni antigen and the HCV-NS4 antigen. In conclusion, the presence of S. mansoni antigen in different liver fibrosis stages of CHC patients confirming that concomitant schistosome infection aggravates liver disease.

  7. The underlying mechanisms for the "isolated positivity for the hepatitis B surface antigen (HBsAg)" serological profile.

    PubMed

    Pondé, Robério Amorim de Almeida

    2011-02-01

    During HBV infection, four structural antigen/antibody systems are observed: hepatitis B surface antigen (HBsAg) and its antibody (anti-HBs); the pre-S antigens associated with HBsAg particles and their antibodies; the particulate nucleocapsid antigen (HBcAg) and anti-HBc; and an antigen structurally related to HBcAg, namely HBeAg and its antibody (anti-HBe). Through the examination of this antigen-antibodies system, hepatitis B infection is diagnosed and the course of the disorder may be observed. Isolated HBsAg seropositivity is a peculiar serological pattern in HBV infection observed some times in routine laboratory. In most cases is not clear how this profile should be interpreted neither its significance. This pattern, however, may be associated with some clinical and laboratorial situations of great relevance, some of which will be addressed in this article.

  8. Characterization of antibody binding to cell surface antigens using a plasma membrane-bound plate assay.

    PubMed

    Vater, C A; Reid, K; Bartle, L M; Goldmacher, V S

    1995-01-01

    A procedure has been developed for measuring antibody binding to cell surface antigens using an immobilized plasma membrane fraction. In this method, isolated plasma membranes are dried onto wells of a 96-well microtiter plate and incubated with antibodies that recognize a cell surface protein. Bound antibody is detected indirectly using an enzyme-linked or fluorescently tagged second antibody. Alternatively, the primary antibody itself can be labeled and its binding can be detected directly. The assay is simple and fast and provides several advantages over whole cell binding assays currently in widespread use.

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

    PubMed

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

    1999-06-01

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

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

    PubMed

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

    2017-02-17

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

  11. [The potentials for immunization against influenza using liposome-incorporated viral surface antigens].

    PubMed

    Burducea, O; Marcheş, F; Duţu, C; Grancea, C; Nicolau, A; Păun, C

    1989-01-01

    Studies were conducted using uni- and multilamellar liposomes to establish optimum conditions for influenza antigen incorporation in view of their transport to the target cells for experimental influenza prophylaxis in hybrid white mice. Radiometric determinations showed a good level of preparation purification, a good efficiency of incorporation in liposomes of the active biological material, the liposome linked radioactivity distribution among different organs. Charged liposomes induced solid and long lasting resistance against influenza control infection.

  12. Elevated levels of a glycoprotein antigen (P-80) in gray and white matter of brain from victims of multiple sclerosis.

    PubMed

    Cruz, T F; Quackenbush, E J; Letarte, M; Moscarello, M A

    1986-06-01

    The levels of a glycoprotein reactive with monoclonal antibody (MAb) 44D10 in white and gray matter from brains of victims of several neurological diseases, including Multiple Sclerosis, Alzheimer's, Parkinson's and Huntington's diseases, were compared to that of normal individuals. The concentration of antigen reactive with MAb 44D10 was elevated in both gray and white matter of all MS brains examined, but not in brains with other neurological diseases. The increase in the concentration of antigen varied amongst the MS brains, such that the levels of antigen were only slightly increased in 2 of the 6 MS brains whereas 2 to 4 fold higher levels were found in the other 4 brains. Increased levels of antigen were detected in gray matter of MS brains, whereas this antigen was either not detected or present in very low levels in gray matter homogenates prepared from age-matched normal brains. MAb Leu 1, which reacts with T lymphocytes, was not absorbed by normal and MS brain tissue suggesting the increase in antigen reactive with MAb 44D10 in MS brain homogenates was not associated with non-specific infiltration by T lymphocytes. Comparison of the purified antigen from MS gray matter and normal white matter by gel electrophoresis demonstrated that MAb 44D10 was reacting with a similar protein in both tissues with an apparent molecular weight of 80K. We have named this molecule P-80 glycoprotein.

  13. Ciliated hepatic foregut cyst with high intra-cystic carbohydrate antigen 19-9 level.

    PubMed

    Ben Ari, Ziv; Cohen-Ezra, Oranit; Weidenfeld, Jonathan; Bradichevsky, Tania; Weitzman, Ella; Rimon, Uri; Inbar, Yael; Amitai, Michal; Bar-Zachai, Barak; Eshkenazy, Roni; Ariche, Arie; Azoulay, Daniel

    2014-11-21

    A ciliated hepatic foregut cyst (CHFC) is a rare foregut developmental malformation usually diagnosed in adulthood. Five percent of reported cases of CHFC transform into squamous cell carcinoma. We report the presentation, evaluation, and surgical management of a symptomatic 45-year-old male found to have a 6.2 cm CHFC. Contrast tomography-guided fine-needle aspiration demonstrated columnar, ciliated epithelium consistent with the histologic diagnosis of CHFC. The intracystic levels of carbohydrate antigen (CA) 19-9 and carcinoembryonic antigen (CEA) were extremely high (978118 U/mL and 973 μg/L, respectively). Histologically, the wall of the cyst showed characteristic pseudopapillae lined with a ciliated stratified columnar epithelium, underlying smooth muscle, an outer fibrous layer and no atypia. Immunohistochemistry for CA19-9 and CEA was positive. This is the first case report of a CHFC in which levels of CA 19-9 and CEA were measured. Our findings suggest that a large sized multilocular cyst and elevated cyst CA19-9 and CEA levels do not exclude a CHFC from consideration in the diagnosis. CHFCs should be included in the differential diagnosis of hepatic lesions. Accurate diagnosis of a CHFC is necessary given its potential for malignant transformation, and surgical excision is recommended.

  14. Comparing Assay Performance of ELISA and Chemiluminescence Immunoassay in Detecting Antibodies to Hepatitis B Surface Antigen

    PubMed Central

    Sagar, Siddharth; Vishwanath, Shashidhar; Banerjee, Barnini; Eshwara, Vandana Kalwaje; Chawla, Kiran

    2016-01-01

    Introduction Antibodies to Hepatitis B surface Antigen (Anti-HBs) levels are measured as markers for immune response to vaccination and in decision making for post-exposure prophylaxis against Hepatitis-B. Several immunoassay formats are used to measure Anti-HBs, thus carrying the possibility of variation in measured levels between different assays. This study compares the performance of Chemiluminescence Immunoassay (CLIA) against Enzyme-linked Immunosorbent Assay (ELISA) in measuring Anti-HBs titer by looking into concordance between the two test reports. Aim To compare the agreement between ELISA and CLIA in measurement of Anti–HBs antibody titers. Materials and Methods This prospective comparative study conducted at Kasturba Medical College, Manipal measured consecutive serum samples (69) sent for anti-HBs levels during May-June 2016 using both CLIA (Abbott Architect) and ELISA (Bio-Rad). Anti-HBs values of ≤10mIU/ml was considered as non-protective and >10mIU/ml as protective. The agreement between the tests in classifying the antibody titers as non-protective or protective was computed using Kappa coefficient, and the difference in individual titer values between the tests compared using Bland-Altman plot on SPSS (v.15). Results Out of the 69 samples analysed, 18 samples (26.1%) were of health-care personnel and remaining of patients. Agreement between ELISA and CLIA in identifying the antibody titers as protective and non-protective were 96.5% and 90.9% respectively, resulting in an agreement of 0.84. The coefficient-of-variation of ELISA and CLIA were 74.5% and 113.1%, respectively. Three value based discordant results were noted; two samples deemed protective by ELISA were reported as non-protective by CLIA. One non-protective titer by ELISA was reported as protective by CLIA. Conclusion Analytical agreement is good between the two immunoassays. However there are some discrepancies in quantitative measurement. This may have been due the variation in

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

    PubMed Central

    Limozin, Laurent; Bongrand, Pierre; Robert, Philippe

    2016-01-01

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

  16. Novel surface antigen based impedimetric immunosensor for detection of Salmonella typhimurium in water and juice samples.

    PubMed

    Mutreja, Ruchi; Jariyal, Monu; Pathania, Preeti; Sharma, Arunima; Sahoo, D K; Suri, C Raman

    2016-11-15

    A specific surface antigen, OmpD has been reported first time as a surface biomarker in the development of selective and sensitive immunosensor for detecting Salmonella typhimurium species. The OmpD surface antigen extraction was done from Salmonella typhimurium serovars, under the optimized growth conditions for its expression. Anti-OmpD antibodies were generated and used as detector probe in immunoassay format on graphene-graphene oxide (G-GO) modified screen printed carbon electrodes. The water samples were spiked with standard Salmonella typhimurium cells, and detection was done by measuring the change in impedimetric response of developed immunosensor to know the concentration of serovar Salmonella typhimurium. The developed immunosensor was able to specifically detect S. typhimurium in spiked water and juice samples with a sensitivity upto 10(1)CFUmL(-1), with high selectivity and very low cross-reactivity with other strains. This is the first report on the detection of Salmonella typhimurum species using a specific biomarker, OmpD. The developed technique could be very useful for the detection of nontyphoidal Salmonellosis and is also important from an epidemiological point of view.

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

    NASA Astrophysics Data System (ADS)

    Limozin, Laurent; Bongrand, Pierre; Robert, Philippe

    2016-10-01

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

  18. Native surface association of a recombinant 38-kilodalton Treponema pallidum antigen isolated from the Escherichia coli outer membrane.

    PubMed Central

    Fehniger, T E; Radolf, J D; Walfield, A M; Cunningham, T M; Miller, J N; Lovett, M A

    1986-01-01

    A recombinant plasmid designated pAW305, containing a 6-kilobase insert of Treponema pallidum DNA, directed the expression of a 38-kilodalton (kDa) treponemal antigen in Escherichia coli. The 38-kDa antigen copurified with the outer membrane fraction of the E. coli cell envelope after treatment with nonionic detergents or sucrose density gradient centrifugation. Rabbits immunized with the recombinant 38-kDa antigen developed antibodies which reacted specifically with a 38-kDa T. pallidum antigen on immunoblots, and 38-kDa antisera specifically immobilized T. pallidum in a complement-dependent manner in the T. pallidum immobilization test. Antisera to the 38-kDa recombinant antigen were also used to demonstrate its native surface association on T. pallidum by immunoelectron microscopy. Images PMID:3516880

  19. Evaluation of a novel chemiluminescent microplate enzyme immunoassay for hepatitis B surface antigen detection.

    PubMed

    Yang, Lin; Song, Liu-Wei; Fang, Lin-Lin; Wu, Yong; Ge, Sheng-Xiang; Li, Hui; Yuan, Quan; Zhang, Jun; Xia, Ning-Shao

    2016-02-01

    Hepatitis B virus surface antigen (HBsAg) is an important biomarker used in the diagnosis of hepatitis B virus (HBV) infection, but false-negative results are still reported in the detection of HBsAg using commercial assays. In this study, we evaluated the qualitative properties of a novel HBsAg chemiluminescence enzyme immunoassay (CLEIA) assay--WTultra. WHO standard sample dilution series and samples from low-level HBsAg carriers (<1 ng/mL) were used to evaluate the sensitivity of the WTultra assay. Boston Biomedica, Inc. (BBI) hepatitis B seroconversion panels were used to assess the ability of the WTultra assay to detect the window period. In addition, dilution series of 22 serum samples with different genotypes, serotypes and HBsAg mutations were used to assess the WTultra assay, and these were compared with other commercial assays. The lower detection limit of the WTultra assay was 0.012 IU/mL, and it showed a high sensitivity (97.52%, 95% CI, 94.95-99.00) in the detection of 282 low-level HBsAg carriers (<1 ng/mL). In samples with various HBV genotypes, serotypes and HBsAg mutations, the WTultra assay yielded 117 positive results in 132 samples, which was significantly higher than the results with the other four commercial assays (89, 83, 65 and 45, respectively, p<0.01). In the assays of mutant strains, the WTultra assay detected 82 positive results in 90 samples, which was significantly better than the results for the Hepanostika HBsAg Ultra (58 positive) and Architect (55 positive) (p<0.01) assays, which in turn were significantly better than the Murex V.3 (41 positive, p=0.026) and AxSYM V2 (29 positive, p<0.01) assays. However, in the detection of 42 samples of wild-type strains with various genotypes and serotypes, no significant differences were observed among the WTultra (35 positive), Architect (28 positive) and Hepanostika HBsAg Ultra (31 positive) assays. However, the WTultra assay detected significantly more samples than the Murex V.3 (24

  20. Elastic liposome-mediated transdermal immunization enhanced the immunogenicity of P. falciparum surface antigen, MSP-119.

    PubMed

    Tyagi, Rajeev K; Garg, Neeraj K; Jadon, Rajesh; Sahu, Tejram; Katare, Om Prakash; Dalai, Sarat K; Awasthi, Amit; Marepally, Srujan K

    2015-08-26

    Transdermal immunization results in poor immunogenicity, which can be attributed to poor permeability of antigens through the skin. Therefore, elastic liposome, ultradeformable lipid vesicles, may overcome the challenges faced during transdermal immunization. This versatile carrier proves better vehicle for transcutaneous delivery of protein, peptide and nucleic acid antigens. The present results are suggestive of improved immunogenicity of carboxyl-terminal 19 kDa fragment of merozoite surface protein-1 (PfMSP-119) of Plasmodium falciparum when administered subcutaneously through elastic liposomes. The prepared elastic liposomes were characterized with respect to vesicles shape and surface morphology, size and size distribution, entrapment efficiency, elasticity, stability and in vitro release. Humoral and cell-mediated immune (CMI) response elicited by topically applied PfMSP-119-loaded elastic liposomes, intramuscularly administered alum-adsorbed PfMSP-119 solution, and topically applied PfMSP-119-loaded conventional liposomes were compared and normalized with vehicle control. Results suggest greater transcutaneous immunization via elastic liposomes, and induced robust and perdurable IgG-specific antibody and cytophilic isotype responses. We report to have achieved sizeable CMI activating factor (IFNγ), a crucial player in conferring resistance to asexual blood stage malaria, responses with elastic liposomes when compared with other formulations. The fluorescence microscopy and histopathology results are suggestive of prominent skin permeation and biodistribution, and demonstrate efficient delivery of malaria antigen via elastic liposomes to immunocompetent Langerhans cells (LC) and lymphatics. In conclusion, elastic liposomal formulation provided greater entrapment efficiency, enhanced penetration and heightened and long-lasting immune response. Moreover, effective immunoadjuvant property of this carrier justifies its potential for improved vaccine delivery

  1. Identification of a cell-surface antigen selectively expressed on the natural killer cell

    PubMed Central

    1977-01-01

    We have studied the cell-surface phenotype of natural killer (NK) cells of NZB and B6 mice which react to an MuLV+ lymphoid tumor. (a) NK cells do not express Thy1, Ly2, or Ig surface markers. (b) NK cells express an antigen recognized by C3H anti-CE antiserum ('anti-Ly1.2 antiserum'). Inasmuch as NK activity of spleen cells from B6 and B6/Ly1.1 congenic strains were both equally sensitive to C3H anti-CE antiserum, the NK antigen is distinct from Ly1.2. This point was confirmed by the observation that alphaNK activity was removed by absorption of C3H anti-CE antiserum with spleen cells from either B6 or B6/Ly1.1 congenic strains. Absorption of C3H alphaCE serum with BALB/c thymocytes and spleen cells (which are Ly1.2+NK-) removed anti-Ly1.2 activity and left anti-NK activity intact. This absorption step could be circumvented by inserting the BALB/c genotype into the recipient immunized to CE cells (i.e., (C3H X BALB/c)F1 alphaCE spleen cells). This antiserum, provisionally termed 'anti-NK', defines a new subclass of lymphocytes which may play a central role in the immunosurveillance against tumors. PMID:187714

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

    PubMed

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

    2016-06-01

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

  3. Rapid formation of cell-particle complexes via dielectrophoretic manipulation for the detection of surface antigens.

    PubMed

    Horii, Takuma; Yamamoto, Masashi; Yasukawa, Tomoyuki; Mizutani, Fumio

    2014-11-15

    A rapid and simple method for the fabrication of the island patterns with particles and cells was applied to detect the presence of specific antigens on the cell surface. An upper interdigitated microband array (IDA) electrode was mounted on a lower substrate with the same design to fabricate a microfluidic-channel device for dielectrophoretic manipulation. The electrode grid structure was fabricated by rotating the upper template IDA by 90° relative to the lower IDA. A suspension of anti-CD33 modified particles and HL-60 cells was introduced into the channel. An AC electrical signal (typically 20 V peak-to-peak, 100 kHz) was then applied to the bands of the upper and lower IDAs, resulting in the formation of island patterns at the intersections with low electric fields. Immunoreactions between the antibodies immobilized on the accumulated particles and the CD33 present on the surface of the cells led to the formation of complexes comprising corresponding antigen-antibody pairs. Non-specific pairs accumulated at the intersection, which did not form complexes, were then dispersed after removal of the applied field. The time required for the detection of the formation/dispersion of the complexes is as short as 6 min in the present procedure. Furthermore, this novel cell binding assay does not require pretreatment such as target labeling or washing of the unbound cells.

  4. Resolving low-expression cell surface antigens by time-gated orthogonal scanning automated microscopy.

    PubMed

    Lu, Jie; Martin, Jody; Lu, Yiqing; Zhao, Jiangbo; Yuan, Jingli; Ostrowski, Martin; Paulsen, Ian; Piper, James A; Jin, Dayong

    2012-11-20

    We report a highly sensitive method for rapid identification and quantification of rare-event cells carrying low-abundance surface biomarkers. The method applies lanthanide bioprobes and time-gated detection to effectively eliminate both nontarget organisms and background noise and utilizes the europium containing nanoparticles to further amplify the signal strength by a factor of ∼20. Of interest is that these nanoparticles did not correspondingly enhance the intensity of nonspecific binding. Thus, the dramatically improved signal-to-background ratio enables the low-expression surface antigens on single cells to be quantified. Furthermore, we applied an orthogonal scanning automated microscopy (OSAM) technique to rapidly process a large population of target-only cells on microscopy slides, leading to quantitative statistical data with high certainty. Thus, the techniques together resolved nearly all false-negative events from the interfering crowd including many false-positive events.

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

    PubMed

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

    2013-02-01

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

  6. Staining of surface antigens of Chlamydia trachomatis L2 in tissue culture.

    PubMed Central

    Baumann, M; Brade, L; Fasske, E; Brade, H

    1992-01-01

    Surface labeling of chlamydial elementary and reticulate bodies in L929 cells infected with Chlamydia trachomatis serotype L2 was monitored by using monoclonal antibodies (MAb) against the major outer membrane protein and lipopolysaccharide (LPS). Different staining and fixation procedures were used to detect these surface antigens during the developmental cycle. Anti-major outer membrane protein MAb yielded a clear staining pattern of exclusively chlamydial inclusions independent of the fixation or staining technique used. Anti-LPS MAb gave a faint staining pattern of reticulate bodies when methanol fixation was used and showed that LPS was released from chlamydiae into the host cell cytoplasm and into the surroundings of the infected host cell. However, when paraformaldehyde-glutardialdehyde fixation was used, extracellular LPS staining was not observed. The data show that chlamydial LPS is loosely bound in the bacterial outer membrane but suggest that shedding of LPS is a fixation artifact. Images PMID:1398957

  7. Greek rheumatoid arthritis patients have elevated levels of antibodies against antigens from Proteus mirabilis.

    PubMed

    Christopoulos, Georgios; Christopoulou, V; Routsias, J G; Babionitakis, A; Antoniadis, C; Vaiopoulos, G

    2017-03-01

    Patients with rheumatoid arthritis (RA) from different ethnic groups present elevated levels of antibodies against Proteus mirabilis. This finding implicates P. mirabilis in the development of RA. The aim of this study was to investigate the importance of P. mirabilis in the etiopathogenesis of RA in Greek RA patients. In this study, 63 patients with RA and 38 healthy controls were included. Class-specific antibodies IgM, IgG, and IgA against three human cross-reactive and non-cross-reactive synthetic peptides from P. mirabilis-hemolysin (HpmB), urease C (UreC), and urease F (UreF)-were performed in all subjects, using the ELISA method. RA patients had elevated levels of IgM, IgG, and IgA antibodies against HpmB and UreC Proteus peptide which are significantly different compared to healthy controls: p = 0.005, p < 0.001, and p = 0.003 and p = 0.007, p = 0.002, and p < 0.001, correspondingly. Also, elevated levels of IgM, IgG, and IgA antibodies against the UreF Proteus peptide-which are non-cross-reactive with human tissue antigens-were observed and their significant difference compared to healthy controls (p = 0.007, p < 0.001, p < 0.001). Anti-peptide antibodies in RA patients showed a significant correlation with rheumatoid factors (Rf), erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP), especially when patients were divided into subgroups according to the receiving treatment. Greek RA patients present elevated levels of antibodies against P. mirabilis antigenic epitopes, such as in North European populations, albeit Greek RA patients presenting the cross-reaction antigen in a low percentage. These results indicate that P. mirabilis through the molecular mimicry mechanism leads to inflammation and damage of the joints in RA.

  8. Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA) Induce Protective Immune Responses in Dogs

    PubMed Central

    Petitdidier, Elodie; Pagniez, Julie; Papierok, Gérard; Vincendeau, Philippe; Lemesre, Jean-Loup; Bras-Gonçalves, Rachel

    2016-01-01

    Preventive vaccination is a highly promising strategy for interrupting leishmaniasis transmission that can, additionally, contribute to elimination. A vaccine formulation based on naturally excreted secreted (ES) antigens was prepared from L. infantum promastigote culture supernatant. This vaccine achieved successful results in Phase III trials and was licensed and marketed as CaniLeish. We recently showed that newly identified ES promastigote surface antigen (PSA), from both viable promastigotes and axenically-grown amastigotes, represented the major constituent and the highly immunogenic antigen of L. infantum and L. amazonensis ES products. We report here that three immunizations with either the recombinant ES LaPSA-38S (rPSA) or its carboxy terminal part LaPSA-12S (Cter-rPSA), combined with QA-21 as adjuvant, confer high levels of protection in naive L. infantum-infected Beagle dogs, as checked by bone marrow parasite absence in respectively 78.8% and 80% of vaccinated dogs at 6 months post-challenge. The parasite burden in infected vaccinated dogs was significantly reduced compared to placebo group, as measured by q-PCR. Moreover, our results reveal humoral and cellular immune response clear-cut differences between vaccinated and control dogs. An early increase in specific IgG2 antibodies was observed in rPSA/QA-21- and Cter-rPSA/QA-21-immunized dogs only. They were found functionally active in vitro and were highly correlated with vaccine protection. In vaccinated protected dogs, IFN-γ and NO productions, as well as anti-leishmanial macrophage activity, were increased. These data strongly suggest that ES PSA or its carboxy-terminal part, in recombinant forms, induce protection in a canine model of zoonotic visceral leishmaniasis by inducing a Th1-dominant immune response and an appropriate specific antibody response. These data suggest that they could be considered as important active components in vaccine candidates. PMID:27223609

  9. Surface expression of Mo3e antigen by activated human monocytes and U-937 cells

    SciTech Connect

    Todd R.F. III; Bury, M.J.; Liu, D.Y.

    1986-03-05

    The surface expression of a protease-sensitive antigen, Mo3e, by activated human monocytes and U-937 cells is a plasma membrane feature of the activated state. Mo3e, which is an 80 kD protein on Western blot analysis, may represent the surface receptor for migration inhibitory factor (MIF), as evidenced by inhibition of MIF responsiveness produced by anti-Mo3e monoclonal antibody. Mo3e is barely detectable (by surface immunofluorescence) on freshly isolated monocytes but becomes expressed in high antigen density during 18-24 hrs culture in medium containing E. coli lipopolysaccharide (> 1 ng/ml), 4..beta..-phorbol 12-myristate 13-acetate (PMA) (5-10 nM), or muramyl dipeptide (0.1-1 ..mu..M). In U-937 cells, Mo3e surface expression is detectable after 24 hrs exposure to PMA and other pharmacological activators of protein kinase C: 4..beta..-phorbol 12, 13 dibutyrate, 4..beta..-phorbol 12, 13 didecanoate, mezerein, or Sn-1,2-dioctanoylglycerol. The biologically-inactivate phorbol compounds, 4..cap alpha..-phorbol 12, 13 didecanoate and 4/sub ..beta../-phorbol do not stimulate Mo3e expression. The calcium ionophore, ionomycin, has a synergistic effect on Mo3e expression stimulated by PMA; conversely, calcium antagonists block PMA-induced Mo3e expression. These results suggest the involvement of protein kinase C activation and intracellular calcium mobilization in the stimulated expression of Mo3e by activated human mononuclear phagocytes.

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

    PubMed

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

    1995-05-01

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

  11. Hepatitis B virus surface antigen as delivery vector can enhance Chlamydia trachomatis MOMP multi-epitope immune response in mice.

    PubMed

    Zhu, Shanli; Feng, Yan; Rao, Pinhuan; Xue, Xiangyang; Chen, Shao; Li, Wenshu; Zhu, Guanbao; Zhang, Lifang

    2014-05-01

    Chlamydia trachomatis is the leading cause of sexually transmitted infections worldwide. There is currently no commercially available vaccine against C. trachomatis. Major outer membrane protein (MOMP) of C. trachomatis is considered to be an ideal candidate for prophylactic vaccine. We designed a MOMP multi-epitope containing T- and B-cell epitope-rich peptides and developed hepatitis B surface antigen (HBsAg) as antigen delivery vehicle. In order to study the immunogenicity and efficacy of the candidate vaccine in a murine model of chlamydial genital infection, we engineered a recombinant plasmid expressing HBsAg and MOMP multi-epitope genes. Results of reverse transcription polymerase chain reaction and immunofluorescence assay revealed successful expression of the recombinant HBsAg/MOMP multi-epitope gene at both the transcription and translation levels. Intramuscular administration in mice was able to elicit not only antibodies against Chlamydia and HBsAg but also cytotoxic T lymphocyte activity against Chlamydia. In addition, mice inoculated with the rHBsAg were highly resistant to C. trachomatis genital infection. The rHBsAg DNA with MOMP multi-epitope appended at the C terminus of the HBsAg stimulated a stronger immune response and protective response than that appended at the N terminus. Together, our results suggested that use of a recombinant HBsAg encoding the MOMP multi-epitope could be a powerful approach to developing a safe and immunogenic C. trachomatis vaccine.

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

    PubMed

    Lai, Zengzu; Schreiber, John R

    2009-05-21

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

  13. King cobra (Ophiophagus hannah) bites in Myanmar: venom antigen levels and development of venom antibodies.

    PubMed

    Tun-Pe; Aye-Aye-Myint; Warrell, D A; Tin-Myint

    1995-03-01

    Venom, venom IgG and IgM antibody and total serum IgG levels following king cobra bites in two reptile handlers were measured by enzyme immunoassay. The patient in case 1 received antivenom while the patient in case 2 did not. Case 1 made a complete recovery following the bite and produced a high titre short-lived antibody. Venom antigen was not detected in the sample taken 11 hr after antivenom. Case 2 had experienced two recent minor king cobra bites and had received traditional immunization 4 weeks before the accident reported here. He had developed only local swelling and suffered no neurological symptoms. Venom antigen measured at 1.45 hr after the bite was 132 ng/ml; this rapidly fell to 45 ng/ml over the next 30 min, and was no longer detectable 14 hr after the bite. The pattern of venom IgG and IgM antibody responses in both cases was comparable, except that in case 2 the venom IgG peak was maintained for 13 days, compared with 1 day in case 1; in case 2 it subsequently fell to low levels 8 weeks after the bite. Venom IgM appeared 1 day after the bite, peaked at day 7-9, rapidly tailed off on day 12-16 and was then undetectable from day 20 onwards in both. Total IgG level remained within normal limits in both. It is possible that previous bites and recent immunization contributed to the boosting of the venom IgG response in case 2.

  14. Surface plasmon resonance label-free monitoring of antibody antigen interactions in real time

    SciTech Connect

    Kausaite, A.; van Dijk, M.; Castrop, J.; Ramanaviciene, A.; Baltrus, J.P.; Acaite, J.; Ramanavicius, A.

    2007-01-01

    Detection of biologically active compounds is one of the most important topics in molecular biology and biochemistry. One of the most promising detection methods is based on the application of surface plasmon resonance for label-free detection of biologically active compounds. This method allows one to monitor binding events in real time without labeling. The system can therefore be used to determine both affinity and rate constants for interactions between various types of molecules. Here, we describe the application of a surface plasmon resonance biosensor for label-free investigation of the interaction between an immobilized antigen bovine serum albumin (BSA) and antibody rabbit anti-cow albumin IgG1 (anti-BSA). The formation of a self-assembled monolayer (SAM) over a gold surface is introduced into this laboratory training protocol as an effective immobilization method, which is very promising in biosensing systems based on detection of affinity interactions. In the next step, covalent attachment via artificially formed amide bonds is applied for the immobilization of proteins on the formed SAM surface. These experiments provide suitable experience for postgraduate students to help them understand immobilization of biologically active materials via SAMs, fundamentals of surface plasmon resonance biosensor applications, and determination of non-covalent biomolecular interactions. The experiment is designed for master and/or Ph.D. students. In some particular cases, this protocol might be adoptable for bachelor students that already have completed an extended biochemistry program that included a background in immunology.

  15. SNSAG5 IS AN ALTERNATIVE SURFACE ANTIGEN OF SARCOCYSTIS NEURONA STRAINS THAT IS MUTUALLY EXCLUSIVE TO SNSAG1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sarcocystis neurona is an obligate intracellular parasite that causes equine protozoal myeloencephalitis (EPM). Previous work has identified a gene family of paralogous surface antigens in S. neurona called SnSAGs. These surface proteins are immunogenic in their host animals, and are therefore can...

  16. Surface-enhanced Raman scattering (SERS) detection of multiple viral antigens using magnetic capture of SERS-active nanoparticles

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A highly sensitive immunoassay based on surface-enhanced Raman scattering (SERS) spectroscopy has been developed for multiplex detection of surface envelope and capsid antigens of the viral zoonotic pathogens West Nile virus (WNV) and Rift Valley fever virus (RVFV). Detection was mediated by antibo...

  17. Hepatitis B surface antigen (HBsAg) expression in plant cell culture: Kinetics of antigen accumulation in batch culture and its intracellular form.

    PubMed

    Smith, Mark L; Mason, Hugh S; Shuler, Michael L

    2002-12-30

    The production of edible vaccines in transgenic plants and plant cell culture may be improved through a better understanding of antigen processing and assembly. The hepatitis B surface antigen (HBsAg) was chosen for study because it undergoes substantial and complex post-translational modifications, which are necessary for its immunogenicity. This antigen was expressed in soybean (Glycine max L. Merr. cv Williams 82) and tobacco NT1 (Nicotiana tabacum L.) cell suspension cultures, and HBsAg production in batch culture was characterized. The plant-derived antigen consisted predominantly of disulfide cross-linked HBsAg protein (p24(s)) dimers, which were all membrane associated. Similar to yeast, the plant-expressed HBsAg was retained intracellularly. The maximal HBsAg titers were obtained with soybean suspension cultures (20-22 mg/L) with titers in tobacco cultures being approximately 10-fold lower. For soybean cells, electron microscopy and immunolocalization demonstrated that all the HBsAg was localized to the endoplasmic reticulum (ER) and provoked dilation and proliferation of the ER network. Sucrose gradient analysis of crude extracts showed that HBsAg had a complex size distribution uncharacteristic of the antigen's normal structure of uniform 22-nm virus-like particles. The extent of authentic epitope formation was assessed by comparing total p24(s) synthesized to that reactive by polyclonal and monoclonal immunoassays. Depending on culture age, between 40% and 100% of total p24(s) was polyclonal antibody reactive whereas between 6% and 37% was recognized by a commercial monoclonal antibody assay. Possible strategies to increase HBsAg production and improve post-translational processing are discussed.

  18. Exposition of hepatitis B surface antigen (HBsAg) on the surface of HEK293T cell and evaluation of its expression

    PubMed Central

    Mirian, Mina; Taghizadeh, Razieh; Khanahmad, Hossein; Salehi, Mansour; Jahanian-Najafabadi, Ali; Sadeghi-aliabadi, Hojjat; Kouhpayeh, Shirin

    2016-01-01

    Hepatitis B virus (HBV) is considered as a global health concern and hepatitis B surface antigen (HBsAg) is the most immunogenic protein of HBV. The purpose of this study was to evaluate the expression of HBsAg on the cell surface of human embryonic kidney cell line (HEK293T). After transformation of expression vector pcDNA/HBsAg to E.coli TOP10F’, plasmid was extracted and digested with BglII. Afterwards, the linearized vector was transfected to cells and treated with hygromycin B for 5 weeks to expand the resulted clonies. The permanent expression of HBsAg followed by flow cytometry uptill now about one year. Genomic DNA was extracted from transfected cells and the existence of HBsAg gene was assessed by PCR. Real-time RT-PCR was utilized to measure the expression at the RNA level and flow cytometery was carried out to assess protein expression. Insertion of HBsAg cDNA in HEK293T genome was confirmed by PCR. The results of real-time RT-PCR illustrated that each cell expresses 2275 copies of mRNA molecule. Flow cytometry showed that compared with negative control cells, 99.9% of transfected cells express HBsAg on their surface. In conclusion, stable expression of hepatitis B surface antigen on the membrane of HEK293T provides an accurate post-translational modification, proper structure, and native folding in contrast with purified protein from prokaryotic expression systems. Therefore, these exposing HBsAg cells are practical in therapeutic, pharmaceutical, and biological sets of research. PMID:27920818

  19. DNA-mediated immunization and the energetic immune response to hepatitis B surface antigen.

    PubMed

    Whalen, R G; Davis, H L

    1995-04-01

    A new and unusual approach for evoking an immune response has recently been introduced--that of DNA-based immunization. Purified plasmid DNA, containing protein coding sequences and the necessary regulatory elements to express them, can be introduced into tissues of the organism by means of a parenteral injection or by particle bombardment. The number of cells transfected and the amount of protein produced is sufficient to produce a remarkably strong and broad-based immune response to a wide variety of foreign proteins. The absence of an exogenous infectious agent or immunogen results in the abrupt appearance of a foreign protein within the normal cells of an immunologically mature and healthy animal and provokes an energetic and efficient reaction to this form of antigen presentation. This review summarizes the results obtained with the various experimental models that have been described to date and considers in greater depth the immune response to the surface antigen of the human hepatitis B virus that has been achieved using DNA-based immunization. Several issues are addressed in a prospective manner in order to anticipate some future developments and to point out topics likely to be pertinent to this field. DNA-mediated induction of immune responses may soon be applied as a form of therapeutic treatment. Although this method may constitute a revolution for vaccination, many issues must first be dealt with, especially concerning the safety of using DNA as an immunizing molecule.

  20. Sequence and immunogenicity of the Taenia saginata homologue of the major surface antigen of Echinococcus spp.

    PubMed

    Benitez, L; Harrison, L J; Parkhouse, R M; Gonzalez, L M; Gottstein, B; Garate, T

    1998-05-01

    A clone (R-Tso18) was isolated from a Taenia saginata oncosphere cDNA library by screening with sera from rabbits immunised with oncosphere extract. It contained a full-length cDNA sequence of 1893 bp with an open reading frame of 1680 bp, corresponding to 559 amino acids with a deduced molecular mass of 65.173 kDa and an isoelectric point of 6.08. The R-Tso18 protein showed 80-84% nucleotide identity with the major protoscolex surface antigens of Echinococcus multilocularis (EM10) and E. granulosus (EG10). Preliminary immunogenicity studies employing the radiolabeled R-Tso18 protein in immune co-precipitation assays indicated sero-positivity for T. saginata-infected calf sera (6/13), T. solium cysticercosis human (7/22) and pig (2/2) sera and E. multilocularis (6/10)- and E. granulosus (1/12)-infected human sera, whereas other helminth-infection sera were negative. As immuno-precipitation is a relatively insensitive assay, it was concluded that further studies on the diagnostic potential of the purified recombinant R-Tso18 antigen, or its peptides, are merited.

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

    PubMed Central

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

    2014-01-01

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

  2. Overview of expression of hepatitis B surface antigen in transgenic plants.

    PubMed

    Guan, Zheng-jun; Guo, Bin; Huo, Yan-lin; Guan, Zheng-ping; Wei, Ya-hui

    2010-10-28

    Hepatitis B virus (HBV), a pathogen for chronic liver infection, afflicts more than 350 million people world-wide. The effective way to control the virus is to take HBV vaccine. Hepatitis B surface antigen (HBsAg) is an effective protective antigen suitable for vaccine development. At present, "edible" vaccine based on transgenic plants is one of the most promising directions in novel types of vaccines. HBsAg production from transgenic plants has been carried out, and the transgenic plant expression systems have developed from model plants (such as tobacco, potato and tomato) to other various plant platforms. Crude or purified extracts of transformed plants have been found to conduct immunological responses and clinical trials for hepatitis B, which gave the researches of plant-based HBsAg production a big boost. The aim of this review was to summarize the recent data about plant-based HBsAg development including molecular biology of HBsAg gene, selection of expression vector, the expression of HBsAg gene in plants, as well as corresponding immunological responses in animal models or human.

  3. Reduced Hepatic Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 Level in Obesity

    PubMed Central

    Heinrich, Garrett; Muturi, Harrison T.; Rezaei, Khadijeh; Al-Share, Qusai Y.; DeAngelis, Anthony M.; Bowman, Thomas A.; Ghadieh, Hilda E.; Ghanem, Simona S.; Zhang, Deqiang; Garofalo, Robert S.; Yin, Lei; Najjar, Sonia M.

    2017-01-01

    Impairment of insulin clearance is being increasingly recognized as a critical step in the development of insulin resistance and metabolic disease. The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes insulin clearance. Null deletion or liver-specific inactivation of Ceacam1 in mice causes a defect in insulin clearance, insulin resistance, steatohepatitis, and visceral obesity. Immunohistological analysis revealed reduction of hepatic CEACAM1 in obese subjects with fatty liver disease. Thus, we aimed to determine whether this occurs at the hepatocyte level in response to systemic extrahepatic factors and whether this holds across species. Northern and Western blot analyses demonstrate that CEACAM1 mRNA and protein levels are reduced in liver tissues of obese individuals compared to their lean age-matched counterparts. Furthermore, Western analysis reveals a comparable reduction of CEACAM1 protein in primary hepatocytes derived from the same obese subjects. Similar to humans, Ceacam1 mRNA level, assessed by quantitative RT-PCR analysis, is significantly reduced in the livers of obese Zucker (fa/fa, ZDF) and Koletsky (f/f) rats relative to their age-matched lean counterparts. These studies demonstrate that the reduction of hepatic CEACAM1 in obesity occurs at the level of hepatocytes and identify the reduction of hepatic CEACAM1 as a common denominator of obesity across multiple species.

  4. Suppression of hamster lymphocyte reactivity to simian virus 40 tumor surface antigens by spleen cells from pregnant hamsters

    SciTech Connect

    Weppner, W.A.; Adkinson, L.R.; Coggin, J.H.Jr

    1980-09-01

    SV40-transformed tumor cells in hamsters have been found to have cell surface antigens cross-reactive with antigens temporally expressed on fetal tissues. Using a lymphocyte transformation assay, spleen cells from pregnant hamsters were found to be incapable of responding to preparations of either hamster fetal tissue or SV40-transformed cells. However, a suppressor component can be demonstrated in spleen cell populations of both primi-and multiparous hamsters during pregnancy that is capable of reducing the response of lymphocytes sensitized against SV40 tumor-associated antigens. The degree of suppression is proportional to the ratio of responder cells to spleen cells from pregnant animals. These results suggest there is a subpopulation of spleen cells involved in immunoregulation during pregnancy that has the ability to suppress the reactivity of lymphocytes sensitized against SV40-associated oncofetal antigens.

  5. Crystallization and preliminary crystallographic analysis of a surface antigen glycoprotein, SAG19, from Eimeria tenella.

    PubMed

    Ramly, Nur Zazarina; Rouzheinikov, Sergey N; Sedelnikova, Svetlana E; Baker, Patrick J; Chow, Yock Ping; Wan, Kiew Lian; Nathan, Sheila; Rice, David W

    2013-12-01

    Coccidiosis in chickens is caused by the apicomplexan parasite Eimeria tenella and is thought to involve a role for a superfamily of more than 20 cysteine-rich surface antigen glycoproteins (SAGs) in host-parasite interactions. A representative member of the family, SAG19, has been overexpressed in Escherichia coli, purified and crystallized by the hanging-drop method of vapour diffusion using ammonium sulfate as the precipitant. Crystals of SAG19 diffracted to beyond 1.50 Å resolution and belonged to space group I4, with unit-cell parameters a = b = 108.2, c = 37.5 Å. Calculation of possible values of VM suggests that there is a single molecule in the asymmetric unit.

  6. Detection of dengue NS1 antigen using long-range surface plasmon waveguides.

    PubMed

    Wong, Wei Ru; Sekaran, Shamala Devi; Adikan, Faisal Rafiq Mahamd; Berini, Pierre

    2016-04-15

    The non-structural 1 (NS1) protein of the dengue virus circulates in infected patients' blood samples and can be used for early diagnosis of dengue infection. In this paper, we present the detection of naturally-occurring dengue NS1 antigen in infected patient blood plasma using straight long-range surface plasmon waveguides. Three commercially-available anti-NS1 monoclonal antibodies were used for recognition and their performance was compared and discussed. A similar figure of merit to the one used in conventional dengue NS1 capture using an enzyme-linked immunosorbent assay (ELISA) was applied to our results. In general, the positive patient samples can be clearly differentiated from the negative ones and the results agree with those obtained using ELISA. The largest signal-to-noise ratio observed during the experiments was 356 and the best detection limit observed is estimated as 5.73 pg/mm(2).

  7. 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.

  8. Studies on the surface antigenicity and susceptibility to antibody-dependent killing of developing schistosomula using sera from chronically infected mice and mice vaccinated with irradiated cercariae

    SciTech Connect

    Bickle, Q.D.; Ford, M.J.

    1982-05-01

    Changes in the surface antigenicity and susceptibility to in vitro killing during development of schistosomula of Schistosoma mansoni were studied using serum from chronically infected mice (CIS) and from mice vaccinated with highly irradiated (20 krad) cercariae (VS). Binding of these sera was quantitated by counting the number of P388D/sub 1/ cells (a transformed, macrophage-like cell of mouse origin, bearing Fc receptors for IgG) binding to the parasite surface. Compared with schistosomula derived in vitro by mechanical transformation (MS), schistosomula recovered 3 hr after skin penetration in vitro (SS) showed a significant loss in surface binding of CIS. Schistosomula recovered 3 hr after skin penetration in vivo (SRS) showed even less binding, and this trend continued such that parasites recovered from the lungs 5 days after infection (LS) showed only minimal binding, and 10-day-old worms from the portal system showed no significant binding. In contrast, VS, which bound significantly less well to MS than CIS, showed enhanced binding to SS, and in the face of their declining antigenicity with respect to CIS, 3- to 24-hr SRS maintained this raised level of antigenicity. Although there appeared to be a decline in binding of VS thereafter, LS remained antigenic, still binding as many cells as MS did despite the fact that they also expressed host antigens detected usng antisera raised against mouse RBC. In spite of this persistence of VS binding up to the lung stage, resistance to eosinophil-mediated killing in vitro had developed by 48 hr post-infection, and LS were totally resistant to both eosinophil- and C-mediated killing.

  9. Small-angle neutron scattering study of recombinant yeast-derived human hepatitis B virus surface antigen vaccine particle

    NASA Astrophysics Data System (ADS)

    Sato, M.; Ito, Y.; Kameyama, K.; Imai, M.; Ishikawa, N.; Takagi, T.

    1995-02-01

    The overall and internal structure of recombinant yeast-derived human hepatitis B virus surface antigen vaccine particles was investigated by small-angle neutron scattering using the contrast variation method. The vaccine is a nearly spherical particle, and its contrast-matching point was determined to be at about 24% D 2O content, indicating that a large part of the vaccine particle is occupied by lipids and carbohydrates from the yeast. The Stuhrmann plot suggests that the surface antigens exist predominantly in the peripheral region of the particle, which is favorable to the induction of anti-virus antibodies.

  10. Surface plasmon-enhanced fluorescence on Au nanohole array for prostate-specific antigen detection

    PubMed Central

    Zhang, Qingwen; Wu, Lin; Wong, Ten It; Zhang, Jinling; Liu, Xiaohu; Zhou, Xiaodong; Bai, Ping; Liedberg, Bo; Wang, Yi

    2017-01-01

    Localized surface plasmon (LSP) has been widely applied for the enhancement of fluorescence emission for biosensing owing to its potential for strong field enhancement. However, due to its small penetration depth, LSP offers limited fluorescence enhancement over a whole sensor chip and, therefore, insufficient sensitivity for the detection of biomolecules, especially large molecules. We demonstrate the simultaneous excitation of LSP and propagating surface plasmon (PSP) on an Au nanohole array under Kretschmann configuration for the detection of prostate-specific antigen with a sandwich immunoassay. The proposed method combines the advantages of high field enhancement by LSP and large surface area probed by PSP field. The simulated results indicated that a maximum enhancement of electric field intensity up to 1,600 times can be achieved under the simultaneous excitation of LSP and PSP modes. The sandwich assay of PSA carried out on gold nanohole array substrate showed a limit of detection of 140 fM supporting coexcitation of LSP and PSP modes. The limit of detection was approximately sevenfold lower than that when only LSP was resonantly excited on the same substrate. The results of this study demonstrate high fluorescence enhancement through the coexcitation of LSP and PSP modes and pave a way for its implementation as a highly sensitive bioassay. PMID:28392689

  11. Surface plasmon-enhanced fluorescence on Au nanohole array for prostate-specific antigen detection.

    PubMed

    Zhang, Qingwen; Wu, Lin; Wong, Ten It; Zhang, Jinling; Liu, Xiaohu; Zhou, Xiaodong; Bai, Ping; Liedberg, Bo; Wang, Yi

    2017-01-01

    Localized surface plasmon (LSP) has been widely applied for the enhancement of fluorescence emission for biosensing owing to its potential for strong field enhancement. However, due to its small penetration depth, LSP offers limited fluorescence enhancement over a whole sensor chip and, therefore, insufficient sensitivity for the detection of biomolecules, especially large molecules. We demonstrate the simultaneous excitation of LSP and propagating surface plasmon (PSP) on an Au nanohole array under Kretschmann configuration for the detection of prostate-specific antigen with a sandwich immunoassay. The proposed method combines the advantages of high field enhancement by LSP and large surface area probed by PSP field. The simulated results indicated that a maximum enhancement of electric field intensity up to 1,600 times can be achieved under the simultaneous excitation of LSP and PSP modes. The sandwich assay of PSA carried out on gold nanohole array substrate showed a limit of detection of 140 fM supporting coexcitation of LSP and PSP modes. The limit of detection was approximately sevenfold lower than that when only LSP was resonantly excited on the same substrate. The results of this study demonstrate high fluorescence enhancement through the coexcitation of LSP and PSP modes and pave a way for its implementation as a highly sensitive bioassay.

  12. Influence of polymer architecture on antigens camouflage, CD47 protection and complement mediated lysis of surface grafted red blood cells.

    PubMed

    Chapanian, Rafi; Constantinescu, Iren; Rossi, Nicholas A A; Medvedev, Nadia; Brooks, Donald E; Scott, Mark D; Kizhakkedathu, Jayachandran N

    2012-11-01

    Hyperbranched polyglycerol (HPG) and polyethylene glycol (PEG) polymers with similar hydrodynamic sizes in solution were grafted to red blood cells (RBCs) to investigate the impact of polymer architecture on the cell structure and function. The hydrodynamic sizes of polymers were calculated from the diffusion coefficients measured by pulsed field gradient NMR. The hydration of the HPG and PEG was determined by differential scanning calorimetry analyses. RBCs grafted with linear PEG had different properties compared to the compact HPG grafted RBCs. HPG grafted RBCs showed much higher electrophoretic mobility values than PEG grafted RBCs at similar grafting concentrations and hydrodynamic sizes indicating differences in the structure of the polymer exclusion layer on the cell surface. PEG grafting impacted the deformation properties of the membrane to a greater degree than HPG. The complement mediated lysis of the grafted RBCs was dependent on the type of polymer, grafting concentration and molecular size of grafted chains. At higher molecular weights and graft concentrations both HPG and PEG triggered complement activation. The magnitude of activation was higher with HPG possibly due to the presence of many hydroxyl groups per molecule. HPG grafted RBCs showed significantly higher levels of CD47 self-protein accessibility than PEG grafted RBCs at all grafting concentrations and molecular sizes. PEG grafted polymers provided, in general, a better shielding and protection to ABO and minor antigens from antibody recognition than HPG polymers, however, the compact HPGs provided greater protection of certain antigens on the RBC surface. Our data showed that HPG 20 kDa and HPG 60 kDa grafted RBCs exhibited properties that are more comparable to the native RBC than PEG 5 kDa and PEG 10 kDa grafted RBCs of comparable hydrodynamic sizes. The study shows that small compact polymers such as HPG 20 kDa have a greater potential in the generation of functional RBC for therapeutic

  13. The T cell antigen receptor CD3:CD4 molecular complex is diminished on the surface of pulmonary lymphocytes.

    PubMed Central

    Marathias, K.; Pinto, C.; Rodberg, G.; Preffer, F.; Wong, J.; Kradin, R.

    1994-01-01

    CD4, a 55-kd cell surface glycoprotein, binds to class II major histocompatibility complex (MHC) (Ia) antigens and functions as a coreceptor for the T cell antigen receptor (Ti alpha beta)-CD3 complex. We have observed that critical elements of the T cell antigen multireceptor complex, including Ti alpha beta, CD3, CD4, but not CD8, were diminished on CD45RO+ pulmonary T lymphocytes but not CD45RO+ peripheral blood T lymphocytes (PBL). Epitopes mapping from the first (D1) to the fourth (D4) extracytoplasmic Ig-like domains of CD4 were expressed to a lesser degree on pulmonary T cells than on PBL (P = 0.002). CD4 expression on pulmonary T cells did not increase after 72 hours of ex vivo culture in complete medium but was restored toward control levels by stimulation with phytohemagglutinin, anti-CD3, or interleukin-2. CD4 mRNA isolated from lung T cells and PBL co-migrated on Northern blots and the total levels of CD4 mRNA were comparable, suggesting that diminished CD4 expression by pulmonary T cells might reflect a posttranscriptional change. To determine whether CD4bright T cells convert with mitogen stimulation to CD4dim cells, PBLs were stimulated with immobilized anti-CD3, anti-CD4, or a molecularly engineered anti-CD3:CD4 bispecific monoclonal antibody and the ratio of the CD4:CD3 mean fluorescence staining intensities was calculated at days 3 and 13. The CD4:CD3 ratio decreased primarily for cells stimulated with anti-CD3:CD4, suggesting that co-ligation of CD3 and CD4 is required for the generation of CD4dim T cells. We conclude that diminished Ti alpha beta-CD3:CD4 expression is a characteristic of T cells in lung that is not shared by peripheral blood T cells in vivo, and speculate that this change reflects T cell activation in a millieu of limited interleukin-2 availability. Images Figure 8 Figure 9 PMID:7977652

  14. cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

    SciTech Connect

    Torian, B.E.; Stroeher, V.L.; Stamm, W.E. ); Flores, B.M. ); Hagen, F.S. )

    1990-08-01

    A {lambda}gt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically {sup 35}S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-{sup 125}I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4{degree}C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested.

  15. Intramuscular myxoma associated with an increased carbohydrate antigen 19.9 level in a woman: a case report

    PubMed Central

    2011-01-01

    Introduction Intramuscular myxoma is a rare benign soft tissue tumor. The lack of specific symptoms and widely used laboratory tests makes the diagnosis quite difficult. We present a case of an Intramuscular myxoma associated with an increased carbohydrate antigen 19.9 level. To the best of our knowledge, there have not been any reported cases of an association of Intramuscular myxoma with tumor markers in the literature. Case presentation A 45-year-old Caucasian woman presented to our department for resection of a mass in her left groin area, discovered incidentally on a triplex ultrasonography of her lower extremities. The diagnosis of Intramuscular myxoma was confirmed on histopathology after the complete surgical excision of the tumor. On laboratory examination, the serum level of carbohydrate antigen 19.9 was found to be elevated, but it returned to normal six months after resection of the mass. Conclusion Carbohydrate antigen 19.9 is a tumor marker that increases in a variety of malignant and benign conditions. After the exclusion of all other possible reasons for carbohydrate antigen 19.9 elevation, we assumed a possible connection of carbohydrate antigen 19.9 elevation and Intramuscular myxoma, an issue that requires needs further investigation. PMID:21569608

  16. Role of surface protective antigen A in the pathogenesis of Erysipelothrix rhusiopathiae strain C43065.

    PubMed

    Borrathybay, Entomack; Gong, Feng-Juan; Zhang, Lei; Nazierbieke, Wulumuhan

    2015-02-01

    To clarify the role of surface protective antigen A (SpaA) in the pathogenesis of Erysipelothrix rhusiopathiae C43065 (serotype 2), the spaA deletion mutant of E. rhusiopathiae ΔspaA was constructed by homologous recombination. The virulence of the ΔspaA mutant decreased more than 76-fold compared with that of the wild-type strain C43065 in mice. The mutant strain was sensitive to the bactericidal action of swine serum, whereas the wild-type strain was resistant. The adhesion of wild-type strain to MEF cells was inhibited significantly by treatment with rabbit antiserum against recombinant SpaA (rSpaA) as compared with the treatment with normal rabbit serum, but the mutant strain was not affected. The mutant strain was readily taken up by mouse peritoneal macrophages in the normal rabbit serum, whereas the wild-type strain was resistant. Whereas the rabbit antiserum against rSpaA promoted the phagocytosis of wild-type strain by macrophages, the mutant strain was not affected. In addition, mice vaccinated with the formalin-killed mutant strain were provided 40% protection against challenge by the homologous virulent strain as compared with those with wild-type strain, NaOH-extracted antigen, or rSpaA, which provided more than 80% protection against the same infection. These suggested that SpaA has an important role in the pathogenesis of E. rhusiopathiae infection and could be a target for vaccination against swine erysipelas.

  17. Identification of new meningococcal serogroup B surface antigens through a systematic analysis of neisserial genomes.

    PubMed

    Pajon, Rolando; Yero, Daniel; Niebla, Olivia; Climent, Yanet; Sardiñas, Gretel; García, Darién; Perera, Yasser; Llanes, Alejandro; Delgado, Maité; Cobas, Karem; Caballero, Evelin; Taylor, Stephen; Brookes, Charlotte; Gorringe, Andrew

    2009-12-11

    The difficulty of inducing an effective immune response against the Neisseria meningitidis serogroup B capsular polysaccharide has lead to the search for vaccines for this serogroup based on outer membrane proteins. The availability of the first meningococcal genome (MC58 strain) allowed the expansion of high-throughput methods to explore the protein profile displayed by N. meningitidis. By combining a pan-genome analysis with an extensive experimental validation to identify new potential vaccine candidates, genes coding for antigens likely to be exposed on the surface of the meningococcus were selected after a multistep comparative analysis of entire Neisseria genomes. Eleven novel putative ORF annotations were reported for serogroup B strain MC58. Furthermore, a total of 20 new predicted potential pan-neisserial vaccine candidates were produced as recombinant proteins and evaluated using immunological assays. Potential vaccine candidate coding genes were PCR-amplified from a panel of representative strains and their variability analyzed using maximum likelihood approaches for detecting positive selection. Finally, five proteins all capable of inducing a functional antibody response vs N. meningitidis strain CU385 were identified as new attractive vaccine candidates: NMB0606 a potential YajC orthologue, NMB0928 the neisserial NlpB (BamC), NMB0873 a LolB orthologue, NMB1163 a protein belonging to a curli-like assembly machinery, and NMB0938 (a neisserial specific antigen) with evidence of positive selection appreciated for NMB0928. The new set of vaccine candidates and the novel proposed functions will open a new wave of research in the search for the elusive neisserial vaccine.

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

    PubMed

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

    2015-03-24

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

  19. 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

  20. Total and antigen-specific Ige levels in umbilical cord blood

    PubMed Central

    2009-01-01

    The present study was conducted to learn whether the perinatal and environmental factors could influence the total and antigen-specific IgE levels in umbilical cord blood. Retrospective data were obtained from 173 mother-infant pairs. Total and specific (for children's food, wheat/grass and house dust mite-HDM) cord blood IgE levels were determined using the immunoassay test. The total cord blood IgE was between 0.0-23.08 IU/ml (mean 0.55 ± 2.07 IU/ml; median 0.16 IU/ml). Total IgE levels were significantly higher in boys compared with girls (OR = 2.2; P = 0.007), and in newborns with complicated pregnancy (OR = 2.7; P = 0.003). A greater number of siblings correlated with increases in the total cord blood IgE (P < 0.02). We detected specific IgE in 34 newborns (40 positive tests). A long-standing contact with a cat during pregnancy decreased the specific IgE level for wheat/grass (OR = 3.2; P < 0.07) and for children's food (OR = 5.0; P < 0.04), and the contact with a dog decreased the specific-IgE for wheat/grass (OR = 0.3; P < 0.05). Exposure to tobacco smoke correlated with the positive specific IgE toward house dust mite (OR = 4.7; P = 0.005). PMID:20156762

  1. Carcinoembryonic antigen expression level as a predictive factor for response to 5-fluorouracil in colorectal cancer.

    PubMed

    Eftekhar, Ebrahim; Naghibalhossaini, Fakhraddin

    2014-01-01

    Carcinoembryonic antigen (CEA) expression has been shown to protect cancer cell lines from apoptosis and anoikis. The aim of this study was to further elucidate the role of CEA expression on resistance to anticancer drugs in human colorectal cancer (CRC). We transfected CEA negative CRC cell line SW742 as well as CHO cells to overexpress CEA and their chemoresistance were assessed by MTT assay. In comparison to the parental cell lines, transfected cells had significantly increased resistance to 5-fluorouracil (5-FU). The results also showed a direct correlation between the amount of cellular CEA protein and 5-FU resistance in CEA expressing cells. We found no significant difference in sensitivity to cisplatin and methotrexate between CEA-transfected cells and their counter parental cells. We also compared the association between CEA expression and chemoresistance of 4 CRC cell lines which differed in the levels of CEA production. The CEA expression levels in monolayer cultures of these cell lines did not correlate with the 5-FU resistance. However, 5-FU treatment resulted in the selection of sub-populations of resistant cells that displayed increased CEA expression levels by increasing drug concentration. We analyzed the effect of 5-FU in a 3D multicellular culture generated from the two CRC cell lines, LS180 and HT29/219. Compared with monolayer culture, CEA production and 5-FU resistance in both cell lines were stimulated by 3D growth. In comparison to the 3D spheroids of parental CHO, we observed a significantly elevated 5-FU resistance in 3D culture of the CEA-expressing CHO transfectants. Our findings suggest that the CEA level may be a suitable biomarker for predicting tumor response to 5-FU-based chemotherapy in CRC.

  2. Study of the immunogenicity of hepatitis B surface antigen synthesized in transgenic potato plants with increased biosafety.

    PubMed

    Rukavtsova, Elena B; Rudenko, Natalya V; Puchko, Elena N; Zakharchenko, Natalya S; Buryanov, Yaroslav I

    2015-06-10

    Oral immunogenicity of the hepatitis B surface antigen (HBsAg) synthesized in the tubers of marker-free potato plants has been demonstrated. Experiments were performed in the two groups of outbred NMRI mice. At the beginning of investigations, the mice of experimental group were fed the tubers of transgenic potato synthesizing the HBsAg three times. The mice of control group were fed nontransgenic potato. Intraperitoneal injection of the commercial vaccine against hepatitis B (0.5μg/mouse) was made on day 71 of the experiment. Enzyme-linked immunoassay (ELISA) of the serum of immunized animals showed an increase in the level of HBsAg antibodies significantly above the protective value, which was maintained for 1 year after the immunization. In 1 year, the experimental group of mice underwent additional oral immunization with HBsAg-containing potato tubers. As a result, the level of antibodies against the HBsAg increased and remained at a high protective level for several months. The findings show the possibility of using transgenic plants as a substance for obtaining a safe edible vaccine against hepatitis B.

  3. Surface plasmon resonance measurements of plasma antibody avidity during primary and secondary responses to anthrax protective antigen.

    PubMed

    Lynch, Heather E; Stewart, Shelley M; Kepler, Thomas B; Sempowski, Gregory D; Alam, S Munir

    2014-02-01

    Establishment of humoral immunity against pathogens is dependent on events that occur in the germinal center and the subsequent induction of high-affinity neutralizing antibodies. Quantitative assays that allow monitoring of affinity maturation and duration of antibody responses can provide useful information regarding the efficacy of vaccines and adjuvants. Using an anthrax protective antigen (rPA) and alum model antigen/adjuvant system, we describe a methodology for monitoring antigen-specific serum antibody concentration and avidity by surface plasmon resonance during primary and secondary immune responses. Our analyses showed that following a priming dose in mice, rPA-specific antibody concentration and avidity increases over time and reaches a maximal response in about six weeks, but gradually declines in the absence of antigenic boost. Germinal center reactions were observed early with maximal development achieved during the primary response, which coincided with peak antibody avidity responses to primary immunization. Boosting with antigen resulted in a rapid increase in rPA-specific antibody concentration and five-fold increase in avidity, which was not dependent on sustained GC development. The described methodology couples surface plasmon resonance-based plasma avidity measurements with germinal center analysis and provides a novel way to monitor humoral responses that can play a role in facilitating vaccine and adjuvant development.

  4. Surface antigen-negative hepatitis B virus infection in Dutch blood donors.

    PubMed

    Lieshout-Krikke, R W; Molenaar-de Backer, M W A; van Swieten, P; Zaaijer, H L

    2014-01-01

    Hepatitis B virus (HBV) surface antigen (HBsAg) is a reliable marker for HBV infection, but HBsAg-negative forms of HBV infection occur. The introduction of HBV DNA screening of Dutch blood donors, which were not preselected for absence of HBV core antibodies, enabled the characterization of HBsAg-negative HBV infection in healthy persons and a comparison of the HBV genomes involved. The screening of 4.4 million Dutch blood donations identified 23 HBsAg-negative, HBV DNA-positive persons. Serological testing of the index donations, follow-up samples and archived earlier samples was performed to determine the nature of each HBV DNA-only case. Despite low viral loads HBV DNA could be sequenced in 14 out of 23 donors, allowing HBV genotyping and the analysis of mutations in the HBV surface gene. Four types of HBsAg-negative HBV infection were detected: infection in the early stage before occurrence of HBsAg; suppressed infection after vaccination; HBV genotype G infection with decreased HBsAg production; and chronic occult (HBsAg negative) HBV infection. In the donors with occult HBV genotype D infection the HBV surface gene showed multiple "escape" mutations in the HBsAg a-determinant and CTL epitopes, while in an occult genotype A case the surface gene showed no mutations. HBsAg-negative forms of HBV infection in healthy blood donors explain the ongoing transmission of HBV via blood transfusion, if donor screening is limited to HBsAg. The screening of blood donors for HBV DNA and HBV core antibodies seems to cover all stages and variants of HBV infection.

  5. Major surface antigen, P30, of Toxoplasma gondii is anchored by a glycolipid

    SciTech Connect

    Nagel, S.D.; Boothroyd, J.C.

    1989-04-05

    P30, the major surface antigen of the parasitic protozoan Toxoplasma gondii, can be specifically labeled with (/sup 3/H)palmitic acid and with myo-(2-/sup 3/H)inositol. The fatty acid label can be released by treatment of P30 with phosphatidylinositol-specific phospholipase C (PI-PLC). Such treatment exposes an immunological cross-reacting determinant first described on Trypanosoma brucei variant surface glycoprotein. PI-PLC cleavage of intact parasites metabolically labeled with (/sup 35/S)methionine results in the release of intact P30 polypeptide in a form which migrates faster in polyacrylamide gel electrophoresis. These results argue that P30 is anchored by a glycolipid. Results from thin layer chromatography analysis of purified (/sup 3/H) palmitate-labeled P30 treated with PI-PLC, together with susceptibility to mild alkali hydrolysis and to cleavage with phospholipase A2, suggest that the glycolipid anchor of T. gondii P30 includes a 1,2-diacylglycerol moiety.

  6. Association of gross virus-associated cell-surface antigen with liposomes.

    PubMed Central

    Sakai, F.; Gerlier, D.; Doré, J. F.

    1980-01-01

    Gross Cell-Surface Antigen (GCSAa) was obtained from W/Fu (C58NT)D lymphoma cells by Nonidet P40(NP40) or 3M KCl extraction and further purified by Sephadex G200 filtration. GCSAa was associated with lipids (dipalmitoylphosphatidycholine, cholesterol and dicetylphosphate, in molar ratios of 7:2:1) to form multilamellar liposomes. The amount of protein associated with liposomes was found to be proportional to the protein concentration of the sensitizing cellular extract and to the amount of phospholipids used and, under defined conditions, 22-55% of the protein of the cellular extract could be associated with liposomes. Analysis of disrupted sensitized liposomes showed that the GCSAa-specific activity of the liposome-associated proteins was quite similar to that of the proteins of the sensitizing cellular extract. Ultracentrifugation of disrupted liposomes showed that about 75% of the liposome-associated GCSAa activity was firmly associated with lipids and that little GCSAa was trapped within aqueous compartments between lipidic lamellae. 1.8--8.0% of the liposome-associated GCSAa was expressed at the liposome surface. No striking differences in degree of GCSAa association were found between liposomes sensitized by NP40 or by 3M KCl extracts. Storage experiments at +4 degrees C showed that GCSAa-sensitized liposomes were fairly stable. Images Fig. 2 PMID:7370162

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

    PubMed Central

    2012-01-01

    Background Cell disruption strategies by high pressure homogenizer for the release of recombinant Hepatitis B surface antigen (HBsAg) from Pichia pastoris expression cells were optimized using response surface methodology (RSM) based on the central composite design (CCD). The factors studied include number of passes, biomass concentration and pulse pressure. Polynomial models were used to correlate the above mentioned factors to project the cell disruption capability and specific protein release of HBsAg from P. pastoris cells. Results The proposed cell disruption strategy consisted of a number of passes set at 20 times, biomass concentration of 7.70 g/L of dry cell weight (DCW) and pulse pressure at 1,029 bar. The optimized cell disruption strategy was shown to increase cell disruption efficiency by 2-fold and 4-fold for specific protein release of HBsAg when compared to glass bead method yielding 75.68% cell disruption rate (CDR) and HBsAg concentration of 29.20 mg/L respectively. Conclusions The model equation generated from RSM on cell disruption of P. pastoris was found adequate to determine the significant factors and its interactions among the process variables and the optimum conditions in releasing HBsAg when validated against a glass bead cell disruption method. The findings from the study can open up a promising strategy for better recovery of HBsAg recombinant protein during downstream processing. PMID:23039947

  8. The Prevalence of Hepatitis B Surface Antigen and Antibody in Home-Reared Individuals with Down Syndrome.

    ERIC Educational Resources Information Center

    Pueschel, Siegfried M.; And Others

    1991-01-01

    This study found no significant difference in the prevalence of hepatitis B surface antigens or antibodies between 180 noninstitutionalized subjects (ages 1-29) with Down's syndrome and 155 subjects without Down's syndrome. This finding contrasts with previous findings with institutionalized Down's syndrome subjects. (Author/DB)

  9. Expression and structural characterization of anti-T-antigen single-chain antibodies (scFvs) and analysis of their binding to T-antigen by surface plasmon resonance and NMR spectroscopy.

    PubMed

    Yuasa, Noriyuki; Koyama, Tsubasa; Subedi, Ganesh P; Yamaguchi, Yoshiki; Matsushita, Misao; Fujita-Yamaguchi, Yoko

    2013-12-01

    T-antigen (Galβ1-3GalNAcα-1-Ser/Thr), also known as Thomsen-Friedenreich antigen (TF antigen), is an oncofetal antigen commonly found in cancerous tissues. Availability of anti-T-antigen human antibodies could lead to the development of cancer diagnostics and therapeutics. Four groups of single-chain variable fragment (scFv) genes were previously isolated from a phage library (Matsumoto-Takasaki et al. (2009) Isolation and characterization of anti-T-antigen single chain antibodies from a phage library. BioSci Trends 3:87-95.). Here, four anti-T-antigen scFv genes belonging to Group 1-4 were expressed and produced in a Drosophila S2 cell expression system. ELISA and surface plasmon resonance (SPR) analyses confirmed the binding activity of 1E8 scFv protein to various T-antigen presenting conjugates. NMR experiments provided evidence of the folded nature of the 1E8 scFv protein. ScFv-ligand contact was identified by STD NMR, indicating that the galactose unit of T-antigen at the non-reducing end was primarily recognized by 1E8 scFv. This thus provides direct evidence of T-antigen specificity.

  10. Body mass index in relation to serum prostate-specific antigen levels and prostate cancer risk.

    PubMed

    Bonn, Stephanie E; Sjölander, Arvid; Tillander, Annika; Wiklund, Fredrik; Grönberg, Henrik; Bälter, Katarina

    2016-07-01

    High Body mass index (BMI) has been directly associated with risk of aggressive or fatal prostate cancer. One possible explanation may be an effect of BMI on serum levels of prostate-specific antigen (PSA). To study the association between BMI and serum PSA as well as prostate cancer risk, a large cohort of men without prostate cancer at baseline was followed prospectively for prostate cancer diagnoses until 2015. Serum PSA and BMI were assessed among 15,827 men at baseline in 2010-2012. During follow-up, 735 men were diagnosed with prostate cancer with 282 (38.4%) classified as high-grade cancers. Multivariable linear regression models and natural cubic linear regression splines were fitted for analyses of BMI and log-PSA. For risk analysis, Cox proportional hazards regression models were used to estimate hazard ratios (HR) and 95% confidence intervals (CI) and natural cubic Cox regression splines producing standardized cancer-free probabilities were fitted. Results showed that baseline Serum PSA decreased by 1.6% (95% CI: -2.1 to -1.1) with every one unit increase in BMI. Statistically significant decreases of 3.7, 11.7 and 32.3% were seen for increasing BMI-categories of 25 < 30, 30 < 35 and ≥35 kg/m(2), respectively, compared to the reference (18.5 < 25 kg/m(2)). No statistically significant associations were seen between BMI and prostate cancer risk although results were indicative of a positive association to incidence rates of high-grade disease and an inverse association to incidence of low-grade disease. However, findings regarding risk are limited by the short follow-up time. In conclusion, BMI was inversely associated to PSA-levels. BMI should be taken into consideration when referring men to a prostate biopsy based on serum PSA-levels.

  11. Immunological evaluation of colonic delivered Hepatitis B surface antigen loaded TLR-4 agonist modified solid fat nanoparticles.

    PubMed

    Sahu, Kantrol Kumar; Pandey, Ravi Shankar

    2016-10-01

    Hepatitis B is one of the leading liver diseases and remains a major global health problem. Currently available vaccines provide protection but often results in weaker/minimal mucosal immunity. Thus the present study is devoted to the development and in-vivo exploration of the colonically delivered biomimetic nanoparticles which capably enhance humoral as well as cellular immune response. In present work, Hepatitis B surface antigen (HBsAg) entrapped nanoparticles containing Monophosphoryl lipid A (MPLA) (HB+L-NP) were prepared by solvent evaporation method and characterized for particle size (~210nm), shape, zeta potential (-24mV±0.68), entrapment efficiency (58.45±1.68%), in-vitro release and antigen integrity. Dose escalation study was done to confirm prophylactic immune response following defined doses of prepared nanoparticulate formulations with or without MPLA. Intramuscular administered alum based marketed HBsAg (Genevac B) was used as standard (10μg) and were able to induce significant systemic (IgG) but remarkably low mucosal immune (IgA) response. Notably, HB+L-NP (0.5ml-10μg) induced strong systemic and robust mucosal immunity (510 and 470 mIU/ml respectively, p<0.001) from which mucosal was more significant due to the involvement of Common Mucosal Immune System (CMIS). Likewise, significant cellular immune response was elicited by HB+L-NP through T-cell activation (mixed Th1 and Th2) as confirmed by significantly increased cytokines level (IL-2 and Interferon-γ) in spleen homogenates. This study supports that delivery of HBsAg to the colon may open new vista in designing oral vaccines later being one of most accepted route for potential vaccines in future.

  12. Seroprevalence of hepatitis B surface antigen in pregnant women attending antenatal clinic in Honiara Solomon Islands, 2015

    PubMed Central

    Getahun, Aneley; Baekalia, Margaret; Panda, Nixon; Lee, Alice; Puiahi, Elliot; Khan, Sabiha; Tahani, Donald; Manongi, Doris

    2016-01-01

    AIM To determine the seroprevalence of hepatitis B surface antigen (HBsAg) among pregnant women attending antenatal clinic in Honiara, Solomon Islands. METHODS This descriptive cross-sectional study was carried out in seven area health centers in Honiara. From March to June 2015, identification of eligible pregnant women in each site was conducted using systematic random sampling technique. A total of 243 pregnant women who gave written informed consent were enrolled. Standardized tool was used to record demographics, obstetric history and serology results. HBsAg and hepatitis B e antigen (HBeAg) were tested using point-of-care rapid diagnostic test. All HBsAg positive samples were verified using enzyme-linked immunosorbent assay. RESULTS The mean age of participants was 26 ± 6 years. The overall hepatitis HBsAg prevalence was 13.8% with higher rate (22%) reported in women between 30-34 years of age. Majority of HBsAg positive participants were Melanesians (29 out for 33). None of the pregnant women in the 15-19 years and ≥ 40 years tested positive for HBsAg. There was no statistically significant difference in HBsAg prevalence by age, ethnicity, education and residential location. The overall HBeAg seroprevalence was 36.7%. Women between 20-24 years of age had the highest rate of 54.5%. Low level of knowledge about hepatitis B vaccination was reputed. Overall, 54.6% of participants were not aware of their hepatitis B vaccination status and only 65.2% of mothers reported their child had been vaccinated. CONCLUSION Hepatitis B is a disease of public health importance in Solomon Islands and emphasize the need for integrated preventative interventions for its control. PMID:28008343

  13. [Diagnostic value of IgG antibody levels against 38 kDa mycobacterial antigen].

    PubMed

    Demkow, U; Zielonka, T M; Strzałkowski, J; Michałowska-Mitczuk, D; Augustynowicz-Kopeć, E; Białas-Chromiec, B; Kuś, J; Skopińska-Rózewska, E; Zwolska, Z

    1998-01-01

    Tuberculosis diagnosis bases on clinical and radiological symptoms and identification of mycobacteria. Accuracy of both methods is limited. Therefore reliable serological test would have considerable advantage. The present study was aimed at evaluating IgG-mediated immune response against specific mycobacterial antigens 38 kDa in group of 200 patients and control subjects. Our material consisted of 104 tuberculosis patients, 25 with sarcoidosis, 24 with lung cancer, 13 with bacterial or fungal pulmonary infection, 8 with mycobacterial infections other than tuberculosis and 26 healthy persons. We used commercially available ELISA based kits (Pathozyme TB-complex). Specificity of 100% and sensitivity of 49% was achieved. Sensitivity increased to 59% in chronic cases and to 52% in culture positive cases. Sensitivity decreased to only 14% in group of new culture negative cases. Measurement of IgG serum level against 38 kDa can be helpful in tuberculosis diagnosis. As the test lacks falsely positive results it indicates its high positive predictive value.

  14. Hepatitis B Surface Antigen Loss and Hepatocellular Carcinoma Development in Patients With Dual Hepatitis B and C Infection.

    PubMed

    Yang, Wan-Ting; Wu, Li-Wei; Tseng, Tai-Chung; Chen, Chi-Ling; Yang, Hung-Chih; Su, Tung-Hung; Wang, Chia-Chi; Kuo, Stephanie Fang-Tzu; Liu, Chen-Hua; Chen, Pei-Jer; Chen, Ding-Shinn; Liu, Chun-Jen; Kao, Jia-Horng

    2016-03-01

    Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are 2 major causes of chronic viral hepatitis. It is still unclear how HCV coinfection affects HBV replication and clinical outcomes in HBV/HCV coinfected patients.We conducted a longitudinal study, which enrolled 111 patients with HBV/HCV coinfection and 111 propensity score-matched controls with HBV monoinfection. Both groups had comparable baseline age, sex, fibrosis stage, levels of HBV DNA, and HBV surface antigen (HBsAg). The HCV coinfection and other host/viral factors were correlated with various outcomes, including HBsAg loss and cirrhosis/hepatocellular carcinoma (HCC) development.After a 10-year follow-up, we found that HCV coinfection itself was not associated with HBsAg loss. However, coinfected patients with alanine aminotransferase (ALT) level >80 U/L had a higher chance of HBsAg loss than those with ALT level ≤80 U/L [hazard ratio (95% confidence interval): 4.41 (1.75-11.15)] or matched controls with HBV monoinfection [hazard ratio (95% confidence interval): 3.40 (1.54-7.50)]. Besides, both HCV coinfection and higher ALT levels were associated with higher HCC risks and the HCC risks remained even after HBsAg loss in HBV/HCV con-infected patient.HCV coinfection is not associated with HBsAg loss. A higher ALT level is a major determinant of HBsAg loss in patients with HBV/HCV coinfection. Both HCV coinfection and a higher ALT level were independent risk factors of HCC.

  15. 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

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

    PubMed

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

    2015-05-01

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

  17. Posttransfusion hepatitis B transmitted by blood from a hepatitis B surface antigen-negative hepatitis B virus carrier.

    PubMed

    Larsen, J; Hetland, G; Skaug, K

    1990-06-01

    A female blood donor at our institution was implicated in three cases of clinical posttransfusion hepatitis B. She had given blood 25 times in 8 years. Twenty-seven recipients were followed up, and about one-half of those who were still living had serologic markers of hepatitis B virus (HBV) infection. The donor was repeatedly negative for hepatitis B surface antigen (HBsAg) and antibody, but she had high titers of antibody to hepatitis B core antigen. We conclude that blood from this HBsAg-negative blood donor was capable of transmitting hepatitis B.

  18. Contraband drug surface chemistry at nanogram levels

    NASA Astrophysics Data System (ADS)

    Homstead, Juliana; Poziomek, Edward J.

    1997-02-01

    The surface chemistry of contraband drugs is very important in many detection techniques. It is also important in choosing materials for sampling and sample handling. The chemical nature of surfaces may facilitate drug decomposition or serve to stabilize the drugs. We have developed a simple technique to study the chemistry of contraband drugs such as cocaine HCl at nanogram levels. The normal operating modes of an IONSCAN 400 ion mobility spectrometer were adjusted to allow the chemistry of the drugs to be examined in the sample chamber of the spectrometer. For example, a membrane with deposited drug is held in the sample chamber at a specified temperature up to 20 seconds with no air flow. An ON-OFF valve was placed in- line just before the carrier gas enters the desorption chamber where samples are heated. This modification allows the gas flow to be manually turned off while the sample is being heated. We have used this technique to examine the pyrolysis of cocaine hydrochloride under a variety of conditions. At the end of the designated reaction time, the air flow is turned on allowing the reaction products and any starting materials to flow into the spectrometer for analysis. This technique has allowed studies of the stability of the drugs at various temperatures on different surfaces. For example, evidence was obtained of cocaine HCl decomposition at 75 degrees for 5 seconds using Teflon as the support material. The use of this technique has also assisted us in choosing materials for pyrolysis studies in which the goal is the decompose target drugs quickly and efficiently for detection applications.

  19. The world's highest levels of surface UV.

    PubMed

    Cordero, Raul R; Seckmeyer, Gunther; Damiani, Alessandro; Riechelmann, Stefan; Rayas, Juan; Labbe, Fernando; Laroze, David

    2014-01-01

    Chile's northern Atacama Desert has been pointed out as one of the places on earth where the world's highest surface ultraviolet (UV) may occur. This area is characterized by its high altitude, prevalent cloudless conditions and relatively low total ozone column. Aimed at detecting those peak UV levels, we carried out in January 2013 ground-based spectral measurements on the Chajnantor Plateau (5100 m altitude, 23°00'S, 67°45'W) and at the Paranal Observatory (2635 m altitude, 24°37'S, 70°24'W). The UV index computed from our spectral measurements peaked at 20 on the Chajnantor Plateau (under broken cloud conditions) and at 16 at the Paranal Observatory (under cloudless conditions). Spectral measurements carried out in June 2005 at the Izaña Observatory (2367 m altitude, 28°18'N, 16°30'W) were used for further comparisons. Due to the differences in sun-earth separation, total ozone column, altitude, albedo, aerosols and clouds, peak UV levels are expected to be significantly higher at southern hemisphere sites than at their northern hemisphere counterparts.

  20. Purification and identification of cell surface antigens using lamprey monoclonal antibodies

    PubMed Central

    Yu, Cuiling; Ali, Shabab; St. Germain, Jonathan; Liu, Yanling; Yu, Xuecong; Jaye, David L.; Moran, Michael F.; Cooper, Max D.; Ehrhardt, Götz R.A.

    2013-01-01

    Variable lymphocyte receptor (VLR) B antibodies of the evolutionary distant sea lamprey are structurally distinct from conventional mammalian antibodies. The different protein architecture and large evolutionary distance of jawless vertebrates suggest that VLR antibodies may represent promising tools for biomarker discovery. Here we report the generation of panels of monoclonal VLR antibodies from lamprey larvae immunized with human T cells and the use of a recombinant monoclonal VLR antibody for antigen purification and mass spectrometric identification. We demonstrate that despite predicted low affinity of individual VLR antigen binding units to the antigen, the high avidity resulting from decameric assembly of secreted VLR antibodies allows for efficient antigen capture and subsequent identification by mass spectometry. We show that VLR antibodies detect their antigens with high specificity and can be used in various standard laboratory application techniques. The lamprey antibodies are novel reagents that can complement conventional monoclonal antibodies in multiple scientific research disciplines. PMID:22964555

  1. Effect of hepatitis B immunisation in newborn infants of mothers positive for hepatitis B surface antigen: systematic review and meta-analysis

    PubMed Central

    Lee, Chuanfang; Gong, Yan; Brok, Jesper; Boxall, Elizabeth H; Gluud, Christian

    2006-01-01

    Objective To evaluate the effects of hepatitis B vaccine and immunoglobulin in newborn infants of mothers positive for hepatitis B surface antigen. Design Systematic review and meta-analysis of randomised clinical trials. Data sources Electronic databases and hand searches. Review methods Randomised clinical trials were assessed for methodological quality. Meta-analysis was undertaken on three outcomes: the relative risks of hepatitis B occurrence, antibody levels to hepatitis B surface antigen, and adverse events. Results 29 randomised clinical trials were identified, five of which were considered high quality. Only three trials reported inclusion of mothers negative for hepatitis B e antigen. Compared with placebo or no intervention, vaccination reduced the occurrence of hepatitis B (relative risk 0.28, 95% confidence interval 0.20 to 0.40; four trials). No significant difference in hepatitis B occurrence was found between recombinant vaccine and plasma derived vaccine (1.00, 0.71 to 1.42; four trials) and between high dose versus low dose vaccine (plasma derived vaccine 0.97, 0.55 to 1.68, three trials; recombinant vaccine 0.78, 0.31 to 1.94, one trial). Compared with placebo or no intervention, hepatitis B immunoglobulin or the combination of plasma derived vaccine and hepatitis B immunoglobulin reduced hepatitis B occurrence (immunoglobulin 0.50, 0.41 to 0.60, one trial; vaccine and immunoglobulin 0.08, 0.03 to 0.17, three trials). Compared with vaccine alone, vaccine plus hepatitis B immunoglobulin reduced hepatitis B occurrence (0.54, 0.41 to 0.73; 10 trials). Hepatitis B vaccine and hepatitis B immunoglobulin seem safe, but few trials reported adverse events. Conclusion Hepatitis B vaccine, hepatitis B immunoglobulin, and vaccine plus immunoglobulin prevent hepatitis B occurrence in newborn infants of mothers positive for hepatitis B surface antigen. PMID:16443611

  2. Cell surface phenotype and ultramicroscopic analysis of purified human enterocytes: a possible antigen-presenting cell in the intestine.

    PubMed

    Martín-Villa, J M; Ferre-López, S; López-Suárez, J C; Corell, A; Pérez-Blas, M; Arnaiz-Villena, A

    1997-12-01

    Epithelial cells of the intestine seem to act as antigen-presenting cells to surrounding lymphoid tissue and may be crucial to maintain the pool of peripheral T lymphocytes. The scope of this study was to carry out an immunophenotypic and ultramicroscopic analysis of purified human enterocytes to elucidate their role as antigen-presenting cells, in the immune responses in the gut-associated lymphoid tissue. A method has been developed to obtain purified and viable human enterocyte populations, later labeled with relevant monoclonal antibodies directed to leukocyte antigens and subjected to cytofluorometric analysis. Phenotypic analysis revealed the presence of markers common to "classical" antigen-presenting cells (CD14, CD35, CD39, CD43, CD63 and CD64), reinforcing the idea that enterocytes may act as such. Moreover, several integrins (CD11b, CD11c, CD18, CD41a, CD61 and CD29) were also found. CD25 (IL-2 receptor alpha chain) and CD28, characteristic of T cells, were detected on the surface of these cells; this latter finding rises the possibility that enterocytes could be activated by IL-2 and/or via CD28 through binding to its ligands CD80 or CD86. Finally, the presence of CD21, CD32, CD35 and CD64 that may bind immune complexes via Fc or C3, suggests their participation in the metabolism of immune complexes. Furthermore, the finding of a Birbeck's-like granule in the cytoplasm of the cells, shows that enterocytes contain an ultramicroscopic feature previously thought to be characteristic of Langerhans' cells, an antigen-presenting cell. The phenotype detected on the surface of enterocytes, along with their ultramicroscopic characteristics, suggests that they may play an important role in the immune responses elicited in the gut, presenting antigens to surrounding lymphoid cells, and establishing cognate interactions with them.

  3. Production of hepatitis B surface antigen in transgenic plants for oral immunization.

    PubMed

    Richter, L J; Thanavala, Y; Arntzen, C J; Mason, H S

    2000-11-01

    Here we present data showing oral immunogenicity of recombinant hepatitis B surface antigen (HBsAg) in preclinical animal trials. Mice fed transgenic HBsAg potato tubers showed a primary immune response (increases in HBsAg-specific serum antibody) that could be greatly boosted by intraperitoneal delivery of a single subimmunogenic dose of commercial HBsAg vaccine, indicating that plants expressing HBsAg in edible tissues may be a new means for oral hepatitis B immunization. However, attainment of such a goal will require higher HBsAg expression than was observed for the potatoes used in this study. We conducted a systematic analysis of factors influencing the accumulation of HBsAg in transgenic potato, including 5' and 3' flanking elements and protein targeting within plant cells. The most striking improvements resulted from (1) alternative polyadenylation signals, and (2) fusion proteins containing targeting signals designed to enhance integration or retention of HBsAg in the endoplasmic reticulum (ER) of plant cells.

  4. Flow cytometric analysis of cell-surface and intracellular antigens in the diagnosis of acute leukemia.

    PubMed

    Paredes-Aguilera, R; Romero-Guzman, L; Lopez-Santiago, N; Burbano-Ceron, L; Camacho-Del Monte, O; Nieto-Martinez, S

    2001-10-01

    To evaluate the usefulness of flow cytometric detection of intracellular antigens (Ags) in establishing proper lineage affiliation and its contribution to the diagnosis of acute leukemia, we studied 100 consecutive patients in whom acute leukemia was diagnosed between January 1997 and July 1998. Immunological classification was assessed using a three-line panel of monoclonal antibodies for phenotypic characterization of leukemic blast cells as proposed at the First Latin American Consensus Conference for Flow Cytometric Immunophenotyping of Leukemia. We found 74 cases of B-cell lineage acute lymphoblastic leukemia (ALL), seven cases of T-cell ALL, and 19 cases of acute myeloid leukemia (AML). In this study cytoplasmic (cy) CD79a, cyCD22, cyCD3, and cyMPO were highly sensitive, specific B, T, and myeloid markers that were expressed in virtually all cases of B and T cell ALL and in all subtypes of AML. Applied in combination with immunophenotyping this knowledge led to improvement in diagnostic precision and refinement of immunological classification, ensuring the selection of the most appropriate therapy for the patients studied. In conclusion, intracellular Ags detection was of utmost importance in establishing correct lineage affiliation in cases lacking expression of B, T, or myeloid surface Ags or disclosing equivocal or ambiguous immunophenotypic features and in identifying biphenotypic acute leukemia. In combination with FAB morphology and immunophenotyping, we were able to reliably classify all patients with acute leukemia in this study.

  5. Hepatitis B surface antigen seroprevalence among children in Papua New Guinea, 2012-2013.

    PubMed

    Kitau, Russel; Datta, Siddhartha Sankar; Patel, Minal K; Hennessey, Karen; Wannemuehler, Kathleen; Sui, Gerard; Lagani, William

    2015-03-01

    Approximately 8% of the population in Papua New Guinea (PNG) has chronic hepatitis B virus (HBV) infection. To decrease the burden of chronic HBV infection, a national 3-dose infant hepatitis B vaccination program was implemented starting in 1989, with a birth dose (BD) added to the schedule in 1992. To assess the impact of the hepatitis B vaccination program, we conducted a serosurvey among children born after vaccine introduction. During 2012-2013, a cross-sectional stratified four-stage cluster survey was conducted to estimate hepatitis B surface antigen (HBsAg) prevalence among children 4-6 years of age. We collected demographic data, vaccination history, and tested children for HBsAg. Of 2,133 participants, 2,130 children had vaccination data by either card or recall: 28% received a BD; 81% received ≥ 3 vaccine doses. Of 2,109 children providing a blood sample, 60 (2.3%) tested positive for HBsAg. This is the largest, most geographically diverse survey of hepatitis B vaccination and HBsAg seroprevalence done in PNG. Progress has been made in PNG toward the Western Pacific Regional goal to reduce the prevalence of chronic HBV infection to < 1% by 2017 among 5-year-old children. Vaccination efforts should be strengthened, including increasing BD coverage and completing the 3-dose series.

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

    PubMed

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

    2016-11-01

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

  7. [Validation of a ultramicroELISA for detecting antibodies against hepatitis B surface antigen].

    PubMed

    Rodríguez, L; Balmaseda, A; Bravo, J; Trujillo, J; Martínez, L; Ochoa, R; Díaz, M; Laferté, J; Ramos, F

    1996-01-01

    The results of a validation study of the ultramicroanalitical assay for the detection of antibodies against the hepatitis B surface antigen (UMELISA anti-HBsAg), which was carried out by comparing the results obtained with the Hepanostika anti-HBsAg, commercial diagnosis kit are presented. For this purpose, sera from the clinical assays of the Cuban recombinant vaccine against hepatitis B were used. With the first sera group (n = 30) it was obtained, 93.1% of sensitivity, 98.5% of specificity and a concordance of 94.3%. The correlation coefficient showed a similar trend of the results (p < 0.01) and no significant differences were found in the average geometrical titre (TPG) between both assays (p > 0.05). With the second group (n = 100), whose assays were carried out at the "Pedro Kouri" Institute of Tropical Medicine (PKI) and at the Immunoassay Center (IAC) simultaneously, it was observed a sensitivity of 96.25% in both centers, a specificity of 75% at the PKI and of 90% at the IAC, and a coincidence of 92% and 95%, respectively. The correlation coefficient presented similar values and there were no significant differences between the TPG obtained by the two methods (p > 0.05). The results attained show in general the validity of the new assay and the feasibility to put it into practice either for following up the infection, or for carrying out clinical assays of vaccine evaluations.

  8. Characterization of cell surface antigens reactive with autologous antibodies from human ovarian neoplasms

    SciTech Connect

    Kutteh, W.H.

    1986-03-01

    Autologous antibodies eluted from membrane fragments of ovarian epithelial neoplasms have been prepared from cyst and ascites fluids. The predominant membrane-bound immunoglobulin, IgG, was present in a range of 18 to 4275 ng of membrane-bound IgG/ml fluid. The autologous antibodies were strongly reactive with human ovarian neoplastic cell lines and fresh ovarian tumor tissue but not with normal human ovaries, other non-ovarian normal or neoplastic tissue or non-ovarian human cell lines. Human ovarian serous cystadenocarcinoma cell lined number2774 was surface labeled with /sup 125/Iodine using lactoperoxidose. Cells were washed and solubilized with Triton X-100. Membrane antigens were prepared and precipitated with autologous antibodies. Precipitates were washed, electrophoresed on 7.5% polyacrylamide gels and analyzed for radioactivity. Three major bands of activity (molecular weights: 180,000; 160,000 and 120,000) were precipitated with autologous antibodies from two patients with serous cystadenocarcinoma and two patients with papillary adenocarcinoma, but not with normal serum or autologous antibodies from a plural effusion of a patient with colon disease.

  9. Epidermal surface antigen (MS17S1) is highly conserved between mouse and human

    SciTech Connect

    Cho, Y.J.; Chema, D.; Cho, M.

    1995-05-20

    A mouse monoclonal antibody ECS-1 raised to human keratinocytes detects a 35-kDa epidermal surface antigen (ESA) and causes keratinocyte dissociation in vitro. ECS-1 stains skin of 16-day mouse embryo and 8- to 9-week human fetus. Mouse Esa cDNA encodes a 379-amino-acid protein that is 99.2% identical to the human, differing at only 3 amino acids. The gene (M17S1) was mapped to mouse chromosome 11, highlighting the conserved linkage synteny existing between human chromosome 17 and mouse chromosome 11. Although the nude locus has been mapped to the same region of chromosome 11, no abnormalities in protein, mRNA, or cDNA or genomic sequences were detected in nude mice. However, both nude and control mice were found to have a second Esa mRNA transcript that conserves amino acid sequence and molecular weight. The mouse and human 5{prime} and 3{prime} untranslated sequences are conserved. Similar RNA folding patterns of the 5{prime} untranslated region are predicted despite a 91-bp insertion in the mouse. These data suggest that both the function and the regulation of ESA protein are of importance and that Esa (M17S1) is not the nude locus gene. 42 refs., 7 figs., 3 tabs.

  10. Detection of cell surface and intracellular antigens by human monoclonal antibodies. Hybrid cell lines derived from lymphocytes of patients with malignant melanoma

    PubMed Central

    1983-01-01

    This study represents an initial attempt to analyze the humoral immune reactions of patients with malignant melanoma by hybridoma methodology. Using lymphocytes from regional lymph nodes, peripheral blood and tumor infiltrates, 158 fusions were performed with SKO-007 (human myeloma line), LICR-LON-HMy2 (LICR-2), GM 4672 (human lymphoblastoid lines), or NS-1 (mouse myeloma line). Fusion of lymph node lymphocytes with NS-1 resulted in a 3-4 times higher frequency of clones than fusion with LICR-2, and a 10 times higher frequency than fusion with SKO-007 or GM 4672. In the case of peripheral blood lymphocytes, fusion with NS-1 gave greater than 25 times higher frequency of clones than fusion with LICR-2 or SKO-007. Production of human mu, gamma, or alpha heavy chains was detected in 50-80% of wells containing growing clones, and the levels of immunoglobulin ranged from 0.3 micrograms to 40 micrograms/ml. NS-1-derived clones could be easily subcultured, while LICR-2 and SKO-007 clones grew more slowly on subculturing. In this study, Ig secretion appeared to be a more stable property of LICR-2- derived clones than NS-1-derived clones. A panel of 20 human cancer cell lines was used to screen 771 Ig-secreting cultures for antibody to cell surface or intracellular antigens. Reactivity with cell surface antigens was found infrequently (6 cultures), whereas reactivity with intracellular antigens was more common (27 cultures). A new cell surface antigen with properties of a glycolipid was defined with an IgM monoclonal antibody secreted by a tetraploid cell derived from a fusion of LICR-2 with lymphocytes from the axillary lymph node of a patient with melanoma. The hybrid cell line has been subcloned four times and secretes 5 micrograms IgM/ml. The antigen detected by this IgM antibody was found on 5 of 23 melanoma cell lines and 12 of 30 epithelial cancer cell lines. No reactions were found with 11 cultures derived from normal cells. Stable cell lines secreting human

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

    PubMed Central

    2012-01-01

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

  12. Antigenic variation in Giardia lamblia.

    PubMed

    Prucca, Cesar G; Lujan, Hugo D

    2009-12-01

    Giardia lamblia undergoes antigenic variation, both in vitro and within the intestines of infected individuals. Variant-specific surface proteins (VSPs) cover the entire surface of the trophozoites and are the main antigens recognized by the host. Only 1 of about 200 VSP genes encoded by the Giardia genome is expressed on the surface of individual Giardia cells at any time; however, VSP antigen switching occurs spontaneously. In the recent year, significant advances in the knowledge of the antigen switching process have been achieved, which strongly suggests that antigenic variation in Giardia is regulated at the post-transcriptional level by a mechanism similar to RNA interference (RNAi). Several enzymes of the RNAi pathway are directly involved in VSP mRNA silencing and/or translational repression. Although several questions remain regarding how individual VSP antigens are selected for expression on the parasite surface, it is clear that an epigenetic mechanism is involved. In this review, we summarize the characteristics of this fascinating mechanism, analyse conflicting information regarding the structure of VSPs as it relates to the host's immune response, and highlight the major issues that need to be resolved to fully understand antigenic variation in this important pathogen.

  13. In Vitro Evaluation of a Soluble Leishmania Promastigote Surface Antigen as a Potential Vaccine Candidate against Human Leishmaniasis

    PubMed Central

    Bahi-Jaber, Narges; Petitdidier, Elodie; Markikou-Ouni, Wafa; Aoun, Karim; Moreno, Javier; Carrillo, Eugenia; Salotra, Poonam; Kaushal, Himanshu; Negi, Narender Singh; Arevalo, Jorge; Falconi-Agapito, Francesca; Privat, Angela; Cruz, Maria; Pagniez, Julie; Papierok, Gérard-Marie; Rhouma, Faten Bel Haj; Torres, Pilar; Lemesre, Jean-Loup; Chenik, Mehdi; Meddeb-Garnaoui, Amel

    2014-01-01

    PSA (Promastigote Surface Antigen) belongs to a family of membrane-bound and secreted proteins present in several Leishmania (L.) species. PSA is recognized by human Th1 cells and provides a high degree of protection in vaccinated mice. We evaluated humoral and cellular immune responses induced by a L. amazonensis PSA protein (LaPSA-38S) produced in a L. tarentolae expression system. This was done in individuals cured of cutaneous leishmaniasis due to L. major (CCLm) or L. braziliensis (CCLb) or visceral leishmaniasis due to L. donovani (CVLd) and in healthy individuals. Healthy individuals were subdivided into immune (HHR-Lm and HHR-Li: Healthy High Responders living in an endemic area for L. major or L. infantum infection) or non immune/naive individuals (HLR: Healthy Low Responders), depending on whether they produce high or low levels of IFN-γ in response to Leishmania soluble antigen. Low levels of total IgG antibodies to LaPSA-38S were detected in sera from the studied groups. Interestingly, LaPSA-38S induced specific and significant levels of IFN-γ, granzyme B and IL-10 in CCLm, HHR-Lm and HHR-Li groups, with HHR-Li group producing TNF-α in more. No significant cytokine response was observed in individuals immune to L. braziliensis or L. donovani infection. Phenotypic analysis showed a significant increase in CD4+ T cells producing IFN-γ after LaPSA-38S stimulation, in CCLm. A high positive correlation was observed between the percentage of IFN-γ-producing CD4+ T cells and the released IFN-γ. We showed that the LaPSA-38S protein was able to induce a mixed Th1 and Th2/Treg cytokine response in individuals with immunity to L. major or L. infantum infection indicating that it may be exploited as a vaccine candidate. We also showed, to our knowledge for the first time, the capacity of Leishmania PSA protein to induce granzyme B production in humans with immunity to L. major and L. infantum infection. PMID:24786587

  14. Proper exercise decreases plasma carcinoembryonic antigen levels with the improvement of body condition in elderly women.

    PubMed

    Ko, Il-Gyu; Park, Eung-Mi; Choi, Hye-Jung; Yoo, Jaehyun; Lee, Jong-Kyun; Jee, Yong-Seok

    2014-01-01

    Aging increases the risk of chronic diseases including cancers. Physical exercise has the beneficial effects for the elderly susceptible to the development of cancers, through maintaining a healthy body condition and improving the immune system. However, excessive or insufficient exercise might increase the risk for cancer. In the present study, we investigated what exercise frequency improves cancer-related biomarkers, such as carcinoembryonic antigen (CEA), alpha fetoprotein (AFP), red blood cell (RBC), and white blood cell (WBC), and the body composition of elderly women. Fifty-four females, aged 70 to 77 years, were divided into 4 groups: control, 1-day exercise (1E), 2-3-day exercise (2-3E), and 5-day exercise (5E) groups. The control group did not participate in any physical activity, while the subjects in the exercise groups underwent the exercise program for 12 weeks. As results, CEA was significantly decreased in the exercise groups, with the lowest values in 2-3E group. In contrast, AFP, RBC and WBC were not significantly changed. CEA is an oncofetal glycoprotein that is overexpressed in adenocarcinomas. Although the function of CEA has not been fully understood, CEA has been suggested to be involved in the release of pro-inflammatory cytokines via stimulating monocytes and macrophages. Moreover, body weight and body mass index were improved in the exercise groups, with the lowest levels in 5E group. Thus, we suggest that exercise for 2-3 days per week decreases the expression of CEA and improves body condition, without loading fatigue or stress, which may contribute to preventing cancer in the elderly women.

  15. 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

  16. Early hepatitis B surface antigen decline predicts treatment response to entecavir in patients with chronic hepatitis B

    PubMed Central

    Peng, Cheng-Yuan; Lai, Hsueh-Chou; Su, Wen-Pang; Lin, Chia-Hsin; Chuang, Po-Heng; Chen, Sheng-Hung; Chen, Ching-Hsiang

    2017-01-01

    Early declines in serum hepatitis B surface (HBsAg) levels, their optimal cutoffs, and association with therapeutic endpoints in chronic hepatitis B (CHB) patients receiving entecavir treatment remain unclear. We prospectively enrolled 529 patients (195 hepatitis B e antigen [HBeAg]-positive and 334 HBeAg-negative) with a median treatment duration of 49.2 months. Median HBsAg levels declined significantly in both groups at Month 3, but only at Months 6–12 in the HBeAg-negative group. Both groups exhibited a significant HBsAg decline with each successive year of treatment. An HBsAg decline of ≥75% from baseline, assessed at Months 3 and 12 of treatment in the HBeAg-positive and -negative patients, respectively, independently predicted a virological response and HBeAg seroconversion in the HBeAg-positive patients, an HBsAg level of <100 IU/mL in the HBeAg-negative patients, and HBsAg loss in all the patients during treatment. HBsAg levels of <3,000 IU/mL at baseline combined with an HBsAg decline of ≥75% from baseline provided a predictive algorithm for HBsAg loss (positive and negative predictive values: 70% and 100%, respectively) during 5 years of treatment. The proposed cutoffs for defining an HBsAg decline may assist clinicians in early assessments of treatment responses in genotype B-infected or C-infected CHB patients receiving entecavir therapy. PMID:28220833

  17. Identification of surface proteins and antigens from larval stages of Ascaris suum by two-dimensional electrophoresis.

    PubMed

    Kasuga-Aoki, H; Tsuji, N; Suzuki, K; Isobe, T; Yoshihara, S

    2000-12-01

    An understanding of the biology of the cuticle in the larval stages of Ascariodea is of importance since the cuticle molecules not only possess a variety of functions related to survival but also have a potential role as a target for immunoprophylaxis. Thus, we made a preliminary characterization of surface proteins and antigens from 3rd-stage larvae (L3) and lung-stage larvae of Ascaris suum using two biotin-derivatives and two-dimen sional polyacrylamide gel electrophoresis (2D-PAGE). The proteins labelled with biotin comprised a total of 37 and 32 spots, with molecular weights (Air) ranging from 15 to 101 kDa and isoelectric points (pI) from 3.8 to 7.6, in L3 and lung-stage larvae, respectively. The profiles revealed that the individual spots bound to one or both biotin derivatives. In addition, stage-common and stage-specific spots were found in L3 and lung-stage larvae. 2D-PAGE/immunoblotting analysis was performed with antisera from rabbits repeatedly inoculated with A. suum L3. Nineteen spots were recognized as surface antigens, with Mr ranging from 32 to 66 kDa and pI from 4.9 to 7.6, from L3 and lung-stage larvae after alignment of the immunoblots with the profile of the surface proteins. These spots were found to include stage-common and stage-specific antigens. Identification of surface proteins by biotin labelling combined with 2D-PAGE allows a substantial shortening of sample preparation time for the target proteins, and will be a viable method for protein analysis of surface proteins and antigens of A. suum L3 and lung-stage larvae.

  18. Detection of Rubisco and mycotoxins as potential contaminants of a plantibody against the hepatitis B surface antigen purified from tobacco.

    PubMed

    Geada, Déborah; Valdés, Rodolfo; Escobar, Arturo; Ares, Dulce M; Torres, Edel; Blanco, Reinaldo; Ferro, Williams; Dorta, Dayamí; González, Marcos; Alemán, María R; Padilla, Sigifredo; Gómez, Leonardo; Del Castillo, Norma; Mendoza, Otto; Urquiza, Dioslaida; Soria, Yordanka; Brito, José; Leyva, Alberto; Borroto, Carlos; Gavilondo, Jorge V

    2007-10-01

    Antibodies have been one of the proteins widely expressed in tobacco plants for pharmaceutical purposes, which demand contaminant free preparations. Rubisco constitutes 40-60% of tobacco leaf soluble proteins; therefore it is the major potential protein contaminant of plantibodies, while mycotoxins are toxic compounds that could be introduced during the biomass production and post-harvest stages with important consequences to human health. The objective of this paper was to investigate whether Rubisco and mycotoxins are present in Plantibody HB-01 preparations used in the immunopurification of the hepatitis B surface antigen. Rubisco was purified from Nicotiana tabacum yielding 154 microg of protein per gram of leaves and purity over 95%. Among mouse monoclonal antibodies generated against this enzyme, the CBSS.Rub-2 was selected for its immunodetection. It recognizes a conserved sequential epitope of Rubisco large subunit with an affinity constant of 0.13 x 10(8)M(-1). Rubisco quantification limit was 1 microg spreading to the measurement of this contaminant less than 4% of plantibodies samples. Additionally, according to a Reverse Phase-HPLC used to measure the level of adventitiously introduced contaminants, it can be concluded that aflatoxins B1, B2, G1 and G2 were undetected in the purified Plantibody HB-01 samples.

  19. B-cell surface antigen B7 provides a costimulatory signal that induces T cells to proliferate and secrete interleukin 2.

    PubMed Central

    Gimmi, C D; Freeman, G J; Gribben, J G; Sugita, K; Freedman, A S; Morimoto, C; Nadler, L M

    1991-01-01

    Occupancy of the T-cell receptor complex does not appear to be a sufficient stimulus to induce a T-cell-mediated immune response. Increasing evidence suggests that cognate cell-cell interaction between an activated T cell and an antigen-presenting cell may provide such a stimulus. A candidate T-cell surface molecule for this costimulatory signal is the T-cell-restricted CD28 antigen. Following crosslinking with anti-CD28 mAb, suboptimally stimulated CD28+ T cells show increased proliferation and markedly increased secretion of a subset of lymphokines. Recently, the B-cell surface activation antigen B7 was shown to be a natural ligand for the CD28 molecule, and both B7 and CD28 are members of the immunoglobulin superfamily. Here we report that B7-transfected CHO cells can induce suboptimally activated CD28+ T cells to proliferate and secrete high levels of interleukin 2. The response is identical whether T cells are submitogenically stimulated with either phorbol myristate acetate or anti-CD3 to activate the T cells. This response is specific and can be totally abrogated with anti-B7 monoclonal antibody. As has previously been observed for anti-CD28 monoclonal antibody, B7 ligation induced secretion of interleukin 2 but not interleukin 4. We have previously demonstrated that B7 expression is restricted to activated B lymphocytes and interferon gamma-activated monocytes. Since these two cellular populations are involved in antigen presentation as well as cognate interaction with T lymphocytes, B7 is likely to represent a central constimulatory signal that is capable of amplifying an immune response. PMID:1650475

  20. Role of quantitative hepatitis B surface antigen in predicting inactive carriers and HBsAg seroclearance in HBeAg-negative chronic hepatitis B patients

    PubMed Central

    Ungtrakul, Teerapat; Sriprayoon, Tassanee; Kusuman, Pattama; Chunnuan, Pitchayachuda; Soonklang, Kamonwan; Sornsamdang, Gaidganok; Auewarakul, Chirayu U.; Tanwandee, Tawesak

    2017-01-01

    Abstract To evaluate quantitative hepatitis B surface antigen (qHBsAg) as a diagnostic marker for inactive carriers (ICs) and hepatitis B surface antigen (HBsAg) seroclearance in hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (CHB) patients. We retrospectively studied 300 HBeAg-negative CHB patients with initial serum hepatitis B virus (HBV) Deoxyribonucleic acid (DNA) levels <2000 IU/mL. Serum HBV DNA and alanine aminotransferase (ALT) levels were monitored every 6 months for 24 months. ICs were identified as having persistent HBV DNA levels <2000 IU/mL and normal ALT levels, whereas active carriers (ACs) were identified as having HBV DNA levels ≥2000 IU/mL, with or without elevated ALT levels. The serum qHBsAg level was defined at baseline and evaluated as a diagnostic predictor using a receiver-operating characteristic curve. The study group comprised 134 men and 166 women with a median age of 41.5 years. At baseline, 200 ICs displayed lower levels of qHBsAg (1492 IU/mL) compared with 100 ACs (2936 IU/mL) (P = 0.005). The qHBsAg level was independently associated with the IC state and HBsAg seroclearance. Baseline qHBsAg levels <1000 IU/mL and HBV DNA levels <2000 IU/mL, when detected simultaneously, allowed for identification of ICs with 41% sensitivity and 72% specificity. Fifteen patients (5%) displayed HBsAg seroclearance after 24 months. A qHBsAg cutoff value of <50 IU/mL provided 100% sensitivity and 92% specificity in predicting HBsAg seroclearance. The qHBsAg level at a single timepoint among HBeAg-negative CHB patients with low HBV DNA levels at baseline was not a predictive marker for ICs; however, it accurately predicted spontaneous HBsAg seroclearance at 24 months. PMID:28353619

  1. Serological responses to Cryptosporidium antigens in inhabitants of Hungary using conventionally filtered surface water and riverbank filtered drinking water.

    PubMed

    Farkas, K; Plutzer, J; Moltchanova, E; Török, A; Varró, M J; Domokos, K; Frost, F; Hunter, P R

    2015-10-01

    In this study the putative protective seroprevalence (PPS) of IgG antibodies to the 27-kDa and 15/17-kDa Cryptosporidium antigens in sera of healthy participants who were and were not exposed to Cryptosporidium oocysts via surface water-derived drinking water was compared. The participants completed a questionnaire regarding risk factors that have been shown to be associated with infection. The PPS was significantly greater (49-61%) in settlements where the drinking water originated from surface water, than in the control city where riverbank filtration was used (21% and 23%). Logistic regression analysis on the risk factors showed an association between bathing/swimming in outdoor pools and antibody responses to the 15/17-kDa antigen complex. Hence the elevated responses were most likely due to the use of contaminated water. Results indicate that waterborne Cryptosporidium infections occur more frequently than reported but may derive from multiple sources.

  2. Physicochemical properties of recombinant hepatitis B surface antigen expressed in mammalian cell (C127).

    PubMed

    Lee, Y S; Kim, B K; Choi, E C

    1998-10-01

    The physicochemical properties of recombinant hepatitis B surface antigen (r-HBsAg), which was expressed in C127 mammalian cell were studied. Using roller bottle culture in DMEM supplemented with fetal bovine serum, 10-15 mg/L of r-HBsAg was produced with about 31% of purification yield. The purity of r-HBsAg by HPLC was 99.8% and electron microscopic examination showed homogeneous spherical particle with 22 nm in diameter, a morphological characteristic of HBsAg. The density of r-HBsAg by CsCl density gradient method was 1.19 g/ml and the isoelectric point by Mono P HR 5/20 column was 4.6. The analysis of subunit protein pattern using SDS-PAGE followed by scanning densitometry gave 81.3% of S protein and 18.7% of pre-S protein. Fluorophore-assisted-carbohydrate-electrophoresis analysis showed the relative amount of carbohydrate to protein was 1.7% and its major component was N-acetyl glucosamine, which was about 39% of total carbohydrate. The relative amount of lipid to protein determined by vanillin phosphoric acid method was 32.5% and its major component was phospholipid, which was about 70% of total lipid. The physicochemical properties of C127 mammalian cell-derived r-HBsAg are similar to those of p-HBsAg, suggesting that the r-HBsAg can be used in developing a new preventive vaccine against hepatitis B.

  3. 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.

  4. Localization of immunodominant epitopes within the "a" determinant of hepatitis B surface antigen using monoclonal antibodies.

    PubMed

    Golsaz-Shirazi, Forough; Mohammadi, Hamed; Amiri, Mohammad Mehdi; Khoshnoodi, Jalal; Kardar, Gholam Ali; Jeddi-Tehrani, Mahmood; Shokri, Fazel

    2016-10-01

    The common "a" determinant is the major immunodominant region of hepatitis B surface antigen (HBsAg) shared by all serotypes and genotypes of hepatitis B virus (HBV). Antibodies against this region are thought to confer protection against HBV and are essential for viral clearance. Mutations within the "a" determinant may lead to conformational changes in this region, which can affect the binding of neutralizing antibodies. There is an increasing concern about identification and control of mutant viruses which is possible by comprehensive structural investigation of the epitopes located within this region. Anti-HBs monoclonal antibodies (mAbs) against different epitopes of HBsAg are a promising tool to meet this goal. In the present study, 19 anti-HBs mAbs were employed to map epitopes localized within the "a" determinant, using a panel of recombinant mutant HBsAgs. The topology of the epitopes was analyzed by competitive enzyme-linked immunosorbent assay (ELISA). Our results indicate that all of the mAbs seem to recognize epitopes within or in the vicinity of the "a" determinant of HBsAg. Different patterns of binding with mutant forms were observed with different mAbs. Amino acid substitutions at positions 123, 126, 129, 144, and 145 dramatically reduced the reactivity of antibodies with HBsAg. The T123N mutation had the largest impact on antibody binding to HBsAg. The reactivity pattern of our panel of mAbs with mutant forms of HBsAg could have important clinical implications for immunoscreening, diagnosis of HBV infection, design of a new generation of recombinant HB vaccines, and immunoprophylaxis of HBV infection as an alternative to therapy with hepatitis B immune globulin (HBIG).

  5. Erysipelothrix spp. genotypes, serotypes, and surface protective antigen types associated with abattoir condemnations.

    PubMed

    Bender, Joseph S; Irwin, Christa K; Shen, Hui-Gang; Schwartz, Kent J; Opriessnig, Tanja

    2011-01-01

    The objective of the current study was to investigate characteristics of Erysipelothrix spp. from slaughter condemnations. Specimens from 70 carcasses with lesions suspect for swine erysipelas were collected at an abattoir in Iowa from October 2007 to February 2009. Erysipelothrix spp. were isolated from 59 of 70 carcasses (84.3%). Abattoir inspectors classified lesions as acute, subacute, or chronic; 8 of 8 (100%) were acute cases, 31 of 32 (96.9%) were subacute cases, and 20 of 30 (66.6%) were chronic cases that were isolation positive. The following serotypes were identified: 1a (40.7%; 24/59), 2 (49.2%; 29/59), 7 (1/59), 10 (1/59), 11 (1/59), and untypeable (5.1%; 3/59). Serotypes 1a and 2 were identified in pigs with acute, subacute, or chronic clinical manifestations, whereas serotypes 7, 10, and 11 were only present in chronic cases. Fifty-seven of the 59 isolates were determined to belong to E. rhusiopathiae, and 2 of 59 of the isolates were determined to be E. tonsillarum by multiplex real-time polymerase chain reaction. Surface protective antigen (spa) A was detected in all E. rhusiopathiae isolates but not in E. tonsillarum serotypes 7 and 10. The results of the present study indicate that E. rhusiopathiae serotypes 1a and 2 continue to be commonly isolated from condemned pig carcasses and that spaA is the exclusive spa type in U.S. abattoir isolates. Interestingly, E. tonsillarum, thought to be avirulent for swine, was isolated from systemic sites from 3.4% of the carcasses that were negative for E. rhusiopathiae, indicating the potential importance of this genotype in erysipelas pathogenesis.

  6. Prevalence of Hepatitis B Surface Antigen in US-Born and Foreign-Born Asian/Pacific Islander College Students

    ERIC Educational Resources Information Center

    Quang, Yen N.; Vu, Joanne; Yuk, Jihey; Li, Chin-Shang; Chen, Moon; Bowlus, Christopher L.

    2010-01-01

    The prevalence of chronic hepatitis B (HBV) among college-age US-born Asian and Pacific Islanders (A/PI) is not well known. Objectives: To compare the prevalence of hepatitis B surface antigen (HBsAg) seropositivity in US-born to A/PI-born students at a public university. Participants: Undergraduate who self-identified themselves as A/PI. Results:…

  7. The SnSAG merozoite surface antigens of Sarcocystis neurona are expressed differentially during the bradyzoite and sporozoite life cycle stages.

    PubMed

    Gautam, A; Dubey, J P; Saville, W J; Howe, D K

    2011-12-29

    Sarcocystis neurona is a two-host coccidian parasite whose complex life cycle progresses through multiple developmental stages differing at morphological and molecular levels. The S. neurona merozoite surface is covered by multiple, related glycosylphosphatidylinositol-linked proteins, which are orthologous to the surface antigen (SAG)/SAG1-related sequence (SRS) gene family of Toxoplasma gondii. Expression of the SAG/SRS proteins in T. gondii and another related parasite Neospora caninum is life-cycle stage specific and seems necessary for parasite transmission and persistence of infection. In the present study, the expression of S. neurona merozoite surface antigens (SnSAGs) was evaluated in the sporozoite and bradyzoite stages. Western blot analysis was used to compare SnSAG expression in merozoites versus sporozoites, while immunocytochemistry was performed to examine expression of the SnSAGs in merozoites versus bradyzoites. These analyses revealed that SnSAG2, SnSAG3 and SnSAG4 are expressed in sporozoites, while SnSAG5 was appeared to be downregulated in this life cycle stage. In S. neurona bradyzoites, it was found that SnSAG2, SnSAG3, SnSAG4 and SnSAG5 were either absent or expression was greatly reduced. As shown for T. gondii, stage-specific expression of the SnSAGs may be important for the parasite to progress through its developmental stages and complete its life cycle successfully. Thus, it is possible that the SAG switching mechanism by these parasites could be exploited as a point of intervention. As well, the alterations in surface antigen expression during different life cycle stages may need to be considered when designing prospective approaches for protective vaccination.

  8. Association of high levels of serum antibody to staphylococcal toxic shock antigen with nasal carriage of toxic shock antigen-producing strains of Staphylococcus aureus.

    PubMed Central

    Ritz, H L; Kirkland, J J; Bond, G G; Warner, E K; Petty, G P

    1984-01-01

    Forty-four asymptomatic male subjects were examined for their nasal carriage of strains of Staphylococcus aureus capable of producing staphylococcal toxic shock antigen (TSA), an exotoxin implicated in the pathogenesis of toxic shock syndrome. In addition, the levels of antibody to TSA in sera from these subjects were determined by an enzyme-linked immunosorbent assay. S. aureus was isolated from the anterior nares of 23 subjects. Of those 23 isolates of S. aureus, 9 were found to produce TSA. All individuals carrying strains of S. aureus capable of producing TSA had high to moderate levels of antibody to TSA. In contrast, those individuals carrying strains not producing TSA had levels of antibody to TSA ranging from high to nondetectable. A second examination of nasal samples from 42 of these subjects revealed that 86% of those carrying S. aureus initially still carried S. aureus after a period of 3 months; all subjects found to carry TSA-producing strains initially and that were examined a second time yielded TSA-producing strains once again. PMID:6698614

  9. Physiological level production of antigen-specific human immunoglobulin in cloned transchromosomic cattle.

    PubMed

    Sano, Akiko; Matsushita, Hiroaki; Wu, Hua; Jiao, Jin-An; Kasinathan, Poothappillai; Sullivan, Eddie J; Wang, Zhongde; Kuroiwa, Yoshimi

    2013-01-01

    Therapeutic human polyclonal antibodies (hpAbs) derived from pooled plasma from human donors are Food and Drug Administration approved biologics used in the treatment of a variety of human diseases. Powered by the natural diversity of immune response, hpAbs are effective in treating diseases caused by complex or quickly-evolving antigens such as viruses. We previously showed that transchromosomic (Tc) cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin heavy-chain (hIGH) and kappa-chain (hIGK) germline loci (named as κHAC) are capable of producing functional hpAbs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, are homozygously inactivated (double knockouts or DKO). However, B lymphocyte development in these Tc cattle is compromised, and the overall production of hpAbs is low. Here, we report the construction of an improved HAC, designated as cKSL-HACΔ, by incorporating all of the human immunoglobulin germline loci into the HAC. Furthermore, for avoiding the possible human-bovine interspecies incompatibility between the human immunoglobulin mu chain protein (hIgM) and bovine transmembrane α and β immunoglobulins (bIgα and bIgβ) in the pre-B cell receptor (pre-BCR) complex, we partially replaced (bovinized) the hIgM constant domain with the counterpart of bovine IgM (bIgM) that is involved in the interaction between bIgM and bIgα/Igβ; human IgM bovinization would also improve the functionality of hIgM in supporting B cell activation and proliferation. We also report the successful production of DKO Tc cattle carrying the cKSL-HACΔ (cKSL-HACΔ/DKO), the dramatic improvement of B cell development in these cattle and the high level production of hpAbs (as measured for the human IgG isotype) in the plasma. We further demonstrate that, upon immunization by tumor immunogens, high titer tumor immunogen-specific human IgG (hIgG) can be produced from such Tc cattle.

  10. [Changes of ADAMTS13 activity and vWF antigen level in patients with acute myelogenous leukemia and their significance].

    PubMed

    Zhang, Wen-Juan; Han, Yue; Ma, Zhen-Ni; Wang, Qian; Tang, Ya-Qiong; Wang, Jie; Su, Jian; Sun, Ai-Ning; Wang, Zhao-Yue; Ruan, Chang-Geng; Wu, De-Pei

    2014-12-01

    This study was purposed to investigate the changes of von Willebrand factor cleaving protease (ADAMTS13) activity and vWF antigen level in patients with acute myelogenous leukemia (AML) before and after treatment and evaluate their clinical significance. Seventy-three AML patients were enrolled in this study, the sodium citrate anticoagulated plasma was collected before and after their induction chemotherapy. Fluorescence resonance energy transfer substrate vWF73 (FRETS-vWF73) assay was established to detect the plasma ADAMTS13 activity while vWF antigen level was measured by ELISA. The results showed that the ADAMTS13 activity in newly diagnosed patients with AML before induction therapy was obviously lower than that in normal controls (63.3 ± 25.5)% vs (105.1 ± 37.7)(P < 0.01), while the vWF antigen level was higher than that in normal controls (226.6 ± 127.0)% vs (111.4 ± 39.7)% (P < 0.01). After standard induction chemotherapy, the ADAMTS13 activity of AML patients in complete remission period was higher than that in AML patients before therapy (P < 0.01), and was not significant difference with that in normal controls; the vWF antigen was significantly lower than that in AML patients before therapy (P < 0.01), but it still was higher than that in controls (P < 0.05). The ADAMTS13 activity in newly diagnosed AML patients complicated with infection before therapy was obviously lower than that in AML patients without infection (52.2 ± 20.6)% vs (73.9 ± 24.7)% (P < 0.01), while the vWF antigen level was significantly higher than that in AML patients without infection (262.2 ± 135.7)% vs (193.8 ± 110.2)% (P < 0.05). The ADAMTS13 activity in AML patients with disseminated intravascular coagulation (DIC) was significantly lower than that in AML patients without DIC (42.0 ± 14.5)% vs (73.4 ± 22.7)% (P < 0.01), while the vWF antigen level was obviously higher that in AML patients without DIC (274.2 ± 140.0)% vs (204.7 ± 115.5)% (P < 0.01). It is concluded

  11. Low-dose oral immunization with lyophilized tissue of herbicide-resistant lettuce expressing hepatitis B surface antigen for prototype plant-derived vaccine tablet formulation.

    PubMed

    Pniewski, Tomasz; Kapusta, Józef; Bociąg, Piotr; Wojciechowicz, Jacek; Kostrzak, Anna; Gdula, Michał; Fedorowicz-Strońska, Olga; Wójcik, Piotr; Otta, Halina; Samardakiewicz, Sławomir; Wolko, Bogdan; Płucienniczak, Andrzej

    2011-05-01

    Efficient immunization against hepatitis B virus (HBV) and other pathogens with plant-based oral vaccines requires appropriate plant expressors and the optimization of vaccine compositions and administration protocols. Previous immunization studies were mainly based on a combination of the injection of a small surface antigen of HBV (S-HBsAg) and the feeding with raw tissue containing the antigen, supplemented with an adjuvant, and coming from plants conferring resistance to kanamycin. The objective of this study was to develop a prototype oral vaccine formula suitable for human immunization. Herbicide-resistant lettuce was engineered, stably expressing through progeny generation micrograms of S-HBsAg per g of fresh weight and formed into virus-like particles (VLPs). Lyophilized tissue containing a relatively low, 100-ng VLP-assembled antigen dose, administered only orally to mice with a long, 60-day interval between prime and boost immunizations and without exogenous adjuvant, elicited mucosal and systemic humoral anti-HBs responses at the nominally protective level. Lyophilized tissue was converted into tablets, which preserved S-HBsAg content for at least one year of room temperature storage. The results of the study provide indications on immunization methodology using a durable, efficacious, and convenient plant-derived prototype oral vaccine against hepatitis B.

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

    PubMed

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

    2010-08-01

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

  13. Episome-carried Surface Antigen K88 of Escherichia coli II. Isolation and Chemical Analysis

    PubMed Central

    Stirm, Stephan; Ørskov, Frits; Ørskov, Ida; Mansa, Bendt

    1967-01-01

    The K88 antigen was carried by episomal transfer to D282, a nonmotile Escherichia coli strain without K antigen. D520, obtained by this episomal transfer, was used for the extraction of K88 antigen. It was shown by the agar gel precipitation technique that some K88 antigen was released from D520 into suspending aqueous medium. The amount of liberated material was increased by gentle heating (60 C) or treatment in a Waring Blendor. The antigen was obtained from the extracts in a purified form by making use of its insolubility between pH 3.5 and 5.5 and of its high sedimentation rate (S020,w = 36.7S). The homogeneity of the material was demonstrated by agar gel precipitation with D520 antiserum, by analytical ultracentrifugation, and by moving-boundary electrophoresis. Chemical analysis revealed that K88 is a pure protein containing all the common amino acids with the exception of cysteine-cystine. Purified K88 selectively precipitated the K88 antibodies from D520 antiserum and was shown to be immunogenic in rabbits. Images PMID:4960185

  14. Predictive and prognostic values of cancer-associated serum antigen (CASA) and cancer antigen 125 (CA 125) levels prior to second-look laparotomy for ovarian cancer.

    PubMed

    Kierkegaard, O; Mogensen, O; Mogensen, B; Jakobsen, A

    1995-11-01

    CA 125 and cancer-associated serum antigen (CASA) were measured prior to second-look laparotomy (SLL) to investigate their predictive and prognostic values in 93 patients treated for epithelial ovarian cancer FIGO stage II, III, or IV. Residual tumor was diagnosed at the SLL in 58 patients (62%). The optimal cutoff level was 15 U/ml for CA 125 and 8 U/ml for CASA. Using these levels, the sensitivity for detection of residual tumor was 40% for CA 125 and 22% for CASA. The combined use of the markers resulted in a sensitivity of 47% (diagnostic gain 6.9%; 95% confidence interval (CI), 0.14-13.44%). Microscopic tumor volumes were equally diagnosed by CASA and CA 125. The independent prognostic value of CA 125 (RR = 2.6; 95% CI, 2.0-3.2) and CASA (RR = 2.2; CI, 1.5-2.9) was established by means of Cox regression analysis of the covariation between survival, age, FIGO stage, histopathology, tumor grade, and bulk of residual tumor at the primary operation and CA 125 and CASA before the SLL. In conclusion, we found that CASA could supplement CA 125 measurement prior to SLL and reduce the number of SLLs. Furthermore, CASA had an independent prognostic value for survival which may be used together with other information in the planning of further treatment of the individual patient.

  15. T cell ontogeny. Organ location of maturing populations as defined by surface antigen markers is similar in neonates and adults

    PubMed Central

    1981-01-01

    Earlier studies have suggested that splenic T cell populations in nursling mice (less than 18 d of age) have Lyt cell surface antigens that identify them as less mature than their adult counterparts. Studies presented here, however, demonstrate that the expression of the Thy-1, Lyt-1, Lyt-2, and Lyt-3 T cell antigens is virtually identical in 14-d-old and adult T cell populations even though at 14 d, T cells constitute less than 10% of the total spleen cell population. Because the expression of these antigens on the immature (cortical) thymocyte population differs substantially from their expression on peripheral T cells, the maturity of splenic T cells as judged by these criteria is similar in nurslings and adults. Very few cells in the neonatal thymus 4 h after birth correspond, in terms of antigen expression, to the more mature (medullary) thymocyte population of adults, but such cells develop rapidly during the first few days of life. They are present, therefore, sufficiently early to serve as the immediate source of peripheral T cells, as they apparently do in the adult. This then suggests that the locations for the major T cell maturational events are established within the first 2 wk of life of the mouse and maintained as such thereafter. The use of monoclonal antibodies and quantitative immunofluorescence analysis in our studies probably explains the differences between our findings and those reported previously, which relied on cytotoxic depletion by alloantisera and complement to estimate the frequencies of cells carrying the Lyt differentiation antigens in nurslings. PMID:6972987

  16. T cell ontogeny. Organ location of maturing populations as defined by surface antigen markers is similar in neonates and adults.

    PubMed

    Haaijman, J J; Micklem, H S; Ledbetter, J A; Dangl, J L; Herzenberg, L A; Herzenberg, L A

    1981-03-01

    Earlier studies have suggested that splenic T cell populations in nursling mice (less than 18 d of age) have Lyt cell surface antigens that identify them as less mature than their adult counterparts. Studies presented here, however, demonstrate that the expression of the Thy-1, Lyt-1, Lyt-2, and Lyt-3 T cell antigens is virtually identical in 14-d-old and adult T cell populations even though at 14 d, T cells constitute less than 10% of the total spleen cell population. Because the expression of these antigens on the immature (cortical) thymocyte population differs substantially from their expression on peripheral T cells, the maturity of splenic T cells as judged by these criteria is similar in nurslings and adults. Very few cells in the neonatal thymus 4 h after birth correspond, in terms of antigen expression, to the more mature (medullary) thymocyte population of adults, but such cells develop rapidly during the first few days of life. They are present, therefore, sufficiently early to serve as the immediate source of peripheral T cells, as they apparently do in the adult. This then suggests that the locations for the major T cell maturational events are established within the first 2 wk of life of the mouse and maintained as such thereafter. The use of monoclonal antibodies and quantitative immunofluorescence analysis in our studies probably explains the differences between our findings and those reported previously, which relied on cytotoxic depletion by alloantisera and complement to estimate the frequencies of cells carrying the Lyt differentiation antigens in nurslings.

  17. Detection of a cell surface antigen found on rat peripheral nervous system neurons and multiple glia: astrocytes, oligodendrocytes, and Schwann cells.

    PubMed

    Akeson, R; Warren, S L

    1984-01-01

    A cell surface component has been identified that is found on cultured rat dorsal root ganglion neurons and Schwann cells and also cultured brain astrocytes and oligodendrocytes. This component was detected with a monoclonal antibody originally generated to the NG108 (N18 mouse neuroblastoma X C6 rat astrocytoma) hybrid cell line. The antibody, designated B2C11, binds to cultured peripheral nervous system cells: intact dorsal root ganglion and trigeminal neurons and cultured dorsal root ganglion and sciatic nerve Schwann cells. The binding of B2C11 to dorsal root ganglion neurons in vivo was confirmed by immunofluorescence analysis of cryostat sections. However, cultured embryonic rat central neurons showed no detectable binding of B2C11. Cultured brain cells containing glial fibrillary acidic protein (astrocytes) and also oligodendrocytes cultured from corpus collosum did bind B2C11 on their surfaces. B2C11 immunoprecipitation of detergent-solubilized membrane proteins from both lactoperoxidase iodinated C6 and PC12 rat pheochromocytoma cells indicated a single band with an apparent molecular weight of 21,000-23,000. Analysis of B2C11 binding to particulate protein preparations from adult rat organs showed highest specific activity in dorsal root ganglia. Other neural tissues had substantial binding. Some nonneural tissues (lung, kidney, and small intestine) expressed significant antigen levels, whereas others (heart, liver, and skeletal muscle) had a B2C11 antigen-specific activity less than 5% of that of dorsal root ganglia. Thus the B2C11 antigen is enriched in neural tissues, where it is found on the surfaces of a unique set of neuronal and glial cells.

  18. Attempts to protect severe combined immunodeficient (scid) mice with antibody enriched for reactivity to Cryptosporidium parvum surface antigen-1.

    PubMed

    Tatalick, L M; Perryman, L E

    1995-07-01

    Cryptosporidium parvum is a protozoal pathogen which infects the gastrointestinal epithelium of mammals causing diarrhoea, the duration and severity of which is determined by the immunocompetency of the host. Currently, there is no effective treatment or prevention. We evaluated the ability of surface antigen-1 (SA-1), defined as those antigens recognized by neutralizing mAb 17.41, to elicit a protective antibody response when used as an immunogen. A SA-1 enriched fraction was obtained by immunoaffinity chromatography and was used to immunize a naive Holstein calf. SA-1 immune serum from this calf detected C. parvum epitopes to a 1:10,000 dilution in a dot blot assay, and sporozoite surface epitopes at a 1:10,000 dilution in a live immunofluorescence assay. Western blot analysis showed that SA-1 immune bovine serum recognized a similar pattern of C. parvum antigens as the defining mAb 17.41. Oral passive transfer of SA-1 immune bovine serum did not protect severe combined immunodeficient (scid) mice or suckling BALB/c mice from initial infection with C. parvum, or terminate a persistent infection in scid mice.

  19. Cellular distribution and molecular heterogeneity of MAC393 antigen (clusterin, beta-chain) on the surface membrane of bull spermatozoa.

    PubMed

    Howes, E A; Hurst, S; Laslop, A; Jones, R

    1998-07-01

    The distribution and size of a surface membrane antigen identified by a monoclonal antibody (MAC9393) have been examined in testicular and epididymal bovine sperm preparations. Western blots indicated a substantial decrease in molecular mass of the antigen during epididymal maturation from approximately 87 kDa in the testis to approximately 35 kDa in the cauda epididymidis. This was accompanied by a change in its cellular localization from the neck and whole head to the acrosomal region. N-terminal microsequencing identified MAC393 antigen as the beta-chain of clusterin. A polyclonal antiserum to the alpha-chain of clusterin recognized both testicular and epididymal forms and revealed that the heterodimer was present on the sperm tail as well as the acrosome. These findings are explained by the co-existence of dimeric and monomeric pools of clusterin on spermatozoa. The polyclonal antiserum recognizes both testicular and epididymal forms of the heterodimer and although the monoclonal antibody binds to the testicular heterodimer, it only recognizes the beta-chain monomer of epididymal clusterin. These findings support previous observations made on human spermatozoa that two forms of clusterin, the beta-chain monomer and the heterodimer, are present on the surface membrane and in seminal plasma.

  20. The Murine Humoral Immune Response to Hepatitis B Surface Antigen: Idiotype Network Pathways.

    NASA Astrophysics Data System (ADS)

    Schick, Michael Roy

    Recognition of a wide spectrum in disease outcomes following Hepatitis B Virus (HBV) infection has led to the suggestion that individual differences may be due to characteristics of the immune response. HBV, a hepatotropic virus, is not directly cytopathic to the host hepatocytes but the cellular damage which does not occur may be due to the host's own immune response. It is this variety in immune response capabilities following natural infection or vaccination which led to the present study in which the murine humoral immune response to hepatitis B surface antigen (HBsAg) was examined. Following immunization with purified HBsAg an anti-HBs response could be detected in 19 inbred strains of mice. The response, which varied among the strains, was linked to the major histocompatibility complex (MHC). Among high responders to HBsAg were two strains in which a poor response to a single epitope could be detected. Although quantitatively serum from these strains resembled serum from other high responders, there was a major difference in the qualitative aspects. Included within this study was the role of idotype networks within the murine anti-HBs response. By directly targeting HBsAg-specific B cells within the framework of an idiotype network by an Ab-2, it was possible to circumvent T cell-dependent regulation of an immune response. In each of five inbred strains of mice immunized with a polyclonal rabbit Ab-2 an Ab-3 population with HBsAg-specificity (Ab -1^') was induced. These mice were also immunized with HBsAg resulting in a higher anti-HBs response as compared to HBsAg immunization alone in all of the strains tested except for one. The response in this strain, normally a low responder to HBsAg, indicated that the mechanisms for genetic restriction of the anti -HBs response was still active, although it was not apparent during anti-Id immunization. The effects of an anti-Id on the murine antibody response to HBsAg may lead to insights on the presence of idiotype

  1. Rapid screening and identification of dominant B cell epitopes of HBV surface antigen by quantum dot-based fluorescence polarization assay

    NASA Astrophysics Data System (ADS)

    Meng, Zhongji; Song, Ruihua; Chen, Yue; Zhu, Yang; Tian, Yanhui; Li, Ding; Cui, Daxiang

    2013-03-01

    A method for quickly screening and identifying dominant B cell epitopes was developed using hepatitis B virus (HBV) surface antigen as a target. Eleven amino acid fragments from HBV surface antigen were synthesized by 9-fluorenylmethoxy carbonyl solid-phase peptide synthesis strategy, and then CdTe quantum dots were used to label the N-terminals of all peptides. After optimizing the factors for fluorescence polarization (FP) immunoassay, the antigenicities of synthetic peptides were determined by analyzing the recognition and combination of peptides and standard antibody samples. The results of FP assays confirmed that 10 of 11 synthetic peptides have distinct antigenicities. In order to screen dominant antigenic peptides, the FP assays were carried out to investigate the antibodies against the 10 synthetic peptides of HBV surface antigen respectively in 159 samples of anti-HBV surface antigen-positive antiserum. The results showed that 3 of the 10 antigenic peptides may be immunodominant because the antibodies against them existed more widely among the samples and their antibody titers were higher than those of other peptides. Using three dominant antigenic peptides, 293 serum samples were detected for HBV infection by FP assays; the results showed that the antibody-positive ratio was 51.9% and the sensitivity and specificity were 84.3% and 98.2%, respectively. In conclusion, a quantum dot-based FP assay is a very simple, rapid, and convenient method for determining immunodominant antigenic peptides and has great potential in applications such as epitope mapping, vaccine designing, or clinical disease diagnosis in the future.

  2. Expression of hepatitis B virus surface antigens induces defective gonad phenotypes in Caenorhabditis elegans

    PubMed Central

    Chen, Yi-Yin; Lee, Li-Wei; Hong, Wei-Ning; Lo, Szecheng J

    2017-01-01

    AIM To test whether a simple animal, Caenorhabditis elegans (C. elegans), can be used as an alternative model to study the interaction between hepatitis B virus antigens (HBsAg) and host factors. METHODS Three plasmids that were able to express the large, middle and small forms of HBsAgs (LHBsAg, MHBsAg, and SHBsAg, respectively) driven by a ubiquitous promoter (fib-1) and three that were able to express SHBsAg driven by different tissue-specific promoters were constructed and microinjected into worms. The brood size, egg-laying rate, and gonad development of transgenic worms were analyzed using microscopy. Levels of mRNA related to endoplasmic reticulum stress, enpl-1, hsp-4, pdi-3 and xbp-1, were determined using reverse transcription polymerase reaction (RT-PCRs) in three lines of transgenic worms and dithiothreitol (DTT)-treated wild-type worms. RESULTS Severe defects in egg-laying, decreases in brood size, and gonad retardation were observed in transgenic worms expressing SHBsAg whereas moderate defects were observed in transgenic worms expressing LHBsAg and MHBsAg. RT-PCR analysis revealed that enpl-1, hsp-4 and pdi-3 transcripts were significantly elevated in worms expressing LHBsAg and MHBsAg and in wild-type worms pretreated with DTT. By contrast, only pdi-3 was increased in worms expressing SHBsAg. To further determine which tissue expressing SHBsAg could induce gonad retardation, we substituted the fib-1 promoter with three tissue-specific promoters (myo-2 for the pharynx, est-1 for the intestines and mec-7 for the neurons) and generated corresponding transgenic animals. Moderate defective phenotypes were observed in worms expressing SHBsAg in the pharynx and intestines but not in worms expressing SHBsAg in the neurons, suggesting that the secreted SHBsAg may trigger a cross-talk signal between the digestive track and the gonad resulting in defective phenotypes. CONCLUSION Ectopic expression of three forms of HBsAg that causes recognizable phenotypes in

  3. Eimeria bovis meront I-carrying host cells express parasite-specific antigens on their surface membrane.

    PubMed

    Badawy, Ahmed Ibrahem I; Lutz, Kathleen; Taubert, Anja; Zahner, Horst; Hermosilla, Carlos

    2010-02-01

    Host immune responses conducted against antigens of Eimeria bovis are key factors for the development of protective immunity against this protozoan disease. In this study we investigated the expression of E. bovis-derived antigens on the host cell surface membrane during E. bovis first merogony in vitro. Host cells carrying E. bovis-meront I stages expressed E. bovis host cell surface antigens (EbHCSAg) on their surface membrane which were recognised by hyperimmune sera of calves and by sera from rats immunized with E. bovis merozoites I, when tested by indirect immune fluorescent antibody test (IIFAT), laser scanning confocal microscopy (LSCM) and immune electron microscopy. Expression of EbHCSAg on permissive host cells was earliest detected 7 days p. i., thus coinciding with the onset of the parasite replication. Membrane-associated EbHCSAg were removed from infected host cells by proteinase K, partially by Triton X-100, Triton X-114 and Triton X-405, but not by 1 M NaCl, CHAPS or phospholipase C treatment. Antibodies, affinity-purified on paraformaldehyde/glutardialdehyde (PAGA)-fixed E. bovis meront I-infected bovine host cells bound to the surface meront I-carrying cells and to merozoites I (IIFAT, LSCM) but, in contrast to untreated sera, not to sporozoites. When tested on methanol-fixed merozoites I and sporozoites by IIFAT, affinity-purified antibodies bound to structures in the apical complex area of merozoites I, but not to sporozoites, whilst untreated sera caused diffuse labelling of internal structures of both parasite stages. Immune electron microscopy demonstrated binding of affinity-purified antibodies to micronemes and dense granules of merozoites I. Although the function of EbHCSAg is still unknown, results of this study might suggest an involvement in the development of protective immunity against E. bovis infections.

  4. [Effect of pH of Adsorption Buffers on the Number and Antigen-Binding Activity of Monoclonal Antibodies Immobilized on the Surface of Polystyrene Microplates].

    PubMed

    Tarakanova, Yu N; Dmitriev, A D; Massino, Yu S; Pechelulko, A A; Segal, O L; Skoblov, Yu O; Ulanova, T I; Lavrov, V F; Dmitriev, D A

    2015-01-01

    The change in the concentration and antigen-binding activity of 28 monoclonal antibodies was studied after their adsorption on the surface of polystyrene microplates in buffers with different pH values (1.0, 2.8, 7.5, 9.6, and 11.9). We used 16 clones to the HIV p24 protein and 12 clones to the surface antigen of Hepatitis B Virus. The binding efficiency of adsorbed antibodies to the labeled antigen was evaluated by the slope of the linear region of the binding curve to the concentration axis. It was shown that the antigen-binding activity of six antibodies (21.5%) statistically significantly increased after adsorption at pH 2.8 and 11.9 as compared to pH 7.5 and 9.5. The maximum amount of antibodies was found to be adsorbed on the solid surface at pH 7.5. The analysis of the binding of 125I-HBs-antigen to adsorbed antibodies made it possible to evaluate the concentration of active antibodies on the polystyrene surface. It was shown that the increase in the antigen-binding activity was due to an increase in the proportion of antibodies with retained activity after adsorption at pH 2.8 and 11.9. Under these conditions, about 20% of the antibodies retained their antigen-binding activity, and 6% did so after immobilization at pH 7.5.

  5. Glycoproteins, antigens, and regulation of complement activation on the surface of the protozoan parasite Trypanosoma lewisi: implications for immune evasion

    SciTech Connect

    Sturtevant, J.E.

    1985-01-01

    The surface antigens and glycoproteins of the rat parasitic protozoan, Trypanosoma lewisi were characterized. Radioiodination with /sup 125/I identified 10 out of more 40 polypeptides separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis. All of these components were identified as glycoproteins by peroxidase-conjugated Conconavalin A (HR-Con A) lectin affinoblotting. This analysis detected that quantitative but not qualitative changes occurred during infection. Localization of most of the reactive determinants was indicated by immunoblotting extracts of radioiodinated T. lewisi. Changes in the antigenicity as related to survival in the host are discussed. The presence of IgG and IgM on the surface of T. lewisi isolated from intact and ..gamma..-irradiated rats (irr.) and that determinants bind Ig from uninfected rat sera (NRS) was indicated by flow cytometric analysis. Immunoblotting identified the major NRS IgG binding component as the 74 kd surface glycoprotein. Complement component C3 deposition during infection was indicated by flow cytometric analysis and immunoblotting. Incubation of intact T. lewisi with normal human sera indicated that C3, C5, and factor B deposition was Mg/sup 2 +/ dependent, Ca/sup 2 +/ independent and deposited C3 was rapidly processed to hemolytically inactive fragments. Radioiodination of intact and protease T. lewisi after cultivation identified three components which correlate with resistance to lysis. This suggests that surface moieties on intact T. lewisi modulate host complement activity by restricting C3/C5 convertase activity.

  6. Supercritical fluid extraction provides an enhancement to the immune response for orally-delivered hepatitis B surface antigen.

    PubMed

    Hayden, Celine A; Smith, Emily M; Turner, Debra D; Keener, Todd K; Wong, Jeffrey C; Walker, John H; Tizard, Ian R; Jimenez-Flores, Rafael; Howard, John A

    2014-03-05

    The hepatitis B virus continues to be a major pathogen worldwide despite the availability of an effective parenteral vaccine for over 20 years. Orally-delivered subunit vaccines produced in maize may help to alleviate the disease burden by providing a low-cost, heat-stable alternative to the parenteral vaccine. Oral subunit vaccination has been an elusive goal due to the large amounts of antigen required to induce an immunologic response when administered through the digestive tract. Here we show that high levels of HBsAg were obtained in maize grain, the grain was formed into edible wafers, and wafers were fed to mice at a concentration of approximately 300 μg/g. When these wafers were made with supercritical fluid extraction (SFE)-treated maize material, robust IgG and IgA responses in sera were observed that were comparable to the injected commercial vaccine (Recombivax(®)). In addition, all mice administered SFE wafers showed high secretory IgA titers in fecal material whereas Recombivax(®) treated mice showed no detectable titer. Increased salivary IgA titers were also detected in SFE-fed mice but not in Recombivax(®) treated mice. Wafers made from hexane-treated or full fat maize material induced immunologic responses, but fecal titers were attenuated relative to those produced by SFE-treated wafers. These responses demonstrate the feasibility of using a two-dose oral vaccine booster in the absence of an adjuvant to induce immunologic responses in both sera and at mucosal surfaces, and highlight the potential limitations of using an exclusively parenteral dosing regime.

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

    PubMed

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

    2013-01-01

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

  8. Mechanism of inactivation of hepatitis B surface antigen by N alpha-cocoyl-L-arginine ethyl ester.

    PubMed Central

    Sugimoto, Y; Toyoshima, S

    1981-01-01

    The mechanism of N alpha-cocoyl-L-arginine ethyl ester (CAE) in the inactivation of hepatitis B surface antigen (HBsAg) was investigated. The CAE increased the density of HBsAg, and particles of the antigen were destroyed in amorphous clusters, suggesting that CAE influences the lipid components of HBsAg. The lipid components such as cholesterol and phospholipid were mostly removed from the antigen by the treatment with CAE. N alpha-Lauroyl-L-[U-14C] arginine ethyl ester (LAE), a principal component of CAE, became tightly bound to HBsAg in place of the lipid components. The binding amounts of LAE in the HBsAg-LAE complex reached 3.04 +/- 0.44 microgram/mg of protein. The formation of the complex was not influenced by the presence of CAE-related compounds such as L-arginine, L-arginine ethyl ester, and N alpha-cocoyl-L-arginine. Treatment with mercaptoethanolurea, guanidine hydrochloride, and some detergents failed to resolve appreciably the labeled LAE from the labeled complex. All attempts to reactivate the CAE-treated HBsAg and to restore it morphologically from the denatured aggregates were unsuccessful. These results indicate that CAE tightly binds to HBsAg, followed by formation of stable aggregates of the denatured HBsAg-CAE complex. Images PMID:7283411

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

    PubMed

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

    2004-08-24

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

  10. Investigation of spore surface antigens in the genus Bacillus by the use of polyclonal antibodies in immunofluorescence tests.

    PubMed

    Phillips, A P; Martin, K L

    1988-01-01

    Fluorescein-conjugated rabbit antibodies to formalized spores of Bacillus anthracis were tested against strains of B. anthracis and other Bacillus species in a subjective immunofluorescence test. The lack of reaction of B. anthracis Vollum spores with conjugated antibody raised against B. anthracis Sterne spores indicated that spores of the Vollum strain lacked a major surface antigen present in most of the other anthrax strains tested, including the non-encapsulated strains Sterne and the Soviet ST1, variants cured of the pX01 plasmid that codes for the toxin, and several virulent strains. Four other antibody preparations, raised against B. anthracis Vollum, New Hampshire, Ames and Strain 15, reacted to an approximately similar degree with spores of all four strains and of Sterne, indicating that Vollum has at least one spore antigen in common with these other strains. The anti-Sterne and anti-Vollum conjugates both displayed cross-reactions with spores of strains of B. cereus, B. coagulans, B. subtilis, B. megaterium, B. polymyxa, B. pumilus and B. thuringiensis. Absorption of the anti-anthrax conjugates with B. cereus NCTC 8035 and NCTC 10320 removed all these cross-reactions, demonstrating the existence of spore antigens specific for anthrax.

  11. Nonspecific Presentation of a Multiloculated Prostatic Abscess After Transurethral Prostatic Biopsy for Elevated Prostate-specific Antigen Level

    PubMed Central

    Gandhi, Nilay M.; Lin, Joseph; Schaeffer, Edward

    2014-01-01

    Prostate postbiopsy infectious complications typically present in the form of prostatitis and uncommonly urosepsis. Prostatic abscesses are generally found after multiple bouts of prostatitis and are associated with a clinically septic picture requiring intensive care unit admission and resuscitation. We report the case of a 65-year-old man who presented with prostatic abscess in the setting of nonspecific urinary symptoms after transrectal ultrasonography–guided prostate biopsy. At 4-month follow-up, he is currently free of disease with undetectable prostate-specific antigen level and negative imaging. PMID:26958487

  12. Probing Surface Chemistry at the Nanoscale Level

    NASA Astrophysics Data System (ADS)

    Rene-Boisneuf, Laetitia

    Studies various nanostructured materials have gained considerable interest within the past several decades. This novel class of materials has opened up a new realm of possibilities, both for the fundamental comprehension of matter, but also for innovative applications. The size-dependent effect observed for these systems often lies in their interaction with the surrounding environment and understanding such interactions is the pivotal point for the investigations undertaken in this thesis. Three families of nanoparticles are analyzed: semiconductor quantum dots, metallic silver nanoparticles and rare-earth oxide nanomaterials. The radical scavenging ability of cerium oxide nanoparticles (CeO 2) is quite controversial since they have been labeled as both oxidizing and antioxidant species for biological systems. Here, both aqueous and organic stabilized nanoparticles are examined in straightforward systems containing only one reactive oxygen species to ensure a controlled release. The apparent absence of their direct radical scavenging ability is demonstrated despite the ease at which CeO2 nanoparticles generate stable surface Ce 3+ clusters, which is used to explain the redox activity of these nanomaterials. On the contrary, CeO2 nanoparticles are shown to have an indirect scavenging effect in Fenton reactions by annihilating the reactivity of Fe 2+ salts. Cadmium selenide quantum dots (CdSe QD) constitute another highly appealing family of nanocolloids in part due to their tunable, size-dependent luminescence across the visible spectrum. The effect of elemental sulfur treatment is investigated to overcome one of the main drawbacks of CdSe QD: low fluorescence quantum yield. Herein, we report a constant and reproducible quantum yield of 15%. The effect of sulfur surface treatment is also assessed following the growth of a silica shell, as well as the response towards a solution quencher (4-amino-TEMPO). The sulfur treated QD is also tested for interaction with

  13. Isolation of a T-cell clone that reacts with both antigen and anti-idiotype: evidence for anti-idiotype as internal image for antigen at the T-cell level.

    PubMed Central

    Singhai, R; Levy, J G

    1987-01-01

    T-cell lines were derived from ferredoxin nonresponder B10.D2 mice that share an idiotype expressed by a monoclonal antibody (Fd-B2) with specificity for one of the two major antigenic determinants (the C determinant) of the antigen. The T-cell line and T-cell clones derived from it release interleukin 2 not only in the presence of anti-Fd-B2 idiotype antibody but in the presence of ferredoxin. The line was shown to be major histocompatibility complex-restricted in that it would respond to the anti-idiotype and antigen only in the context of presentation by cells of the H-2d haplotype. This observation also establishes that the nonresponder status of H-2d animals cannot be attributed to a lesion at the level of antigen presentation. Analysis of the fine specificity of one idiotypic clone showed that it responded only to the anti-idiotype or products of the antigen containing the C determinant, since enzymatically degraded peptides devoid of this determinant did not stimulate these cells. Furthermore, it was found that presentation of both the antigen and the anti-idiotype to the specific clone could be blocked by the Fd-B2 monoclonal antibody. PMID:3495798

  14. 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.

  15. Prostate specific antigen biosensor based on long range surface plasmon-enhanced fluorescence spectroscopy and dextran hydrogel binding matrix.

    PubMed

    Wang, Yi; Brunsen, Annette; Jonas, Ulrich; Dostálek, Jakub; Knoll, Wolfgang

    2009-12-01

    A new biosensor based on surface plasmon-enhanced fluorescence spectroscopy (SPFS), which employs long-range surface plasmons (LRSP) and a photo-cross-linkable carboxymethyl dextran (PCDM) hydrogel binding matrix, is reported. LRSPs are surface plasmon modes that propagate along a thin metallic film with orders of magnitude lower damping compared to regular surface plasmons. Therefore, their excitation provides strong enhancement of the intensity of the electromagnetic field and a greatly increased fluorescence signal measured upon binding of fluorophore-labeled molecules on the sensor surface. In addition, these modes exhibit highly extended evanescent fields penetrating up to micrometers in distance from the metallic sensor surface. Therefore, a PCDM hydrogel with approximately micrometer thickness was anchored on the sensor surface to serve as the binding matrix. We show that this approach provides large binding capacity and allows for the ultrasensitive detection. In a model experiment, the developed biosensor platform was applied for the detection of free prostate specific antigen (f-PSA) in buffer and human serum by using a sandwich immunoassay. The limit of detection at the low femtomolar range was achieved, which is approximately 4 orders of magnitude lower than that for direct detection of f-PSA based on the monitoring of binding-induced refractive index changes.

  16. Circulating Gut-Homing (α4β7+) Plasmablast Responses against Shigella Surface Protein Antigens among Hospitalized Patients with Diarrhea

    PubMed Central

    Sinha, Anuradha; Dey, Ayan; Saletti, Giulietta; Samanta, Pradip; Chakraborty, Partha Sarathi; Bhattacharya, M. K.; Ghosh, Santanu; Ramamurthy, T.; Kim, Jae-Ouk; Yang, Jae Seung; Kim, Dong Wook

    2016-01-01

    Developing countries are burdened with Shigella diarrhea. Understanding mucosal immune responses associated with natural Shigella infection is important to identify potential correlates of protection and, as such, to design effective vaccines. We performed a comparative analysis of circulating mucosal plasmablasts producing specific antibodies against highly conserved invasive plasmid antigens (IpaC, IpaD20, and IpaD120) and two recently identified surface protein antigens, pan-Shigella surface protein antigen 1 (PSSP1) and PSSP2, common to all virulent Shigella strains. We examined blood and stool specimens from 37 diarrheal patients admitted to the Infectious Diseases & Beliaghata General Hospital, Kolkata, India. The etiological agent of diarrhea was investigated in stool specimens by microbiological methods and real-time PCR. Gut-homing (α4β7+) antibody-secreting cells (ASCs) were isolated from patient blood by means of combined magnetic cell sorting and two-color enzyme-linked immunosorbent spot (ELISPOT) assay. Overall, 57% (21 of 37) and 65% (24 of 37) of the patients were positive for Shigella infection by microbiological and real-time PCR assays, respectively. The frequency of α4β7+ IgG ASC responders against Ipas was higher than that observed against PSSP1 or PSSP2, regardless of the Shigella serotype isolated from these patients. Thus, α4β7+ ASC responses to Ipas may be considered an indirect marker of Shigella infection. The apparent weakness of ASC responses to PSSP1 is consistent with the lack of cross-protection induced by natural Shigella infection. The finding that ASC responses to IpaD develop in patients with recent-onset shigellosis indicates that such responses may not be protective or may wane too rapidly and/or be of insufficient magnitude. PMID:27193041

  17. Well-known surface and extracellular antigens of pathogenic microorganisms among the immunodominant proteins of the infectious microalgae Prototheca zopfii

    PubMed Central

    Irrgang, Alexandra; Murugaiyan, Jayaseelan; Weise, Christoph; Azab, Walid; Roesler, Uwe

    2015-01-01

    Microalgae of the genus Prototheca (P.) are associated with rare but severe infections (protothecosis) and represent a potential zoonotic risk. Genotype (GT) 2 of P. zopfii has been established as pathogenic agent for humans, dogs, and cattle, whereas GT1 is considered to be non-pathogenic. Since pathogenesis is poorly understood, the aim of this study was to determine immunogenic proteins and potential virulence factors of P. zopfii GT2. Therefore, 2D western blot analyses with sera and isolates of two dogs naturally infected with P. zopfii GT2 have been performed. Cross-reactivity was determined by including the type strains of P. zopfii GT2, P. zopfii GT1, and P. blaschkeae, a close relative of P. zopfii, which is known to cause subclinical forms of bovine mastitis. The sera showed a high strain-, genotype-, and species-cross-reactivity. A total of 198 immunogenic proteins have been analyzed via MALDI—TOF MS. The majority of the 86 identified proteins are intracellularly located (e.g., malate dehydrogenase, oxidoreductase, 3-dehydroquinate synthase) but some antigens and potential virulence factors, known from other pathogens, have been found (e.g., phosphomannomutase, triosephosphate isomerase). One genotype-specific antigen could be identified as heat shock protein 70 (Hsp70), a well-known antigen of eukaryotic pathogens with immunological importance when located extracellularly. Both sera were reactive to glyceraldehyde-3-phosphate-dehydrogenase of all investigated strains. This house-keeping enzyme is found to be located on the surface of several pathogens as virulence factor. Flow-cytometric analysis revealed its presence on the surface of P. blaschkeae. PMID:26484314

  18. 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.

  19. Toxocara canis: monoclonal antibodies to larval excretory-secretory antigens that bind with genus and species specificity to the cuticular surface of infective larvae.

    PubMed

    Bowman, D D; Mika-Grieve, M; Grieve, R B

    1987-12-01

    When maintained in culture, the infective-stage larvae of Toxocara canis produce a group of excretory-secretory antigens. Monoclonal antibodies to these antigens have been produced and partially characterized. Hybridomas were made using spleens from mice that had been given 250 embryonated eggs of T. canis followed by immunization with excretory-secretory antigens. Monoclonal antibodies were first screened against excretory-secretory antigens using an indirect enzyme-linked immunosorbent assay. Those antibodies positive in this assay were then screened against the surfaces of formalin-fixed, infective-stage larvae using an indirect fluorescent antibody assay. The two monoclonal antibodies showing fluorescence were also tested against the surfaces of infective-stage larvae of Toxocara cati, Baylisascaris procyonis, Toxascaris leonina, Ascaris suum, a Porrocaecum sp., and Dirofilaria immitis. One of these two antibodies bound to the surface of T. canis and T. cati while the other bound only to the surface of T. canis; neither were reactive with the other ascaridoid larvae or the larvae of D. immitis. Enzyme-linked immunoelectrotransfer blotting techniques were used to demonstrate that the cross-reactive antibody recognized antigens with molecular weights of about 200 kDa while the more specific monoclonal antibody recognized antigens with approximate molecular weights of 80 kDa. The specificity of these two antibodies for T. canis and T. cati should prove helpful in the development of more specific assays for the diagnosis of visceral and ocular larva migrans.

  20. A single point in protein trafficking by Plasmodium falciparum determines the expression of major antigens on the surface of infected erythrocytes targeted by human antibodies.

    PubMed

    Chan, Jo-Anne; Howell, Katherine B; Langer, Christine; Maier, Alexander G; Hasang, Wina; Rogerson, Stephen J; Petter, Michaela; Chesson, Joanne; Stanisic, Danielle I; Duffy, Michael F; Cooke, Brian M; Siba, Peter M; Mueller, Ivo; Bull, Peter C; Marsh, Kevin; Fowkes, Freya J I; Beeson, James G

    2016-11-01

    Antibodies to blood-stage antigens of Plasmodium falciparum play a pivotal role in human immunity to malaria. During parasite development, multiple proteins are trafficked from the intracellular parasite to the surface of P. falciparum-infected erythrocytes (IEs). However, the relative importance of different proteins as targets of acquired antibodies, and key pathways involved in trafficking major antigens remain to be clearly defined. We quantified antibodies to surface antigens among children, adults, and pregnant women from different malaria-exposed regions. We quantified the importance of antigens as antibody targets using genetically engineered P. falciparum with modified surface antigen expression. Genetic deletion of the trafficking protein skeleton-binding protein-1 (SBP1), which is involved in trafficking the surface antigen PfEMP1, led to a dramatic reduction in antibody recognition of IEs and the ability of human antibodies to promote opsonic phagocytosis of IEs, a key mechanism of parasite clearance. The great majority of antibody epitopes on the IE surface were SBP1-dependent. This was demonstrated using parasite isolates with different genetic or phenotypic backgrounds, and among antibodies from children, adults, and pregnant women in different populations. Comparisons of antibody reactivity to parasite isolates with SBP1 deletion or inhibited PfEMP1 expression suggest that PfEMP1 is the dominant target of acquired human antibodies, and that other P. falciparum IE surface proteins are minor targets. These results establish SBP1 as part of a critical pathway for the trafficking of major surface antigens targeted by human immunity, and have key implications for vaccine development, and quantifying immunity in populations.

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

    PubMed

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

    2016-03-01

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

  2. An improved method for the detection of cell surface antigens in samples of low viability using flow cytometry.

    PubMed

    Wing, M G; Montgomery, A M; Songsivilai, S; Watson, J V

    1990-01-24

    A high non-specific background fluorescence signal was observed when cell surface antigen analysis was carried out using flow cytometry on a cell sample which contained a high proportion of dead and dying cells. To overcome this problem it was necessary to analyse the cells in three stages. First the intact cells were identified by their forward (FWD) and 90 degree scatter profile. These cells were gated-on, then analysed on the basis of their FWD scatter and propidium iodide (PI) signal, allowing the dead PI positive cells to be gated out. The PI negative cells were then displayed using their 90 degree scatter and fluorescence signals following staining with the irrelevant antibody control. This revealed a population of dead cells, which despite being PI negative, were non-specifically binding antibody molecules. Such multiparameter analysis permitted the successful analysis of cell surface antigens in preparations of low viability by gating out the high background fluorescence associated with dead PI positive and negative cells.

  3. Cloning and Characterization of Surface-Localized α-Enolase of Streptococcus iniae, an Effective Protective Antigen in Mice.

    PubMed

    Wang, Jun; Wang, Kaiyu; Chen, Defang; Geng, Yi; Huang, Xiaoli; He, Yang; Ji, Lili; Liu, Tao; Wang, Erlong; Yang, Qian; Lai, Weimin

    2015-06-25

    Streptococcus iniae is a major fish pathogen that can also cause human bacteremia, cellulitis and meningitis. Screening for and identification of protective antigens plays an important role in developing therapies against S. iniae infections. In this study, we indicated that the α-enolase of S. iniae was not only distributed in the cytoplasm and associated to cell walls, but was also secreted to the bacterial cell surface. The functional identity of the purified recombinant α-enolase protein was verified by its ability to catalyze the conversion of 2-phosphoglycerate (2-PGE) to phosphoenolpyruvate (PEP), and both the recombinant and native proteins interacted with human plasminogen. The rabbit anti-rENO serum blockade assay shows that α-enolase participates in S. iniae adhesion to and invasion of BHK-21 cells. In addition, the recombinant α-enolase can confer effective protection against S. iniae infection in mice, which suggests that α-enolase has potential as a vaccine candidate in mammals. We conclude that S. iniae α-enolase is a moonlighting protein that also associates with the bacterial outer surface and functions as a protective antigen in mice.

  4. Identifying Plasmodium falciparum merozoite surface antigen 3 (MSP3) protein peptides that bind specifically to erythrocytes and inhibit merozoite invasion

    PubMed Central

    Rodríguez, Luis E.; Curtidor, Hernando; Ocampo, Marisol; Garcia, Javier; Puentes, Alvaro; Valbuena, John; Vera, Ricardo; López, Ramses; Patarroyo, Manuel E.

    2005-01-01

    Receptor–ligand interactions between synthetic peptides and normal human erythrocytes were studied to determine Plasmodium falciparum merozoite surface protein-3 (MSP-3) FC27 strain regions that specifically bind to membrane surface receptors on human erythrocytes. Three MSP-3 protein high activity binding peptides (HABPs) were identified; their binding to erythrocytes became saturable, had nanomolar affinity constants, and became sensitive on being treated with neuraminidase and trypsin but were resistant to chymotrypsin treatment. All of them specifically recognized 45-, 55-, and 72-kDa erythrocyte membrane proteins. They all presented α-helix structural elements. All HABPs inhibited in vitro P. falciparum merozoite invasion of erythrocytes by ~55%–85%, suggesting that MSP-3 protein’s role in the invasion process probably functions by using mechanisms similar to those described for other MSP family antigens. PMID:15987906

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

    PubMed

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

    2015-03-01

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

  6. Early dutasteride monotherapy in men with detectable serum prostate-specific antigen levels following radical prostatectomy: A prospective trial

    PubMed Central

    Shin, Yu Seob; Lee, Jea Whan; Kim, Myung Ki; Jeong, Young Beom

    2017-01-01

    Purpose To investigate the effects of early administration of dutasteride in patients with detectable serum prostate-specific antigen (PSA) levels after radical prostatectomy (RP). Materials and Methods A prospective open-label study, with a cumulative analysis of asymptomatic increase in PSA following RP, was conducted from January 2005 to December 2013. An early increase in PSA level was defined as detectable serum PSA level> 0.04 ng/mL. Patients with PSA level>0.04 ng/mL were treated with dutasteride 0.5 mg daily. Serum PSA level and biochemical recurrence (BCR) were monitored. We divided the patients into 2 groups based on the serum PSA response after dutasteride treatment. Results Eighty patients were included in the study. At the median follow-up of 51.8 months, 56 patients (70.0%) showed a decrease of greater than 10% in serum PSA level, and 24 showed increased PSA levels. Twelve of the 56 patients with PSA response showed subsequently increased PSA. Intergroup differences in preoperative PSA levels, PSA nadir levels, and Gleason score of 6 or less were significant (p=0.028, p=0.030, and p=0.035, respectively). A multivariate analysis revealed that Gleason score of 6 or less (p=0.018) and PSA nadir levels (p=0.011) were predictive factors for PSA response after early dutasteride treatment in men with increased PSA levels following RP. Conclusions Early monotherapy of dutasteride showed a decline in serum PSA levels in men with lower nadir PSA levels, and a Gleason score 6, when the serum PSA was detected after RP. PMID:28261678

  7. Influence of dioxin exposure upon levels of prostate-specific antigen and steroid hormones in Vietnamese men.

    PubMed

    Sun, Xian Liang; Kido, Teruhiko; Honma, Seijiro; Okamoto, Rie; Manh, Ho Dung; Maruzeni, Shoko; Nishijo, Muneko; Nakagawa, Hideaki; Nakano, Takeshi; Koh, Eitetsu; Takasuga, Takumi; Nhu, Dang Duc; Hung, Nguyen Ngoc; Son, Le Ke

    2016-04-01

    Most studies on the relationship between Agent Orange and prostate cancer have focused on US veterans of the Vietnam War. There have been few studies focusing on the relationship between levels of prostate-specific antigen (PSA) and dioxins or steroid hormones in Vietnamese men. In 2009-2011, we collected blood samples from 97 men who had resided in a "dioxin hotspot" and 85 men from a non-sprayed region in Vietnam. Then levels of PSA, dioxins, and steroid hormones were analyzed. Levels of most dioxins, furans, and non-ortho polychlorinated biphenyls were higher in the hotspot than those in the non-sprayed region. Levels of testosterone, dehydroepiandrosterone, and estradiol differed significantly between the hotspot and the non-sprayed region, but there were no correlations between levels of PSA and steroid hormones and dioxins in either of the two regions. Our findings suggest that PSA levels in Vietnamese men are not associated with levels of dioxin or steroid hormones in these two regions.

  8. Improved detection of equine antibodies against Sarcocystis neurona using polyvalent ELISAs based on the parasite SnSAG surface antigens.

    PubMed

    Yeargan, Michelle R; Howe, Daniel K

    2011-02-28

    Equine protozoal myeloencephalitis (EPM) is a common neurologic disease of horses that is caused by the apicomplexan pathogen Sarcocystis neurona. To help improve serologic diagnosis of S. neurona infection, we have modified existing enzyme-linked immunosorbent assays (ELISAs) based on the immunogenic parasite surface antigens SnSAG2, SnSAG3, and SnSAG4 to make the assays polyvalent, thereby circumventing difficulties associated with parasite antigenic variants and diversity in equine immune responses. Two approaches were utilized to achieve polyvalence: (1) mixtures of the individual recombinant SnSAGs (rSnSAGs) were included in single ELISAs; (2) a collection of unique SnSAG chimeras that fused protein domains from different SnSAG surface antigens into a single recombinant protein were generated for use in the ELISAs. These new assays were assessed using a defined sample set of equine sera and cerebrospinal fluids (CSFs) that had been characterized by Western blot and/or were from confirmed EPM horses. While all of the polyvalent ELISAs performed relatively well, the highest sensitivity and specificity (100%/100%) were achieved with assays containing the rSnSAG4/2 chimera (Domain 1 of SnSAG4 fused to SnSAG2) or using a mixture of rSnSAG3 and rSnSAG4. The rSnSAG4 antigen alone and the rSnSAG4/3 chimera (Domain 1 of SnSAG4 fused to Domain 2 of SnSAG3) exhibited the next best accuracy at 95.2% sensitivity and 100% specificity. Binding ratios and percent positivity (PP) ratios, determined by comparing the mean values for positive versus negative samples, showed that the most advantageous signal to noise ratios were provided by rSnSAG4 and the rSnSAG4/3 chimera. Collectively, our results imply that a polyvalent ELISA based on SnSAG4 and SnSAG3, whether as a cocktail of two proteins or as a single chimeric protein, can give optimal results in serologic testing of serum or CSF for the presence of antibodies against S. neurona. The use of polyvalent SnSAG ELISAs will

  9. MAGE-A Cancer/Testis Antigens Inhibit MDM2 Ubiquitylation Function and Promote Increased Levels of MDM4

    PubMed Central

    Marcar, Lynnette; Ihrig, Bianca; Hourihan, John; Bray, Susan E.; Quinlan, Philip R.; Jordan, Lee B.; Thompson, Alastair M.; Hupp, Ted R.; Meek, David W.

    2015-01-01

    Melanoma antigen A (MAGE-A) proteins comprise a structurally and biochemically similar sub-family of Cancer/Testis antigens that are expressed in many cancer types and are thought to contribute actively to malignancy. MAGE-A proteins are established regulators of certain cancer-associated transcription factors, including p53, and are activators of several RING finger-dependent ubiquitin E3 ligases. Here, we show that MAGE-A2 associates with MDM2, a ubiquitin E3 ligase that mediates ubiquitylation of more than 20 substrates including mainly p53, MDM2 itself, and MDM4, a potent p53 inhibitor and MDM2 partner that is structurally related to MDM2. We find that MAGE-A2 interacts with MDM2 via the N-terminal p53-binding pocket and the RING finger domain of MDM2 that is required for homo/hetero-dimerization and for E2 ligase interaction. Consistent with these data, we show that MAGE-A2 is a potent inhibitor of the E3 ubiquitin ligase activity of MDM2, yet it does not have any significant effect on p53 turnover mediated by MDM2. Strikingly, however, increased MAGE-A2 expression leads to reduced ubiquitylation and increased levels of MDM4. Similarly, silencing of endogenous MAGE-A expression diminishes MDM4 levels in a manner that can be rescued by the proteasomal inhibitor, bortezomid, and permits increased MDM2/MDM4 association. These data suggest that MAGE-A proteins can: (i) uncouple the ubiquitin ligase and degradation functions of MDM2; (ii) act as potent inhibitors of E3 ligase function; and (iii) regulate the turnover of MDM4. We also find an association between the presence of MAGE-A and increased MDM4 levels in primary breast cancer, suggesting that MAGE-A-dependent control of MDM4 levels has relevance to cancer clinically. PMID:26001071

  10. Surface antigens of Xenorhabdus nematophila (F. Enterobacteriaceae) and Bacillus subtilis (F. Bacillaceae) react with antibacterial factors of Malacosoma disstria (C. Insecta: O. Lepidoptera) hemolymph.

    PubMed

    Giannoulis, Paschalis; Brooks, Cory L; Dunphy, Gary B; Niven, Donald F; Mandato, Craig A

    2008-03-01

    Previous research established different interactions of the insect pathogen, Xenorhabdus nematophila and nonpathogen, Bacillus subtilis, with antimicrobial hemocytes and humoral factors of larval Malacosoma disstria [Giannoulis, P., Brooks, C.L., Dunphy, G.B., Mandato, C.A., Niven, D.F., Zakarian, R.J., 2007. Interaction of the bacteria Xenorhabdus nematophila (Enterobacteriaceae) and Bacillus subtilis (Bacillaceae) with the hemocytes of larval Malacosoma disstria (Insecta: Lepidoptera: Lasicocampidae). J. Invertebr. Pathol. 94, 20-30]. The antimicrobial systems were inhibited by X. nematophila and stimulated by B. subtilis. The bacterial surface antigens participating in these reactions were unknown. Thus, herein the effects of lipopolysaccharide (endotoxin) from X. nematophila and lipoteichoic acid from B. subtilis on the larval M. disstria immune factors, the hemocytes and phenoloxidase, were determined. Endotoxin elevated the level of damaged hemocytes limiting the removal of X. nematophila from the hemolymph and enhancing the rapid release of bacteria trapped by nodulation. Similar effects were observed with the lipid A moiety of the endotoxin. The effects of lipopolysaccharide and lipid A on the hemocyte activities were abrogated by polymyxin B (an antibiotic that binds to lipid A) confirming lipopolysaccharide as the hemocytotoxin by virtue of the lipid A moiety. Lipoteichoic acid elicited nodulation and enhanced phenoloxidase activation and/or activity. Although lipoidal endotoxin and lipid A inhibited phenoloxidase activation they enhanced the activity of the enzyme. Apolipophorin-III precluded the effects of lipopolysaccharide, lipid A, and lipoteichoic acid on the hemocytes and prophenoloxidase until the antigens exceeded a critical threshold.

  11. Characterization of a cDNA clone encoding the carboxy-terminal domain of a 90-kilodalton surface antigen of Trypanosoma cruzi metacyclic trypomastigotes.

    PubMed Central

    Franco, F R; Paranhos-Bacallà, G S; Yamauchi, L M; Yoshida, N; da Silveira, J F

    1993-01-01

    We have cloned and sequenced a cDNA for a metacyclic trypomastigote-specific glycoprotein with a molecular mass of 90 kDa, termed MTS-gp90. By immunoblotting, antibodies to the MTS-gp90 recombinant protein reacted exclusively with a 90-kDa antigen of metacyclic trypomastigotes. The insert of the MTS-gp90 cDNA clone strongly hybridized with a single 3.0-kb mRNA of metacyclic forms, whereas the hybridization signal with epimastigote mRNA was weak and those with RNAs from other developmental stages were negative, indicating that transcription of the MTS-gp90 gene is developmentally regulated. A series of experiments showed that the MTS-gp90 gene is present in multiple copies in the Trypanosoma cruzi genome, arranged in a nontandem manner, and that there are at least 40 copies of the gene per haploid genome. Sequence analysis of recombinant MTS-gp90 revealed 40 to 60% identity at the amino acid level with members of a family of mammalian stage-specific, 85-kDa surface antigens of T. cruzi. However, there are considerable differences in the amino acid compositions outside the homology region. Images PMID:8406808

  12. High antigen levels induce an exhausted phenotype in a chronic infection without impairing T cell expansion and survival

    PubMed Central

    Alfei, Francesca; Roelli, Patrick; Delorenzi, Mauro; Pinschewer, Daniel D.

    2016-01-01

    Chronic infections induce T cells showing impaired cytokine secretion and up-regulated expression of inhibitory receptors such as PD-1. What determines the acquisition of this chronic phenotype and how it impacts T cell function remain vaguely understood. Using newly generated recombinant antigen variant-expressing chronic lymphocytic choriomeningitis virus (LCMV) strains, we uncovered that T cell differentiation and acquisition of a chronic or exhausted phenotype depend critically on the frequency of T cell receptor (TCR) engagement and less significantly on the strength of TCR stimulation. In fact, we noted that low-level antigen exposure promotes the formation of T cells with an acute phenotype in chronic infections. Unexpectedly, we found that T cell populations with an acute or chronic phenotype are maintained equally well in chronic infections and undergo comparable primary and secondary expansion. Thus, our observations contrast with the view that T cells with a typical chronic infection phenotype are severely functionally impaired and rapidly transition into a terminal stage of differentiation. Instead, our data unravel that T cells primarily undergo a form of phenotypic and functional differentiation in the early phase of a chronic LCMV infection without inheriting a net survival or expansion deficit, and we demonstrate that the acquired chronic phenotype transitions into the memory T cell compartment. PMID:27455951

  13. Monitoring human leukocyte antigen class I molecules by micro-Raman spectroscopy at single-cell level

    NASA Astrophysics Data System (ADS)

    Das, Gobind; La Rocca, Rosanna; Lakshmikanth, Tadepally; Gentile, Francesco; Tallerico, Rossana; Zambetti, Lia P.; Devitt, J.; Candeloro, Patrizio; de Angelis, Francesco; Carbone, Ennio; di Fabrizio, Enzo

    2010-03-01

    Human leukocyte antigen (HLA) class I molecules are formed by three immunoglobulin-like domains (α1, α2, and α3) once folded by peptide and β2-microglobulin show the presence of two α-helix streams and one β-sheet limiting the pocket for the antigenic peptide. The loss of HLA class I expression in tumors and virus-infected cells, on one hand, prevents T cell recognition, while on the other hand, it leads to natural killer (NK) cell mediated cytotoxicity. We propose the possibility of using Raman spectroscopy to measure the relative expression of HLA class I molecules at the single-cell level. Raman spectra are recorded for three cell lines (K562, T2, and T3) and monomers (HLA class I folded, unfolded and peptide+β2-microlobulin refolded) using 830 nm laser line. Our data are consistent with the hypothesis that in the Raman spectra, ranging from 1600 to 1800 cm-1, the intensity variation of cells associated with HLA class I molecules could be measured.

  14. Occurrence of hepatitis and hepatitis B surface antigen in adult patients with acute leukemia.

    PubMed Central

    Cowan, D. H.; Kouroupis, G. M.; Leers, W. D.

    1975-01-01

    Fifty-eight adult patients with acute leukemia were screened at the onset of the disease for hepatitis B antigen (HBSAg) in the serum, and during the course of the disease for the development of hepatitis B. One patient had a positive test for HBSAg by the radioimmunoassay technique only at the time leukemia was diagnosed; this patient had received transfusions some years before. In six patients icteric hepatitis B developed; five recovered completely and one died of leukemia during the course of hepatitis. All patients in whom hepatitis developed had received transfusions as a part of supportive therapy for leukemia. The hepatitis risk for patients who received transfusions of blood found to be negative for HBSAg by counterimmunoelectrophoresis was 0.26 percent per unit of blood administered. PMID:1054615

  15. A vaccine prepared from the 22 nm particles of surface hepatitis B antigen (HBsAg)

    SciTech Connect

    Karelin, V.P.; Babaeva, E.E.; Gubenko, E.F.; Kaulen, D.K.; Zhdanov, V.M.

    1980-01-01

    A method for obtaining a subunit inactivated vaccine preparation from the 22-nm particles of HBsAg is proposed. For inactivation of the residual infectious hepatitis B virus (HBV) the preparations were successively treated with 1% sodium dodecyl sulfate (SDS) and nucleases. In addition, thermal denaturation and ultraviolet irradiation of HBV DNA were used. As a control the biologic activity of a reference virus (SV40) was tested after the same treatment. The effectiveness of DNA inactivation was monitored by adding 3H-thymidine labeled reference virus to the vaccine preparations. The purified and inactivated HBsAg was adsorbed on Al2O3. Antigenicity was calculated on the basis of the determination of antibody in guinea pigs immunized with various doses of the vaccine, and the release of /sup 125/I- HBsAg from blood and kidneys in immunized and control mice was analyzed. Possible methods of inactivation and control of HBV vaccine is discussed.

  16. Major role for carbohydrate epitopes preferentially recognized by chronically infected mice in the determination of Schistosoma mansoni schistosomulum surface antigenicity

    SciTech Connect

    Omer-ali, P.; Magee, A.I.; Kelly, C.; Simpson, A.J.G.

    1986-12-01

    A radioimmunoassay that makes use of whole Schistosomula and /sup 125/I-labeled protein A has been used to characterize and to quantify the binding of antisera to the surface of 3 hr mechanically transformed schistosomula of Schistosoma mansoni. This technique facilitates the determination of epitopes on the schistosomula in addition to those detected by surface labeling and immunoprecipitation. By using this technique, it has been demonstrated that there is a much greater binding to the parasite surface of antibodies from chronically infected mice (CMS) than of antibodies from mice infected with highly irradiated cercariae (VMS), and CMS recognizes epitopes that VMS does not. Treatment of the surface of the schistosomula with trifluoromethanesulphonic acid and sodium metaperiodate has suggested that the discrepancy of the binding between the two sera is due to the recognition of a large number of additional epitopes by CMS, which are carbohydrate in nature. Some of the carbohydrate epitopes are expressed on the previously described surface glycoprotein antigens of M/sub r/ 200,000, 38,000, and 17,000.

  17. In silico predicted conserved B-cell epitopes in the Merozoite Surface Antigen -2 family of B. bovis are neutralization-sensitive

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Merozoite Surface Antigens-2 of Babesia bovis conform a family of GPI-anchored glycoproteins located at the parasite cell surface, that contain neutralization-sensitive B-cell epitopes, thus constituting putative vaccine candidates for bovine babesiosis. It was previously shown that (i) the MSA-...

  18. The H Antigen at Epithelial Surfaces Is Associated with Susceptibility to Asthma Exacerbation

    PubMed Central

    Innes, Anh L.; McGrath, Kelly Wong; Dougherty, Ryan H.; McCulloch, Charles E.; Woodruff, Prescott G.; Seibold, Max A.; Okamoto, Kimberly S.; Ingmundson, Kelsey J.; Solon, Margaret C.; Carrington, Stephen D.; Fahy, John V.

    2011-01-01

    Rationale: Acute asthma exacerbations, precipitated by viral infections, are a significant cause of morbidity, but not all patients with asthma are equally susceptible. Objectives: To explore susceptibility factors for asthma exacerbations, we considered a role for histoblood group antigens because they are implicated in mechanisms of gastrointestinal viral infection, specifically the O-secretor mucin glycan phenotype. We investigated if this phenotype is associated with susceptibility to asthma exacerbation. Methods: We performed two consecutive case-control studies in subjects with asthma who were either prone or resistant to asthma exacerbations. Exacerbation-prone cases had frequent use of prednisone for an asthma exacerbation and frequent asthma-related healthcare utilization, whereas exacerbation-resistant control subjects had rarely reported asthma exacerbations. The frequency of different mucin glycan phenotypes, defined by the presence or absence of H (O), A, B, or AB antigens, was compared in cases and control subjects. Measurements and Main Results: In an initial study consisting of 49 subjects with asthma (23 cases and 26 control subjects), we found that having the O-secretor phenotype was associated with a 5.8-fold increase in the odds of being a case (95% confidence interval, 1.7–21.0; P = 0.006). In a replication study consisting of 204 subjects with asthma (101 cases and 103 control subjects), we found that having the O-secretor phenotype was associated with a 2.3-fold increased odds of being a case (95% confidence interval, 1.2–4.4; P = 0.02). Conclusions: The O-secretor mucin glycan phenotype is associated with susceptibility to asthma exacerbation. Clinical trial registered at www.clinicaltrials.gov (NCT00201266). PMID:20732988

  19. An alternative to reduction of surface pressure to sea level

    NASA Technical Reports Server (NTRS)

    Deardorff, J. W.

    1982-01-01

    The pitfalls of the present method of reducing surface pressure to sea level are reviewed, and an alternative, adjusted pressure, P, is proposed. P is obtained from solution of a Poisson equation over a continental region, using the simplest boundary condition along the perimeter or coastline where P equals the sea level pressure. The use of P would avoid the empiricisms and disadvantages of pressure reduction to sea level, and would produce surface pressure charts which depict the true geostrophic wind at the surface.

  20. The Relationship Between Metformin and Serum Prostate‐Specific Antigen Levels

    PubMed Central

    Jayalath, Viranda H.; Ireland, Christopher; Fleshner, Neil E.; Hamilton, Robert J.

    2016-01-01

    BACKGROUND Metformin is the first‐line oral antihyperglycemic of choice for individuals with type 2 diabetes. Recent evidence supports a role for metformin in prostate cancer chemoprotection. However, whether metformin indeed influences prostate biology is unknown. We aimed to study the association between metformin and serum prostate‐specific antigen (PSA) levels—the primary prostate cancer biomarker. METHODS We conducted a cross‐sectional study of 326 prostate cancer‐free men with type 2 diabetes were recruited between 2004 and 2013 at St. Michael's Hospital. Men were excluded if they had a PSA ≥10‐ng/ml, or used >2,550‐mg/d metformin or supplemental androgens. Multivariate linear regressions quantified the association between metformin dose and log‐PSA. Secondary analyses quantified the association between other antihyperglycemics (sulfonylureas, thiazolidinediones) and PSA; sensitivity analyses tested covariate interactions. RESULTS Median PSA was 0.9‐ng/ml (IQR: 0.5–1.6‐ng/ml). Metformin dose associated positively with BMI, HbA1c, diabetes duration, and number of statin, acetylsalicylic acid, diuretic users, and number of antihyperglycemics used, and negatively with LDL‐C. In multivariate models, PSA changed by −8% (95%CI: −13 to −2%, P = 0.011) per 500‐mg/d increase in metformin. Men with diabetes for ≥6 years (n = 163) saw a greater difference in PSA per 500‐mg/d metformin (−12% [95% CI: −19 to −4%, P = 0.002], P‐interaction = 0.018). Serum PSA did not relate with sulfonylureas, thiazolidinediones, or total number of antihyperglycemic agents used. Our findings are limited by the cross‐sectional design of this study. CONCLUSIONS Metformin dose‐dependently inversely associated with serum PSA, independent of other antihyperglycemic medications. Whether metformin confers a dose‐dependent benefit on prostate tumorigenesis and progression warrants investigation. Prostate 76:1445–1453, 2016.

  1. Humoral immune response against proteophosphoglycan surface antigens of Entamoeba histolytica elicited by immunization with synthetic mimotope peptides.

    PubMed

    Melzer, Helen; Baier, Karin; Felici, Franco; von Specht, Bernd Ulrich; Wiedermann, Gerhard; Kollaritsch, Herwig; Wiedermann, Ursula; Duchêne, Michael

    2003-07-15

    The protozoan parasite Entamoeba histolytica, which is responsible for intestinal amebiasis and amebic liver abscess, is causing significant morbidity and mortality worldwide. Proteophosphoglycans (PPGs, also known as lipophosphoglycans, LPGs, or lipopeptidophosphoglycans, LPPGs) are major surface components of E. histolytica. Passive immunization with a monoclonal antibody (EH5) directed against the PPGs protected severe combined immune-deficient mice from amebic liver abscess. The structure of the PPGs is very complex and only known in part. To find peptide mimics of E. histolytica PPG antigens, we had screened phage-displayed random peptide libraries with the antibody EH5. We identified various peptide mimics of E. histolytica PPGs, all sharing a consensus sequence Gly-Thr-His-Pro-X-Leu. Several of the phage clones induced a significant, specific IgG response against membrane antigens of E. histolytica after immunization of mice with whole phage particles. In the present work, in order to avoid the use of phage particles for immunization, we coupled two selected chemically synthesized peptides to keyhole limpet hemocyanin (KLH). The two KLH-conjugated peptides were immunogenic in mice and induced the production of high titers of anti-peptide antibodies, and one of the two peptides was also able to induce significant titers of antibodies against E. histolytica PPGs. Our results demonstrate that the KLH-conjugated peptides are able to mimic the EH5 epitope without the M13 phage sequences flanking the peptide inserts and independent of the structural framework of the phage.

  2. Surface swarming motility by Pectobacterium atrosepticum is a latent phenotype that requires O antigen and is regulated by quorum sensing.

    PubMed

    Bowden, Steven D; Hale, Nicola; Chung, Jade C S; Hodgkinson, James T; Spring, David R; Welch, Martin

    2013-11-01

    We describe a previously cryptic phenotype associated with the opportunistic phytopathogen Pectobacterium atrosepticum (Pca): surface swarming. We found that when Pca was spotted onto plates containing <0.5% (w/v) agar, the culture produced copious amounts of extracellular matrix material containing highly motile cells. Once produced, this 'slime layer' spread rapidly across the plate either as an advancing front or as tendrils. Transposon mutagenesis was used to identify mutants that were affected in swarming. Hypo-swarmer mutants mostly carried insertions in a horizontally acquired island (HAI5), which encodes a cluster of genes involved in O antigen biosynthesis. Hyper-swarmer mutants mostly carried insertions in hexY, a known antagonist of the class I flagellar master regulator, FlhD4C2. In addition, we found that the nucleoid protein, histone-like nuclear structuring protein 2 (H-NS2), also regulated swarming behaviour. A mutant in which hns2 was overexpressed displayed a hyper-swarming phenotype, whereas a mutant in which the hns2 ORF was inactivated had a hypo-swarming phenotype. Swarming was also regulated by quorum sensing (QS) and by the carbon source being utilized. We show, using a range of epistasis experiments, that optimal swarming requires both motility and O antigen biosynthesis, and that H-NS2 and QS both promote swarming through their effects on motility.

  3. MetQ of Neisseria gonorrhoeae Is a Surface-Expressed Antigen That Elicits Bactericidal and Functional Blocking Antibodies

    PubMed Central

    Semchenko, Evgeny A.; Day, Christopher J.

    2016-01-01

    ABSTRACT Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection (STI) gonorrhea, is a growing public health threat for which a vaccine is urgently needed. We characterized the functional role of the gonococcal MetQ protein, which is the methionine binding component of an ABC transporter system, and assessed its potential as a candidate antigen for inclusion in a gonococcal vaccine. MetQ has been found to be highly conserved in all strains investigated to date, it is localized on the bacterial surface, and it binds l-methionine with a high affinity. MetQ is also involved in gonococcal adherence to cervical epithelial cells. Mutants lacking MetQ have impaired survival in human monocytes, macrophages, and serum. Furthermore, antibodies raised against MetQ are bactericidal and are able to block gonococcal adherence to epithelial cells. These data suggest that MetQ elicits both bactericidal and functional blocking antibodies and is a valid candidate antigen for additional investigation and possible inclusion in a vaccine for prevention of gonorrhea. PMID:27895130

  4. Immunological fine structure of the variable and constant regions of a polymorphic malarial surface antigen from Plasmodium falciparum.

    PubMed

    Jones, G L; Edmundson, H M; Lord, R; Spencer, L; Mollard, R; Saul, A J

    1991-09-01

    The 51-kDa merozoite surface antigen MSA2 of Plasmodium falciparum shows considerable strain-dependent polymorphism. Although marked sequence variation occurs in the central region of the molecule, the N and C-terminal sequences are highly conserved. A number of monoclonal antibodies directed against MSA2 have been described which inhibit parasite growth in vitro, but these are all directed against variable regions. In an attempt to raise strain independent antibodies we have prepared peptide-diphtheria toxoid (DT) constructs from 36 N-terminal octapeptides spanning the constant region and extending into the variable region of the FCQ/27 PNG variant staggered by one amino acid at either end. Similarly, we prepared 26 C-terminal octapeptides spanning the C-terminal constant region as well as 10 octapeptides from the variable region of the Indochina I variant MSA2. Most of the peptides elicited antipeptide titres in excess of 1/10(4) when administered to mice as peptide-DT adducts emulsified with Freund's complete adjuvant. Only 3 of the 43 N- and C-terminal constant region peptides elicited antibodies which reacted appropriately on immunofluorescence (IFA) or immunoblotting analysis with the intact MSA2 of both strains studied (FCQ/27 and Indochina I), whereas 3 other peptides from the variable region elicited antibodies reactive with the parent MSA2 only. Peptide constructs eliciting antibodies recognising the intact protein corresponded to elements in the cognate sequence of high antigenicity as predicted by the Jameson and Wolf algorithm.

  5. A self-amplified transistor immunosensor under dual gate operation: highly sensitive detection of hepatitis B surface antigen

    NASA Astrophysics Data System (ADS)

    Lee, I.-K.; Jeun, M.; Jang, H.-J.; Cho, W.-J.; Lee, K. H.

    2015-10-01

    Ion-sensitive field-effect transistors (ISFETs), although they have attracted considerable attention as effective immunosensors, have still not been adopted for practical applications owing to several problems: (1) the poor sensitivity caused by the short Debye screening length in media with high ion concentration, (2) time-consuming preconditioning processes for achieving the highly-diluted media, and (3) the low durability caused by undesirable ions such as sodium chloride in the media. Here, we propose a highly sensitive immunosensor based on a self-amplified transistor under dual gate operation (immuno-DG ISFET) for the detection of hepatitis B surface antigen. To address the challenges in current ISFET-based immunosensors, we have enhanced the sensitivity of an immunosensor by precisely tailoring the nanostructure of the transistor. In the pH sensing test, the immuno-DG ISFET showed superior sensitivity (2085.53 mV per pH) to both standard ISFET under single gate operation (58.88 mV per pH) and DG ISFET with a non-tailored transistor (381.14 mV per pH). Moreover, concerning the detection of hepatitis B surface antigens (HBsAg) using the immuno-DG ISFET, we have successfully detected trace amounts of HBsAg (22.5 fg mL-1) in a non-diluted 1× PBS medium with a high sensitivity of 690 mV. Our results demonstrate that the proposed immuno-DG ISFET can be a biosensor platform for practical use in the diagnosis of various diseases.Ion-sensitive field-effect transistors (ISFETs), although they have attracted considerable attention as effective immunosensors, have still not been adopted for practical applications owing to several problems: (1) the poor sensitivity caused by the short Debye screening length in media with high ion concentration, (2) time-consuming preconditioning processes for achieving the highly-diluted media, and (3) the low durability caused by undesirable ions such as sodium chloride in the media. Here, we propose a highly sensitive immunosensor

  6. Genomic Characterization of Variable Surface Antigens Reveals a Telomere Position Effect as a Prerequisite for RNA Interference-Mediated Silencing in Paramecium tetraurelia

    PubMed Central

    Baranasic, Damir; Oppermann, Timo; Cheaib, Miriam; Cullum, John; Schmidt, Helmut

    2014-01-01

    ABSTRACT Antigenic or phenotypic variation is a widespread phenomenon of expression of variable surface protein coats on eukaryotic microbes. To clarify the mechanism behind mutually exclusive gene expression, we characterized the genetic properties of the surface antigen multigene family in the ciliate Paramecium tetraurelia and the epigenetic factors controlling expression and silencing. Genome analysis indicated that the multigene family consists of intrachromosomal and subtelomeric genes; both classes apparently derive from different gene duplication events: whole-genome and intrachromosomal duplication. Expression analysis provides evidence for telomere position effects, because only subtelomeric genes follow mutually exclusive transcription. Microarray analysis of cultures deficient in Rdr3, an RNA-dependent RNA polymerase, in comparison to serotype-pure wild-type cultures, shows cotranscription of a subset of subtelomeric genes, indicating that the telomere position effect is due to a selective occurrence of Rdr3-mediated silencing in subtelomeric regions. We present a model of surface antigen evolution by intrachromosomal gene duplication involving the maintenance of positive selection of structurally relevant regions. Further analysis of chromosome heterogeneity shows that alternative telomere addition regions clearly affect transcription of closely related genes. Consequently, chromosome fragmentation appears to be of crucial importance for surface antigen expression and evolution. Our data suggest that RNAi-mediated control of this genetic network by trans-acting RNAs allows rapid epigenetic adaptation by phenotypic variation in combination with long-term genetic adaptation by Darwinian evolution of antigen genes. PMID:25389173

  7. Malaria vaccine candidate antigen targeting the pre-erythrocytic stage of Plasmodium falciparum produced at high level in plants.

    PubMed

    Voepel, Nadja; Boes, Alexander; Edgue, Güven; Beiss, Veronique; Kapelski, Stephanie; Reimann, Andreas; Schillberg, Stefan; Pradel, Gabriele; Fendel, Rolf; Scheuermayer, Matthias; Spiegel, Holger; Fischer, Rainer

    2014-11-01

    Plants have emerged as low-cost production platforms suitable for vaccines targeting poverty-related diseases. Besides functional efficacy, the stability, yield, and purification process determine the production costs of a vaccine and thereby the feasibility of plant-based production. We describe high-level plant production and functional characterization of a malaria vaccine candidate targeting the pre-erythrocytic stage of Plasmodium falciparum. CCT, a fusion protein composed of three sporozoite antigens (P. falciparum cell traversal protein for ookinetes and sporozoites [PfCelTOS], P. falciparum circumsporozoite protein [PfCSP], and P. falciparum thrombospondin-related adhesive protein [PfTRAP]), was transiently expressed by agroinfiltration in Nicotiana benthamiana leaves, accumulated to levels up to 2 mg/g fresh leaf weight (FLW), was thermostable up to 80°C and could be purified to >95% using a simple two-step procedure. Reactivity of sera from malaria semi-immune donors indicated the immunogenic conformation of the purified fusion protein consisting of PfCelTOS, PfCSP_TSR, PfTRAP_TSR domains (CCT) protein. Total IgG from the CCT-specific mouse immune sera specifically recognized P. falciparum sporozoites in immunofluorescence assays and induced up to 35% inhibition in hepatocyte invasion assays. Featuring domains from three promising sporozoite antigens with different roles (attachment and cell traversal) in the hepatocyte invasion process, CCT has the potential to elicit broader immune responses against the pre-erythrocytic stage of P. falciparum and represents an interesting new candidate, also as a component of multi-stage, multi-subunit malaria vaccine cocktails.

  8. Localization of hepatitis B surface antigen in conventional paraffin sections of the liver. Comparison of immunofluorescence, immunoperoxidase, and orcein staining methods with regard to their specificity and reliability as antigen marker.

    PubMed Central

    Nayak, N. C.; Sachdeva, R.

    1975-01-01

    Hepatitis B antigen (HBAg) has been demonstrated in conventional formalin-fixed paraffin-embedded liver tissue by peroxidase and fluorescent immunostaining as well as by orcein. Complete locational and morphologic identity is seen between material stained by specific immunologic methods and by orcein. The antigen is restricted to the cytoplasm and is generally observed in the hepatocyte; it is present in three morphologic forms. Certain morphologic forms can even be identified in hematoxylin and eosin-stained tissue. Results of immunostaining procedures indicate that the antigen demonstrated in this study consists entirely of surface coat of hepatitis B virus (HBsAg). This seems to be the only component revealed by orcein staining. The latter is considered to be a good marker of the surface antigen and to have certain advantages over immunostaining. It is suggested that suitability of conventional paraffin sections for the detection of HBAg has wide and important implications. Images Figures 1-5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 PMID:55076

  9. MISR Level 3 Land Surface and Aerosol Versioning

    Atmospheric Science Data Center

    2016-11-04

      MISR Level 3 Land Surface and Aerosol Versioning Component Global Land Surface Product (CGLS) and Component Global Aerosol Product (CGAS) - ... to small, medium, large, spherical, non-spherical particles; LAND - DHRPAR, spectral DHR, FPAR (excluding needleleaf forest biome type), LAI ...

  10. Hookah smoking and cancer: carcinoembryonic antigen (CEA) levels in exclusive/ever hookah smokers

    PubMed Central

    Sajid, Khan Mohammad; Chaouachi, Kamal; Mahmood, Rubaida

    2008-01-01

    Background We have recently published some work on CEA levels in hookah (also called narghile, shisha elsewhere) and cigarette smokers. Hookah smokers had higher levels of CEA than non-smokers although mean levels were low compared to cigarette smokers. However some of them were also users of other tobacco products (cigarettes, bidis, etc.). Objectives To find serum CEA levels in ever/exclusive hookah smokers, i.e. those who smoked only hookah (no cigarettes, bidis, etc.), prepared between 1 and 4 times a day with a quantity of up to 120 g of a tobacco-molasses mixture each (i.e. the tobacco weight equivalent of up to 60 cigarettes of 1 g each) and consumed in 1 to 8 sessions. Methods Enhanced chemiluminescent immunometric technique was applied to measure CEA levels in serum samples from 59 exclusive male smokers with age ranging from 20–80 years (mean = 58.8 ± 14.7 years) and 8–65 years of smoking (mean = 37.7 ± 16.8). 36 non-smokers served as controls. Subjects were divided into 3 groups according to the number of preparations; the number of sessions and the total daily smoking time: Light (1; 1; ≤ 20 minutes); Medium (1–3; 1–3; >20 min to ≤ 2 hrs) and Heavy smokers (2–4; 3–8; >2 hrs to ≤ 6 hrs). Because of the nature of distribution of CEA levels among our individuals, Wilcoxon's rank sum two-sample test was applied to compare the variables. Results The overall CEA levels in exclusive hookah smokers (mean: 3.58 ± 2.61 ng/ml; n = 59) were not significantly different (p ≤ 0.0937) from the levels in non-smokers (2.35 ± 0.71 ng/ml). Mean levels in light, medium and heavy smokers were: 1.06 ± 0.492 ng/ml (n = 5); 2.52 ± 1.15 ng/ml (n = 28) and 5.11 ± 3.08 ng/ml (n = 26) respectively. The levels in medium smokers and non-smokers were also not significantly different (p ≤ 0.9138). In heavy smokers, the CEA levels were significantly higher than in non-smokers (p ≤ 0.0001567). Conclusion Overall CEA levels in exclusive hookah smokers were

  11. Synthetic antigens reveal dynamics of BCR endocytosis during inhibitory signaling.

    PubMed

    Courtney, Adam H; Bennett, Nitasha R; Zwick, Daniel B; Hudon, Jonathan; Kiessling, Laura L

    2014-01-17

    B cells detect foreign antigens through their B cell antigen receptor (BCR). The BCR, when engaged by antigen, initiates a signaling cascade. Concurrent with signaling is endocytosis of the BCR complex, which acts to downregulate signaling and facilitate uptake of antigen for processing and display on the cell surface. The relationship between signaling and BCR endocytosis is poorly defined. Here, we explore the interplay between BCR endocytosis and antigens that either promote or inhibit B cell activation. Specifically, synthetic antigens were generated that engage the BCR alone or both the BCR and the inhibitory co-receptor CD22. The lectin CD22, a member of the Siglec family, binds sialic acid-containing glycoconjugates found on host tissues, inhibiting BCR signaling to prevent erroneous B cell activation. At low concentrations, antigens that can cocluster the BCR and CD22 promote rapid BCR endocytosis; whereas, slower endocytosis occurs with antigens that bind only the BCR. At higher antigen concentrations, rapid BCR endocytosis occurs upon treatment with either stimulatory or inhibitory antigens. Endocytosis of the BCR, in response to synthetic antigens, results in its entry into early endocytic compartments. Although the CD22-binding antigens fail to activate key regulators of antigen presentation (e.g., Syk), they also promote BCR endocytosis, indicating that inhibitory antigens can be internalized. Together, our observations support a functional role for BCR endocytosis in downregulating BCR signaling. The reduction of cell surface BCR levels in the absence of B cell activation should raise the threshold for BCR subsequent activation. The ability of the activating synthetic antigens to trigger both signaling and entry of the BCR into early endosomes suggests strategies for targeted antigen delivery.

  12. Identification of sporozoite surface proteins and antigens of Eimeria nieschulzi (Apicomplexa)

    SciTech Connect

    Tilley, M.; Upton, S.J. )

    1990-03-01

    Sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting, lectin binding, and {sup 125}I surface labeling of sporozoites were used to probe sporozoites of the rat coccidian, Eimeria nieschulzi. Analysis of silver stained gels revealed greater than 50 bands. Surface iodination revealed about 14 well labeled, and about 10 weakly labeled but potential, surface proteins. The most heavily labeled surface proteins had molecular masses of 60, 53-54, 45, 28, 23-24, 17, 15, 14, 13, and 12 kD. Following electrophoresis and Western blotting, 2 of the 12 125I labeled lectin probes bound to two bands on the blots, which collectively indicated that two bands were glycosylated. Concanavalin A (ConA) specifically recognized a band at 53 kD, which may represent a surface glycoprotein, and a lectin derived from Osage orange (MPA) bound to a single band at 82-88 kD, that may also be a surface molecule. Immunoblotting using sera collected from rats inoculated orally with oocysts, as well as sera from mice hyperimmunized with sporozoites, revealed that many surface molecules appear to be immunogenic.

  13. Development of Lateral Flow Assay Based on Size-Controlled Gold Nanoparticles for Detection of Hepatitis B Surface Antigen

    PubMed Central

    Kim, Dong Seok; Kim, Yong Tae; Hong, Seok Bok; Kim, Jinwoon; Heo, Nam Su; Lee, Moon-Keun; Lee, Seok Jae; Kim, Byeong Il; Kim, In Soo; Huh, Yun Suk; Choi, Bong Gill

    2016-01-01

    In this study, we developed lateral flow assay (LFA) biosensors for the detection of hepatitis B surface antigens using well-controlled gold nanoparticles (AuNPs). To enhance colorimetric signals, a seeded growth method was used for the preparation of size-controlled AuNPs with a narrow size distribution. Different sizes of AuNPs in the range of 342–137.8 nm were conjugated with antibodies and then optimized for the efficient detection of LFA biosensors. The conjugation stability was investigated by UV-vis spectroscopy of AuNP dispersion at various pH values and concentrations of antibody. Based on optimized conjugation conditions, the use of 42.7 ± 0.8 nm AuNPs exhibited superior performance for the detection of LFAs relative to other sizes of AuNPs. PMID:27999291

  14. A molecular assembly system for presentation of antigens on the surface of HBc virus-like particles

    SciTech Connect

    Blokhina, Elena A.; Kuprianov, Victor V.; Stepanova, Ludmila A.; Tsybalova, Ludmila M.; Kiselev, Oleg I.; Ravin, Nikolai V.; Skryabin, Konstantin G.

    2013-01-20

    Hepatitis B virus-like particles, icosahedral structures formed by multiple core protein dimers, are promising immune-enhancing vaccine carriers for foreign antigens. Insertions into the surface-exposed immunodominant loop are especially immunogenic. However, the need to conserve the particulate structure to ensure high immunogenicity imposes restraints on the nature of the heterologous sequence that can be inserted. We propose a new approach to constructing HBc particles linked to the target epitopes that relies on non-covalent interactions between the epitope and pre-assembled unmodified HBc particles. Interaction was enabled by fusion of the epitope to the GSLLGRMKGA peptide, binding to the spike tips. This peptide may be used as a 'binding tag' allowing in vitro construction of HBc particles carrying the target peptide. Such virus-like particles carrying multiple copies of the extracellular domain of the M2 protein of different influenza strains appeared to be highly immunogenic and protected immunised mice against a lethal influenza challenge.

  15. The Effect of a Polyvalent Antivenom on the Serum Venom Antigen Levels of Naja sputatrix (Javan Spitting Cobra) Venom in Experimentally Envenomed Rabbits.

    PubMed

    Yap, Michelle Khai Khun; Tan, Nget Hong; Sim, Si Mui; Fung, Shin Yee; Tan, Choo Hock

    2015-10-01

    The treatment protocol of antivenom in snake envenomation remains largely empirical, partly due to the insufficient knowledge of the pharmacokinetics of snake venoms and the effects of antivenoms on the blood venom levels in victims. In this study, we investigated the effect of a polyvalent antivenom on the serum venom antigen levels of Naja sputatrix (Javan spitting cobra) venom in experimentally envenomed rabbits. Intravenous infusion of 4 ml of Neuro Polyvalent Snake Antivenom [NPAV, F(ab')2 ] at 1 hr after envenomation caused a sharp decline of the serum venom antigen levels, followed by transient resurgence an hour later. The venom antigen resurgence was unlikely to be due to the mismatch of pharmacokinetics between the F(ab')2 and venom antigens, as the terminal half-life and volume of distribution of the F(ab')2 in serum were comparable to that of venom antigens (p > 0.05). Infusion of an additional 2 ml of NPAV was able to prevent resurgence of the serum venom antigen level, resulting in a substantial decrease (67.1%) of the total amount of circulating venom antigens over time course of envenomation. Our results showed that the neutralization potency of NPAV determined by neutralization assay in mice may not be an adequate indicator of its capability to modulate venom kinetics in relation to its in vivo efficacy to neutralize venom toxicity. The findings also support the recommendation of giving high initial dose of NPAV in cobra envenomation, with repeated doses as clinically indicated in the presence of rebound antigenemia and symptom recurrence.

  16. A self-amplified transistor immunosensor under dual gate operation: highly sensitive detection of hepatitis B surface antigen.

    PubMed

    Lee, I-K; Jeun, M; Jang, H-J; Cho, W-J; Lee, K H

    2015-10-28

    Ion-sensitive field-effect transistors (ISFETs), although they have attracted considerable attention as effective immunosensors, have still not been adopted for practical applications owing to several problems: (1) the poor sensitivity caused by the short Debye screening length in media with high ion concentration, (2) time-consuming preconditioning processes for achieving the highly-diluted media, and (3) the low durability caused by undesirable ions such as sodium chloride in the media. Here, we propose a highly sensitive immunosensor based on a self-amplified transistor under dual gate operation (immuno-DG ISFET) for the detection of hepatitis B surface antigen. To address the challenges in current ISFET-based immunosensors, we have enhanced the sensitivity of an immunosensor by precisely tailoring the nanostructure of the transistor. In the pH sensing test, the immuno-DG ISFET showed superior sensitivity (2085.53 mV per pH) to both standard ISFET under single gate operation (58.88 mV per pH) and DG ISFET with a non-tailored transistor (381.14 mV per pH). Moreover, concerning the detection of hepatitis B surface antigens (HBsAg) using the immuno-DG ISFET, we have successfully detected trace amounts of HBsAg (22.5 fg mL(-1)) in a non-diluted 1× PBS medium with a high sensitivity of 690 mV. Our results demonstrate that the proposed immuno-DG ISFET can be a biosensor platform for practical use in the diagnosis of various diseases.

  17. Definition of the surface antigens of Mycobacterium malmoense and use in studying the etiology of a form of mycobacteriosis.

    PubMed Central

    McNeil, M; Tsang, A Y; McClatchy, J K; Stewart, C; Jardine, I; Brennan, P J

    1987-01-01

    Mycobacterium malmoense is the latest of a roster of atypical mycobacteria implicated in pulmonary infections. Yet it lacks recognizable phenotypic features to allow its ready identification. Some 23 clinical isolates of M. malmoense were examined for homologous seroagglutination reactions and characteristic surface antigens. One group showed concordant agglutination interreactions and an identical spectrum of glycolipids and are regarded as M. malmoense sensu stricto. The glycolipids are of the newly found, trehalose-containing lipooligosaccharide class. De-O-acylation followed by high-pressure liquid chromatography revealed one major and several minor oligosaccharides. Partial acidic cleavage to release glycosidically linked trehalose, alpha-mannosidase digestion to demonstrate the presence of a non-reducing-end mannobiose, perdeuteriomethylation, partial acid hydrolysis, reduction, and O ethylation, combined with 1H nuclear magnetic resonance and electron impact and fast-atom bombardment mass spectrometry revealed the structure of the major oligosaccharide as alpha-D-Manp-(1----3) -alpha-D-Manp-(1----[2-alpha-L-Rhap-(1--]4--3)-alpha-L-Rh ap- (1----3)-alpha-D-Glcp-(1----1)-alpha-D-Glcp, in which two of the 2-alpha-L-Rhap residues are O methylated at C-3. (Man, mannose; Rha, rhamnose; Glc, glucose; p, pyranosyl). The structures of the minor oligosaccharides were also determined; they differ at the distal nonreducing end. The dominant oligosaccharide was acylated by octanoate, 2-methyleicosanoate, and 2,4-dimethylpentacosanoate to yield the major species-specific surface antigen of M. malmoense, which we regard as the most characteristic feature of the pathogen. Images PMID:3597323

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

    PubMed Central

    1987-01-01

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

  19. Production and Characterization of a Monoclonal Antibody Raised Against Surface Antigens from Mycelium of Gaeumannomyces graminis var. tritici: Evidence for an Extracellular Polyphenol Oxidase.

    PubMed

    Thornton, C R; Dewey, F M; Gilligan, C A

    1997-01-01

    ABSTRACT A murine monoclonal antibody (MAb) of immunoglobulin class M (IgM) was raised against surface antigens from Gaeumannomyces graminis var. tritici and, by enzyme-linked immunosorbent assay, recognized isolates of G. graminis var. tritici, G. graminis var. avenae and G. graminis var. graminis. Characterization of the antigen by heat and protease treatments showed that the epitope recognized by the MAb was a protein. Antigen production was detected only in live mycelia. Immunofluorescence studies showed that the antigen was associated with both the broad melanized macrohyphae and hyaline mycelia of G. graminis var. tritici. Secretion of antigen into an aqueous minimal medium was promoted only by exposure of live mycelia to certain phenolic substrates, including monophenols ortho-, para-, and meta-cresol; 3,4,5-trihydroxybenzoic acid (gallic acid); and phenolic amino acid L-3-(3,4-dihydroxyphenyl) alanine (L-DOPA). Antigen secretion was not promoted by 3-(4-hydroxyphenyl) alanine (L-tyrosine). The MAb reacted strongly with purified enzyme laccase (polyphenol oxidase, EC 1.10.3.2) but did not recognize purified tyrosinase (monophenol oxidase, EC 1.14.18.1). Moreover, chemicals that bind to copper and inhibit copper-containing enzymes such as laccase completely inhibited antigen secretion in response to L-DOPA. The MAb was tested for specificity against a wide range of fungi, common yeast species, and gram positive and negative bacteria. It did not recognize antigens from a broad range of unrelated fungi, including Gliocladium roseum, Fusarium sp., Phoma exigua, Phialophora fastigiata, Penicillium crustosum, Pythium ultimum, Rhizopus stolonifer, Rhizoctonia carotae, R. oryzae, R. tuliparum, and Trichoderma viride, nor did it recognize surface antigens from yeasts or bacteria. The MAb cross-reacted with antigens from Botrytis spp., Chaetomium globosum, R. cerealis, and R. solani. However, secretion of antigen by R. solani and R. cerealis was not promoted by L

  20. Cloning of a cDNA encoding a surface antigen of Schistosoma mansoni schistosomula recognized by sera of vassinated mice

    SciTech Connect

    Dalton, J.P.; Tom, T.D.; Strand, M.

    1987-06-01

    Spleen cells of mice vaccinated with radiation-attenuated Schistosoma mansoni cercariae were used to produce monoclonal antibodies directed against newly transformed schistosomular surface antigens. One of these monoclonal antibodies recognized a polypeptide of 18 kDa. Binding was measured by radioimmunoassay. This glycoprotein was purified by monoclonal antibody immunoaffinity chromatography and a polyclonal antiserum was prepared against it. Immunofluorescence assays showed that the polyclonal antiserum bound to the surface of newly transformed schistosomula and lung-stage organisms but not to the surface of liver-stage and adult worms. Using this polyclonal antiserum we isolated recombinant clones from an adult worm cDNA expression library constructed in lambdagt11. Clone 654.2 contained an insert of 0.52 kilobase and hybridized to a 1.2-kilobase mRNA species from adult worms. Most importantly, clone 654.2 produced a fusion protein of 125 kDa that was reactive with sera of vaccinated mice that are capable of transferring resistance. This result encourages future vaccination trials with the fusion protein.

  1. The surface core level shift for lithium at the surface of lithium borate

    NASA Astrophysics Data System (ADS)

    Wooten, David; Ketsman, I.; Xiao, Jie; Losovyj, Ya. B.; Petrosky, J.; McClory, J.; Burak, Ya. V.; Adamiv, V. T.; Dowben, P. A.

    2010-01-01

    The shallow Li 1s core level exhibits a surface-to-bulk core level shift for the stoichiometric Li 2B 4O 7(1 1 0) surface. Angle-resolved photoemission spectroscopy was used to indentify Li 1s bulk and surface core level components at binding energies -56.5±0.4 and -53.7±0.5 eV, respectively. We find photoemission evidence for surface states of Li 2B 4O 7(1 1 0) that exist in the gap of the projected bulk density of states. The existence of surface states is consistent with the large surface-to-bulk core level shift for the Li 1s core.

  2. Lesion Size Correlates with Leishmania Antigen-Stimulated TNF-Levels in Human Cutaneous Leishmaniasis

    PubMed Central

    Oliveira, Fabiano; Bafica, Andre; Rosato, Andrea B.; Favali, Cecilia B. F.; Costa, Jackson M.; Cafe, Virginia; Barral-Netto, Manoel; Barral, Aldina

    2011-01-01

    Cutaneous leishmaniasis (CL) is a worldwide disease endemic in several regions of the globe. The hallmark of CL is skin ulcers likely driven by efforts of the immune system to control Leishmania growth. Cytokines, such as tumor necrosis factor (TNF) and interferon-gamma can control disease progression in animal models. Nevertheless, the impact of these cytokines in CL ulcer outcome is not well established in humans. In this study, 96 CL patients from an endemic area of Leishmania braziliensis were enrolled for a follow-up study that consisted of clinical and immunological evaluations in a 2-year period. Statistical analysis revealed that healing time (P = 0.029), age (P = 0.002), and TNF levels (P = 0.0002) positively correlate with ulcer size at the time of the first clinical evaluation. Our findings suggest that ulcer size correlates with healing time and TNF levels support the use of TNF inhibitors combined with standard therapy to improve healing in CL patients with severe lesions. PMID:21734128

  3. Surface Plasmon Resonance Label-Free Monitoring of Antibody Antigen Interactions in Real Time

    ERIC Educational Resources Information Center

    Kausaite, Asta; van Dijk, Martijn; Castrop, Jan; Ramanaviciene, Almira; Baltrus, John P.; Acaite, Juzefa; Ramanavicius, Arunas

    2007-01-01

    Detection of biologically active compounds is one of the most important topics in molecular biology and biochemistry. One of the most promising detection methods is based on the application of surface plasmon resonance for label-free detection of biologically active compounds. This method allows one to monitor binding events in real time without…

  4. Time to lowest postoperative carcinoembryonic antigen level is predictive on survival outcome in rectal cancer

    PubMed Central

    Yu, Huichuan; Luo, Yanxin; Wang, Xiaolin; Bai, Liangliang; Huang, Pinzhu; Wang, Lei; Huang, Meijin; Deng, Yanhong; Wang, Jianping

    2016-01-01

    This study was to investigate whether the time to the lowest postoperative CEA can predict cancer survival. We enrolled 155 rectal cancer patients in this retrospective and longitudinal cohort study. Deepness of response (DpR) of CEA refers to the relative change of the lowest postoperative CEA level from baseline, and time to DpR (TTDpR) refers to the time from surgery to the lowest postoperative CEA level. The median of TTDpR and DpR was 4.5 (range, 3.0–18.0) weeks and −67% (range, −99% to 114%) respectively. Patients with TTDpR 4.5 weeks. Using TTDpR as a continuous variable, the HR of DFS and OS was 1.13 (95% CI 1.06–1.22, P = 0.001) and 1.17 (95% CI 1.07–1.29, P = 0.001) respectively. On multivariate analysis, the predictive value of prolonged TTDpR remained [adjusted HRs: 1.12 (95% CI 1.03–1.21, P = 0.006) and 1.17 (95% CI 1.06–1.28, P = 0.001)]. These findings remained significant in patients with normal preoperative CEA. Our results showed prolonged TTDpR of CEA independently predicted unfavorable survival outcomes, regardless of whether preoperative CEA was elevated or not. PMID:27658525

  5. Surface levelling of thermosetting powder coatings: theory and experiment

    NASA Astrophysics Data System (ADS)

    Andrei, D. C.; Hay, J. N.; Keddie, J. L.; Sear, R. P.; Yeates, S. G.

    2000-08-01

    The deposition of protective coatings from thermosetting polymer powders is an ecological, economic and energy-efficient technology. A frequent problem encountered with powder coatings is a rough surface (with undulations on a length scale much greater than the powder particle size) that detracts from the visual appearance. The levelling (i.e. flattening) of the surface of a polymer melt is driven by the minimization of the surface energy but opposed by the (possibly time-dependent) viscosity of the melt. We address the problem by developing a model of surface levelling to consider flow in two directions, building upon a one-dimensional model already in the literature. We have performed simulations to predict the final coating profiles starting with a Gaussian profile and using experimentally determined values of polymer viscosity. To compare to the simulations, we have measured experimentally the dimensions of surface undulations on coatings formed from thermosetting acrylic powder layers having purposely created features of known dimensions. There is good agreement between simulation and experiment. Both find that the levelling proceeds to a greater extent with increasing coating thickness and with decreasing lateral dimension of the surface undulation. Our results open up the possibility of predicting final surface topography given the rheological properties of a polymer.

  6. Beryllium surface levels in a military ammunition plant.

    PubMed

    Sanderson, Wayne T; Leonard, Stephanie; Ott, Darrin; Fuortes, Laurence; Field, William

    2008-07-01

    This study evaluated the presence of beryllium surface contamination in a U.S. conventional munitions plant as an indicator of possible past beryllium airborne and skin exposure and used these measurements to classify job categories by potential level of exposure. Surface samples were collected from production and nonproduction areas of the plant and at regional industrial reference sites with no known history of beryllium use. Surface samples of premoistened wiping material were analyzed for beryllium mass content using inductively coupled plasma-atomic emission spectroscopy (ICP-AES) and results expressed as micrograms of beryllium per 100 square centimeters (micro g/100 cm(2)). Beryllium was detected in 87% of samples collected at the munitions plant and in 72% of the samples collected at regional reference sites. Two munitions plant samples from areas near sanders and grinders were above 3.0 micro g/100 cm(2) (U.S. Department of Energy surface contamination limit). The highest surface level found at the reference sites was 0.44 micro g/100 cm(2). Workers in areas where beryllium-containing alloy tools were sanded or ground, but not other work areas, may have been exposed to airborne beryllium concentrations above levels encountered in other industries where metal work is conducted. Surface sampling provided information useful for categorizing munitions plant jobs by level of past beryllium airborne and skin exposure and, subsequently, for identifying employees within exposure strata to be screened for beryllium sensitization.

  7. Increased serum IL-2R levels in coeliac disease are related to CD4 but not CD8 antigens.

    PubMed

    Blanco, A; Garrote, J A; Arranz, E; Alonso, M; Clavo, C

    1992-11-01

    Forty-three coeliac children, ranging from 1 year and 3 months to 14 years and 9 months, were studied. Twenty-eight patients were in an active phase of the disease, and 15 were in remission. The criteria of coeliac disease (CD) activity were established according to the results of IgA anti-endomysial antibodies (IgA-AEm). Interleukin 2 receptor (IL-2R) and CD4 and CD8 antigens were measured in serum samples by an ELISA technique using two noncompetitive monoclonal antibodies. Antigliadin antibodies of IgG (IgG-AGA) and IgA (IgA-AGA) classes were also measured. The AEm-positive coeliac patient group showed values of 1,860 +/- 948 U/ml for IL-2R, 430 +/- 228 U/ml for CD8, and 36.8 +/- 25.1 U/ml for CD4. AEm-negative patients showed values of 980 +/- 436 U/ml, 350 +/- 243 U/ml, and 24.1 +/- 20 U/ml, respectively. IL-2R levels were the only ones significantly elevated (p < 0.005) in the active coeliac group. On the other hand, IgG-AGA and IgA-AGA were both clearly increased (p < 0.001). IL-2R levels in active coeliac patients correlated with CD4 levels (p < 0.05), but not with CD8, IgG-AGA, and IgA-AGA levels. We also found a surprising negative correlation between AEm antibodies of IgA2 class with both IL-2R (r = 0.471; p < 0.05) and CD8 (r = 0.616; p < 0.05). The results show that in CD there is a lymphocyte activation affecting mainly CD4+ cells and not correlated with serum AGA levels, suggesting an independence of both immunological phenomena and probably with different locations of origin.

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

    PubMed

    Yeo, Seung-Gu

    2016-04-01

    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.

  9. Hepatitis B surface antigen nanoparticles coated with chitosan and trimethyl chitosan: Impact of formulation on physicochemical and immunological characteristics.

    PubMed

    Tafaghodi, Mohsen; Saluja, Vinay; Kersten, Gideon F A; Kraan, Heleen; Slütter, Bram; Amorij, Jean-Pierre; Jiskoot, Wim

    2012-08-03

    Mucosal immunization offers various advantages over parenteral vaccination, but typically requires potent delivery systems and/or adjuvants to result in protective immunity. Here we report on the preparation of trimethylated chitosan (TMC) and chitosan (CHT) nanoparticles (NPs) loaded with hepatitis B surface antigen (HB), by a simple and scalable method. TMC:HB and CHT:HB NPs were prepared by direct coating of antigen by polymer. The impact of buffer, pH and tonicity of the dispersion medium on NPs' polydispersity, zeta potential and association percentage of polymer with antigen was evaluated. Moreover, biological properties of both NPs were addressed in vitro by studying their effect on cell viability, transepithelial electrical resistance (TEER) and dendritic cell (DC) maturation. Finally, immunogenicity was assessed by evaluating IgG, IgG1, IgG2a, IgA titers and sIgA after both mucosal (nasal) as well intramuscular (i.m.) vaccination in a murine model. TMC:HB and CHT:HB NPs, prepared in acetate buffer pH 6.7 of three different tonicities, had comparable size, polydispersity, zeta potential and association percentage. TMC:HB NPs, but not CHT:HB NPs, had a mild negative effect on cell viability and TEER, and a considerable positive effect on DC maturation. After nasal and i.m. immunization, TMC:HB NPs in hypotonic medium and CHT:HB NPs in all media induced higher serum and nasal antibody titers compared with HB solution (P<0.001). After i.m. injection, both TMC:HB and CHT:HB NPs induced higher IgG and IgG2a titers compared with alum adsorbed HB (P<0.001). For CHT:HB NPs, the tonicity of the dispersion medium did not affect the mucosal and systemic immune responses. In conclusion, TMC NPs and CHT NPs are similarly potent mucosal immunoadjuvants for HB. Moreover, both polymers are potent immunoadjuvants for i.m. administered isotonic HB, resulting in higher IgG2a/IgG1 ratios compared with alum adjuvanted HB.

  10. Bovine leukocyte antigen major histocompatibility complex class II DRB3*2703 and DRB3*1501 alleles are associated with variation in levels of protection against Theileria parva challenge following immunization with the sporozoite p67 antigen.

    PubMed

    Ballingall, Keith T; Luyai, Anthony; Rowlands, G John; Sales, Jill; Musoke, Anthony J; Morzaria, Subash P; McKeever, Declan J

    2004-05-01

    Initial laboratory trials of an experimental subunit vaccine against Theileria parva based on the 67-kDa major sporozoite surface antigen revealed a range of responses to challenge. We have analyzed convergence in seven sets of monozygotic twins which suggests that genetic factors may have an influence in determining the degree of protection provided by p67 immunization. In addition, we have examined whether allelic diversity at major histocompatibility complex class II loci influences protection. Analysis of bovine leukocyte antigen DRB3 diversity in 201 animals identified significant associations with vaccine success (DRB3*2703; P = 0.027) and vaccine failure (DRB3*1501; P = 0.013). Furthermore, DRB3*2703 was associated with the likelihood of immunized animals showing little to no clinical signs of disease following challenge. We discuss the acquired and innate immune mechanisms that may be behind the associations described here.

  11. Hepatitis B Surface Antigen Reduction by Switching from Long-term Nucleoside/nucleotide Analog Administration to Pegylated Interferon.

    PubMed

    Tamaki, Nobuharu; Kurosaki, Masayuki; Kusakabe, Atsunori; Orito, Etsuro; Joko, Kouji; Kojima, Yuji; Kimura, Hiroyuki; Uchida, Yasushi; Hasebe, Chitomi; Asahina, Yasuhiro; Izumi, Namiki

    2017-02-15

    Hepatitis B surface antigen (HBsAg) reduction during nucleoside/nucleotide analog (NA) therapy is slow and an alternative strategy for patients receiving ongoing NA to facilitate HBsAg reduction is required. We investigated whether switching to pegylated interferon (PEG-IFN) after long-term NA administration enhances HBsAg reduction. Forty-nine patients who switched from long-term NA to 48 weeks of PEG-IFN alfa-2a were studied. The mean duration of previous NA was 48 months (sequential group). A total of 147 patients who continued NA and matched for baseline characteristics were analyzed for comparison (NA continuation group). The treatment response was defined as HBsAg reduction ≥1.0 logIU/ml at the end of PEG-IFN. HBsAg reduction at week 48 was 0.81 ± 1.1 logIU/ml in the sequential group, which was significantly higher than that in the NA continuation group (0.11 ± 0.3 logIU/ml, p <0.001). The treatment response was achieved in 29% and 2% of the sequential group and NA continuation group (p <0.001), and the odds ratio of sequential therapy for treatment response was 19 compared with NA continuation (p <0.001). In patients positive for hepatitis B e antigen (HBeAg), HBeAg seroconversion was higher in the sequential group (44% vs. 8%, p <0.001). In HBeAg negative patients, only patients in the sequential group achieved HBsAg loss. No patient needed to resume NA administration because of HBV DNA increase accompanied by alanine aminotransferase flares. In summary, sequential therapy with PEG-IFN after long-term NA enhances the reduction of HBsAg and may represent a treatment option to promote HBsAg loss. This article is protected by copyright. All rights reserved.

  12. Molecular basis for variable expression of merozoite surface antigen gp45 among American isolates of Babesia bigemina.

    PubMed

    Fisher, T G; McElwain, T F; Palmer, G H

    2001-06-01

    Immunization with the merozoite surface glycoprotein gp45 induces protection against challenge using the homologous Babesia bigemina strain. However, gp45 B-cell epitopes are highly polymorphic among B. bigemina strains isolated from different geographical locations within North and South America. The molecular basis for this polymorphism was investigated using the JG-29 biological clone of a Mexico strain of B. bigemina and comparison with the Puerto Rico, St. Croix, and Texcoco strains. The molecular size and antibody reactivity of gp45 expressed by the JG-29 clone were identical to those of the parental Mexico strain. gp45 cDNA and the genomic locus encompassing gp45 were cloned and sequenced from JG-29. The locus sequence and Southern blot data were consistent with a single gp45 copy in the JG-29 genome. The JG-29 cDNA expressed the full-length protein recognized by the gp45-specific monoclonal antibody 14/1.3.2. The genomes of the Puerto Rico and St. Croix strains of B. bigemina were shown to lack a closely related gp45-like gene by PCR using multiple primer sets and by Southern blots using both full-length and region-specific gp45 probes. This genomic difference was confirmed using unpassaged isolates from a 1999 disease outbreak in Puerto Rico. In contrast, the Texcoco strain retains a gp45 gene, encoding an open reading frame identical to that of JG-29. However, the Texcoco gp45 gene is not transcribed. These two mechanisms, lack of a closely related gp45-like gene and failure to transcribe gp45, result in generation of antigenic polymorphism among B. bigemina strains, and the latter mechanism is unique compared to prior mechanisms of antigenic polymorphism identified in babesial parasites.

  13. Association between Duffy antigen receptor for chemokines expression and levels of inflammation markers in sickle cell anemia patients.

    PubMed

    Nebor, Danitza; Durpes, Marie Claude; Mougenel, Danielle; Mukisi-Mukaza, Martin; Elion, Jacques; Hardy-Dessources, Marie-Dominique; Romana, Marc

    2010-07-01

    Since inflammation plays a prominent role in the pathogenesis of sickle cell anemia (SCA) and Duffy antigen receptor for chemokines (DARC) modulates the function of inflammatory processes, we analyzed the relationship between the erythrocyte DARC phenotype and clinical expression of SCA. DARC locus was genotyped in 212 SS adult patients followed by the sickle cell center of Guadeloupe (French West Indies). After patients' stratification according to RBC DARC expression, the prevalence of renal disease, leg ulcers, priapism and osteonecrosis was compared between patient groups as well as hematological variables and plasma levels of chemokines. Duffy-positive patients exhibited higher counts of white blood cells (9.95+/-2.36 vs 8.88+/-2.32 10(9)/L, p=0.0066), polynuclear neutrophils (5.1+/-1.73 vs 4.51+/-1.71 10(9)/L, p=0.0227), higher plasma levels of IL-8 (4.46+/-1.22 vs 1.47+/-0.5 pg/mL, p=0.0202) and RANTES (27.8+/-4.3 vs 18.1+/-2.3 ng/mL, p=0.04) than Duffy-negative patients. No association was detected between RBC expression of DARC and the studied complications.

  14. Genome-wide association study of prostate-specific antigen levels identifies novel loci independent of prostate cancer

    PubMed Central

    Hoffmann, Thomas J.; Passarelli, Michael N.; Graff, Rebecca E.; Emami, Nima C.; Sakoda, Lori C.; Jorgenson, Eric; Habel, Laurel A.; Shan, Jun; Ranatunga, Dilrini K.; Quesenberry, Charles P.; Chao, Chun R.; Ghai, Nirupa R.; Aaronson, David; Presti, Joseph; Nordström, Tobias; Wang, Zhaoming; Berndt, Sonja I.; Chanock, Stephen J.; Mosley, Jonathan D.; Klein, Robert J.; Middha, Mridu; Lilja, Hans; Melander, Olle; Kvale, Mark N.; Kwok, Pui-Yan; Schaefer, Catherine; Risch, Neil; Van Den Eeden, Stephen K.; Witte, John S.

    2017-01-01

    Prostate-specific antigen (PSA) levels have been used for detection and surveillance of prostate cancer (PCa). However, factors other than PCa—such as genetics—can impact PSA. Here we present findings from a genome-wide association study (GWAS) of PSA in 28,503 Kaiser Permanente whites and 17,428 men from replication cohorts. We detect 40 genome-wide significant (P<5 × 10−8) single-nucleotide polymorphisms (SNPs): 19 novel, 15 previously identified for PSA (14 of which were also PCa-associated), and 6 previously identified for PCa only. Further analysis incorporating PCa cases suggests that at least half of the 40 SNPs are PSA-associated independent of PCa. The 40 SNPs explain 9.5% of PSA variation in non-Hispanic whites, and the remaining GWAS SNPs explain an additional 31.7%; this percentage is higher in younger men, supporting the genetic basis of PSA levels. These findings provide important information about genetic markers for PSA that may improve PCa screening, thereby reducing over-diagnosis and over-treatment. PMID:28139693

  15. Genotypic Characterization of Egypt Enterotoxigenic Escherichia coli Isolates Expressing Coli Surface Antigen 6

    DTIC Science & Technology

    2013-02-01

    USA Abstract Introduction: One approach to control enterotoxigenic Escherichia coli (ETEC) infections has been to develop vaccines focused on...results show a lack of clonality among Egypt CS6 E. coli isolates and supports the use and the further research on vaccines targeting this cell surface...has received considerable attention as a target for vaccine development [11-14]. CS6 is immunogenic in humans both after natural infection and

  16. Age-dependent B cell autoimmunity to a myelin surface antigen in pediatric multiple sclerosis.

    PubMed

    McLaughlin, Katherine A; Chitnis, Tanuja; Newcombe, Jia; Franz, Bettina; Kennedy, Julia; McArdel, Shannon; Kuhle, Jens; Kappos, Ludwig; Rostasy, Kevin; Pohl, Daniela; Gagne, Donald; Ness, Jayne M; Tenembaum, Silvia; O'Connor, Kevin C; Viglietta, Vissia; Wong, Susan J; Tavakoli, Norma P; de Seze, Jerome; Idrissova, Zhannat; Khoury, Samia J; Bar-Or, Amit; Hafler, David A; Banwell, Brenda; Wucherpfennig, Kai W

    2009-09-15

    Multiple sclerosis (MS) typically manifests in early to mid adulthood, but there is increasing recognition of pediatric-onset MS, aided by improvements in imaging techniques. The immunological mechanisms of disease are largely unexplored in pediatric-onset MS, in part because studies have historically focused on adult-onset disease. We investigated autoantibodies to myelin surface Ags in a large cohort of pediatric MS cases by flow cytometric labeling of transfectants that expressed different myelin proteins. Although Abs to native myelin oligodendrocyte glycoprotein (MOG) were uncommon among adult-onset patients, a subset of pediatric patients had serum Abs that brightly labeled the MOG transfectant. Abs to two other myelin surface Ags were largely absent. Affinity purification of MOG Abs as well as competition of binding with soluble MOG documented their binding specificity. Such affinity purified Abs labeled myelin and glial cells in human CNS white matter as well as myelinated axons in gray matter. The prevalence of such autoantibodies was highest among patients with a very early onset of MS: 38.7% of patients less than 10 years of age at disease onset had MOG Abs, compared with 14.7% of patients in the 10- to 18-year age group. B cell autoimmunity to this myelin surface Ag is therefore most common in patients with a very early onset of MS.

  17. Retention of structure, antigenicity, and biological function of pneumococcal surface protein A (PspA) released from polyanhydride nanoparticles

    PubMed Central

    Haughney, Shannon L.; Petersen, Latrisha K.; Schoofs, Amy D.; Ramer-Tait, Amanda E.; King, Janice; Briles, David; Wannemuehler, Michael J.; Narasimhan, Balaji

    2013-01-01

    Pneumococcal surface protein A (PspA) is a choline-binding protein which is a virulence factor found on the surface of all Streptococcus pneumoniae strains. Vaccination with PspA has been shown to be protective against a lethal challenge with S. pneumoniae, making it a promising immunogen for use in vaccines. Herein, the design of a PspA-based subunit vaccine using polyanhydride nanoparticles as a delivery platform is described. Nanoparticles based on sebacic acid (SA), 1,6-bis-(p-carboxyphenoxy)hexane (CPH) and 1,8-bis-(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG), specifically 50:50 CPTEG:CPH and 20:80 CPH:SA, were used to encapsulate and release PspA. The protein released from the nanoparticle formulations retained its primary and secondary structure as well as its antigenicity. The released PspA was also biologically functional based on its ability to bind to apolactoferrin and prevent its bactericidal activity towards Escherichia coli. When the PspA nanoparticle formulations were administered subcutaneously to mice, the animals elicited a high titer and high avidity anti-PspA antibody response. Together, these studies provide a framework for the rational design of a vaccine against S. pneumoniae based on polyanhydride nanoparticles. PMID:23774257

  18. Locus NMB0035 codes for a 47-kDa surface-accessible conserved antigen in Neisseria.

    PubMed

    Arenas, Jesús; Abel, Ana; Sánchez, Sandra; Alcalá, Belén; Criado, María T; Ferreirós, Carlos M

    2006-12-01

    A47 kDa neisserial outer-membrane antigenic protein (P47) was purified to homogeneity and used to prepare polyclonal anti-P47 antisera. Protein P47 was identified by MALDI-TOF fingerprinting analysis as the hypothetical lipoprotein NMB0035. Two-dimensional diagonal SDS-PAGE results suggested that, contrary to previous findings, P47 is not strongly associated with other proteins in membrane complexes. Western blotting with the polyclonal monospecific serum showed that linear P47 epitopes were expressed in similar amounts in the 27 Neisseria meningitidis strains tested and, to a lesser extent, in commensal Neisseria, particularly N. lactamica. However, dot-blotting assays with the same serum demonstrated binding variability between meningococcal strains, indicating differences in surface accessibility or steric hindrance by other surface structures. Specific anti-P47 antibodies were bactericidal against the homologous strain but had variable activity against heterologous strains, consistent with the results from dot-blotting experiments. An in-depth study of P47 is necessary to evaluate its potential as a candidate for new vaccine designs.

  19. Protection of rats against Mycoplasma arthritidis-induced arthritis by active and passive immunizations with two surface antigens.

    PubMed

    Washburn, L R; Weaver, E J

    1997-05-01

    We previously identified two surface-exposed Mycoplasma arthritidis protein antigens, designated MAA1 and MAA2, that may be involved in cytadherence. Since adherence to host tissues is an important first step in most bacterial infections, we suggest that MAA1 and MAA2 may be virulence factors for M. arthritidis. In order to provide evidence for such a role, we conducted a series of experiments in which rats were actively immunized with each of these proteins purified from sodium dodecyl sulfate-polyacrylamide gels or passively immunized with poly- or monoclonal antibodies against MAA1 and MAA2. In each case, immunity against MAA1 and MAA2 conferred at least partial protection against M. arthritidis-induced disease. The greatest protection was achieved by passive immunization with monoclonal antibody A9a, directed against a surface-exposed epitope of putative adhesin MAA1. Because protective immunity in most bacterial infections is directed against major virulence factors, these results suggest that MAA1 and MAA2 may play a role in the pathogenesis of M. arthritidis-induced arthritis of rats, possibly by mediating initial colonization of joint tissues.

  20. The prevalence of mutations in the major hydrophilic region of the surface antigen of hepatitis B virus varies with subgenotype.

    PubMed

    Wang, X Y; Harrison, T J; He, X; Chen, Q Y; Li, G J; Liu, M H; Li, H; Yang, J Y; Fang, Z L

    2015-12-01

    Mutations in the major hydrophilic region (MHR) of the surface antigen of hepatitis B virus (HBV) may result in vaccine escape, failure of immunotherapy and antiviral resistance. These mutants may be transmitted and constitute a public health threat. We aimed to determine the prevalence of MHR mutations of HBV in areas of high endemicity in Guangxi, China. HBV surface gene was analysed from 278 HBsAg-positive asymptomatic individuals recruited from Guangxi using cluster sampling. Three genotypes, B, C and I, were identified. The overall prevalence of MHR mutations is 17·6%. The prevalence of MHR mutations in genotype B (15·1%) is not significantly different from that in genotype C (16·4%). However, the prevalence in subgenotype C5 (31·1%) is significantly higher than in subgenotype C2 (13·0%) (χ 2 = 6·997, P < 0·05). The prevalence of escape mutations and overlapping polymerase substitutions in subgenotype C5 is significantly higher than in subgenotypes B2 and C2. In total, 7·9% of MHR mutants are escape mutations and 72·1% of MHR mutations produced amino-acid changes in the overlapping polymerase, including resistance mutations to entecavir. Our results suggest that the prevalence of MHR mutations varies with subgenotype. The prevalence of escape mutations and polymerase mutations may be associated with subgenotype.

  1. Characterization of hepatitis B virus surface antigen variability and impact on HBs antigen clearance under nucleos(t)ide analogue therapy.

    PubMed

    Velay, A; Jeulin, H; Eschlimann, M; Malvé, B; Goehringer, F; Bensenane, M; Frippiat, J-P; Abraham, P; Ismail, A M; Murray, J M; Combet, C; Zoulim, F; Bronowicki, J-P; Schvoerer, E

    2016-05-01

    For hepatitis B virus (HBV)-related chronic infection under treatment by nucleos(t)ide analogues (NUCs), HBsAg clearance is the ultimate therapeutic goal but very infrequent. We investigated how HBV envelope protein variability could lead to differential HBsAg clearance on NUCs. For 12 HBV genotype D patients receiving NUCs, six resolvers (HBsAg clearance) were compared to six matched nonresolvers (HBsAg persistence). PreS/S amino acid (aa) sequences were analysed with bioinformatics to predict HBV envelope antigenicity and aa covariance. To enrich our analyses on very rare resolvers, these were compared with other HBV genotype D strains in three characterized clinical cohorts including common chronically infected patients. The sT125M+sP127T combination was observed in four nonresolvers of six, corroborated by aa covariance analysis, associated with a lower predicted antigenicity than sT125T+sP127P. Concordant features within this HBV key functional domain, at positions 125 and 127, were reported from two of the three comparative cohorts. In our hands, a lower ELISA reactivity of HBV-vaccinated mice sera was observed against the sT125M mutant. In the S gene, 56 aa changes in minor variants were detected in non-resolvers, mainly in the major hydrophilic region, vs 28 aa changes in resolvers. Molecular features in patients showing HBsAg persistence on NUCs argue in favour of a different aa pattern in the HBV S gene compared to those showing HBsAg clearance. In nonresolvers, a decrease in HBs 'a' determinant antigenicity and more frequent mutations in the S gene suggest a role for the HBV envelope characteristics in HBsAg persistence.

  2. Characterization of the antigenicity of Cpl1, a surface protein of Cryptococcus neoformans var. neoformans.

    PubMed

    Cai, Jian-Piao; Liu, Ling-Li; To, Kelvin K W; Lau, Candy C Y; Woo, Patrick C Y; Lau, Susanna K P; Guo, Yong-Hui; Ngan, Antonio H Y; Che, Xiao-Yan; Yuen, Kwok-Yung

    2015-01-01

    Cryptococcus neoformans var. neoformans is an important fungal pathogen. The capsule is a well established virulence factor and a target site for diagnostic tests. The CPL1 gene is required for capsular formation and virulence. The protein product Cpl1 has been proposed to be a secreted protein, but the characteristics of this protein have not been reported. Here we sought to characterize Cpl1. Phylogenetic analysis showed that the Cpl1 of C. neoformans var. neoformans and the Cpl1 orthologs identified in C. neoformans var. grubii and C. gattii formed a distinct cluster among related fungi; while the putative ortholog found in Trichosporon asahii was distantly related to the Cryptococcus cluster. We expressed Cpl1 abundantly as a secreted His-tagged protein in Pichia pastoris. The protein was used to immunize guinea pigs and rabbits for high titer mono-specific polyclonal antibody that was shown to be highly specific against the cell wall of C. neoformans var. neoformans and did not cross react with C. gattii, T. asahii, Aspergillus spp., Candida spp. and Penicillium spp. Using the anti-Cpl1 antibody, we detected Cpl1 protein in the fresh culture supernatant of C. neoformans var. neoformans and we showed by immunostaining that the Cpl1 protein was located on the surface. The Cpl1 protein is a specific surface protein of C. neoformans var. neoformans.

  3. Confidence sets for optimal factor levels of a response surface.

    PubMed

    Wan, Fang; Liu, Wei; Bretz, Frank; Han, Yang

    2016-12-01

    Construction of confidence sets for the optimal factor levels is an important topic in response surfaces methodology. In Wan et al. (2015), an exact (1-α) confidence set has been provided for a maximum or minimum point (i.e., an optimal factor level) of a univariate polynomial function in a given interval. In this article, the method has been extended to construct an exact (1-α) confidence set for the optimal factor levels of response surfaces. The construction method is readily applied to many parametric and semiparametric regression models involving a quadratic function. A conservative confidence set has been provided as an intermediate step in the construction of the exact confidence set. Two examples are given to illustrate the application of the confidence sets. The comparison between confidence sets indicates that our exact confidence set is better than the only other confidence set available in the statistical literature that guarantees the (1-α) confidence level.

  4. Identification of surface antigens of schistosomula of Schistosoma mansoni recognized by antibodies from mice immunized by chronic infection and by exposure to highly irradiated cercariae

    SciTech Connect

    Simpson, A.J.; James, S.L.; Sher, A.

    1983-08-01

    Surface components of mechanically transformed schistosomula of Schistosoma mansoni were labeled by lactoperoxidase-catalyzed iodination. After solubilization with Triton X-100, antigens were identified by immunoprecipitation. Serum from chronically infected Swiss mice reproducibly precipitated seven major polypeptides with approximate molecular weights (X 10/sup 3/) of 94, 68, 45, 40 to 32, 22, and 16. The antigens of molecular weights (X 10/sup 3/) of 94, 40 to 32, 22, and 16 were shown to be exposed on the parasite surface by interaction of the antibodies with intact labeled schistosomula. Sera from several strains of infected inbred mice precipitated the same polypeptides. The antibodies produced during chronic infection were found to be stimulated by adult worms since sera from 6-week-infected animals precipitated none of the surface antigens, and the pattern produced by precipitation with antibodies from a mouse infected with male worms only was indistinguishable from the pattern obtained with sera from mice with bisexual infections. Antibodies from mice immunized with highly irradiated cercariae reproducibly precipitated major polypeptides of approximately (X 10/sup 3/) 94, 68, 45, 32, 22, 19, and 15 daltons. The antigens of (X 10/sup 3/) 94, 43, 32, 22, and 15 daltons were shown to be exposed on the parasite surface by interaction of the antibodies with intact labeled schistosomula. The 15 X 10(3)-dalton surface protein was recognized by sera from vaccinated, but not chronically infected, mice, suggesting that it represents a stage-specific immunogen present on schistosomula but not on adult worms. Sera from two inbred strains of mice which develop different degrees of immunity recognized the same antigens.

  5. A siphon gage for monitoring surface-water levels

    USGS Publications Warehouse

    McCobb, T.D.; LeBlanc, D.R.; Socolow, R.S.

    1999-01-01

    A device that uses a siphon tube to establish a hydraulic connection between the bottom of an onshore standpipe and a point at the bottom of a water body was designed and tested for monitoring surface-water levels. Water is added to the standpipe to a level sufficient to drive a complete slug of water through the siphoning tube and to flush all air out of the system. The water levels in the standpipe and the water body equilibrate and provide a measurable static water surface in the standpipe. The siphon gage was designed to allow quick and accurate year-round measurements with minimal maintenance. Currently available devices for monitoring surface-water levels commonly involve time-consuming and costly installation and surveying, and the movement of reference points and the presence of ice cover in cold regions cause discontinuity and inaccuracy in the data collected. Installation and field testing of a siphon gage using 0.75-in-diameter polyethylene tubing at Ashumet Pond in Falmouth, Massachusetts, demonstrated that the siphon gage can provide long-term data with a field effort and accuracy equivalent to measurement of ground-water levels at an observation well.A device that uses a siphon tube to establish a hydraulic connection between the bottom of an onshore standpipe and a point at the bottom of a water body was designed and tested for monitoring surface-water levels. Water is added to the standpipe to a level sufficient to drive a complete slug of water through the siphoning tube and to flush all air out of the system. The water levels in the standpipe and the water body equilibrate and provide a measurable static water surface in the standpipe. The siphon gage was designed to allow quick and accurate year-round measurements with minimal maintenance. Currently available devices for monitoring surface-water levels commonly involve time-consuming and costly installation and surveying, and the movement of reference points and the presence of ice cover in cold

  6. HIV-1 Adaptation to Antigen Processing Results in Population-Level Immune Evasion and Affects Subtype Diversification

    PubMed Central

    Tenzer, Stefan; Crawford, Hayley; Pymm, Phillip; Gifford, Robert; Sreenu, Vattipally B.; Weimershaus, Mirjana; de Oliveira, Tulio; Burgevin, Anne; Gerstoft, Jan; Akkad, Nadja; Lunn, Daniel; Fugger, Lars; Bell, John; Schild, Hansjörg; van Endert, Peter; Iversen, Astrid K.N.

    2014-01-01

    Summary The recent HIV-1 vaccine failures highlight the need to better understand virus-host interactions. One key question is why CD8+ T cell responses to two HIV-Gag regions are uniquely associated with delayed disease progression only in patients expressing a few rare HLA class I variants when these regions encode epitopes presented by ∼30 more common HLA variants. By combining epitope processing and computational analyses of the two HIV subtypes responsible for ∼60% of worldwide infections, we identified a hitherto unrecognized adaptation to the antigen-processing machinery through substitutions at subtype-specific motifs. Multiple HLA variants presenting epitopes situated next to a given subtype-specific motif drive selection at this subtype-specific position, and epitope abundances correlate inversely with the HLA frequency distribution in affected populations. This adaptation reflects the sum of intrapatient adaptations, is predictable, facilitates viral subtype diversification, and increases global HIV diversity. Because low epitope abundance is associated with infrequent and weak T cell responses, this most likely results in both population-level immune evasion and inadequate responses in most people vaccinated with natural HIV-1 sequence constructs. Our results suggest that artificial sequence modifications at subtype-specific positions in vitro could refocus and reverse the poor immunogenicity of HIV proteins. PMID:24726370

  7. The effect of finasteride on the prostate gland in men with elevated serum prostate-specific antigen levels.

    PubMed Central

    Cote, R. J.; Skinner, E. C.; Salem, C. E.; Mertes, S. J.; Stanczyk, F. Z.; Henderson, B. E.; Pike, M. C.; Ross, R. K.

    1998-01-01

    Prostate cancer is a disease associated with androgens. It has been hypothesized that reducing the conversion of testosterone (T) to dihydrotestosterone (DHT) in the prostate by the use of the drug finasteride, a 5alpha-reductase inhibitor, will reduce the incidence of prostate cancer. We investigated the chemopreventive potential of finasteride by evaluating its effect on the prostate gland of men with elevated serum prostate-specific antigen (PSA). Fifty-two men with elevated PSA and prostate sextant biopsies negative for cancer were randomized to receive finasteride 5 mg day(-1) (27 patients) or no medication (25 patients) for 12 months and were rebiopsied at 12 months. The biopsies were evaluated for the presence of cancer, the proportion of glandular and hyperplastic tissue, and the presence of high-grade prostatic intraepithelial neoplasia (PIN). Epithelial proliferation was assessed in the prestudy and 12-month biopsies by immunohistochemistry using antibody to proliferating cell nuclear antigen (PCNA). Serum blood samples were drawn at baseline and after 1, 3, 6 and 12 months of study. In the control group, serum levels of PSA and T were unchanged throughout the 12 months. In the finasteride group, PSA decreased 48% (P < 0.001), DHT decreased 67% (P < 0.001) and T increased 21% (P < 0.001). Histological evaluation of prestudy and 12-month biopsy specimens revealed that the finasteride group had a 30% reduction in the percentage of hyperplastic epithelial tissue (P = 0.002), although this decrease was not statistically significantly different between the finasteride and control groups (P = 0.11). In patients with PIN on prestudy biopsy, no change occurred in the PIN lesions with finasteride treatment. Finasteride also had no effect on the proliferation index of prostatic epithelial cells. Of the 27 patients treated with finasteride, eight (30%) had adenocarcinoma of the prostate detected on the 12-month biopsy, compared with one (4%) of the control patients

  8. Opium consumption is negatively associated with serum prostate-specific antigen (PSA), free PSA, and percentage of free PSA levels.

    PubMed

    Safarinejad, Mohammad Reza; Asgari, Seyyed Alaeddin; Farshi, Alireza; Iravani, Shahrokh; Khoshdel, Alireza; Shekarchi, Babak

    2013-01-01

    Addiction to opium continues to be a major worldwide medical and social problem. The study addressing the association between opium consumption and serum prostate-specific antigen (PSA) level is lacking. We determined the effects of opium consumption on serum PSA levels in opium-addict men. Our study subjects comprised 438 opium-addict men with a mean age of 52.2 ± 6.4 years (group 1). We compared these men with 446 men who did not indicate current or past opium use (group 2). Serum total PSA (tPSA), free PSA (fPSA), % fPSA, and sex hormones were compared between the 2 groups. The mean serum tPSA level was significantly lower in group 1 (1.05 ng/mL) than in controls (1.45 ng/mL) (P = 0.001). Opium consumption was also associated with lower fPSA (P = 0.001) and % fPSA (P = 0.001). Serum free testosterone level in opium-addict patients (132.5 ± 42 pg/mL) was significantly lower than that in controls (156.2 ± 43 pg/mL) (P = 0.03). However, no significant correlation existed between tPSA and free testosterone levels (r = 0.28, 95% CI, -0.036 to 0.51, P = 0.34). Among the patients with cancer in group 1, 35% were found to have high-grade tumor (Gleason score ≥ 7) compared with 26.7% in group 2 (P = 0.02). Total PSA and fPSA were strongly correlated with duration of opium use (r = -0.06, 95% CI, -0.04 to -0.08, P = 0.0001; and r = -0.05, 95% CI, -0.03 to -0.07, P = 0.0001, respectively). Opium consumption is independently and negatively associated with serum tPSA, fPSA, and % fPSA levels.

  9. Automatic Measurement of Low Level Contamination on Concrete Surfaces

    SciTech Connect

    Tachibana, M.; Itoh, H.; Shimada, T.; Yanagihara, S.

    2002-02-28

    Automatic measurement of radioactivity is necessary for considering cost effectiveness in final radiological survey of building structures in decommissioning nuclear facilities. The RAPID (radiation measuring pilot device for surface contamination) was developed to be applied to automatic measurement of low level contamination on concrete surfaces. The RAPID has a capability to measure contamination with detection limit of 0.14 Bq/cm2 for 60Co in 30 seconds of measurement time and its efficiency is evaluated to be 5 m2/h in a normal measurement option. It was confirmed that low level contamination on concrete surfaces could be surveyed by the RAPID efficiently compared with direct measurement by workers through its actual application.

  10. Bone Marrow Transplantation Results in Human Donor Blood Cells Acquiring and Displaying Mouse Recipient Class I MHC and CD45 Antigens on Their Surface

    PubMed Central

    Yamanaka, Nobuko; Wong, Christine J.; Gertsenstein, Marina; Casper, Robert F.; Nagy, Andras; Rogers, Ian M.

    2009-01-01

    Background Mouse models of human disease are invaluable for determining the differentiation ability and functional capacity of stem cells. The best example is bone marrow transplants for studies of hematopoietic stem cells. For organ studies, the interpretation of the data can be difficult as transdifferentiation, cell fusion or surface antigen transfer (trogocytosis) can be misinterpreted as differentiation. These events have not been investigated in hematopoietic stem cell transplant models. Methodology/Principal Findings In this study we investigated fusion and trogocytosis involving blood cells during bone marrow transplantation using a xenograft model. We report that using a standard SCID repopulating assay almost 100% of the human donor cells appear as hybrid blood cells containing both mouse and human surface antigens. Conclusion/Significance Hybrid cells are not the result of cell-cell fusion events but appear to be due to efficient surface antigen transfer, a process referred to as trogocytosis. Antigen transfer appears to be non-random and includes all donor cells regardless of sub-type. We also demonstrate that irradiation preconditioning enhances the frequency of hybrid cells and that trogocytosis is evident in non-blood cells in chimera mice. PMID:20046883

  11. Serological reactivity to the ring-infected erythrocyte surface antigen and circumsporozoite protein in gravid and nulligravid women infected with Plasmodium falciparum.

    PubMed

    Deloron, P; Steketee, R W; Campbell, G H; Peyron, F; Kaseje, D C; Brandling-Bennett, A D

    1989-01-01

    To investigate potential mechanisms for pregnancy-associated alterations in the immune response to malaria, we tested plasma samples from Plasmodium falciparum-infected nulligravida (42), primigravida (23) and multigravida (38) Kenyan women for reactivity to the ring-infected erythrocyte surface antigen (RESA) by a modified indirect fluorescent antibody assay and to synthetic peptides derived from amino acid sequences of RESA and the circumsporozoite (CS) protein of P. falciparum by an enzyme-linked immunosorbent assay. Reactivity to RESA showed the lowest titres in primigravid women, intermediate titres in nulligravid women and the highest titres in multigravid women (loge mean antibody = 3.28, 4.64, and 5.28, respectively, P less than 0.03), but was not associated with initial parasite density or response to chloroquine treatment. No relationship in antibody reactivity to the 3 synthetic peptides of the RESA molecule was observed by gravidity (0, 1, or greater than or equal to 2), age, initial parasite density or response to treatment. Levels of antibody to the synthetic peptides of the CS protein increased with age and were higher in gravid than in nulligravid women in the 15-19 year age group. The increased malaria prevalence and parasite density and the decreased response to antimalarial treatment in pregnant women is not explained by lower levels of antibody to RESA or CS protein during pregnancy.

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

    PubMed

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

    2008-11-05

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

  13. Naturally Occurring Surface Antigen Variants of Hepatitis B Virus in Tunisian Patients.

    PubMed

    Chaouch, Houda; Taffon, Stefania; Villano, Umbertina; Equestre, Michele; Bruni, Roberto; Belhadj, Manel; Hannachi, Naila; Aouni, Mahjoub; Letaief, Amel; Ciccaglione, Anna Rita

    2016-01-01

    In Tunisia, the prevalence of naturally occurring surface (S) gene variants of hepatitis B virus (HBV) has not been determined. In the present study, the prevalence of these variants was examined in terms of the clinical and viral state in a series of 99 Tunisian patients with HBV infection. The S genes were amplified and directly sequenced. Genotype D was predominant (98%), 40.4% isolates belonged to subgenotypes D7 and 1 to subgenotype D2. The most common subtype was ayw2 (95.9%). In total, 60.6% of the studied strains harbored S mutations. Several novel mutation patterns were detected. Interestingly, the presence of S mutations was significantly correlated with the D7 subgenotype, low HBV DNA and advancing age (≥35 years), and tended to be higher in liver cirrhosis than in chronic infection. The global prevalence of the major hydrophilic region variants was 12.1%, with substitution S143L/T as the most frequent (4%). Only 33.9% of S substitutions produced amino acid changes in the polymerase gene. In conclusion, a high prevalence of naturally occurring HBsAg variants was observed among Tunisian HBV carriers. Natural viral variability in a geographical region and duration of infection are among the major factors associated with the occurrence of S mutations.

  14. Bivalent kinetic binding model to surface plasmon resonance studies of antigen-antibody displacement reactions.

    PubMed

    Gelinsky-Wersing, Dagmar; Wersing, Wolfram; Pompe, Wolfgang

    2017-02-01

    Molecular and functional analysis of small molecule binding to protein can provoke insights into cellular signaling and regulatory systems as well as facilitate pharmaceutical drug discovery. In label free small molecule detection the displacement assay format can be applied. This is beneficial because displacement of high molecular weight receptors is detected instead of low molecular weight ligand as in classical binding analysis. Thus, detection limit is potentially lowered. Using the influenza haemagglutinin (HA) peptide binding to mono or bivalent anti-haemagglutinin peptide antibody displacement assay formats could be established. The exact time resolved analysis of binding and dissolution of ligand HA and anti-Haemagglutinin peptide antibody was achieved with surface plasmon resonance (SPR) spectroscopy. Mathematical models could be developed from kinetic equations of ligand binding to mono or bivalent antibodies. With this, an accurate simulation of the SPR results was reached. The simulation plot had to be exactly adjusted to the SPR results to determine all kinetic rate constants defining ligand and receptor binding kinetics. Large variations in receptor concentration gave almost identical rate constants in binding. It became obvious that rebinding is in any case not necessary to understand the binding kinetics of our model system HA/anti-HA. Maximum decline of SPR response could be used to determine ligand concentrations in analyte.

  15. WI-1, a novel 120-kilodalton surface protein on Blastomyces dermatitidis yeast cells, is a target antigen of cell-mediated immunity in human blastomycosis.

    PubMed Central

    Klein, B S; Sondel, P M; Jones, J M

    1992-01-01

    A large body of experimental data has demonstrated the central role of T cells in acquired resistance to the dimorphic fungus Blastomyces dermatitidis. We examined the human T-cell response to WI-1, a 120-kDa B. dermatitidis yeast cell surface protein recently shown to be an immunodominant antigen of the B-cell response in infected humans. Peripheral blood lymphocytes from 10 blastomycosis patients studied proliferated in response to WI-1 (mean, 19,431 cpm) and to the standard, crude cell wall antigen, Blastomyces alkali- and water-soluble antigen (B-ASWS) (mean, 19,131 cpm); lymphocytes from 10 histoplasmosis patients and 10 normal control subjects did not respond to WI-1. WI-1 stimulation of patient lymphocytes and rechallenge with WI-1 or B-ASWS showed that the antigens share immunodominant epitopes. Of 100 WI-1-responsive T-cell clones derived from peripheral blood, 10 were studied in detail to assess the phenotype, function, and ligands recognized. The clones exhibit the CD3+ CD4+ phenotype of helper T cells; 2 of 10 clones (and 21% of antigen-stimulated peripheral blood lymphocytes) use the V beta 8 T-cell receptor gene element to respond to WI-1. All the clones proliferate in response to both WI-1 and B-ASWS but not other fungal antigens, and some mediate potent cytolytic effects on WI-1- and B-ASWS-labeled targets. WI-1 recognition requires antigen processing and presentation of epitopes in association with HLA-DR (to noncytolytic clones) and HLA-DP (to cytolytic clones). From these findings, we conclude that CD4+ T cells with regulatory and cytolytic properties are involved in the development of acquired resistance of B. dermatitidis, that the cells are directed against WI-1, and that the manner of display of WI-1 peptide epitopes in conjunction with major histocompatibility complex class II may influence the profile of the immune response. PMID:1383148

  16. 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.

  17. Characterization of surface antigen protein 1 (SurA1) from Acinetobacter baumannii and its role in virulence and fitness.

    PubMed

    Liu, Dong; Liu, Zeng-Shan; Hu, Pan; Cai, Ling; Fu, Bao-Quan; Li, Yan-Song; Lu, Shi-Ying; Liu, Nan-Nan; Ma, Xiao-Long; Chi, Dan; Chang, Jiang; Shui, Yi-Ming; Li, Zhao-Hui; Ahmad, Waqas; Zhou, Yu; Ren, Hong-Lin

    2016-04-15

    Acinetobacter baumannii is a Gram-negative bacillus that causes nosocomial infections, such as bacteremia, pneumonia, and meningitis and urinary tract and wound infections. In the present study, the surface antigen protein 1 (SurA1) gene of A. baumannii strain CCGGD201101 was identified, cloned and expressed, and then its roles in fitness and virulence were investigated. Virulence was observed in the human lung cancer cell lines A549 and HEp-2 at one week after treatment with recombinant SurA1. One isogenic SurA1 knock-out strain, GR0015, which was derived from the A. baumannii strain CCGGD201101 isolated from diseased chicks in a previous study, highlighted the effect of SurA1 on fitness and growth. Its growth rate in LB broth and killing activity in human sera were significantly decreased compared with strain CCGGD201101. In the Galleria mellonella insect model, the isogenic SurA1 knock-out strain exhibited a lower survival rate and decreased dissemination. These results suggest that SurA1 plays an important role in the fitness and virulence of A. baumannii.

  18. Long term follow-up and outcome of liver transplantation from hepatitis B surface antigen positive donors

    PubMed Central

    Ballarin, Roberto; Cucchetti, Alessandro; Russo, Francesco Paolo; Magistri, Paolo; Cescon, Matteo; Cillo, Umberto; Burra, Patrizia; Pinna, Antonio Daniele; Di Benedetto, Fabrizio

    2017-01-01

    Liver transplant for hepatitis B virus (HBV) currently yields excellent outcomes: it allows to rescue patients with an HBV-related advanced liver disease, resulting in a demographical modification of the waiting list for liver transplant. In an age of patient-tailored treatments, in liver transplantation as well the aim is to offer the best suitable graft to the patient who can benefit from it, also expanding the criteria for organ acceptance and allocation. With the intent of developing strategies to increase the donor pool, we set-up a multicenter study involving 3 Liver Transplant Centers in Italy: patients undergoing liver transplantation between March 03, 2004, and May 21, 2010, were retrospectively evaluated. 1408 patients underwent liver transplantation during the study period, 28 (2%) received the graft from hepatitis B surface antigen positive (HBsAg)-positive deceased donors. The average follow-up after liver transplantation was 63.7 mo [range: 0.1-119.4; SD ± 35.8]. None Primary non-function, re-liver transplantation, early or late hepatic artery thrombosis occurred. The 1-, 3- and 5-year graft and patient survival resulted of 85.7%, 82.1%, 78.4%. Our results suggest that the use of HBsAg-positive donors liver grafts is feasible, since HBV can be controlled without affecting graft stability. However, the selection of grafts and the postoperative antiviral therapy should be managed appropriately.

  19. Purification and properties of a 75-kilodalton major protein, an immunodominant surface antigen, from the oral anaerobe Bacteroides gingivalis.

    PubMed Central

    Yoshimura, F; Watanabe, K; Takasawa, T; Kawanami, M; Kato, H

    1989-01-01

    A 75-kilodalton major protein (75K protein) was purified to homogeneity from the cell lysate fraction and the envelope of Bacteroides gingivalis 381. The 75K protein was originally present in the outer membrane or the outermost part of this organism as a large, stable complex with an apparent molecular weight of about 2,000,000. Heating at 80 degrees C and at higher temperatures in the presence of sodium dodecyl sulfate was needed to completely dissociate it to monomers. Amino acid analysis revealed that the 75K protein had about 50% nonpolar amino acids. Various strains of B. gingivalis but not other bacteria, including oral Bacteroides species tested, contained serologically related 75K proteins when tested in Western blotting (immunoblotting) analysis. The abundance and localization of the 75K protein in this organism suggest that it has the potential to participate in the host-parasite interaction in infection. The 75K protein was, indeed, strongly recognized in patients with adult periodontal diseases. Immunoblotting with sera from patients and with rabbit antisera generated by intravenous inoculations of whole B. gingivalis cells revealed that the 75K protein was an immunodominant antigen on the surface of B. gingivalis. Images PMID:2553610

  20. Primary structure of the merozoite surface antigen 1 of Plasmodium vivax reveals sequences conserved between different Plasmodium species.

    PubMed Central

    del Portillo, H A; Longacre, S; Khouri, E; David, P H

    1991-01-01

    Merozoite surface antigen 1 (MSA1) of several species of plasmodia has been shown to be a promising candidate for a vaccine directed against the asexual blood stages of malaria. We report the cloning and characterization of the MSA1 gene of the human malaria parasite Plasmodium vivax. This gene, which we call Pv200, encodes a polypeptide of 1726 amino acids and displays features described for MSA1 genes of other species, such as signal peptide and anchoring sequences, conserved cysteine residues, number of potential N-glycosylation sites, and repeats consisting here of 23 glutamine residues in a row. When the nucleotide and deduced amino acid sequences of the MSA1 of P. vivax are compared to those of another human malaria parasite, Plasmodium falciparum, and to those of the rodent parasite Plasmodium yoelii, 10 regions of high amino acid similarity are observed despite the very different dG + dC contents of the corresponding genes. All of the interspecies conserved regions reside within the conserved or semiconserved blocks delimited by the sequences of different alleles of the MSA1 gene of P. falciparum. Images PMID:2023952

  1. [Development of a new hydrophobic interaction chromatography absorbent and its application to the purification of recombinant hepatitis B surface antigen].

    PubMed

    Wang, Yang-Mu; Bi, Jing-Xiu; Zhao, Lan; Zhou, Wei-Bin; Li, Yan; Huang, Yong-Dong; Zhang, Yan; Lin, Hai; Su, Zhi-Guo

    2006-03-01

    A new hydrophobic absorbent based on homemade highly cross-linked agarose beads was synthesized by immobilizing butyl derivative onto the matrix linkage. The density of ligand was controlled by adjusting the concentration of butanethiol and the synthesis route was optimized by evaluating the purification efficiency of recombinant Hepatitis B surface antigen (HBsAg) expressed by Chinese hamster ovary (CHO) cell line. A high performance absorbent was finally screened out with up to 80% of HBsAg recovery and purification-fold (PF) about 20. Furthermore, the column pressure was about 0.06 MPa under the flow rate of 500cm/h, and no leaked butyl were detected after exposing the gel in common buffers, chaotropic agents, high concentrations of denaturing agents such as guanidine hydrochloride, urea and polar organic solvents. These results demonstrated that the absorbent have high physico-chemical stability, so it was available for the downstream process. Finally, after scaled up to 2L wet gel/batch, the absorbent was applied to the integration of three-step chromatography and obtained the purified CHO-HBsAg with 95% purity by SDS-PAGE and HPLC, which meet the requirements of SFDA. The purification efficiency and the reproducible ability of the absorbents were also evaluated from batch-to-batch. The results demonstrated that the absorbent met the requirement of scalable, reproducible, economic effect as well. This absorbent is a promising alternative exported HIC gel for wildly being used in Chinese pharmaceutical industries.

  2. Europium nanoparticle-based simple to perform dry-reagent immunoassay for the detection of hepatitis B surface antigen.

    PubMed

    Talha, Sheikh M; Salminen, Teppo; Juntunen, Etvi; Spangar, Anni; Gurramkonda, Chandrasekhar; Vuorinen, Tytti; Khanna, Navin; Pettersson, Kim

    2016-03-01

    Hepatitis B infection, caused by hepatitis B virus (HBV), presents a huge global health burden. Serological diagnosis of HBV mainly relies on the detection of hepatitis B surface antigen (HBsAg). Although there are high sensitivity commercial HBsAg enzyme immunoassays (EIAs) available, many low-resource laboratories lacking trained technicians continue to use rapid point-of-care assays with low sensitivities for HBsAg detection, due to their simplicity to operate. We developed a time-resolved fluorometric dry-reagent HBsAg immunoassay which meets the detection limit of high sensitivity EIAs but is simple to operate. To develop the assay, anti-HBsAg monoclonal antibody coated on europium nanoparticles was dried atop of biotinylated anti-HBsAg polyclonal antibody immobilized on streptavidin-coated microtiter wells. To test a sample in dry-reagent assay, serum sample and assay buffer were added to the wells, incubated, washed and europium signals were measured. The assay showed a detection limit of 0.25 ng/ml using HBsAg spiked in serum sample. When evaluated with 24 HBV positive and 37 negative serum samples, assay showed 100% sensitivity and specificity. Assay wells are stable for at least 26 weeks when stored at 4°C, and can tolerate elevated temperatures of up to 35°C for two weeks. The developed assay has high potential to be used in low-resource laboratories.

  3. Structural characterization of the N-glycans of a recombinant hepatitis B surface antigen derived from yeast

    SciTech Connect

    Ip, C.C.Y.; Miller, W.J.; Kubek, D.J. ); Strang, A.M.; van Halbeek, H. ); Piesecki, S.J.; Alhadeff, J.A. )

    1992-01-14

    The N-glycans of purified recombinant middle surface protein (preS2+S) from hepatitis B virus, a candidate vaccine antigen expressed in a mnn9 mutant strain of Saccharomyces cerevisiae, have been characterized structurally. The glycans were released by N-glycanase treatment, isolated by size-exclusion chromatography on Sephadex G-50 and Bio-Gel P-4 columns, and analyzed by 500-MHz {sup 1}H NMR spectroscopy and fast atom bombardment mass spectrometry. The mixture of oligosaccharides was fractionated by HPLC, the major subfractions were isolated, and their carbohydrate compositions were determined by high-pH anion-exchange chromatography with pulsed amperometric detection. The combined results suggest that high-mannose oligosaccharides account for all the N-glycans released from preS2+S: structures include Man{sub 7}GlcNAc{sub 2}, Man{sub 8}GlcNAc{sub 2} isomers in the ratios of 3:6:1. Approximately 80% of the oligosaccharides contain the C2, C6-branched trimannosyl structural element typical of yeast high-mannose oligosaccharides but not usually found in high-mannose oligosaccharides in animal glycoproteins.

  4. Comprehensive Analysis of Pathogen-specific Antibody Response in Vivo Based on an Antigen Library Displayed on Surface of Yeast*

    PubMed Central

    Zuo, Teng; Shi, Xuanling; Liu, Zhonghua; Guo, Linlin; Zhao, Qing; Guan, Tianxia; Pan, Xianming; Jia, Na; Cao, Wuchun; Zhou, Boping; Goldin, Mark; Zhang, Linqi

    2011-01-01

    Host antibody response is a crucial defense against pathogenic infection. Here, we report a novel technique allowing quantitative measurement of polyclonal antibody response in vivo. This involves expression of a combinatorial library of target proteins from a candidate pathogen on the surface of yeast Saccharomyces cerevisiae. After mixing with serum/plasma from infected or immunized subjects, positive yeast clones were isolated via fluorescence-activated cell sorting (FACS). Using this technique, we have studied mouse immunized serum with recombinant hemagglutinin (HA) protein from a human influenza H5N1 strain (A/Anhui/1/2005) and convalescent plasma from an infected human in China. Our technique has identified novel antigenic domains targeted by serum/plasma and allowed calculation of the relative proportion of the antibody response against each domain. We believe such systematic measurement of an antibody response is unprecedented, and applying this method to different pathogens will improve understanding of protective immunity and guide development of vaccines and therapeutics. PMID:21795672

  5. Hepatitis B surface antigen vector delivers protective cytotoxic T-lymphocyte responses to disease-relevant foreign epitopes.

    PubMed

    Woo, Wai-Ping; Doan, Tracy; Herd, Karen A; Netter, Hans-Jürgen; Tindle, Robert W

    2006-04-01

    Although hepatitis B surface antigen (HBsAg) per se is highly immunogenic, its use as a vector for the delivery of foreign cytotoxic T-lymphocyte (CTL) epitopes has met with little success because of constraints on HBsAg stability and secretion imposed by the insertion of foreign sequence into critical hydrophobic/amphipathic regions. Using a strategy entailing deletion of DNA encoding HBsAg-specific CTL epitopes and replacement with DNA encoding foreign CTL epitopes, we have derived chimeric HBsAg DNA immunogens which elicited effector and memory CTL responses in vitro, and pathogen- and tumor-protective responses in vivo, when the chimeric HBsAg DNAs were used to immunize mice. We further show that HBsAg DNA recombinant for both respiratory syncytial virus and human papillomavirus CTL epitopes elicited simultaneous responses to both pathogens. These data demonstrate the efficacy of HBsAg DNA as a vector for the delivery of disease-relevant protective CTL responses. They also suggest the applicability of the approach of deriving chimeric HBsAg DNA immunogens simultaneously encoding protective CTL epitopes for multiple diseases. The DNAs we tested formed chimeric HBsAg virus-like particles (VLPs). Thus, our results have implications for the development of vaccination strategies using either chimeric HBsAg DNA or VLP vaccines. HBsAg is the globally administered vaccine for hepatitis B virus infection, inviting its usage as a vector for the delivery of immunogens from other diseases.

  6. Quantification of hepatitis B surface antigen: a new concept for the management of chronic hepatitis B.

    PubMed

    Moucari, Rami; Marcellin, Patrick

    2011-01-01

    HBsAg is a very important clinical test that might not only indicate active hepatitis B virus (HBV) infection but might also be used to predict clinical and treatment outcome. Clearance of HBsAg in patients with chronic HBV infection is associated with a much better clinical outcome, although surveillance for early detection of hepatocellular carcinoma (HCC) should continue. HBV DNA quantification is currently used for selecting candidates for therapy, monitoring response to therapy and detecting the emergence of drug resistance. Assays for HBsAg quantification are less expensive than HBV DNA and fully automated with a high throughput capacity. HBsAg titering may be a useful tool to manage patients with chronic HBV, to more clearly define which patients may, and more importantly, may not, benefit from treatment. Baseline and on-treatment HBsAg quantification may help to refine future treatment algorithms for both immune-modulator therapy and nucleos(t)ide analogues. Both HBV markers provide complementary information on the status of HBV infection. However, the relevance of serum HBsAg levels and its use as a reliable replacement for both covalently closed circular DNA and HBV DNA remain unclear.

  7. Functional characterization of human CD34+ cells that express low or high levels of the membrane antigen CD111 (nectin 1).

    PubMed

    Belaaloui, G; Imbert, A-M; Bardin, F; Tonnelle, C; Dubreuil, P; Lopez, M; Chabannon, C

    2003-06-01

    Nectins are recently described adhesion molecules that are widely expressed on many tissues, including the hematopoietic tissue. Nectin 1 (CD111) is expressed on a higher proportion of mobilized peripheral blood (mPB) than cord blood (CB) CD34+ cells, and of CD34+/CD38+ cells when compared with CD34+/CD38- cells. We studied functional properties of human CB and mPB CD34+ cells that express low or high levels of CD111. CD34+/CD111(dim) cells contain a higher proportion of cells in G0/G1 phase than CD34+/CD111(bright) cells. CD34+/CD111(bright) cells contain more erythroid progenitors: CFU-E, than their counterparts, which on the opposite contain more HPP-CFC. Limiting dilution analyses demonstrate a higher frequency of immature progenitors: cobblestone-area colony-forming cells, CD34+/CD111(dim) than in CD34+/CD111(bright) cells. In vitro differentiation assays demonstrate a higher frequency of B-, T- and dendritic-cell precursors, but less NK-cell precursors in CD34+/CD111(dim) cells. Evaluation of engraftment in NOD-SCID mice shows that SCID repopulating cells are more frequent among mPB CD34+/CD111(dim) cells. Liquid culture of CD34+/CD111(dim) cells with erythropoietin shows that CD111 expression increases simultaneously with CD36, following CD71 and before glycophorin A expression. In conclusion, immature human hematopoietic progenitors express low levels of CD111 on their surface. During erythroid differentiation CD34+ cells acquire higher levels of the CD111 antigen.

  8. Interface Surface Area Tracking for the Conservative Level Set Method

    NASA Astrophysics Data System (ADS)

    Firehammer, Stephanie; Desjardins, Olivier

    2015-11-01

    One key question in liquid-gas flows is how to model the interface between phases in a way that is mass, momentum, and energy conserving. The accurate conservative level set (ACLS) method of Desjardins et al. provides a tool for tracking a liquid-gas interface with minimal mass conservation issues; however, it does not explicitly compute the interface surface area and thus nothing can be said a priori about the balance between kinetic energy and surface energy. This work examines an equation for the transport of interface surface area density, which can be written in terms of the gradient of the volume fraction. Furthermore this presentation will outline a numerical method for jointly transporting a conservative level set and surface area density. Finally, we will explore oppportunities for energy conservation via the accurate exchange of energy between the flow field and the interface through surface tension, with test cases to show the results of our extended ACLS method. Funding from the National Science Foundation is gratefully acknowledged.

  9. Two Independent Regions of Simian Virus 40 T Antigen Increase CBP/p300 Levels, Alter Patterns of Cellular Histone Acetylation, and Immortalize Primary Cells

    PubMed Central

    Sáenz Robles, Maria Teresa; Shivalila, Chikdu; Wano, Jeremy; Sorrells, Shelly; Roos, Alison

    2013-01-01

    Simian virus 40 (SV40) large T antigen (SVT) interferes with normal cell regulation and thus has been used to identify cellular components controlling proliferation and homeostasis. We have previously shown that SVT-mediated transformation requires interaction with the histone acetyltransferases (HATs) CBP/p300 and now report that the ectopic expression of SVT in several cell types in vivo and in vitro results in a significant increase in the steady-state levels of CBP/p300. Furthermore, SVT-expressing cells contain higher levels of acetylated CBP/p300, a modification that has been linked to increased HAT activity. Concomitantly, the acetylation levels of histone residues H3K56 and H4K12 are markedly increased in SVT-expressing cells. Other polyomavirus-encoded large T antigens also increase the levels of CBP/p300 and sustain a rise in the acetylation levels of H3K56 and H4K12. SVT does not affect the transcription of CBP/p300, but rather, alters their overall levels through increasing the loading of CBP/p300 mRNAs onto polysomes. Two distinct regions within SVT, one located in the amino terminus and one in the carboxy terminus, can independently alter both the levels of CBP/p300 and the loading of CBP/p300 transcripts onto polysomes. Within the amino-terminal fragment, a functional J domain is necessary for increasing CBP/p300 and specific histone acetylation levels, as well as for immortalizing primary cells. These studies uncover the action of polyomavirus T antigens on cellular CBP/p300 and suggest that additional mechanisms are used by T antigens to induce cell immortalization and transformation. PMID:24089570

  10. Development of a Highly Sensitive Bioluminescent Enzyme Immunoassay for Hepatitis B Virus Surface Antigen Capable of Detecting Divergent Mutants

    PubMed Central

    Takehara, Shizuka; Takahashi, Masaharu

    2013-01-01

    Hepatitis B virus (HBV) infections are sometimes overlooked when using commercial kits to measure hepatitis B virus surface antigen (HBsAg) due to their low sensitivities and reactivities to mutant strains of various genotypes. We developed an ultrasensitive bioluminescent enzyme immunoassay (BLEIA) for HBsAg using firefly luciferase, which is adaptable to a variety of HBsAg mutants, by combining four monoclonal antibodies with a polyclonal antibody against HBsAg. The measurement of seroconversion panels showed trace amounts of HBsAg during the early infection phase by the BLEIA because of its high sensitivity of 5 mIU/ml. The BLEIA detected HBsAg as early as did PCR in five of seven series and from 2.1 to 9.4 days earlier than commercial immunoassay methods. During the late infection phase, the BLEIA successfully detected HBsAg even 40 days after the disappearance of HBV DNA and the emergence of antibodies against HBsAg. The HBsAg BLEIA successfully detected all 13 recombinant HBsAg and 45 types of HBsAg mutants with various mutations within amino acids 90 to 164 in the S gene product. Some specimens had higher values determined by the BLEIA than those by a commercial chemiluminescent immunoassay; this suggests that such discrepancies were caused by the dissociation of preS1/preS2 peptides from the particle surface. With its highly sensitive detection of low-titer HBsAg, including various mutants, the HBsAg BLEIA is considered to be useful for the early diagnosis and prevention of HBV infection because of the shorter window of infection prior to detection, which facilitates early prediction of recurrence in HBV-infected individuals. PMID:23761660

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  13. Successful Antiparasitic Treatment for Cysticercosis Is Associated with a Fast and Marked Reduction of Circulating Antigen Levels in a Naturally Infected Pig Model

    PubMed Central

    Gonzalez, Armando E.; Bustos, Javier A.; Garcia, Hector H.; Rodriguez, Silvia; Zimic, Mirko; Castillo, Yesenia; Praet, Nicolas; Gabriël, Sarah; Gilman, Robert H.

    2015-01-01

    Taenia solium cysticercosis is a common parasitic infection of humans and pigs. We evaluated the posttreatment evolution of circulating parasite-specific antigen titers in 693 consecutive blood samples from 50 naturally infected cysticercotic pigs, which received different regimes of antiparasitic drugs (N = 39, 7 groups), prednisone (N = 5), or controls (N = 6). Samples were collected from baseline to week 10 after treatment, when pigs were euthanized and carefully dissected at necropsy. Antigen levels decreased proportionally to the efficacy of treatment and correlated with the remaining viable cysts at necropsy (Pearson's p = 0.67, P = 0.000). A decrease of 5 times in antigen levels (logarithmic scale) compared with baseline was found in 20/26 pigs free of cysts at necropsy, compared with 1/24 of those who had persisting viable cysts (odds ratio [OR] = 76.7, 95% confidence interval [CI] = 8.1–3308.6, P < 0.001). Antigen monitoring reflects the course of infection in the pig. If a similar correlation exists in infected humans, this assay may provide a minimally invasive and easy monitoring assay to assess disease evolution and efficacy of antiparasitic treatment in human neurocysticercosis. PMID:26392159

  14. Successful Antiparasitic Treatment for Cysticercosis is Associated with a Fast and Marked Reduction of Circulating Antigen Levels in a Naturally Infected Pig Model.

    PubMed

    Gonzalez, Armando E; Bustos, Javier A; Garcia, Hector H; Rodriguez, Silvia; Zimic, Mirko; Castillo, Yesenia; Praet, Nicolas; Gabriël, Sarah; Gilman, Robert H; Dorny, Pierre

    2015-12-01

    Taenia solium cysticercosis is a common parasitic infection of humans and pigs. We evaluated the posttreatment evolution of circulating parasite-specific antigen titers in 693 consecutive blood samples from 50 naturally infected cysticercotic pigs, which received different regimes of antiparasitic drugs (N = 39, 7 groups), prednisone (N = 5), or controls (N = 6). Samples were collected from baseline to week 10 after treatment, when pigs were euthanized and carefully dissected at necropsy. Antigen levels decreased proportionally to the efficacy of treatment and correlated with the remaining viable cysts at necropsy (Pearson's p = 0.67, P = 0.000). A decrease of 5 times in antigen levels (logarithmic scale) compared with baseline was found in 20/26 pigs free of cysts at necropsy, compared with 1/24 of those who had persisting viable cysts (odds ratio [OR] = 76.7, 95% confidence interval [CI] = 8.1-3308.6, P < 0.001). Antigen monitoring reflects the course of infection in the pig. If a similar correlation exists in infected humans, this assay may provide a minimally invasive and easy monitoring assay to assess disease evolution and efficacy of antiparasitic treatment in human neurocysticercosis.

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

    NASA Astrophysics Data System (ADS)

    Stefan, V. Alexander

    2014-03-01

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

  16. Outcome After Conformal Salvage Radiotherapy in Patients With Rising Prostate-Specific Antigen Levels After Radical Prostatectomy

    SciTech Connect

    Geinitz, Hans; Riegel, Martina G.; Thamm, Reinhard; Astner, Sabrina T.; Lewerenz, Carolin; Zimmermann, Frank; Molls, Michael; Nieder, Carsten

    2012-04-01

    Purpose: This study attempts to improve our understanding of the role of salvage radiotherapy (SRT) in patients with prostate-specific antigen (PSA) relapse after radical prostatectomy with regard to biochemical control, rate of distant metastasis, and survival. Methods and Materials: We performed a retrospective analysis of 96 men treated with conformal prostate bed SRT (median, 64.8 Gy) at a single institution (median follow-up, 70 months). The majority had intermediate- or high-risk prostate cancer. Fifty-four percent underwent a resection with positive margins (R1 resection). The median time interval between surgery and SRT was 22 months. Results: After SRT, 66% of patients reached a PSA nadir of less than 0.2 ng/mL. However, the 5-year biochemical no evidence of disease rate was 35%. Seminal vesicle involvement was predictive for a significantly lower biochemical no evidence of disease rate. All patients with a preoperative PSA level greater than 50 ng/mL relapsed biochemically within 2 years. The 5-year distant metastasis rate was 18%, the 5-year prostate cancer-specific survival rate was 90%, and the 5-year overall survival rate was 88%. Significantly more distant metastases developed in patients with a PSA nadir greater than 0.05 ng/mL after SRT, and they had significantly inferior prostate cancer-specific and overall survival rates. Resection status (R1 vs. R0) was not predictive for any of the endpoints. Conclusions: Men with postoperative PSA relapse can undergo salvage treatment by prostate bed radiotherapy, but durable PSA control is maintained only in about one-third of the patients. Despite a high biochemical failure rate after SRT, prostate cancer-specific survival does not decrease rapidly.

  17. Functions of Cell Surface-Anchored Antigen I/II Family and Hsa Polypeptides in Interactions of Streptococcus gordonii with Host Receptors

    PubMed Central

    Jakubovics, Nicholas S.; Kerrigan, Steven W.; Nobbs, Angela H.; Strömberg, Nicklas; van Dolleweerd, Craig J.; Cox, Dermot M.; Kelly, Charles G.; Jenkinson, Howard F.

    2005-01-01

    Streptococcus gordonii colonizes multiple sites within the human oral cavity. This colonization depends upon the initial interactions of streptococcal adhesins with host receptors. The adhesins that bind salivary agglutinin glycoprotein (gp340) and human cell surface receptors include the antigen I/II (AgI/II) family polypeptides SspA and SspB and a sialic acid-binding surface protein designated Hsa or GspB. In this study we determined the relative functions of the AgI/II polypeptides and Hsa in interactions of S. gordonii DL1 (Challis) with host receptors. For an isogenic mutant with the sspA and sspB genes deleted the levels of adhesion to surface-immobilized gp340 were reduced 40%, while deletion of the hsa gene alone resulted in >80% inhibition of bacterial cell adhesion to gp340. Adhesion of S. gordonii DL1 cells to gp340 was sialidase sensitive, verifying that Hsa has a major role in mediating sialic acid-specific adhesion to gp340. Conversely, aggregation of S. gordonii cells by fluid-phase gp340 was not affected by deletion of hsa but was eliminated by deletion of the sspA and sspB genes. Deletion of the AgI/II polypeptide genes had no measurable effect on hsa mRNA levels or Hsa surface protein expression, and deletion of hsa did not affect AgI/II polypeptide expression. Further analysis of mutant phenotypes showed that the Hsa and AgI/II proteins mediated adhesion of S. gordonii DL1 to human HEp-2 epithelial cells. Hsa was also a principal streptococcal cell surface component promoting adhesion of human platelets to immobilized streptococci, but Hsa and AgI/II polypeptides acted in concert in mediating streptococcal cell-platelet aggregation. The results suggest that Hsa directs primary adhesion events for S. gordonii DL1 (Challis) with immobilized gp340, epithelial cells, and platelets. AgI/II polypeptides direct gp340-mediated aggregation, facilitate multimodal interactions necessary for platelet aggregation, and modulate S. gordonii-host engagements

  18. Sea level: measuring the bounding surfaces of the ocean

    PubMed Central

    Tamisiea, Mark E.; Hughes, Chris W.; Williams, Simon D. P.; Bingley, Richard M.

    2014-01-01

    The practical need to understand sea level along the coasts, such as for safe navigation given the spatially variable tides, has resulted in tide gauge observations having the distinction of being some of the longest instrumental ocean records. Archives of these records, along with geological constraints, have allowed us to identify the century-scale rise in global sea level. Additional data sources, particularly satellite altimetry missions, have helped us to better identify the rates and causes of sea-level rise and the mechanisms leading to spatial variability in the observed rates. Analysis of all of the data reveals the need for long-term and stable observation systems to assess accurately the regional changes as well as to improve our ability to estimate future changes in sea level. While information from many scientific disciplines is needed to understand sea-level change, this review focuses on contributions from geodesy and the role of the ocean's bounding surfaces: the sea surface and the Earth's crust. PMID:25157196

  19. Sea level: measuring the bounding surfaces of the ocean.

    PubMed

    Tamisiea, Mark E; Hughes, Chris W; Williams, Simon D P; Bingley, Richard M

    2014-09-28

    The practical need to understand sea level along the coasts, such as for safe navigation given the spatially variable tides, has resulted in tide gauge observations having the distinction of being some of the longest instrumental ocean records. Archives of these records, along with geological constraints, have allowed us to identify the century-scale rise in global sea level. Additional data sources, particularly satellite altimetry missions, have helped us to better identify the rates and causes of sea-level rise and the mechanisms leading to spatial variability in the observed rates. Analysis of all of the data reveals the need for long-term and stable observation systems to assess accurately the regional changes as well as to improve our ability to estimate future changes in sea level. While information from many scientific disciplines is needed to understand sea-level change, this review focuses on contributions from geodesy and the role of the ocean's bounding surfaces: the sea surface and the Earth's crust.

  20. Combined Linkage and Association Studies Show that HLA Class II Variants Control Levels of Antibodies against Epstein-Barr Virus Antigens

    PubMed Central

    Cobat, Aurélie; Guergnon, Julien; Brice, Pauline; Fermé, Christophe; Carde, Patrice; Hermine, Olivier; Pendeven, Catherine Le-; Amiel, Corinne; Taoufik, Yassine; Alcaïs, Alexandre; Theodorou, Ioannis; Besson, Caroline; Abel, Laurent

    2014-01-01

    Over 95% of the adult population worldwide is infected with Epstein-Barr virus (EBV). EBV infection is associated with the development of several cancers, including Hodgkin lymphoma (HL). Elevated levels of anti-EBV antibodies have been associated with increased risk of HL. There is growing evidence that genetic factors control the levels of antibodies against EBV antigens. Here, we conducted linkage and association studies to search for genetic factors influencing either anti-viral capsid antigen (VCA) or anti-Epstein Barr nuclear antigen-1 (EBNA-1) IgG levels in a unique cohort of 424 individuals of European origin from 119 French families recruited through a Hodgkin lymphoma (HL) patient. No major locus controlling anti-VCA antibody levels was identified. However, we found that the HLA region influenced anti-EBNA-1 IgG titers. Refined association studies in this region identified a cluster of HLA class II variants associated with anti-EBNA-1 IgG titers (e.g. p = 5×10–5 for rs9268403). The major allele of rs9268403 conferring a predisposition to high anti-EBNA-1 antibody levels was also associated with an increased risk of HL (p = 0.02). In summary, this study shows that HLA class II variants influenced anti-EBNA-1 IgG titers in a European population. It further shows the role of the same variants in the risk of HL. PMID:25025336

  1. Context affects lightness at the level of surfaces.

    PubMed

    Maertens, Marianne; Wichmann, Felix A; Shapley, Robert

    2015-01-14

    Visual perception of object attributes such as surface lightness is crucial for successful interaction with the environment. How the visual system assigns lightness to image regions is not yet understood. It has been shown that the context in which a surface is embedded influences its perceived lightness, but whether that influence involves predominantly low-, mid-, or high-level visual mechanisms has not been resolved. To answer this question, we measured whether perceptual attributes of target image regions affected their perceived lightness when they were placed in different contexts. We varied the sharpness of the edge while keeping total target flux fixed. Targets with a sharp edge were consistent with the perceptual interpretation of a surface, and in that case, observers perceived significant brightening or darkening of the target. Targets with blurred edges rather appeared to be spotlights instead of surfaces; for targets with blurred edges, there was much less of a contextual effect on target lightness. The results indicate that the effect of context on the lightness of an image region is not fixed but is strongly affected by image manipulations that modify the perceptual attributes of the target, implying that a mid-level scene interpretation affects lightness perception.

  2. Expression of the surface antigen 4F2hc affects system-L-like neutral-amino-acid-transport activity in mammalian cells.

    PubMed Central

    Bröer, S; Bröer, A; Hamprecht, B

    1997-01-01

    Mammalian cells possess a variety of amino acid-transport systems with overlapping substrate specificity. System L is one of the major amino acid-transport systems of non-epithelial cells. By expression cloning we have recently demonstrated that the surface antigen 4F2hc (CD98) is a necessary component for expression of system-L-like amino acid-transport activity in C6-BU-1 rat glioma cells [Bröer, Bröer and Hamprecht (1995) Biochem. J. 312, 863-870]. 4F2hc mRNA was detected in CHO cells, COS cells, activated lymphocytes isolated from mouse spleen and primary cultures of astrocytes. In all these cell types, Na+-independent isoleucine transport was mediated by system L. No contribution of system y+L to isoleucine or arginine transport was detected in C6-BU-1 cells. In lymphocytes, both system-L-like amino acid-transport activity and 4F2hc mRNA levels increased after treatment with phorbol ester plus ionomycin. Antisense oligonucleotides caused modest inhibition of Na+-independent isoleucine transport in C6-BU-1 cells and primary cultures of astroglial cells, whereas arginine transport was unaffected. Overexpression of 4F2hc cDNA in CHO cells resulted in an increase in Na+-independent isoleucine transport. PMID:9182715

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

    PubMed

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

    2016-09-13

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

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

    PubMed Central

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

    2016-01-01

    Circulating tumor cells (CTCs) have emerged as promising tools for noninvasive cancer detection and prognosis. Most conventional approaches for capturing CTCs use an EpCAM-based enrichment strategy, which does not work well in cancers that show low or no expression of EpCAM, such as renal cell carcinoma (RCC). In this study, we developed a new set of cell surface markers including CA9 and CD147 as alternative CTC-capture antigens specifically designed for RCC patients. We showed that the expression of both CA9 and CD147 was prevalent in a RCC patient cohort (n=70) by immunohistochemical analysis, with both molecules in combination covering 97.1% of cases. The NanoVelcro platform combined with CA9-/CD147-capture antibodies demonstrated significantly higher efficiency for capturing both CTC-mimicking renal cancer cells and RCC CTCs in peripheral blood, compared to the conventional EpCAM-based method. Using immunofluorescence cytological validation at the single-cell level, we were able to identify bona fide CTCs in RCC patient blood following the well-accepted criteria in our CTC-capture system. We further demonstrated a significant association of CTC numbers as well as the CTC expression status of Vimentin, a mesenchymal marker, with disease progression, including pathologic features and clinical staging. These results provide new insights into developing novel, effective targets/approaches for capturing CTCs, making CTCs a valuable tool for improved cancer detection, prognosis and treatment in RCC. PMID:27494883

  5. Interaction of Plasmodium vivax Tryptophan-rich Antigen PvTRAg38 with Band 3 on Human Erythrocyte Surface Facilitates Parasite Growth.

    PubMed

    Alam, Mohd Shoeb; Choudhary, Vandana; Zeeshan, Mohammad; Tyagi, Rupesh K; Rathore, Sumit; Sharma, Yagya D

    2015-08-14

    Plasmodium tryptophan-rich proteins are involved in host-parasite interaction and thus potential drug/vaccine targets. Recently, we have described several P. vivax tryptophan-rich antigens (PvTRAgs), including merozoite expressed PvTRAg38, from this noncultivable human malaria parasite. PvTRAg38 is highly immunogenic in humans and binds to host erythrocytes, and this binding is inhibited by the patient sera. This binding is also affected if host erythrocytes were pretreated with chymotrypsin. Here, Band 3 has been identified as the chymotrypsin-sensitive erythrocyte receptor for this parasite protein. Interaction of PvTRAg38 with Band 3 has been mapped to its three different ectodomains (loops 1, 3, and 6) exposed at the surface of the erythrocyte. The binding region of PvTRAg38 to Band3 has been mapped to its sequence, KWVQWKNDKIRSWLSSEW, present at amino acid positions 197-214. The recombinant PvTRAg38 was able to inhibit the parasite growth in in vitro Plasmodium falciparum culture probably by competing with the ligand(s) of this heterologous parasite for the erythrocyte Band 3 receptor. In conclusion, the host-parasite interaction at the molecular level is much more complicated than known so far and should be considered during the development of anti-malarial therapeutics.

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

    PubMed

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

    1990-08-01

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

  7. The effect of polarity and surface states on the Fermi level at III-nitride surfaces

    SciTech Connect

    Reddy, P; Bryan, I; Bryan, Z; Guo, W; Hussey, L; Collazo, R; Sitar, Z

    2014-09-28

    Surface states and their influence on the Fermi level at the surface of GaN and AlN are studied using x-ray photoelectron spectroscopy (XPS). The effect of polarity on surface electronic properties was studied. Accurate modeling of the valence band edge and comparison with XPS data revealed the presence of donor surface states at 1.4 eV and acceptor states at energies > 2.7 eV from the valence band in GaN. Al polar AlN showed acceptor states at energies > 3.3 eV. Density of acceptor surface states was estimated to be between 10(13) and 10(14) eV(-1) cm(-2) in both GaN and AlN. The shift in charge neutrality levels and barrier heights due to polarity and the density of surface states on AlN and GaN were estimated from XPS measurements. Theoretical modeling and comparison with XPS data implied full compensation of spontaneous polarization charge by charged surface states. Barrier height measurements also reveal a dependence on polarity with phi(metal-polar)>phi(non-polar)>phi(nitrogen-polar) suggesting that the N-polar surface is the most suitable for Ohmic contacts. (C) 2014 AIP Publishing LLC.

  8. Human methicillin-sensitive Staphylococcus aureus biofilms: potential associations with antibiotic resistance persistence and surface polysaccharide antigens.

    PubMed

    Babra, Charlene; Tiwari, Jully; Costantino, Paul; Sunagar, Raju; Isloor, Shrikrishna; Hegde, Nagendra; Mukkur, Trilochan

    2014-07-01

    The development of persistent antibiotic resistance by human methicillin-sensitive Staphylococcus aureus (MSSA) strains and substantial association with poly-N-acetyl glucosamine (PNAG) in biofilms is reported in this investigation. Sixteen of 31 MSSA strains under study were found to have developed resistance to one or more antibiotics, with four strains, two of which did not produce biofilms, showing resistance to cefoxitin, undetectable by mecA amplification. Antibiotic resistance displayed by 13/14 biofilm-forming S. aureus isolates remained persistent for 4 weeks prior to reverting back to the original antibiotic susceptibility, prompting a suggestion of determining antibiograms for clinical S. aureus isolates subcultured from biofilms developed in vitro as well as planktonic subcultures prepared from the site of infection. While there was correlation of antibiotic resistance with biofilm formation confirming previous reports, this is the first time that persistence of the biofilm-associated antibiotic resistance by S. aureus as planktonic cells is reported. Among the two methods used for assessment of biofilm formation, the tissue culture plate (TCP) method revealed that almost all strains were strong or moderate biofilm producers whereas only 19/31 strains were biofilm producers using the Congo Red agar (CRA) method indicating the superiority of the TCP method in detecting biofilm producers. We also observed no association between biofilm formation and major capsule types. However, substantial, although not absolute, association of biofilm formation with PNAG was observed, warranting continued identification of additional surface-associated polysaccharide and/or protein antigens associated with biofilm formation for development of an effective vaccine against S. aureus infections regardless of capsular phenotype.

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

    PubMed

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

    2015-03-01

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

  10. Hepatocellular carcinoma. A study of 50 autopsy cases with detection of hepatitis B surface antigen in fixed tissues.

    PubMed

    Perez-Barrios, A; Colina-Ruizdelgado, F; Gallego, I; Martinez-Tello, F J

    1983-03-01

    Fifty patients who died of hepatocellular carcinoma (HCC) were autopsied at the Ciudad Sanitaria "1 degree de Octubre" and the Hospital de la Cruz Roja (Madrid) from 1974 to 1980. Formalin fixed paraffin-embedded autopsy tissue of liver and tumor from the 50 HCC and liver tissue from 50 liver cirrhosis (LC) and from 50 autopsy of non cirrhotic control cases were examined for the presence of cytoplasmic hepatitis B surface antigen (HBsAg). The study was carried out using orcein staining, immunoperoxidase technique (IP) and indirect immunofluorescence (IF). In livers with HCC the HBsAg was detected in the cytoplasm of the hepatocytes in 10 cases (20%) with the orcein staining and in 11 (22%) with the IP and IF techniques. In one case (2%) HBsAg was found in the cytoplasm of tumor cells with the three methods--In four cases (8%) of LC and 2 (4%) control cases cytoplasmic positive cells were found. In 41 patients with HCC HBsAg was studied in the serum by radio-immunoassay (RIA) (13 cases) and immunodiffussion (28 cases). 5 patients (12,1%) were positive and 36 (72%) were negative. In the 5 serum positive HBsAg HCC the staining methods for cytoplasmic HBsAg were positive (100%). In 36 serum negative HBsAg HCC the staining method were positive in 2 cases. The results let us to conclude that HBV is a probable important etiologic factor of HCC in our milieu. 54% of the patients with HCC had a previous history of alcohol abuse; however, histologic features compatible with an alcoholic etiology were found in only 5 cases. Nevertheless we consider that the described histopathologic findings do not exclude excess alcohol consumption as a possible etiologic factor for HCC in our series.

  11. Protection against Invasive Amebiasis by a Single Monoclonal Antibody Directed against a Lipophosphoglycan Antigen Localized on the Surface of Entamoeba histolytica

    PubMed Central

    Marinets, Alexandra; Zhang, Tonghai; Guillén, Nancy; Gounon, Pierre; Bohle, Barbara; Vollmann, Ute; Scheiner, Otto; Wiedermann, Gerhard; Stanley, Samuel L.; Duchêne, Michael

    1997-01-01

    A panel of monoclonal antibodies was raised from mice immunized with a membrane preparation from Entamoeba histolytica, the pathogenic species causing invasive amebiasis in humans. Antibody EH5 gave a polydisperse band in immunoblots from membrane preparations from different E. histolytica strains, and a much weaker signal from two strains of the nonpathogenic species Entamoeba dispar. Although the exact chemical structure of the EH5 antigen is not yet known, the ability of the antigen to be metabolically radiolabeled with [32P]phosphate or [3H]glucose, its sensitivity to digestion by mild acid and phosphatidylinositol-specific phospholipase C, and its specific extraction from E. histolytica trophozoites by a method used to prepare lipophosphoglycans from Leishmania showed that it could be classified as an amebal lipophosphoglycan. Confocal immunofluorescence and immunogold labeling of trophozoites localized the antigen on the outer face of the plasma membrane and on the inner face of internal vesicle membranes. Antibody EH5 strongly agglutinated amebas in a similar way to concanavalin A (Con A), and Con A bound to immunoaffinity-purified EH5 antigen. Therefore, surface lipophosphoglycans may play an important role in the preferential agglutination of pathogenic amebas by Con A. The protective ability of antibody EH5 was tested in a passive immunization experiment in a severe combined immunodeficient (SCID) mouse model. Intrahepatic challenge of animals after administration of an isotype-matched control antibody or without treatment led to the development of a liver abscess in all cases, whereas 11 out of 12 animals immunized with the EH5 antibody developed no liver abscess. Our results demonstrate the importance and, for the first time, the protective capacity of glycan antigens on the surface of the amebas. PMID:9348313

  12. A Novel Pan-Genome Reverse Vaccinology Approach Employing a Negative-Selection Strategy for Screening Surface-Exposed Antigens against leptospirosis.

    PubMed

    Zeng, LingBing; Wang, Dongliang; Hu, NiYa; Zhu, Qing; Chen, Kaishen; Dong, Ke; Zhang, Yan; Yao, YuFeng; Guo, XiaoKui; Chang, Yung-Fu; Zhu, YongZhang

    2017-01-01

    Reverse vaccinology (RV) has been widely used for screening of surface-exposed proteins (PSEs) of important pathogens, including outer membrane proteins (OMPs), and extracellular proteins (ECPs) as potential vaccine candidates. In this study, we applied a novel RV negative strategy and a pan-genome analysis for screening of PSEs from 17 L. interrogans strains covering 11 predominately epidemic serovars and 17 multilocus typing (MLST) sequence types (STs) worldwide. Our results showed, for instance, out of a total of 633 predicted PSEs in strain 56601, 92.8% were OMPs or ECPs (588/633). Among the 17 strains, 190 core PSEs, 913 dispensable PSEs and 861 unique PSEs were identified. Of the 190 PSEs, 121 were further predicted to be highly antigenic and thus may serve as potential vaccine candidates against leptospirosis. With the exception of LipL45, OmpL1, and LigB, the majority of the 121 PSEs were newly identified antigens. For example, hypothetical proteins BatC, LipL71, and the OmpA family proteins sharing many common features, such as surface-exposed localization, universal conservation, and eliciting strong antibody responses in patients, are regarded as the most promising vaccine antigens. Additionally, a wide array of potential virulence factors among the predicted PSEs including TonB-dependent receptor, sphingomyelinase 2, leucine-rich repeat protein, and 4 neighboring hypothetical proteins were identified as potential antigenicity, and deserve further investigation. Our results can contribute to the prediction of suitable antigens as potential vaccine candidates against leptospirosis and also provide further insights into mechanisms of leptospiral pathogenicity. In addition, our novel negative-screening strategy combined with pan-genome analysis can be a routine RV method applied to numerous other pathogens.

  13. A Novel Pan-Genome Reverse Vaccinology Approach Employing a Negative-Selection Strategy for Screening Surface-Exposed Antigens against leptospirosis

    PubMed Central

    Zeng, LingBing; Wang, Dongliang; Hu, NiYa; Zhu, Qing; Chen, Kaishen; Dong, Ke; Zhang, Yan; Yao, YuFeng; Guo, XiaoKui; Chang, Yung-Fu; Zhu, YongZhang

    2017-01-01

    Reverse vaccinology (RV) has been widely used for screening of surface-exposed proteins (PSEs) of important pathogens, including outer membrane proteins (OMPs), and extracellular proteins (ECPs) as potential vaccine candidates. In this study, we applied a novel RV negative strategy and a pan-genome analysis for screening of PSEs from 17 L. interrogans strains covering 11 predominately epidemic serovars and 17 multilocus typing (MLST) sequence types (STs) worldwide. Our results showed, for instance, out of a total of 633 predicted PSEs in strain 56601, 92.8% were OMPs or ECPs (588/633). Among the 17 strains, 190 core PSEs, 913 dispensable PSEs and 861 unique PSEs were identified. Of the 190 PSEs, 121 were further predicted to be highly antigenic and thus may serve as potential vaccine candidates against leptospirosis. With the exception of LipL45, OmpL1, and LigB, the majority of the 121 PSEs were newly identified antigens. For example, hypothetical proteins BatC, LipL71, and the OmpA family proteins sharing many common features, such as surface-exposed localization, universal conservation, and eliciting strong antibody responses in patients, are regarded as the most promising vaccine antigens. Additionally, a wide array of potential virulence factors among the predicted PSEs including TonB-dependent receptor, sphingomyelinase 2, leucine-rich repeat protein, and 4 neighboring hypothetical proteins were identified as potential antigenicity, and deserve further investigation. Our results can contribute to the prediction of suitable antigens as potential vaccine candidates against leptospirosis and also provide further insights into mechanisms of leptospiral pathogenicity. In addition, our novel negative-screening strategy combined with pan-genome analysis can be a routine RV method applied to numerous other pathogens. PMID:28352257

  14. Cyclosporine inhibits macrophage-mediated antigen presentation

    SciTech Connect

    Ziegler, H.K.; Palay, D.; Wentworth, P.; Cluff, C.

    1986-03-01

    The influence of cyclosporine on antigen-specific, macrophage-dependent T cell activation was analyzed in vitro. Murine T cell activation by antigens derived from Listeria monocytogenes was monitored by the production of interleukin-2. Pretreatment (2 hrs., 37/sup 0/C) of macrophages with cyclosporine resulted in a population of macrophages with a markedly diminished capacity to support the activation of T lymphocytes. When cyclosporine-pretreated macrophages were added to cultures of antigen and untreated T cells, the dose of cyclosporine which produced 50% inhibition was 1.5 ..mu..g/ml. Appropriate control experiments indicated that cyclosporine was indeed inhibiting at the macrophage level. The addition of interleukin-1 or indomethacin to the cultures did not alter the inhibitory effect of cyclosporine. Under conditions which produced >90% inhibition of antigen presentation, macrophage surface Ia expression was not altered, and the uptake and catabolism of radiolabelled antigen was normal. Thus, cyclosporine inhibits antigen presentation by a mechanism which appears unrelated to changes in Il-1 elaboration, prostaglandin production, Ia expression, or antigen uptake and catabolism.

  15. The Taenia saginata homologue of the major surface antigen of Echinococcus spp. is immunogenic and 97% identical to its Taenia solium homologue.

    PubMed

    González, Luis Miguel; Ferrer, Elizabeth; Spickett, Andrea; Michael, Lynne M; Vatta, Adriano F; Gárate, Teresa; Harrison, Leslie J S; Parkhouse, R Michael E

    2007-11-01

    The TEG-Tsag gene of Taenia saginata is homologous to the genes expressing the two major surface antigens of Echinococcus spp. (EM10 and EG10). Surface antigens of parasites are logical candidates for vaccines, and in this paper we demonstrate that cattle vaccinated with the recombinant TEG-Tsag protein, either used singly or in conjunction with the recombinant HP6-Tsag protein, the major 18 kDa surface/secreted antigen of T. saginata oncospheres, produce excellent antibody responses to both these recombinant proteins. Thus TEG-Tsag may have utility as a vaccine and also as a diagnostic tool for bovine cysticercosis. In addition, as we now demonstrate a 97% homology between TEG-Tsag and its Taenia solium homologue, TEG-Tsol, this latter molecule may have similar potential in the control of human and porcine cysticercosis. The TEG molecule is characterized by an N-terminal FERM domain and a C-terminal ERM domain which are found in a number of cytoskeletal-associated proteins located at the interface between the plasma membrane and the cytoskeleton and in proteins that interact with lipid membranes. The FERM domain is also postulated to bind to adhesion proteins, in a PIP2-regulated fashion, providing a link between cytoskeletal signals and membrane dynamics. Thus TEG protein may play a role in tegument function and interaction with the host.

  16. Induction of cell surface human leukocyte antigen-G expression in pandemic H1N1 2009 and seasonal H1N1 influenza virus-infected patients.

    PubMed

    Chen, Hai-Xiao; Chen, Bao-Guo; Shi, Wei-Wu; Zhen, Rui; Xu, Dan-Ping; Lin, Aifen; Yan, Wei-Hua

    2011-02-01

    A novel H1N1 virus of swine origin (H1N1v) recently caused a pandemic; however, knowledge of immunologic aspects of the virus infection are limited. Human leukocyte antigen-G (HLA-G) was speculated to play critical roles in viral infection, although its clinical relevance in H1N1 infection remains unknown. In this study, HLA-G expression in peripheral T lymphocytes, monocytes, and CD4(+) CD25(+) FoxP3+ regulatory T (Treg) cells (in 50 H1N1v-infected and 41 seasonal H1N1-infected patients and 27 control subjects) were analyzed by flow cytometry. Plasma-soluble HLA-G (sHLA-G, in 28 H1N1v-infected, 29 seasonal H1N1-infected patients and 85 control subjects) were determined with enzyme-linked immunosorbent assay. The percentage of HLA-G-positive T lymphocytes and monocytes among patients with H1N1v and seasonal H1N1 infections was dramatically increased compared with controls (all p < 0.001). Treg was markedly increased among H1N1v- infected patients compared with normal controls (p = 0.041), but not for the seasonal H1N1-infected patients. Meanwhile, no significant difference was observed for sHLA-G levels between the groups. Together, cell surface HLA-G expression was markedly induced in H1N1v-infected and seasonal H1N1-infected patients, and increased Treg was observed only in H1N1v-infected patients. Given its immune-suppressive property, elevated cell surface HLA-G expression may help to explain the virus escaping from host immune responses.

  17. Comprehensive Analysis and Characterization of Linear Antigenic Domains on HN Protein from Genotype VII Newcastle Disease Virus Using Yeast Surface Display System.

    PubMed

    Li, Tao; Wang, Gaoling; Shi, Bingtian; Liu, Peixin; Si, Wei; Wang, Bin; Jiang, Li; Zhou, Lunjiang; Xiu, Jinsheng; Liu, Henggui

    2015-01-01

    Circulation of genotype VII Newcastle disease virus (NDV) has posed a great threat for the poultry industry worldwide. Antibodies against Hemagglutinin-neuraminidase (HN), a membrane protein of NDV with critical roles in NDV infection, have been reported to provide chickens protection from NDV infection. In this study, we comprehensively analyzed the in vivo antibody responses against the linear antigenic domains of the HN protein from genotype VII NDV using a yeast surface display system. The results revealed four distinct regions of HN, P1 (1-52aa), P2 (53-192aa), P3 (193-302aa) and P4 (303-571aa), respectively, according to their antigenic potency. Analysis by FACS and ELISA assay indicated P2 to be the dominant linear antigenic domain, with the immunogenic potency to protect the majority of chickens from NDV challenge. In contrast, the P1, P3 and P4 domains showed weak antigenicity in vivo and could not protect chickens from NDV challenge. These results provide important insight into the characteristic of humoral immune responses elicited by HN of NDV in vivo.

  18. Comprehensive Analysis and Characterization of Linear Antigenic Domains on HN Protein from Genotype VII Newcastle Disease Virus Using Yeast Surface Display System

    PubMed Central

    Li, Tao; Wang, Gaoling; Shi, Bingtian; Liu, Peixin; Si, Wei; Wang, Bin; Jiang, Li; Zhou, Lunjiang; Xiu, Jinsheng; Liu, Henggui

    2015-01-01

    Circulation of genotype VII Newcastle disease virus (NDV) has posed a great threat for the poultry industry worldwide. Antibodies against Hemagglutinin-neuraminidase (HN), a membrane protein of NDV with critical roles in NDV infection, have been reported to provide chickens protection from NDV infection. In this study, we comprehensively analyzed the in vivo antibody responses against the linear antigenic domains of the HN protein from genotype VII NDV using a yeast surface display system. The results revealed four distinct regions of HN, P1 (1-52aa), P2 (53-192aa), P3 (193-302aa) and P4 (303-571aa), respectively, according to their antigenic potency. Analysis by FACS and ELISA assay indicated P2 to be the dominant linear antigenic domain, with the immunogenic potency to protect the majority of chickens from NDV challenge. In contrast, the P1, P3 and P4 domains showed weak antigenicity in vivo and could not protect chickens from NDV challenge. These results provide important insight into the characteristic of humoral immune responses elicited by HN of NDV in vivo. PMID:26121247

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

    PubMed

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

    2013-12-01

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

  20. Pasteurella haemolytica antigens associated with resistance to pneumonic pasteurellosis.

    PubMed Central

    Mosier, D A; Simons, K R; Confer, A W; Panciera, R J; Clinkenbeard, K D

    1989-01-01

    Antigens associated with whole Pasteurella haemolytica biotype A serotype 1, a capsular carbohydrate-protein extract of the organism, and P. haemolytica leukotoxin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antigens of the electrophoresed preparations were detected by Western blotting (immunoblotting) with sera from cattle which were either nonvaccinated or vaccinated with live or killed P. haemolytica vaccines and had variable degrees of resistance to experimental pneumonic pasteurellosis. Distinct, easily recognizable antigens of these preparations were identified, and the antibody responses to these antigens were quantified by densitometry. To determine their importance to disease resistance, we then compared antibody responses with experimental lesion scores. Antibody reactivity to surface antigens which were significantly correlated with resistance and present in two or more of the preparations were detected at 86, 66, 51, 49, 34, 31, and 16 kilodaltons (kDa). Of these, antibody responses to antigens at 86, 49, and 31 kDa appeared most important based on their concentration and significance levels. Antibody reactivity to leukotoxin antigens which were significantly correlated with resistance and common with important surface antigens were detected at 86, 66, and 49 kDa. Antibody responses to unique leukotoxin antigens which were significantly correlated with resistance were present at 92 and 58 kDa. Images PMID:2917783

  1. The half-filled Landau level and topological insulator surfaces

    NASA Astrophysics Data System (ADS)

    Senthil, T.

    The metallic state of the half-filled Landau level - described originally in pioneering work by Halperin , Lee, and Read as a liquid of composite fermions - was proposed recently by Son to be described by a particle-hole symmetric effective field theory distinct from that in the prior literature. This talk will develop a simple picture of the particle-hole symmetric composite fermion through a modification of older pictures as electrically neutral ``dipolar'' particles. This picture, and the proposed particle-hole symmetric theory, will be further substantiated through a recently developed deep connection between the half-filled Landau level and correlated surface states of certain three dimensional topological insulators. The phenomenology of composite fermi liquids (with or without particle-hole symmetry) will be revisited. It will be shown that their heat/electrical transport dramatically violates the conventional Wiedemann-Franz law but satisfies a modified one. References: 1. Chong Wang and T. Senthil, ``Half-filled Landau Level, Topological Insulator Surfaces, and Three Dimensional Quantum Spin Liquids,'' cond-mat arXiv:1507.08290 (2015).

  2. Heterozygous Mutation in IκBNS Leads to Reduced Levels of Natural IgM Antibodies and Impaired Responses to T-Independent Type 2 Antigens

    PubMed Central

    Pedersen, Gabriel K.; Ádori, Monika; Stark, Julian M.; Khoenkhoen, Sharesta; Arnold, Carrie; Beutler, Bruce; Karlsson Hedestam, Gunilla B.

    2016-01-01

    Mice deficient in central components of classical NF-κB signaling have low levels of circulating natural IgM antibodies and fail to respond to immunization with T-independent type 2 (TI-2) antigens. A plausible explanation for these defects is the severely reduced numbers of B-1 and marginal zone B (MZB) cells in such mice. By using an ethyl-N-nitrosourea mutagenesis screen, we identified a role for the atypical IκB protein IκBNS in humoral immunity. IκBNS-deficient mice lack B-1 cells and have severely reduced numbers of MZB cells, and thus resemble several other strains with defects in classical NF-κB signaling. We analyzed mice heterozygous for the identified IκBNS mutation and demonstrate that these mice have an intermediary phenotype in terms of levels of circulating IgM antibodies and responses to TI-2 antigens. However, in contrast to mice that are homozygous for the IκBNS mutation, the heterozygous mice had normal frequencies of B-1 and MZB cells. These results suggest that there is a requirement for IκBNS expression from two functional alleles for maintaining normal levels of circulating natural IgM antibodies and responses to TI-2 antigens. PMID:26973645

  3. DNA vaccines expressing pneumococcal surface protein A (PspA) elicit protection levels comparable to recombinant protein.

    PubMed

    Ferreira, Daniela M; Miyaji, Eliane N; Oliveira, Maria Leonor S; Darrieux, Michelle; Arêas, Ana Paula M; Ho, Paulo L; Leite, Luciana C C

    2006-04-01

    Pneumococcal surface protein A (PspA) is a promising candidate for the development of cost-effective vaccines against Streptococcus pneumoniae. In the present study, BALB/c mice were immunized with DNA vaccine vectors expressing the N-terminal region of PspA. Animals immunized with a vector expressing secreted PspA developed higher levels of antibody than mice immunized with the vector expressing the antigen in the cytosol. However, both immunogens elicited similar levels of protection against intraperitoneal challenge. Furthermore, immunization with exactly the same fragment in the form of a recombinant protein, with aluminium hydroxide as an adjuvant, elicited even higher antibody levels, but this increased humoral response did not correlate with enhanced protection. These results show that DNA vaccines expressing PspA are able to elicit protection levels comparable to recombinant protein, even though total anti-PspA IgG response is considerably lower.

  4. Plant-based strategies aimed at expressing HIV antigens and neutralizing antibodies at high levels. Nef as a case study.

    PubMed

    Marusic, Carla; Vitale, Alessandro; Pedrazzini, Emanuela; Donini, Marcello; Frigerio, Lorenzo; Bock, Ralph; Dix, Philip J; McCabe, Matthew S; Bellucci, Michele; Benvenuto, Eugenio

    2009-08-01

    The first evidence that plants represent a valid, safe and cost-effective alternative to traditional expression systems for large-scale production of antigens and antibodies was described more than 10 years ago. Since then, considerable improvements have been made to increase the yield of plant-produced proteins. These include the use of signal sequences to target proteins to different cellular compartments, plastid transformation to achieve high transgene dosage, codon usage optimization to boost gene expression, and protein fusions to improve recombinant protein stability and accumulation. Thus, several HIV/SIV antigens and neutralizing anti-HIV antibodies have recently been successfully expressed in plants by stable nuclear or plastid transformation, and by transient expression systems based on plant virus vectors or Agrobacterium-mediated infection. The current article gives an overview of plant expressed HIV antigens and antibodies and provides an account of the use of different strategies aimed at increasing the expression of the accessory multifunctional HIV-1 Nef protein in transgenic plants.

  5. Effect of context and adjuvant on the immunogenicity of recombinant proteins and peptide conjugates derived from the polymorphic malarial surface antigen MSA2.

    PubMed

    Jones, G L; Spencer, L; Lord, R; Saul, A J

    1996-01-01

    We have identified a 51 kDa glycosylated myristylated merozoite surface antigen (MSA2) as the target of a number of monoclonal antibodies which inhibit in vitro invasion of the human malarial parasite Plasmodium falciparum. This antigen has been shown to exist in a limited number of strain specific forms but despite wide variation in the sequences of the internal repeat regions both N and C terminal elements of the protein are almost totally conserved. Accordingly, we prepared a large number of overlapping peptide constructs and demonstrated that one peptide SNTFINNA (E71) from the N terminus and two peptides, QHGHMHGS (G5) and NTSDSQKE (G12) from the C terminus could, when suitably conjoined to the carrier protein diphtheria toxoid (DT), elicit antibodies reactive with MSA2 from diverse strains of P. falciparum. Here we compare the immunogenicity of these peptide constructs with two recombinant proteins containing the entire amino acid sequence of MSA2 from the FCQ-27/PNG strain (1609) and the 3D7 strain (1623). We have formulated these recombinant and peptide antigens with Freund's adjuvant, Alum and Algammulin. Both recombinant and peptide antigens elicit high titre antibodies when tested by ELISA against the immunogens themselves. Although both recombinant proteins include the constant region peptide sequences E71, G5 and G12, the extent of ELISA cross reaction between antibody raised against recombinant and peptide antigen or antibody raised against peptide and recombinant antigen is small and sporadic, and depends to an extent on the adjuvant employed. Antisera against both recombinant proteins 1609 and 1623 detected either recombinant on Western blots, as well as detecting native MSA2 in whole protein extracts from both FCQ-27/PNG and 3D7 strains. Antisera against peptide construct E71 recognized recombinant 1609 but not 1623 but recognized the native MSA2 in both strains studied. Antisera against peptide construct G5 showed a similar pattern of recognition

  6. Immunochromatographic assay for quantitative and sensitive detection of hepatitis B virus surface antigen using highly luminescent quantum dot-beads.

    PubMed

    Shen, Jun; Zhou, Yaofeng; Fu, Fen; Xu, Hengyi; Lv, Jiaofeng; Xiong, Yonghua; Wang, Andrew

    2015-09-01

    Hepatitis B virus infection is one of the major causes of hepatitis, liver cirrhosis and liver cancer. In this study, we used highly luminescent quantum dot-beads (QBs) as signal amplification probes in the sandwich immunochromatographic assay (ICA) for ultrasensitive and quantitative detection of hepatitis B virus surface antigen (HBsAg) in human serum. Various parameters that influenced the sensitivity and stability of the QB-based ICA (QB-ICA) sensor were investigated. Two linear independent regression equations for detection of serum HBsAg were expressed with Y=0.3361X-0.0059 (R(2)=0.9983) for low HBsAg concentrations between 75 pg mL(-1) and 4.8 ng mL(-1), and Y=0.8404 X-2.9364 (R(2)=0.9939) for high HBsAg concentrations in the range from 4.8 ng mL(-1) to 75 ng mL(-1). The detection limit of the proposed ICA sensor achieved was 75 pg mL(-1), which is much higher than that of the routinely-used gold nanoparticle based ICA. The intra- and inter-assays recovery rates for spiked serum samples at HBsAg concentrations of 75 pg mL(-1), 3.75 ng mL(-1) and 18.75 ng mL(-1) ranged from 90.14% to 97.6%, and coefficients of variation were all below 7%, indicating that the QB-ICA sensor has an acceptable accuracy for HBsAg detection. Additionally, the quantitative method developed showed no false positive results in an analysis of 49 real HBsAg-negative serum samples, and exhibited excellent agreement (R(2)=0.9209) with a commercial chemiluminescence immunoassay kit in identifying 47 HBsAg-positive serum samples. In summary, due to its high fluorescence intensity, the sandwich QB-ICA sensor is a very promising point-of-care test for rapid, simple and ultrasensitive detection of HBsAg, as well as other disease-related protein biomarkers.

  7. [Characterization of surface antigens of the nematode parasite Trichinella spiralis: study of its role in protection mechanisms and their usefulness in the diagnosis of trichinosis].

    PubMed

    Ortega-Pierres, M G

    1995-01-01

    Among the most important aspects in the study of trichinosis are the development of specific and sensitive diagnostic methods for the detection of the infection by the parasite as well as the characterization of antigens from Trichinella spiralis that induce protection in the host. In the context, the characterization of surface stichosome and excretory secretory antigens of T. spiralis muscle larvae has been an important issue due to the high immunogenicity of such components in most hosts so far studied. In this work, we have been able to identify and characterize molecules from both compartments of muscle larvae. These components have been used in assays for specific detection of T. spiralis infections particularly in pigs, as well as in assays to evaluate their role in the induction of protection in mice.

  8. Surface plasmon resonance imaging for ABH antigen detection on red blood cells and in saliva: secretor status-related ABO subgroup identification.

    PubMed

    Peungthum, Patjaree; Sudprasert, Krisda; Amarit, Ratthasart; Somboonkaew, Armote; Sutapun, Boonsong; Vongsakulyanon, Apirom; Seedacoon, Wuttigrai; Kitpoka, Pimpun; Kunakorn, Mongkol; Srikhirin, Toemsak

    2017-03-27

    Low antigenic expression of ABO subgroup system on red blood cell (RBC) is cause of discrepancy between forward and reverse blood typing in the standard agglutination technique. Neutralization agglutination is employed for verification of the detection of ABH substances in saliva. However, the neutralization technique is complicated, time-consuming and requires expertise. To overcome these drawbacks, surface plasmon resonance (SPR) imaging was developed for ABH antigen detection on RBCs and in saliva. An antibody array was designed to classify the ABO subgroups by anti-A, anti-B, and anti-H antibodies; the array was immobilized on a carboxymethyl-dextran sensor-surface. RBCs and saliva specimens from sixty-four donors were analysed by passing them over the antibody array, where the secretor status and blood group could be simultaneously identified. Consequently, the immobilized antibodies could specifically and quantitatively detect the ABH antigen on RBCs. Using the direct assay, the SPR signal of saliva detection was weaker than that of RBC detection. However, a sandwich assay with a mixture of anti-A, anti-B, and anti-H antibodies could efficiently enhance the signal. The sensor chip provided high specificity (cut-off at 100 to 175 micro refractive index units) and high precision at 0.06%-4.9% CV. The blood group results of the sixty-four donor specimens obtained by SPR agreed with the standard agglutination test with 100% accuracy. SPR could indicate different ABH antigen densities on the RBCs and nearly the same amounts of ABH substances in the saliva of strong and weak subgroups. Finally, we also demonstrated reduced assay time and fewer complications with the SPR imaging platform compared to the neutralization technique.

  9. The monoclonal antibody GZS-1 detects a maturation-associated antigen of human spermatozoa that is also present on the surface of human mononuclear blood cells.

    PubMed

    Hutter, H; Hammer, A; Blaschitz, A; Hartmann, M; Mahnert, W; Sedlmayr, P; Primus, G; Rosenkranz, C; Gebru, G; Henkel, R; Dohr, G

    1996-05-01

    A monoclonal antibody (GZS-1) has been generated by fusion of mouse myeloma cells with spleen cells from BALB/c mice immunised with human sperm cells. The antibody was determined to be an IgG1. The corresponding antigen is present on the whole surface of ejaculated human spermatozoa. It is not detectable on spermatozoa of other mammalian species (rabbit, cat, dog, sheep, boar, bull, horse). In human male genital organs, immunostaining with GZS-1 is observed on sperm cells in the epididymis and the ductus deferens together with the lining epithelium of those organs. No reactivity of sperm cells or germ cell precursors in the testis has been detected. Functional tests using the antibody show a strong inhibitory effect of human sperm in the hamster egg penetration assay. Furthermore, the GZS-1 antigen is detectable on the surface of human lymphocytes and monocytes by immunogold electron microscopy and FACS analysis. By Western blotting of human sperm and seminal plasma performed under reducing conditions immunostaining was detected at 21-25, 31, 51-54, and 62 kDa. The reaction with human lymphocytes shows one major band at 62 kDa and additional bands at 31 and 54 kDa. The results suggest that the monoclonal antibody GZS-1 may recognise an antigen which is secreted from the epithelial cells of the epididymis and binds to ejaculated spermatozoa as a sperm coating antigen. This component may be involved in the capacitation of the sperm and the acrosome reaction. Molecules that are expressed both on sperm and on immunocompetent cells may be relevant for the regulation of immunological processes or for the development of the related immunological tolerance of sperm in the female reproductive tract.

  10. DNA vaccine encoding the Toxoplasma gondii bradyzoite-specific surface antigens SAG2CDX protect BALB/c mice against type II parasite infection.

    PubMed

    Zhang, Min; Zhao, Lingxiao; Song, Jing; Li, Ying; Zhao, Qunli; He, Shenyi; Cong, Hua

    2013-09-23

    The surface antigens SAG2C, SAG2D, and SAG2X, which expressed specifically on bradyzoite stage of Toxoplasma gondii, have been demonstrated to be important for persistence of cyst in the brain. In this study, DNA vaccines expressing SAG2C, SAG2D, and SAG2X of T. gondii were constructed and their protective efficacy were evaluated in BALB/c mice. Mice vaccinated with pVAX1-SAG2C (pSAG2C), pVAX1-2D (pSAG2D) or pVAX1-2X (pSAG2C) showed higher levels of serum IgG antibodies and lymphocyte proliferation response compared to PBS and pVAX1 treated mice (p<0.05). The immune response was characterized by a strong Th1 response and increased cytokine production of IL-2 and IFN-γ. Vaccinated mice displayed significant protection against the challenge with the cyst of T. gondii genotype II strain of PRU (cyst-forming in mouse). A significant reduction in the brain cyst burden was detected in the mice immunized with pSAG2C (72%), pSAG2D (23%), pSAG2X (69%) alone and even more reduction rate, 77%, was achieved in the combination group compared to PBS treated mice. The results implied that immunization with DNA vaccines expressing SAG2C, SAG2D, and SAG2X, and, in particular, a combination of all three DNA plasmids, could effectively protect the mice against T. gondii chronic infection.

  11. Effects of Serial Skin Testing with Purified Protein Derivative on the Level and Quality of Antibodies to Complex and Defined Antigens in Mycobacterium bovis-Infected Cattle

    PubMed Central

    Palmer, Mitchell V.; Stafne, Molly R.; Bass, Kristin E.; Maggioli, Mayara F.; Thacker, Tyler C.; Linscott, Rick; Lawrence, John C.; Nelson, Jeffrey T.; Esfandiari, Javan; Greenwald, Rena; Lyashchenko, Konstantin P.

    2015-01-01

    Several serological tests designed to detect antibodies to immunodominant Mycobacterium bovis antigens have recently emerged as ancillary tests for the detection of bovine tuberculosis in cattle, particularly when used after the injection of purified protein derivative (PPD) for skin testing, which significantly boosts M. bovis-specific antibody responses. The present findings demonstrate the onset and duration of boosted antibody responses after the injection of M. bovis PPD for the caudal fold test (CFT) and Mycobacterium avium and M. bovis PPDs for the comparative cervical test (CCT), administered in series in cattle experimentally infected with M. bovis. While skin tests boosted the responses to certain antigens (i.e., MPB83 and MPB70), they did not affect the responses to other antigens (e.g., ESAT-6, CFP10, MPB59, and MPB64). Administration of the CCT 105 days after the CFT resulted in an even greater secondary boost in antibody responses to MPB83 and MPB70 and to a proteinase K-digested whole-cell sonicate (WCS-PK) of M. bovis. Both IgM and IgG contributed to the initial boost in the MPB83/MPB70-specific antibody response after the CFT. The secondary boost after the CCT was primarily due to increased IgG levels. Also, the avidity of antibodies to MPB83 and MPB70 increased after the CCT in M. bovis-infected cattle. The avidity of antibodies to the WCS-PK antigens increased in the interval between the CFT and the CCT but did not increase further after the CCT. Together, these findings demonstrate that the administration of PPDs for skin tests results in additive enhancement (i.e., when the CFT and CCT are performed in series), both qualitative and quantitative, of MPB83/MPB70-specific antibody responses. PMID:25855555

  12. Measurement of Circulating Filarial Antigen Levels in Human Blood with a Point-of-Care Test Strip and a Portable Spectrodensitometer

    PubMed Central

    Chesnais, Cédric B.; Vlaminck, Johnny; Kunyu-Shako, Billy; Pion, Sébastien D.; Awaca-Uvon, Naomi-Pitchouna; Weil, Gary J.; Mumba, Dieudonné; Boussinesq, Michel

    2016-01-01

    The Alere Filariasis Test Strip (FTS) is a qualitative, point-of-care diagnostic tool that detects Wuchereria bancrofti circulating filarial antigen (CFA) in human blood, serum, or plasma. The Global Program to Eliminate Lymphatic Filariasis employs the FTS for mapping filariasis-endemic areas and assessing the success of elimination efforts. The objective of this study was to explore the relationship between the intensity of positive test lines obtained by FTS with CFA levels as determined by enzyme-linked immunosorbent assay (ELISA) with blood and plasma samples from 188 individuals who live in a filariasis-endemic area. The intensity of the FTS test line was assessed visually to provide a semiquantitative score (visual Filariasis Test Strip [vFTS]), and line intensity was measured with a portable spectrodensitometer (quantitative Filariasis Test Strip [qFTS]). These results were compared with antigen levels measured by ELISA in plasma from the same subjects. qFTS measurements were highly correlated with vFTS scores (ρ = 0.94; P < 0.001) and with plasma CFA levels (ρ = 0.91; P < 0.001). Thus, qFTS assessment is a convenient method for quantifying W. bancrofti CFA levels in human blood, which are correlated with adult worm burdens. This tool may be useful for assessing the impact of treatment on adult filarial worms in individuals and communities. PMID:27114288

  13. Oral immunization with recombinant hepatitis E virus antigen displayed on the Lactococcus lactis surface enhances ORF2-specific mucosal and systemic immune responses in mice.

    PubMed

    Gao, Shenyang; Li, Dandan; Liu, Ying; Zha, Enhui; Zhou, Tiezhong; Yue, Xiqing

    2015-01-01

    Hepatitis E virus (HEV) as a recognized zoonotic pathogen has posed global burden on public health, which is exacerbated by lack of efficient vaccine. In this study, we constructed a recombinant (inaQ-ORF2 gene fusion) Lactococcus lactis (L. lactis) strain NZ3900 that expresses and displays the hepatitis E virus antigen ORF2 utilizing an ice uncleation protein-based anchor system. After oral vaccination of BALB/c mice, significantly higher levels of ORF2-specific mucosal IgA and serum IgG were detected and cellular immunity was also induced. These findings further support that L. lactis-based HEV antigen vaccines could be used for human and animal protection against infection.

  14. Process for leveling film surfaces and products thereof

    DOEpatents

    Birkmire, R.W.; McCandless, B.E.

    1990-03-20

    Semiconductor films and photovoltaic devices prepared therefrom are provided wherein the semiconductor films have a specular surface with a texture less than about 0.25 micron greater than the average planar film surface and wherein the semiconductor films are surface modified by exposing the surface to an aqueous solution of bromine containing an acid or salt and continuing such exposure for a time sufficient to etch the surface. 8 figs.

  15. Process for levelling film surfaces and products thereof

    DOEpatents

    Birkmire, Robert W.; McCandless, Brian E.

    1990-03-20

    Semiconductor films and photovoltaic devices prepared therefrom are provided wherein the semiconductor films have a specular surface with a texture less than about 0.25 micron greater than the average planar film surface and wherein the semiconductor films are surface modified by exposing the surface to an aqueous solution of bromine containing an acid or salt and continuing such exposure for a time sufficient to etch the surface.

  16. Selection of scFv Antibody Fragments Binding to Human Blood versus Lymphatic Endothelial Surface Antigens by Direct Cell Phage Display

    PubMed Central

    Keller, Thomas; Kalt, Romana; Raab, Ingrid; Schachner, Helga; Mayrhofer, Corina; Kerjaschki, Dontscho; Hantusch, Brigitte

    2015-01-01

    The identification of marker molecules specific for blood and lymphatic endothelium may provide new diagnostic tools and identify new targets for therapy of immune, microvascular and cancerous diseases. Here, we used a phage display library expressing human randomized single-chain Fv (scFv) antibodies for direct panning against live cultures of blood (BECs) and lymphatic (LECs) endothelial cells in solution. After six panning rounds, out of 944 sequenced antibody clones, we retrieved 166 unique/diverse scFv fragments, as indicated by the V-region sequences. Specificities of these phage clone antibodies for respective compartments were individually tested by direct cell ELISA, indicating that mainly pan-endothelial cell (EC) binders had been selected, but also revealing a subset of BEC-specific scFv antibodies. The specific staining pattern was recapitulated by twelve phage-independently expressed scFv antibodies. Binding capacity to BECs and LECs and differential staining of BEC versus LEC by a subset of eight scFv antibodies was confirmed by immunofluorescence staining. As one antigen, CD146 was identified by immunoprecipitation with phage-independent scFv fragment. This antibody, B6-11, specifically bound to recombinant CD146, and to native CD146 expressed by BECs, melanoma cells and blood vessels. Further, binding capacity of B6-11 to CD146 was fully retained after fusion to a mouse Fc portion, which enabled eukaryotic cell expression. Beyond visualization and diagnosis, this antibody might be used as a functional tool. Overall, our approach provided a method to select antibodies specific for endothelial surface determinants in their native configuration. We successfully selected antibodies that bind to antigens expressed on the human endothelial cell surfaces in situ, showing that BECs and LECs share a majority of surface antigens, which is complemented by cell-type specific, unique markers. PMID:25993332

  17. Analysis of the binding loops configuration and surface adaptation of different crystallized single-domain antibodies in response to various antigens.

    PubMed

    Al Qaraghuli, Mohammed M; Ferro, Valerie A

    2017-04-01

    Monoclonal antibodies have revolutionized the biomedical field through their ubiquitous utilization in different diagnostics and therapeutic applications. Despite this widespread use, their large size and structural complexity have limited their versatility in specific applications. The antibody variable region that is responsible for binding antigen is embodied within domains that can be rescued individually as single-domain antibody (sdAb) fragments. Because of the unique characteristics of sdAbs, such as low molecular weight, high physicochemical stability, and the ability to bind antigens inaccessible to conventional antibodies, they represent a viable alternative to full-length antibodies. Consequently, 149 crystal structures of sdAbs, originating from human (VH), camelids (VHH), or sharks (VNAR), were retrieved from the Protein Data Bank, and their structures were compared. The 3 types of sdAbs displayed complementarity determining regions (CDRs) with different lengths and configurations. CDR3 of the VHH and VNAR domains were dominated by pleated and extended orientations, respectively. Although VNAR showed the smallest average molecular weight and molecular surface area compared with VHH and VH antibodies. However, the solvent accessible surface area measurements of the 3 tested sdAbs types were very similar. All the antihapten VHH antibodies showed pleated CDR3, which were sufficient to create a binding pocket to accommodate haptens (methotrexate and azo dyes) in terms of shape and electrostatic potential. The sdAbs that recognized lysozyme showed more diversity in their CDR3 orientation to enable them to recognize various topographies of lysozyme. Subsequently, the three sdAb classes were different in size and surface area and have shown distinguishable ability to optimize their CDR length and orientation to recognize different antigen classes.

  18. Energy filtering transmission electron microscopy immunocytochemistry and antigen retrieval of surface layer proteins from Tannerella forsythensis using microwave or autoclave heating with citraconic anhydride.

    PubMed

    Moriguchi, K; Mitamura, Y; Iwami, J; Hasegawa, Y; Higuchi, N; Murakami, Y; Maeda, H; Yoshimura, F; Nakamura, H; Ohno, N

    2012-11-01

    Tannerella forsythensis (Bacteroides forsythus), an anaerobic Gram-negative species of bacteria that plays a role in the progression of periodontal disease, has a unique bacterial protein profile. It is characterized by two unique protein bands with molecular weights of more than 200 kDa. It also is known to have a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane. We examined the relationship between high molecular weight proteins and the S-layer using electron microscopic immunolabeling with chemical fixation and an antigen retrieval procedure consisting of heating in a microwave oven or autoclave with citraconic anhydride. Immunogold particles were localized clearly at the outermost cell surface. We also used energy-filtering transmission electron microscopy (EFTEM) to visualize 3, 3'-diaminobenzidine tetrahydrochloride (DAB) reaction products after microwave antigen retrieval with 1% citraconic anhydride. The three-window method for electron spectroscopic images (ESI) of nitrogen by the EFTEM reflected the presence of moieties demonstrated by the DAB reaction with horseradish peroxidase (HRP)-conjugated secondary antibodies instead of immunogold particles. The mapping patterns of net nitrogen were restricted to the outermost cell surface.

  19. Underwater and surface strategies of 200 m world level swimmers.

    PubMed

    Veiga, Santiago; Roig, Andreu

    2016-01-01

    Pacing strategies of elite swimmers have been consistently characterised from the average lap velocities. In the present study, we examined the racing strategies of 200 m world class-level swimmers with regard to their underwater and surface lap components. The finals and semi-finals of the 200 m races at the 2013 World Swimming Championships (Barcelona, Spain) were analysed by an innovative image-processing system (InThePool® 2.0). Free swimming velocities of elite swimmers typically decreased throughout the 200 m race laps (-0.12 m · s(-1), 95% CI -0.11 to -0.14 m · s(-1), P = 0.001, η(2) = 0.81), whereas underwater velocities, which were faster than free swimming, were not meaningfully affected by the race progress (0.02 m · s(-1), -0.01 to 0.04 m · s(-1), P = 0.01, η(2) = 0.04). When swimming underwater, elite swimmers typically travelled less distance (-0.66 m, -0.83 to -0.49 m, P = 0.001, η(2) = 0.34) from the first to the third turn of the race, although underwater distances were maintained on the backstroke and butterfly races. These strategies allowed swimmers to maintain their average velocity in the last lap despite a decrease in the free swimming velocity. Elite coaches and swimmers are advised to model their racing strategies by considering both underwater and surface race components.

  20. SnSAG5 is an alternative surface antigen of Sarcocystis neurona strains that is mutually exclusive to SnSAG1.

    PubMed

    Crowdus, Carolyn A; Marsh, Antoinette E; Saville, Willliam J; Lindsay, David S; Dubey, J P; Granstrom, David E; Howe, Daniel K

    2008-11-25

    Sarcocystis neurona is an obligate intracellular parasite that causes equine protozoal myeloencephalitis (EPM). Previous work has identified a gene family of paralogous surface antigens in S. neurona called SnSAGs. These surface proteins are immunogenic in their host animals, and are therefore candidate molecules for development of diagnostics and vaccines. However, SnSAG diversity exists in strains of S. neurona, including the absence of the major surface antigen gene SnSAG1. Instead, sequence for an alternative SnSAG has been revealed in two of the SnSAG1-deficient strains. Herein, we present data characterizing this new surface protein, which we have designated SnSAG5. The results indicated that the protein encoded by the SnSAG5 sequence is indeed a surface-associated molecule that has characteristics consistent with the other SAGs identified in S. neurona and related parasites. Importantly, Western blot analyses of a collection of S. neurona strains demonstrated that 6 of 13 parasite isolates express SnSAG5 as a dominant surface protein instead of SnSAG1. Conversely, SnSAG5 was not detected in SnSAG1-positive strains. One strain, which was isolated from the brain of a sea otter, did not express either SnSAG1 or SnSAG5. Genetic analysis with SnSAG5-specific primers confirmed the presence of the SnSAG5 gene in Western blot-positive strains, while also suggesting the presence of a novel SnSAG sequence in the SnSAG1-deficient, SnSAG5-deficient otter isolate. The findings provide further indication of S. neurona strain diversity, which has implications for diagnostic testing and development of vaccines against EPM as well as the population biology of Sarcocystis cycling in the opossum definitive host.

  1. Antigen-specific Treg regulate Th17-mediated lung neutrophilic inflammation, B cell recruitment and polymeric IgA and IgM levels in the airways

    PubMed Central

    Jaffar, Zeina; Ferrini, Maria E.; Girtsman, Teri A.; Roberts, Kevan

    2010-01-01

    Summary Th17 cells play key roles in mediating autoimmunity, inflammation and mucosal host defense against pathogens. To determine whether naturally occurring Treg (nTreg) limit Th17-mediated pulmonary inflammation, OVA-specific CD4+ Th17 cells and expanded CD4+CD25+Foxp3+ nTreg were cotransferred into BALB/c mice that were then exposed to OVA aerosols. Th17 cells, when transferred alone, accumulated in the lungs and posterior mediastinal LN and evoked a pronounced airway hyperreactivity (AHR) and neutrophilic inflammation, characterized by B cell recruitment and elevated IgA and IgM levels. Cotransfer of antigen-specific nTreg markedly reduced the Th17-induced pulmonary inflammation and associated neutrophilia, B cell influx and polymeric Ig levels in the airways, but did not inhibit AHR. Moreover, the regulation appeared restricted to the site of mucosal inflammation, since transfer of nTreg did not affect the Th17 response developing in the lung draining LN, as evidenced by unaltered levels of IL-17 production and low numbers of Foxp3+ Treg. Our findings suggest a crucial role for Th17 cells in mediating airway B cell influx and IgA response and demonstrate that antigen-specific nTreg suppress Th17-mediated lung inflammation. These results provide new insights into how Th17 responses are limited and may facilitate development of novel approaches for controlling Th17-induced inflammation. PMID:19830731

  2. Airborne Interferometry using GNSS Reflections for Surface Level Estimation

    NASA Astrophysics Data System (ADS)

    Semmling, Maximilian; Beyerle, Georg; Schön, Steffen; Stosius, Ralf; Gerber, Thomas; Beckheinrich, Jamila; Markgraf, Markus; Ge, Maorong; Wickert, Jens

    2013-04-01

    The interferometric use of GNSS reflections for ocean altimetry can fill the gap in coverage of ocean observations. Today radar altimeters are used for large scale ocean observations to monitor e.g. global sea level change or circulation processes like El Niño. Spacial and temporal resolution of a single radar altimeter, however, is insufficient to observe mesoscale ocean phenomena like large oceanic eddies that are important indicators of climate change. The high coverage expected for a spaceborne altimeter based on GNSS reflections stimulated investigations on according interferometric methods. Several airborne experiments have been conducted using code observations. Carrier observations have a better precision but are severely affected by noise and have mostly been used in ground-based experiments. A new interferometric approach is presented using carrier observations for airborne application. Implementing a spectral retrieval noise reduction is achieved. A flight experiment was conducted with a Zeppelin airship on 2010/10/12 over Lake Constance at the border between Austria, Germany and Switzerland. The lake surface with an area of 536km2 is suitable for altimetric study as its decimeter range Geoid undulations are well-known. Three GNSS receiver were installed on the airship. A Javad Delta receiver recording direct signals for navigation. The DLR G-REX receiver recording reflected signals for scatterometry and the GORS (GNSS Occultation Reflectometry Scatterometry) receiver recording direct and reflected signals for interferometry. The airship's trajectory is determined from navigation data with a precision better than 10cm using regional augmentation. This presentation focuses on the interferometric analysis of GORS observations. Ray tracing calculations are used to model the difference of direct and reflected signals' path. Spectral retrieval is applied to determine Doppler residuals of modelled path difference and interferometric observations. Lake level

  3. Antigen-Antibody Interaction Database (AgAbDb): a compendium of antigen-antibody interactions.

    PubMed

    Kulkarni-Kale, Urmila; Raskar-Renuse, Snehal; Natekar-Kalantre, Girija; Saxena, Smita A

    2014-01-01

    Antigen-Antibody Interaction Database (AgAbDb) is an immunoinformatics resource developed at the Bioinformatics Centre, University of Pune, and is available online at http://bioinfo.net.in/AgAbDb.htm. Antigen-antibody interactions are a special class of protein-protein interactions that are characterized by high affinity and strict specificity of antibodies towards their antigens. Several co-crystal structures of antigen-antibody complexes have been solved and are available in the Protein Data Bank (PDB). AgAbDb is a derived knowledgebase developed with an objective to compile, curate, and analyze determinants of interactions between the respective antigen-antibody molecules. AgAbDb lists not only the residues of binding sites of antigens and antibodies, but also interacting residue pairs. It also helps in the identification of interacting residues and buried residues that constitute antibody-binding sites of protein and peptide antigens. The Antigen-Antibody Interaction Finder (AAIF), a program developed in-house, is used to compile the molecular interactions, viz. van der Waals interactions, salt bridges, and hydrogen bonds. A module for curating water-mediated interactions has also been developed. In addition, various residue-level features, viz. accessible surface area, data on epitope segment, and secondary structural state of binding site residues, are also compiled. Apart from the PDB numbering, Wu-Kabat numbering and explicit definitions of complementarity-determining regions are provided for residues of antibodies. The molecular interactions can be visualized using the program Jmol. AgAbDb can be used as a benchmark dataset to validate algorithms for prediction of B-cell epitopes. It can as well be used to improve accuracy of existing algorithms and to design new algorithms. AgAbDb can also be used to design mimotopes representing antigens as well as aid in designing processes leading to humanization of antibodies.

  4. Adiponectin, C-reactive protein, fibrinogen and tissue plasminogen activator antigen levels among glucose-intolerant women with and without histories of gestational diabetes

    PubMed Central

    Kim, C.; Christophi, C. A.; Goldberg, R. B.; Perreault, L.; Dabelea, D.; Marcovina, S. M.; Pi-Sunyer, X.; Barrett-Connor, E.

    2015-01-01

    Aim To examine concentrations of biomarkers (adiponectin, C-reactive protein, fibrinogen and tissue plasminogen-activator antigen) associated with glucose homeostasis and diabetes risk by history of gestational diabetes. Methods We conducted a secondary analysis of the Diabetes Prevention Program, a randomized trial of lifestyle intervention or metformin for diabetes prevention. At baseline, participants were overweight and had impaired glucose tolerance. Biomarkers at baseline and 1 year after enrolment were compared between parous women with (n=350) and without a history of gestational diabetes (n=1466). Cox proportional hazard models evaluated whether history of gestational diabetes was associated with diabetes risk, after adjustment for baseline biomarker levels as well as for change in biomarker levels, demographic factors and anthropometrics. Results At baseline, women with histories of gestational diabetes had lower adiponectin (7.5 μg/ml vs. 8.7 μg/ml; p<0.0001) and greater log C-reactive protein (−0.90 mg/l vs. −0.78 mg/l, p=0.04) levels than women without histories of gestational diabetes, but these associations did not persist after adjustment for demographic factors. Fibrinogen and tissue plasminogen-activator antigen were similar between women with and without histories of gestational diabetes. Women with and without histories of gestational diabetes had a similar pattern of changes in biomarkers within randomization arm. Adjustment for age, race/ethnicity, baseline weight, change in weight, baseline biomarker level and change in biomarker level did not significantly alter the association between history of gestational diabetes and diabetes risk. Conclusions Among women with impaired glucose tolerance, biomarkers in women with and without histories of gestational diabetes are similar and respond similarly to lifestyle changes and metformin. Adjustment for biomarker levels did not explain the higher risk of diabetes observed in women with

  5. Intranasal immunization of mice with Echinococcus granulosus surface antigens iscoms evokes a strong immune response, biased towards glucidic epitopes.

    PubMed

    Carol, H; Nieto, A; Villacres-Eriksson, M; Morein, B

    1997-05-01

    The present work describes the preparation and characterization of iscoms from tegumental antigens of Echinococcus granulosus protoscoleces, and their use as immunogens in mice by intranasal and subcutaneous routes. Iscoms given at 10 mg doses evoke a significant antibody response when administered i.n. and a strong and long lasting antibody response by s.c. route. The Ag administered i.n. in a non-adjuvanted form as a booster was not immunogenic. The i.n. route of immunization induced higher IgA serum titre in relation to IgG than the s.c. route and antisera of slightly higher avidity. Also, ten fold lower IgG2a/IgG1 and ten fold higher IgG3/IgG1 ratios were observed for the i.n. protocol compared to the s.c. one. Moreover, the mucosal route of immunization induced more efficiently antibodies of both isotypes directed to carbohydrate epitopes. The differences between immune response generated by i.n. and parenteral immunizations should be taken into consideration, particularly in those cases in which protective antigens are glycosylated.

  6. Development and characterization of surface modified PLGA nanoparticles for nasal vaccine delivery: effect of mucoadhesive coating on antigen uptake and immune adjuvant activity.

    PubMed

    Pawar, Dilip; Mangal, Sharad; Goswami, Roshan; Jaganathan, K S

    2013-11-01

    In this study, the efficacy of mucoadhesive polymers, i.e., chitosan and glycol chitosan as a mucoadhesive coating material in nasal vaccine delivery was investigated. The Hepatitis B surface Antigen (HBsAg) encapsulated PLGA, chitosan coated PLGA (C-PLGA), and Glycol chitosan coated PLGA (GC-PLGA) nanoparticles (NPs) were prepared. The formulations were characterized for particle size, shape, surface charge, and entrapment efficiency. The mucoadhesive ability of coated and non-coated NPs was determined using in vitro mucoadhesion and nasal clearance test. In addition, the systemic uptake and bio-distribution were also evaluated to understand the fate of NPs following nasal delivery. The immuno-adjuvant ability of various formulations was compared by measuring specific antibody titer in serum and secretory. The results indicated that PLGA NPs exhibit negative surface charge, whereas C-PLGA and GC-PLGA NPs exhibited positive surface charge. The GC-PLGA NPs demonstrated lower clearance and better local and systemic uptake compared to chitosan coated and uncoated PLGA NPs. In vivo immunogenicity studies indicated that GC-PLGA NPs could induce significantly higher systemic and mucosal immune response compared to PLGA and C-PLGA NPs. In conclusion, GC-PLGA NPs could be a promising carrier adjuvant for the nasal vaccine delivery for inducing a potent immune response at mucosal surface(s) and systemic circulation.

  7. Effect of monoclonal antibodies anti-2H9, anti-IRac, and anti-HeFi-1 on the surface antigens of Reed-Sternberg cells.

    PubMed

    Hsu, S M; Ho, Y S; Hsu, P L

    1987-11-01

    The interaction of 3 monoclonal antibodies (MAbs)--anti-HeFi-1, anti-2H9, and anti-IRac--with Reed-Sternberg (RS) cells was examined in vitro. MAb anti-2H9 induced specific modulation of the corresponding antigen (Ag) from the cell surface. Cell-bound anti-2H9 antibody (Ab)-Ag complexes rapidly disappeared during incubation of cells at 37 degrees C for 2-4 hours. This early loss was followed by reappearance of the Ag on the cell surface within 4-12 hours. In contrast, anti-HeFi-1 and anti-IRac bound avidly to the surface of RS cells and persisted for more than 3 days. During this period, there were no significant changes in the expression of HeFi-1 or IRac Ag on the surface of tumor cells. Cell surface-bound Abs were distributed uniformly, and there was no evidence of microaggregation, as determined by electron microscopy. None of the 3 MAbs was directly cytotoxic or exhibited complement-mediated cytotoxicity. On the basis of these findings (persistence of anti-HeFi-1 and anti-IRac on the cell surface), these 2 MAbs may be suitable for immunoimaging and immunotherapy for Hodgkin's disease.

  8. Plasma Carotenoids and Tocopherols in Relation to Prostate-specific Antigen (PSA) Levels Among Men with Biochemical Recurrence of Prostate Cancer

    PubMed Central

    Antwi, Samuel; Steck, Susan E.; Zhang, Hongmei; Stumm, Lareissa; Zhang, Jiajia; Hurley, Thomas G.; Hebert, James R.

    2015-01-01

    Background Although men presenting with clinically localized prostate cancer (PrCA) often are treated with radical prostatectomy or radiation therapy with curative intent, about 25–40% develop biochemically recurrent PrCA within 5 years of treatment, which has no known cure. Studies suggest that carotenoid and tocopherol intake may be associated with PrCA risk and progression. We examined plasma carotenoid and tocopherol levels in relation to prostate-specific antigen (PSA) levels among men with PSA-defined biochemical recurrence of PrCA. Methods Data analyzed were from a 6-month diet, physical activity and stress-reduction intervention trial conducted in South Carolina among biochemically recurrent PrCA patients (n=39). Plasma carotenoids and tocopherol levels were measured using high-performance liquid chromatography (HPLC). Linear regression was used to estimate least-square means comparing PSA levels of men with high versus low carotenoid/tocopherol levels, adjusting for covariates. Results After adjusting for baseline PSA level, plasma cis-lutein/zeaxanthin level at 3 months was related inversely to PSA level at 3 months (P=0.0008), while α-tocopherol (P=0.01), β-cryptoxanthin (P=0.01), and all-trans-lycopene (P=0.004) levels at 3 months were related inversely to PSA levels at 6-months. Percent increase in α-tocopherol and trans-β-carotene levels from baseline to month 3 were associated with lower PSA levels at 3 and 6 months. Percent increase in β−cryptoxanthin, cislutein/zeaxanthin and all-trans-lycopene were associated with lower PSA levels at 6 months only. Conclusions Certain plasma carotenoids and tocopherols were related inversely to PSA levels at various timepoints, suggesting that greater intake of foods containing these micronutrients might be beneficial to men with PSA-defined PrCA recurrence. PMID:26165176

  9. Immunity to onchocerciasis: cells from putatively immune individuals produce enhanced levels of interleukin-5, gamma interferon, and granulocyte-macrophage colony-stimulating factor in response to Onchocerca volvulus larval and male worm antigens.

    PubMed

    Turaga, P S; Tierney, T J; Bennett, K E; McCarthy, M C; Simonek, S C; Enyong, P A; Moukatte, D W; Lustigman, S

    2000-04-01

    Antigen-specific interleukin-5 (IL-5), gamma interferon (IFN-gamma), and granulocyte-macrophage colony-stimulating factor (GM-CSF) responses in individuals living in an area of hyperendemicity for onchocerciasis in Cameroon were examined. The responses against antigens prepared from Onchocerca volvulus third-stage larvae (L3), molting L3 (mL3), and crude extract from adult males (M-OvAg) were compared to the responses against antigens from adult female worms and skin microfilariae. Cytokine responses for the putatively immune individuals (PI) and the infected individuals (INF) were compared. A differential cytokine profile of IL-5 (Th2 phenotype) and IFN-gamma (Th1 phenotype) was found in these individuals in response to the antigens. In both the PI and the INF, Th2 responses against all the antigens tested were dominant. However, in the PI group as a whole, there was an enhanced Th2 response against the larval antigens and the adult male and adult female antigens, and a Th1 response in a subgroup of the PI (27 to 54.5%) against L3, mL3, and M-OvAg antigens was present. While the PI produced significantly higher levels of GM-CSF against L3, mL3, and M-OvAg antigens than the INF, there was no difference in the GM-CSF responses of the groups against the other antigens. The present study indicated that, in comparison to the INF, the PI have distinct larva-specific and adult male-specific cytokine responses, thus supporting the premise that immunological studies of the PI would lead to the identification of immune mechanisms and the target genes that play a role in protective immunity.

  10. The major parasite surface antigen associated with human resistance to schistosomiasis is a 37-kD glyceraldehyde-3P-dehydrogenase

    PubMed Central

    1989-01-01

    Schistosomiasis, due to Schistosoma mansoni, is a major health problem in many subtropical countries, and major efforts are being made to define a vaccine. In this regard, we have reported that sera from subjects with low susceptibility to infection by S. mansoni react with a major larval surface antigen (P-37), having an apparent molecular mass of 37 kD, against which sera of susceptible individuals show little reactivity. We have now cloned the cDNA for this antigen by screening a schistosome cDNA expression library with antibodies against the purified protein. The selected cDNAs encode a protein that is specifically identified by immune human sera containing antibodies against P-37, while sera exhibiting low or no reactivity toward P-37 fail to recognize the recombinant protein. The cloned cDNAs hybridize with a 1.2-kb RNA that is the transcript of a single copy gene. This RNA directs the synthesis of a 36.5-kD polypeptide that is precipitated by sera from the most resistant subjects. The amino acid sequence of the encoded polypeptide shows homology with the glycolytic enzyme Glyceraldehyde-3P-dehydrogenase (72.5% of positional identity with human Glyceraldehyde-3P-dehydrogenase). Antibodies against the recombinant protein identified P-37 on the larva. These findings, together with other reports, indicate that a number of conserved proteins may be major targets of host-protective immunity against S. mansoni. The hypothesis is discussed that genetic restriction of the immune response to these antigens may occur in heterogeneous human populations because of the limited number of T cell epitopes carried by these host-like proteins. Such genetic effects might allow parasite transmission through nonresponder (susceptible) individuals. This hypothesis and the protective properties of P-37 can now be tested using the recombinant protein and synthetic peptides derived from selected regions of the polypeptide chain. PMID:2584935

  11. Truncated surface protective antigen (SpaA) of Erysipelothrix rhusiopathiae serotype 1a elicits protection against challenge with serotypes 1a and 2b in pigs.

    PubMed

    Imada, Y; Goji, N; Ishikawa, H; Kishima, M; Sekizaki, T

    1999-09-01

    Erysipelothrix rhusiopathiae is a causal agent of swine erysipelas, which is of economic importance in the swine industry by virtue of causing acute septicemia, chronic arthritis, and endocarditis. However, little is known about the genetic properties of its protective antigens. Recently, a surface protective antigen (SpaA) gene was identified from serotype 2 in a mouse model. We cloned spaA from virulent strain Fujisawa (serotype 1a) and determined that the N-terminal 342 amino acids without C-terminal repeats of 20 amino acids have the ability to elicit protection in mice. Fusions of 342 amino acids of Fujisawa SpaA and histidine hexamer (HisSpa1.0) protected pigs against challenge with both serotype 1 and serotype 2, the most important serotypes in the swine industry. Pigs immunized with HisSpa1.0 reacted well with both HisSpa1.0 and intact SpaA by enzyme-linked immunosorbent assay and immunoblotting. Serum collected at the time of challenge from a pig immunized with HisSpa1. 0 markedly enhanced the in vitro phagocytic and killing activity of pig neutrophils against the bacteria. DNA sequences of protective regions of spaA genes from five strains of serotypes 1 and 2 were almost identical. The full DNA sequences also seemed to be conserved among strains of all 12 serotype reference strains harboring the spaA gene by restriction fragment length polymorphism analysis of PCR products. These results indicates that SpaA is a common protective antigen of serotypes 1 and 2 of E. rhusiopathiae in swine and will be a useful tool for development of new types of vaccines and diagnostic tools for effective control of the disease.

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

    SciTech Connect

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

    2013-08-23

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

  13. Expressing Redundancy among Linear-Epitope Sequence Data Based on Residue-Level Physicochemical Similarity in the Context of Antigenic Cross-Reaction

    PubMed Central

    2016-01-01

    Epitope-based design of vaccines, immunotherapeutics, and immunodiagnostics is complicated by structural changes that radically alter immunological outcomes. This is obscured by expressing redundancy among linear-epitope data as fractional sequence-alignment identity, which fails to account for potentially drastic loss of binding affinity due to single-residue substitutions even where these might be considered conservative in the context of classical sequence analysis. From the perspective of immune function based on molecular recognition of epitopes, functional redundancy of epitope data (FRED) thus may be defined in a biologically more meaningful way based on residue-level physicochemical similarity in the context of antigenic cross-reaction, with functional similarity between epitopes expressed as the Shannon information entropy for differential epitope binding. Such similarity may be estimated in terms of structural differences between an immunogen epitope and an antigen epitope with reference to an idealized binding site of high complementarity to the immunogen epitope, by analogy between protein folding and ligand-receptor binding; but this underestimates potential for cross-reactivity, suggesting that epitope-binding site complementarity is typically suboptimal as regards immunologic specificity. The apparently suboptimal complementarity may reflect a tradeoff to attain optimal immune function that favors generation of immune-system components each having potential for cross-reactivity with a variety of epitopes. PMID:27274725

  14. Laparoscopic resection of a benign true cyst of the spleen with the harmonic scalpel producing high levels of CA 19-9 and carcinoembryonic antigen.

    PubMed

    Sardi, A; Ojeda, H F; King, D

    1998-12-01

    Primary true cysts of the spleen constitute 10 per cent of all nonparasitic cysts of the spleen. These cysts have been managed with total or partial splenectomy or marsupialization. Previous series reported elevated serum levels of CA 19-9 and carcinoembryonic antigen (CEA). A 16-year-old male presented with left upper quadrant pain and was found to have a mass on palpation. An ultrasound and a CT scan of the abdomen showed a 20-cm cystic lesion in the spleen. The CA 19-9 and CEA serum levels were 1264 units/ml (nl <33 units/ml) and 7.5 ng/ml (nl <3.1 ng/ml), respectively. Laparoscopic resection of the cyst using the harmonic scalpel was performed. The pathology showed a benign true epithelial cyst of the spleen, with strongly immunoperoxidase stains for keratin, CA 19-9, and CEA. Levels of CA 19-9 and CEA in the cystic fluid were 48,275 units/ml and 96.6 ng/ml, respectively. This is the first case reported in the literature of a true benign splenic cyst producing high levels of CEA and CA 19-9 in serum and cystic fluid. The patient was discharged home on the 1st postoperative day without problems and returned to his normal daily activities with the 1st week after surgery. The CA 19-9 and CEA serum levels returned to normal levels.

  15. Serum heat shock protein 27 antigen and antibody levels appear to be related to the macrovascular complications associated with insulin resistance: a pilot study.

    PubMed

    Burut, Dayangku Fatiha Pengiran; Borai, Anwar; Livingstone, Callum; Ferns, Gordon

    2010-07-01

    Heat shock protein 27 (Hsp27) is over-expressed when cells are exposed to stressful conditions that include oxidative stress. Oxidative stress has been implicated in the pathogenesis of cardiovascular disease (CVD), diabetes and insulin resistance. We have investigated the concentrations of serum Hsp27 antigen and antibodies in subjects from different glycaemic categories, who either did or did not have established CVD. Serum Hsp27 antigen and antibody levels (immunoglobulins M and G (IgM and IgG)) were determined by enzyme-linked immunosorbent assays (ELISAs) in 68 individuals: 26 with normal glucose tolerance (NGT), 10 with (+) and 16 without (-) a history of CVD and 42 individuals with varying degrees of glucose intolerance (GI; 21 with and 21 without a history of CVD). Insulin sensitivity was determined in each subject using indices derived from the homeostasis model assessment of sensitivity and the insulin sensitivity index for glycaemia. Serum Hsp27 concentrations were significantly higher in GI (+CVD) subjects compared to GI (-CVD) subjects (p = 0.03), NGT (-CVD) subjects (p = 0.02) and NGT (+CVD) subjects (p = 0.04) and were positively correlated to fasting plasma glucose for all subjects (r = 0.28, p = 0.03). IgM antibody levels were significantly higher in GI (+CVD) subjects compared to NGT (-CVD) group (p = 0.02) and were inversely related to fasting insulin concentrations (r = -0.27, p = 0.04) and the 2-h insulin concentrations (r = -0.29, p = 0.03) for all subjects. Serum IgG antibody levels were higher in GI (+CVD) group compared to GI (-CVD) group (p = 0.06). In conclusion, Hsp27 and its antibody concentrations appear to relate to the presence of cardiovascular complications in patients with GI.

  16. A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface.

    PubMed

    de Jong, Rob N; Beurskens, Frank J; Verploegen, Sandra; Strumane, Kristin; van Kampen, Muriel D; Voorhorst, Marleen; Horstman, Wendy; Engelberts, Patrick J; Oostindie, Simone C; Wang, Guanbo; Heck, Albert J R; Schuurman, Janine; Parren, Paul W H I

    2016-01-01

    IgG antibodies can organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell killing. Here, we translated this natural concept into a novel technology platform (HexaBody technology) for therapeutic antibody potentiation. We identified mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from hematological and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. Both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD20 antibody that was ineffective in complement activation, while retaining its ability to induce apoptosis. The identified IgG1 Fc backbones provide a novel platform for the generation of therapeutics with enhanced effector functions that only become activated upon binding to target cell-expressed antigen.

  17. A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface

    PubMed Central

    Verploegen, Sandra; Strumane, Kristin; van Kampen, Muriel D.; Voorhorst, Marleen; Horstman, Wendy; Engelberts, Patrick J.; Oostindie, Simone C.; Wang, Guanbo; Heck, Albert J. R.; Schuurman, Janine; Parren, Paul W. H. I.

    2016-01-01

    IgG antibodies can organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell killing. Here, we translated this natural concept into a novel technology platform (HexaBody technology) for therapeutic antibody potentiation. We identified mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from hematological and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. Both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD20 antibody that was ineffective in complement activation, while retaining its ability to induce apoptosis. The identified IgG1 Fc backbones provide a novel platform for the generation of therapeutics with enhanced effector functions that only become activated upon binding to target cell–expressed antigen. PMID:26736041

  18. Application and expression of Toxoplasma gondii surface antigen 2 (SAG2) and rhoptry protein 2 (ROP2) from recombinant Escherichia coli strain.

    PubMed

    Yan, Hua; Yan, Huishen; Tao, Yong; Chen, Hongju; Li, Guocai; Gong, Weijuan; Jiao, Hongmei; Tian, Fang; Ji, Mingchun

    2012-06-01

    The gene encoding surface antigen 2 (SAG2) or rhoptry protein 2 (ROP2) of Toxoplasma gondii was cloned into the plasmid pGEX-4T-1 and subsequently expressed in Escherichia coli as a glutathione-s-transferase (GST) fusion protein. The characteristics of purified GST-SAG2 or GST-ROP2 were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. The specific IgG of a panel of serum samples provided by the National Institute for the Control of Pharmaceutical and Biological Products were tested with commercial ELISA and the lateral flow immunoassay (LFIA) based on GST-SAG2, GST-ROP2 or GST-SAG2+ROP2. A total of 1096 sera and saliva samples from pregnant women were tested by GST-SAG2+ROP2-LFIA. In total, 20 T. gondii IgM positive sera (1.82%), 81 T. gondii IgG positive sera (7.4%) and 23 T. gondii IgA positive saliva (2.1%) were finally confirmed. The SAG2+ROP2 specific IgG and IFN-γ producing CD8+ T cells were induced in mice immunised with GST-SAG2+ROP2. The results indicate that GST-SAG2+ROP2 protein can be used as an antigen for diagnosing T. gondii infection and provide a strategy for development of subunit vaccines for protection against T. gondii infection.

  19. Hepatitis B virus-associated diffuse large B-cell lymphoma: unique clinical features, poor outcome, and hepatitis B surface antigen-driven origin.

    PubMed

    Deng, Lijuan; Song, Yuqin; Young, Ken H; Hu, Shimin; Ding, Ning; Song, Weiwei; Li, Xianghong; Shi, Yunfei; Huang, Huiying; Liu, Weiping; Zheng, Wen; Wang, Xiaopei; Xie, Yan; Lin, Ningjing; Tu, Meifeng; Ping, Lingyan; Ying, Zhitao; Zhang, Chen; Sun, Yingli; Zhu, Jun

    2015-09-22

    While the epidemiologic association between hepatitis B virus (HBV) infection and diffuse large B-cell lymphoma (DLBCL) is established, little is known more than this epidemiologic evidence. We studied a cohort of 587 patients with DLBCL for HBV infection status, clinicopathologic features, and the immunoglobulin variable region in HBV surface antigen (HBsAg)-positive patients. Eighty-one (81/587, 13.8%) patients were HBsAg-positive. Compared with HBsAg-negative DLBCL, HBsAg-positive DLBCL displayed a younger median onset age (45 vs. 55 years), more frequent involvement of spleen or retroperitoneal lymph node (40.7% vs. 16.0% and 61.7% vs. 31.0% respectively, both p < 0.001), more advanced disease (stage III/IV: 76.5% vs 59.5%, p = 0.003), and significantly worse outcome (2-year overall survival: 47% versus 70%, p < 0.001). In HBsAg-positive DLBCL patients, almost all (45/47, 96%) amino acid sequences of heavy and light chain complementarity determining region 3 exhibited a high homology to antibodies specific for HBsAg, and the majority (45/50, 90%) of IgHV and IgLV genes were mutated. We conclude that 13.8% of DLBCL cases are HBV-associated in HBV-endemic China and show unique clinical features and poor outcomes. Furthermore, our study strongly suggests that HBV-associated DLBCL might arise from HBV antigen-selected B cells.

  20. Array-in-well platform-based multiplex assay for the simultaneous detection of anti-HIV- and treponemal-antibodies, and Hepatitis B surface antigen.

    PubMed

    Talha, Sheikh M; Saviranta, Petri; Hattara, Liisa; Vuorinen, Tytti; Hytönen, Jukka; Khanna, Navin; Pettersson, Kim

    2016-02-01

    Multiplex assays detecting sets of related clinical analytes simultaneously can save considerable amount of time and resources. Array-in-well (AIW) is a powerful platform for the multiplex detection of different analytes where microarrays can be printed at the bottom of microtiter wells, thus combining the potential of microarrays with the ease of handling microtiter wells. We have developed a single-step AIW assay for the simultaneous screening of HIV, Treponema pallidum subspecies pallidum (causing syphilis) and Hepatitis B virus infections targeting the specific detection of anti-HIV- and treponemal-antibodies and Hepatitis B surface antigen (HBsAg), respectively, using two different fluorescent label technologies i.e. DyLight 633 and europium nanoparticle. Double-antigen assay formats were used for anti-HIV- and treponemal-antibody detection that can simultaneously detect both IgG and IgM, and thus reduce the window period of detection. AIW assay was evaluated with well characterized serum/plasma samples (n=111), and the qualitative results were in near complete agreement with those of the reference assays. The AIW assay exhibited 100% sensitivities for all three analytes, and 100% specificities for anti-HIV antibodies and HBsAg, and 98.6% specificity for treponemal antibodies. The limit of detection of HBsAg in AIW assay was 0.18 ng/ml. This high performing AIW assay has the potential to be used as a multiplex screening test for these three infections.

  1. In silico design, cloning and high level expression of L7/L12-TOmp31 fusion protein of Brucella antigens

    PubMed Central

    Golshani, Maryam; Rafati, Sima; Jahanian-Najafabadi, Ali; Nejati-Moheimani, Mehdi; Siadat, Seyed Davar; Shahcheraghi, Fereshteh; Bouzari, Saeid

    2015-01-01

    Globally, Brucella melitensis and B. abortus are the most common cause of human brucellosis. The outer membrane protein 31 (Omp31) and L7/L12 are immunodominant and protective antigens conserved in human Brucella pathogens which are considered as potential vaccine candidates. We aimed to design the fusion protein from Brucella L7/L12 and truncated Omp31proteins, in silico, clone the fusion in pET28a vector, and express it in Escherichia coli host. Two possible fusion forms, L7/L12-TOmp31 and TOmp31-L7/L12 were subjected to in silico modeling and analysis. Analysis and validation of the fusion proteins with three dimensional (3D) models showed that both models are in the range of native proteins. However, L7/L12-Tomp31 structure was more valid than the TOmp31-L7/L12 model and subjected to in vitro production. The major histocompatibility complex (MHC II) epitope mapping using IEDB database indicated that the model contained good MHC II binders. The L7/L12-TOmp31 coding sequence was cloned in pET28a vector. The integrity of the construct was confirmed by polymerase chain reaction, restriction enzyme mapping, and sequencing. The fusion was successfully expressed in E. coli BL21 (DE3) by induction with isopropyl β-D-thiogalactopyranoside. The rL7/L12-TOmp31 was purified with Ni-NTA column. The yield of the purified rL7/L12-TOmp31 was estimated by Bradford method and found to be 40 mg/L of the culture. Western blotting with anti-His antibody revealed a specific reactivity with purified rL7/L12-TOmp31 produced in E. coli and showed the functional expression in the prokaryotic system. In this study, a new protein vaccine candidate against brucellosis was constructed with the help of bioinformatics tools and the construct was expressed in the bacterial host. Studies evaluating the immunogenicity and cross-protection of this fusion protein against B. melitensis and B. abortus are underway. PMID:26752992

  2. In silico design, cloning and high level expression of L7/L12-TOmp31 fusion protein of Brucella antigens.

    PubMed

    Golshani, Maryam; Rafati, Sima; Jahanian-Najafabadi, Ali; Nejati-Moheimani, Mehdi; Siadat, Seyed Davar; Shahcheraghi, Fereshteh; Bouzari, Saeid

    2015-01-01

    Globally, Brucella melitensis and B. abortus are the most common cause of human brucellosis. The outer membrane protein 31 (Omp31) and L7/L12 are immunodominant and protective antigens conserved in human Brucella pathogens which are considered as potential vaccine candidates. We aimed to design the fusion protein from Brucella L7/L12 and truncated Omp31proteins, in silico, clone the fusion in pET28a vector, and express it in Escherichia coli host. Two possible fusion forms, L7/L12-TOmp31 and TOmp31-L7/L12 were subjected to in silico modeling and analysis. Analysis and validation of the fusion proteins with three dimensional (3D) models showed that both models are in the range of native proteins. However, L7/L12-Tomp31 structure was more valid than the TOmp31-L7/L12 model and subjected to in vitro production. The major histocompatibility complex (MHC II) epitope mapping using IEDB database indicated that the model contained good MHC II binders. The L7/L12-TOmp31 coding sequence was cloned in pET28a vector. The integrity of the construct was confirmed by polymerase chain reaction, restriction enzyme mapping, and sequencing. The fusion was successfully expressed in E. coli BL21 (DE3) by induction with isopropyl β-D-thiogalactopyranoside. The rL7/L12-TOmp31 was purified with Ni-NTA column. The yield of the purified rL7/L12-TOmp31 was estimated by Bradford method and found to be 40 mg/L of the culture. Western blotting with anti-His antibody revealed a specific reactivity with purified rL7/L12-TOmp31 produced in E. coli and showed the functional expression in the prokaryotic system. In this study, a new protein vaccine candidate against brucellosis was constructed with the help of bioinformatics tools and the construct was expressed in the bacterial host. Studies evaluating the immunogenicity and cross-protection of this fusion protein against B. melitensis and B. abortus are underway.

  3. A protective immune response is generated in rainbow trout by an OmpH-like surface antigen (P18) of Flavobacterium psychrophilum.

    PubMed

    Dumetz, Fabien; Duchaud, Eric; LaPatra, Scott E; Le Marrec, Claire; Claverol, Stéphane; Urdaci, Maria-C; Le Hénaff, Michel

    2006-07-01

    Investigations of the surface characteristics of Flavobacterium psychrophilum, an important pathogen of fish, assisted us in identifying a surface protein termed P18. In the current study, we developed a simple and efficient procedure for the purification of this protein by a two-step method. First, P18 was selectively released from flavobacteria by a heat-HEPES treatment of the cells and then subjected to anion-exchange high-performance liquid chromatography. De novo sequencing was used to generate a fragmented peptide spectrum from purified P18. Comparison of two obtained peptide sequences with a partial genome sequence of F. psychrophilum (INRA, Jouy-en-Josas, France) identified one gene encoding a 166-amino-acid OmpH-like protein that mostly likely undergoes N-terminal cleavage of the 23-residue signal peptide. The susceptibility of the OmpH-like protein to proteinase K treatment and the bacteriostatic/bactericidal activities of anti-OmpH-like protein antibodies indicated that this protein is actually exposed on the surface of F. psychrophilum. Vaccination trials showed that the OmpH-like protein can induce a high titer of anti-OmpH-like protein antibodies which are protective. Taken together, these results suggest that this surface protein produced by F. psychrophilum could be used in future vaccine development as a promising candidate antigen.

  4. A Protective Immune Response Is Generated in Rainbow Trout by an OmpH-Like Surface Antigen (P18) of Flavobacterium psychrophilum

    PubMed Central

    Dumetz, Fabien; Duchaud, Eric; LaPatra, Scott E.; Le Marrec, Claire; Claverol, Stéphane; Urdaci, Maria-C.; Le Hénaff, Michel

    2006-01-01

    Investigations of the surface characteristics of Flavobacterium psychrophilum, an important pathogen of fish, assisted us in identifying a surface protein termed P18. In the current study, we developed a simple and efficient procedure for the purification of this protein by a two-step method. First, P18 was selectively released from flavobacteria by a heat-HEPES treatment of the cells and then subjected to anion-exchange high-performance liquid chromatography. De novo sequencing was used to generate a fragmented peptide spectrum from purified P18. Comparison of two obtained peptide sequences with a partial genome sequence of F. psychrophilum (INRA, Jouy-en-Josas, France) identified one gene encoding a 166-amino-acid OmpH-like protein that mostly likely undergoes N-terminal cleavage of the 23-residue signal peptide. The susceptibility of the OmpH-like protein to proteinase K treatment and the bacteriostatic/bactericidal activities of anti-OmpH-like protein antibodies indicated that this protein is actually exposed on the surface of F. psychrophilum. Vaccination trials showed that the OmpH-like protein can induce a high titer of anti-OmpH-like protein antibodies which are protective. Taken together, these results suggest that this surface protein produced by F. psychrophilum could be used in future vaccine development as a promising candidate antigen. PMID:16820479

  5. The perception of surface layout during low level flight

    NASA Technical Reports Server (NTRS)

    Perrone, John A.

    1991-01-01

    Although it is fairly well established that information about surface layout can be gained from motion cues, it is not so clear as to what information humans can use and what specific information they should be provided. Theoretical analyses tell us that the information is in the stimulus. It will take more experiments to verify that this information can be used by humans to extract surface layout from the 2D velocity flow field. The visual motion factors that can affect the pilot's ability to control an aircraft and to infer the layout of the terrain ahead are discussed.

  6. Cyclosporine A inhibits colorectal cancer proliferation probably by regulating expression levels of c-Myc, p21(WAF1/CIP1) and proliferating cell nuclear antigen.

    PubMed

    Masuo, Takashige; Okamura, Shinichi; Zhang, Yajing; Mori, Masatomo

    2009-11-18

    The present study investigated the role of calcineurin (CaN) in the proliferation of human colorectal cancers. CaN activity and protein expression were increased in human colorectal cancers. Nuclear transcription factor NFAT, a physiological substrate for CaN, was activated in human colon cancer specimen as well as in the human colon cancer cell lines. CaN inhibitor cyclosporine A (CsA) reduced cell growth in these cell lines. CsA decreased the expressions of c-Myc and the proliferating cell nuclear antigen (PCNA) but also increased p21(WAF1/CIP1) expression. Our results suggest that CaN promotes colorectal cancer proliferation probably by regulating levels of c-Myc, p21(WAF1/CIP1), and PCNA.

  7. Sero-Prevalence of Hepatitis B Surface Antigen Amongst Pregnant Women Attending an Antenatal Clinic, Volta Region, Ghana

    PubMed Central

    Dassah, Sylvester; Lokpo, Sylvester; Ameke, Louise; Noagbe, Mark; Adatara, Peter; Hagan, Oheneba; Binka, Fred

    2017-01-01

    Hepatitis B virus (HBV) infection remains a global challenge, although there is currently a safe and effective vaccine available. HBV prevalence in Ghana is not well documented, but vary regionally from 4.8% to 12.3% in the general population, 10.8% to 12.7% in blood donors and about 10.6% in pregnant women. This puts Ghana among the high endemic countries in Africa. The study objective was to determine the sero-prevalence of HBs antigen (Ag) and HBeAg among pregnant women in the Ho municipality. Two hundred and eigh participants (pregnant women), attending Ho Municipal antenatal clinic were enrolled into the study. This study recorded a HBsAg sero-prevalence rate of 2.4% among the pregnant women, with primigravida pregnant women recording (0.98%) and multigravida (1.42%). The prevalence of HBsAg among the pregnant women can be classified as Low Intermediate; therefore there is still the need for routine screening of pregnant women during antenatal visits. Amongst HBsAg positives, HBeAg positivity was significantly high (40% of all HBsAg positive women), which suggests high chances of carrier and vertical transmission (mother to child) state. PMID:28299162

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

    PubMed

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

    2002-02-01

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

  9. Solitary recurrence of castration-resistant prostate cancer with low or undetectable levels of prostate specific antigen salvaged with local ablative radiation therapy: A case report

    PubMed Central

    WANG, CHIACHIEN JAKE; YING, JAMES; KAPUR, PAYAL; WOHLFELD, BRYAN; ROEHRBORN, CLAUS; KIM, DONG W. NATHAN

    2016-01-01

    Prostate cancer recurrences are usually first detected by increased levels of prostate specific antigen (PSA), and systemic therapy is often initiated if distant metastasis is confirmed. However, low or nearly undetectable levels of PSA in the modern era of ultrasensitive PSA assay may be difficult to interpret in patients with a history of prostate cancer. Deciding whether to initiate additional systemic therapy in limited indolent metastatic disease while balancing the quality of life of the patient and ensuring the oncologic control of the disease may be challenging. In the present study, the case of a biopsy-confirmed solitary spine recurrence of prostate cancer with nearly undetectable but persistent levels of PSA (0.05 ng/ml) is reported. Treatment of the recurrence with local ablative radiotherapy improved the pain experienced by the patient, and reduced his levels of PSA to undetectable limits (<0.05 ng/ml). Repeated imaging analysis, PSA assay and clinical assessment demonstrated durable control of the disease without the requirement for additional systemic treatments. The present case highlighted the importance of initiating appropriate work-up according to the clinical scenario. Local treatment for solitary or oligometastatic recurrence of prostate cancer may enhance the effectiveness of current therapeutic strategies and benefit certain patients. PMID:26870272

  10. Changes in Serum IgA Antibody Levels against the Glycopeptidolipid Core Antigen during Antibiotic Treatment of Mycobacterium avium Complex Lung Disease.

    PubMed

    Jhun, Byung Woo; Kim, Su-Young; Park, Hye Yun; Jeon, Kyeongman; Shin, Sung Jae; Koh, Won-Jung

    2017-03-28

    We evaluated serial changes in the levels of serum immunoglobulin A (IgA) antibody to the glycopeptidolipid (GPL) core antigen during antibiotic treatment in 57 patients with M. avium complex (MAC) lung disease, at baseline (T0) and after 3 months (T3) and 6 months (T6) of treatment. The median patient age was 59 years and 37 (65%) were female. Etiologic organisms included M. avium in 32 (56%) patients and M. intracellulare in 25 (44%). Seven (12%) patients had the fibrocavitary form of the disease on computed tomography. After 12 months of treatment, 42 (74%) patients achieved favorable responses, whereas 15 (26%) patients had unfavorable responses defined as no sputum culture conversion within 12 months of treatment. The initial median serum anti-GPL IgA levels in the 57 patients was 3.50 U/mL, and measurements at T0 (median 3.50 U/mL), T3 (median 2.71 U/mL), and T6 (median 2.61 U/mL) revealed significant decreases following treatment (P < 0.001). Multivariate analysis showed that an initially elevated anti-GPL IgA level (> 3.50 U/mL) was associated with an unfavorable response (P = 0.049). Our data suggest that elevated anti-GPL IgA levels may reflect disease activity, which may help to predict treatment response in patients with MAC lung disease.

  11. Atomic-level imaging, processing and characterization of semiconductor surfaces

    DOEpatents

    Kazmerski, L.L.

    1995-08-22

    A method for selecting and removing single specific atoms from a solid material surface uses photon biasing to break down bonds that hold the selected atom in the lattice and to reduce barrier effects that hold the atom from transferring to a probe. The photon bias is preferably light or other electromagnetic radiation with a wavelength and frequency that approximately matches the wave function of the target atom species to be removed to induce high energy, selective thermionic-like vibration. An electric field potential is then applied between the probe and the surface of the solid material to pull the atom out of the lattice and to transfer the atom to the probe. Different extrinsic atoms can be installed in the lattice sites that are vacated by the removed atoms by using a photon bias that resonates the extrinsic atom species, reversing polarity of the electric field, and blowing gas comprising the extrinsic atoms through a hollow catheter probe. 8 figs.

  12. Atomic-level imaging, processing and characterization of semiconductor surfaces

    DOEpatents

    Kazmerski, Lawrence L.

    1995-01-01

    A method for selecting and removing single specific atoms from a solid material surface uses photon biasing to break down bonds that hold the selected atom in the lattice and to reduce barrier effects that hold the atom from transferring to a probe. The photon bias is preferably light or other electromagnetic radiation with a wavelength and frequency that approximately matches the wave function of the target atom species to be removed to induce high energy, selective thermionic-like vibration. An electric field potential is then applied between the probe and the surface of the solid material to pull the atom out of the lattice and to transfer the atom to the probe. Different extrinsic atoms can be installed in the lattice sites that are vacated by the removed atoms by using a photon bias that resonates the extrinsic atom species, reversing polarity of the electric field, and blowing gas comprising the extrinsic atoms through a hollow catheter probe.

  13. Is there any association between National Institute of Health category IV prostatitis and prostate-specific antigen levels in patients with low-risk localized prostate cancer?

    PubMed Central

    Doluoglu, Omer Gokhan; Ceylan, Cavit; Kilinc, Fatih; Gazel, Eymen; Resorlu, Berkan; Odabas, Oner

    2016-01-01

    ABSTRACT Purpose We investigated the association between National Institute of Health category IV prostatitis and prostate-specific antigen levels in patients with low-risk localized prostate cancer. Materials and Methods The data of 440 patients who had undergone prostate biopsies due to high PSA levels and suspicious digital rectal examination findings were reviewed retrospectively. The patients were divided into two groups based on the presence of accompanying NIH IV prostatitis. The exclusion criteria were as follows: Gleason score>6, PSA level>20ng/mL, >2 positive cores, >50% cancerous tissue per biopsy, urinary tract infection, urological interventions at least 1 week previously (cystoscopy, urethral catheterization, or similar procedure), history of prostate biopsy, and history of androgen or 5-alpha reductase use. All patient's age, total PSA and free PSA levels, ratio of free to total PSA, PSA density and prostate volume were recorded. Results In total, 101 patients were included in the study. Histopathological examination revealed only PCa in 78 (77.2%) patients and PCa+NIH IV prostatitis in 23 (22.7%) patients. The median total PSA level was 7.4 (3.5–20.0) ng/mL in the PCa+NIH IV prostatitis group and 6.5 (0.6–20.0) ng/mL in the PCa group (p=0.67). The PSA level was≤10ng/mL in 60 (76.9%) patients in the PCa group and in 16 (69.6%) patients in the PCa+NIH IV prostatitis group (p=0.32). Conclusions Our study showed no statistically significant difference in PSA levels between patients with and without NIH IV prostatitis accompanying PCa. PMID:27256190

  14. The impact of hypoxemia on serum total and free prostate-specific antigen levels in patients with chronic obstructive pulmonary disease.

    PubMed

    Ozge, Cengiz; Bozlu, Murat; Ozgur, Eylem Sercan; Tek, Mesut; Tunckiran, Ahmet; Muslu, Necati; Ilvan, Ahmet

    2015-05-01

    Prostate-specific antigen (PSA) is the most important biochemical marker in the diagnosis and follow-up of patients with prostate cancer. In recent years, a relationship between PSA levels and hypoxic conditions has been described. However, no study has investigated the PSA levels in patients with chronic obstructive pulmonary disease (COPD). The aim of the present study was to investigate the impact of hypoxemia on serum total (tPSA) and free PSA (fPSA) levels in patients with COPD. Between January 2010 and January 2014, 95 male patients who hospitalized for acute exacerbations of COPD and 80 control subjects were enrolled in the study. Serum tPSA and fPSA levels and f/tPSA ratios were determined in all patients on the first day of hospitalization (exacerbation) and 7 days after the treatment (stable state). Statistical analysis included paired t test and Mann-Whitney U test. No statistically significant differences were found between COPD and control groups with regard to the baseline characteristics, except for smoking status. The levels of serum tPSA and fPSA during exacerbation of COPD were significantly higher than the levels of the stable period (p < 0.01), whereas f/tPSA ratio did not change (p > 0.05). Hypoxemia during acute exacerbation of COPD can cause a rise in serum tPSA and fPSA levels, but f/tPSA ratio is not affected. Acute exacerbation of COPD may be added to list of the events in which PSA measurements must be interpreted with caution.

  15. Increase of the prevalence of hepatitis B virus surface antigen related to immunodeficiency inherent in acquired immune deficiency syndrome (AIDS).

    PubMed

    Ouattara, S A; Meite, M; Aron, Y; Akran, V; Gody, M; Manlan, L K; de-The, G

    1990-01-01

    During the period from January to December 1987, 2,191 serum samples were collected from different groups of the population in the Ivory Coast (1,126 healthy people selected from the general population, 416 blood donors, 112 healthy anti-HIV carriers, 173 AIDS patients, and 364 patients suffering from icterigenic hepatitis) and tested for anti-HIV (HIV-1 and HIV-2) antibodies, HBsAg, HBeAg, delta antigen (HDAg), and anti-delta (anti-HD) antibody. Anti-HIV antibodies were found in 30 (2.6%) of the general population. 55 (13.2%) blood donors, and 93 (25.5%) patients suffering from icterigenic hepatitis. HBsAg was observed in 103 (9.1%) of the general population, 45 (10.8%) blood donors, 15 (13.4%) healthy anti-HIV carriers, 59 (34.1%) AIDS patients, and 45 (40%) icterigenous hepatitis patients. The simultaneous presence of anti-HIV and HBsAg was noted in 4 (0.4%) of the general population, 8 (2%) blood donors, 15 (13.4%) healthy anti-HIV carriers, 59 (34.1%) AIDS cases, and 36 (10%) patients suffering from icterigenic hepatitis. A high prevalence of HBeAg and serological markers of infection by the delta agent were noted in the different groups. HDAg was noted only among AIDS patients or those suffering from icterigenic hepatitis, with a higher frequency among anti-HIV carriers. Our conclusion from this study is that healthy anti-HIV carriers are no more likely to be HBsAg carriers than the HIV-seronegative subjects. However, immunodeficiency induced previously by HIV infection is likely to be responsible for the high prevalence of HBsAg among AIDS patients.

  16. Nonatopic asthma: in vivo airway hyperreactivity adoptively transferred to naive mice by THY-1(+) and B220(+) antigen-specific cells that lack surface expression of CD3.

    PubMed Central

    Geba, G P; Wegner, C D; Wolyniec, W W; Li, Y; Askenase, P W

    1997-01-01

    To investigate the cellular immune events contributing to airway hyperreactivity (AHR), we studied an in vivo mouse model induced by the hapten picryl (trinitrophenyl) chloride (PCl). Mice were immunized by cutaneous contact sensitization with PCl and airway challenged subsequently with picryl sulfonic acid (PSA) antigen (Ag). Increased airway resistance was produced late (24 h) after Ag challenge, disappeared by 48 h, and was associated with no decrease in diffusion capacity. AHR could be produced in PCl immune/ PSA challenged mice on day 7 or even, with challenge, as early as 1 d after contact sensitization, after adoptive transfer of immune cells lacking CD3(+) contact sensitivity effector T cells, or after transfer of Ag-specific lymphoid cells depleted of conventional T lymphocytes with surface determinants for CD3, CD4, CD8, TCR-beta, or TCR-delta molecules. Further experiments showed that development of AHR depended upon transfer of immune cells expressing surface membrane Thy-1 and B220 (CD45RA) determinants. We concluded that a novel population of Ag-specific lymphoid cells with a defined surface phenotype (Thy-1(+), CD3(-), CD4(-), CD8(-), TCR-alphabeta-, TCR-gammadelta-, and CD45RA+) is required in a mouse model for the development of AHR. PMID:9241124

  17. Thermodynamic and kinetic analysis of the interaction between hepatitis B surface antibody and antigen on a gold electrode modified with cysteamine and colloidal gold via electrochemistry.

    PubMed

    He, Yan Bin; Luo, Hong Qun; Li, Nian Bing

    2007-06-15

    Hepatitis B surface antibody (HBsAb) was immobilized to the surface of a gold electrode modified with cysteamine and colloidal gold as matrices to detect hepatitis B surface antigen (HBsAg). Differential pulse voltammetry (DPV) method was used for the investigation of the specific interaction between the immobilized HBsAb and HBsAg in solution, which was followed as a change of peak current in DPV with time. With the modified gold electrode, the differences in affinity of HBsAb with HBsAg at the temperatures of 37 and 40 degrees C were easily distinguished and the kinetic rate constants (k(ass) and k(diss)) and kinetic affinity constant K were determined from the curves of current versus time. In addition, the thermodynamic constants, DeltaG, DeltaH and DeltaS, of the interaction at 37 degrees C were calculated, which were -56.65, -64.54 and -25.45 kJ mol(-1), respectively.

  18. Determination of antigenicity-altering patches on the major surface protein of human influenza A/H3N2 viruses.

    PubMed

    Kratsch, Christina; Klingen, Thorsten R; Mümken, Linda; Steinbrück, Lars; McHardy, Alice C

    2016-01-01

    Human influenza viruses are rapidly evolving RNA viruses that cause short-term respiratory infections with substantial morbidity and mortality in annual epidemics. Uncovering the general principles of viral coevolution with human hosts is important for pathogen surveillance and vaccine design. Protein regions are an appropriate model for the interactions between two macromolecules, but the currently used epitope definition for the major antigen of influenza viruses, namely hemagglutinin, is very broad. Here, we combined genetic, evolutionary, antigenic, and structural information to determine the most relevant regions of the hemagglutinin of human influenza A/H3N2 viruses for interaction with human immunoglobulins. We estimated the antigenic weights of amino acid changes at individual sites from hemagglutination inhibition data using antigenic tree inference followed by spatial clustering of antigenicity-altering protein sites on the protein structure. This approach determined six relevant areas (patches) for antigenic variation that had a key role in the past antigenic evolution of the viruses. Previous transitions between successive predominating antigenic types of H3N2 viruses always included amino acid changes in either the first or second antigenic patch. Interestingly, there was only partial overlap between the antigenic patches and the patches under strong positive selection. Therefore, besides alterations of antigenicity, other interactions with the host may shape the evolution of human influenza A/H3N2 viruses.

  19. Carcinoembryonic Antigen-Related Cell Adhesion Molecule 5 Is an Important Surface Attachment Factor That Facilitates Entry of Middle East Respiratory Syndrome Coronavirus

    PubMed Central

    Chan, Che-Man; Chu, Hin; Wang, Yixin; Wong, Bosco Ho-Yin; Zhao, Xiaoyu; Zhou, Jie; Yang, Dong; Leung, Sze Pui; Chan, Jasper Fuk-Woo; Yeung, Man-Lung; Yan, Jinghua; Lu, Guangwen; Gao, George Fu

    2016-01-01

    ABSTRACT The spike proteins of coronaviruses are capable of binding to a wide range of cellular targets, which contributes to the broad species tropism of coronaviruses. Previous reports have demonstrated that Middle East respiratory syndrome coronavirus (MERS-CoV) predominantly utilizes dipeptidyl peptidase 4 (DPP4) for cell entry. However, additional cellular binding targets of the MERS-CoV spike protein that may augment MERS-CoV infection have not been further explored. In the current study, using the virus overlay protein binding assay (VOPBA), we identified carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) as a novel cell surface binding target of MERS-CoV. CEACAM5 coimmunoprecipitated with the spike protein of MERS-CoV in both overexpressed and endogenous settings. Disrupting the interaction between CEACAM5 and MERS-CoV spike with anti-CEACAM5 antibody, recombinant CEACAM5 protein, or small interfering RNA (siRNA) knockdown of CEACAM5 significantly inhibited the entry of MERS-CoV. Recombinant expression of CEACAM5 did not render nonpermissive baby hamster kidney (BHK21) cells susceptible to MERS-CoV infection. Instead, CEACAM5 overexpression significantly enhanced the attachment of MERS-CoV to the BHK21 cells. More importantly, the entry of MERS-CoV was increased when CEACAM5 was overexpressed in permissive cells, which suggested that CEACAM5 could facilitate MERS-CoV entry in conjunction with DPP4 despite not being able to support MERS-CoV entry independently. Taken together, the results of our study identified CEACAM5 as a novel cell surface binding target of MERS-CoV that facilitates MERS-CoV infection by augmenting the attachment of the virus to the host cell surface. IMPORTANCE Infection with the Middle East respiratory syndrome coronavirus (MERS-CoV) is associated with the highest mortality rate among all known human-pathogenic coronaviruses. Currently, there are no approved vaccines or therapeutics against MERS-CoV infection. The

  20. Deviating the level of proliferating cell nuclear antigen in Trypanosoma brucei elicits distinct mechanisms for inhibiting proliferation and cell cycle progression.

    PubMed

    Valenciano, Ana L; Ramsey, Aaron C; Mackey, Zachary B

    2015-01-01

    The DNA replication machinery is spatially and temporally coordinated in all cells to reproduce a single exact copy of the genome per division, but its regulation in the protozoan parasite Trypanosoma brucei is not well characterized. We characterized the effects of altering the levels of proliferating cell nuclear antigen, a key component of the DNA replication machinery, in bloodstream form T. brucei. This study demonstrated that tight regulation of TbPCNA levels was critical for normal proliferation and DNA replication in the parasite. Depleting TbPCNA mRNA reduced proliferation, severely diminished DNA replication, arrested the synthesis of new DNA and caused the parasites to accumulated in G2/M. Attenuating the parasite by downregulating TbPCNA caused it to become hypersensitive to hydroxyurea. Overexpressing TbPCNA in T. brucei arrested proliferation, inhibited DNA replication and prevented the parasite from exiting G2/M. These results indicate that distinct mechanisms of cell cycle arrest are associated with upregulating or downregulating TbPCNA. The findings of this study validate deregulating intra-parasite levels of TbPCNA as a potential strategy for therapeutically exploiting this target in bloodstream form T. brucei.

  1. Cell surface Glut1 levels distinguish human CD4 and CD8 T lymphocyte subsets with distinct effector functions

    PubMed Central

    Cretenet, Gaspard; Clerc, Isabelle; Matias, Maria; Loisel, Severine; Craveiro, Marco; Oburoglu, Leal; Kinet, Sandrina; Mongellaz, Cédric; Dardalhon, Valérie; Taylor, Naomi

    2016-01-01

    CD4 and CD8 T lymphocyte activation requires the generation of sufficient energy to support new biosynthetic demands. Following T cell receptor (TCR) engagement, these requirements are met by an increased glycolysis, due, at least in part, to induction of the Glut1 glucose transporter. As Glut1 is upregulated on tumor cells in response to hypoxia, we assessed whether surface Glut1 levels regulate the antigen responsiveness of human T lymphocytes in both hypoxic and atmospheric oxygen conditions. Notably, Glut1 upregulation in response to TCR stimulation was significantly higher in T lymphocytes activated under hypoxic as compared to atmospheric oxygen conditions. Furthermore, TCR-stimulated human T lymphocytes sorted on the basis of Glut1-Lo and Glut1-Hi profiles maintained distinct characteristics, irrespective of the oxygen tension. While T cells activated in hypoxia divided less than those activated in atmospheric oxygen, Glut1-Hi lymphocytes exhibited increased effector phenotype acquisition, augmented proliferation, and an inverted CD4/CD8 ratio in both oxygen conditions. Moreover, Glut1-Hi T lymphocytes exhibited a significantly enhanced ability to produce IFN-γ and this secretion potential was completely dependent on continued glycolysis. Thus, Glut1 surface levels identify human T lymphocytes with distinct effector functions in both hypoxic and atmospheric oxygen tensions. PMID:27067254

  2. Fine mapping of quantitative trait nucleotides underlying thrombin-activatable fibrinolysis inhibitor antigen levels by a transethnic study.

    PubMed

    Frère, Corinne; Tregouet, David-Alexandre; Morange, Pierre-Emmanuel; Saut, Noémie; Kouassi, Dinar; Juhan-Vague, Irène; Tiret, Laurence; Alessi, Marie-Christine

    2006-09-01

    Recent studies revisiting the association between plasma thrombin-activatable fibrinolysis inhibitor (TAFI) Ag levels and polymorphisms of the CPB2 gene (coding for TAFI) suggested that TAFI Ag levels were influenced by 2 major quantitative trait nucleotides (QTNs) in European whites. However, the strong linkage disequilibrium (LD) between CPB2 polymorphisms in European whites did not allow one to distinguish which polymorphisms could be the putative QTNs. To get a better insight into the identification of QTNs, a transethnic haplotype analysis contrasting 2 populations of African and European subjects was performed using 13 CPB2 polymorphisms. Results of the haplotype analyses suggested that 3 QTNs had independent effects and explained about 15% of the TAFI variability, consistently in the 2 populations. The lower LD observed in the African population enabled us to identify the 1583T>A SNP located in 3'UTR as one of these QTNs, whereas the -2599C>G and -2345--2344insG SNPs located in the 5' region might be the 2 other QTNs. A phylogenetic study suggested that these 3 polymorphisms occurred before the period of migration "out of Africa." Although this transethnic comparison contributed to better map the putative CPB2 QTNs, further studies are required to clarify the role of the promoter region.

  3. Neutrophil surface presentation of the anti-neutrophil cytoplasmic antibody-antigen proteinase 3 depends on N-terminal processing

    PubMed Central

    von Vietinghoff, S; Eulenberg, C; Wellner, M; Luft, F C; Kettritz, R

    2008-01-01

    The neutrophil serine protease proteinase 3 (PR3) is a main autoantigen in anti-neutrophil cytoplasmic antibody-associated vasculitis. PR3 surface presentation on neutrophilic granulocytes, the main effector cells, is pathogenically important. PR3 is presented by the NB1 (CD177) glycoprotein, but how the presentation develops during neutrophil differentiation is not known. An N-terminally unprocessed PR3 (proPR3) is produced early during neutrophil development and promotes myeloid cell differentiation. We therefore investigated if PR3 presentation depended on NB1 during neutrophil differentiation and if PR3 and proPR3 could both be presented by NB1. In contrast to mature neutrophils, differentiating neutrophils showed an early NB1-independent PR3 surface display that was recognized by only two of four monoclonal anti-PR3 antibodies and occurred in parallel with proPR3, but not PR3 secretion, suggesting that the NB1-independent surface PR3 was proPR3. PR3 gene expression preceeded NB1. When the NB1 receptor was detected on the surface, a mode of PR3 surface display similar to mature neutrophils developed together with the degranulation system. Ectopic expression studies showed that NB1 was a sufficient receptor for PR3 but not proPR3. ProPR3 display on the plasma membrane may influence the bone marrow microenvironment. NB1-mediated PR3 presentation depended on PR3 N-terminal processing implicating the PR3–N-terminus as NB1-binding site. PMID:18462208

  4. Heterogeneous distribution of Candida albicans cell-surface antigens demonstrated with an Als1-specific monoclonal antibody

    PubMed Central

    Coleman, David A.; Oh, Soon-Hwan; Zhao, Xiaomin; Hoyer, Lois L.

    2010-01-01

    Despite an abundance of data describing expression of genes in the Candida albicans ALS (agglutinin-like sequence) gene family, little is known about the production of Als proteins on individual cells, their spatial localization or stability. Als proteins are most commonly discussed with respect to function in adhesion of C. albicans to host and abiotic surfaces. Development of a mAb specific for Als1, one of the eight large glycoproteins encoded by the ALS family, provided the opportunity to detect Als1 during growth of yeast and hyphae, both in vitro and in vivo, and to demonstrate the utility of the mAb in blocking C. albicans adhesion to host cells. Although most C. albicans yeast cells in a saturated culture are Als1-negative by indirect immunofluorescence, Als1 is detected on the surface of nearly all cells shortly after transfer into fresh growth medium. Als1 covers the yeast cell surface, with the exception of bud scars. Daughters of the inoculum cells, and sometimes granddaughters, also have detectable Als1, but Als1 is not detectable on cells from subsequent generations. On germ tubes and hyphae, most Als1 is localized proximal to the mother yeast. Once deposited on yeasts or hyphae, Als1 persists long after the culture has reached saturation. Growth stage-dependent production of Als1, coupled with its persistence on the cell surface, results in a heterogeneous population of cells within a C. albicans culture. Anti-Als1 immunolabelling patterns vary depending on the source of the C. albicans cells, with obvious differences between cells recovered from culture and those from a murine model of disseminated candidiasis. Results from this work highlight the temporal parallels for ALS1 expression and Als1 production in yeasts and germ tubes, the specialized spatial localization and persistence of Als1 on the C. albicans cell surface, and the differences in Als1 localization that occur in vitro and in vivo. PMID:20705663

  5. A bicistronic DNA vaccine containing apical membrane antigen 1 and merozoite surface protein 4/5 can prime humoral and cellular immune responses and partially protect mice against virulent Plasmodium chabaudi adami DS malaria.

    PubMed

    Rainczuk, A; Scorza, T; Spithill, T W; Smooker, P M

    2004-10-01

    The ultimate malaria vaccine will require the delivery of multiple antigens from different stages of the complex malaria life cycle. In order to efficiently deliver multiple antigens with use of DNA vaccine technology, new antigen delivery systems must be assessed. This study utilized a bicistronic vector construct, containing an internal ribosome entry site, expressing a combination of malarial candidate antigens: merozoite surface protein 4/5 (MSP4/5) (fused to a monocyte chemotactic protein 3 chemoattractant sequence) and apical membrane antigen 1 (AMA-1) (fused to a tissue plasminogen activator secretion signal). Transfection of COS 7 cells with bicistronic plasmids resulted in