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1

Search and analysis of identical reverse octapeptides in unrelated proteins.  

PubMed

For the past few decades, intensive studies have been carried out in an attempt to understand how the amino acid sequences of proteins encode their three dimensional structures to perform their specific functions. In order to understand the sequence-structure relationship of proteins, several sub-sequence search studies in non-redundant sequence-structure databases have been undertaken which have given some fruitful clues. In our earlier work, we analyzed a set of 3124 non-redundant protein sequences from the Protein Data Bank (PDB) and retrieved 30 identical octapeptides having different secondary structures. These octapeptides were characterized by using different computational procedures. This prompted us to explore the presence of octapeptides with reverse sequences and to analyze whether these octapeptides would adopt similar structures as that of their parent octapeptides. Our identical reverse octapeptide search resulted in the finding of eight octapeptide pairs (octapeptide and reverse octapeptide) with similar secondary structure and 23 octapeptide pairs with different secondary structures. In the present work, the geometrical and biophysical characteristics of identical reverse octapeptides were explored and compared with unrelated octapeptide pairs by using various computational tools. We thus conclude that proteins containing identical reverse octapeptides are not very abundant and residues in the octapeptide pairs do not contribute to the stability of the protein. Furthermore, compared to unrelated octapeptides, identical reverse octapeptides do not show certain biophysical and geometrical properties. PMID:23523652

Saravanan, Konda Mani; Selvaraj, Samuel

2013-04-01

2

Quantitating T Cell Cross-Reactivity for Unrelated Peptide Antigens1  

PubMed Central

Quantitating the frequency of T cell cross-reactivity to unrelated peptides is essential to understanding T cell responses in infectious and autoimmune diseases. Here we used 15 mouse or human CD8+ T cell clones (11 antiviral, 4 anti-self) in conjunction with a large library of defined synthetic peptides to examine nearly 30,000 TCR-peptide MHC class I interactions for cross-reactions. We identified a single cross-reaction consisting of an anti-self TCR recognizing a poxvirus peptide at relatively low sensitivity. We failed to identify any cross-reactions between the synthetic peptides in the panel and polyclonal CD8+ T cells raised to viral or alloantigens. These findings provide the best estimate to date of the frequency of T cell cross-reactivity to unrelated peptides (?1/30,000), explaining why cross-reactions between unrelated pathogens are infrequently encountered and providing a critical parameter for understanding the scope of self-tolerance. PMID:19734234

Ishizuka, Jeffrey; Grebe, Kristie; Shenderov, Eugene; Peters, Bjoern; Chen, Qiongyu; Peng, YanChun; Wang, Lili; Dong, Tao; Pasquetto, Valerie; Osroff, Carla; Sidney, John; Hickman, Heather; Cerundolo, Vincenzo; Sette, Alessandro; Bennink, Jack R.; McMchael, Andrew; Yewdell, Jonathan W.

2009-01-01

3

C-Reactive Protein and Cognition Are Unrelated to Leukoaraiosis  

PubMed Central

Elevated serum levels of C-reactive protein (CRP) have been associated with leukoaraiosis in elderly brain. However, several studies indicate that leukoaraiosis is associated with an increased risk of cognitive impairment. It is unknown how the effect of CRP on cognition is mediated by leukoaraiosis. The purpose of this study is to assess the relationship between serum levels of CRP, the presence of leukoaraiosis, and cognitive impairment in a population of coronary patients over 50 years old. CRP levels explained 7.18% (P: 0.002) of the variance of the MMSE. The adjustment for the presence of leukoaraiosis little changed this variance (5.98%, P: 0.005), indicating that only a small portion of the CRP influence on cognition was mediated via leukoaraiosis. Patients with CRP levels ?5.0 had 2.9 (95% CI: 1.26–6.44) times more chance to present cognitive impairment (P: 0.012). We found that elevated serum levels of CRP were associated with increased risk of cognitive impairment in elderly and it was not mediated by presence of leukoaraiosis. PMID:24587705

Marques, Fabricio Correia; Rosset, Idiane; Moriguchi, Emilio Hideyuki; Picon, Paulo Dornelles; Chaves, Marcia Lorena Fagundes; Roriz-Cruz, Matheus

2014-01-01

4

Immune recognition of protein antigens  

SciTech Connect

This book contains 33 papers. Some of the titles are: Antigenic Structure of Influenze Virus Hemagglutinin; Germ-line and Somatic Diversity in the Antibody Response to the Influenza Virus A/PR/8/34 Hemagglutinin; Recognition of Cloned Influenza A Virus Gene Products by Cytotoxic T Lymphocytes; Antigenic Structure of the Influenza Virus N2 Neuraminidase; and The Molecular and Genetic Basis of Antigenic Variation in Gonococcal Pillin.

Laver, W.G.; Air, G.M.

1985-01-01

5

Antigenic properties of N protein of hantavirus.  

PubMed

Hantavirus causes two important rodent-borne viral zoonoses, hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus pulmonary syndrome (HPS) in North and South America. Twenty-four species that represent sero- and genotypes have been registered within the genus Hantavirus by the International Committee on Taxonomy of Viruses (ICTV). Among the viral proteins, nucleocapsid (N) protein possesses an immunodominant antigen. The antigenicitiy of N protein is conserved compared with that of envelope glycoproteins. Therefore, N protein has been used for serological diagnoses and seroepidemiological studies. An understanding of the antigenic properties of N protein is important for the interpretation of results from serological tests using N antigen. N protein consists of about 430 amino acids and possesses various epitopes. The N-terminal quarter of N protein bears linear and immunodominant epitopes. However, a serotype-specific and multimerization-dependent antigenic site was found in the C-terminal half of N protein. In this paper, the structure, function, and antigenicity of N protein are reviewed. PMID:25123683

Yoshimatsu, Kumiko; Arikawa, Jiro

2014-08-01

6

Antigenic Properties of N Protein of Hantavirus  

PubMed Central

Hantavirus causes two important rodent-borne viral zoonoses, hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus pulmonary syndrome (HPS) in North and South America. Twenty-four species that represent sero- and genotypes have been registered within the genus Hantavirus by the International Committee on Taxonomy of Viruses (ICTV). Among the viral proteins, nucleocapsid (N) protein possesses an immunodominant antigen. The antigenicitiy of N protein is conserved compared with that of envelope glycoproteins. Therefore, N protein has been used for serological diagnoses and seroepidemiological studies. An understanding of the antigenic properties of N protein is important for the interpretation of results from serological tests using N antigen. N protein consists of about 430 amino acids and possesses various epitopes. The N-terminal quarter of N protein bears linear and immunodominant epitopes. However, a serotype-specific and multimerization-dependent antigenic site was found in the C-terminal half of N protein. In this paper, the structure, function, and antigenicity of N protein are reviewed. PMID:25123683

Yoshimatsu, Kumiko; Arikawa, Jiro

2014-01-01

7

Interaction of Vi Antigen with Proteins  

PubMed Central

Whiteside, Roberta E. (Boston University School of Medicine, Boston, Mass.), and Edgar E. Baker. Interaction of Vi antigen with proteins. J. Bacteriol. 92:1597–1603. 1966.—Purified Vi antigen (Vi) mixed in equal amounts with bovine serum albumin (BSA) or human ? globulin (HGG) at pH values above 4.7 formed a complex which was not precipitated by trichloroacetic acid or tungstic acid. At pH values below 4.7, the interaction between Vi and either BSA or HGG produced insoluble complexes except when excess Vi antigen was present. When sufficient Vi was present at the lower pH values, the soluble complex was not precipitated by trichloroacetic acid. Other acid polysaccharides tested did not form trichloroacetic acid-soluble complexes with BSA. When subjected to immunoelectrophoresis, the Vi-BSA complex migrated in agar at a rate different from that of either BSA or Vi alone. The complex reacted with both Vi and BSA antiserum. The addition of either BSA or Vi antiserum to a Vi-BSA complex resulted in dissociation of the complex and precipitation of either Vi or BSA, depending upon the antiserum used. Vi antigen mixed with purified O antigen from Salmonella typhosa formed a complex which migrated in agar at a rate different from that of either component alone when subjected to immunoelectrophoresis. Images PMID:4163411

Whiteside, Roberta E.; Baker, Edgar E.

1966-01-01

8

Immunogenic disparities of 11 minor histocompatibility antigens (mHAs) in HLA-matched unrelated allogeneic hematopoietic SCT.  

PubMed

We determined the alleles of 11 mHAs and investigated the association of immunogenic mHA mismatches between a donor and a recipient with a course of allogeneic hematopoietic SCT (allo-HSCT) from 10/10 alleles HLA-matched unrelated donors in 92 recipients after myeloablative conditioning between 2004 and 2006. The frequency analysis of mHA alleles, genotypes and phenotypes accompanied by appropriate restriction HLA Ags allowed for an estimation of the probability of immunogenic mismatches, which was the highest for HA-1, HA-8 and HY. GVH-directed disparity of mHAs with broad tissue distribution, especially of the sex-related HY Ag, influenced the results of allo-HSCT from HLA-matched unrelated donors by not only increasing the probability of chronic GVHD (cGVHD) but also by decreasing the relapse rate. PMID:18850018

Markiewicz, M; Siekiera, U; Karolczyk, A; Szymszal, J; Helbig, G; Wojnar, J; Dzierzak-Mietla, M; Kyrcz-Krzemien, S

2009-02-01

9

Phospholipase C-gamma contains introns shared by src homology 2 domains in many unrelated proteins.  

PubMed Central

Many proteins with novel functions were created by exon shuffling around the time of the metazoan radiation. Phospholipase C-gamma (PLC-gamma) is typical of proteins that appeared at this time, containing several different modules that probably originated elsewhere. To gain insight into both PLC-gamma evolution and structure-function relationships within the Drosophila PLC-gamma encoded by small wing (sl), we cloned and sequenced the PLC-gamma homologs from Drosophila pseudoobscura and D. virilis and compared their gene structure and predicted amino acid sequences with PLC-gamma homologs in other animals. PLC-gamma has been well conserved throughout, although structural differences suggest that the role of tyrosine phosphorylation in enzyme activation differs between vertebrates and invertebrates. Comparison of intron positions demonstrates that extensive intron loss has occurred during invertebrate evolution and also reveals the presence of conserved introns in both the N- and C-terminal PLC-gamma SH2 domains that are present in SH2 domains in many other genes. These and other conserved SH2 introns suggest that the SH2 domains in PLC-gamma are derived from an ancestral domain that was shuffled not only into PLC-gamma, but also into many other unrelated genes during animal evolution. PMID:12807765

Manning, Charlene M; Mathews, Wendy R; Fico, Leah P; Thackeray, Justin R

2003-01-01

10

The human leucocyte antigen-G 14-basepair polymorphism correlates with graft-versus-host disease in unrelated bone marrow transplantation for thalassaemia.  

PubMed

The presence of the 14-bp insertion polymorphism of the human leucocyte antigen (HLA)-G gene (HLA-G) promotes immune tolerance through increased synthesis of HLA-G molecules. We investigated this polymorphism in a large cohort of 53 thalassaemia patients transplanted from an unrelated donor. Sixteen patients (30.2%) homozygous for the 14-bp deletion had a higher risk of developing acute graft-versus-host disease (aGvHD) than patients homozygous for the 14-bp insertion (-14-bp/-14-bp vs +14-bp/+14-bp: Relative Risk = 15.0; 95% confidence interval 1.59-141.24; P = 0.008). Therefore, the 14-bp polymorphism could be an important predictive factor for aGvHD following bone marrow transplantation. PMID:17897304

La Nasa, Giorgio; Littera, Roberto; Locatelli, Franco; Lai, Sara; Alba, Francesco; Caocci, Giovanni; Lisini, Daniela; Nesci, Sonia; Vacca, Adriana; Piras, Eugenia; Bernardo, Maria Ester; Di Cesare-Merlone, Alessandra; Orrù, Sandro; Carcassi, Carlo

2007-10-01

11

Protection of chickens to antigenically variant avian influenza virus challenge after immunization with two antigenically unrelated strains of the same subtype  

Technology Transfer Automated Retrieval System (TEKTRAN)

The antigenic diversity of avian influenza virus (AIV) within a subtype has been well established and is believed to be driven by the selection of immunologic escape mutants. In regions where vaccination against AIV has been implemented for prolonged periods (e.g. Vietnam and Egypt), vaccines which...

12

Original article Permeability of milk protein antigens across the intestinal  

E-print Network

Original article Permeability of milk protein antigens across the intestinal epithelium in vitro D by proteolytic enzymes and intestinal epithelial permeability represent two major drawbacks to the transfer-cas. These results suggest a selective intestinal permeability for milk protein antigens. This selectivity

Paris-Sud XI, Université de

13

Protein Folding Activity of Ribosomal RNA Is a Selective Target of Two Unrelated Antiprion Drugs  

Microsoft Academic Search

Background6-Aminophenanthridine (6AP) and Guanabenz (GA, a drug currently in use for the treatment of hypertension) were isolated as antiprion drugs using a yeast-based assay. These structurally unrelated molecules are also active against mammalian prion in several cell-based assays and in vivo in a mouse model for prion-based diseases.Methodology\\/Principal FindingsHere we report the identification of cellular targets of these drugs. Using

Déborah Tribouillard-Tanvier; Suzana Dos Reis; Fabienne Gug; Cécile Voisset; Vincent Béringue; Raimon Sabate; Ema Kikovska; Nicolas Talarek; Stéphane Bach; Chenhui Huang; Nathalie Desban; Sven J. Saupe; Surachai Supattapone; Jean-Yves Thuret; Stéphane Chédin; Didier Vilette; Hervé Galons; Suparna Sanyal; Marc Blondel; Stefan Wölfl

2008-01-01

14

Protein Folding Activity of Ribosomal RNA Is a Selective Target of Two Unrelated Antiprion Drugs  

Microsoft Academic Search

Background: 6-Aminophenanthridine (6AP) and Guanabenz (GA, a drug currently in use for the treatment of hypertension) were isolated as antiprion drugs using a yeast-based assay. These structurally unrelated molecules are also active against mammalian prion in several cell-based assays and in vivo in a mouse model for prion-based diseases. Methodology\\/Principal Findings: Here we report the identification of cellular targets of

Déborah Tribouillard-Tanvier; Suzana Dos Reis; Fabienne Gug; Cécile Voisset; Vincent Béringue; Raimon Sabate; Ema Kikovska; Nicolas Talarek; Stéphane Bach; Chenhui Huang; Nathalie Desban; Sven J. Saupe; Surachai Supattapone; Jean-Yves Thuret; Stéphane Chédin; Didier Vilette; Hervé Galons; Suparna Sanyal; Marc Blondel

2008-01-01

15

Prediction of protein antigenic determinants from amino acid sequences  

SciTech Connect

A method is presented for locating protein antigenic determinants by analyzing amino acid sequences in order to find the point of greatest local hydrophilicity. This is accomplished by assigning each amino acid a numerical value (hydrophilicity value) and then repetitively averaging these values along the peptide chain. The point of highest local average hydrophilicity is invariably located in, or immediately adjacent to, an antigenic determinant. It was found that the prediction success rate depended on averaging group length, with hexapeptide averages yielding optimal results. The method was developed using 12 proteins for which extensive immunochemical analysis has been carried out and subsequently was used to predict antigenic determinants for the following proteins: hepatitis B surface antigen, influenza hemagglutinis, fowl plague virus hemagglutinin, human histocompatibility antigen HLA-B7, human interferons, Escherichia coli and cholera enterotoxins, ragweed allergens Ra3 and Ra5, and streptococcal M protein. The hepatitis B surface antigen sequence was synthesized by chemical means and was shown to have antigenic activity by radioimmunoassay.

Hopp, T.P.; Woods, K.R.

1981-06-01

16

Changes in structural and antigenic properties of proteins by radiation  

NASA Astrophysics Data System (ADS)

Radiation effect on structural and antigenic properties of proteins (0.2% in 0.01 M phosphate buffer, pH 7.4) were investigated using ovalbumin (OVA) and bovine serum albumin (BSA). Aggregation of OVA and BSA was induced by radiation and the molecular mass increased significantly in N 2. Significant changes in surface hydrophobicity and [ ?] 222 nm of CD were also observed by radiation showing the destruction of secondary structure of proteins. Antigenicity of irradiated OVA measured by the method of immunodiffusion was decreased by radiation, and the reactivity to anti-OVA antibody was almost diminished at 8 kGy in N 2 and 4 kGy in O 2, respectively. The reactivity of BSA was diminished at 4 kGy both in N 2 and O 2. Changes in hydrophobicity of OVA did not correspond to the decrease in antigenicity, whereas the changes in [ ?] 222 nm relatively well corresponded to the antigenicity. The SDS-PAGE and immunoblotting analysis showed that radiation at higher doses induced the production of protein aggregates and degraded fragments with reactivity to the specific antibodies. These results suggest that the main part of conformation-dependent antigenic structure (conformational epitope) is easily lost by radiation, but some antigenicity, which is mostly due to the amino acid sequence-dependent antigenic structures (sequential epitopes), remains even at higher dose.

Kume, Tamikazu; Matsuda, Tsukasa

1995-08-01

17

[Antigenic structure of Ebola virus VP35 protein].  

PubMed

Antigenic structure of Ebola virus (EV) (strain Mayinga) nucleocapsid protein VP35 was analyzed using monoclonal antibodies to EV VP35 and polyclonal antibodies to EV. EV protein VP35 was shown to have antigenic sites inducing the production of antibodies in animals. For better characterization of protein VP35 antigenic structure. EV gene encoding the full-length VP35 was cloned in vector pQE31 as a recombinant fusion protein (rec.VP35). The antigenic and immunogenic properties of rec.VP35 and EV VP35 were compared by ELISA and Western blot analysis with polyclonal and monoclonal antibodies. Antibodies of positive sera and VP35 MAbs cross reacted with the analyzed antigens. The topography of epitopes on EV VP35 and rec.VP35 was studied using MAbs and polyclonal antibodies to rec.VP35 in a competitive antibody binding assay. Two epitopes of one site were identified on these proteins. These epitopes are present on infectious virion protein VP35 and are stable during physicochemical exposures. PMID:11715705

Kazachinskaia, E I; Ternovo?, V A; Rudzevich, T N; Netesov, S V; Chepurnov, A A; Razumov, I A

2001-01-01

18

An analysis of bison erythrocyte antigens and blood proteins  

E-print Network

-chairmen of Advisory Committee: Or. J. Caldwell Or. J. Templeton Six hundred and sixty four blood samples were collected from eight herds of American b1son. Hemolyt1c tests were made using 140 cattle blood typing reagents specific for 55 antigenic Factors and 20... experimental bison blood typ1ng reagents specific for 5 antigenic factors. Red cell hemolysates and plasma proteins were subjected to electrophoresis to test for transferri n, hemoglobin and carbonic anhydrase polymorphi sms. Frequencies of each blood...

Zamora, Linda Elia

1983-01-01

19

Antigenic analysis of fimbrial proteins from Moraxella bovis.  

PubMed Central

Fimbrial proteins were extracted from 15 isolates of Moraxella bovis, and antisera to each of the preparations were raised in rabbits. The antigenic relationships of the fimbriae were investigated by an enzyme-linked immunosorbent assay, tandem crossed immunoelectrophoresis, and a slide agglutination test. With all three methods there was a similar pattern of antigenic cross-reactivity among the fimbriae. The 15 isolates, together with 23 additional isolates, could be grouped into seven fimbrial serogroups. Images PMID:2891723

Moore, L J; Rutter, J M

1987-01-01

20

Considering scores between unrelated proteins in the search database improves profile comparison  

PubMed Central

Background Profile-based comparison of multiple sequence alignments is a powerful methodology for the detection remote protein sequence similarity, which is essential for the inference and analysis of protein structure, function, and evolution. Accurate estimation of statistical significance of detected profile similarities is essential for further development of this methodology. Here we analyze a novel approach to estimate the statistical significance of profile similarity: the explicit consideration of background score distributions for each database template (subject). Results Using a simple scheme to combine and analytically approximate query- and subject-based distributions, we show that (i) inclusion of background distributions for the subjects increases the quality of homology detection; (ii) this increase is higher when the distributions are based on the scores to all known non-homologs of the subject rather than a small calibration subset of the database representatives; and (iii) these all known non-homolog distributions of scores for the subject make the dominant contribution to the improved performance: adding the calibration distribution of the query has a negligible additional effect. Conclusion The construction of distributions based on the complete sets of non-homologs for each subject is particularly relevant in the setting of structure prediction where the database consists of proteins with solved 3D structure (PDB, SCOP, CATH, etc.) and therefore structural relationships between proteins are known. These results point to a potential new direction in the development of more powerful methods for remote homology detection. PMID:19961610

2009-01-01

21

Structural basis for recognition of AT-rich DNA by unrelated xenogeneic silencing proteins  

PubMed Central

H-NS and Lsr2 are nucleoid-associated proteins from Gram-negative bacteria and Mycobacteria, respectively, that play an important role in the silencing of horizontally acquired foreign DNA that is more AT-rich than the resident genome. Despite the fact that Lsr2 and H-NS proteins are dissimilar in sequence and structure, they serve apparently similar functions and can functionally complement one another. The mechanism by which these xenogeneic silencers selectively target AT-rich DNA has been enigmatic. We performed high-resolution protein binding microarray analysis to simultaneously assess the binding preference of H-NS and Lsr2 for all possible 8-base sequences. Concurrently, we performed a detailed structure-function relationship analysis of their C-terminal DNA binding domains by NMR. Unexpectedly, we found that H-NS and Lsr2 use a common DNA binding mechanism where a short loop containing a “Q/RGR” motif selectively interacts with the DNA minor groove, where the highest affinity is for AT-rich sequences that lack A-tracts. Mutations of the Q/RGR motif abolished DNA binding activity. Netropsin, a DNA minor groove-binding molecule effectively outcompeted H-NS and Lsr2 for binding to AT-rich sequences. These results provide a unified molecular mechanism to explain findings related to xenogeneic silencing proteins, including their lack of apparent sequence specificity but preference for AT-rich sequences. Our findings also suggest that structural information contained within the DNA minor groove is deciphered by xenogeneic silencing proteins to distinguish genetic material that is self from nonself. PMID:21673140

Gordon, Blair R. G.; Li, Yifei; Cote, Atina; Weirauch, Matthew T.; Ding, Pengfei; Hughes, Timothy R.; Navarre, William Wiley; Xia, Bin; Liu, Jun

2011-01-01

22

Immunological Properties of Hepatitis B Core Antigen Fusion Proteins  

NASA Astrophysics Data System (ADS)

The immunogenicity of a 19 amino acid peptide from foot-and-mouth disease virus has previously been shown to approach that of the inactivated virus from which it was derived after multimeric particulate presentation as an N-terminal fusion with hepatitis B core antigen. In this report we demonstrate that rhinovirus peptide-hepatitis B core antigen fusion proteins are 10-fold more immunogenic than peptide coupled to keyhole limpet hemocyanin and 100-fold more immunogenic than uncoupled peptide with an added helper T-cell epitope. The fusion proteins can be readily administered without adjuvant or with adjuvants acceptable for human and veterinary application and can elicit a response after nasal or oral dosing. The fusion proteins can also act as T-cell-independent antigens. These properties provide further support for their suitability as presentation systems for "foreign" epitopes in the development of vaccines.

Francis, Michael J.; Hastings, Gillian Z.; Brown, Alan L.; Grace, Ken G.; Rowlands, David J.; Brown, Fred; Clarke, Berwyn E.

1990-04-01

23

High-Affinity Reactions Between Antigen-Specific T-Cell Receptors and Peptides Associated with Allogeneic and Syngeneic Major Histocompatibility Complex Class I Proteins  

Microsoft Academic Search

We report here that the intrinsic affinities of the antigen-specific T-cell receptors (TCR) of two unrelated CD8^+ T-cell clones for their respective peptide-major histocompatibility complex (MHC) ligands are higher than the values generally thought to prevail for TCR. The TCR of one clone (2C) binds an allogeneic class I MHC protein (L^d) in association with an alpha-ketoglutarate dehydrogenase nonapeptide (QLSPFPFDL,

Yuri Sykulev; Anders Brunmark; Theodore J. Tsomides; Shigeki Kageyama; Michael Jackson; Per A. Peterson; Herman N. Eisen

1994-01-01

24

Antigenic structure of the hepatitis C virus envelope 2 protein.  

PubMed Central

The antigenic structure of the envelope 2 (e2) protein of the hepatitis C virus (HCV) was characterized by the use of 70 synthetic peptides and 131 human sera from persons with antibodies to HCV. Among 34 overlapping peptides spanning the e2 protein of HCV, two major antigenic regions were located to residues 484-499 and residues 554-569. The sequence of the two major antigenic regions of the e2 protein are partly well conserved within the described types of HCV. Both regions contain two Cys residues in close proximity, and the region at residues 554-569 contains a putative N-glycosylation site, which are factors that previously have been suggested to affect the immune recognition of the e2 protein. Using substitution peptide analogues where each position within residues 484-499 and 554-569 were sequentially substituted by Ala or Gly, the most essential residues for antibody binding were found to be the Pro-498, Ala-499, Ala-566, Pro-567, and Pro-568. All of these, except for the Pro-498 and Ala-566, are conserved among different HCV strains. Also, according to previous studies, position 496 often shows variations, which could be explained by position 496 being contained within the antigenic region at residues 484-499. Interestingly, none of the Cys residues at positions 486, 494, 564 and 569 were found to be essential for antibody binding, indicating that these are not essential in maintaining the e2 antigenicity of the peptides. In a material of 114 confirmed anti-HCV positive sera, derived from patients during the acute or the chronic phase of HCV infection, the prevalence of antibodies to the two major linear antigenic regions of the e2 protein was found to be 55% among HCV RNA-positive sera, and 53% among HCV RNA-negative sera. In conclusion, we have identified and characterized two major linear antigenic regions outside the two hypervariable regions of the e2 protein. Since these regions are accessible to the B cells of the infected host, these two regions are likely to be surface exposed either on the precursor polyprotein or the native e2 protein. Also, we could confirm that antibodies to the e2 protein co-exist with HCV viraemia. PMID:7527739

Zhang, Z X; Sönnerborg, A; Sällberg, M

1994-01-01

25

Prediction of protein antigenic determinants from amino acid sequences  

Microsoft Academic Search

A method is presented for locating protein antigenic determinants by analyzing amino acid sequences in order to find the point of greatest local hydrophilicity. This is accomplished by assigning each amino acid a numerical value (hydrophilicity value) and then repetitively averaging these values along the peptide chain. The point of highest local average hydrophilicity is invariably located in, or immediately

T. P. Hopp; K. R. Woods

1981-01-01

26

Antigenicity and immunogenicity of Plasmodium vivax merozoite surface protein-3.  

PubMed

A recent clinical trial in African children demonstrated the potential utility of merozoite surface protein (MSP)-3 as a vaccine against Plasmodium falciparum malaria. The present study evaluated the use of Plasmodium vivax MSP-3 (PvMSP-3) as a target antigen in vaccine formulations against malaria caused by P. vivax. Recombinant proteins representing MSP-3? and MSP-3? of P. vivax were expressed as soluble histidine-tagged bacterial fusions. Antigenicity during natural infection was evaluated by detecting specific antibodies using sera from individuals living in endemic areas of Brazil. A large proportion of infected individuals presented IgG antibodies to PvMSP-3? (68.2%) and at least 1 recombinant protein representing PvMSP-3? (79.1%). In spite of the large responder frequency, reactivity to both antigens was significantly lower than was observed for the immunodominant epitope present on the 19-kDa C-terminal region of PvMSP-1. Immunogenicity of the recombinant proteins was studied in mice in the absence or presence of different adjuvant formulations. PvMSP-3?, but not PvMSP-3?, induced a TLR4-independent humoral immune response in the absence of any adjuvant formulation. The immunogenicity of the recombinant antigens were also tested in formulations containing different adjuvants (Alum, Salmonella enterica flagellin, CpG, Quil A,TiterMax® and incomplete Freunds adjuvant) and combinations of two adjuvants (Alum plus flagellin, and CpG plus flagellin). Recombinant PvMSP-3? and PvMSP-3? elicited higher antibody titers capable of recognizing P. vivax-infected erythrocytes harvested from malaria patients. Our results confirm that P. vivax MSP-3 antigens are immunogenic during natural infection, and the corresponding recombinant proteins may be useful in elucidating their vaccine potential. PMID:23457498

Bitencourt, Amanda R; Vicentin, Elaine C; Jimenez, Maria C; Ricci, Ricardo; Leite, Juliana A; Costa, Fabio T; Ferreira, Luis C; Russell, Bruce; Nosten, François; Rénia, Laurent; Galinski, Mary R; Barnwell, John W; Rodrigues, Mauricio M; Soares, Irene S

2013-01-01

27

Antigenicity and Immunogenicity of Plasmodium vivax Merozoite Surface Protein-3  

PubMed Central

A recent clinical trial in African children demonstrated the potential utility of merozoite surface protein (MSP)-3 as a vaccine against Plasmodium falciparum malaria. The present study evaluated the use of Plasmodium vivax MSP-3 (PvMSP-3) as a target antigen in vaccine formulations against malaria caused by P. vivax. Recombinant proteins representing MSP-3? and MSP-3? of P. vivax were expressed as soluble histidine-tagged bacterial fusions. Antigenicity during natural infection was evaluated by detecting specific antibodies using sera from individuals living in endemic areas of Brazil. A large proportion of infected individuals presented IgG antibodies to PvMSP-3? (68.2%) and at least 1 recombinant protein representing PvMSP-3? (79.1%). In spite of the large responder frequency, reactivity to both antigens was significantly lower than was observed for the immunodominant epitope present on the 19-kDa C-terminal region of PvMSP-1. Immunogenicity of the recombinant proteins was studied in mice in the absence or presence of different adjuvant formulations. PvMSP-3?, but not PvMSP-3?, induced a TLR4-independent humoral immune response in the absence of any adjuvant formulation. The immunogenicity of the recombinant antigens were also tested in formulations containing different adjuvants (Alum, Salmonella enterica flagellin, CpG, Quil A,TiterMax® and incomplete Freunds adjuvant) and combinations of two adjuvants (Alum plus flagellin, and CpG plus flagellin). Recombinant PvMSP-3? and PvMSP-3? elicited higher antibody titers capable of recognizing P. vivax-infected erythrocytes harvested from malaria patients. Our results confirm that P. vivax MSP-3 antigens are immunogenic during natural infection, and the corresponding recombinant proteins may be useful in elucidating their vaccine potential. PMID:23457498

Bitencourt, Amanda R.; Vicentin, Elaine C.; Jimenez, Maria C.; Ricci, Ricardo; Leite, Juliana A.; Costa, Fabio T.; Ferreira, Luis C.; Russell, Bruce; Nosten, François; Rénia, Laurent; Galinski, Mary R.; Barnwell, John W.; Rodrigues, Mauricio M.; Soares, Irene S.

2013-01-01

28

Antigenic response to topically applied proteins.  

PubMed Central

Six different proteins varying widely in molecular weight, ribonuclease, lysostaphin, ovalbumin, penicillinase, collagenase, and Varidase were tested for their ability to induce circulating antibody formation in rabbits after repeated topical application of the proteins in a water-soluble gel vehicle. After a 12-week exposure period, significant hemagglutinin titers were noted in rabbits treated with ovalbumin, lysostaphin, or ribonuclease; markedly elevated, passive cutaneous anaphylaxis-reacting sera were obtained only from collagenase- or lysostaphin-treated animals. Precipitin antibodies as evidenced by gel diffusion were also found in sera from collagenas- and lysostaphin-treated animals. Topical application of penicillinase was only marginally effective and Varidase was totally ineffective in elicting a positive circulating antibody response. In all cases, topical application of proteins for periods in excess of 3 weeks was required for induction of circulating antibody formation. PMID:163218

Harrison, E F; Fuquay, M E; Zygmunt, W A

1975-01-01

29

Changes in the clinical impact of high-risk human leukocyte antigen allele mismatch combinations on the outcome of unrelated bone marrow transplantation.  

PubMed

Several high-risk HLA allele mismatch combinations (HR-MMs) for severe acute graft-versus-host disease (GVHD) have been identified by analyzing transplantation outcomes in Japanese unrelated hematopoietic stem cell transplant recipients. In this study, we analyzed the effects of HR-MMs in 3 transplantation time periods. We confirmed that the incidence of grade III to IV acute GVHD in the HR-MM group was significantly higher than that in the low-risk (LR) MM group (hazard ratio [HR], 2.74; P < .0001) in the early time period (1993 to 2001). However, the difference in the incidence of grade III to IV acute GVHD between the HR-MM and LR-MM groups was not statistically significant (HR, 1.06; P = .85 and HR, .40; P = .21, respectively) in the mid (2002 to 2007) and late (2008 to 2011) time periods. Similarly, survival in the HR-MM group was significantly inferior to that in the LR-MM group (HR, 1.46; P = .019) in the early time period, whereas the difference in survival between the 2 groups was not statistically significant in the mid and late time periods (HR, 1.06; P = .75 and HR, .82; P = .58, respectively). In conclusion, the adverse impact of HR-MM has become less significant over time. Unrelated transplantation with a single HR-MM could be a viable option in the absence of a matched unrelated donor or an unrelated donor with a single LR-MM. PMID:24417871

Kanda, Yoshinobu; Kanda, Junya; Atsuta, Yoshiko; Fuji, Shigeo; Maeda, Yoshinobu; Ichinohe, Tastuo; Takanashi, Minoko; Ohashi, Kazuteru; Fukuda, Takahiro; Miyamura, Koichi; Mori, Takehiko; Sao, Hiroshi; Kobayashi, Naoki; Iwato, Koji; Sawada, Akihisa; Mori, Shinichiro

2014-04-01

30

Ia antigen-bearing B cell tumor lines can present protein antigen and alloantigen in a major histocompatibility complex-restricted fashion to antigen-reactive T cells  

PubMed Central

Several Ia-positive BALB/c B cell tumor lines were screened for their ability to present alloantigen and protein antigens to alloreactive and antigen-reactive T cells. Of six Ia-positive tumor lines studied, three were found to be effective as antigen presenting cells (APC). Indeed, on a per cell basis, one of the stimulatory lines, A20.3, was substantially more effective than whole spleen cells. The other three lines, although Ia-positive, were nonstimulatory. A20.3 was chosen for further study. This tumor appeared to behave like the conventional APC because (a) the tumor cells presented alloantigen, (b) they presented protein antigen in an MHC-restricted fashion to both primed donor T cells and to long-term continuous T cell lines, (c) alloantigen presentation was blocked by the inclusion of an anti-Ia antibody in the culture system, and (d) A20.3 cells could be effectively pulsed with antigen, although the continuous presence of antigen in the culture system resulted in a superior response. The addition of an exogenous source of interleukin 1 proved necessary to obtain an alloreactive but not an antigen-specific T cell response, although its inclusion did enhance the magnitude of antigen-stimulated proliferation. These tumor cells should prove useful in studying the biochemical events that occur during antigen processing and the requirements for T cell triggering by processed antigen in association with Ia molecules. PMID:6460073

1982-01-01

31

The antigenicity and allergenicity of microparticulated proteins: Simplesse.  

PubMed

New technologies are allowing the food industry to develop products from standard foods which may not be recognized in its modified form by food allergic patients. One such product, Simplesse, has been formulated by microparticulation of egg white and/or cows' milk proteins and is used as a fat substitute in many fat-laden foods. The purpose of this study was to determine whether the process of microparticulation altered the allergenicity/antigenicity of egg white and cows' milk proteins compared to the starting materials. Soluble protein fractions of Simplesse and its respective starting materials were compared to egg white, cows' milk protein, an ultra-filtered egg white/condensed milk mixture, and/or a whey concentrate by SDS-polyacrylamide gel electrophoresis. In addition, sera from 16 patients with documented egg and/or cows' milk hypersensitivity and two controls who were not allergic to egg or milk were used to assess potential allergenicity/antigenicity of these products by immunoblot (Western blot) analysis. There were heterogeneous IgE and IgG binding patterns to the food fractions among these food allergic patients suggesting differing sensitivity patterns among the individuals tested. However, utilizing both SDS-PAGE and immunoblot analyses, the major allergens in the microparticulated products were the same as those found in the starting materials, egg and cows' milk. In addition, there was no evidence of 'novel' protein fractions in the Simplesse test materials compared to the starting materials. PMID:1464052

Sampson, H A; Cooke, S

1992-10-01

32

Quantitating protein synthesis, degradation, and endogenous antigen processing.  

PubMed

Using L929 cells, we quantitated the macroeconomics of protein synthesis and degradation and the microeconomics of producing MHC class I associated peptides from viral translation products. To maintain a content of 2.6 x 10(9) proteins, each cell's 6 x 10(6) ribosomes produce 4 x 10(6) proteins min(-1). Each of the cell's 8 x 10(5) proteasomes degrades 2.5 substrates min(-1), creating one MHC class I-peptide complex for each 500-3000 viral translation products degraded. The efficiency of complex formation is similar in dendritic cells and macrophages, which play a critical role in activating T cells in vivo. Proteasomes create antigenic peptides at different efficiencies from two distinct substrate pools: rapidly degraded newly synthesized proteins that clearly represent defective ribosomal products (DRiPs) and a less rapidly degraded pool in which DRiPs may also predominate. PMID:12648452

Princiotta, Michael F; Finzi, Diana; Qian, Shu-Bing; Gibbs, James; Schuchmann, Sebastian; Buttgereit, Frank; Bennink, Jack R; Yewdell, Jonathan W

2003-03-01

33

Identification of major antigenic proteins of Pasteurella piscicida.  

PubMed

Two different antigenic protein-coding clones (PPA1 and PPA2) were isolated using anti-Pasteurella piscicida rabbit serum from a genomic DNA library of P. piscicida strain KP9038. The PPA1 and PPA2 expressed 7 kDa and 45 kDa proteins in Escherichia coli, respectively, and the molecular sizes of these expressed proteins are the same as these of the major antigenic proteins of P. piscicida. PPA1 encodes a protein of 83 amino acids residues, which is similar to the bacterial lipoprotein. Comparison of the predicted amino acid sequence of the PPA1-encoded 7 kDa protein of P. piscicida with previously reported bacterial lipoprotein sequence data revealed that it shares about 40% amino acid sequence identity. PPA2 has two large open reading frame (ORFs). The larger ORF (encoding 452 amino acid residues) encodes a homolog of DegQ protease, and the smaller ORF (371 amino acid residues) encodes a homolog of DegS protease. The antibodies reacted with the larger ORF-encoded 45 kDa DegQ homolog protein. The DegQ and DegS homolog proteins contain an export signal and a serine protease active site. The structural features of the PPA2-coding locus are similar to those of the loci in E. coli for the degQ and degS serine protease genes. A sequence in the 3' non-coding region of Vibrio hollisae thermostable hemolysin gene that is highly homologous with a similar located sequence in the Pseudomonas putida p-cresol methylhydroxylase gene is also found in the 3' non-coding region of the degS homolog gene of the PPA2. PMID:9441863

Hirono, I; Kato, M; Aoki, T

1997-12-01

34

Factor H-binding protein, a unique meningococcal vaccine antigen.  

PubMed

GNA1870, also named factor H-binding protein (fHbp) or rLP-2086, is a genome-derived antigen and one of the components of a rationally designed vaccine against Neisseria meningitidis serogroup B, which has entered phase III clinical trials. It has been classified into three main non-cross-protective variant groups. GNA1870 has also been termed fHbp because of its ability to bind factor H, a key regulatory component of the alternative complement pathway. fHbp is important for survival in human blood, human sera, and in presence of antimicrobial peptides, independently of its expression level. All these properties make fHbp a unique vaccine antigen. PMID:19388164

Pizza, Mariagrazia; Donnelly, John; Rappuoli, Rino

2008-12-30

35

Immunogenicity and antigenicity of Plasmodium vivax merozoite surface protein 10.  

PubMed

Among the proteins involved in the invasion by merozoite, the glycosylphosphatidylinositol-anchored proteins (GPI-APs) are suggested as potential vaccine candidates because of their localization to apical organelles and the surface; these candidates are predicted to play essential roles during invasion. As a GPI-AP, Plasmodium vivax merozoite surface protein 10 (PvMSP-10) induces high antibody titers. However, such high antibody titers have shown no protective efficacy for animals challenged with P. vivax parasites in a previous study. To adequately evaluate the immunogenicity and further characterize PvMSP-10 in order to understand its vaccine potential, we assessed its immunogenicity by immunizing BALB/c mice with cell-free expressed recombinant PvMSP-10 protein. The antigenicity of MSP-10 was analyzed, and we found 42% sensitivity and 95% specificity using serum samples from P. vivax-infected Korean patients. The IgG1 and IgG3 were the predominant immunoreactive antibodies against PvMSP-10 in vivax patient sera, and IgG1 and IgG3 and Th1-type cytokines were predominantly secreted in PvMSP-10-immunized mice. We conclude that the immunogenicity and antigenicity of MSP-10 may serve as a potential vaccine against vivax malaria. PMID:24764159

Cheng, Yang; Wang, Bo; Sattabongkot, Jetsumon; Lim, Chae Seung; Tsuboi, Takafumi; Han, Eun-Taek

2014-07-01

36

[Location of the surface protein antigen I/II on Mutans Streptococci with immunogold electron microscope].  

PubMed

Mutans Streptococci possess a number of surface protein antigens. The surface protein antigen I/II with a molecular mass of 190,000 is considered to play an important role in the initial attachment to tooth surface. The antigen is highly immunogenic and has been successfully used as a vaccine against dental caries. The object of this study is to locate the surface protein antigen I/II of Mutans Streptococci with immunogold electron microscope. The results suggest that (1) antigen I/II locate on the cell wall surface of serotype c, e, f; (2) antigen I/II locate on the "fuzz coat" of the cell wall of serotype d, g; (3) some antigen I/II locate at the surface of cell wall of serotype a; (4) antigen I/II are absent on the cell wall surface of serotype b strain. PMID:9592282

Fang, M; Bian, Z; Du, M

1996-11-01

37

Elicitation of T Cell Responses to Histologically Unrelated Tumors by Immunization with the Novel Cancer-Testis Antigen, Brother of the Regulator of Imprinted Sites1  

PubMed Central

Brother of the regulator of imprinted sites (BORIS) was previously described as a transcription factor for epigenetic reprogramming the expression of which is strictly confined to germ cells of adult testes but is aberrantly activated in the vast majority of neoplastic cells. Considering the critical role of BORIS in cancerogenesis and the fact that its expression pattern may preclude thymic tolerance, we generated DNA- and protein-based mouse BORIS antitumor vaccines using a non-DNA-binding version of the BORIS molecule. Clinical use of BORIS as a vaccine Ag would require that certain safety concerns be met. Specifically, administration of the functional BORIS protein would hypothetically pose a risk of BORIS accelerating the progression of cancer. To alleviate such safety concerns, we have developed vaccines based on the BORIS molecule lacking the DNA-binding zinc fingers domain. To enhance anti-BORIS cellular immune responses, we used a standard molecular adjuvant approach. It consisted of plasmids encoding murine IL-12 and IL-18 for a DNA-based vaccine and conventional Th1 type adjuvant, Quil A, for a protein-based vaccine. Both DNA- and protein-based vaccines induced Ag-specific CD4+ T cell proliferation with Th1 and Th2 cytokine profiles, respectively. Protein-based, but not DNA-based, BORIS vaccine induced a significant level of Ab production in immunized animals. Importantly, potent anticancer CD8+-cytotoxic lymphocytes were generated after immunization with the DNA-based, but not protein-based, BORIS vaccine. These cytolytic responses were observed across a wide range of different mouse cancers including mammary adenocarcinoma, glioma, leukemia, and mastocytoma. PMID:17182597

Ghochikyan, Anahit; Mkrtichyan, Mikayel; Loukinov, Dmitri; Mamikonyan, Gregory; Pack, Svetlana D.; Movsesyan, Nina; Ichim, Thomas E.; Cribbs, David H.; Lobanenkov, Victor V.; Agadjanyan, Michael G.

2008-01-01

38

Elicitation of T cell responses to histologically unrelated tumors by immunization with the novel cancer-testis antigen, brother of the regulator of imprinted sites.  

PubMed

Brother of the regulator of imprinted sites (BORIS) was previously described as a transcription factor for epigenetic reprogramming the expression of which is strictly confined to germ cells of adult testes but is aberrantly activated in the vast majority of neoplastic cells. Considering the critical role of BORIS in cancerogenesis and the fact that its expression pattern may preclude thymic tolerance, we generated DNA- and protein-based mouse BORIS antitumor vaccines using a non-DNA-binding version of the BORIS molecule. Clinical use of BORIS as a vaccine Ag would require that certain safety concerns be met. Specifically, administration of the functional BORIS protein would hypothetically pose a risk of BORIS accelerating the progression of cancer. To alleviate such safety concerns, we have developed vaccines based on the BORIS molecule lacking the DNA-binding zinc fingers domain. To enhance anti-BORIS cellular immune responses, we used a standard molecular adjuvant approach. It consisted of plasmids encoding murine IL-12 and IL-18 for a DNA-based vaccine and conventional Th1 type adjuvant, Quil A, for a protein-based vaccine. Both DNA- and protein-based vaccines induced Ag-specific CD4(+) T cell proliferation with Th1 and Th2 cytokine profiles, respectively. Protein-based, but not DNA-based, BORIS vaccine induced a significant level of Ab production in immunized animals. Importantly, potent anticancer CD8(+)-cytotoxic lymphocytes were generated after immunization with the DNA-based, but not protein-based, BORIS vaccine. These cytolytic responses were observed across a wide range of different mouse cancers including mammary adenocarcinoma, glioma, leukemia, and mastocytoma. PMID:17182597

Ghochikyan, Anahit; Mkrtichyan, Mikayel; Loukinov, Dmitri; Mamikonyan, Gregory; Pack, Svetlana D; Movsesyan, Nina; Ichim, Thomas E; Cribbs, David H; Lobanenkov, Victor V; Agadjanyan, Michael G

2007-01-01

39

Targeting and activation of antigen-specific B-cells by calcium phosphate nanoparticles loaded with protein antigen.  

PubMed

Cross-linking of the B-cell receptors of an antigen-specific B-cell is the initial signal for B-cell activation, proliferation, and differentiation into antibody secreting plasma cells. Since multivalent particulate structures are efficient activators of antigen-specific B-cells, we developed biodegradable calcium phosphate nanoparticles displaying protein antigens on their surface and explored the efficacy of the B-cell activation after exposure to these nanoparticles. The calcium phosphate nanoparticles were functionalized with the model antigen Hen Egg Lysozyme (HEL) to take advantage of a HEL-specific B-cell receptor transgenic mouse model. The nanoparticles were characterized by scanning electron microscopy and dynamic light scattering. The functionalized calcium phosphate nanoparticles were preferentially bound and internalized by HEL-specific B-cells. Co-cultivation of HEL-specific B-cells with the functionalized nanoparticles also increased surface expression of B-cell activation markers. Functionalized nanoparticles were able to effectively cross-link B-cell receptors at the surface of antigen-matched B-cells and were 100-fold more efficient in the activation of B-cells than soluble HEL. Thus, calcium phosphate nanoparticles coated with protein antigens are promising vaccine candidates for induction humoral immunity. PMID:24776487

Temchura, Vladimir V; Kozlova, Diana; Sokolova, Viktoriya; Uberla, Klaus; Epple, Matthias

2014-07-01

40

A major allogenic leukocyte antigen in the agnathan hagfish  

PubMed Central

All vertebrates, from jawless fish to mammals, possess adaptive immune systems that can detect and inactivate non-self-antigens through a vast repertoire of antigen receptors. Unlike jawed vertebrates, the hagfish utilizes variable lymphocyte receptors (VLRs) that are unrelated to immunoglobulin molecules but are diversified by copy-choice gene conversion mechanism. Here, we report that hagfish VLRs react with allogenic leukocyte antigens but not with self-antigens. We found that a highly polymorphic membrane protein, NICIR3, is recognized by VLRs as an allogenic leukocyte antigen (ALA). In a serological cross-reactivity test, a close correlation was observed between the amino acid differences in the protein sequences and the VLR cross-reactivities. This leukocyte antigen was predominantly expressed in phagocytic leukocytes, where it was associated with phagocytosed protein antigens. These findings suggest that a polymorphic leukocyte antigen, NICIR3/ALA, plays a pivotal role in jawless vertebrate adaptive immunity. PMID:23612706

Takaba, Hiroyuki; Imai, Takeshi; Miki, Shoji; Morishita, Yasuyuki; Miyashita, Akihiro; Ishikawa, Naoko; Nishizumi, Hirofumi; Sakano, Hitoshi

2013-01-01

41

A pentatricopeptide repeat protein acts as a site-specificity factor at multiple RNA editing sites with unrelated cis-acting elements in plastids.  

PubMed

In plant organelles, RNA editing alters specific cytidine residues to uridine in transcripts. All of the site-specificity factors of RNA editing identified so far are pentatricopeptide repeat (PPR) proteins. A defect in a specific PPR protein often impairs RNA editing at multiple sites, at which the cis-acting elements are not highly conserved. The molecular mechanism for sharing a single PPR protein over multiple sites is still unclear. We focused here on the PPR proteins OTP82 and CRR22, the putative target elements of which are, respectively, partially and barely conserved. Recombinant OTP82 specifically bound to the -15 to 0 regions of its target sites. Recombinant CRR22 specifically bound to the -20 to 0 regions of the ndhB-7 and ndhD-5 sites and to the -17 to 0 region of the rpoB-3 site. Taking this information together with the genetic data, we conclude that OTP82 and CRR22 act as site-specificity factors at multiple sites in plastids. In addition, the high-affinity binding of CRR22 to unrelated cis-acting elements suggests that only certain specific nucleotides in a cis-acting element are sufficient for high-affinity binding of a PPR protein. The cis-acting elements can therefore be rather divergent and still be recognized by a single PPR protein. PMID:22362750

Okuda, Kenji; Shikanai, Toshiharu

2012-06-01

42

Characterization of partially purified 8 kDa antigenic protein of Clonorchis sinensis  

Microsoft Academic Search

The 8 kDa antigenic protein of Clonorchis sinensis was partially purified by ammonium sulfate precipitation and subsequently by a column chromatographic steps. The purified protein was separated into 7 and 8 kDa protein bands through SDS-tricine gel electrophoresis, while the protein was found to migrate to a 8 kDa band in 7.5-15% SDS-PAGE. The molecular weight of the antigen was

Young-Bae Chung; Mejeong Lee; Hyun-Jong Yang; Byung-Suk Chung; Shun-Yu Lee; Min-Ho Choi; Sung-Tae Hong

2002-01-01

43

Consensus classification of human leukocyte antigen class II proteins.  

PubMed

Class II human leukocyte antigens (HLA II) are proteins involved in the human immunological adaptive response by binding and exposing some pre-processed, non-self peptides in the extracellular domain in order to make them recognizable by the CD4+ T lymphocytes. However, the understanding of HLA-peptide binding interaction is a crucial step for designing a peptide-based vaccine because the high rate of polymorphisms in HLA class II molecules creates a big challenge, even though the HLA II proteins can be grouped into supertypes, where members of different class bind a similar pool of peptides. Hence, first we performed the supertype classification of 27 HLA II proteins using their binding affinities and structural-based linear motifs to create a stable group of supertypes. For this purpose, a well-known clustering method was used, and then, a consensus was built to find the stable groups and to show the functional and structural correlation of HLA II proteins. Thus, the overlap of the binding events was measured, confirming a large promiscuity within the HLA II-peptide interactions. Moreover, a very low rate of locus-specific binding events was observed for the HLA-DP genetic locus, suggesting a different binding selectivity of these proteins with respect to HLA-DR and HLA-DQ proteins. Secondly, a predictor based on a support vector machine (SVM) classifier was designed to recognize HLA II-binding peptides. The efficiency of prediction was estimated using precision, recall (sensitivity), specificity, accuracy, F-measure, and area under the ROC curve values of random subsampled dataset in comparison with other supervised classifiers. Also the leave-one-out cross-validation was performed to establish the efficiency of the predictor. The availability of HLA II-peptide interaction dataset, HLA II-binding motifs, high-quality amino acid indices, peptide dataset for SVM training, and MATLAB code of the predictor is available at http://sysbio.icm.edu.pl/HLA . PMID:23229472

Saha, Indrajit; Mazzocco, Giovanni; Plewczynski, Dariusz

2013-02-01

44

Discovery of novel Schistosoma japonicum antigens using a targeted protein microarray approach  

PubMed Central

Background Novel vaccine candidates against Schistosoma japonicum are required, and antigens present in the vulnerable larval developmental stage are attractive targets. Post-genomic technologies are now available which can contribute to such antigen discovery. Methods A schistosome-specific protein microarray was probed using the local antibody response against migrating larvae. Antigens were assessed for their novelty and predicted larval expression and host-exposed features. One antigen was further characterised and its sequence and structure were analysed in silico. Real-time polymerase chain reaction was used to analyse transcript expression throughout development, and immunoblotting and enzyme-linked immunosorbent assays employed to determine antigen recognition by antibody samples. Results Several known and novel antigens were discovered, two of which showed up-regulated transcription in schistosomula. One novel antigen, termed S. japonicum Ly-6-like protein 1 (Sj-L6L-1), was further characterised and shown to share structural and sequence features with the Ly-6 protein family. It was found to be present in the worm tegument and expressed in both the larval and adult worms, but was found to be antigenic only in the lungs that the larvae migrate to and traverse. Conclusions This study represents a novel approach to vaccine antigen discovery and may contribute to schistosome vaccine development against this important group of human and veterinary pathogens. PMID:24964958

2014-01-01

45

Analysis of transcutaneous antigenic protein delivery by a hydrogel patch formulation.  

PubMed

We have developed a hydrogel patch, which could promote antigen penetration through stratum corneum (SC), and have demonstrated its safety and efficacy in animals and humans. For the availability improvement of our system, it is important to develop a device, which enhances antigen penetration through SC more efficiently. In this study, we have tried to collect the basic information involved in transcutaneous antigen delivery by investigating the immune event induced by our system and examining the effect of physical property of antigens or patch component on antigen penetration. A hydrogel patch delivered antigens through SC into skin, and some of Langerhans cells captured antigens, activated, and migrated to regional lymph nodes. We also showed that protein distribution into SC was regulated by various complexly-intertwined factors of proteins but not one particular parameter. Additionally, glycerin as the patch component contributed to the formation of SC hydration by patch application, which might be one of the factors of acceleration of protein penetration. On the basis of the present information, we are planning to modify the patch composition and establish the antigen modification technology for improvement in the efficacy of transcutaneous immunization. PMID:23585300

Matsuo, Kazuhiko; Ishii, Yumiko; Kawai, Yasuaki; Saiba, Yuki; Quan, Ying-Shu; Kamiyama, Fumio; Hirobe, Sachiko; Okada, Naoki; Nakagawa, Shinsaku

2013-06-01

46

Correlation between segmental mobility and the location of antigenic determinants in proteins  

Microsoft Academic Search

Most continuous antigenic determinants of tobacco mosaic virus protein (TMVP), myoglobin and lysozyme correspond to those surface regions in the protein structure, as determined by X-ray crystallography, which possess a run of high-temperature factors along the polypeptide backbone, that is, a high segmental mobility. The mobility of an antigenic determinant may make it easier to adjust to a pre-existing antibody

E. Westhof; D. Altschuh; D. Moras; A. C. Bloomer; A. Mondragon; A. Klug; M. H. V. van Regenmortel

1984-01-01

47

A structurally distinct human mycoplasma protein that generically blocks antigen-antibody union.  

PubMed

We report the discovery of a broadly reactive antibody-binding protein (Protein M) from human mycoplasma. The crystal structure of the ectodomain of transmembrane Protein M differs from other known protein structures, as does its mechanism of antibody binding. Protein M binds with high affinity to all types of human and nonhuman immunoglobulin G, predominantly through attachment to the conserved portions of the variable region of the ? and ? light chains. Protein M blocks antibody-antigen union, likely because of its large C-terminal domain extending over the antibody-combining site, blocking entry to large antigens. Similar to the other immunoglobulin-binding proteins such as Protein A, Protein M as well as its orthologs in other Mycoplasma species could become invaluable reagents in the antibody field. PMID:24503852

Grover, Rajesh K; Zhu, Xueyong; Nieusma, Travis; Jones, Teresa; Boero, Isabel; MacLeod, Amanda S; Mark, Adam; Niessen, Sherry; Kim, Helen J; Kong, Leopold; Assad-Garcia, Nacyra; Kwon, Keehwan; Chesi, Marta; Smider, Vaughn V; Salomon, Daniel R; Jelinek, Diane F; Kyle, Robert A; Pyles, Richard B; Glass, John I; Ward, Andrew B; Wilson, Ian A; Lerner, Richard A

2014-02-01

48

HIV Immune Evasion: Disruption of antigen presentation by the HIV Nef protein  

PubMed Central

The Human Immunodeficiency Virus (HIV) Nef protein is necessary for high viral loads and for timely progression to AIDS. Nef plays a number of roles but its effect on antigen presentation and immune evasion are among the best characterized. Cytotoxic T Lymphocytes (CTLs) recognize and lyse virally infected cells by detecting viral antigens in complex with host major histocompatibility complex class I molecules (MHC-I) on the infected cell surface. The HIV Nef protein disrupts antigen presentation at the cell surface by interfering with the normal trafficking pathway of MHC-I and thus reduces CTL recognition and lysis of infected cells. The molecular mechanism by which Nef causes MHC-I downmodulation is becoming more clear, but some questions remain. A better understanding of how Nef disrupts antigen presentation may lead to the development of drugs that enhance the ability of the anti-HIV CTLs to control HIV disease. PMID:21762823

Wonderlich, Elizabeth R.; Leonard, Jolie A.; Collins, Kathleen L.

2013-01-01

49

Antigenic characterization of influenza A virus matrix protein with monoclonal antibodies  

SciTech Connect

Monoclonal antibodies were used to study antigenic variation in three distinct epitopes on the matrix protein of influenza A viruses. The authors found that two of these epitopes underwent antigenic variation, but in a very limited number of virus strains. A third epitope appeared to be an invariant type-specific determinant for influenza A viruses. Competitive antibody binding assays and Western blot analysis of proteolytically digested matrix protein indicated that at least two of three epitopes are located in nonoverlapping domains on the matrix protein molecule.

van Wyke, K.L.; Yewdell, J.W.; Reck, L.J.; Murphy, B.R.

1984-01-01

50

Antigenic domains on the peplomer protein of avian infectious bronchitis virus: correlation with biological functions  

Microsoft Academic Search

Monoclonal antibodies (MAbs) directed against struc- tural proteins of infectious bronchitis virus (IBV) were produced to analyse the antigenic structure of this virus. Competitive binding of enzyme-labelled and unlabelled MAbs to IBV peplomer protein was ana- lysed in an antibody binding assay to test the relatedness of the epitopes defined by the MAbs. Based on the competition groups, eight epitope

G. Koch; L. Hartog; A. Kant; D. J. van Roozelaar

1990-01-01

51

Structure of signal-regulatory protein alpha: a link to antigen receptor evolution.  

PubMed

Signal-regulatory protein alpha (SIRPalpha) is a myeloid membrane receptor that interacts with the membrane protein CD47, a marker of self. We have solved the structure of the complete extracellular portion of SIRPalpha, comprising three immunoglobulin superfamily domains, by x-ray crystallography to 2.5 A resolution. These data, together with previous data on the N-terminal domain and its ligand CD47 (possessing a single immunoglobulin superfamily domain), show that the CD47-SIRPalpha interaction will span a distance of around 14 nm between interacting cells, comparable with that of an immunological synapse. The N-terminal (V-set) domain mediates binding to CD47, and the two others are found to be constant (C1-set) domains. C1-set domains are restricted to proteins involved in vertebrate antigen recognition: T cell antigen receptors, immunoglobulins, major histocompatibility complex antigens, tapasin, and beta2-microglobulin. The domains of SIRPalpha (domains 2 and 3) are structurally more similar to C1-set domains than any cell surface protein not involved in antigen recognition. This strengthens the suggestion from sequence analysis that SIRP is evolutionarily closely related to antigen recognition proteins. PMID:19628875

Hatherley, Deborah; Graham, Stephen C; Harlos, Karl; Stuart, David I; Barclay, A Neil

2009-09-25

52

Envelope protein complexes of Mycobacterium avium subsp. paratuberculosis and their antigenicity.  

PubMed

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic enteric disease of ruminant animals. In the present study, blue native PAGE electrophoresis and 2D SDS-PAGE were used to separate MAP envelope protein complexes, followed by mass spectrometry (MS) to identify individual proteins within the complexes. Identity of individual proteins within complexes was further confirmed by MS upon excision of spots from 2D SDS-PAGE gels. Among the seven putative membrane complexes observed, major membrane protein (MAP2121c), a key MAP antigen involved in invasion of epithelial cells, was found to form a complex with cysteine desulfurase (MAP2120c). Other complexes found included those involved in energy metabolism (succinate dehydrogenase complex) as well as a complex formed by Cfp29, a characterized T cell antigen of Mycobacterium tuberculosis. To determine antigenicity of proteins, Western blot was performed on replicate 2D SDS-PAGE gels with sera from noninfected control cows (n=9) and naturally infected cows in the subclinical (n=10) and clinical (n=13) stages of infection. Clinical animals recognized MAP2121c in greater proportion than subclinical and control cows, whereas cysteine desulfurase recognition was not differentiated by infection status. To further characterize antigenicity, recombinant proteins were expressed for 10 of the proteins identified and evaluated in an interferon-gamma (IFN-?) release assay as well as immunoblots. This study reveals the presence of protein complexes in the cell envelope of MAP, suggesting protein interactions in the envelope of this pathogen. Furthermore the identification of antigenic proteins with potential as diagnostic targets was characterized. PMID:25500374

Leite, Fernando L; Reinhardt, Timothy A; Bannantine, John P; Stabel, Judith R

2015-02-25

53

Protein quality, antigenicity, and antioxidant activity of soy-based foodstuffs.  

PubMed

Commercial soy-based foodstuffs, including beverages ( n = 15), cow's milk supplemented with soy isoflavones ( n = 1), snacks ( n = 1), and biscuits ( n = 2), were analyzed to find any link between alterations in protein quality, safety (antigenicity), functionality (antioxidant activity), and food processing. Protein content was analyzed by the Kjeldhal method and available lysine by OPA assay. Chromatographic (RP-HPLC) and electrophoretic (SDS-PAGE) protein profiles were obtained to monitor modifications in the structure of soy allergens. The antigenicity was estimated by immunoblotting against soy total antibodies. Total phenol content was measured by Folin-Ciocalteu, while peroxyl radical scavenging activity of the sample was determined by ORAC FL assay. Protein content did not differ of those declared by the producers. Lysine availability was higher in liquid soy beverages compared to that in other soy foodstuffs studied here. 7S and 11S soy allergens were detected by RP-HPLC and SDS-PAGE, respectively. Both data indicated changes in soy protein patterns due to processing of instant powdered soymilk, soy snacks, and biscuits. Immunoblotting assay showed modifications in the antigenic response of these foodstuffs based on soy, suggesting that their processing had altered the structure of soy allergens. RP-HPLC, SDS-PAGE, and immunoblotting resulted in adequate analytical approaches for detecting changes in protein structure due to processing and adulteration. Protein quality, antigenicity, and antioxidant activity of soy products can be affected as a function of the intensity of the thermal processing. PMID:18620400

Amigo-Benavent, Miryam; Silván, Jose Manuel; Moreno, Francisco Javier; Villamiel, Mar; Del Castillo, M Dolores

2008-08-13

54

Mapping of Antigenic Sites on the Nucleocapsid Protein of the Severe Acute Respiratory Syndrome Coronavirus  

PubMed Central

Antigenic sites on the nucleocapsid (N) protein of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) were mapped by Pepscan analysis with overlapping peptides that span the N protein sequence. Two major immunodominant epitopes located in the C-terminal region (amino acids [aa] 362 to 412) and middle region (aa 153 to 178) reacted with more than 75% of sera from SARS patients. Several minor immunodominant epitopes were reactive with about 50% of the SARS sera. Antisera from mice immunized with inactivated SARS-CoV recognized the two major immunodominant epitopes and one antigenic site located adjacent to the N-terminal region (aa 76 to 101), which did not react with the sera from SARS patients. Several monoclonal antibodies against SARS-CoV bound to the N- or C-terminal antigenic sites. These results suggest that the above antigenic sites on the N protein are important in eliciting humoral immune response against SARS-CoV in humans and animals and can be used as antigens for developing diagnostic tests. PMID:15528730

He, Yuxian; Zhou, Yusen; Wu, Hao; Kou, Zhihua; Liu, Shuwen; Jiang, Shibo

2004-01-01

55

A plasmid DNA immunogen expressing fifteen protein antigens and complex virus-like particles (VLP +) mimicking naturally occurring HIV  

Microsoft Academic Search

We describe here a single plasmid DNA immunogen representing the broadest antigen repertoire among HIV vaccine candidates. This pDNA was “ANTIGENeered” for the regulated expression of thirteen complete and two non-functional HIV protein antigens. These proteins self assemble into complex virus-like particles (VLP+). Multiple irreversible safety features were introduced by genetic modifications including the complete impairment of integration, reverse transcription,

Eszter Somogyi; Jianqing Xu; Ágnes Gudics; József Tóth; Attila L. Kovács; Franco Lori; Julianna Lisziewicz

2011-01-01

56

Analysis of Known Bacterial Protein Vaccine Antigens Reveals Biased Physical Properties and Amino Acid Composition  

PubMed Central

Many vaccines have been developed from live attenuated forms of bacterial pathogens or from killed bacterial cells. However, an increased awareness of the potential for transient side-effects following vaccination has prompted an increased emphasis on the use of sub-unit vaccines, rather than those based on whole bacterial cells. The identification of vaccine sub-units is often a lengthy process and bioinformatics approaches have recently been used to identify candidate protein vaccine antigens. Such methods ultimately offer the promise of a more rapid advance towards preclinical studies with vaccines. We have compared the properties of known bacterial vaccine antigens against randomly selected proteins and identified differences in the make-up of these two groups. A computer algorithm that exploits these differences allows the identification of potential vaccine antigen candidates from pathogenic bacteria on the basis of their amino acid composition, a property inherently associated with sub-cellular location. PMID:18629010

Mayers, Carl; Rowe, Sonya; Miller, Julie; Lingard, Bryan; Hayward, Sarah; Titball, Richard W.

2003-01-01

57

Human Leukocyte Antigen Class II Alleles Influence Levels of Antibodies to the Plasmodium falciparum Asexual-Stage Apical Membrane Antigen 1 but Not to Merozoite Surface Antigen 2 and Merozoite Surface Protein 1  

Microsoft Academic Search

The apical membrane antigen 1 (AMA1), merozoite surface antigen 2 (MSA2), and merozoite surface protein 1 (MSP1) are asexual-stage proteins currently being evaluated for inclusion in a vaccine for Plasmodium falciparum. Accordingly, it is important to understand factors that control antibody responses to these anti- gens. Antibody levels in plasma from residents of Etoa, Cameroon, between the ages of 5

Armead H. Johnson; Rose G. F. Leke; Nancy R. Mendell; D. Shon; Y. J. Suh; D. Bomba-Nkolo; V. Tchinda; S. Kouontchou; L. W. Thuita; A. M. van der Wel; A. Thomas; A. Stowers; A. Saul; A. Zhou; D. W. Taylor; I. A. Quakyi

2004-01-01

58

The identification of a Clonorchis sinensis gene encoding an antigenic egg protein.  

PubMed

The cDNA library of Clonorchis sinensis was screened for genes encoding antigenic proteins by using sera from clonorchiasis patients. A gene of 888 bp encoding a 28-kDa protein (Cs28) was cloned and found to contain a high percentage of glycine (20%), tyrosine (11%), and lysine (11%). The amino acid sequence of Cs28 showed 60% homology with the vitelline B precursor protein of Opisthorchis viverrini and of 33% homology with the vitelline B1 and B2 proteins of Fasciola hepatica. A strong positive reaction was observed in the intrauterine eggs of adult C. sinensis by immunohistochemical analysis using specific immune sera against recombinant Cs28 protein (rCs28). By immunoblot analysis, rCs28 displayed an antigenic reaction with 73% of the serum samples from 115 cases of clonorchiasis. In addition, it cross-reacted with the sera of 77.5% of 40 opisthorchiasis cases, 90% of 20 schistosomiasis cases, and 50% of 10 paragonimiasis cases. However, no cross-reactions were observed with the sera of sparganosis or cysticercosis patients. In conclusion, the Cs28 protein was identified as an egg protein of C. sinensis and as an antigen common to the trematode species examined. PMID:15616856

Lee, Mejeong; Chung, Young-Bae; Lee, Suk-Keun; Chung, Byung-Suk; Li, Shunyu; Choi, Min-Ho; Hong, Sung-Tae

2005-02-01

59

Comparison of antigen presentation by lymph node cells from protein and peptide-primed mice.  

PubMed Central

Lymph node cells from mice primed with peptides from the allergens Der p I and Der p II (the group I and II allergens of Dermatophagoides pteronyssinus) were unable to recall responses to the protein antigen when cultured in vitro despite being able to mount large responses to the peptides. The T cells could however recall responses to the protein when spleen-adherent cells were added into culture. Treating the spleen accessory cells with the monoclonal antibody (mAb) 33D1 and complement largely abrogated the protein response of peptide-primed T cells which indicates that dendritic cells were mainly responsible for the antigen-presenting function. If mice were primed with two injections of peptide the lymph node cells obtained could respond to both protein and peptides in vitro without the need for exogenous accessory cells. Using either negative depletion with the J11D mAb or positive purification, it was found that the presentation of protein antigen to lymph node T cells primed with either protein or peptide was limited to antigen-specific B cells. Peptide antigens could however be presented by both B and non-B populations. In one case the peptide 105-129 from Der p II which contains a T-cell epitope could not be shown to induce T-cell responses in the lymph node unless presentation was mediated by spleen-adherent or B-specific cells. These results are important for peptide-based immunomodulation and in interpreting results obtained from lymph node cultures. PMID:8436404

Hoyne, G F; Callow, M G; Kuo, M C; Thomas, W R

1993-01-01

60

CREATING A MULTIVALENT SUBUNIT VACCINE USING TYPE III SECRETION SYSTEM TIP PROTEINS AS ANTIGENS  

E-print Network

CREATING A MULTIVALENT SUBUNIT VACCINE USING TYPE III SECRETION SYSTEM TIP PROTEINS AS ANTIGENS BY Copyright 2009 Aaron Paul Markham Submitted to the graduate degree program in Pharmaceutical Chemistry and the Graduate...* ___________________________ ___________________________ ___________________________ ___________________________ Date Defended June 29, 2009_ ii The Dissertation Committee for Aaron P. Markham certifies that this is the approved version of the following dissertation: CREATING A MULTIVALENT SUBUNIT...

Markham, Aaron Paul

2009-07-06

61

Detection of common antigenic sites in lethal proteins of non-related animal venoms.  

PubMed

Monoclonal antibodies neutralizing specific coelenterate lethal toxins were used to determine the presence of homologous antigenic sites on toxin proteins of a rattlesnake (Crotalus durissus terrificus), a hornet (Vespa orientalis) and the sea wasp (Chironex fleckeri). An anti-Portuguese man-o'war toxin antibody was found useful for isolating a C. d. terrificus toxin. PMID:6623490

Russo, A J; Cobbs, C S; Calton, G J; Burnett, J W

1983-01-01

62

ankA: an Ehrlichia phagocytophila Group Gene Encoding a Cytoplasmic Protein Antigen with Ankyrin Repeats  

Microsoft Academic Search

Human granulocytic ehrlichiosis (HGE) is a potentially fatal, tick-borne disease caused by a bacterium related or identical to Ehrlichia phagocytophila. To identify and characterize E. phagocytophila group-specific protein antigen genes, we prepared and screened HGE agent and Ehrlichia equi genomic DNA expression libraries using polyclonal equine E. equi antibodies. Two clones, one each from HGE agent and E. equi, that

PATRIZIO CATUREGLI; KRISTIN M. ASANOVICH; JENNIFER J. WALLS; JOHAN S. BAKKEN; JOHN E. MADIGAN; VSEVOLOD L. POPOV; J. STEPHEN DUMLER

2000-01-01

63

Identification of Novel Mycobacterium bovis Antigens by Dissection of Crude Protein Fractions? †  

PubMed Central

Culture filtrate and cell extracts from Mycobacterium bovis cultures contain molecules which could promote protective immunity to tuberculosis in animals. Different protein fractions of M. bovis cultures were obtained by elution electrophoresis and were tested in experimentally infected cattle. The fractions that elicited gamma interferon (IFN-?) responses were resolved by two-dimensional gel electrophoresis, and individual proteins were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The open reading frames were cloned, expressed as their recombinant forms, and retested with naturally and experimentally infected animals. Eleven protein fractions were highly reactive, from which the Rv1636, HspX, Rv0138, Rv2524, EsxI, and Rv3740 recombinant proteins were obtained. EsxI and HspX were the antigens most recognized by the IFN-? release assay. In summary, a proteomic approach allowed the identification of novel antigens useful for the diagnosis of bovine tuberculosis. PMID:19641100

Meikle, V.; Alito, A.; Llera, A. S.; Gioffré, A.; Peralta, A.; Buddle, B. M.; Cataldi, A.

2009-01-01

64

Antigen-receptor induced clonal expansion and deletion of lymphocytes are impaired in mice lacking HS1 protein, a substrate of the antigen-receptor-coupled tyrosine kinases.  

PubMed Central

HS1, an intracellular protein expressed specifically in hematopoietic cells, is rapidly tyrosine phosphorylated after cross-linking of antigen receptors on B and T lymphocytes, implicating involvement of this molecule in the signal transduction pathways from the antigen receptors as a substrate of membrane-associated tyrosine kinase(s). The development of lymphoid cells in HS1-deficient mice, generated through gene targeting, appeared normal. However, antibody production to T-independent antigen and proliferative responses of splenic B and T cells after cross-linking of the antigen receptors were impaired in these mutant mice. Furthermore, B cells in the peritoneal cavity of the mutant mice were resistant to multivalent cross-linking of the antigen receptor, which causes apoptosis of such cells in normal mice. Crossing the HS1-deficient mice with the mice harboring transgenes encoding alpha and beta chains of T-cell antigen receptor against a male H-Y antigen resulted in a progeny that demonstrated a significantly impaired ability of thymic negative selection. These results indicate that HS1 is a novel molecule involved in the antigen-receptor-derived signaling pathways and plays important roles not only in clonal expansion, but also in clonal deletion of B and T cells. Images PMID:7641686

Taniuchi, I; Kitamura, D; Maekawa, Y; Fukuda, T; Kishi, H; Watanabe, T

1995-01-01

65

Mapping of Antigenic Sites on the Nucleocapsid Protein of the Severe Acute Respiratory Syndrome Coronavirus  

Microsoft Academic Search

Received 9 June 2004\\/Returned for modification 18 July 2004\\/Accepted 27 July 2004 Antigenic sites on the nucleocapsid (N) protein of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) were mapped by Pepscan analysis with overlapping peptides that span the N protein sequence. Two major immunodominant epitopes located in the C-terminal region (amino acids (aa) 362 to 412) and middle region (aa

Yuxian He; Yusen Zhou; Hao Wu; Zhihua Kou; Shuwen Liu; Shibo Jiang

2004-01-01

66

The polycomb group proteins, BMI1 and EZH2, are tumour-associated antigens  

Microsoft Academic Search

We used SEREX technology to identify novel tumour-associated antigens in patients with primary hepatocellular carcinoma and found serological responses to the polycomb group (PcG) protein BMI-1, which is overexpressed in a range of different tumour types. Further studies identified T-cell responses to both BMI-1 and another PcG protein, EZH2, in cancer patients and at relatively lower levels in some normal

J C Steele; E E Torr; K L Noakes; E Kalk; P A Moss; G M Reynolds; S G Hubscher; M van Lohuizen; D H Adams; L S Young

2006-01-01

67

Determinants of antigenicity and specificity in immune response for protein sequences  

Microsoft Academic Search

Background  Target specific antibodies are pivotal for the design of vaccines, immunodiagnostic tests, studies on proteomics for cancer\\u000a biomarker discovery, identification of protein-DNA and other interactions, and small and large biochemical assays. Therefore,\\u000a it is important to understand the properties of protein sequences that are important for antigenicity and to identify small\\u000a peptide epitopes and large regions in the linear sequence

Yulong Wang; Wenjun Wu; Nicolas N Negre; Kevin P White; Cheng Li; Parantu K Shah

2011-01-01

68

Neisseria meningitidis antigen NMB0088: sequence variability, protein topology and vaccine potential.  

PubMed

The significance of Neisseria meningitidis serogroup B membrane proteins as vaccine candidates is continually growing. Here, we studied different aspects of antigen NMB0088, a protein that is abundant in outer-membrane vesicle preparations and is thought to be a surface protein. The gene encoding protein NMB0088 was sequenced in a panel of 34 different meningococcal strains with clinical and epidemiological relevance. After this analysis, four variants of NMB0088 were identified; the variability was confined to three specific segments, designated VR1, VR2 and VR3. Secondary structure predictions, refined with alignment analysis and homology modelling using FadL of Escherichia coli, revealed that almost all the variable regions were located in extracellular loop domains. In addition, the NMB0088 antigen was expressed in E. coli and a procedure for obtaining purified recombinant NMB0088 is described. The humoral immune response elicited in BALB/c mice was measured by ELISA and Western blotting, while the functional activity of these antibodies was determined in a serum bactericidal assay and an animal protection model. After immunization in mice, the recombinant protein was capable of inducing a protective response when it was administered inserted into liposomes. According to our results, the recombinant NMB0088 protein may represent a novel antigen for a vaccine against meningococcal disease. However, results from the variability study should be considered for designing a cross-protective formulation in future studies. PMID:19141737

Sardiñas, Gretel; Yero, Daniel; Climent, Yanet; Caballero, Evelin; Cobas, Karem; Niebla, Olivia

2009-02-01

69

Application of a Novel Radioimmunoassay to Identify Baculovirus Structural Proteins That Share Interspecies Antigenic Determinants  

PubMed Central

Immunological comparisons were made of baculovirus structural proteins by using a modification of the radioimmunological techniques described by Renart et al. (Proc. Natl. Acad. Sci. U.S.A. 76: 3116-3120, 1979) and Towbin et al. (Proc. Natl. Acad. Sci. U.S.A. 76: 4350-4354, 1979). Viral proteins were electrophoresed in polyacrylamide gels, transferred to nitrocellulose, and incubated with viral antisera, and the antibodies were detected with 125I-labeled Staphylococcus aureus protein A. Antisera were prepared to purified and intact virions from five baculoviruses: Autographa californica, Porthetria dispar, Trichoplusia ni, and Heliothis zea nuclear polyhedrosis viruses (NPVs) and T. ni granulosis virus (GV). These antisera were tested against the virion structural polypeptides of 17 different species of baculoviruses. Specific multiple-nucleocapsid NPV (MNPV), single-nucleocapsid NPV (SNPV), and GV virion polypeptides were shown to have similar antigenic determinants and thus be immunologically related. The molecular weights of the virion polypeptides with cross-reacting antigenic determinants were identified. Antisera prepared to purified A. californica and H. zea MNPV polyhedrin (the occlusion body protein from NPVs) recognized antigenic determinants on all the polyhedrins and granulins (occlusion body protein from GVs) that were tested. No immunological relationship was detected between A. californica MNPV polyhedrin and any of the A. californica MNPV virion structural polypeptides present on either the virus isolated from occlusion bodies or A. californica MNPV extracellular virus from infected-cell cultures. Images PMID:16789210

Smith, Gale E.; Summers, Max D.

1981-01-01

70

Specific Nongluten Proteins of Wheat Are Novel Target Antigens in Celiac Disease Humoral Response  

PubMed Central

While the antigenic specificity and pathogenic relevance of immunologic reactivity to gluten in celiac disease have been extensively researched, the immune response to nongluten proteins of wheat has not been characterized. We aimed to investigate the level and molecular specificity of antibody response to wheat nongluten proteins in celiac disease. Serum samples from patients and controls were screened for IgG and IgA antibody reactivity to a nongluten protein extract from the wheat cultivar Triticum aestivum Butte 86. Antibodies were further analyzed for reactivity to specific nongluten proteins by two-dimensional gel electrophoresis and immunoblotting. Immunoreactive molecules were identified by tandem mass spectrometry. Compared with healthy controls, patients exhibited significantly higher levels of antibody reactivity to nongluten proteins. The main immunoreactive nongluten antibody target proteins were identified as serpins, purinins, ?-amylase/protease inhibitors, globulins, and farinins. Assessment of reactivity toward purified recombinant proteins further confirmed the presence of antibody response to specific antigens. The results demonstrate that, in addition to the well-recognized immune reaction to gluten, celiac disease is associated with a robust humoral response directed at a specific subset of the nongluten proteins of wheat. PMID:25329597

2014-01-01

71

Determinants of antigenicity and specificity in immune response for protein sequences  

PubMed Central

Background Target specific antibodies are pivotal for the design of vaccines, immunodiagnostic tests, studies on proteomics for cancer biomarker discovery, identification of protein-DNA and other interactions, and small and large biochemical assays. Therefore, it is important to understand the properties of protein sequences that are important for antigenicity and to identify small peptide epitopes and large regions in the linear sequence of the proteins whose utilization result in specific antibodies. Results Our analysis using protein properties suggested that sequence composition combined with evolutionary information and predicted secondary structure, as well as solvent accessibility is sufficient to predict successful peptide epitopes. The antigenicity and the specificity in immune response were also found to depend on the epitope length. We trained the B-Cell Epitope Oracle (BEOracle), a support vector machine (SVM) classifier, for the identification of continuous B-Cell epitopes with these protein properties as learning features. The BEOracle achieved an F1-measure of 81.37% on a large validation set. The BEOracle classifier outperformed the classical methods based on propensity and sophisticated methods like BCPred and Bepipred for B-Cell epitope prediction. The BEOracle classifier also identified peptides for the ChIP-grade antibodies from the modENCODE/ENCODE projects with 96.88% accuracy. High BEOracle score for peptides showed some correlation with the antibody intensity on Immunofluorescence studies done on fly embryos. Finally, a second SVM classifier, the B-Cell Region Oracle (BROracle) was trained with the BEOracle scores as features to predict the performance of antibodies generated with large protein regions with high accuracy. The BROracle classifier achieved accuracies of 75.26-63.88% on a validation set with immunofluorescence, immunohistochemistry, protein arrays and western blot results from Protein Atlas database. Conclusions Together our results suggest that antigenicity is a local property of the protein sequences and that protein sequence properties of composition, secondary structure, solvent accessibility and evolutionary conservation are the determinants of antigenicity and specificity in immune response. Moreover, specificity in immune response could also be accurately predicted for large protein regions without the knowledge of the protein tertiary structure or the presence of discontinuous epitopes. The dataset prepared in this work and the classifier models are available for download at https://sites.google.com/site/oracleclassifiers/. PMID:21693021

2011-01-01

72

Plasmodium vivax Antigen Discovery Based on Alpha-Helical Coiled Coil Protein Motif  

PubMed Central

Protein ?-helical coiled coil structures that elicit antibody responses, which block critical functions of medically important microorganisms, represent a means for vaccine development. By using bioinformatics algorithms, a total of 50 antigens with ?-helical coiled coil motifs orthologous to Plasmodium falciparum were identified in the P. vivax genome. The peptides identified in silico were chemically synthesized; circular dichroism studies indicated partial or high ?-helical content. Antigenicity was evaluated using human sera samples from malaria-endemic areas of Colombia and Papua New Guinea. Eight of these fragments were selected and used to assess immunogenicity in BALB/c mice. ELISA assays indicated strong reactivity of serum samples from individuals residing in malaria-endemic regions and sera of immunized mice, with the ?-helical coiled coil structures. In addition, ex vivo production of IFN-? by murine mononuclear cells confirmed the immunogenicity of these structures and the presence of T-cell epitopes in the peptide sequences. Moreover, sera of mice immunized with four of the eight antigens recognized native proteins on blood-stage P. vivax parasites, and antigenic cross-reactivity with three of the peptides was observed when reacted with both the P. falciparum orthologous fragments and whole parasites. Results here point to the ?-helical coiled coil peptides as possible P. vivax malaria vaccine candidates as were observed for P. falciparum. Fragments selected here warrant further study in humans and non-human primate models to assess their protective efficacy as single components or assembled as hybrid linear epitopes. PMID:24959747

Céspedes, Nora; Habel, Catherine; Lopez-Perez, Mary; Castellanos, Angélica; Kajava, Andrey V.; Servis, Catherine; Felger, Ingrid; Moret, Remy; Arévalo-Herrera, Myriam; Corradin, Giampietro; Herrera, Sócrates

2014-01-01

73

Analysis of sperm antigens by sodium dodecyl sulfate gel/protein blot radioimmunobinding method  

SciTech Connect

A radioimmunobinding method based on the blotting of renatured proteins from sodium dodecyl sulfate gels on to nitrocellulose filter papers was developed to analyze the sperm antigens that elicit serum anti-sperm antibodies. In rabbits, serum anti-sperm antibodies were raised by immunization with homologous epididymal spermatozoa mixed with complete Freund's adjuvant. The raised antisera from either male or female rabbits were shown to react with three major sperm protein bands on sodium dodecyl sulfate gels with the corresponding molecular weights of about 70,000 +/- 5000, 14,000, and 13,000, respectively. In humans, the monoclonal antibodies against human sperm were raised by a hybridoma technique. Out of six independent hybrid cell lines that were generated, three of them were shown to secrete immunoglobulins that react with the same two protein bands on sodium dodecyl sulfate gels, which have the approximate molecular weight of 10,000. The same procedure was also used to analyze human serum samples that were shown to contain anti-sperm antibodies by the known techniques. Unique sperm antigens that elicit anti-sperm antibodies in humans were identified and correlated. The results of this study suggest that sodium dodecyl sulfate gel/protein blot radioimmunobinding method may be a sensitive and useful tool for the study of sperm antigens that elicit autoimmune responses and their association with human infertility.

Lee, C.Y.G. (Univ. of British Columbia, Vancouver, Canada); Huang, Y.S.; Hu, P.C.; Gomel, V.; Menge, A.C.

1982-06-01

74

A protective protein antigen of Rickettsia rickettsii has tandemly repeated, near-identical sequences.  

PubMed Central

The nucleotide sequence of a Rickettsia rickettsii gene that encodes a high-molecular-mass surface antigen (190 kilodaltons), which elicits protective immunity, was determined. The 6,747-nucleotide gene coded for a 2,249-amino-acid protein with a calculated molecular weight of 224,321. A 3.8-kilobase PstI fragment proximal to the 5' end of the gene was found to consist of 13 highly related tandem repeats which constituted over 40% of the coding region. The repeated sequences could be divided into either a 225-nucleotide, 75-amino-acid unit (type I) or a 216-nucleotide, 72-amino-acid unit (type II), with extensive homology between the two types of repeating units. The deduced amino acid sequence for these repeat units, overall, was slightly hydrophobic with short hydrophilic domains. The carboxy-terminal (nonrepetitive) portion of the deduced protein sequence was hydrophilic, with potential surface-exposed epitopes. The full-length reading frame was reconstructed in Escherichia coli, and transient expression of the 190-kilodalton antigen was demonstrated; however, the protein appeared to be severely degraded by proteases and was apparently toxic to E. coli. The conservation of this unique repetitive gene structure, coupled with results from previous reports showing the protective properties of the 190-kilodalton antigen, suggests that this protein plays an important role in the pathogenesis of and immunity to Rocky Mountain spotted fever. Images PMID:2117568

Anderson, B E; McDonald, G A; Jones, D C; Regnery, R L

1990-01-01

75

Serological expression cloning of novel immunoreactive antigens of Babesia microti.  

PubMed

Increased recognition of the prevalence of human babesiosis in the United States, together with rising concern about the potential for transmission of this infection by blood transfusion, has provided motivation to develop definitive serologic and molecular tests for the causative agent, Babesia microti. To develop more sensitive and specific assays for B. microti, we screened a genomic expression library with patient serum pools. This screening resulted in the identification of three classes of novel genes and an additional two novel, unrelated genes, which together encode a total of 17 unique B. microti antigens. The first class (BMN1-2 family) of genes encodes seven closely related antigens with a degenerate six-amino-acid repeat that shows limited homology to Plasmodium sp. merozoite and sporozoite surface antigens. A second class (BMN1-8 family) of genes encodes six related antigens, and the third class (BMN1-17 family) of genes encodes two related antigens. The two remaining genes code for novel and unrelated sequences. Among the three classes of antigens and remaining novel sequences, five were chosen to code for the most immunodominant antigens (BMN1-2, -9, -15, and -17 and MN-10). Western blot analysis with the resulting recombinant proteins indicated that these antigens were targets of humoral immune responses during B. microti infection in humans. PMID:10768973

Lodes, M J; Houghton, R L; Bruinsma, E S; Mohamath, R; Reynolds, L D; Benson, D R; Krause, P J; Reed, S G; Persing, D H

2000-05-01

76

Antigenic polymorphism of the LamB protein among members of the family Enterobacteriaceae.  

PubMed Central

In this study we demonstrate that most members of the family Enterobacteriaceae possess a maltose-inducible outer membrane protein homologous to the LamB protein of Escherichia coli K-12. These proteins react with polyclonal antibodies raised against the LamB protein of E. coli K-12. We compared the antigenic structure of the LamB protein in members of the family Enterobacteriaceae with six monoclonal antibodies raised against the LamB protein of E. coli K-12. Four of them reacted with epitopes located at the outer face of the membrane, and two reacted with epitopes located at the inner face of the membrane. A great degree of variability was observed for the external epitopes. Even in a single species, such as E. coli, an important polymorphism was present. In contrast, the internal epitopes were more conserved. Images PMID:4040134

Bloch, M A; Desaymard, C

1985-01-01

77

Interactions between killer immunoglobulin-like receptors and their human leucocyte antigen Class I ligands influence the outcome of unrelated haematopoietic stem cell transplantation for thalassaemia: a novel predictive algorithm.  

PubMed

In a study conducted on 114 patients undergoing unrelated donor haematopoietic stem cell transplantation (HSCT) for thalassaemia, we observed that the lack of activating killer immunoglobulin-like receptors (KIRs) on donor natural killer (NK) cells significantly increased the risk of graft-versus-host disease (GvHD) [hazard risk (HR) 4.2, 95% confidence interval (CI) 1.7-10.1, P = 0.002] and transplantation-related mortality (HR 4.7, 95% CI 1.6-14.2, P = 0.01). The risk of GvHD furthermore increased when recipients heterozygous for HLA-C KIR ligand groups (C1/C2) were transplanted from donors completely lacking activating KIRs (HR 6.1, 95% CI 1.9-19.2, P = 0.002). We also found that the risk of rejection was highest when the recipient was homozygous for the C2 HLA-KIR ligand group and the donor carried two or more activating KIRs (HR 6.8, 95% CI 1.9-24.4, P = 0.005). By interpolating the number of donor activating KIRs with recipient HLA-C KIR ligands, we created an algorithm capable of stratifying patients according to the immunogenetic risk of complications following unrelated HSCT. In clinical practice, this predictive tool could serve as an important supplement to clinical judgement and decision-making. PMID:22077388

Littera, Roberto; Orrù, Nicola; Caocci, Giovanni; Sanna, Marco; Mulargia, Marina; Piras, Eugenia; Vacca, Adriana; Giardini, Claudio; Orofino, Maria G; Visani, Giuseppe; Bertaina, Alice; Giorgiani, Giovanna; Locatelli, Franco; Carcassi, Carlo; La Nasa, Giorgio

2012-01-01

78

Immuno-electronmicroscopic identification and localization of the antigenic proteins of tree pollen grains.  

PubMed

The localization of antigenic proteins on ultrathin sections of pollen grains represents an interesting approach to understanding the release mechanisms of these antigens when the pollen grains come in contact with various physiological fluids. Using different rabbit antibodies we have demonstrated the locations of these antigens in the various structures of pollen grains. We further demonstrated the cross-reactivities between alder (Alnus incana), birch (Betula verrucosa) and hazel (Corylus avellana) pollen allergens. Ultrathin sections of the pollen grains were prepared and allowed to react with two individually raised rabbit antibodies, (Ab-BV and Ab-ALK), against birch pollen. The sites of the Ag/Ab complex on the sections were labelled by protein A/gold, and identified in a transmission electron microscope. The two birch antibodies showed either quantitative or qualitative differences regarding their binding to various structures on the pollen sections. Using Ab-BV, the antigen-binding sites were located in the apertural region of the pollen grain and in the cytoplasm, while almost no gold labelling could be seen on the pollen surface. With the other antibodies (Ab-ALK), we could visualize the antigen-binding locations on the surface material of the pollen grains, particularly in the exine part of the wall and in the cytoplasm. A few gold particles could also be seen in the apertural region of the pollen. In hazel and alder pollen the exine part of the wall was the most densely labelled, whereas the cytoplasm and the aperture bound smaller numbers of gold particles. Cross-incubations: birch pollen incubated with antibodies against hazel (Ab-CA), or alder (Ab-AI), showed various intensities of gold labelling for each of the three species. Statistically, the differences in the number of gold particles bound per micron 2 grain section between birch, hazel and alder, were highly significant. The cross-reactivities between these antigens from the three pollen species were further tested using house-produced rabbit antisera against antigens of the three species by means of electrophoretic and autoradiographic techniques (CIE and CRIE). The three antibodies could precipitate the major IgE-binding antigen from all three pollen species. PMID:3207183

Grote, M; Vik, H; Elsayed, S

1988-11-01

79

Interaction of the p53-Regulated Protein Gadd45 with Proliferating Cell Nuclear Antigen  

Microsoft Academic Search

GADD45 is a ubiquitously expressed mammalian gene that is induced by DNA damage and certain other stresses. Like another p53-regulated gene, p21WAF1\\/CIP1, whose product binds to cyclin-dependent kinases (Cdk's) and proliferating cell nuclear antigen (PCNA), GADD45 has been associated with growth suppression. Gadd45 was found to bind to PCNA, a normal component of Cdk complexes and a protein involved in

Martin L. Smith; I.-Tsuen Chen; Qimin Zhan; Insoo Bae; Chaw-Yuan Chen; Tona M. Gilmer; Michael B. Kastan; Patrick M. O'Connor; Albert J. Fornace Jr.

1994-01-01

80

Mapping Antigenic Domains Expressed by Chlamydia trachomatis Major Outer Membrane Protein Genes  

Microsoft Academic Search

Chlamydia trachomatis is an obligate prokaryotic intracellular pathogen of humans that infects mucosal epithelial cells. Exposed domains of its major outer membrane protein (MOMP) are both serotyping and protective antigenic determinants. To identify these domains, we have cloned and epitope-mapped the genes of serovars A, C (C serogroup) and L2, B (B serogroup) with a panel of monoclonal antibodies (mAbs).

Wolfgang Baehr; You-Xun Zhang; Theresa Joseph; Hua Su; Francis E. Nano; Karin D. E. Everett; Harlan D. Caldwell

1988-01-01

81

Antigenicity and Immunogenicity of a Synthetic Oligosaccharide-Protein Conjugate Vaccine against Haemophilus influenzae Type b  

Microsoft Academic Search

Polysaccharide-protein conjugates as vaccines have proven to be very effective in preventing Haemophilus influenzae type b infections in industrialized countries. However, cost-effective technologies need to be devel- oped for increasing the availability of anti-H. influenzae type b vaccines in countries from the developing world. Consequently, vaccine production with partially synthetic antigens is a desirable goal for many reasons. They may

V. Fernandez-Santana; F. Cardoso; A. Rodriguez; T. Carmenate; L. Pena; Y. Valdes; E. Hardy; F. Mawas; L. Heynngnezz; M. C. Rodriguez; I. Figueroa; J. Chang; M. E. Toledo; A. Musacchio; I. Hernandez; M. Izquierdo; K. Cosme; R. Roy; V. Verez-Bencomo

2004-01-01

82

Application of a novel radioimmunoassay to identify baculovirus structural proteins that share interspecies antigenic determinants  

SciTech Connect

Immunological comparisons were made of baculovirus structural proteins by using a modification of the radioimmunological techniques described by Renart et al. and Towbin et al. Viral proteins were electrophoresed in polyacrylamide gels, transferred to nitrocellulose, and incubated with viral antisera, and the antibodies were detected with /sup 125/I-labeled Staphylococcus aureus protein A. Antisera were prepared to purified and intact virions from five baculoviruses: Autographa californica, Porthetria dispar, Trichoplusia ni, and Heliothis zea nuclear polyhedrosis viruses (NPVs) and T. ni granulosis virus (GV). These antisera were tested against the virion structural polypeptides of 17 different species of baculoviruses. Specific multiple-nucleocapsid NPV (MNPV), single-nucleocapsid NPV (SNPV), and GV virion polypeptides were shown to have similar antigenic determinants and thus be immunologically related. The molecular weights of the virion polypeptides with cross-reacting antigenic determinants were identified. Antisera prepared to purified A. californica and H. zea MNPV polyhedrin recognized antigenic determinants on all the polyhedrins and granulins that were tested. No immunological relationship was detected between A. californica MNPV polyhedrin and any of the A. californica MNPV virion structural polypeptides present on either the virus isolated from occlusion bodies or A. californica MNPV extracellular virus from infected-cell cultures.

Smith, G.E.; Summers, M.D.

1981-07-01

83

The inner centromere protein (INCENP) antigens: movement from inner centromere to midbody during mitosis  

PubMed Central

We describe a novel set of polypeptide antigens that shows a dramatic change in structural localization during mitosis. Through metaphase these antigens define a new chromosomal substructure that is located between the sister chromatids. Because the antigens are concentrated in the pericentromeric region, we have provisionally termed them the INCENPs (inner centromere proteins). The INCENPs (two polypeptides of 155 and 135 kD) were identified with a monoclonal antibody that was raised against the bulk proteins of the mitotic chromosome scaffold fraction. These two polypeptides are the most tightly bound chromosomal proteins known. When scaffolds are prepared, 100% of the detectable INCENPs remain scaffold associated. We were therefore unprepared for the fate of the INCENPs at anaphase. As the sister chromatids separate, the INCENPs dissociate fully from them, remaining behind at the metaphase plate as the chromatids migrate to the spindle poles. During anaphase the INCENPs are found on coarse fibers in the central spindle, and also in close apposition to the cell membrane in the region of the forming contractile ring. During telophase, the INCENPs gradually become focused onto the forming midbody, together with which they are ultimately discarded. Several possible in vivo roles for the INCENPs are suggested by these data: regulation of sister chromatid pairing, stabilization of the plane of cleavage, and separation of spindle poles at anaphase. PMID:3316246

1987-01-01

84

Synergistic effect of major histocompatibility complex class I-related chain a and human leukocyte antigen-DPB1 mismatches in association with acute graft-versus-host disease after unrelated donor hematopoietic stem cell transplantation.  

PubMed

The clinical relevance of mismatches at the MHC class I-related chain A (MICA) in hematopoietic stem cell transplantation (HSCT) remains unclear. We investigated the association of MICA donor/recipient mismatch and whether there is an interaction between these and HLA-DPB1 mismatch on clinical outcomes after unrelated donor HSCT. Our study included 227 patients who underwent unrelated donor allogeneic HSCT at our institution between 2000 and 2010. Among these, 177 (78%) received HSCT from a 10/10 HLA-matched donor. MICA genotyping was performed using commercially available kits. In univariable analysis, the risk of grade II to IV acute graft-versus-host disease (GVHD) was greater for patients with MICA mismatch (hazard ratio [HR], 1.73; P = .02) than for those with HLA-DPB1 mismatch (HR, 1.62; P = .07). When MICA and HLA-DPB1 were assessed simultaneously, patients mismatched at both loci had the greatest risk (HR, 2.51; P < .01) and those mismatched at only 1 locus had somewhat greater risk (HR, 1.53; P = .12) than patients matched at both loci; this remained significant in multivariable analysis. The 100-day incidence was 66%, 45%, and 31%, respectively (P = .03). Results were similar for grade III and IV acute GVHD, with 100-day incidence 34%, 16%, and 8% (P = .01). These results are clinically pertinent to donor selection strategies and indicate that patients with mismatch at both MICA and HLA-DPB1 are at increased risk for acute GVHD. PMID:25064744

Askar, Medhat; Sun, Yuchu; Rybicki, Lisa; Zhang, Aiwen; Thomas, Dawn; Kalaycio, Matt; Pohlman, Brad; Dean, Robert; Duong, Hien; Hanna, Rabi; Maciejewski, Jaroslaw; Majhail, Navneet S; Bolwell, Brian; Sobecks, Ronald

2014-11-01

85

Comparison of the Proteosomes and Antigenicities of Secreted and Cellular Proteins Produced by Mycobacterium paratuberculosis  

PubMed Central

The protein expression profiles and antigenicities of both culture filtrates (CF) and cellular extracts (CE) of Mycobacterium paratuberculosis were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), one-dimensional electrophoresis (1-DE) and 2-DE immunoblotting, and enzyme-linked immunosorbent assay (ELISA). The CF proteins were harvested from supernatants of stationary-phase liquid cultures and concentrated by size exclusion filtration. The CE proteins were extracted by mechanical disruption of cells using glass beads and a high-speed agitator. Analysis of SDS-PAGE gels showed that the majority of CF proteins had low molecular masses (<50 kDa), whereas CE protein mass ranged more evenly over a broader range up to 100 kDa. By 2-DE, CF proteins had a narrow array of pI values, with most being between pH 4.0 and 5.5; CE proteins spanned pI values from pH 4.0 to 7.0. The antigenicities of CF and CE proteins were first determined by 1-DE and 2-DE immunoblotting with serum from a cow naturally infected with M. paratuberculosis. The serum reacted strongly to more proteins in the CF than the CE. Sera from 444 infected and 412 uninfected cattle were tested by ELISA with CF and CE as solid-phase antigens. Receiver-operator characteristic curve analysis of the ELISA results showed a significantly greater area under the curve for CF compared to CE (P < 0.05). A high degree of variability in protein binding patterns was shown with 1-DE immunoblot analysis with 31 sera from M. paratuberculosis-infected cattle. Collectively, these results indicate that serologic tests for bovine paratuberculosis may be improved by using proteins derived from CF instead of CE. To maximize the diagnostic sensitivity of serologic tests, multiple proteins will be required. Even so, a CF ELISA may not be able to detect all M. paratuberculosis-infected cattle, in particular those in the early stages of infection that have yet to mount an antibody response. PMID:17028217

Cho, Donghee; Collins, Michael T.

2006-01-01

86

Potential role for Duffy antigen chemokine-binding protein in angiogenesis and maintenance of homeostasis in response to stress  

Microsoft Academic Search

CXC chemokines, which induce angio- genesis, have glutamine-leucine-arginine amino acid residues (ELR motif) in the amino terminus and bind CXCR2 and the Duffy antigen chemokine- binding protein. Duffy, a seven transmembrane protein that binds CXC and CC chemokines, has not been shown to couple to trimeric G proteins or to transduce intracellular signals, although it is highly expressed on red

Jianguo Du; Jing Luan; Hua Liu; Thomas O. Daniel; Stephen Peiper; Theresa S. Chen; Yingchun Yu; Linda W. Horton; Lillian B. Nanney; Robert M. Strieter; Ann Richmond

87

Overexpression, purification and validation of antigenic Salmonella enterica serovar Typhi proteins identified from LC-MS/MS.  

PubMed

In our earlier study, an immunoblot analysis using sera from febrile patients revealed that a 50-kDa band from an outer membrane protein fraction of Salmonella enterica serovar Typhi was specifically recognized only by typhoid sera and not sera from other febrile illnesses. Here, we investigated the identities of the proteins contained in the immunogenic 50-kDa band to pinpoint antigens responsible for its immunogenicity. We first used LC-MS/MS for protein identification, then used the online tool ANTIGENpro for antigenicity prediction and produced recombinant proteins of the lead antigens for validation in an enzyme-linked immunosorbent assay (ELISA). We found that proteins TolC, GlpK and SucB were specific to typhoid sera but react to antibodies differently under native and denatured conditions. This difference suggests the presence of linear and conformational epitopes on these proteins. PMID:25149461

Chin, Chai Fung; Teh, Boon Aun; Anthony, Amy Amilda; Aziah, Ismail; Ismail, Asma; Ong, Eugene Boon Beng; Lim, Theam Soon

2014-11-01

88

Antigen detection using recombinant, bifunctional single-chain Fv fusion proteins synthesised in Escherichia coli.  

PubMed

A gene fusion approach has been used to produce antibody conjugates for use in immunoassays. Escherichia coli expression vectors encoding fusions between the outer membrane protein A signal peptide, an anti-phytochrome single-chain Fv protein, and either Escherichia coli alkaline phosphatase or Staphylococcal protein A downstream of the T7 O10 promoter were constructed. A crude lysate from cells expressing the single-chain Fv-alkaline phosphatase fusion protein could be used directly for the sensitive and specific staining of phytochrome on protein blots by a single-step immunoassay procedure. Following purification by immunoglobulin G affinity chromatography, the Staphylococcal protein A-single-chain Fv fusion protein was also used for selective immunostaining of phytochrome on protein blots by a two-step procedure in which a rabbit immunoglobulin-alkaline phosphatase conjugate was used to detect antigen-bound Staphylococcal protein A. Recombinant antibody conjugates of the types described here are simple and inexpensive to produce and are a realistic alternative to conventional antibody conjugates. PMID:7950386

Gandecha, A; Owen, M R; Cockburn, W; Whitelam, G C

1994-08-01

89

Recombinant dengue 2 virus NS3 protein conserves structural antigenic and immunological properties relevant for dengue vaccine design.  

PubMed

The NS3 protein is a multifunctional non-structural protein of flaviviruses implicated in the polyprotein processing. The predominance of cytotoxic T cell lymphocytes epitopes on the NS3 protein suggests a protective role of this protein in limiting virus replication. In this work, we studied the antigenicity and immunogenicity of a recombinant NS3 protein of the Dengue virus 2. The full-length NS3 gene was cloned and expressed as a His-tagged fusion protein in Escherichia coli. The pNS3 protein was purified by two chromatography steps. The recombinant NS3 protein was recognized by anti-protease NS3 polyclonal antibody and anti-DENV2 HMAF by Western Blot. This purified protein was able to stimulate the secretion of high levels of gamma interferon and low levels of interleukin-10 and tumor necrosis factor-? in mice splenocytes, suggesting a predominantly Th-1-type T cell response. Immunized BALB/c mice with the purified NS3 protein showed a strong induction of anti-NS3 IgG antibodies, essentially IgG2b, as determined by ELISA. Immunized mice sera with recombinant NS3 protein showed specific recognition of native dengue protein by Western blotting and immunofluorescence techniques. The successfully purified recombinant protein was able to preserv the structural and antigenic determinants of the native dengue protein. The antigenicity shown by the recombinant NS3 protein suggests its possible inclusion into future DENV vaccine preparations. PMID:24854144

Ramírez, Rosa; Falcón, Rosabel; Izquierdo, Alienys; García, Angélica; Alvarez, Mayling; Pérez, Ana Beatriz; Soto, Yudira; Muné, Mayra; da Silva, Emiliana Mandarano; Ortega, Oney; Mohana-Borges, Ronaldo; Guzmán, María G

2014-10-01

90

Comparative evaluation of the diagnostic potential of recombinant envelope proteins and native cell culture purified viral antigens of Chikungunya virus.  

PubMed

Despite the fact that Chikungunya resurgence is associated with epidemic of unprecedented magnitude, there are challenges in the field of its clinical diagnosis. However, serological tests in an ELISA format provide a rapid tool for the diagnosis of Chikungunya infection. Indeed, ELISAs based on recombinant proteins hold a great promise as these methods are cost effective and are free from the risk of handling biohazardous material. In this study, the performance of recombinant CHIKV antigens was compared in various ELISA formats for the diagnosis of Chikungunya. Two recombinant antigens derived from the envelope proteins of Chikungunya virus were prepared and evaluated by comparing their competence for detecting circulating antibodies in serum samples of patients infected with CHIKV using MAC-ELISA and indirect IgM-ELISA. The efficacy of the recombinant antigens was also compared with the native antigen. The indirect antibody capture IgM microplate ELISA revealed ?90% concordance with the native antigen in detecting the CHIKV specific IgM antibodies whereas the recombinant antigen based MAC-ELISA showed 100% specificity. The recombinant antigens used in this study were effective and reliable targets for the diagnosis of CHIKV infection and also provide an alternative for native antigen use which is potentially biohazardous. PMID:24105844

Khan, Mohsin; Dhanwani, Rekha; Kumar, Jyoti S; Rao, P V Lakshmana; Parida, Manmohan

2014-07-01

91

Characterization of Treponema denticola Mutants Defective in the Major Antigenic Proteins, Msp and TmpC  

PubMed Central

Treponema denticola, a gram-negative and anaerobic spirochete, is associated with advancing severity of chronic periodontitis. In this study, we confirmed that two major antigenic proteinswere Msp and TmpC, and examined their physiological and pathological roles using gene-deletion mutants. Msp formed a large complex that localized to the outer membrane, while TmpC existed as a monomer and largely localized to the inner membrane. However, TmpC was also detected in the outer membrane fraction, but its cell-surface exposure was not detected. Msp defects increased cell-surface hydrophobicity and secretion of TNF-? from macrophage-like cells, whereas TmpC defects decreased autoagglutination and chymotrypsin-like protease activities. Both mutants adhered to gingival epithelial cells similarly to the wild-type and showed slightly decreased motility. In addition, in Msp-defective mutants, the TDE1072 protein, which is a major membrane protein, was abolished; therefore, phenotypic changes in the mutant can be, at least in part, attributed to the loss of the TDE1072 protein. Thus, the major antigenic proteins, Msp and TmpC, have significant and diverse impacts on the characteristics of T. denticola, especially cell surface properties. PMID:25401769

Abiko, Yuki; Nagano, Keiji; Yoshida, Yasuo; Yoshimura, Fuminobu

2014-01-01

92

A peptide binding protein having a role in antigen presentation is a member of the HSP70 heat shock family.  

PubMed

The T cell recognition of globular protein antigens requires the processing and presentation of the antigen by Ia-expressing APCs. Processing is believed to involve the uptake of antigen into an acidic compartment where proteolysis occurs. The resulting peptides containing the T cell antigenic determinant are associated with Ia and presented at the cell surface to the specific T cells. The mechanisms by which antigenic peptides become associated with Ia is not known. We previously described a peptide binding protein of 72/74 x 10(3) Mr (PBP72/74) that plays a role in antigen presentation as shown by the ability of an antiserum raised in rabbits to affinity-purified PBP72/74 to block presentation of cytochrome c to a cytochrome c-specific T cell hybrid. Here we show that PBP72/74 is recognized by mAbs specific for members of the HSP70 family of proteins. In Western blots PBP72/74 is bound by mAb 7.10, specific for an evolutionarily conserved epitope of HSP proteins and by mAb N27, specific for both the constitutively expressed and inducible 72/73 x 10(3) Mr HSP70 proteins. In addition, PBP72/74 shares a second common feature of the HSP proteins, that of binding to ATP. Indeed, ATP causes the release of PBP72/74 from binding to a peptide fragment of cytochrome c (Pc 81-104) and PBP72/74 can be eluted from ATP columns by Pc 81-104. Finally, a portion of PBP72/74 is shown to be present on B cell surfaces by immunofluorescence staining. Thus, it appears that characteristics of the heat shock proteins are shared by a protein playing a role in antigen presentation, suggesting some commonality in function. PMID:2584924

Vanbuskirk, A; Crump, B L; Margoliash, E; Pierce, S K

1989-12-01

93

Antigenic heterogeneity of capsid protein VP1 in foot-and-mouth disease virus (FMDV) serotype Asia 1  

PubMed Central

Foot and mouth disease virus (FMDV), with its seven serotypes, is a highly contagious virus infecting mainly cloven-hoofed animals. The serotype Asia1 occurs mainly in Asian regions. An in-silico approach was taken to reveal the antigenic heterogeneities within the capsid protein VP1 of Asia1. A total of 47 VP1 sequences of Asia1 isolates from different countries of South Asian regions were selected, retrieved from database, and were aligned. The structure of VP1 protein was modeled using a homology modeling approach. Several antigenic sites were identified and mapped onto the three-dimensional protein structure. Variations at these antigenic sites were analyzed by calculating the protein variability index and finding mutation combinations. The data suggested that vaccine escape mutants have derived from only few mutations at several antigenic sites. Five antigenic peptides have been identified as the least variable epitopes, with just fewer amino acid substitutions. Only a limited number of serotype Asia1 antigenic variants were found to be circulated within the South Asian region. This emphasizes a possibility of formulating synthetic vaccines for controlling foot-and-mouth disease by Asia1 serotypes. PMID:23983476

Alam, SM Sabbir; Amin, Ruhul; Rahman, Mohammed Ziaur; Hossain, M Anwar; Sultana, Munawar

2013-01-01

94

Antigen-driven bystander effect accelerates epicutaneous sensitization with a new protein allergen.  

PubMed

Exposure to protein allergen epicutaneously, inducing a Th2-dominant immune response, sensitizes the host to the development of atopic disease. Antigen-driven bystander effect demonstrates that polarized T cells could instruct naïve T cells to differentiate into T cells with similar phenotype. In this study, we aimed to determine the contribution of antigen-driven bystander effect on epicutaneous sensitization with a newly introduced protein allergen. BALB/c mice were immunized intraperitoneally with BSA emulsified in alum, known to induce a Th2 response, three weeks before given BSA and OVA epicutaneously. Lymph node cells from these mice restimulated with OVA secreted higher levels IL-4, IL-5 and IL-13 as compared with cells from mice without BSA immunization. In addition, BALB/c mice immunized subcutaneously with BSA emulsified in complete Freund's adjuvant, known to induce a Th1-predominant response, also induced higher Th1 as well as Th2 cytokine response when restimulated with OVA as compared with mice without immunization. We demonstrated that subcutaneous immunization with BSA in CFA induced Th2 as well as Th1 response. The threshold of epicutaneous sensitization to OVA was also reduced, possibly due to increased expressions of IL-4 and IL-10 in the draining lymph nodes during the early phase of sensitization. In conclusion, antigen-driven bystander effect, whether it is of Th1- or Th2-predominant nature, can accelerate epicutaneous sensitization by a newly introduced protein allergen. These results provide a possible explanation for mono- to poly-sensitization spread commonly observed in atopic children. PMID:19272128

Wang, Li-Fang; Chen, Jau-Shiuh; Hsu, Chih-Jung; Liu, Ching-Yi; Yu, Jhang-Sian; Miaw, Shi-Chuen

2009-01-01

95

Major Antigenic Proteins of the Agent of Human Granulocytic Ehrlichiosis Are Encoded by Members of a Multigene Family  

Microsoft Academic Search

Western blot analysis of proteins from a cell culture isolate (USG3) of the human granulocytic ehrlichiosis (HGE) agent has identified a number of immunoreactive proteins, including major antigenic proteins of 43 and 45 kDa. Peptides derived from the 43- and 45-kDa proteins were sequenced, and degenerate PCR primers based on these sequences were used to amplify DNA from USG3. Sequencing

CHERYL I. MURPHY; JAMES R. STOREY; JOANNE RECCHIA; LINDA A. DOROS-RICHERT; CINDY GINGRICH-BAKER; KENNETH MUNROE; JOHAN S. BAKKEN; RICHARD T. COUGHLIN; GERALD A. BELTZ

1998-01-01

96

Mapping antigenic domains expressed by Chlamydia trachomatis major outer membrane protein genes.  

PubMed Central

Chlamydia trachomatis is an obligate prokaryotic intracellular pathogen of humans that infects mucosal epithelial cells. Exposed domains of its major outer membrane protein (MOMP) are both serotyping and protective antigenic determinants. To identify these domains, we have cloned and epitope-mapped the genes of serovars A, C (C serogroup) and L2, B (B serogroup) with a panel of monoclonal antibodies (mAbs). Predominantly conserved regions of the genes of both serogroups are interspersed with four short variable domains (I-IV). Recombinant phage clones expressing specific MOMP antigenic determinants revealed that protective serotype-specific recognized epitopes in variable domains I and II. Protective subspecies and serogroup-specific mAbs recognized overlapping determinants in variable domain IV near the C terminus. A nonprotective species-specific mAb mapped to an invariant peptide of nine residues contained within variable domain IV. In the intact chlamydial organism of serovar B, variable domains II and IV were susceptible to proteolytic digestion, whereas both N and C termini were protected. These results suggest an arrangement of MOMP in the outer membrane in which three of the four variable domains are exposed to the outside and in which both N and C termini are presumably oriented toward the periplasmic space. This molecular analysis of MOMP antigenic determinants and their surface topology on intact chlamydiae will be useful toward the development of a recombinant subunit or synthetic chlamydial vaccine. Images PMID:2453883

Baehr, W; Zhang, Y X; Joseph, T; Su, H; Nano, F E; Everett, K D; Caldwell, H D

1988-01-01

97

Outcomes after Transplantation of Cord Blood or Bone Marrow from Unrelated Donors in Adults with Leukemia  

Microsoft Academic Search

background Data regarding the outcome of cord-blood transplantation in adults are scant, despite the fact that these grafts are increasingly used in adults. methods We compared the outcomes of the transplantation of hematopoietic stem cells from unrelated donors in adults with leukemia who had received cord blood that was mis- matched for one HLA antigen (34 patients) or two antigens

Mary J. Laughlin; Mary Eapen; Pablo Rubinstein; John E. Wagner; Mei-Jei Zhang; Richard E. Champlin; Cladd Stevens; Juliet N. Barker; Robert P. Gale; Hillard M. Lazarus; David I. Marks; Jon J. van Rood; Andromachi Scaradavou; Mary M. Horowitz

2004-01-01

98

Expression and Binding Properties of a Soluble Chimeric Protein Containing the N-Terminal Domain of the Duffy Antigen  

Microsoft Academic Search

The blood group Duffy antigen of human erythrocytes, which exists in two allelic forms, Fya and Fyb, is a promiscuous chemokine receptor. In this report we describe the expression and purification of a chimeric protein composed of the amino-terminal extracellular domain of the Duffy antigen (aa 3–60), C-terminal intracellular fragment of glycophorin A (GPA, aa 104–131), and the hexahistydyl tag.

Kazimiera Wa?niowska; Marcin Czerwi?ski; Wojciech Jachymek; Elwira Lisowska

2000-01-01

99

Adjuvant effect of bacterial LPS and/or alum precipitation in responses to polysaccharide and protein antigens.  

PubMed Central

A conjugate of a hapten (NIP) and a strongly antigenic protein chicken gamma globulin (CGG), when injected in soluble form into mice, induced weak primary responses, as weak as responses induced by conjugates of NIP (or other haptens) to polysaccharides Ficoll or alpha (-1-6) dextran. Mean concentrations of anti-hapten antibodies on day 14 varied within the range of 37-105 micrograms/ml (C57BL mice) or 14-38 micrograms/ml (CBA mice). The NIP-protein conjugate administered in alum-precipitated form induced 100 times higher primary antibody responses. Alum-precipitation of NIP-Ficoll made it a modestly stronger antigen than soluble NIP-Ficoll. When lipopolysaccharide (LPS) was injected together with any of the soluble antigens, the mice produced plenty of anti-hapten antibodies regardless of whether the antigen was hapten-polysaccharide or hapten-protein conjugate. Concentrations on day 14 varied from around 400 micrograms/ml to approximately 1600 micrograms/ml. LPS had a similar adjuvant effect on antidextran responses. LPS alone induced a polyclonal immunoglobulin production, and the immunoglobulin produced included 'anti-NIP' or 'anti-dextran' detectable in the solid phase antibody assay. These 'antibodies' induced by LPS alone were almost totally mercapto-ethanol-sensitive and poorly detectable by Farr assay or the bacteriophage assay. The response to the LPS+antigen combination was specific for the antigen and included both mercapto-ethanol-sensitive and resistant antibodies. PMID:6209209

Seppälä, I J; Mäkelä, O

1984-01-01

100

Chlamydia trachomatis polymorphic membrane protein D is a species-common pan-neutralizing antigen  

PubMed Central

Infections caused by the obligate intracellular pathogen Chlamydia trachomatis have a marked impact on human health. C. trachomatis serovariants are the leading cause of bacterial sexually transmitted disease and infectious preventable blindness. Despite decades of effort, there is no practical vaccine against C. trachomatis diseases. Here we report that all C. trachomatis reference serotypes responsible for sexually transmitted disease and blinding trachoma synthesize a highly conserved surface-exposed antigen termed polymorphic membrane protein D (PmpD). We show that Ab specific to PmpD are neutralizing in vitro. We also present evidence that Ab against serovariable-neutralizing targets, such as the major outer membrane protein, block PmpD neutralization. This finding suggests that a decoy-like immune evasion strategy may be active in vivo whereby immunodominant type-specific surface antigens block the neutralizing ability of species-common PmpD Ab. Collectively, these results show that PmpD is a previously uncharacterized C. trachomatis species-common pan-neutralizing target. Moreover, a vaccine protocol using recombinant PmpD to elicit neutralizing Ab in the absence of immunodominant type-specific Ab might be highly efficacious and surpass the level of protection achieved through natural immunity. PMID:16446444

Crane, Deborah D.; Carlson, John H.; Fischer, Elizabeth R.; Bavoil, Patrik; Hsia, Ru-ching; Tan, Chun; Kuo, Cho-chou; Caldwell, Harlan D.

2006-01-01

101

Antigenic Protein In Microgravity-Grown Human Mixed Mullerian Tumor (LN1) Cells Preserved In RNA Stabilizing Agent  

NASA Technical Reports Server (NTRS)

Cells treated with RNAlater(TradeMark) have previously been shown to contain antigenic proteins that can be visualized using Western blot analysis. These proteins seem to be stable for several months when stored in RNA stabilizer at 4 C. Antigenic protein can be recovered from cells that have been processed using an Ambion RNAqueous(Registered TradeMark) kit to remove RNA. In this set of experiments, human mixed Mullerian tumor (LN1) cells grown on the International Space Station during Expedition 3 were examined for antigenic stability after removal of RNA. The cells were stored for three months in RNAlater(TradeMark) and RNA was extracted. The RNA filtrate Containing the protein was precipitated, washed, and suspended in buffer containing sodium dodecyl sulfate (SDS). Samples containing equal concentrations of protein were loaded onto SDS-polyacrylamide gels. Proteins were separated by electrophoresis and transferred by Western blot to polyvinylidene fluoride (PVDF) membrane. The Western blots were stained with an enhanced chemiluminescent ECL(Registered TradeMark)Plus detection kit (Amersham) and scanned using a Storm 840 gel image analyzer (Amersham, Molecular Dynamics). ImageQuant(Registered TradeMark)a software was used to quantify the densities of the protein bands. The ground control and flight LN1 cell samples showed a similar staining pattern over time with antibodies to vimentin, glyceraldehyde-3-phosphate dehydrogenase, and epithelial membrane antigens.

Hammond, Dianne K.; Becker, Jeanne; Holubec, K.; Baker, T. L.; Love, J. E.

2004-01-01

102

Identification of Immunogenic Hot Spots within Plum Pox Potyvirus Capsid Protein for Efficient Antigen Presentation  

PubMed Central

PEPSCAN analysis has been used to characterize the immunogenic regions of the capsid protein (CP) in virions of plum pox potyvirus (PPV). In addition to the well-known highly immunogenic N- and C-terminal domains of CP, regions within the core domain of the protein have also shown high immunogenicity. Moreover, the N terminus of CP is not homogeneously immunogenic, alternatively showing regions frequently recognized by antibodies and others that are not recognized at all. These results have helped us to design efficient antigen presentation vectors based on PPV. As predicted by PEPSCAN analysis, a small displacement of the insertion site in a previously constructed vector, PPV-?, turned the derived chimeras into efficient immunogens. Vectors expressing foreign peptides at different positions within a highly immunogenic region (amino acids 43 to 52) in the N-terminal domain of CP were the most effective at inducing specific antibody responses against the foreign sequence. PMID:12438590

Fernández-Fernández, M. Rosario; Martínez-Torrecuadrada, Jorge L.; Roncal, Fernando; Domínguez, Elvira; García, Juan Antonio

2002-01-01

103

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

PubMed Central

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

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

2013-01-01

104

Characterization and antigenicity of recombinant Campylobacter jejuni flagellar capping protein FliD.  

PubMed

Campylobacter jejuni, a flagellated, spiral-rod, Gram-negative bacterium, is the leading pathogen of human acute bacterial gastroenteritis worldwide, and chickens are regarded as a major reservoir of this micro-organism. Bacterial flagella, composed of more than 35 proteins, play important roles in colonization and adhesion to the mucosal surface of chicken caeca. In this study, the flagellar capping protein, FliD, encoded by the fliD gene, from the Campylobacter jenuni D1-39 isolate was expressed and characterized, and its antigenicity determined. The fliD gene comprised 1929 nt, potentially encoding a 642 aa peptide with a calculated molecular mass of 69.6 kDa. This gene was PCR amplified and overexpressed in Escherichia coli. The recombinant FliD protein was purified by cobalt-chelating affinity chromatography and confirmed by nucleotide sequencing of the expression plasmid, SDS-PAGE analysis, His tag detection and matrix-assisted laser desorption/ionization time of flight mass spectrometry. The immunoblot data showed that the purified recombinant FliD protein reacted strongly to sera from broiler chickens older than 4 weeks, indicating that anti-FliD antibody may be prevalent in the poultry population. These results provide a rationale for further evaluation of the FliD protein as a vaccine candidate for broiler chickens to improve food safety for poultry. PMID:24445509

Yeh, Hung-Yueh; Hiett, Kelli L; Line, John E; Seal, Bruce S

2014-04-01

105

Expression of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus in soybean seed yields an immunogenic antigenic protein.  

PubMed

Porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV), is a serious disease of swine and contributes to severe worldwide economic losses in swine production. Current vaccines against PRRS rely on the use of an attenuated-live virus; however, these are unreliable. Thus, alternative effective vaccines against PRRS are needed. Plant-based subunit vaccines offer viable, safe, and environmentally friendly alternatives to conventional vaccines. In this study, efforts have been undertaken to develop a soybean-based vaccine against PRRSV. A construct carrying a synthesized PRRSV-ORF7 antigen, nucleocapsid N protein of PRRSV, has been introduced into soybean, Glycine max (L.) Merrill. cvs. Jack and Kunitz, using Agrobacterium-mediated transformation. Transgenic plants carrying the sORF7 transgene have been successfully generated. Molecular analyses of T(0) plants confirmed integration of the transgene and transcription of the PRRSV-ORF7. Presence of a 15-kDa protein in seeds of T(1) transgenic lines was confirmed by Western blot analysis using PRRSV-ORF7 antisera. The amount of the antigenic protein accumulating in seeds of these transgenic lines was up to 0.65% of the total soluble protein (TSP). A significant induction of a specific immune response, both humoral and mucosal, against PRRSV-ORF7 was observed following intragastric immunization of BALB/c female mice with transgenic soybean seeds. These findings provide a 'proof of concept', and serve as a critical step in the development of a subunit plant-based vaccine against PRRS. PMID:21971995

Vimolmangkang, Sornkanok; Gasic, Ksenija; Soria-Guerra, Ruth; Rosales-Mendoza, Sergio; Moreno-Fierros, Leticia; Korban, Schuyler S

2012-03-01

106

Human Leukocyte Antigen Class II Alleles Influence Levels of Antibodies to the Plasmodium falciparum Asexual-Stage Apical Membrane Antigen 1 but Not to Merozoite Surface Antigen 2 and Merozoite Surface Protein 1  

PubMed Central

The apical membrane antigen 1 (AMA1), merozoite surface antigen 2 (MSA2), and merozoite surface protein 1 (MSP1) are asexual-stage proteins currently being evaluated for inclusion in a vaccine for Plasmodium falciparum. Accordingly, it is important to understand factors that control antibody responses to these antigens. Antibody levels in plasma from residents of Etoa, Cameroon, between the ages of 5 and 70 years, were determined using recombinant AMA1, MSA2, and the N-terminal region of MSP1 (MSP1-190L). In addition, antibody responses to four variants of the C-terminal region of MSP1 (MSP119) were assessed. Results showed that all individuals produced antibodies to AMA1, MSA2, and MSP1-190L; however, a proportion of individuals never produced antibodies to the MSP119 variants, although the percentage of nonresponders decreased with age. The influence of age and human leukocyte antigen (HLA)-DRB1/DQB1 alleles on antibody levels was evaluated using two-way analysis of variance. Age was correlated with levels of antibodies to AMA1 and MSP119 but not with levels of antibodies to MSA2 and MSP1-190L. No association was found between a single HLA allele and levels of antibodies to MSA2, MSP1-190L, or any of the MSP119 variants. However, individuals positive for DRB1*1201 had higher levels of antibodies to the variant of recombinant AMA1 tested than did individuals of all other HLA types. Since the effect was seen across all age groups, HLA influenced the level but not the rate of antibody acquisition. This association for AMA1, combined with the previously reported association between HLA class II alleles and levels of antibodies to rhoptry-associated protein 1 (RAP1) and RAP2, indicates that HLA influences the levels of antibodies to three of the five vaccine candidate antigens that we have evaluated. PMID:15102786

Johnson, Armead H.; Leke, Rose G. F.; Mendell, Nancy R.; Shon, Dewon; Suh, Young Ju; Bomba-Nkolo, Dennis; Tchinda, Viviane; Kouontchou, Samuel; Thuita, Lucy W.; van der Wel, Anne Marie; Thomas, Alan; Stowers, Anthony; Saul, Allan; Zhou, Ainong; Taylor, Diane W.; Quakyi, Isabella A.

2004-01-01

107

Identifying large sets of unrelated individuals and unrelated markers  

PubMed Central

Background Genetic Analyses in large sample populations are important for a better understanding of the variation between populations, for designing conservation programs, for detecting rare mutations which may be risk factors for a variety of diseases, among other reasons. However these analyses frequently assume that the participating individuals or animals are mutually unrelated which may not be the case in large samples, leading to erroneous conclusions. In order to retain as much data as possible while minimizing the risk of false positives it is useful to identify a large subset of relatively unrelated individuals in the population. This can be done using a heuristic for finding a large set of independent of nodes in an undirected graph. We describe a fast randomized heuristic for this purpose. The same methodology can also be used for identifying a suitable set of markers for analyzing population stratification, and other instances where a rapid heuristic for maximal independent sets in large graphs is needed. Results We present FastIndep, a fast random heuristic algorithm for finding a maximal independent set of nodes in an arbitrary undirected graph along with an efficient implementation in C++. On a 64 bit Linux or MacOS platform the execution time is a few minutes, even with a graph of several thousand nodes. The algorithm can discover multiple solutions of the same cardinality. FastIndep can be used to discover unlinked markers, and unrelated individuals in populations. Conclusions The methods presented here provide a quick and efficient method for identifying sets of unrelated individuals in large populations and unlinked markers in marker panels. The C++ source code and instructions along with utilities for generating the input files in the appropriate format are available at http://taurus.ansci.iastate.edu/wiki/people/jabr/Joseph_Abraham.html PMID:24635884

2014-01-01

108

The immunity and protective effects of antigen 85A and heat-shock protein X against progressive tuberculosis.  

PubMed

The anti-tuberculosis vaccine, Mycobacterium bovis BCG, has been used worldwide, but its protective efficacy is variable against adult pulmonary tuberculosis. In this study, immune responses of antigen 85A (Ag85A) and heat-shock protein X (HspX) antigen of Mycobacterium tuberculosis were investigated during acute and stationary stage of infection in the murine aerosol TB challenge model and their protective effects were evaluated against progressive tuberculosis. A high level of Ag85A-specific IFN-? production was induced from the early stage of the infection, whereas HspX-specific IFN-? production was increased in the later stationary stage. As a subunit vaccine, Ag85A and HspX antigen vaccine induced high levels of IFN-?, and a vaccine comprising both antigens induced the highest level of IFN-?. At 30 days post-challenge, the Ag85A subunit vaccine was protective against M. tuberculosis challenge, but the HspX subunit vaccine was not. Interestingly, the HspX antigen vaccine induced significant protective efficacy at 90 days post-challenge. Moreover, the combined antigen vaccine induced the highest protective efficacy against M. tuberculosis challenge both at 30 days and 90 days post-challenge. These results suggest that the vaccine comprising Ag85A and HspX antigen which react in different stages of infection is highly protective against progressive tuberculosis. PMID:21093603

Jeon, Bo-Young; Kim, Seung-Cheol; Eum, Seok-Yong; Cho, Sang-Nae

2011-03-01

109

Molecular characterization of a Spirometra mansoni antigenic polypeptide gene encoding a 28.7 kDa protein.  

PubMed

The Spirometra mansoni antigenic polypeptide (SmAP) gene was expressed in Escherichia coli, and its characteristics and value as an antigen for the serodiagnosis of sparganosis were investigated. The recombinant SmAP protein (rSmAP) has the molecular weight of 28.7 kDa. On Western blotting analysis, the rSmAP strongly reacted with the sera of mice infected with spargana, but not with normal sera; the anti-rSmAP serum obviously recognized the 28.7-kDa band in the crude antigens and excretory-secretory (ES) antigens of spargana. The immunofluorescence test (IFT) results showed that the positive staining was observed at different stages of spargana from the infected frogs and mice, but not adult worm of S. mansoni. An immunolocalization analysis identified SmAP in the teguments and parenchymal tissues of spargana. ELISA with rSmAP antigen or sparganum ES antigens were evaluated for the serodiagnosis of sparganosis. The results showed that the sensitivity of rSmAP-ELISA and ES-ELISA was 83.3% (25/30) and 100% (30/30), respectively, for the detection of anti-sparganum IgG antibodies in sera of the experimentally infected mice (P?>?0.05), the specificities of both ELISA were 100% (67/67). It is suggested that the rSmAP might be a potential candidate antigen for serodiagnosis of sparganosis. PMID:25096536

Cui, Jing; Wei, Tong; Liu, Li Na; Zhang, Xi; Qi, Xin; Zhang, Zi Fang; Wang, Zhong Quan

2014-09-01

110

Cloning and Characterization of a Novel Membrane-Associated Antigenic Protein of Helicobacter pylori  

PubMed Central

Infection by Helicobacter pylori, a noninvasive bacterium, induces chronic leukocyte infiltration in the stomach by still largely unknown molecular mechanisms. We investigated the possibility that a membrane protein of H. pylori induces an inflammatory reaction in the subepithelial tissue of the stomach. By generating an expression library of H. pylori chromosomal DNA and screening with rabbit antiserum raised to a membrane fraction of H. pylori and sera of infected patients, we cloned a 16.0-kDa protein (HP-MP1) which appeared to attach to the inner membrane of the H. pylori in a homodimeric form. Anti-HP-MP1 antibodies were detected in the sera of infected patients but not in those of uninfected controls. Coincubation of monocytes with recombinant HP-MP1 led to cell activation and production of interleukin-1? (IL-1?), tumor necrosis factor alpha, IL-8, and macrophage inflammatory protein 1?. The results indicate that HP-MP1 is an antigenic membrane-associated protein of H. pylori which potentially activates monocytes. This suggests that HP-MP1 may play roles in the pathogenesis of perpetual tissue inflammation associated with H. pylori infection. PMID:9864228

Yoshida, Masaru; Wakatsuki, Yoshio; Kobayashi, Yoshinao; Itoh, Toshiyuki; Murakami, Kazuhisa; Mizoguchi, Akira; Usui, Takashi; Chiba, Tsutomu; Kita, Toru

1999-01-01

111

Lineage specific antigenic differences in porcine torovirus hemagglutinin-esterase (PToV-HE) protein  

PubMed Central

Hemagglutinin-esterases (HE) are viral envelope proteins present in some members from the toro-, corona- and orthomyxovirus families, all related with enteric and/or respiratory tract infections. HE proteins mediate reversible binding to sialic acid receptor determinants, very abundant glycan residues in the enteric and respiratory tracts. The role of the HE protein during the torovirus infection cycle remains unknown, although it is believed to be important in the natural infection process. The phylogenetic analysis of HE coding sequences from porcine torovirus (PToV) field strains revealed the existence of two distinct HE lineages. In a previous study, PToV virus strains with HE proteins from the two lineages were found coexisting in a pig herd, and they were even obtained from the same animal at two consecutive sampling time points. In this work, we report antigenic differences between the two HE lineages, and discuss the possible implications that the coexistence of viruses belonging to both lineages might have on the spread and sustainment of PToV infection in the farms. PMID:24364900

2013-01-01

112

Plasmodium vivax and Plasmodium chabaudi: intraerythrocytic traffic of antigenically homologous proteins involves a brefeldin A-sensitive secretory pathway.  

PubMed

We have used a monoclonal antibody (mAb 7C5B71) raised against the erythrocytic stages of Plasmodium vivax to identify a 148-kDa P vivax protein antigen (Pv-148) which crossreacts with an antigenically homologous 190-kDa protein of P. chabaudi (Pc-190). During parasite intraerythrocytic development Pv-148 and Pc-190 are exported into the host cell cytosol and become located in the surface membrane of the infected erythrocyte. Immunofluorescence confocal microscopy and immunoelectron microscopy studies showed that both Pv-148 and Pc-190 are released from the parasite and exported to the host cell cytoplasm in association with tubovesicular membrane (TVM) structures. Fluorescent in vivo labelling of P. chabaudi with Bodipy-ceramide followed by immunofluorescence staining with the mAb supported the association of antigenically homologous Pc-190 with TVM structures. In the presence of brefeldin A (BFA), secretion of antigenically homologous Pc-190 into the host cell cytoplasm was inhibited and the antigen remained in the parasite cytoplasm. BFA also arrested the maturation of the parasite. Taken together these results suggest that Pv-148 and Pc-190 are related parasite proteins that are transported into the host cell through a BFA-sensitive secretory pathway. PMID:11302521

Bracho, C; Dunia, I; Romano, M; Benedetti, E L; Perez, H A

2001-02-01

113

Nanogel antigenic protein-delivery system for adjuvant-free intranasal vaccines  

NASA Astrophysics Data System (ADS)

Nanotechnology is an innovative method of freely controlling nanometre-sized materials. Recent outbreaks of mucosal infectious diseases have increased the demands for development of mucosal vaccines because they induce both systemic and mucosal antigen-specific immune responses. Here we developed an intranasal vaccine-delivery system with a nanometre-sized hydrogel (`nanogel') consisting of a cationic type of cholesteryl-group-bearing pullulan (cCHP). A non-toxic subunit fragment of Clostridium botulinum type-A neurotoxin BoHc/A administered intranasally with cCHP nanogel (cCHP-BoHc/A) continuously adhered to the nasal epithelium and was effectively taken up by mucosal dendritic cells after its release from the cCHP nanogel. Vigorous botulinum-neurotoxin-A-neutralizing serum IgG and secretory IgA antibody responses were induced without co-administration of mucosal adjuvant. Importantly, intranasally administered cCHP-BoHc/A did not accumulate in the olfactory bulbs or brain. Moreover, intranasally immunized tetanus toxoid with cCHP nanogel induced strong tetanus-toxoid-specific systemic and mucosal immune responses. These results indicate that cCHP nanogel can be used as a universal protein-based antigen-delivery vehicle for adjuvant-free intranasal vaccination.

Nochi, Tomonori; Yuki, Yoshikazu; Takahashi, Haruko; Sawada, Shin-Ichi; Mejima, Mio; Kohda, Tomoko; Harada, Norihiro; Kong, Il Gyu; Sato, Ayuko; Kataoka, Nobuhiro; Tokuhara, Daisuke; Kurokawa, Shiho; Takahashi, Yuko; Tsukada, Hideo; Kozaki, Shunji; Akiyoshi, Kazunari; Kiyono, Hiroshi

2010-07-01

114

Genetically modified anthrax lethal toxin safely delivers whole HIV protein antigens into the cytosol to induce T cell immunity  

NASA Astrophysics Data System (ADS)

Bacillus anthrax lethal toxin can be engineered to deliver foreign proteins to the cytosol for antigen presentation to CD8 T cells. Vaccination with modified toxins carrying 8-9 amino acid peptide epitopes induces protective immunity in mice. To evaluate whether large protein antigens can be used with this system, recombinant constructs encoding several HIV antigens up to 500 amino acids were produced. These candidate HIV vaccines are safe in animals and induce CD8 T cells in mice. Constructs encoding gag p24 and nef stimulate gag-specific CD4 proliferation and a secondary cytotoxic T lymphocyte response in HIV-infected donor peripheral blood mononuclear cells in vitro. These results lay the foundation for future clinical vaccine studies.

Lu, Yichen; Friedman, Rachel; Kushner, Nicholas; Doling, Amy; Thomas, Lawrence; Touzjian, Neal; Starnbach, Michael; Lieberman, Judy

2000-07-01

115

Bacterial ghosts as carriers of protein subunit and DNA-encoded antigens for vaccine applications.  

PubMed

Bacterial ghosts (BGs) represent vaccine delivery systems gifted with outstanding natural adjuvant properties. BGs are empty cell envelopes of Gram-negative bacteria lacking cytoplasmic content yet retaining all unaltered morphological and structural features of their living counterparts. The intact surface make-up of BGs is easily recognized by professional APCs through pattern-recognition receptors, making them ideal for mucosal administration through oral, ocular, intranasal or aerogenic routes, which represent the most desirable methods of application in advanced vaccine use. BGs have been designed to be used as carriers of active substances and foreign antigens (protein and/or DNA) for vaccine development. This review highlights the salient features of the BGs' versatile multipurpose vaccine platform for application in a wide range of human and veterinary medicines. PMID:22149712

Muhammad, Abbas; Champeimont, Jonathan; Mayr, Ulrike Beate; Lubitz, Werner; Kudela, Pavol

2012-01-01

116

Challenges for the evaluation of Staphylococcus aureus protein based vaccines: monitoring antigenic diversity.  

PubMed

Clumping factors A (ClfA) and B (ClfB) are Staphylococcus aureus virulence proteins that are displayed on the cell surface of the organism and have potential as vaccine antigens for the prevention of S. aureus disease. Here we evaluate the phylogeny of S. aureus in the context of antigenic variation of these two surface proteins. ClfA and ClfB gene sequences, along with epidemiological markers (MLST, spa and capsule genotype) were obtained for 224 S. aureus isolates including both historical strains and a collection representative of current MRSA isolates from the United States. Variation within ClfA and ClfB was consistent with the established population biology of S. aureus, namely, that S. aureus strains belong to a relatively small number of clonal lineages, with evolution proceeding mainly by mutation and with little to no recombination between clades. Thus most variation in ClfA and ClfB occurs between but not within lineages, and particular groups of ClfA and ClfB variants are closely linked. This has important implications for vaccine development and assessment as it suggests that a relatively small survey of strains will be representative of the total population variation, whereas for species that evolve mainly by recombination, such as Neisseria meningitidis, analysis of a much larger number of strains is needed to accomplish the same purpose. Our study also revealed evidence for the de-evolution of ClfB and therefore its reduced suitability as a target for vaccine development compared to ClfA. PMID:21245656

Murphy, Ellen; Lin, Shuo L; Nunez, Lorna; Andrew, Lubomira; Fink, Pamela S; Dilts, Deborah A; Hoiseth, Susan K; Jansen, Kathrin U; Anderson, Annaliesa S

2011-01-01

117

Design and immunological properties of topographic immunogenic determinants of a protein antigen (LDH-C4) as vaccines.  

PubMed

Antibodies elicited by immunization with short peptides containing antigenic determinants have been shown, in general, to bind with greatly reduced affinity to the corresponding region in the native proteins. Thus, contiguous linear peptides have not proven to be effective immunogens in generating high affinity neutralizing or protective antibodies and consequently appear to be poor prospects for vaccines. The molecular basis for such reduced reactivity is clear from the crystal structure determination of antibody Fabs bound to protein antigens, which showed the complementarity between interfaces to be lock-and-key-like and extending over a large area (750 A2) involving discontinuous segments of the polypeptide chain. Thus, small perturbations in the secondary and tertiary structure of the antigen have profound effects on the fit of the antigen and its corresponding antibody. Because short peptides are unlikely to assume any particular conformation in solution, the fit is likely to be poor. New strategies are therefore required to produce conformationally stable peptides that mimic the critical structural features of the protein antigenic site. Here we show that a putative topographic determinant of the testis-specific isozyme of lactate dehydrogenase C4 (LDH-C4), designed and synthesized to adopt a well defined alpha-helical secondary and tertiary structure (four-helix bundle motif) in aqueous solutions, is highly immunogenic in both rabbits and mice, inducing IgG antibodies that bind to native LDH-C4. This engineered conformational 40-residue peptide is considerably more effective in inducing antibodies, as compared with the corresponding linear peptide. The antibody response is obtained without coupling the peptide to a carrier protein, suggesting that the peptide contains a T-cell antigenic determinant. The strategy described here to produce a conformationally stable peptide that mimics the native structure may have general applications in vaccine design. PMID:1372905

Kaumaya, P T; VanBuskirk, A M; Goldberg, E; Pierce, S K

1992-03-25

118

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

PubMed Central

Merkel cell carcinoma (MCC) is a highly aggressive nonmelanoma skin cancer arising from epidermal mechanoreceptor Merkel cells. In 2008, a novel human polyomavirus, Merkel cell polyomavirus (MCPyV), was identified and is strongly implicated in MCC pathogenesis. Currently, little is known regarding the virus-host cell interactions which support virus replication and virus-induced mechanisms in cellular transformation and metastasis. Here we identify a new function of MCPyV small T antigen (ST) as an inhibitor of NF-?B-mediated transcription. This effect is due to an interaction between MCPyV ST and the NF-?B essential modulator (NEMO) adaptor protein. MCPyV ST expression inhibits I?B kinase ? (IKK?)/IKK?-mediated I?B phosphorylation, which limits translocation of the NF-?B heterodimer to the nucleus. Regulation of this process involves a previously undescribed interaction between MCPyV ST and the cellular phosphatase subunits, protein phosphatase 4C (PP4C) and/or protein phosphatase 2A (PP2A) A?, but not PP2A A?. Together, these results highlight a novel function of MCPyV ST to subvert the innate immune response, allowing establishment of early or persistent infection within the host cell. PMID:24109239

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

2013-01-01

119

Detection of proteins antigenically related to Bothrops asper myotoxin in crotaline snake venoms.  

PubMed

The presence of components antigenically related to Bothrops asper myotoxin was investigated by Western blotting and immunoelectrophoretic techniques. B. asper myotoxin is a non-glycosylated monomeric phospholipase A with a molecular weight by SDS-PAGE of 16,000 and isoelectric point of pH 9.8-10.0. Results showed that proteins in the venoms of B. nummifer, B. godmani, B. schlegelii, B. picadoi, and Agkistrodon bilineatus were recognized by monospecific antibodies to B. asper myotoxin raised in rabbit and sheep. Western blotting indicated that cross-reacting proteins have a molecular weight of 16,000, with the exception of that of B. picadoi, which is of 24,000 mol. wt. However, immunoelectrophoresis indicated that these components are highly heterogeneous in charge, ranging from basic to acidic proteins. The cross-reacting component(s) present in newborn B. asper venom has a different charge from that of the 'adult-type'. Venoms from newborn specimens showed an additional cross-reacting band of 18,000 mol. wt. Myotoxin is an abundant component in adult B. asper venom. Myotoxin-antimyotoxin complexes had different electrophoretic mobilities in rocket immunoelectrophoresis depending upon the species in which monospecific immune sera were produced. PMID:2448918

Lomonte, B; Moreno, E; Gutiérrez, J M

1987-01-01

120

Detection of pancreatic cancer with normal carbohydrate antigen 19-9 using protein chip technology  

PubMed Central

AIM: To develop a method to differentiate pancreatic cancer patients from healthy or benign individuals when carbohydrate antigen (CA) 19-9 is normal. METHODS: Forty-one serum samples from patients with pancreatic lesions and blood samples from 20 healthy individuals were collected at the first stage of the experiment according to the enrolment criteria. General characteristics and some clinical features were carefully compared to ensure that the results were reasonable. All the blood samples were analyzed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) combined with CM10 chips and a related bioinformatics analysis program to generate diagnostic models with different proteins. Forty-seven consecutive samples were tested at the next stage to verify the veracity and efficiency of the models. RESULTS: The sex, age, and serum CA19-9 levels among the three groups (malignant, benign, and healthy) were statistically matched (P values were 0.957, 0.145, and 0.382, respectively). Two patterns were generated. Pattern 1 with four proteins theoretically had a specificity and sensitivity of 100% in distinguishing pancreatic cancer from healthy individuals, while it was 86.7% and 86.4%, respectively, in the subsequent practical verification. The positive predictive value (PPV) of the model was 86.4%. One of the four proteins was expressed highly in pancreatic cancer while the other three were expressed weakly. Pattern 2 consisted of six proteins that showed a specificity of 70.0% and sensitivity of 77.3% for differentiating malignancy from benign tumors. Its PPV reached 85.0%. Only one of these six proteins showed high expression in the malignant group. CONCLUSION: SELDI-TOF-MS may facilitate diagnosis or differential diagnosis of pancreatic cancer when CA19-9 is normal. Pattern 1 may serve as a useful screening tool. PMID:25356057

Jin, Xiao-Li; Xu, Bin; Wu, Yu-Lian

2014-01-01

121

Discovery of GAMA, a Plasmodium falciparum Merozoite Micronemal Protein, as a Novel Blood-Stage Vaccine Candidate Antigen ? ‡  

PubMed Central

One of the solutions for reducing the global mortality and morbidity due to malaria is multivalent vaccines comprising antigens of several life cycle stages of the malarial parasite. Hence, there is a need for supplementing the current set of malaria vaccine candidate antigens. Here, we aimed to characterize glycosylphosphatidylinositol (GPI)-anchored micronemal antigen (GAMA) encoded by the PF08_0008 gene in Plasmodium falciparum. Antibodies were raised against recombinant GAMA synthesized by using a wheat germ cell-free system. Immunoelectron microscopy demonstrated for the first time that GAMA is a microneme protein of the merozoite. Erythrocyte binding assays revealed that GAMA possesses an erythrocyte binding epitope in the C-terminal region and it binds a nonsialylated protein receptor on human erythrocytes. Growth inhibition assays revealed that anti-GAMA antibodies can inhibit P. falciparum invasion in a dose-dependent manner and GAMA plays a role in the sialic acid (SA)-independent invasion pathway. Anti-GAMA antibodies in combination with anti-erythrocyte binding antigen 175 exhibited a significantly higher level of invasion inhibition, supporting the rationale that targeting of both SA-dependent and SA-independent ligands/pathways is better than targeting either of them alone. Human sera collected from areas of malaria endemicity in Mali and Thailand recognized GAMA. Since GAMA in P. falciparum is refractory to gene knockout attempts, it is essential to parasite invasion. Overall, our study indicates that GAMA is a novel blood-stage vaccine candidate antigen. PMID:21896773

Arumugam, Thangavelu U.; Takeo, Satoru; Yamasaki, Tsutomu; Thonkukiatkul, Amporn; Miura, Kazutoyo; Otsuki, Hitoshi; Zhou, Hong; Long, Carole A.; Sattabongkot, Jetsumon; Thompson, Jennifer; Wilson, Danny W.; Beeson, James G.; Healer, Julie; Crabb, Brendan S.; Cowman, Alan F.; Torii, Motomi; Tsuboi, Takafumi

2011-01-01

122

CD1d-mediated presentation of endogenous lipid antigens by adipocytes requires microsomal triglyceride transfer protein.  

PubMed

Obesity-induced adipose tissue (AT) dysfunction results in a chronic low-grade inflammation that predisposes to the development of insulin resistance and type 2 diabetes. During the development of obesity, the AT-resident immune cell profile alters to create a pro-inflammatory state. Very recently, CD1d-restricted invariant (i) natural killer T (NKT) cells, a unique subset of lymphocytes that are reactive to so called lipid antigens, were implicated in AT homeostasis. Interestingly, recent data also suggest that human and mouse adipocytes can present such lipid antigens to iNKT cells in a CD1d-dependent fashion, but little is known about the lipid antigen presentation machinery in adipocytes. Here we show that CD1d, as well as the lipid antigen loading machinery genes pro-saposin (Psap), Niemann Pick type C2 (Npc2), ?-galactosidase (Gla), are up-regulated in early adipogenesis, and are transcriptionally controlled by CCAAT/enhancer-binding protein (C/EBP)-? and -?. Moreover, adipocyte-induced Th1 and Th2 cytokine release by iNKT cells also occurred in the absence of exogenous ligands, suggesting the display of endogenous lipid antigen-D1d complexes by 3T3-L1 adipocytes. Furthermore, we identified microsomal triglyceride transfer protein, which we show is also under the transcriptional regulation of C/EBP? and -?, as a novel player in the presentation of endogenous lipid antigens by adipocytes. Overall, our findings indicate that adipocytes can function as non-professional lipid antigen presenting cells, which may present an important aspect of adipocyte-immune cell communication in the regulation of whole body energy metabolism and immune homeostasis. PMID:24966328

Rakhshandehroo, Maryam; Gijzel, Sanne M W; Siersbæk, Rasmus; Broekema, Marjoleine F; de Haar, Colin; Schipper, Henk S; Boes, Marianne; Mandrup, Susanne; Kalkhoven, Eric

2014-08-01

123

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

PubMed Central

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

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

2014-01-01

124

Alphavirus Replicon DNA Expressing HIV Antigens Is an Excellent Prime for Boosting with Recombinant Modified Vaccinia Ankara (MVA) or with HIV gp140 Protein Antigen  

PubMed Central

Vaccination with DNA is an attractive strategy for induction of pathogen-specific T cells and antibodies. Studies in humans have shown that DNA vaccines are safe, but their immunogenicity needs further improvement. As a step towards this goal, we have previously demonstrated that immunogenicity is increased with the use of an alphavirus DNA-launched replicon (DREP) vector compared to conventional DNA vaccines. In this study, we investigated the effect of varying the dose and number of administrations of DREP when given as a prime prior to a heterologous boost with poxvirus vector (MVA) and/or HIV gp140 protein formulated in glucopyranosyl lipid A (GLA-AF) adjuvant. The DREP and MVA vaccine constructs encoded Env and a Gag-Pol-Nef fusion protein from HIV clade C. One to three administrations of 0.2 ?g DREP induced lower HIV-specific T cell and IgG responses than the equivalent number of immunizations with 10 ?g DREP. However, the two doses were equally efficient as a priming component in a heterologous prime-boost regimen. The magnitude of immune responses depended on the number of priming immunizations rather than the dose. A single low dose of DREP prior to a heterologous boost resulted in greatly increased immune responses compared to MVA or protein antigen alone, demonstrating that a mere 0.2 ?g DREP was sufficient for priming immune responses. Following a DREP prime, T cell responses were expanded greatly by an MVA boost, and IgG responses were also expanded when boosted with protein antigen. When MVA and protein were administered simultaneously following multiple DREP primes, responses were slightly compromised compared to administering them sequentially. In conclusion, we have demonstrated efficient priming of HIV-specific T cell and IgG responses with a low dose of DREP, and shown that the priming effect depends on number of primes administered rather than dose. PMID:25643354

Knudsen, Maria L.; Ljungberg, Karl; Tatoud, Roger; Weber, Jonathan; Esteban, Mariano; Liljeström, Peter

2015-01-01

125

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

PubMed Central

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

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

2013-01-01

126

p34cdc2-mediated phosphorylation at T124 inhibits nuclear import of SV- 40 T antigen proteins  

PubMed Central

The nuclear import of transcription regulatory proteins appears to be used by the cell to trigger transitions in cell cycle, morphogenesis, and transformation. We have previously observed that the rate at which SV-40 T antigen fusion proteins containing a functional nuclear localization sequence (NLS; residues 126-132) are imported into the nucleus is enhanced in the presence of the casein kinase II (CK-II) site S111/112. In this study purified p34cdc2 kinase was used to phosphorylate T antigen proteins specifically at T124 and kinetic measurements at the single-cell level performed to assess its effect on nuclear protein import. T124 phosphorylation, which could be functionally simulated by a T-to-D124 substitution, was found to reduce the maximal extent of nuclear accumulation whilst negligibly affecting the import rate. The inhibition of nuclear import depended on the stoichiometry of phosphorylation. T124 and S111/112 could be phosphorylated independently of one another. Two alternative mechanisms were considered to explain the inhibition of nuclear import by T124 phosphorylation: inactivation of the NLS and cytoplasmic retention, respectively. Furthermore, we speculate that in vivo T124 phosphorylation may regulate the small but functionally significant amount of cytoplasmic SV-40 T antigen. A sequence comparison showed that many transcription regulatory proteins contain domains comprising potential CK-II-sites, cdc2-sites, and NLS. This raises the possibility that the three elements represent a functional unit regulating nuclear protein import. PMID:1659575

1991-01-01

127

Proteomic analysis of embryonic Fasciola hepatica: characterization and antigenic potential of a developmentally regulated heat shock protein.  

PubMed

Fasciola hepatica is responsible for human disease and economic livestock loss on a global scale. We report the first post-genomic investigation of cellular proteins expressed by embryonic F. hepatica via two-dimensional electrophoresis, image analysis and tandem mass spectrometry. Antioxidant proteins and protein chaperones are prominently expressed by embryonic F. hepatica. Molecular differences between the egg and other characterized F. hepatica lifecycle stages were noted. Furthermore, proteins expressed within liver fluke eggs differ to those isolated from the well-characterized eggs of the human blood flatworm Schistosoma mansoni were revealed. Plasticity in expression of major proteins, particularly a prominently expressed 65kDa protein cluster was seen between natural populations of embryonating F. hepatica eggs suggesting that liver fluke embryogenisis is a plastic process. Immunoblotting revealed that the abundant 65kDa protein cluster is recognised by infection sera from three F. hepatica challenged host species. Mass spectrometry and BLAST analyses demonstrated that the 65kDa antigen shows homology to egg antigens of other flatworm parasites, and is represented in a F. hepatica EST database constructed from adult fluke transcripts. EST clones encoding the egg antigen were re-sequenced, predicting two forms of the protein. Four clones predict a 312 aa polypeptide, three clones encode a putative 110 amino acid extension at the N-terminus which may be involved in protein secretion, although this extension was not expressed by natively extracted proteins. Consistent expression of alpha crystallin domains confirmed the protein to be a member of the alpha crystallin containing small heat shock protein (AC/sHSP) superfamily. AC/sHSPs are ubiquitous in nature, however, this is the first time a member of this protein superfamily has been described from F. hepatica. The antigenic AC/sHSP was named Fh-HSP35alpha based on predictions of molecular weight. Production of recombinant Fh-HSP35alpha reveals considerable mass discrepancy between native and recombinant proteins, although descriptions of other characterized flatworm AC/sHSPs, suggest that the native form is a dimer. Immunoblot analyses confirm that the recombinant protein is recognised by F. hepatica challenged hosts, but does not react with sera from non-infected animals. We discuss the potential of recombinant Fh-HSP35alpha as an egg-based diagnostic marker for liver fluke infection. PMID:20089359

Moxon, Joseph V; LaCourse, E James; Wright, Hazel A; Perally, Samirah; Prescott, Mark C; Gillard, Jennifer L; Barrett, John; Hamilton, Joanne V; Brophy, Peter M

2010-04-19

128

Isolation and characterization of Bacteroides nodosus fimbriae: structural subunit and basal protein antigens.  

PubMed Central

We examined the isolation of fimbriae from Bacteroides nodosus. It was found that the best preparations were obtained from the supernatant of washed cells cultured on solid medium, from which fimbriae could be recovered in high yield and purity by a simple one-step procedure. Analysis of such preparations by sodium dodecyl sulfate gel electrophoresis showed that greater than 98% of the protein consisted of fimbrial structural subunits whose molecular weight was ca. 17,000. These preparations also usually exhibited minor contamination with a polypeptide of ca. 80,000 molecular weight, as well as trace amounts of lipopolysaccharide. Attempts to release additional fimbriae by the traditional means of subjecting the bacterial cells to physical stress, such as shearing or heating, resulted primarily in an increase in the level of contamination, without significant gain in the yield of fimbriae. Removal of the 80,000-dalton component could not be achieved by any of a variety of techniques normally used in fimbriae purification, including isoelectric precipitation, MgCl2 precipitation, and CsCl gradient ultracentrifugation, implying a direct physical association with the fimbrial strand. Electron micrographs of fractions containing this protein show cap-shaped structures attached to the ends of what appeared to be fimbrial stubs. These observations suggest that the 80,000-dalton polypeptide may actually constitute the basal attachment site which anchors the fimbria to the outer membrane, analogous to a similar protein recently described in enterotoxigenic strains of Escherichia coli. In B. nodosus, this 80,000-dalton protein is a major surface antigen, and like the fimbrial subunit, exhibited variation in electrophoretic mobility between serotypically different isolates. Images PMID:6150024

Mattick, J S; Anderson, B J; Mott, M R; Egerton, J R

1984-01-01

129

Molecular cloning of a Mycoplasma meleagridis-specific antigenic domain endowed with a serodiagnostic potential.  

PubMed

A recombinant phage library harbouring Mycoplasma meleagridis (MM) genomic DNA fragments was generated in the bacteriophage lambda gt11 expression vector. The library was screened for expression of MM specific antigens with a polyclonal antiserum that had been preadsorbed with antigens of the most common unrelated avian mycoplasma species. A 49-amino acid antigenic domain unique to MM was isolated, expressed in Escherichia coli, and its serodiagnostic potential was demonstrated. An antiserum raised against this MM-specific antigenic domain recognized a cluster of seven membrane-associated MM proteins with molecular masses ranging from 34 to 75 kDa. Overall, this study resulted in the identification of a potent serodiagnostic tool and revealed the complex antigenic nature of MM. PMID:16973309

Mardassi, B Ben Abdelmoumen; Béjaoui Khiari, A; Oussaief, L; Landoulsi, A; Brik, C; Mlik, B; Amouna, F

2007-01-17

130

Development of antigen capture ELISA for the quantification of EIAV p26 protein.  

PubMed

An antigen capture enzyme-linked immunosorbent assay (AC-ELISA) was established based on two monoclonal antibodies (mAbs) for the quantification of equine infectious anemia virus (EIAV). Two p26-specific monoclonal antibodies were developed in mice. The mAb 9H8 was coated in microtiter plates as the capture antibody; the other mAb, 1G11, was coupled to horseradish peroxidase (HRP) and used as the detection antibody. The limit of detection for the EIAV p26 protein was 0.98 ng/ml, and the linearity range was 3.9-62.5 ng/ml. The sensitivity of p26 AC-ELISA for the detection of the virus (EIAV infectious clone, FDDVcmv3-8) was the same as that for the purified p26 protein. No cross-reaction with other equine viruses was observed by this method. The intra- and inter-assay coefficients of variation were below 8.3 and 10.3 % for testing p26 and FDDVcmv3-8, respectively. The AC-ELISA was also compared to Western blotting (WB) and reverse transcriptase (RT) assays, validating the sensitivity, accuracy, and reliability of this method. Both the AC-ELISA and RT assay showed good agreement, with a correlation coefficient of R (2)?=0.9946. Sample analysis showed that this AC-ELISA is a useful tool for quantifying EIAV p26 in cell lysates and culture medium. PMID:25256618

Hu, Zhe; Chang, Hao; Ge, Man; Lin, Yuezhi; Wang, Xuefeng; Guo, Wei; Wang, Xiaojun

2014-11-01

131

Murine carcinoma expressing carcinoembryonic antigen-like protein is restricted by antibody against neem leaf glycoprotein.  

PubMed

We have generated a polyclonal antibody against a novel immunomodulator, neem leaf glycoprotein (NLGP) that can react to a specific 47kDa subunit of NLGP. Generated anti-NLGP antibody (primarily IgG2a) was tested for its anti-tumor activity in murine carcinoma (EC, CT-26), sarcoma (S180) and melanoma (B16Mel) tumor models. Surprisingly, tumor growth restriction was only observed in CT-26 carcinoma models, without any alteration in other tumor systems. Comparative examination of antigenicity between four different tumor models revealed high expression of CEA-like protein on the surface of CT-26 tumors. Subsequent examination of the cross-reactivity of anti-NLGP antibody with purified or cell bound CEA revealed prominent recognition of CEA by anti-NLGP antibody, as detected by ELISA, Western Blotting and immunohistochemistry. This recognition seems to be responsible for anti-tumor function of anti-NLGP antibody only on CEA-like protein expressing CT-26 tumor models, as confirmed by ADCC reaction in CEA(+) tumor systems where dependency to anti-NLGP antibody is equivalent to anti-CEA antibody. Obtained result with enormous therapeutic potential for CEA(+) tumors may be explained in view of the epitope spreading concept, however, further investigation is crucial. PMID:25128841

Das, Arnab; Barik, Subhasis; Bose, Anamika; Roy, Soumyabrata; Biswas, Jaydip; Baral, Rathindranath; Pal, Smarajit

2014-11-01

132

Evaluation of Multiple Antigenic Peptides Based on the Chikungunya E2 Protein for Improved Serological Diagnosis of Infection.  

PubMed

Abstract In recent years, Chikungunya virus (CHIKV) reemerged and numerous outbreaks were reported all over the world. After screening CHIKV-positive sera, we had already reported many dominant epitopes within the envelope E2 protein of CHIKV. In the present study, we aimed at developing a highly sensitive immunodiagnostic assay for CHIKV based on a multiple antigenic peptide (MAP) approach using selective epitopes of the E2 protein. MAPs in four different E2 peptide combinations were screened with CHIKV-positive sera. The MAPs reacted with all CHIKV-positive sera and no reactivity was seen with healthy or dengue-positive sera. Our results indicate that MAP 1 seems to be an alternate antigen to full-length protein E2 for immunodiagnosis of CHIKV infections with high sensitivity and specificity. PMID:25412351

Bhatnagar, Santwana; Kumar, Pradeep; Mohan, Teena; Verma, Priyanka; Parida, M M; Hoti, S L; Rao, D N

2014-11-20

133

Improved diagnostic performance of a commercial anaplasma antibody competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5–glutathione S-transferase fusion protein as antigen  

Technology Transfer Automated Retrieval System (TEKTRAN)

This study tested the hypothesis that removal of maltose binding protein from recombinant antigen used for plate coating would improve the specificity of Anaplasma antibody competitive ELISA. Three hundred and eight sera with significant MBP antibody binding (=30%I) in Anaplasma negative herds was 1...

134

Murine dendritic cells loaded in vitro with soluble protein prime cytotoxic T lymphocytes against tumor antigen in vivo  

PubMed Central

The priming of an immune response against a major histocompatibility complex class I-restricted antigen expressed by nonhematopoietic cells involves the transfer of that antigen to a host bone marrow-derived antigen presenting cell (APC) for presentation to CD8+ T lymphocytes. Dendritic cells (DC), as bone marrow-derived APC, are first candidates for presentation of tumor-associated antigens (TAA). The aim of this study was to see whether DC are able to prime in vivo antigen-specific cytotoxic T lymphocytes after exposure to a soluble protein antigen in vitro. Lacking a well-defined murine TAA, we took advantage of beta- galactosidase (beta-gal)-transduced tumor cell lines as a model in which beta-gal operationally functions as TAA. For in vivo priming both a DC line, transduced or not transduced with the gene coding for murine GM-CSF, and fresh bone marrow-derived DC (bm-DC), loaded in vitro with soluble beta-gal, were used. Priming with either granulocyte macrophage colony-stimulating factor-transduced DC line or fresh bm-DC but not with untransduced DC line generated CTL able to lyse beta-gal- transfected target cells. Furthermore, GM-CSF was necessary for the DC line to efficiently present soluble beta-gal as an H-2Ld-restricted peptide to a beta-gal-specific CTL clone. Data also show that a long- lasting immunity against tumor challenge can be induced using beta-gal- pulsed bm-DC as vaccine. These results indicate that effector cells can be recruited and activated in vivo by antigen-pulsed DC, providing an efficient immune reaction against tumors. PMID:8551239

1996-01-01

135

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

PubMed Central

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

Schuenke, K W; Walker, D H

1994-01-01

136

Folded conformations of antigenic peptides from riboflavin carrier protein in aqueous hexafluoroacetone.  

PubMed Central

Riboflavin carrier protein (RCP) plays an important role in transporting vitamin B2 across placental membranes, a process critical for maintenance of pregnancy. Association of the vitamin with the carrier protein ensures optimal bioavailability, facilitating transport. The conformations of three antigenic peptide fragments encompassing residues 4-23 (N21), 170-186 (R18), and 200-219 (Y21) from RCP, which have earlier been studied as potential leads toward a synthetic peptide-based contraceptive vaccine, have been investigated using CD and NMR spectroscopy in aqueous solution and in the presence of the structure-stabilizing cosolvent hexafluoroacetone trihydrate (HFA). In aqueous solution at pH 3.0, all three peptides are largely unstructured, with limited helical population for the peptides R18 and Y21. The percentage of helicity estimated from CD experiments is 10% for both the peptides. A dramatic structural transition from an unstructured state to a helical state is achieved with addition of HFA, as evidenced by intensification of CD bands at 222 nm and 208 nm for Y21 and R18. The structural transition is completed at 50% HFA (v/v) with 40% and 35% helicity for R18 and Y21, respectively. No structural change is evident for the peptide N21, even in the presence of HFA. NMR analysis of the three peptides in 50% HFA confirms a helical conformation of R18 and Y21, as is evident from upfield shifts of CalphaH resonances and the presence of many sequential NH/NH NOEs with many medium-range NOEs. The helical conformation is well established at the center of the sequence, with substantial fraying at the termini for both the peptides. An extended conformation is suggested for the N21 peptide from NMR studies. The helical region of both the peptides (R18, Y21) comprises the core epitopic sequence recognized by the respective monoclonal antibodies. These results shed some light on the issue of structure and folding of antigenic peptides. PMID:9514267

Bhattacharjya, S.; Awasthi, S. K.; Adiga, P. R.; Balaram, P.

1998-01-01

137

Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen 1 Mimics Epstein-Barr Virus EBNA1 Immune Evasion through Central Repeat Domain Effects on Protein Processing  

Microsoft Academic Search

Kaposi's sarcoma-associated herpesvirus (KSHV\\/human herpesvirus 8 (HHV8)) and Epstein-Barr virus (EBV\\/HHV4) are distantly related gammaherpesviruses causing tumors in humans. KSHV latency-associated nuclear antigen 1 (LANA1) is functionally similar to the EBV nuclear antigen-1 (EBNA1) protein expressed during viral latency, although they have no amino acid similarities. EBNA1 escapes cytotoxic lymphocyte (CTL) antigen processing by inhibiting its own proteosomal degradation and

Hyun Jin Kwun; Suzane Ramos da Silva; Ishita M. Shah; Neil Blake; Patrick S. Moore; Yuan Chang

2007-01-01

138

Successful unrelated cord blood transplantation for homozygous ?-thalassemia.  

PubMed

A now 10-year-old Laotian female was delivered at 30-week gestation by cesarean section because of severe hydrops. Fetal blood sampling revealed homozygous ?-thalassemia. After immediate resuscitation, the infant was supported with frequent red cell transfusions. At 44 months of age, she received a 5 of 6 human leukocyte antigen-matched unrelated cord blood transplantation. She was treated with phlebotomy and chelation therapy with Deferasirox for correction of hemosiderosis and has been transfusion-independent since 41 days after transplant. She is currently 6 years after transplantation with stable, 100% donor engraftment, resolved iron overload, and normal growth and development. PMID:23337553

Gumuscu, Burak; Thompson, Elizabeth I; Grovas, Alfred C; Zach, Terrence L; Warkentin, Phyllis I; Coccia, Peter F

2013-10-01

139

Ratio of HCV structural antigens in protein-based vaccine formulations is critical for functional immune response induction.  

PubMed

HCV (hepatitis C virus) infection is among the leading causes of chronic liver disease, but currently there is no vaccine available. Data have accumulated about the importance of targeting different HCV antigens in vaccine candidate preparations. Here, a surface response study to select the optimal ratio of recombinant HCV structural antigens in a vaccine preparation, capable of generating in vivo functional cellular immune response in mice, was performed. The immunogenicity of the selected HCV structural protein mixture (Co-E1-E2) in mice and African green monkeys, after five doses of immunization, was also demonstrated. Specific T-cell proliferative response against HCV structural antigens was induced in vaccinated mice. Moreover, on challenge with recombinant HCV VV (vaccinia virus), all mice controlled the viraemia and 80% were protected. On the other hand, monkeys immunized with Co-E1-E2 developed antibodies, specifically directed to region 412-438 of E2 protein, that include an epitope implicated in HCV neutralization, in addition to a specific proliferative response against HCV Core and E2 proteins. These results indicated that the optimal amount and ratio of HCV recombinant proteins should be taken into account to elicit a successful immune response against HCV and therefore have important implications for vaccine design. PMID:20515441

Martínez-Donato, Gillian; Musacchio, Alexis; Alvarez-Lajonchere, Liz; Acosta-Rivero, Nelson; Amador, Yalena; Guerra, Ivis; Peña, Dilver; Pérez, Angel; Castro, Jorge; Puentes, Pedro; Soria, Yordanka; Cosme, Karelia; Sanchez, Jorge; Dueñas-Carrera, Santiago

2010-07-01

140

Algae as Protein Factories: Expression of a Human Antibody and the Respective Antigen in the Diatom Phaeodactylum tricornutum  

PubMed Central

Microalgae are thought to offer great potential as expression system for various industrial, therapeutic and diagnostic recombinant proteins as they combine high growth rates with all benefits of eukaryotic expression systems. Moreover, microalgae exhibit a phototrophic lifestyle like land plants, hence protein expression is fuelled by photosynthesis, which is CO2-neutral and involves only low production costs. So far, however, research on algal bioreactors for recombinant protein expression is very rare calling for further investigations in this highly promising field. In this study, we present data on the expression of a monoclonal human IgG antibody against the Hepatitis B surface protein and the respective antigen in the diatom Phaeodactylum tricornutum. Antibodies are fully-assembled and functional and accumulate to 8.7% of total soluble protein, which complies with 21 mg antibody per gram algal dry weight. The Hepatitis B surface protein is functional as well and is recognized by algae-produced and commercial antibodies. PMID:22164289

Hempel, Franziska; Lau, Julia; Klingl, Andreas; Maier, Uwe G.

2011-01-01

141

Antigenic Diversity of the Plasmodium vivax Circumsporozoite Protein in Parasite Isolates of Western Colombia  

PubMed Central

Circumsporozoite (CS) protein is a malaria antigen involved in sporozoite invasion of hepatocytes, and thus considered to have good vaccine potential. We evaluated the polymorphism of the Plasmodium vivax CS gene in 24 parasite isolates collected from malaria-endemic areas of Colombia. We sequenced 27 alleles, most of which (25/27) corresponded to the VK247 genotype and the remainder to the VK210 type. All VK247 alleles presented a mutation (Gly ? Asn) at position 28 in the N-terminal region, whereas the C-terminal presented three insertions: the ANKKAGDAG, which is common in all VK247 isolates; 12 alleles presented the insertion GAGGQAAGGNAANKKAGDAG; and 5 alleles presented the insertion GGNAGGNA. Both repeat regions were polymorphic in gene sequence and size. Sequences coding for B-, T-CD4+, and T-CD8+ cell epitopes were found to be conserved. This study confirms the high polymorphism of the repeat domain and the highly conserved nature of the flanking regions. PMID:21292878

Hernández-Martínez, Miguel Ángel; Escalante, Ananías A.; Arévalo-Herrera, Myriam; Herrera, Sócrates

2011-01-01

142

Elastin, a Novel Extracellular Matrix Protein Adhering to Mycobacterial Antigen 85 Complex*  

PubMed Central

The antigen 85 complex (Ag85) consists of three predominantly secreted proteins (Ag85A, Ag85B, and Ag85C), which play a key role in the mycobacterial pathogenesis and also possess enzymatic mycolyltransferase activity involved in cell wall synthesis. Ag85 is not only considered to be a virulence factor because its expression is essential for intracellular survival within macrophages, but also because it contributes to adherence, invasion, and dissemination of mycobacteria in host cells. In this study, we report that the extracellular matrix components, elastin and its precursor (tropoelastin) derived from human aorta, lung, and skin, serve as binding partners of Ag85 from Mycobacterium tuberculosis. The binding affinity of M. tuberculosis Ag85 to human tropoelastin was characterized (KD = 0.13 ± 0.006 ?m), and a novel Ag85-binding motif, AAAKAA(K/Q)(Y/F), on multiple tropoelastin modules was identified. In addition, the negatively charged Glu-258 of Ag85 was demonstrated to participate in an electrostatic interaction with human tropoelastin. Moreover, binding of Ag85 on elastin siRNA-transfected Caco-2 cells was significantly reduced (34.3%), implying that elastin acts as an important ligand contributing to mycobacterial invasion. PMID:23250738

Kuo, Chih-Jung; Ptak, Christopher P.; Hsieh, Ching-Lin; Akey, Bruce L.; Chang, Yung-Fu

2013-01-01

143

Sequence Diversity of Bacillus thuringiensis Flagellin (H Antigen) Protein at the Intra-H Serotype Level? †  

PubMed Central

In Bacillus thuringiensis, the hag gene encodes flagellin, the protein responsible for eliciting the immunological reaction in H serotyping. Specific flagellin amino acid sequences have been correlated to specific B. thuringiensis H serotypes, H1 to H67. Ten H serotypes, however, contain three or more antigenic subfactors, labeled a, b, c, d, or e, and have been subdivided into 23 serovars. In the present study, we set out to analyze the sequence diversity of flagellins among serovars from the same H serotypes. We studied the hag genes in 39 B. thuringiensis strains representing the 23 serovars from the 10 H serotypes mentioned above. A serovar and a biovar from an 11th H serotype were also included. The hag genes were amplified and cloned and their nucleotide sequences were determined and translated into amino acid sequences, or the sequences were retrieved directly from GenBank when available. Strains of the H3 serotype contained two or three copies of the fla gene, an ortholog of the hag gene. Strains of the H6 serotype contained three copies. Strains of all other H serotypes each contained a single copy of the hag gene. Alignments of amino acid sequences from all copies in all strains of the H3 serotype revealed short signature sequences, GGAG and SGG, GPDPDDAVKNLT, and DITTTK, that appeared to be specific to the H3c, H3d, and H3e antigenic subfactors, respectively. Similar short signature sequences, GDIT, AFIK, TSAGKA, and SAPSKG, were revealed for H8b, H8c, H20b, and H20c, respectively. Amino acid sequences in the flagellin central variable region were highly conserved among serovars of the H3, H5, H11, and H20 serotypes and much more divergent among serovars of the H4, H10, H18, H24, and H28 serotypes. Two bootstrapped neighbor-joining trees were respectively generated from the alignments of the amino acid sequences translated from all copies of the hag genes in the B. thuringiensis strains of the H3 and H6 serotypes. Sequence identities and relationships were revealed. A third bootstrapped neighbor-joining tree was generated, this one from the alignment of the flagellin amino acid sequences from all the B. thuringiensis strains in the study. Eight clusters, I to VIII, were revealed. Although most clusters contained strains and serovars from the same H serotype, clusters VII and VIII contained serovars from different H serotypes. PMID:18586969

Xu, Dong; Côté, Jean-Charles

2008-01-01

144

Mutual Conformational Adaptations in Antigen and Antibody upon Complex Formation between an Fab and HIV1 Capsid Protein p24  

Microsoft Academic Search

Background: Elucidating the structural basis of antigen-antibody recognition ideally requires a structural comparison of free and complexed components. To this end we have studied a mouse monoclonal antibody, denoted 13B5, raised against p24, the capsid protein of HIV-1. We have previously described the first crystal structure of intact p24 as visualized in the Fab13B5-p24 complex. Here we report the structure

Stéphanie Monaco-Malbet; Carmen Berthet-Colominas; Armelle Novelli; Nadia Piga; Valérie Cheynet; François Mallet; Stephen Cusack

2000-01-01

145

Epitope mapping of the N-terminal portion of tissue transglutaminase protein antigen to identify linear epitopes in celiac disease.  

PubMed

Celiac disease (CD) is an autoimmune mediated disease with complex and multifactorial etiology. Gluten intake triggers a composite immune response involving T-cells and B-cells and leading to the secretion of autoantibodies if a genetic predisposition is present. Untreated CD patients show high levels of circulating autoantibodies directed to different auto-antigens present in the intestinal mucosa. The most important auto-antigen is the endomysial enzyme tissue transglutaminase (tTG). Both IgA and IgG antibody isotypes to tTG are known, but only the IgA antibodies demonstrate the highest disease specificity and thus are considered disease biomarkers. Because the pathogenicity and exact tTG binding properties of these autoantibodies are still unclear, the characterization of tTG antigenic domains is a crucial step in understanding CD onset and the autoimmune pathogenesis. Overlapping peptide libraries can be used for epitope mapping of selected protein portions to determine antigenic fragments contributing to the immunological activity and possibly develop innovative peptide-based tools with high specificity and sensitivity for CD. We performed an epitope mapping study to characterize putative linear auto-antigenic epitopes present in the tTG N-terminal portion (1-230). A library of 23 overlapping peptides spanning tTG(1-230) was generated by Fmoc/tBu solid-phase peptide synthesis and screened by immunoenzymatic assays employing patients' sera. The results indicate that four synthetic peptides, that is, Ac-tTG(1-15)-NH2 , Ac-tTG(41-55)-NH2 , Ac-tTG(51-65)-NH2 , and Ac-tTG(151-165)-NH2 , are recognized by IgA autoantibodies circulating in CD patients' sera. These results offer important insight on the nature of the antigen-antibody interaction. PMID:24831711

Di Pisa, Margherita; Buccato, Patrick; Sabatino, Giuseppina; Real Fernández, Feliciana; Berti, Brunilde; Cocola, Francesco; Papini, Anna Maria; Rovero, Paolo

2014-09-01

146

DNA, but not protein vaccine based on mutated BORIS antigen significantly inhibits tumor growth and prolongs the survival of mice  

PubMed Central

The ideal immunological target for cancer vaccine development would meet the criteria of tumor specificity, immunogenicity and vital dependency of the tumor on the functional activities of the antigenic target so as to avoid antigenic loss by mutation. Given that at face value the brother of regulator of imprinted sites (BORIS) transcription factor meets these criteria, we have developed a mutant variant of this molecule (mBORIS) that lacks tumorigenic ability, while retaining immunogenic epitopes that elicits responses against histologically irrelevant tumor cells. Here we compared vaccine strategies employing as an immunogen either mBORIS recombinant protein formulated in a strong Th1-type adjuvant, QuilA or DNA encoding this immunogen along with plasmids expressing interleukin (IL)12/IL18 molecular adjuvants. In both groups of vaccinated mice induction of tumor-specific immunity (antibody response, T-cell proliferation, cytokine production, T-cell cytotoxicity) as well as ability to inhibit growth of the aggressive breast cancer cell line and to prolong survival of vaccinated animals have been tested. We determined that DNA, but not recombinant protein vaccine, induced potent Th1-like T-cell recall responses that significantly inhibited tumor growth and prolongs the survival of vaccinated mice. These studies demonstrate that DNA immunization is superior to recombinant protein strategy and provide a clear guidance for clinical development of a cancer vaccine targeting what appears to be a universal tumor antigen. PMID:17972923

Mkrtichyan, M; Ghochikyan, A; Loukinov, D; Davtyan, H; Ichim, TE; Cribbs, DH; Lobanenkov, VV; Agadjanyan, MG

2008-01-01

147

pH6 antigen (PsaA protein) of Yersinia pestis, a novel bacterial Fc-receptor.  

PubMed

It was found that recombinant pH6 antigen (rPsaA protein) forming virulence-associated fimbriae on the surface of Yersinia pestis at pH 6.7 in host macrophage phagolysosomes or extracellularly in abscesses such as buboes, is a novel bacterial Fc-receptor. rPsaA protein displays reactivity with human IgG1, IgG2 and IgG3 subclasses but does not react with rabbit, mouse and sheep IgG. PMID:8804976

Zav'yalov, V P; Abramov, V M; Cherepanov, P G; Spirina, G V; Chernovskaya, T V; Vasiliev, A M; Zav'yalova, G A

1996-05-01

148

Phosphate starvation enhances expression of the immunodominant 38-kilodalton protein antigen of Mycobacterium tuberculosis: demonstration by immunogold electron microscopy.  

PubMed Central

In this work, we grew Mycobacterium tuberculosis in an enriched Proskauer-Beck-Youmans culture medium in the presence and in the absence of phosphate salts. Immunoblot analysis of sonic extracts showed overexpression of the 38-kDa protein antigen by bacilli grown in the medium without phosphate. These observations were confirmed by immunogold electron microscopy, which showed that the number of gold particles was significantly higher in bacilli grown in medium without phosphate than in bacilli grown in medium with phosphate. The 38-kDa protein was located mainly in the wall and on the cell surface. Images PMID:1612766

Espitia, C; Elinos, M; Hernández-Pando, R; Mancilla, R

1992-01-01

149

pH6 antigen (PsaA protein) of Yersinia pestis, a novel bacterial Fc-receptor  

Microsoft Academic Search

It was found that recombinant pH6 antigen (rPsaA protein) forming virulence-associated fimbriae on the surface of Yersinia pestis at pH 6.7 in host macrophage phagolysosomes or extracellularly in abscesses such as buboes, is a novel bacterial Fc-receptor. rPsaA protein displays reactivity with human IgG1, IgG2 and IgG3 subclasses but does not react with rabbit, mouse and sheep IgG.

Vladimir P. Zav'yalov; Vyacheslav M. Abramov; Peter G. Cherepanov; Galina V. Spirina; Tatiana V. Chernovskaya; Anatolii M. Vasiliev; Galina A. Zav'yalova

1996-01-01

150

Synergy between lysosomotropic amines and cyclosporin A on human T cell responses to an exogenous protein antigen, tetanus toxoid.  

PubMed

Previously, it has been shown that the lysosomotropic amine, chloroquine, is effective in the prevention of graft-versus-host disease (GVHD) using murine models. Because chloroquine and hydroxychloroquine suppress MHC class II antigen presentation, their mechanism of action is different to other immune suppressant drugs (cyclosporin A) currently used to control GVHD. It is possible that the use of cyclosporin A and chloroquine in combination may have an additive or synergistic effect on T cell responses to antigens presented in the context of MHC class II. We investigated the effects of chloroquine or hydroxychloroquine in combination with cyclosporin A on human T cell responses in vitro to tetanus toxoid, an exogenous protein antigen dependent on MHC class II presentation for proliferative responses. We demonstrate that similar levels of chloroquine and hydroxychloroquine suppress human T cell responses to tetanus toxoid and that the use of either agent in combination with cyclosporin A results in synergistic suppression. Evaluation for a direct effect by the lysosomotropic amines on T cells, in the absence of antigen presenting cells, revealed that there was inhibition of T cell responses but only at high concentrations. No significant decrease or increase was seen in surface MHC II or invariant chain expression or in cytoplasmic invariant chain after exposure to chloroquine. Thus, lysosomotropic amines in combination with cyclosporin A are synergistic in suppression of T cell proliferation. Use of these agents in combination with cyclosporin A may improve control of graft-versus-host disease. PMID:8879628

Schultz, K R; Nelson, D; Bader, S

1996-09-01

151

Intestinal uptake of macromolecules. Differences in distribution and degradation of protein antigen in control and immunised rats.  

PubMed Central

The present study examined intraluminal events in the in vivo processing of a protein antigen by the intestine of normal and orally immunised rats. One hour after the administration of 125I-bovine serum albumin (125I-BSA) and unlabelled BSA by gavage, the majority of the radioactivity was found in the distal small intestine of control and immunised rats but there was a difference in the distribution of radioactivity. In contrast with controls, immunised rats retained a lesser percentage of radioactivity in the proximal small intestine and a greater percentage of radioactivity in the distal small intestine. Radioactive substances present in intestinal rinse fluids and mucosal extracts were characterised by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), density gradient ultracentrifugation, and by immunochemical methods. Rinse fluids and mucosal extracts from immunised rats fed 125I-BSA by gavage contained high molecular weight components with characteristics of antigen-antibody complexes. Rinse fluids and extracts of normal rats contained more intact BSA and less fragments of BSA than did rinse fluids and extracts from immunised animals. These findings suggest that oral immunisation alters the distribution of antigen administered into the gut and that immunisation enhances the intraluminal degradation of antigen. PMID:7033055

Pang, K Y; Walker, W A; Bloch, K J

1981-01-01

152

JC Virus Small t Antigen Binds Phosphatase PP2A and Rb Family Proteins and Is Required for Efficient Viral DNA Replication Activity  

Microsoft Academic Search

BackgroundThe human polyomavirus, JC virus (JCV) produces five tumor proteins encoded by transcripts alternatively spliced from one precursor messenger RNA. Significant attention has been given to replication and transforming activities of JCV's large tumor antigen (TAg) and three T? proteins, but little is known about small tumor antigen (tAg) functions. Amino-terminal sequences of tAg overlap with those of the other

Brigitte Bollag; Catherine A. Hofstetter; Marta M. Reviriego-Mendoza; Richard J. Frisque; Wang-Shick Ryu

2010-01-01

153

Signaling and Transcriptional Changes Critical for Transformation of Human Cells by Simian Virus 40 Small Tumor Antigen or Protein Phosphatase 2A B56 Knockdown  

Microsoft Academic Search

One set of genes sufficient for transformation of primary human cells uses the combination of Ha-Ras-V12, the telomerase catalytic subunit hTERT, SV40 large tumor antigen (LT), and SV40 small tumor antigen (ST). Whereas SV40 LT inactivates the retinoblastoma protein and p53, the contribution of ST is poorly understood. The essential helper function of ST requires a functional interaction with protein

Carlos S. Moreno; Sumathi Ramachandran; Danita G. Ashby; Noelani Laycock; Courtney A. Plattner; Wen Chen; William C. Hahn; David C. Pallas

2004-01-01

154

Incidental CD8 T cell reactivity against caspase-cleaved apoptotic self-antigens from ubiquitously expressed proteins in islets from prediabetic human leucocyte antigen-A2 transgenic non-obese diabetic mice.  

PubMed

Apoptosis is known as a major mechanism which contributes to beta cell decay in type 1 diabetes. Commitment to this pathway generally involves caspase-mediated protein cleavage and was found to induce cross-presentation of a specific antigen repertoire under certain inflammatory conditions. We aimed to assess the significance of the CD8 T cell population reactive against such caspase-cleaved apoptotic self-antigens in pancreatic islets of prediabetic human leucocyte antigen (HLA)-A2 transgenic non-obese diabetic chimeric monochain transgene construct (NOD.HHD) mice. We have reproduced a unique peptide library consisting of human CD8 T cell-derived apoptosis-specific antigens, all of which belong to structural proteins expressed ubiquitously in human islets. Pancreatic islets from prediabetic NOD.HHD mice, harbouring humanized major histocompatibilty complex (MHC) class I, were isolated and handpicked at various ages, and islet-infiltrating CD8 T cells were expanded in vitro and used as responders in an interferon (IFN)-? enzyme-linked immunospot (ELISPOT) assay. Human T2 cells were used as antigen-presenting cells (APC) to avoid endogenous antigen presentation. Analogous to the interindividual variability found with peptides from known islet autoantigens such as islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP) and insulin, some mice showed variable, low-degree CD8 T cell reactivity against caspase-cleaved self-antigens. Because reactivity was predominantly minor and often undetectable, we conclude that beta cell apoptosis does not routinely provoke the development of dominant cytotoxic T lymphocyte (CTL) reactive against caspase-cleaved self-antigens in the NOD.HHD model. PMID:21605113

Coppieters, K T; Amirian, N; von Herrath, M G

2011-08-01

155

Molecular characterization and antigenic properties of a novel Babesia gibsoni glutamic acid-rich protein (BgGARP).  

PubMed

Identification and molecular characterization of Babesia gibsoni proteins with potential antigenic properties are crucial for the development and validation of the serodiagnostic method. In this study, we isolated a cDNA clone encoding a novel B. gibsoni 76-kDa protein by immunoscreening of the parasite cDNA library. Computer analysis revealed that the protein presents a glutamic acid-rich region in the C-terminal. Therefore, the protein was designated as B. gibsoni glutamic acid-rich protein (BgGARP). A BLASTp analysis of a translated BgGARP polypeptide demonstrated that the peptide shared a significant homology with a 200-kDa protein of Babesia bigemina and Babesia bovis. A truncated BgGARP cDNA (BgGARPt) encoding a predicted 13-kDa peptide was expressed in Escherichia coli (E. coli), and mouse antisera against the recombinant protein were used to characterize a corresponding native protein. The antiserum against recombinant BgGARPt (rBgGARPt) recognized a 140-kDa protein in the lysate of infected erythrocytes, which was detectable in the cytoplasm of the parasites by confocal microscopic observation. In addition, the specificity and sensitivity of enzyme-linked immunosorbent assay (ELISA) with rBgGARPt were evaluated using B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. Moreover, 107 serum samples from dogs clinically diagnosed with babesiosis were examined using ELISA with rBgGARPt. The results showed that 86 (80.4%) samples were positive by rBgGARPt-ELISA, which was comparable to IFAT and PCR as reference test. Taken together, these results demonstrate that BgGARP is a suitable serodiagnostic antigen for detecting antibodies against B. gibsoni in dogs. PMID:23968686

Mousa, Ahmed Abdelmoniem; Cao, Shinuo; Aboge, Gabriel Oluga; Terkawi, Mohamad Alaa; El Kirdasy, Ahmed; Salama, Akram; Attia, Mabrouk; Aboulaila, Mahmoud; Zhou, Mo; Kamyingkird, Ketsarin; Moumouni, Paul Franck Adjou; Masatani, Tatsunori; El Aziz, Sami Ahmed Abd; Moussa, Waheed Mohammed; Chahan, Bayin; Fukumoto, Shinya; Nishikawa, Yoshifumi; El Ballal, Salah Sayed; Xuan, Xuenan

2013-10-01

156

Differentiation of pathogenic Entamoeba histolytica infections from nonpathogenic infections by detection of galactose-inhibitable adherence protein antigen in sera and feces.  

PubMed Central

We determined whether epitope-specific monoclonal antibodies to the galactose-inhibitable adherence protein (GIAP) of Entamoeba histolytica could be used in an enzyme-linked immunosorbent assay (ELISA) to detect antigen in serum and feces and differentiate between nonpathogenic zymodemes and the potentially invasive pathogenic organisms that require treatment. Overall, 57% of subjects from Cairo, Egypt, with symptomatic intestinal amebiasis and 42% with asymptomatic infection possessed GIAP antigen in their sera, whereas 4% of uninfected controls or subjects with other parasitic infections possessed GIAP antigen in their sera (P < 0.001). In subjects from Durban, South Africa, only 6% of uninfected controls or those with nonpathogenic E. histolytica infection were positive for GIAP in serum, whereas 3 of 4 with asymptomatic pathogenic intestinal infection and 75% with amebic liver abscess were positive for GIAP in serum. Fifteen stool samples from patients with intestinal amebiasis were available for study; all had a positive ELISA result for fecal GIAP antigen. Epitope-specific monoclonal antibodies identified 8 of 15 subjects with fecal antigen from pathogenic strains. Seven of those eight subjects had adherence protein antigen in their sera, whereas none of seven with apparent nonpathogenic E. histolytica infection had adherence protein antigen in their sera. In summary, we were able to detect E. histolytica adherence protein antigen directly in serum and fecal samples by ELISA. The presence of amebic antigen in serum demonstrated 94% specificity for pathogenic E. histolytica infection, and amebic antigen is present during asymptomatic intestinal infection. In conjunction with antibody detection, this method should be very useful in the diagnosis and management of intestinal amebiasis. Images PMID:8263165

Abd-Alla, M D; Jackson, T F; Gathiram, V; el-Hawey, A M; Ravdin, J I

1993-01-01

157

Tyrosine-phosphorylated Galectin-3 Protein Is Resistant to Prostate-specific Antigen (PSA) Cleavage*  

PubMed Central

Galectin-3 is a chimeric carbohydrate-binding protein, which interacts with cell surface carbohydrate-containing molecules and extracellular matrix glycoproteins and has been implicated in various biological processes such as cell growth, angiogenesis, motility, and metastasis. It is expressed in a wide range of tumor cells and is associated with tumor progression. The functions of galectin-3 are dependent on its localization and post-translational modifications such as cleavage and phosphorylation. Recently, we showed that galectin-3 Tyr-107 is phosphorylated by c-Abl; concomitantly, it was also shown that galectin-3 can be cleaved at this site by prostate-specific antigen (PSA), a chymotrypsin-like serine protease, after Tyr-107, resulting in loss of galectin-3 multivalency while preserving its carbohydrate binding activity. Galectin-3 is largely a monomer in solution but may form a homodimer by self-association through its carbohydrate recognition domain, whereas, in the presence of a ligand, galectin-3 polymerizes up to pentamers utilizing its N-terminal domain. Oligomerization is a unique feature of secreted galectin-3, which allows its function by forming ordered galectin-glycan structures, i.e. lattices, on the cell surface or through direct engagement of specific cell surface glycoconjugates by traditional ligand-receptor binding. We questioned whether Tyr-107 phosphorylation by c-Abl affects galectin-3 cleavage by PSA. The data suggest a role for galectin-3 in prostate cells associated with increased activity of c-Abl kinase and loss of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) activity. In addition, the ratio of phosphorylated/dephosphorylated galectin-3 might be used as a complementary value to that of PSA for prognosis of prostate cancer and a novel therapeutic target for the treatment of prostate cancer. PMID:22232548

Balan, Vitaly; Nangia-Makker, Pratima; Kho, Dhong Hyo; Wang, Yi; Raz, Avraham

2012-01-01

158

In Vivo and In Vitro studies on transfer factor with a protein antigen system  

E-print Network

subject was possible by transfer of serum. In the tuberculin type sensiti. vity he found that skin testing wi. th speci. fic antigen resulted in a delayed (24 to 48 hours) reaction, that antibodies were not demon- strable, and that passive transfer... leukocytes from sensi- tized donors to a nonsensitive recipient. These recipients, when skin tested with specific antigen, gave a typical delayed response. The passive transfer of the delayed hypersensitivity reaction with cells was later confirmed...

Kelleher, Peter Joseph

1975-01-01

159

Functional identity of proliferating cell nuclear antigen and a DNA polymerase-delta auxiliary protein  

Microsoft Academic Search

The mechanism of replication of the simian virus 40 (SV40) genome closely resembles that of cellular chromosomes, thereby providing an excellent model system for examining the enzymatic requirements for DNA replication1. Only one viral gene product, the large tumour antigen (large-T antigen), is required for viral replication2, so the majority of replication enzymes must be cellular. Indeed, a number of

Gregory Prelich; Cheng-Keat Tan; Matthew Kostura; Michael B. Mathews; Antero G. So; Kathleen M. Downey; Bruce Stillman

1987-01-01

160

Spike Protein VP8* of Human Rotavirus Recognizes Histo-Blood Group Antigens in a Type-Specific Manner  

PubMed Central

Rotaviruses (RVs), an important cause of severe diarrhea in children, have been found to recognize sialic acid as receptors for host cell attachment. While a few animal RVs (of P[1], P[2], P[3], and P[7]) are sialidase sensitive, human RVs and the majority of animal RVs are sialidase insensitive. In this study, we demonstrated that the surface spike protein VP8* of the major P genotypes of human RVs interacts with the secretor histo-blood group antigens (HBGAs). Strains of the P[4] and P[8] genotypes shared reactivity with the common antigens of Lewis b (Leb) and H type 1, while strains of the P[6] genotype bound the H type 1 antigen only. The bindings between recombinant VP8* and human saliva, milk, or synthetic HBGA oligosaccharides were demonstrated, which was confirmed by blockade of the bindings by monoclonal antibodies (MAbs) specific to Leb and/or H type 1. In addition, specific binding activities were observed when triple-layered particles of a P[8] (Wa) RV were tested. Our results suggest that the spike protein VP8* of RVs is involved in the recognition of human HBGAs that may function as ligands or receptors for RV attachment to host cells. PMID:22345472

Huang, Pengwei; Xia, Ming; Zhong, Weiming; Wei, Chao; Wang, Leyi; Morrow, Ardythe

2012-01-01

161

Paracoccidioides brasiliensis 87-Kilodalton Antigen, a Heat Shock Protein Useful in Diagnosis: Characterization, Purification, and Detection in Biopsy Material via Immunohistochemistry  

PubMed Central

The 87-kDa antigen derived from the fungal pathogen Paracoccidioides brasiliensis can be detected in the sera of infected patients, and its levels have been shown to correlate well with response to treatment and with clinical cure. Despite its potential importance, the antigen has been poorly characterized. The 87-kDa antigen was purified to homogeneity via preparative gel electrophoresis; N-terminal amino acid sequencing revealed substantial homology with heat shock proteins (hsps) from a variety of organisms. A monoclonal antibody (MAb) raised against a Histoplasma capsulatum 80-kDa hsp showed cross-reactivity to the purified 87-kDa antigen via Western blotting, and the 87-kDa-specific MAb P1B demonstrated that the antigen was expressed at higher levels in yeast than in mycelia by the same technique. Enzyme-linked immunosorbent assay and immunofluorescence reactivity using P1B confirmed increased expression of the 87-kDa antigen during the temperature-induced transformation of mycelia to yeast. Yeast-to-mycelium transformation was accompanied by a fall in expression, although the 87-kDa antigen was clearly constitutively expressed in both phases. Immunochemical staining of tissues from patients with MAb P1B who were infected with P. brasiliensis confirmed in vivo expression of the 87-kDa antigen by yeasts, and identification of this antigen via this method appears to be a useful adjunct to other methods used to diagnose paracoccidioidomycosis. PMID:11825942

Díez, Soraya; Gómez, Beatriz L.; Restrepo, Angela; Hay, Rod J.; Hamilton, Andrew J.

2002-01-01

162

Growth suppression induced by wild-type p53 protein is accompanied by selective down-regulation of proliferating-cell nuclear antigen expression.  

PubMed Central

The p53 gene is a frequent target of mutation in a wide variety of human cancers. Previously, it was reported that conditional expression of wild-type p53 protein in a cell line (GM47.23) derived from a human glioblastoma multiform tumor had a negative effect on cell proliferation. We have now investigated the effect that induction of wild-type p53 protein in this cell line has on the expression of the proliferating-cell nuclear antigen gene. The proliferating-cell nuclear antigen gene encodes a nuclear protein that is an auxiliary factor of DNA polymerase delta and part of the DNA replication machinery of the cell. We show that inhibition of cell cycle progression into S-phase after induction of wild-type p53 protein is accompanied by selective down-regulation of proliferating-cell nuclear antigen mRNA and protein expression. Images PMID:1705714

Mercer, W E; Shields, M T; Lin, D; Appella, E; Ullrich, S J

1991-01-01

163

Comparison of Colorimetric Assays with Quantitative Amino Acid Analysis for Protein Quantification of Generalized Modules for Membrane Antigens (GMMA).  

PubMed

Genetically induced outer membrane particles from Gram-negative bacteria, called Generalized Modules for Membrane Antigens (GMMA), are being investigated as vaccines. Rapid methods are required for estimating the protein content for in-process assays during production. Since GMMA are complex biological structures containing lipid and polysaccharide as well as protein, protein determinations are not necessarily straightforward. We compared protein quantification by Bradford, Lowry, and Non-Interfering assays using bovine serum albumin (BSA) as standard with quantitative amino acid (AA) analysis, the most accurate currently available method for protein quantification. The Lowry assay has the lowest inter- and intra-assay variation and gives the best linearity between protein amount and absorbance. In all three assays, the color yield (optical density per mass of protein) of GMMA was markedly different from that of BSA with a ratio of approximately 4 for the Bradford assay, and highly variable between different GMMA; and approximately 0.7 for the Lowry and Non-Interfering assays, highlighting the need for calibrating the standard used in the colorimetric assay against GMMA quantified by AA analysis. In terms of a combination of ease, reproducibility, and proportionality of protein measurement, and comparability between samples, the Lowry assay was superior to Bradford and Non-Interfering assays for GMMA quantification. PMID:25223624

Rossi, Omar; Maggiore, Luana; Necchi, Francesca; Koeberling, Oliver; MacLennan, Calman A; Saul, Allan; Gerke, Christiane

2015-01-01

164

Characterization of T cell antigens associated with the cell wall protein-peptidoglycan complex of Mycobacterium tuberculosis.  

PubMed

Mycobacterium tuberculosis cell walls are likely to contain critical T cell Ag capable of inducing protective immunity against the development of tuberculosis in animal models. Therefore, we characterized cell wall-associated Ag that stimulate T lymphocytes in tuberculosis patients and clinically well tuberculin-positive individuals. A protein-peptidoglycan complex isolated from the M. tuberculosis cell wall had potent immunologic activity, evoking PBMC proliferative responses similar to those induced by sonicated whole M. tuberculosis. In order to characterize the immunoreactive protein determinants associated with the protein-peptidoglycan complex, T cell lines were established to cell wall Ag and used to probe M. tuberculosis proteins separated by SDS-PAGE. These T cell lines proliferated primarily to protein Ag of 10, 19, 23, 28, 30, 40 to 50, and 65 kDa. Cell wall-reactive T cell clones that recognized the 10-, 23-, 28-, and 30-kDa proteins as single bands on SDS-PAGE did so under reducing and nonreducing conditions, suggesting that these are not proteolytic fragments or subunits of larger protein aggregates. We propose that these protein monomers, when post-translationally complexed with peptidoglycan, are the key ingredients of the immunogenic protein-peptidoglycan complex. In order to assess the relationship of the cell wall-associated Ag to those secreted proteins from "early culture filtrates" of actively growing M. tuberculosis recently implicated in eliciting protective immunity, cell wall-reactive T cell clones were tested for their ability to recognize early culture filtrates. Results revealed that at least three proteins shared with the cell wall complex are contained within early culture filtrates. Our data indicate that antigenic determinants associated with the protein-peptidoglycan complex of the M. tuberculosis cell wall may be involved in protective immunity and hence are potential candidates for inclusion in an effective antituberculosis vaccine. PMID:2507635

Barnes, P F; Mehra, V; Hirschfield, G R; Fong, S J; Abou-Zeid, C; Rook, G A; Hunter, S W; Brennan, P J; Modlin, R L

1989-10-15

165

Identification of a common antigenic site in the nucleocapsid protein of European and North American isolates of porcine reproductive and respiratory syndrome virus.  

PubMed

Porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid (N) protein has been identified as the most immunodominant viral protein. The N protein genes from two PRRSV isolates Olot/91 (European) and Quebec 807/94 (North American) were cloned and expressed in Escherichia coli using the pET3x system. The antigenic structure of the PRRSV N protein was dissected using seven monoclonal antibodies (MAbs) and overlapping fragments of the protein expressed in E.coli. Three antigenic sites were found. Four MAbs recognized two discontinuous epitopes that were present in the partially folded protein or at least a large fragment comprising the first 78 residues, respectively. The other three MAbs revealed the presence of a common antigenic site localized in the central region of the protein (amino acids 50 to 66). This hydrophillic region is well conserved among different isolates of European and North American origin. However, since this epitope is not recognized by many pig sera, it is not adequate for diagnostic purposes. Moreover, none of the N protein fragments were able to mimic the antigenicity of the entire N protein. PMID:9782317

Casal, J I; Rodriguez, M J; Sarraseca, J; Garcia, J; Plana-Duran, J; Sanz, A

1998-01-01

166

Proteomics reveals differences in protein abundance and highly similar antigenic profiles between Besnoitia besnoiti and Besnoitia tarandi.  

PubMed

Besnoitia besnoiti and Besnoitia tarandi are two cyst-forming apicomplexan parasites of the genus Besnoitia. B. besnoiti uses cattle as an intermediate host, in which it causes a disease that progresses in two sequential phases: the acute anasarca stage and the chronic scleroderma stage. Reindeer and caribou act as intermediate hosts for B. tarandi, which causes clinical signs similar to those caused by B. besnoiti. Previous studies demonstrated high molecular similarity, as determined by 18S and ITS-1 RNA sequences, between these Besnoitia spp., and strong serological cross-reactivity between these species has recently been demonstrated. Thus, a difference gel electrophoresis approach and mass spectrometry analysis were used to describe the proteomes and explore differences in protein abundance between B. besnoiti and B. tarandi in tachyzoite extracts. Immunoproteomes were also compared using 2-DE immunoblotting with polyclonal sera from experimentally infected rabbits. From approximately 1400 spots detected in DIGE-gels, 28 and 29 spots were differentially abundant in B. besnoiti and B. tarandi tachyzoites, respectively (± 1.5-fold, p<0.05). Four and 13 spots were exclusively detected in B. besnoiti and B. tarandi, respectively. Of the 32 differentially abundant spots analyzed by MALDI-TOF/MS, 6 up-regulated B. besnoiti proteins (LDH; HSP90; purine nucleoside phosphorylase and 3 hypothetical proteins) and 6 up-regulated B. tarandi proteins (G3PDH; LDH; PDI; mRNA decapping protein and 2 hypothetical proteins) were identified. Interestingly, no specific antigen spots were recognized by sera on any of the Besnoitia species studied and a similar antigen profile has been observed for B. tarandi and B. besnoiti sera when cross reactions were studied. This fact corroborates the difficulty in discerning Besnoitia infections using current serological assays. The present study underscores the importance of sequencing the B. besnoiti genome for species diversity studies of the genus Besnoitia. PMID:25260331

García-Lunar, P; Regidor-Cerrillo, J; Ortega-Mora, L M; Gutiérrez-Expósito, D; Alvarez-García, G

2014-10-15

167

Partial Purification of Integral Membrane Antigenic Proteins from Trypanosoma evansi That Display Immunological Cross-Reactivity with Trypanosoma vivax  

PubMed Central

Trypanosoma evansi and Trypanosoma vivax, which are the major causative agents of animal trypanosomosis in Venezuela, have shown a very high immunological cross-reactivity. Since the production of T. vivax antigens is a limiting factor as this parasite is difficult to propagate in experimental animal models, our goal has been to identify and isolate antigens from T. evansi that cross-react with T. vivax. Here, we used the Venezuelan T. evansi TEVA1 isolate to prepare the total parasite lysate and its corresponding cytosolic and membranous fractions. In order to extract the T. evansi integral membrane proteins, the particulate portion was further extracted first with Triton X-100, and then with sodium dodecyl sulfate. After discarding the cytosolic and Triton X-100 solubilized proteins, we employed sedimentation by centrifugation on linear sucrose gradients to partially purify the sodium dodecyl sulfate-solubilized proteins from the Triton X-100 resistant particulate fraction of T. evansi. We obtained enriched pools containing polypeptide bands with apparent molecular masses of 27?kDa, 31?kDa, and 53?kDa, which were recognized by anti-T. vivax antibodies from experimentally and naturally infected bovines. PMID:24757558

Velásquez, Norma P.; Camargo, Rocío E.; Uzcanga, Graciela L.; Bubis, José

2014-01-01

168

Immunisation with ID83 fusion protein induces antigen-specific cell mediated and humoral immune responses in cattle  

PubMed Central

In this study we have investigated the potential of mycobacterial proteins as candidate subunit vaccines for bovine tuberculosis. In addition, we have explored the use of TLR-ligands as potential adjuvants in cattle. In vitro screening assays with whole blood from M. bovis-infected and BCG-vaccinated cattle demonstrated that fusion protein constructs were most commonly recognised, and the ID83 fusion protein was selected for further immunisation studies. Furthermore, glucopyranosyl lipid A (GLA) and resiquimod (R848), agonists for TLR4 and TLR7/8 respectively, stimulated cytokine production (IL-12, TNF-?, MIP-1? and IL-10) in bovine dendritic cell cultures, and these were formulated as novel oil-in-water emulsions (GLA-SE and R848-SE) for immunisation studies. Immunisation with ID83 in a water-in-oil emulsion adjuvant (ISA70) induced both cell mediated and humoral immune responses, as characterised by antigen-specific IFN-? production, cell proliferation, IgG1 and IgG2 antibody production. In comparison, ID83 immunisation with the novel adjuvants induced weaker (ID83/R848-SE) or no (ID83/GLA-SE) antigen-specific IFN-? production and cell proliferation. However, both did induce ID83-specific antibody production, which was restricted to IgG1 antibody isotype. Overall, these results provide encouraging preliminary data for the further development of ID83 in vaccine strategies for bovine TB. PMID:24012566

Jones, Gareth J.; Steinbach, Sabine; Clifford, Derek; Baldwin, Susan L.; Ireton, Gregory C.; Coler, Rhea N.; Reed, Steven G.; Vordermeier, H. Martin

2013-01-01

169

TAP-independent human histocompatibility complex-Cw1 antigen processing of an HIV envelope protein conserved peptide.  

PubMed

Individuals with nonfunctional transporters associated with antigen processing (TAP) complexes are not particularly susceptible to viral infections or neoplasms. Therefore, their immune system must be reasonably efficient, and the present, though reduced, cytolytic CD8 ?? T subpopulation specific for TAP-independent antigens may be sufficient to establish an immune defense protecting against viral infections in these individuals. The objective of the present study was to identify TAP-independent ligands from HIV gp160 protein. An analysis and comparison of complex human histocompatibility complex (HLA)-bound peptide pools isolated from large quantities of healthy or HIV gp160-expressing human cells was performed using mass spectrometry and bioinformatics tools. A conserved TAP-independent HLA peptide ligand endogenously processed and presented in infected human cells was identified. This ligand originates from the envelope protein bound to the HLA-Cw1 class I molecule with high affinity. It was concluded that HLA class I peptides derived from a large fraction of the N-terminal HIV envelope protein could be presented even in the absence of the TAP complex. PMID:21099670

Lorente, Elena; Infantes, Susana; Barnea, Eilon; Beer, Ilan; García, Ruth; Lasala, Fátima; Jiménez, Mercedes; Admon, Arie; López, Daniel

2011-01-14

170

Ribosomal Protein S6 Interacts with the Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus ?  

PubMed Central

The latency-associated nuclear antigen (LANA) is central to the maintenance of Kaposi's sarcoma-associated herpesvirus (KSHV) and to the survival of KSHV-carrying tumor cells. In an effort to identify interaction partners of LANA, we purified authentic high-molecular-weight complexes of LANA by conventional chromatography followed by immunoprecipitation from the BC-3 cell line. This is the first analysis of LANA-interacting partners that is not based on forced ectopic expression of LANA. Subsequent tandem mass spectrometry (MS/MS) analysis identified many of the known LANA-interacting proteins. We confirmed LANA's interactions with histones. Three classes of proteins survived our stringent four-step purification procedure (size, heparin, anion, and immunoaffinity chromatography): two heat shock proteins (Hsp70 and Hsp96 precursor), signal recognition particle 72 (SRP72), and 10 different ribosomal proteins. These proteins are likely involved in structural interactions within LANA high-molecular-weight complexes. Here, we show that ribosomal protein S6 (RPS6) interacts with LANA. This interaction is mediated by the N-terminal domain of LANA and does not require DNA or RNA. Depletion of RPS6 from primary effusion lymphoma (PEL) cells dramatically decreases the half-life of full-length LANA. The fact that RPS6 has a well-established nuclear function beyond its role in ribosome assembly suggests that RPS6 (and by extension other ribosomal proteins) contributes to the extraordinary stability of LANA. PMID:21734034

Chen, Wuguo; Dittmer, Dirk P.

2011-01-01

171

Protein and antigen diversity in the vesicular fluid of Taenia solium cysticerci dissected from naturally infected pigs.  

PubMed

Cysticercosis caused by Taenia solium is a health threat for humans and pigs living in developing countries, for which there is neither a flawless immunodiagnostic test nor a totally effective vaccine. Suspecting of individual diversity of hosts and parasites as possible sources of the variations of the parasite loads among cysticercotic animals and of the limited success of such immunological applications as well as, we explored and measured both in nine cases of naturally acquired porcine cysticercosis. For this purpose, 2-Dimensional IgG immunoblots were performed by reacting the sera of each cysticercotic pig with the antigens contained in the vesicular fluid (VF) of their own cysticerci. We found an unexpectedly large diversity among the proteins and antigens contained in each of the nine VFs. Also diverse were the serum IgG antibody responses of the nine pigs, as none of their 2D- immunoblot images exhibited the same number of spots and resembled each other in only 6.3% to 65.3% of their features. So large an individual immunological diversity of the cysticercal antigens and of the infected pigs´ IgG antibody response should be taken into account in the design of immunological tools for diagnosis and prevention of cysticercosis and should also be considered as a possibly significant source of diversity in Taenia solium´s infectiveness and pathogenicity. PMID:22110381

Esquivel-Velázquez, Marcela; Larralde, Carlos; Morales, Julio; Ostoa-Saloma, Pedro

2011-01-01

172

Protein and Antigen Diversity in the Vesicular Fluid of Taenia Solium Cysticerci Dissected from Naturally Infected Pigs  

PubMed Central

Cysticercosis caused by Taenia solium is a health threat for humans and pigs living in developing countries, for which there is neither a flawless immunodiagnostic test nor a totally effective vaccine. Suspecting of individual diversity of hosts and parasites as possible sources of the variations of the parasite loads among cysticercotic animals and of the limited success of such immunological applications as well as, we explored and measured both in nine cases of naturally acquired porcine cysticercosis. For this purpose, 2-Dimensional IgG immunoblots were performed by reacting the sera of each cysticercotic pig with the antigens contained in the vesicular fluid (VF) of their own cysticerci. We found an unexpectedly large diversity among the proteins and antigens contained in each of the nine VFs. Also diverse were the serum IgG antibody responses of the nine pigs, as none of their 2D- immunoblot images exhibited the same number of spots and resembled each other in only 6.3% to 65.3% of their features. So large an individual immunological diversity of the cysticercal antigens and of the infected pigs´ IgG antibody response should be taken into account in the design of immunological tools for diagnosis and prevention of cysticercosis and should also be considered as a possibly significant source of diversity in Taenia solium´s infectiveness and pathogenicity. PMID:22110381

Esquivel-Velázquez, Marcela; Larralde, Carlos; Morales, Julio; Ostoa-Saloma, Pedro

2011-01-01

173

Cell surface antigens of human melanocytes and melanoma. Expression of adenosine deaminase binding protein is extinguished with melanocyte transformation  

PubMed Central

It has been proposed that the pathogenesis of melanoma proceeds through multiple stages, ranging from benign proliferation of melanocytic cells to acquisition of the capacity to invade tissues and metastasize. During investigations of cell surface antigens expressed by melanocytes and melanoma, we identified an antigen system that was expressed by cultured normal melanocytes but not by melanoma cell lines. mAbs against this antigen detected a 120-kD cell surface glycoprotein on melanocytes. This molecule had been identified previously as the binding protein for adenosine deaminase (ADAbp). ADAbp was expressed by 51 melanocyte cell lines derived from normal fetal, newborn, and adult skin and adult choroid, but not by 102 melanoma cell lines derived from primary and metastatic lesions. Studies with radiolabeled bovine adenosine deaminase, confirmed that melanocytes expressed binding sites for adenosine deaminase, but no binding sites were detected on cultured melanoma cells. Further studies showed that ADAbp+ melanocytes became ADAbp- upon malignant transformation in vitro. Immunohistochemical studies on a panel of frozen tissues demonstrated reactivity of anti- ADAbp mAbs with epidermal melanocytes and benign junctional nevi, but not with potentially premalignant dysplastic nevi or primary/metastatic melanoma lesions. These studies demonstrate that ADAbp expression is lost with malignant transformation of melanocytes, presumably at an early stage in the transformation process. PMID:2891780

1988-01-01

174

Exemptions from Unrelated Business Tax: Rental Income  

ERIC Educational Resources Information Center

Section 512(b) of the Internal Revenue Code contains several categorical exemptions from the unrelated business tax including rental income. The article covers various problems faced by nonprofit organizations such as parochial schools in leasing or selling property. (LBH)

Reed, George E.

1975-01-01

175

Antibodies to the Plasmodium falciparum antigens circumsporozoite protein, thrombospondin-related adhesive protein, and liver-stage antigen 1 vary by ages of subjects and by season in a highland area of Kenya.  

PubMed

Immunoglobulin G (IgG) antibodies to three vaccine candidate preerythrocytic Plasmodium falciparum antigens were evaluated in children and adults in an epidemic-prone highland area of Kenya during rainy (high-transmission) and dry (low-transmission) seasons. The frequencies and median levels of IgG antibodies to circumsporozoite protein (CSP) and thrombospondin-related adhesive protein (TRAP) were compared to the frequencies and median levels of IgG antibodies to liver-stage antigen 1 (LSA-1) reported previously. The frequencies and median levels of IgG antibodies to CSP and TRAP were similar in children and adults in the rainy season, but they were lower in children than in adults in the dry season. The frequencies and median levels of antibodies to LSA-1 were lower in children than in adults in both the rainy and dry seasons. Antibodies to CSP and LSA-1 were primarily members of the IgG1 and IgG3 subclasses, while antibodies to TRAP were primarily members of the IgG3 and IgG4 subclasses. In a treatment-reinfection study following dry season testing, antibodies to TRAP were associated with a trend toward protection from infection in children (P = 0.051) but not in adults. Antibodies to LSA-1 and CSP did not correlate with protection in children or adults. In this highland area of Kenya with unstable transmission, IgG antibodies to preerythrocytic P. falciparum antigens vary in subjects by age and season, and the protective effects of these antibodies against infection may be different in adults and children. PMID:12874308

John, Chandy C; Zickafoose, Joseph S; Sumba, P Odada; King, Christopher L; Kazura, James W

2003-08-01

176

The role of protein solubilization in antigen removal from xenogeneic tissue for heart valve tissue engineering  

E-print Network

matrix (ECM), selection of an appropriate tissue-engineered heart valve scaffold material is crucial July 2011 Available online 31 July 2011 Keywords: Antigen removal Xenogeneic scaffold Decellularization Extracellular matrix Heart valve tissue engineering a b s t r a c t Decellularization techniques have been

Athanasiou, Kyriacos

177

Prediction of Major Histocompatibility Complex Binding Regions of Protein Antigens by Sequence Pattern Analysis  

Microsoft Academic Search

We have previously experimentally analyzed the structural requirements for interaction between peptide antigens and mouse major histocompatibility complex (MHC) molecules of the d haplotype. We describe here two procedures devised to predict specifically the capacity of peptide molecules to interact with these MHC class II molecules (IAd and IEd). The accuracy of these procedures has been tested on a large

Alessandro Sette; Soren Buus; Ettore Appella; John A. Smith; Robert Chesnut; Craig Miles; Sonia M. Colon; Howard M. Grey

1989-01-01

178

Quantitation of Serum Prostate-specific Membrane Antigen by a Novel Protein Biochip Immunoassay Discriminates Benign from Malignant Prostate Disease1  

Microsoft Academic Search

The lack of a sensitive immunoassay for quantitating serum prostate- specific membrane antigen (PSMA) hinders its clinical utility as a diagnostic\\/ prognostic biomarker. An innovative protein biochip immunoassay was used to quantitate and compare serum PSMA levels in healthy men and patients with either benign or malignant prostate disease. PSMA was captured from serum by anti-PSMA antibody bound to ProteinChip

Zhen Xiao; Bao-Ling Adam; Lisa H. Cazares; John W. Davis; Paul F. Schellhammer; Enrique A. Dalmasso; George L. Wright

179

Determination of the genus-specific antigens in outer membrane proteins from the strains of Leptospira interrogans and Leptospira biflexa with different virulence  

Microsoft Academic Search

Objective: To determine the existence of genus-specific antigens in outer membrane proteins (OMPs) ofleptospira with different virulence. Methods: Microscope agglutination test (MAT) was applied to detect the agglutination between commercial\\u000a rabbit antiserum against leptospiral genus-specific TR\\/Patoc I antigen and 17 strains ofLeptospira interrongans belonging to 15 serogroups and 2 strains ofLeptospira biflexa belonging to 2 serogroups. The outer envelopes (OEs)

Yi-hui Luo; Jie Yan; Ya-fei Mao; Shu-ping Li

2004-01-01

180

Proteolytic Activity of Prostate-Specific Antigen (PSA) towards Protein Substrates and Effect of Peptides Stimulating PSA Activity  

PubMed Central

Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3) exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA. PMID:25237904

Mattsson, Johanna M.; Ravela, Suvi; Hekim, Can; Jonsson, Magnus; Malm, Johan; Närvänen, Ale; Stenman, Ulf-Håkan; Koistinen, Hannu

2014-01-01

181

Inferring Epitopes of a Polymorphic Antigen Amidst Broadly Cross-Reactive Antibodies Using Protein Microarrays: A Study of OspC Proteins of Borrelia burgdorferi  

PubMed Central

Epitope mapping studies aim to identify the binding sites of antibody-antigen interactions to enhance the development of vaccines, diagnostics and immunotherapeutic compounds. However, mapping is a laborious process employing time- and resource-consuming ‘wet bench’ techniques or epitope prediction software that are still in their infancy. For polymorphic antigens, another challenge is characterizing cross-reactivity between epitopes, teasing out distinctions between broadly cross-reactive responses, limited cross-reactions among variants and the truly type-specific responses. A refined understanding of cross-reactive antibody binding could guide the selection of the most informative subsets of variants for diagnostics and multivalent subunit vaccines. We explored the antibody binding reactivity of sera from human patients and Peromyscus leucopus rodents infected with Borrelia burgdorferi to the polymorphic outer surface protein C (OspC), an attractive candidate antigen for vaccine and improved diagnostics for Lyme disease. We constructed a protein microarray displaying 23 natural variants of OspC and quantified the degree of cross-reactive antibody binding between all pairs of variants, using Pearson correlation calculated on the reactivity values using three independent transforms of the raw data: (1) logarithmic, (2) rank, and (3) binary indicators. We observed that the global amino acid sequence identity between OspC pairs was a poor predictor of cross-reactive antibody binding. Then we asked if specific regions of the protein would better explain the observed cross-reactive binding and performed in silico screening of the linear sequence and 3-dimensional structure of OspC. This analysis pointed to residues 179 through 188 the fifth C-terminal helix of the structure as a major determinant of type-specific cross-reactive antibody binding. We developed bioinformatics methods to systematically analyze the relationship between local sequence/structure variation and cross-reactive antibody binding patterns among variants of a polymorphic antigen, and this method can be applied to other polymorphic antigens for which immune response data is available for multiple variants. PMID:23826301

Zeller, Michael; Barbour, Alan G.

2013-01-01

182

Inferring epitopes of a polymorphic antigen amidst broadly cross-reactive antibodies using protein microarrays: a study of OspC proteins of Borrelia burgdorferi.  

PubMed

Epitope mapping studies aim to identify the binding sites of antibody-antigen interactions to enhance the development of vaccines, diagnostics and immunotherapeutic compounds. However, mapping is a laborious process employing time- and resource-consuming 'wet bench' techniques or epitope prediction software that are still in their infancy. For polymorphic antigens, another challenge is characterizing cross-reactivity between epitopes, teasing out distinctions between broadly cross-reactive responses, limited cross-reactions among variants and the truly type-specific responses. A refined understanding of cross-reactive antibody binding could guide the selection of the most informative subsets of variants for diagnostics and multivalent subunit vaccines. We explored the antibody binding reactivity of sera from human patients and Peromyscus leucopus rodents infected with Borrelia burgdorferi to the polymorphic outer surface protein C (OspC), an attractive candidate antigen for vaccine and improved diagnostics for Lyme disease. We constructed a protein microarray displaying 23 natural variants of OspC and quantified the degree of cross-reactive antibody binding between all pairs of variants, using Pearson correlation calculated on the reactivity values using three independent transforms of the raw data: (1) logarithmic, (2) rank, and (3) binary indicators. We observed that the global amino acid sequence identity between OspC pairs was a poor predictor of cross-reactive antibody binding. Then we asked if specific regions of the protein would better explain the observed cross-reactive binding and performed in silico screening of the linear sequence and 3-dimensional structure of OspC. This analysis pointed to residues 179 through 188 the fifth C-terminal helix of the structure as a major determinant of type-specific cross-reactive antibody binding. We developed bioinformatics methods to systematically analyze the relationship between local sequence/structure variation and cross-reactive antibody binding patterns among variants of a polymorphic antigen, and this method can be applied to other polymorphic antigens for which immune response data is available for multiple variants. PMID:23826301

Baum, Elisabeth; Randall, Arlo Z; Zeller, Michael; Barbour, Alan G

2013-01-01

183

Immunization with a Combination of Integral Chlamydial Antigens and a Defined Secreted Protein Induces Robust Immunity against Genital Chlamydial Challenge ?  

PubMed Central

We have previously demonstrated the efficacy of recombinant chlamydial protease-like activity factor (rCPAF; a secreted chlamydial protein) in inducing antigen-specific CD4+ T cell/gamma interferon (IFN-?)-mediated but not antibody-mediated chlamydial clearance and reduction of upper genital tract (UGT) pathological sequelae. Since chlamydial integral antigens may induce neutralizing antibody protection, we further evaluated induction of protective immunity using a combination of rCPAF and UV-inactivated chlamydial elementary bodies (UV-EB) against vaginal chlamydial challenge in comparison to immunization with the individual components or live EB. The rCPAF-UV-EB immunization induced a significantly enhanced anti-UV-EB cellular and antibody response and a reduced anti-CPAF cellular and antibody response, compared to immunization with the respective individual components. Moreover, vaccination with UV-EB and rCPAF-UV-EB induced serum antibodies that neutralized chlamydial infectivity. The rCPAF-UV-EB immunization resulted in a significant reduction of vaginal chlamydial shedding and induced earlier bacterial clearance than vaccination of mice with the individual components. Importantly, the UGT sequelae were significantly reduced in mice immunized with rCPAF or rCPAF-UV-EB, but not in those immunized with UV-EB alone, and approached the levels of protection induced by live EB. These results collectively suggest that a combination of neutralizing antibodies induced by integral chlamydial antigens and cell-mediated responses induced by secreted proteins such as CPAF induces optimal protective immunity against genital chlamydial infections. PMID:20605976

Li, Weidang; Murthy, Ashlesh K.; Guentzel, M. Neal; Chambers, James P.; Forsthuber, Thomas G.; Seshu, J.; Zhong, Guangming; Arulanandam, Bernard P.

2010-01-01

184

Proteasomal cleavage site prediction of protein antigen using BP neural network based on a new set of amino acid descriptor.  

PubMed

The accurate identification of cytotoxic T lymphocyte epitopes is becoming increasingly important in peptide vaccine design. The ubiquitin-proteasome system plays a key role in processing and presenting major histocompatibility complex class I restricted epitopes by degrading the antigenic protein. To enhance the specificity and efficiency of epitope prediction and identification, the recognition mode between the ubiquitin-proteasome complex and the protein antigen must be considered. Hence, a model that accurately predicts proteasomal cleavage must be established. This study proposes a new set of parameters to characterize the cleavage window and uses a backpropagation neural network algorithm to build a model that accurately predicts proteasomal cleavage. The accuracy of the prediction model, which depends on the window sizes of the cleavage, reaches 95.454% for the N-terminus and 95.011% for the C-terminus. The results show that the identification of proteasomal cleavage sites depends on the sequence next to it and that the prediction performance of the C-terminus is better than that of the N-terminus on average. Thus, models based on the properties of amino acids can be highly reliable and reflect the structural features of interactions between proteasomes and peptide sequences. PMID:23584554

Wang, Yuanqiang; Lin, Yong; Shu, Mao; Wang, Rui; Hu, Yong; Lin, Zhihua

2013-08-01

185

Analysis of Immune Responses Directed toward a Recombinant Early Secretory Antigenic Target Six-Kilodalton Protein-Culture Filtrate Protein 10 Fusion Protein in Mycobacterium bovis-Infected Cattle  

PubMed Central

Cell-mediated immune responses are critical for protective immunity to mycobacterial infections. Recent progress in defining mycobacterial antigens has determined that region of difference 1 (RD1) gene products induce strong T-cell responses, particularly the early secretory antigenic target 6-kDa (ESAT-6) protein and culture filtrate protein 10 (CFP10). However, comprehensive analysis of the immune response towards these antigens is incompletely characterized. To evaluate recall responses to ESAT-6 and CFP10, peripheral blood mononuclear cells from M. bovis-infected cattle were stimulated in vitro with a recombinant ESAT-6 (rESAT-6)-CFP10 fusion protein and compared to responses induced by M. bovis-derived purified protein derivative. Following antigenic stimulation, activation marker expression was evaluated. Significant proliferative responses (P < 0.05) were evident in CD4+, CD8+, immunoglobulin M-positive, and CD172a+ cell fractions after 6 days of culture. Expression of CD25 and CD26 was increased (P < 0.05) on CD4+, CD8+, and ?? T-cell-receptor-positive cells. CD4+ and CD8+ cells also exhibited significant changes (P < 0.05) in expression of CD45 isoforms. Using a flow cytometry-based proliferation assay, it was determined that CD45R expression is downregulated (P < 0.05) and that CD45RO expression is upregulated (P < 0.05) on proliferating (i.e., activated) CD4+ cells. Collectively, data indicate that recall immune responses directed toward the rESAT-6-CFP10 fusion protein or purified protein derivative are comparable and that recall to mycobacterial antigens correlates with a CD45RO+ phenotype. PMID:16177342

Maue, Alexander C.; Waters, W. Ray; Davis, William C.; Palmer, Mitchell V.; Minion, F. Chris; Estes, D. Mark

2005-01-01

186

Characterization of Protective Epitopes in a Highly Conserved Plasmodium falciparum Antigenic Protein Containing Repeats of Acidic and Basic Residues  

PubMed Central

The delineation of putatively protective and immunogenic epitopes in vaccine candidate proteins constitutes a major research effort towards the development of an effective malaria vaccine. By virtue of its role in the formation of the immune clusters of merozoites, its location on the surface of merozoites, and its highly conserved nature both at the nucleotide sequence level and the amino acid sequence level, the antigen which contains repeats of acidic and basic residues (ABRA) of the human malaria parasite Plasmodium falciparum represents such an antigen. Based upon the predicted amino acid sequence of ABRA, we synthesized eight peptides, with six of these (AB-1 to AB-6) ranging from 12 to 18 residues covering the most hydrophilic regions of the protein, and two more peptides (AB-7 and AB-8) representing its repetitive sequences. We found that all eight constructs bound an appreciable amount of antibody in sera from a large proportion of P. falciparum malaria patients; two of these peptides (AB-1 and AB-3) also elicited a strong proliferation response in peripheral blood mononuclear cells from all 11 human subjects recovering from malaria. When used as carrier-free immunogens, six peptides induced a strong, boostable, immunoglobulin G-type antibody response in rabbits, indicating the presence of both B-cell determinants and T-helper-cell epitopes in these six constructs. These antibodies specifically cross-reacted with the parasite protein(s) in an immunoblot and in an immunofluorescence assay. In another immunoblot, rabbit antipeptide sera also recognized recombinant fragments of ABRA expressed in bacteria. More significantly, rabbit antibodies against two constructs (AB-1 and AB-5) inhibited the merozoite reinvasion of human erythrocytes in vitro up to ?90%. These results favor further studies so as to determine possible inclusion of these two constructs in a multicomponent subunit vaccine against asexual blood stages of P. falciparum. PMID:9596765

Sharma, Pawan; Kumar, Anil; Singh, Balwan; Bharadwaj, Ashima; Sailaja, V. Naga; Adak, T.; Kushwaha, Ashima; Malhotra, Pawan; Chauhan, V. S.

1998-01-01

187

Identification and Characterization of a Trypanosoma congolense 46 kDa Protein as a Candidate Serodiagnostic Antigen  

PubMed Central

ABSTRACT Trypanosoma congolense is a major livestock pathogen in Africa, causing large economic losses with serious effects on animal health. Reliable serodiagnostic tests are therefore urgently needed to control T. congolense infection. In this study, we have identified one T. congolense protein as a new candidate serodiagnostic antigen. The 46.4 kDa protein (TcP46, Gene ID: TcIL3000.0.25950) is expressed 5.36 times higher in metacyclic forms than epimastigote forms. The complete nucleotide sequences of TcP46 contained an open reading frame of 1,218 bp. Southern blot analysis indicated that at least two copies of the TcP46 gene were tandemly-arranged in the T. congolense genome. The recombinant TcP46 (rTcP46) was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Western blot analysis and confocal laser scanning microscopy revealed that the native TcP46 protein is expressed in the cytoplasm during all life-cycle stages of the parasite. Moreover, an enzyme-linked immunosorbent assay (ELISA) based on rTcP46 detected the specific antibodies as early as 8 days post-infection from mice experimentally infected with T. congolense. No cross-reactivity was observed in the rTcP46-based ELISA against serum samples from cattle experimentally infected with Babesia bigemina, B. bovis and Anaplasma marginale. These results suggest that rTcP46 could be used as a serodiagnostic antigen for T. congolense infection. PMID:24492330

ZHOU, Mo; SUGANUMA, Keisuke; RUTTAYAPORN, Ngasaman; NGUYEN, Thu-Thuy; YAMASAKI, Shino; IGARASHI, Ikuo; KAWAZU, Shin-ichiro; SUZUKI, Yasuhiko; INOUE, Noboru

2014-01-01

188

Fournier's gangrene after unrelated cord blood stem cell transplantation.  

PubMed

A 16-year-old boy with refractory acute myelogenous leukemia developed Fournier's gangrene as an early complication after two-antigen HLA-mismatched unrelated cord blood stem cell transplantation. On day 25 after the transplantation, he noted abrupt onset of penile swelling with miction pain. The penile inflammation rapidly extended posteriorly to involve the scrotum and perianal tissues, inferiorly to involve the thighs, and superiorly up the lower abdominal region within the next 36 h, and he died from sepsis on day 27. Fournier's gangrene presenting as a genitoperineal necrotizing fasciitis should be considered as a potential complication in umbilical-cord blood recipients in the cytopenic post-transplant phase. PMID:12373358

Yoshida, C; Kojima, K; Shinagawa, K; Hashimoto, D; Asakura, S; Takata, S; Tanimoto, M

2002-09-01

189

Non-major histocompatibility complex-restricted cytotoxic activity of blood mononuclear cells stimulated with secreted mycobacterial proteins and other mycobacterial antigens.  

PubMed Central

Several observations indicate that non-major histocompatibility complex (MHC)-restricted cytotoxicity, mediated for example by natural killer cells and lymphokine-activated killer cells, may serve as an important antimicrobial defense mechanism. The purpose of the present study was to investigate the influences of different mycobacterial antigens on non-MHC-restricted cytotoxicity and further to investigate the ways by which various lymphocyte subpopulations contribute to the development of this cytotoxicity. Non-MHC-restricted cytotoxicity was induced following stimulation of mononuclear cells with tuberculin purified protein derivative, Mycobacterium bovis bacillus Calmette-Guérin (BCG), short- and long-term culture filtrates of virulent Mycobacterium tuberculosis H37Rv, and 30-31-kDa secreted mycobacterial protein. These antigens also induced proliferation and production of gamma interferon. The CD4+ cells proliferated and expressed interleukin-2 receptors following stimulation with mycobacterial antigens. Depletion studies after antigen stimulation showed that the cytotoxic effector cells were CD16+ CD56+ and CD4-; the CD4+ cells alone did not mediate non-MHC-restricted cytotoxicity. To evaluate the influence of CD4+ cells on the development of non-MHC-restricted cytotoxicity, blood mononuclear cells were depleted of CD4+ cells before antigen stimulation. When mononuclear cells were incubated with purified protein derivative or short-term culture filtrate in the absence of CD4+ cells, cytotoxic activity was reduced. This reduction was abolished by interleukin-2 but not by gamma interferon. We conclude that several mycobacterial antigens are able to induce non-MHC-restricted cytotoxicity. This study indicates that non-MHC-restricted cytotoxicity following stimulation with mycobacterial antigens is induced by cytokines released by antigen-specific activated CD4+ cells. PMID:7960109

Ravn, P; Pedersen, B K

1994-01-01

190

Evidence for multiple CD95-CD95 ligand interactions in anteriorchamber-associated immune deviation induced by soluble protein antigen.  

PubMed

We have investigated whether CD95-CD95 ligand interactions are important in anterior chamber-associated immune deviation (ACAID) induced by soluble protein antigen, and if so, to identify the participating cells on which these molecules are expressed. Peritoneal exudate cells as antigen-presenting cells (APC) obtained from B6.lpr/lpr, B6.gld/gld and C57BL/6 mice were cultured with ovalbumin (OVA) and transforming growth factor-beta2 (TGF-beta2) overnight, then injected intravenously into C57BL/6 or B6.lpr/lpr recipients. Some B6.lpr/lpr mice were reconstituted with naive T cells from wild-type C57BL/6 donors. In other experiments, B6. lpr/lpr and B6.gld/gld mice received an anterior chamber injection of OVA followed 7 days later by subcutaneous immunization with OVA plus adjuvant. Delayed hypersensitivity (DH) was assessed with an ear swelling assay. T cells activated in vitro with OVA-pulsed, TGF-beta-treated APC were tested in vivo for their capacity to suppress DH expression in a local adoptive transfer assay. The results indicate that when ACAID was induced by in-vitro generated ACAID-inducing cells, the APC expressed CD95L, and recipient T cells expressed CD95. The capacity of in vitro generated regulatory T cells to suppress DH expression to OVA in vivo was not governed by CD95-CD95L interactions. When OVA was injected into the anterior chamber of naive mice, CD95 expression was required for ACAID induction, although ACAID was readily induced in CD95L-deficient mice. We conclude that CD95-CD95L interactions are required in ACAID for the initial stage of APC presentation of eye-derived antigens to T cells, and that CD95-CD95L interactions participate at one or more additional step in the process by which ACAID is induced by soluble protein antigens. PMID:10712676

Kezuka, T; Streilein, J W

2000-03-01

191

Comparison of antibodies raised against the peptide 10–24 of chicken riboflavin carrier protein (cRCP) by classical and multiple antigen peptide (MAP) approaches  

Microsoft Academic Search

Riboflavin carrier protein is an essential protein required for the growth and development of the embryo and hence for the maintenance of pregnancy. Our efforts to delineate the antigenic determinants of chicken riboflavin carrier protein (cRCP) resulted in the identification of a bioneutralization epitope in the region 10–24 of cRCP. The present work compares the properties of the antibodies raised

S. D. Mahale; J. Pereira; U. Natraj; K. S. N. Iyer

1996-01-01

192

Expression and refolding of truncated recombinant major outer membrane protein antigen (r56) of Orientia tsutsugamushi and its use in enzyme-linked immunosorbent assays.  

PubMed

The variable 56-kDa major outer membrane protein of Orientia tsutsugamushi is the immunodominant antigen in human scrub typhus infections. The gene encoding this protein from Karp strain was cloned into the expression vector pET11a. The recombinant protein (r56) was expressed as a truncated nonfusion protein (amino acids 80 to 456 of the open reading frame) which formed an inclusion body when expressed in Escherichia coli BL21. Refolded r56 was purified and compared to purified whole-cell lysate of the Karp strain of O. tsutsugamushi by immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for reactivity with rabbit sera prepared against eight antigenic prototypes of O. tsutsugamushi as well as several other species of Rickettsiales and nonrickettsial antigens. Refolded r56 exhibited broad reactivity with the rabbit antisera against the Orientia prototypes, and the ELISA reactions with the r56 and Karp whole-cell lysate antigens correlated well (r = 0.81, n = 22, sensitivity compared to that of standard ELISA of 91%). Refolded r56 did not react with most antisera against other rickettsial species or control antigens (specificity = 92%, n = 13) using a positive cutoff value determined with eight uninfected rabbit sera. Refolded r56 was evaluated further by ELISA, using 128 sera obtained from patients with suspected scrub typhus from Korat, Thailand, and 74 serum specimens from healthy Thai soldiers. By using the indirect immunoperoxidase assay as the reference assay, the recombinant antigen exhibited a sensitivity and specificity of 93% or greater for detection of both IgG and IgM in the ELISA at 1:400 serum dilution. These results strongly suggest that purified r56 is a suitable candidate for replacing the density gradient-purified, rickettsia-derived, whole-cell antigen currently used in the commercial dipstick assay available in the United States. PMID:9665960

Ching, W M; Wang, H; Eamsila, C; Kelly, D J; Dasch, G A

1998-07-01

193

Loss of T Cell Antigen Recognition Arising from Changes in Peptide and Major Histocompatibility Complex Protein Flexibility: Implications for Vaccine Design  

SciTech Connect

Modification of the primary anchor positions of antigenic peptides to improve binding to major histocompatibility complex (MHC) proteins is a commonly used strategy for engineering peptide-based vaccine candidates. However, such peptide modifications do not always improve antigenicity, complicating efforts to design effective vaccines for cancer and infectious disease. Here we investigated the MART-1{sub 27-35} tumor antigen, for which anchor modification (replacement of the position two alanine with leucine) dramatically reduces or ablates antigenicity with a wide range of T cell clones despite significantly improving peptide binding to MHC. We found that anchor modification in the MART-1{sub 27-35} antigen enhances the flexibility of both the peptide and the HLA-A*0201 molecule. Although the resulting entropic effects contribute to the improved binding of the peptide to MHC, they also negatively impact T cell receptor binding to the peptide {center_dot} MHC complex. These results help explain how the 'anchor-fixing' strategy fails to improve antigenicity in this case, and more generally, may be relevant for understanding the high specificity characteristic of the T cell repertoire. In addition to impacting vaccine design, modulation of peptide and MHC flexibility through changes to antigenic peptides may present an evolutionary strategy for the escape of pathogens from immune destruction.

Insaidoo, Francis K.; Borbulevych, Oleg Y.; Hossain, Moushumi; Santhanagopolan, Sujatha M.; Baxter, Tiffany K.; Baker, Brian M. (Notre)

2012-05-08

194

APPLICATION OF A NOVEL RADIOIMMUNOASSAY TO IDENTIFY BACULOVIRUS STRUCTURAL PROTEINS THAT SHARE INTERSPECIES ANTIGENIC DETERMINANTS  

EPA Science Inventory

Viral proteins were electrophoresed in polyacrylamide gels, transferred to nitrocellulose, and incubated with viral antisera, and the antibodies were detected with 125 I-labeled Staphylococcus aureus protein A. Antisera were prepared to purified and intact virions from five bacul...

195

Defining a protective epitope on factor H binding protein, a key meningococcal virulence factor and vaccine antigen  

PubMed Central

Mapping of epitopes recognized by functional monoclonal antibodies (mAbs) is essential for understanding the nature of immune responses and designing improved vaccines, therapeutics, and diagnostics. In recent years, identification of B-cell epitopes targeted by neutralizing antibodies has facilitated the design of peptide-based vaccines against highly variable pathogens like HIV, respiratory syncytial virus, and Helicobacter pylori; however, none of these products has yet progressed into clinical stages. Linear epitopes identified by conventional mapping techniques only partially reflect the immunogenic properties of the epitope in its natural conformation, thus limiting the success of this approach. To investigate antigen–antibody interactions and assess the potential of the most common epitope mapping techniques, we generated a series of mAbs against factor H binding protein (fHbp), a key virulence factor and vaccine antigen of Neisseria meningitidis. The interaction of fHbp with the bactericidal mAb 12C1 was studied by various epitope mapping methods. Although a 12-residue epitope in the C terminus of fHbp was identified by both Peptide Scanning and Phage Display Library screening, other approaches, such as hydrogen/deuterium exchange mass spectrometry (MS) and X-ray crystallography, showed that mAb 12C1 occupies an area of ?1,000 Å2 on fHbp, including >20 fHbp residues distributed on both N- and C-terminal domains. Collectively, these data show that linear epitope mapping techniques provide useful but incomplete descriptions of B-cell epitopes, indicating that increased efforts to fully characterize antigen–antibody interfaces are required to understand and design effective immunogens. PMID:23396847

Malito, Enrico; Faleri, Agnese; Lo Surdo, Paola; Veggi, Daniele; Maruggi, Giulietta; Grassi, Eva; Cartocci, Elena; Bertoldi, Isabella; Genovese, Alessia; Santini, Laura; Romagnoli, Giacomo; Borgogni, Erica; Brier, Sébastien; Lo Passo, Carla; Domina, Maria; Castellino, Flora; Felici, Franco; van der Veen, Stijn; Johnson, Steven; Lea, Susan M.; Tang, Christoph M.; Pizza, Mariagrazia; Savino, Silvana; Norais, Nathalie; Rappuoli, Rino; Bottomley, Matthew J.; Masignani, Vega

2013-01-01

196

The ubiquitin-like protein, ISG15, is a novel tumor-associated antigen for cancer immunotherapy.  

PubMed

The recent announcement of the first FDA-approved therapeutic vaccine for prostate cancer, Sipuleucel-T, is a watershed moment for the field of tumor immunotherapy. However, while Sipuleucel-T provides a powerful tool to clinicians for the most prevalent form of cancer in men, there remains an unmet need for a similar therapeutic strategy against breast cancer, the most prevalent cancer in women. While current breast cancer vaccines in development target several antigens, the most prevalent is the tumor-associated antigen, HER2. Initial results with HER2 vaccines appear promising in terms of efficacy; however, the lack of HER2 overexpression by a majority of breast tumors and the safety concerns associated with current HER2-targeted immunotherapy suggest that additional therapeutic strategies would be beneficial. Recently, several studies have identified ISG15 as a molecule highly expressed in numerous malignancies. ISG15 is a small ubiquitin-like protein regulated by type-I interferon and classically associated with viral defense. Elevated ISG15 expression in breast cancer is especially well documented and is independent of HER2, progesterone receptor, and estrogen receptor status. Additionally, high ISG15 expression in breast cancer correlates with an unfavorable prognosis and poor responses to traditional treatment strategies such as chemotherapy and radiation. To overcome these challenges, we employ a novel strategy to specifically target tumor-associated ISG15 expression with immunotherapy. We demonstrate that vaccination against ISG15 results in significant CD8-mediated reductions in both primary and metastatic mammary tumor burden. These results validate ISG15 as a tumor-associated antigen for cancer immunotherapy. PMID:22057675

Wood, Laurence M; Pan, Zhen-Kun; Seavey, Matthew M; Muthukumaran, Geetha; Paterson, Yvonne

2012-05-01

197

Extending the honey bee venome with the antimicrobial peptide apidaecin and a protein resembling wasp antigen 5.  

PubMed

Honey bee venom is a complex mixture of toxic proteins and peptides. In the present study we tried to extend our knowledge of the venom composition using two different approaches. First, worker venom was analysed by liquid chromatography-mass spectrometry and this revealed the antimicrobial peptide apidaecin for the first time in such samples. Its expression in the venom gland was confirmed by reverse transcription PCR and by a peptidomic analysis of the venom apparatus tissue. Second, genome mining revealed a list of proteins with resemblance to known insect allergens or venom toxins, one of which showed homology to proteins of the antigen 5 (Ag5)/Sol i 3 cluster. It was demonstrated that the honey bee Ag5-like gene is expressed by venom gland tissue of winter bees but not of summer bees. Besides this seasonal variation, it shows an interesting spatial expression pattern with additional production in the hypopharyngeal glands, the brains and the midgut. Finally, our immunoblot study revealed that both synthetic apidaecin and the Ag5-like recombinant from bacteria evoke no humoral activity in beekeepers. Also, no IgG4-based cross-reactivity was detected between the honey bee Ag5-like protein and its yellow jacket paralogue Ves v 5. PMID:23350689

Van Vaerenbergh, M; Cardoen, D; Formesyn, E M; Brunain, M; Van Driessche, G; Blank, S; Spillner, E; Verleyen, P; Wenseleers, T; Schoofs, L; Devreese, B; de Graaf, D C

2013-04-01

198

Expression of foreign antigens on the surface of Escherichia coli by fusion to the outer membrane protein traT.  

PubMed

The traT gene is one of the F factor transfer genes and encodes an outer membrane protein which is involved in interactions between an Escherichia coli and its surroundings. This protein was altered so as to permit the expression of foreign proteins on the outer membrane of E. coli in this study. A 729-bp DNA fragment, including the leader and entire structural gene sequence of traT, was amplified and obtained by PCR. This sequence was then subcloned downstream of the tac promoter of pDR540, resulting in a TraT expression vector, pT2. Here, we report that the expression of TraT protein, fused either with a partial pre-S antigen of hepatitis B virus (60 and 98 amino acids, respectively) or with the snake venom rhodostomin (72 amino acids), was successfully achieved on the outer membrane of E. coli, using the pT2 plasmid. This result was demonstrated using dot blot and immunofluorescence analysis. This finding supports the notion that the pT2 plasmid can be used as an E. coli display system. This system can detect a foreign peptide of about 100 amino acid residues in length on the bacterial surface. PMID:9933744

Chang, H J; Sheu, S Y; Lo, S J

1999-01-01

199

Antigenic properties of a transport-competent influenza HA/HIV Env chimeric protein  

SciTech Connect

The transmembrane subunit (gp41) of the HIV Env glycoprotein contains conserved neutralizing epitopes which are not well-exposed in wild-type HIV Env proteins. To enhance the exposure of these epitopes, a chimeric protein, HA/gp41, in which the gp41 of HIV-1 89.6 envelope protein was fused to the C-terminus of the HA1 subunit of the influenza HA protein, was constructed. Characterization of protein expression showed that the HA/gp41 chimeric proteins were expressed on cell surfaces and formed trimeric oligomers, as found in the HIV Env as well as influenza HA proteins. In addition, the HA/gp41 chimeric protein expressed on the cell surface can also be cleaved into 2 subunits by trypsin treatment, similar to the influenza HA. Moreover, the HA/gp41 chimeric protein was found to maintain a pre-fusion conformation. Interestingly, the HA/gp41 chimeric proteins on cell surfaces exhibited increased reactivity to monoclonal antibodies against the HIV Env gp41 subunit compared with the HIV-1 envelope protein, including the two broadly neutralizing monoclonal antibodies 2F5 and 4E10. Immunization of mice with a DNA vaccine expressing the HA/gp41 chimeric protein induced antibodies against the HIV gp41 protein and these antibodies exhibit neutralizing activity against infection by an HIV SF162 pseudovirus. These results demonstrate that the construction of such chimeric proteins can provide enhanced exposure of conserved epitopes in the HIV Env gp41 and may represent a novel vaccine design strategy for inducing broadly neutralizing antibodies against HIV.

Ye Ling [Department of Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, 1510 Clifton Road, Room 3086 Rollins Research Center, Atlanta, GA 30322 (United States); Sun Yuliang [Department of Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, 1510 Clifton Road, Room 3086 Rollins Research Center, Atlanta, GA 30322 (United States); Lin Jianguo [Department of Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, 1510 Clifton Road, Room 3086 Rollins Research Center, Atlanta, GA 30322 (United States); Bu Zhigao [Harbin Veterinary Research Institute, CAAS, 427 Maduan Street, Nangang District, Harbin 150001 (China); Wu Qingyang [Department of Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, 1510 Clifton Road, Room 3086 Rollins Research Center, Atlanta, GA 30322 (United States); Jiang, Shibo [New York Blood Center, 310 E. 67 Street, New York, NY 10021 (United States); Steinhauer, David A. [Department of Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, 1510 Clifton Road, Room 3086 Rollins Research Center, Atlanta, GA 30322 (United States); Compans, Richard W. [Department of Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, 1510 Clifton Road, Room 3086 Rollins Research Center, Atlanta, GA 30322 (United States); Yang Chinglai [Department of Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, 1510 Clifton Road, Room 3086 Rollins Research Center, Atlanta, GA 30322 (United States)]. E-mail: chyang@emory.edu

2006-08-15

200

SEPPA 2.0--more refined server to predict spatial epitope considering species of immune host and subcellular localization of protein antigen.  

PubMed

Spatial Epitope Prediction server for Protein Antigens (SEPPA) has received lots of feedback since being published in 2009. In this improved version, relative ASA preference of unit patch and consolidated amino acid index were added as further classification parameters in addition to unit-triangle propensity and clustering coefficient which were previously reported. Then logistic regression model was adopted instead of the previous simple additive one. Most importantly, subcellular localization of protein antigen and species of immune host were fully taken account to improve prediction. The result shows that AUC of 0.745 (5-fold cross-validation) is almost the baseline performance with no differentiation like all the other tools. Specifying subcellular localization of protein antigen and species of immune host will generally push the AUC up. Secretory protein immunized to mouse can push AUC to 0.823. In this version, the false positive rate has been largely decreased as well. As the first method which has considered the subcellular localization of protein antigen and species of immune host, SEPPA 2.0 shows obvious advantages over the other popular servers like SEPPA, PEPITO, DiscoTope-2, B-pred, Bpredictor and Epitopia in supporting more specific biological needs. SEPPA 2.0 can be accessed at http://badd.tongji.edu.cn/seppa/. Batch query is also supported. PMID:24838566

Qi, Tao; Qiu, Tianyi; Zhang, Qingchen; Tang, Kailin; Fan, Yangyang; Qiu, Jingxuan; Wu, Dingfeng; Zhang, Wei; Chen, Yanan; Gao, Jun; Zhu, Ruixin; Cao, Zhiwei

2014-07-01

201

Antigenic Properties and Diagnostic Potential of Baculovirus-Expressed Infectious Bursal Disease Virus Proteins VPX and VP3  

PubMed Central

The routine technique for detecting antibodies specific to infectious bursal disease virus (IBDV) is a serological evaluation by enzyme-linked immunosorbent assay (ELISA) with preparations of whole virions as the antigens. To avoid using complete virus in the standard technique, we have developed two new antigens through the expression of the VPX and VP3 genes in insect cells. VPX and especially VP3 were expressed at high levels in insect cells and simple to purify. The immunogenicity of both proteins was similar to that of the native virus. VPX was able to elicit neutralizing antibodies but VP3 was not. Purified VPX and VP3 were tested in an indirect ELISA with more than 300 chicken sera. There was an excellent correlation between the results of the ELISA using VPX and those of the two commercial kits. VP3 did not perform as well as VPX, and the linear correlation was significantly lower. A comparison with the standard reference technique, seroneutralization, showed that the indirect ELISA was more sensitive. Therefore, VPX-based ELISA is a good alternative to conventional ELISAs that use whole virions. PMID:10882666

Martínez-Torrecuadrada, Jorge L.; Lázaro, Beatriz; Rodriguez, José F.; Casal, J. Ignacio

2000-01-01

202

Antigenic properties and diagnostic potential of baculovirus-expressed infectious bursal disease virus proteins VPX and VP3.  

PubMed

The routine technique for detecting antibodies specific to infectious bursal disease virus (IBDV) is a serological evaluation by enzyme-linked immunosorbent assay (ELISA) with preparations of whole virions as the antigens. To avoid using complete virus in the standard technique, we have developed two new antigens through the expression of the VPX and VP3 genes in insect cells. VPX and especially VP3 were expressed at high levels in insect cells and simple to purify. The immunogenicity of both proteins was similar to that of the native virus. VPX was able to elicit neutralizing antibodies but VP3 was not. Purified VPX and VP3 were tested in an indirect ELISA with more than 300 chicken sera. There was an excellent correlation between the results of the ELISA using VPX and those of the two commercial kits. VP3 did not perform as well as VPX, and the linear correlation was significantly lower. A comparison with the standard reference technique, seroneutralization, showed that the indirect ELISA was more sensitive. Therefore, VPX-based ELISA is a good alternative to conventional ELISAs that use whole virions. PMID:10882666

Martínez-Torrecuadrada, J L; Lázaro, B; Rodriguez, J F; Casal, J I

2000-07-01

203

Dependence of the localization and function of the human cytomegalovirus protein US6 on the transporter associated with antigen processing.  

PubMed

Human cytomegalovirus protein US6 inhibits the transporter associated with antigen processing (TAP), which transports peptides into the endoplasmic reticulum (ER) for binding to major histocompatibility complex (MHC) class I molecules. We demonstrate that, in TAP-deficient cells, US6 is retained in the ER and binds to calnexin, but does not inhibit cell-surface expression of HLA-A201, an MHC class I allele that binds to peptides whose import into the ER is TAP-independent. Furthermore, in TAP-positive cells, US6 reduces the cell-surface expression of HLA-B2705, an MHC class I allele that is dependent on TAP for peptide binding, to a greater extent than that of HLA-A201. These data demonstrate that US6 has differential effects on the cell-surface expression of MHC class I alleles and are consistent with TAP being the sole inhibitory target of US6 in the MHC class I antigen-presentation pathway. PMID:19439551

Dugan, Gillian E; Hewitt, Eric W

2009-09-01

204

Lymphocyte cytosolic protein 1 is a chronic lymphocytic leukemia membrane-associated antigen critical to niche homing.  

PubMed

Membrane antigens are critical to the pathogenesis of chronic lymphocytic leukemia (CLL) as they facilitate microenvironment homing, proliferation, and survival. Targeting the CLL membrane and associated signaling patterns is a current focus of therapeutic development. Many tumor membrane targets are simultaneously targeted by humoral immunity, thus forming recognizable immunoglobulin responses. We sought to use this immune response to identify novel membrane-associated targets for CLL. Using a novel strategy, we interrogated CLL membrane-specific autologous immunoglobulin G reactivity. Our analysis unveiled lymphocyte cytosolic protein 1 (LCP1), a lymphocyte-specific target that is highly expressed in CLL. LCP1 plays a critical role in B-cell biology by crosslinking F-actin filaments, thereby solidifying cytoskeletal structures and providing a scaffold for critical signaling pathways. Small interfering RNA knockdown of LCP1 blocked migration toward CXCL12 in transwell assays and to bone marrow in an in vivo xenotransplant model, confirming a role for LCP1 in leukemia migration. Furthermore, we demonstrate that the Bruton's tyrosine kinase inhibitor ibrutinib or the PI3K inhibitor idelalisib block B-cell receptor induced activation of LCP1. Our data demonstrate a novel strategy to identify cancer membrane target antigens using humoral anti-tumor immunity. In addition, we identify LCP1 as a membrane-associated target in CLL with confirmed pathogenic significance. This clinical trial was registered at clinicaltrials.gov; study ID number: OSU-0025 OSU-0156. PMID:24009233

Dubovsky, Jason A; Chappell, Danielle L; Harrington, Bonnie K; Agrawal, Kitty; Andritsos, Leslie A; Flynn, Joseph M; Jones, Jeffrey A; Paulaitis, Michael E; Bolon, Brad; Johnson, Amy J; Byrd, John C; Muthusamy, Natarajan

2013-11-01

205

Serological cross-reactivity between sporothrix schenckii and various unrelated fungi  

Microsoft Academic Search

The serological cross-reactivity ofSporothrix schenckii with various unrelated fungi was investigated by use of immunodiffusion tests. A rabbit antiS. schenckii serum was obtained, which reacted withCladosporium werneckii, C. carrionii, C. bantianum, Coccidioides immitis, Phialophora jeanselmei, P. gougerotii, P. dermatitidis, Fonsecaea pedrosoi, Aspergillus fumigatus, Histoplasma capsulatum andTrichophyton mentagrophytes, but not withSaccharomyces cerevisiae antigens. The serological determinants responsible for the cross-reactions were

Hiroshi Ishizaki; Yoichi Nakamura; Robert W. Wheat

1981-01-01

206

Binding of Polyomavirus Small T Antigen to Protein Phosphatase 2A Is Required for Elimination of p27 and Support of S-Phase Induction in Concert with Large T Antigen  

Microsoft Academic Search

Although polyomavirus large T antigen readily transactivates S-phase-specific enzymes in serum-starved Swiss 3T3 mouse fibroblasts, it is incapable by itself to efficiently drive such cells into S phase. We describe here that this inability correlates with a weak proficiency of the viral protein to induce the synthesis of cyclin A and cyclin E and to stimulate the respective cyclin\\/cdk activities.

STEFAN SCHUCHNER; ERHARD WINTERSBERGER

1999-01-01

207

Escherichia coli heat-labile toxin subunit B fusions with Streptococcus sobrinus antigens expressed by Salmonella typhimurium oral vaccine strains: importance of the linker for antigenicity and biological activities of the hybrid proteins.  

PubMed Central

A set of vectors possessing the genes for aspartate semialdehyde dehydrogenase (asd) and the B subunit of the heat-labile enterotoxin of Escherichia coli (LT-B) has been developed. These vectors allow operon or gene fusions of foreign gene epitopes at the C-terminal end of LT-B. Two groups of vectors have been constructed with and without leader sequences to facilitate placing of the foreign antigen in different cell compartments. Two Streptococcus sobrinus genes coding for principal colonization factors, surface protein antigen A (SpaA), and dextranase (Dex), have been fused into the 3' end of the LT-B gene. Resulting protein fusions of approximately 120 to 130 kDa are extremely well recognized by antibodies directed against both SpaA and Dex as well as against LT-B domains and retain the enzymatic activity of dextranase and the biological activity of LT-B in that they bind to GM1 gangliosides. Maximum antigenicity was obtained with the vector possessing an intervening linker of at least six amino acids with two proline residues. Some of the fusion proteins also exhibited another property of LT-B in that they were exported into the periplasm where they oligomerized. LT-B-SpaA and LT-B-Dex hybrid proteins are expressed stably and at a high level in avirulent Salmonella typhimurium vaccine strains which are being used to investigate their immunogenicity and types of induced immune responses. The fusion vectors will also be useful for production and purification of LT-B fusion antigens to be used and evaluated in other vaccine compositions. Images PMID:8432584

Jagusztyn-Krynicka, E K; Clark-Curtiss, J E; Curtiss, R

1993-01-01

208

Characterization of the antigenicity of Cpl1, a surface protein of Cryptococcus neoformans var. neoformans.  

PubMed

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. PMID:25261494

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

209

Cloning of the structural genes of three H8 antigens and of protein III of Neisseria gonorrhoeae.  

PubMed

A bank of gonococcal DNA was constructed in the lambda gt11 expression vector. immunological screening of the bank resulted in the isolation of a clone that contains the structural gene of protein III. In addition, several clones reactive with mAbs specific for the H8 antigen were isolated. DNA hybridization studies revealed that these H8-reactive clones were derived from three different gonococcal genes. When the products produced by these clones were used to absorb antibodies from a rabbit antiserum, and the eluted antibodies were used in immunological studies, it could be shown that the parent gonococcus expressed the product of two of these H8 genes, and in strain R10, these had Mr of approximately 19,700 21,200 respectively. The larger form has not been recognized hitherto because the epitope reactive with the H8 mAb may be masked in this product. PMID:3091756

Gotschlich, E C; Blake, M S; Koomey, J M; Seiff, M; Derman, A

1986-09-01

210

Antigenic reactivity of ribosomal protein S6 and the calcium-binding ATPase inhibitor protein of mammalian mitochondria  

Microsoft Academic Search

Phosphorylation of ribosomal protein S6 of mammals precedes activation of cell growth in numerous biological systems. We have cloned a cDNA for ribosomal protein S6 from T-47D human breast cancer cells by immunoscreening a ?gt11 expression library with antibody raised against the mitochondrial Ca2+-binding ATPase inhibitor protein (CaBI) of bovine heart mitochondria (Yamada & Huzel: J Biol Chem 263: 11498–11503,

C. Gail Penner; Leigh C. Murphy; Norman J. Huzel; Esther W. Yamada

1991-01-01

211

Polypoid cystitis unrelated to indwelling catheters  

Microsoft Academic Search

Since polypoid cystitis (PC) is generally caused by indwelling catheter use, in order to evaluate the patients with PC unrelated to a intravesical catheter, a retrospective analysis of the records of the Pathology Department of Turgut Özal Medical Center was performed and this revealed 8 patients. Mean age of the 2 female and 6 male patients was 48 years (28

Süleyman Kiliç; Rezzan Erguvan; Deniz ?pek; Hasan Gökçe; Ali Güne?; N. Engin Aydin; Can Baydinç

2002-01-01

212

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

PubMed Central

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

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

2013-01-01

213

Retention of structure, antigenicity, and biological function of pneumococcal surface protein A (PspA) released from polyanhydride nanoparticles  

PubMed Central

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

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

214

Vaccination of mice with an antigenic serine protease-like protein elicits a protective immune response against Trichinella spiralis infection.  

PubMed

Trichinellosis has major economic impacts on animal husbandry and food safety, and the control and elimination of trichinellosis is a major objective of veterinary medicine. A gene encoding serine protease of Trichinella spiralis (Ts-Adsp) was identified by immunoscreening an adult T. spiralis cDNA library. In this study, the recombinant Ts-Adsp protein (rTs-Adsp) was cloned and expressed in a prokaryotic expression system and purified by Ni-affinity chromatography. To determine whether the purified rTs-Adsp is a potential vaccine candidate for the control of T. spiralis infection, we immunized BALB/c mice with this protein in combination with an alum adjuvant and subsequently challenged with T. spiralis larvae. The results showed that mice vaccinated with rTs-Adsp exhibited an average reduction in the muscle larvae burden of 46.5% relative to the control group. Immunization with the rTs-Adsp antigen induced both humoral and cellular immune responses, which manifested as elevated specific anti-rTs-Adsp IgG and IgE antibodies and a mixed Th1-Th2 response, as determined by Th1 (IFN-? and IL-2) and Th2 (IL-4, IL-10, and IL-13) cytokine profiling, with the Th2 predominant. Thus, purified rTs-Adsp is able to limit the invasion of T. spiralis , and this protein could be an effective vaccine candidate for trichinellosis. PMID:23252743

Feng, Shuang; Wu, Xiuping; Wang, Xuelin; Bai, Xue; Shi, Haining; Tang, Bin; Liu, Xiaolei; Song, Yanxia; Boireau, Pascal; Wang, Feng; Zhao, Ying; Liu, Mingyuan

2013-06-01

215

The large tumor antigen: a "Swiss Army knife" protein possessing the functions required for the polyomavirus life cycle.  

PubMed

The SV40 large tumor antigen (L-Tag) is involved in the replication and cell transformation processes that take place during the polyomavirus life cycle. The ability of the L-Tag to interact with and to inactivate the tumor suppressor proteins p53 and pRb, makes this polyfunctional protein an interesting target in the search for compounds with antiviral and/or antiproliferative activities designed for the management of polyomavirus-associated diseases. The severe diseases caused by polyomaviruses, mainly in immunocompromised hosts, and the absence of licensed treatments, make the discovery of new antipolyomavirus drugs urgent. Parallels can be made between the SV40 L-Tag and the human papillomavirus (HPV) oncoproteins (E6 and E7) as they are also able to deregulate the cell cycle in order to promote cell transformation and its maintenance. In this review, a presentation of the SV40 L-Tag characteristics, regarding viral replication and cellular transformation, will show how similar these two processes are between the polyoma- and papillomavirus families. Insights at the molecular level will highlight similarities in the binding of polyoma- and papillomavirus replicative helicases to the viral DNA and in their disruptions of the p53 and pRb tumor suppressor proteins. PMID:23201316

Topalis, D; Andrei, G; Snoeck, R

2013-02-01

216

Comparison of Two Human Papovaviruses with Simian Virus 40 by Structural Protein and Antigenic Analysis  

PubMed Central

The proteins of simian virus 40 (SV40) and two human papovaviruses, the hemagglutinating BK virus and the non-hemagglutinating DAR virus, were analyzed and compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The virions of SV40 and DAR contain seven proteins. By molecular weight analysis the constituent proteins of SV40 and DAR are identical. Approximately 84% of the viral protein has a molceular weight of 45,000. The major protein of BK virus is 3,000 to 5,000 daltons lighter than the major proteins of SV40 and DAR viruses. The five most rapidly migrating proteins of BK virus are indistinguishable by molecular weight analysis from the corresponding proteins of SV40 and DAR viruses. Radial immunodiffusion and immunoelectrophoresis of whole virus gave lines of identity between SV40 and DAR when reacted with SV40 antibody. SV40 antiserum tested against BK virus and BK antiserum tested against SV40 virus showed no reactivity by complement fixation, immunodiffusion, or immunoelectrophoresis. Images PMID:4363247

Mullarkey, Michael F.; Hruska, Jerome F.; Takemoto, Kenneth K.

1974-01-01

217

Identification of cytoplasmic membrane protein antigens of Mycobacterium avium, M. intracellulare, and M. scrofulaceum.  

PubMed

The cytoplasmic membrane isolated from representative strains of the Mycobacterium avium, M. intracellulare, and M. scrofulaceum (MAIS) group contained approximately 20 proteins, as identified by SDS - polyacrylamide gel electrophoresis. One membrane protein predominated, comprising up to 50% of the total membrane protein. This major cytoplasmic membrane protein (MCMP) had a molecular weight of 31,000 and was surface accessible based on its susceptibility to proteinase digestion. The composition of the culture medium strongly influenced the amount of MCMP in the membrane fraction. Western blot analysis revealed that the MCMP and several other membrane proteins reacted with serum samples from patients infected with M. avium-intracellulare, M. tuberculosis, or other mycobacteria. PMID:2743223

George, K L; Falkinham, J O

1989-05-01

218

An autoclave treatment reduces the solubility and antigenicity of an allergenic protein found in buckwheat flour.  

PubMed

The effects of an autoclave treatment of buckwheat flour on a 24-kDa allergenic protein were investigated by measuring reduction in solubility and antibody binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the intensity of the major bands, including that of the 24-kDa allergen, was reduced by the autoclave treatment. The protein solubility in buckwheat flour was variably decreased by the autoclave treatment. Enzyme-linked immunosorbent assay analysis using a monoclonal antibody specific for buckwheat 24-kDa protein showed that the reactivity of protein extracts (10 ?g/ml) from buckwheat flour was lowered by the autoclave treatment. The autoclave treatment may reduce the major allergen content of buckwheat. Future studies will determine if autoclaving treatments affect the allergenicity of the 24-kDa buckwheat protein. PMID:22691491

Tomotake, Hiroyuki; Yamazaki, Rikio; Yamato, Masayuki

2012-06-01

219

Synthetic Copolymer I and Myelin Basic Protein Do Not Require Processing Prior to Binding to Class II Major Histocompatibility Complex Molecules on Living Antigen-Presenting Cells  

Microsoft Academic Search

In the present study we attempted to examine whether copolymer 1 (Cop 1), a synthetic basic random copolymer of amino acids (a candidate drug for multiple sclerosis (MS)), and myelin basic protein (MBP) undergo processing prior to their binding to MHC class II molecules on antigen-presenting cells (APC). The direct binding of biotinylated Cop 1 and MBP to living APC

Masha Fridkis-Hareli; Dvora Teitelbaum; Ruth Arnon; Michael Sela

1995-01-01

220

Binding of copolymer 1 and myelin basic protein leads to clustering of class II MHC molecules on antigen-presenting cells  

Microsoft Academic Search

Copolymer 1 (Cop 1), a synthetic copolymer of amino acids, effective in suppression of experimental allergic encephalomyelitis (EAE) and myelin basic protein (MBP), was shown to bind extensively and promiscuously to the class II MHC molecules on antigen-presenting cells (APC) without prior processing. In the case of human APC, binding has earlier been demonstrated to DR but not DQ or

Masha Fridkis-Hareli; Dvora Teitelbaum; Israel Pecht; Ruth Arnon; Michael Sela

1997-01-01

221

Antigen 43/Fc?3 chimeric protein expressed by a novel bacterial surface expression system as an effective asthma vaccine.  

PubMed

The IgE Fc?3 domain is an active immunotherapeutic target for asthma and other allergic diseases. However, previous methods for preparing IgE fusion protein vaccines are complex. Antigen 43 (Ag43) is a surface protein found in Escherichia coli that contains ? and ? subunits (the ? subunit contains multiple T epitopes). Here we constructed a novel Ag43 surface display system (Ag43 system) to express Ag43 chimeric proteins to disrupt immune tolerance against IgE. The Ag43 system was constructed from the E. coli strain Tan109, in which the Ag43 gene was deleted and a recombinant plasmid (pETAg43) expressing a partial Ag43 gene was introduced. The Fc?3 domain of the IgE gene was then subcloned into plasmid pETAg43, resulting in a recombinant plasmid pETAg43/Fc?3, which was used to transform Tan109 for Ag43/Fc?3 surface expression. Thereafter, Ag43/Fc?3 was investigated as an asthma vaccine in a mouse model. Ag43/Fc?3 was expressed on and could be separated from the bacterial surface by heating to 60° while retaining activity. Ag43/Fc?3, as a protein vaccine, produced neutralizing autoantibodies to murine IgE, induced significant anti-asthma effects, and regulated IgE and T helper cytokines in a murine asthma model. Data show that Ag43/Fc?3 chimeric protein is a potential model vaccine for asthma treatment, and that the Ag43 system may be an effective tool for novel vaccine preparation to break immune tolerance to other self-molecules. PMID:24750112

Huang, Feng-Ying; Wang, Cai-Chun; Huang, Yong-Hao; Zhao, Huan-Ge; Guo, Jun-Li; Zhou, Song-Lin; Wang, Hua; Lin, Ying-Ying; Tan, Guang-Hong

2014-10-01

222

dbDiarrhea: the database of pathogen proteins and vaccine antigens from diarrheal pathogens.  

PubMed

Diarrhea occurs world-wide and is most commonly caused by gastrointestinal infections which kill around 2.2 million people globally each year, mostly children in developing countries. We describe here dbDiarrhea, which is currently the most comprehensive catalog of proteins implicated in the pathogenesis of diarrhea caused by major bacterial, viral and parasitic species. The current release of the database houses 820 proteins gleaned through an extensive and critical survey of research articles from PubMed. The major contributors to this compendium of proteins are Escherichia coli and Salmonella enterica. These proteins are classified into different categories such as Type III secretion system effectors, Type III secretion system components, and Pathogen proteins. There is another complementary module called 'Host proteins'. dbDiarrhea also serves as a repository of the research articles describing (1) trials of subunit and whole organism vaccines (2) high-throughput screening of Type III secretion system inhibitors and (3) diagnostic assays, for various diarrheal pathogens. The database is web accessible through an intuitive user interface that allows querying proteins and research articles for different organism, keywords and accession number. Besides providing the search facility through browsing, the database supports sequence similarity search with the BLAST tool. With the rapidly burgeoning global burden of the diarrhea, we anticipate that this database would serve as a source of useful information for furthering research on diarrhea. The database can be freely accessed at http://www.juit.ac.in/attachments/dbdiarrhea/diarrhea_home.html. PMID:22917656

Ramana, Jayashree; Tamanna

2012-12-01

223

Measles virus and canine distemper virus target proteins into a TAP-independent MHC class I-restricted antigen-processing pathway  

Microsoft Academic Search

After infection of CEM174.T2 cells (deficient for the transporter of antigen presentation (TAP)) with measles virus (MV) the nucleocapsid protein is recognized by Ld-restricted cytotoxic T cells in a TAP-independent, chloroquine-sensitive fashion. Presentation via the TAP-independent pathway requires virus replication. During MV infection of the cell the nucleocapsid as well as the matrix protein enter the endolysosomal compartment as indicated

Claudia Neumeister; Ralph Nanan; Tatjana I. Cornu; Carsten G. K. Lu; Hussein Naim; Stefan Niewiesk

224

A Single Cys706to Phe Substitution in the Retinoblastoma Protein Causes the Loss of Binding to SV4O T Antigen  

Microsoft Academic Search

Most naturally occurring mutants of the retinoblastoma (RB) protein contain large deletions or truncations. The small cell lung carcinoma cell line H209 contains a normal-sized but unphosphorylated RB protein (Hensel et al., Cancer Res., 50: 3067-3072, 1990), which fails to form a complex with SV4O T antigen, suggesting that the RB gene of H209 may contain a subtle mutation. To

Yves-Jean Bignon; Jin-Yuh Shew; Dan Rappolee; Susan L Naylor; Eva Y-H; P. Lee; Joachim Schnier; Wen-Hwa Lee

1990-01-01

225

A single Cys sup 706 to Phe substitution in the retinoblastoma protein causes the loss of binding to SV40 T antigen  

Microsoft Academic Search

Most naturally occurring mutants of the retinoblastoma (RB) protein contain large deletions or truncations. The small cell lung carcinoma cell line H209 contains a normal-sized but unphosphorylated RB protein, which fails to form a complex with SV40 T antigen, suggesting that the RB gene of H209 may contain a subtle mutation. To define this mutation, the RB complementary DNA and

Y. J. Bignon; Jinyuh Shew; E. Y. H. P. Lee; J. Schnier; Wenhwa Lee; D. Rappolee; S. L. Naylor

1990-01-01

226

Chinese hamster ovary cells can produce galactose-?-1,3-galactose antigens on proteins  

E-print Network

Chinese hamster ovary (CHO) cells are widely used for the manufacture of biotherapeutics, in part because of their ability to produce proteins with desirable properties, including 'human-like' glycosylation profiles. For ...

Bosques, Carlos J

227

groEL Encodes a Highly Antigenic Protein in Burkholderia pseudomallei  

Microsoft Academic Search

No recombinant protein is available for serodiagnosis of melioidosis. In this study, we report the cloning of the groEL gene, which encodes an immunogenic protein of Burkholderia pseudomallei. Bidirectional DNA sequencing of groEL revealed that the gene contained a single open reading frame encoding 546 amino acid residues with a predicted molecular mass of 57.1 kDa. Basic Local Alignment Search

PATRICK C. Y. WOO; PATRICIA K. L. LEUNG; SAMSON S. Y. WONG; PAK-LEUNG HO; KWOK-YUNG YUEN

2001-01-01

228

Heat shock response and heat shock protein antigens of Vibrio cholerae.  

PubMed Central

Sixteen heat shock proteins (Hsps) have been identified in the hypertoxinogenic strain 569B of Vibrio cholerae which are synthesized in response to small and large elevations of temperature. The induction of the Hsps is necessary for the cells to survive the deleterious effects of heat. There is no difference in the pattern of induction of the Hsps in V. cholerae strains varying in levels of toxinogenicity. One of the major low-molecular-mass Hsps, a 16-kDa protein, is preferentially degraded following shift down of temperature. This protein is induced at a much lower level at high temperatures in cells maintained in the laboratory for a prolonged period. The only Hsp located in the outer membrane of V. cholerae cells is a 23-kDa protein. Western immunoblot analysis with human immune sera collected from convalescent cholera patients revealed that this protein is markedly immunogenic. The human immune serum also reacted with the 69- and 16-kDa major Hsps and the 88-, 66-, and 46-kDa Hsps but not with the 61-kDa major Hsp identified as the groEL gene product. All major Hsps reacted with rabbit anti-V. cholerae sera. Ethanol stress leads to the induction of four of the major Hsps and three additional proteins. Images PMID:7960144

Sahu, G K; Chowdhury, R; Das, J

1994-01-01

229

HLA-B27 antigen  

MedlinePLUS

HLA-B27 is a blood test to look for a protein that is found on the surface of ... The protein is called human leukocyte antigen B27 (HLA-B27). Human leukocyte antigens (HLAs) are proteins that help ...

230

[Prokaryotic expression of the major antigenic domain of equine arteritis virus GL protein and the establishment of putative indirect ELISA assay].  

PubMed

According to the antigenic analysis of equine arteritis virus (EAV) GL protein, one pair of primers were designed, with which the gene fragment coding the high antigenic domain of EAV GL protein was amplified from the EAV genome. The cloned gene was digested with BamH I and Xho I and then inserted into pET-32a and resulted pET-GL1. The pET-GL1 was transformed into the host cell BL21(DE3) and the expression was optimized including cultivation temperature and concentration of IPTG. The aim protein was highly expressed and the obtained recombinant protein manifested well reactiongenicity as was confirmed by Western blot. The recombinant GL1 protein was purified by the means of His * Bind resin protein purification procedure. Then an indirect ELISA was established to detect antibody against EAV with the purified GL1 protein as the coating antigen. The result showed that the optimal concentration of coated antigen was 9.65 microg/mL and the optimal dilution of serum was 1:80. The positive criterion of this ELISA assay is OD (the tested serum) > 0.4 and OD (the tested serum) /OD (the negative serum) > 2.0. The iGL-ELISA was evaluated versus micro-virus neutralization test. The ELISA was performed on 900 sera from which were preserved by this lab during horse entry/exit inspection, the agreement (94.1%) of these test were considered suitable for individual serological detection. In another test which 180 sera samples were detected by iGL-ELISA and INGEZIM ELISA kit respectively. The agreement ratio between the two methods is 95.6%. PMID:16933616

Liang, Cheng-Zhu; Cao, Rui-Bing; Wei, Jian-Chao; Zhu, Lai-Hua; Chen, Pu-Yan

2006-06-01

231

Sequence heterogeneity of the major antigenic protein 1 genes from Cowdria ruminantium isolates from different geographical areas.  

PubMed Central

The genes for the immunodominant major antigenic protein 1 (MAP1) of Cowdria ruminantium from four African and two Caribbean isolates were cloned, restriction mapped, and sequenced to identify conserved epitopes for development of serodiagnostic tools for heartwater. Restriction length polymorphisms were observed among the respective MAP1 genes analyzed and were confirmed by sequencing. The sequence data generated for these isolates were compared with data for the previously reported Senegal isolate MAP1 gene. These sequences were found to differ from each other by 0.6 to 14.0%. These differences translate into a 0.8 to 10.0% variation in the predicted protein sequence. In the entire coding sequence, several amino acid substitutions were identified in addition to deletions or insertions at three regions of the gene. These variable regions are referred to as variable regions I, II, and III. From the sequence data, an evolutionary distance tree was constructed; this tree suggested that at least two genetically distinct C. ruminantium strains exist in the Caribbean: the isolate from Antigua is similar to that from Senegal, while the isolate from Guadeloupe is closely related to that from Sudan. PMID:8807206

Reddy, G R; Sulsona, C R; Harrison, R H; Mahan, S M; Burridge, M J; Barbet, A F

1996-01-01

232

Heat shock protein 90 mediates efficient antigen cross presentation through the scavenger receptor expressed by endothelial cells-I.  

PubMed

Ag cross presentation is an important mechanism for CD8(+) T cell activation by APCs. We have investigated mechanisms involved in heat shock protein 90 (Hsp90) chaperone-mediated cross presentation of OVA-derived Ags. Hsp90-OVA peptide complexes bound to scavenger receptor expressed by endothelial cells (SREC-I) on the surface of APCs. SREC-I then mediated internalization of Hsp90-OVA polypeptide complexes through a Cdc42-regulated, dynamin-independent endocytic pathway known as the GPI-anchored protein-enriched early endosomal compartment to recycling endosomes. Peptides that did not require processing could then be loaded directly onto MHC class I in endosomes, whereas longer peptides underwent endosomal and cytosomal processing by aminopeptidases and proteases. Cross presentation of Hsp90-chaperoned peptides through this pathway to CD8(+) T cells was highly efficient compared with processing of free polypeptides. In addition, Hsp90 also activated c-Src kinase associated with SREC-I, an activity that we determined to be required for effective cross presentation. Extracellular Hsp90 can thus convey antigenic peptides through an efficient endocytosis pathway in APCs and facilitate cross presentation in a highly regulated manner. PMID:20686127

Murshid, Ayesha; Gong, Jianlin; Calderwood, Stuart K

2010-09-01

233

Immunohistochemical Assessment of Proliferating Cell Nuclear Antigen Protein Expression in Plaque, Reticular and Erosive Types of Oral Lichen Planus  

PubMed Central

Background: Proliferating cell nuclear antigen (PCNA) is a nuclear protein synthesized in the late G1 and S-phase of the cell cycle. Immunodetection of this protein represents a useful marker of the proliferation status of lesions. Aims: The aim of this study is to evaluate the immunohistochemical expression of PCNA in oral lichen planus (OLP) and to assess the PCNA expression in a different layer of epithelium in different types of OLP. Subjects and Methods: A total of 96 cases of histologically proven OLP, 32 cases each of erosive, reticular and plaque type were selected. Two sections were taken from each one for H and E. Other sections were stained according to super sensitive polymer horseradish peroxidase method for identifying PCNA expression. Results: Of the three types of OLP, erosive type showed higher expression of PCNA (average 66.8%, minimum of 55% and maximum of 80.3%) followed by reticular (average 37.7%, minimum of 26% and maximum of 47%) and plaque type (average 17%, minimum of 5% and maximum of 25%) indicating increased proliferative activity. The erosive type also showed higher expression of PCNA in all the layers of epithelium followed by reticular and plaque type. Conclusion: PCNA is a good marker to indicate proliferation status of disease. Out of three types, erosive type possess more proliferative ratio, chances of malignant changes is more in this type. PMID:25221712

Pramod, RC; Pandit, S; Desai, D; Suresh, KV; Ingaleshwar, PS; Shetty, SJ; Ahamad, S

2014-01-01

234

Homology of the 70-kilodalton antigens from Mycobacterium leprae and Mycobacterium bovis with the Mycobacterium tuberculosis 71-kilodalton antigen and with the conserved heat shock protein 70 of eucaryotes.  

PubMed Central

Two lambda gt11 recombinant clones, JKL2 and JKL15, each containing an insert coding for part of the highly immunogenic 70-kilodalton (kDa) protein antigen, were isolated from a Mycobacterium leprae genomic library by immunoscreening with the monoclonal antibody L7. Clone JKL2 contained the largest insert, 2.3 kilobase pairs. Nonoverlapping fragments of this insert were used as probes and showed strong hybridization to a number of Mycobacterium tuberculosis-lambda gt11 recombinants producing proteins recognized by an anti-M. tuberculosis 71-kDa monoclonal antibody, IT11. One clone from a recombinant Mycobacterium bovis library was also characterized by using L7, and the insert from this clone, B5bt, hybridized strongly to the M. leprae probes as well. The nucleotide sequence of the 1,037-base-pair coding region of the JKL2 M. leprae clone which encodes the carboxy-terminal half of the 70-kDa protein had extensive homology with genes from a number of species. In all cases, these genes, including the recently described Ag63 and Ag361 of Plasmodium falciparum, were found to be members of the heat shock protein 70 (hsp 70) family of genes. At the amino acid level, homology was maximal between amino acids 83 through 107 and 159 through 184, which showed extreme conservation (92 and 85% identity) with Escherichia coli DnaK amino acids 386 through 409 and 460 through 485, respectively, and was 51% homologous over the entire coding region (amino acids 1 through 344 of JKL2). In contrast, amino acids 129 through 158 had maximal homology with the phylogenetically more distant Xenopus laevis hsp70. Homology declined substantially in the carboxy-terminal 34 amino acids. The predicted ATP-binding functional activity of the 70-kDa antigen from M. bovis was confirmed with affinity purification of the antigen by binding to ATP-agarose and elution with ATP. In view of the conservation of sequences between these mycobacterial antigens and mammalian endogenous cellular enzymes, further evaluation of these molecules in vivo may aid in understanding tolerance to self-antigens as well as provide potentially useful immunodiagnostic reagents. Images PMID:2491836

Garsia, R J; Hellqvist, L; Booth, R J; Radford, A J; Britton, W J; Astbury, L; Trent, R J; Basten, A

1989-01-01

235

Phase I Malaria Vaccine Trial with a Long Synthetic Peptide Derived from the Merozoite Surface Protein 3 Antigen  

PubMed Central

The C-terminal conserved region of Plasmodium falciparum merozoite surface protein 3 (MSP3) is the trigger antigen of a protective immune response mediated by cytophilic antibodies. In an open, randomized, two-adjuvant (Montanide ISA 720, aluminum hydroxide) phase I clinical trial we evaluated the safety and immunogenicity of increasing doses of a long synthetic peptide construct spanning the conserved region of MSP3 targeted by biologically active antibodies (MSP3-LSP). Thirty-five healthy volunteers were randomized to receive three subcutaneous injections on days 0, 30, and 120. Of the 100 injections given, 10 caused severe local reactions, 62 caused transient mild to moderate local reactions, and 28 caused no reaction. On the basis of preestablished exclusion criteria, use of the Montanide formulation led to withdrawal of five volunteers after the second injection. This led to a reduction in the subsequent vaccine doses in four of the groups. No vaccine-related serious adverse events occurred throughout the trial. After the third injection, volunteers displayed a marked specific anti-MSP3-LSP antibody response (23/30 individuals, compared with 29/34 individuals for plasma from an area where malaria is endemic), an anti-native MSP3 antibody response (19/30 individuals), a T-cell-antigen-specific proliferative response (26/30 individuals), and gamma interferon production (25/30 individuals). In conclusion, the MSP3-LSP vaccine was immunogenic with both adjuvants, although it was unacceptably reactogenic when it was combined with Montanide. The potential usefulness of the candidate vaccine is supported by the induction of a strong cytophilic response (i.e., the type of anti-MSP3 antibodies involved in antibody-dependent, monocyte-mediated protective mechanisms in areas where malaria is endemic). PMID:16299295

Audran, Régine; Cachat, Michel; Lurati, Floriana; Soe, Soe; Leroy, Odile; Corradin, Giampietro; Druilhe, Pierre; Spertini, François

2005-01-01

236

The proliferating cell nuclear antigen regulates retinoic acid receptor transcriptional activity through direct protein–protein interaction  

PubMed Central

Retinoic acid receptors (RARs) interact, in a ligand-dependent fashion, with many coregulators that participate in a wide spectrum of biological responses, ranging from embryonic development to cellular growth control. The transactivating function of these ligand-inducible transcription factors reside mainly, but not exclusively, in their ligand-binding domain (AF2), which recruits or dismiss coregulators in a ligand-dependent fashion. However, little is known about AF2-independent function(s) of RARs. We have isolated the proliferating cell nuclear antigen (PCNA) as a repressor of RAR transcriptional activity, able to interact with an AF2-crippled RAR. The N-terminus of PCNA interacts directly with the DNA-binding domain of RAR, and PCNA is recruited to a retinoid-regulated promoter in intact cells. This interaction affects the transcriptional response to retinoic acid in a promoter-specific manner, conferring an unanticipated role to PCNA in transcriptional regulation. Our findings also suggest a role for RAR as a factor coordinating DNA transcription and repair. PMID:16055921

Martin, Perrine J.; Lardeux, Virginie; Lefebvre, Philippe

2005-01-01

237

Antigenic Profiles of Recombinant Proteins from Mycobacterium avium subsp paratuberculosis in Sheep with Johne's Disease  

Technology Transfer Automated Retrieval System (TEKTRAN)

Methods to improve the ELISA test to detect Mycobacterium avium subsp paratuberculosis have been explored over several years. Previously, selected recombinant proteins of M. avium subspecies paratuberculosis were found to be immunogenic in cattle with Johne’s disease. In the present study, antibo...

238

Identification of Leishmania Proteins Preferentially Released in Infected Cells Using Change Mediated Antigen Technology (CMAT)  

PubMed Central

Although Leishmania parasites have been shown to modulate their host cell's responses to multiple stimuli, there is limited evidence that parasite molecules are released into infected cells. In this study, we present an implementation of the change mediated antigen technology (CMAT) to identify parasite molecules that are preferentially expressed in infected cells. Sera from mice immunized with cell lysates prepared from L. donovani or L. pifanoi-infected macrophages were adsorbed with lysates of axenically grown amastigotes of L. donovani or L. pifanoi, respectively, as well as uninfected macrophages. The sera were then used to screen inducible parasite expression libraries constructed with genomic DNA. Eleven clones from the L. pifanoi and the L. donovani screen were selected to evaluate the characteristics of the molecules identified by this approach. The CMAT screen identified genes whose homologs encode molecules with unknown function as well as genes that had previously been shown to be preferentially expressed in the amastigote form of the parasite. In addition a variant of Tryparedoxin peroxidase that is preferentially expressed within infected cells was identified. Antisera that were then raised to recombinant products of the clones were used to validate that the endogenous molecules are preferentially expressed in infected cells. Evaluation of the distribution of the endogenous molecules in infected cells showed that some of these molecules are secreted into parasitophorous vacuoles (PVs) and that they then traffic out of PVs in vesicles with distinct morphologies. This study is a proof of concept study that the CMAT approach can be applied to identify putative Leishmania parasite effectors molecules that are preferentially expressed in infected cells. In addition we provide evidence that Leishmania molecules traffic out of the PV into the host cell cytosol and nucleus. PMID:20957202

Kima, Peter E.; Bonilla, J. Alfredo; Cho, Eumin; Ndjamen, Blaise; Canton, Johnathan; Leal, Nicole; Handfield, Martin

2010-01-01

239

Affinities of human histo-blood group antigens for norovirus capsid protein complexes.  

PubMed

The binding profiles of many human noroviruses (huNoVs) for human histo-blood group antigens have been characterized. However, quantitative-binding data for these important virus-host interactions are lacking. Here, we report on the intrinsic (per binding site) affinities of HBGA oligosaccharides for the huNoV VA387 virus-like particles (VLPs) and the associated subviral P particles measured using electrospray ionization mass spectrometry. The affinities of 13 HBGA oligosaccharides, containing A, B and H epitopes, with variable sizes (disaccharide to tetrasaccharide) and different precursor chain types (types 1, 2, 3, 5 and 6), were measured for the P particle, while the affinities of the A and B trisaccharides and A and B type 6 tetrasaccharides for the VLP were determined. The intrinsic affinities of the HBGA oligosaccharides for the P particle range from 500 to 2300 M(-1), while those of the A and B trisaccharides and the A and B type 6 tetrasaccharides for the VLP range from 1000 to 4000 M(-1). Comparison of these binding data with those measured previously for the corresponding P dimer reveals that the HBGA oligosaccharides tested exhibit similar intrinsic affinities for the P dimer and P particle. The intrinsic affinities for the VLP are consistently higher than those measured for the P particle, but within a factor of three. While the cause of the subtle differences in HBGA oligosaccharide affinities for the P dimer and P particle and those for the VLP remains unknown, the present data support the use of P dimers or P particles as surrogates to the VLP for huNoV-receptor-binding studies. PMID:25395406

Han, Ling; Kitova, Elena N; Tan, Ming; Jiang, Xi; Pluvinage, Benjamin; Boraston, Alisdair B; Klassen, John S

2015-02-01

240

Antigenicity of recombinant maltose binding protein-Mycobacterium avium subsp. paratuberculosis fusion proteins with and without factor Xa cleaving  

Technology Transfer Automated Retrieval System (TEKTRAN)

Mycobacterium avium subsp paratuberculosis (MAP) causes Johne’s disease (JD) in ruminants. Proteomic studies have shown that MAP expresses certain proteins when exposed to in vitro physiological stress conditions similar to the conditions experienced within a host during natural infection. Such prot...

241

A Protein-Conjugate Approach to Develop a Monoclonal Antibody-Based Antigen Detection Test for the Diagnosis of Human Brucellosis  

PubMed Central

Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen. Nucleic acid amplification techniques have not proven capable of reproducibly and sensitively demonstrating the presence of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases. PMID:24901521

Patra, Kailash P.; Saito, Mayuko; Atluri, Vidya L.; Rolán, Hortensia G.; Young, Briana; Kerrinnes, Tobias; Smits, Henk; Ricaldi, Jessica N.; Gotuzzo, Eduardo; Gilman, Robert H.; Tsolis, Renee M.; Vinetz, Joseph M.

2014-01-01

242

A protein-conjugate approach to develop a monoclonal antibody-based antigen detection test for the diagnosis of human brucellosis.  

PubMed

Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen. Nucleic acid amplification techniques have not proven capable of reproducibly and sensitively demonstrating the presence of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases. PMID:24901521

Patra, Kailash P; Saito, Mayuko; Atluri, Vidya L; Rolán, Hortensia G; Young, Briana; Kerrinnes, Tobias; Smits, Henk; Ricaldi, Jessica N; Gotuzzo, Eduardo; Gilman, Robert H; Tsolis, Renee M; Vinetz, Joseph M

2014-06-01

243

Herpes Simplex Virus 2 (HSV-2) Infected Cell Proteins Are among the Most Dominant Antigens of a Live-Attenuated HSV-2 Vaccine  

PubMed Central

Virion glycoproteins such as glycoprotein D (gD) are believed to be the dominant antigens of herpes simplex virus 2 (HSV-2). We have observed that mice immunized with a live HSV-2 ICP0- mutant virus, HSV-2 0?NLS, are 10 to 100 times better protected against genital herpes than mice immunized with a HSV-2 gD subunit vaccine (PLoS ONE 6:e17748). In light of these results, we sought to determine which viral proteins were the dominant antibody-generators (antigens) of the live HSV-2 0?NLS vaccine. Western blot analyses indicated the live HSV-2 0?NLS vaccine elicited an IgG antibody response against 9 or more viral proteins. Many antibodies were directed against infected-cell proteins of >100 kDa in size, and only 10 ± 5% of antibodies were directed against gD. Immunoprecipitation (IP) of total HSV-2 antigen with 0?NLS antiserum pulled down 19 viral proteins. Mass spectrometry suggested 44% of immunoprecipitated viral peptides were derived from two HSV-2 infected cells proteins, RR-1 and ICP8, whereas only 14% of immunoprecipitated peptides were derived from HSV-2’s thirteen glycoproteins. Collectively, the results suggest the immune response to the live HSV-2 0?NLS vaccine includes antibodies specific for infected cell proteins, capsid proteins, tegument proteins, and glycoproteins. This increased breadth of antibody-generating proteins may contribute to the live HSV-2 vaccine’s capacity to elicit superior protection against genital herpes relative to a gD subunit vaccine. PMID:25658852

Geltz, Joshua J.; Gershburg, Edward; Halford, William P.

2015-01-01

244

[Recombinant heat-shock protein (rHSP70) boosts activation of innate and adaptive immunity during simultaneous administration with bacterial antigens in experiment].  

PubMed

Experimental study of adjuvant effect of recombinant mycobacterial heat-shock protein rHSP70 on immune response to antigens of opportunistic microorganisms was performed. Therapeutic poly-component vaccine Immunovac-VP-4, containing ligands for binding with Toll-like receptors (TLRs) 2, 4 and 9, was used as a source of bacterial antigens. Obtained data showed that administration of rHSP70 mixed with bacterial antigens of opportunistic microorganisms leads to maturation of mice dendritic cells obtained from bone marrow precursors, increased expression of TLRs 2, 4, 9 on their surface and production of cytokines IL-1beta, IL-6, IL-10, TNFalpha, as well as to activation of innate immunity in experiments in vivo resulting to increased resistance of mice to experimental Salmonella infection. Simultaneous administration of rHSP70 with bacterial antigens increased titers of antibodies to vaccine's antigenic components in direct hemagglutination reaction. Thus, adjuvant effect of rHSP70 was confirmed on the used model as well as increase of stimulation of innate and adaptive immunity was shown. PMID:19338235

Shevchik, Iu S; Kurbatova, E A; Sveshnikov, P G; Akhmatova, N K; Egorova, N B

2009-01-01

245

Heligmosomoides polygyrus antigens inhibit the intrinsic pathway of apoptosis by overexpression of survivin and Bcl-2 protein in CD4 T cells  

PubMed Central

Many laboratory studies and epidemiological observations confirm that nematodes prevent some immune-mediated diseases. The development of immunologically well-defined laboratory models of intestinal nematode infection has allowed significant advances to be made in understanding the immunological basis of effector mechanisms operating during infection under controlled laboratory conditions. The Heligmosomoides polygyrus- mouse system is used for studies of parasite immunomodulation. H. polygyrus causes a chronic, asymptomatic intestinal infection and effectively maintains both local and systemic tolerance to reduce allergic and autoimmune inflammation. However, exposure of mice to H. polygyrus antigen reduced spontaneous and glucocorticoid-induced apoptosis of CD4- positive T cells in mesenteric lymph node (MLN). In this study we evaluate the proliferation, cytokine secretion, cell cycle progression and expression of apoptosis related genes in MLN CD4 T cells of uninfected and H. polygyrus infected mice ex vivo and in vitro after restimulation with parasite excretory secretory antigen (ESAg), somatic antigen (SAg) and fraction 9 (F9Ag) of somatic antigen. For the first time we explain the influence of H. polygyrus antigens on the intrinsic pathway of apoptosis. We found that the proliferation provoked by fraction 9 and inhibition of apoptosis was dependent on a low Bax/Bcl-2 ratio, dramatical upregulation of survivin, D1 cyclin, P-glycoprotein, and loss of p27Kip1 protein with inhibition of active caspase-3 but not caspase- 8. PMID:23787700

Donskow-?ysoniewska, Katarzyna; Brodaczewska, Klaudia; Doligalska, Maria

2013-01-01

246

Utilization of protein-A in immuno-histochemical techniques for detection of Peste des Petits Ruminants (PPR) virus antigens in tissues of experimentally infected goats.  

PubMed

This paper constitutes the first record of utilizing the S. aureus protein-A (PA), conjugated to peroxidase enzyme, for the detection of the Peste des Petits Ruminants (PPR) virus antigens in tissues of experimentally infected goats. The goats were experimentally infected with a virulent PPR virus, which was previously isolated from a severe natural disease outbreak in gazelles, during 2002 in Saudi Arabia. The technique is rapid, and has the superiority over the peroxidase -anti-peroxidase (PAP) test in that, inactivation of the indigenous peroxidase in the tissues is not required and that it can be used against a wide range of animal species. An advantage over the other immunolabelled conjugates is that PA attaches specifically to the crystalizable fraction (Fc) of the IgG molecule, thus allowing the antigen binding fraction (Fab) of the molecule, free to interact specifically with the antigen. So, it doesn't actually compete with the antigen for the Fab portion of the IgG molecule. In the present study, PA conjugate detected the PPR virus antigens in various tissues of the experimentally infected goats. PMID:19052895

Abu Elzein, E M E; Al-Naeem, A

2009-01-01

247

Expression, Self-Assembly, and Antigenicity of a Snow Mountain Agent-Like Calicivirus Capsid Protein  

Microsoft Academic Search

Virus-like particles were produced in insect cells infected with a recombinant baculovirus containing the capsid gene of MX virus, a Mexican strain of human calicivirus. These recombinant MX (rMX) particles were morphologically similar to recombinant Norwalk virus (rNV) particles as observed under an electron micro- scope and contained a single capsid protein with a molecular weight of 57,000, which was

XI JIANG; DAVID O. MATSON; GUILLERMO M. RUIZ-PALACIOS; JIAN HU; JOHN TREANOR; ANDLARRY K. PICKERING

1995-01-01

248

Iscom, a novel structure for antigenic presentation of membrane proteins from enveloped viruses  

Microsoft Academic Search

We describe here a novel type of immunostimiilating complex, called `iscom', in which virus membrane proteins are presented in a multimeric form1-8. The matrix of the iscom is the glycoside Quil A (Spikoside; Iscotec AB), extracted from the bark of Quillaja saponaria Molina9, which forms micelles at the critical micellar concentration of 0.03%. In micelle form, Quil A probably has

B. Morein; B. Sundquist; S. Höglund; K. Dalsgaard; A. Osterhaus

1984-01-01

249

Autoimmunity to heat shock protein 60 and antigen-specific production of interleukin-10.  

PubMed Central

The immunopathologic sequelae of chlamydial infection are correlated with immune responses to the Chlamydia trachomatis heat shock protein 60 (hsp60). One pathogenic mechanism that may explain this association is the induction of autoimmune responses to self hsp60, since these two proteins share a high degree of amino acid sequence identity. To investigate the conditions under which autoimmune responses can be generated against self hsp60, groups of CBA mice were immunized with recombinant mouse hsp60, recombinant chlamydial hsp60, or both proteins. The data show that autoimmune responses characterized by strong T-cell proliferation and high titers of antibody to self hsp60 are induced only by concurrent immunization with mouse and chlamydial hsp60. Immunization with mouse hsp60 alone induced lymphocytes that secreted high levels of interleukin-10 (IL-10) but did not proliferate in response to in vitro stimulation with mouse hsp60; coimmunization with mouse and chlamydial hsp60s induced lymphocytes that proliferated strongly in response to mouse hsp60, secreted 6-fold less IL-10, and exhibited a 12-fold increase in the ratio of gamma interferon/IL-10 production. Switches in cytokine production patterns may mediate the pathogenesis of hsp60-associated diseases such as C. trachomatis immunopathology. PMID:9125545

Yi, Y; Yang, X; Brunham, R C

1997-01-01

250

Epstein-Barr virus nuclear antigen 2 induces expression of the virus-encoded latent membrane protein.  

PubMed Central

Infection of Epstein-Barr virus-negative human B-lymphoma cell lines with the fully transforming B95.8 Epstein-Barr virus strain was associated with complete virus latent gene expression and a change in the cell surface and growth phenotype toward that of in vitro-transformed lymphoblastoid cell lines. In contrast, the cells infected with the P3HR1 Epstein-Barr virus strain, a deletion mutant that cannot encode Epstein-Barr nuclear antigen 2 (EBNA2) or a full-length EBNA-LP, expressed EBNAs1, 3a, 3b, and 3c but were negative for the latent membrane protein (LMP) and showed no change in cellular phenotype. This suggests that EBNA2 and/or EBNA-LP may be required for subsequent expression of LMP in Epstein-Barr virus-infected B cells. Recombinant vectors capable of expressing the B95.8 EBNA2A protein were introduced by electroporation into two P3HR1-converted B-lymphoma cell lines, BL30/P3 and BL41/P3. In both cases, stable expression of EBNA2A was accompanied by activation of LMP expression from the resident P3HR1 genome; control transfectants that did not express the EBNA2A protein never showed induction of LMP. In further experiments, a recombinant vector capable of expressing the full-length B95.8 EBNA-LP was introduced into the same target lines. Strong EBNA-LP expression was consistently observed in the transfected clones but was never accompanied by induction of LMP. The EBNA2A gene transfectants expressing EBNA2A and LMP showed a dramatic change in cell surface and growth phenotype toward a pattern like that of lymphoblastoid cell lines; some but not all of these changes could be reproduced in the absence of EBNA2A by transfection of P3HR1-converted cell lines with a recombinant vector expressing LMP. These studies suggest that EBNA2 plays an important dual role in the process of B-cell activation to the lymphoblastoid phenotype; the protein can have a direct effect upon cellular gene expression and is also involved in activating the expression of a second virus-encoded effector protein, LMP. Images PMID:2157875

Abbot, S D; Rowe, M; Cadwallader, K; Ricksten, A; Gordon, J; Wang, F; Rymo, L; Rickinson, A B

1990-01-01

251

Isolation of Potentially Useful Antigens from Cyathostomin Third-Stage Larvae by Using a Fast Protein Liquid Chromatography One-Step Method?  

PubMed Central

Three major protein complexes (51, 29, and 15 kDa, named P1 to P3, respectively) were resolved by gel filtration of the excretory/secretory antigens collected from a mixture of horse cyathostomin third-stage larvae (L3s). The potential application for the detection of infected horses was assessed with an enzyme-linked immunosorbent assay (ELISA) by the comparison of the serological and copromicroscopical results. The value of the area under the receiver operating characteristic (ROC) curve was higher than 0.9 when the three peaks were used. Elevated values (>90%) for the sensitivity, specificity, and the positive-likelihood ratio were also observed for all the antigen complexes. A significant increment in the IgG antibody levels 4 weeks prior to the observation of eggs in the feces of weanlings naturally infected was recorded. Our results indicate that the evaluation of chemotherapy is possible by using immunoenzymatic probes and fast protein liquid chromatography (FPLC)-purified antigens. Data collected in the present investigation indicate that FPLC isolation offers a very helpful one-step method for collecting antigens with diagnostic potential to be employed in immunoenzymatic probes. PMID:21775518

Paz-Silva, A.; Francisco, R.; Rodríguez, I.; Francisco, I.; Cazapal-Monteiro, C. F.; Arias, M. S.; Suárez, J. L.; Sánchez-Andrade, R.

2011-01-01

252

Immunoglobulin production in the European pond tortoise, Emys orbicularis, immunized with serum protein antigens  

PubMed Central

The immunological responses of the European pond tortoise, Emys orbicularis, to BGG and sheep serum proteins indicate that in this tortoise there is no serum component corresponding with mammalian albumin and that both ?G (7S) and ?M (19S) immunoglobulins are involved when antibodies are produced. A prolonged period of ?M antibody production occurs after primary immunization. The separation of the ?M and ?G immunoglobulins was performed using starch block electrophoresis and Sephadex G-200 gel filtration. The separated immunoglobulins were characterized by immunodiffusion and by starch gel, agar gel and immunoelectrophoresis. ImagesFIG. 1-3FIG. 6FIG. 8FIG. 9FIG. 10 PMID:4173673

Lykakis, J. J.

1968-01-01

253

Antigenic drift in the ligand domain of Plasmodium vivax duffy binding protein confers resistance to inhibitory antibodies.  

PubMed

Interaction of the Duffy binding protein (DBP) with its erythrocyte receptor is critical for maintaining Plasmodium vivax blood-stage infections, making DBP an appealing vaccine candidate. The cysteine-rich region II is the ligand domain of DBP and a target of vaccine development. Interestingly, most of the allelic diversity observed in DBP is due to the high rate of nonsynonymous polymorphisms in this critical domain for receptor recognition. Similar to the hypervariability in influenza hemagglutinin, this pattern of polymorphisms in the DBP ligand domain suggests that this variation is a mechanism to evade antibody neutralization. To evaluate the role that dbp allelic diversity plays in strain-specific immunity, we examined the ability of an anti-Sal1 DBP serum to inhibit the erythrocyte-binding function of variant dbp alleles expressed on COS cells. We observed that the PNG-7.18 allele was significantly less sensitive to immune inhibition of its erythrocyte-binding activity than were the Sal1 and PNG-27.16 alleles. This result suggested that the unique polymorphisms of resistant PNG-7.18 were part of a protective epitope on the DBP ligand. To confirm this, Sal1 was converted to the refractory phenotype by introduction of 3 polymorphisms unique to PNG-7.18, via site-directed mutagenesis. The results of the present study indicate that linked polymorphisms have an additive, synergistic effect on DBP antigenic character. PMID:15478059

VanBuskirk, Kelley M; Cole-Tobian, Jennifer L; Baisor, Moses; Sevova, Elitza S; Bockarie, Moses; King, Christopher L; Adams, John H

2004-11-01

254

The secreted antigen, HP0175 of Helicobacter pylori links the unfolded protein response (UPR) to autophagy in gastric epithelial cells.  

PubMed

Autophagy is an intracellular catabolic process which is required to maintain cellular homeostasis. Pathogen-elicited host cell autophagy may favour containment of infection or may help in bacterial survival. Pathogens have developed the ability to modulate host autophagy. The secreted antigen HP0175, a peptidyl prolyl cis,trans isomerase of Helicobacter pylori has moonlighting functions with reference to host cells. Here we show that it executes autophagy in gastric epithelial cells. Autophagy is dependent on the unfolded protein response (UPR) which activates the expression of PKR-like ER kinase (PERK). This is accompanied by phosphorylation of eIF2-? and transcriptional activation of ATF4 and CHOP. Knockdown of UPR- related genes inhibits the conversion of LC3I to LC3-II, a marker of autophagy. The autophagy- inducing ability of H. pylori is compromised when cells are infected with an isogenic hp0175 mutant. Autophagy precedes apoptosis. Silencing of BECLIN 1 augments cleavage of caspase 3 as well as apoptosis. Increased apoptosis of gastric epithelial cells is known to be linked to H. pylori- mediated gastric inflammation and carcinogenesis. To the best of our knowledge, this study provides the first demonstration of how HP0175, endowed with moonlighting functions, links UPR- dependent autophagy and apoptosis during H. pylori infection. PMID:25439545

Halder, Priyanka; Datta, Chandreyee; Kumar, Ranjeet; Sharma, Arun Kumar; Basu, Joyoti; Kundu, Manikuntala

2014-12-01

255

Leukotoxin (Leukothera®) targets active leukocyte function antigen-1 (LFA-1) protein and triggers a lysosomal mediated cell death pathway.  

PubMed

Leukotoxin (LtxA) is a protein toxin that is secreted from the oral bacterium, Aggregatibacter actinomycetemcomitans. LtxA targets specifically the ?(2) integrin, leukocyte function antigen-1 (LFA-1) on white blood cells (WBCs) and causes cell death. LtxA preferentially targets activated WBCs and is being developed as a therapeutic agent for the treatment of WBC diseases such as hematologic malignancies and autoimmune/inflammatory diseases. However, the mechanism by which interaction between LtxA and LFA-1 results in cell death is not well understood. Furthermore, how LtxA preferentially recognizes activated WBCs is not known. We show here that LtxA interacts specifically with LFA-1 in the active (exposed) conformation. In THP-1 monocytes, LtxA caused rapid activation of caspases, but LtxA could overcome the inhibition of caspases and still intoxicate. In contrast, inhibiting the vesicular trafficking pathway or cathepsin D release from the lysosome resulted in significant inhibition of LtxA-mediated cytotoxicity, indicating a more potent, lysosomal mediated cell death pathway. LtxA caused rapid disruption of the lysosomal membrane and release of lysosomal contents into the cytosol. Binding of LtxA to LFA-1 resulted in the internalization of both LtxA and LFA-1, with LtxA localizing specifically to the lysosomal compartment. To our knowledge, LtxA represents the first bacterial toxin shown to localize to the lysosome where it induces rapid cell death. PMID:22467872

DiFranco, Kristina M; Gupta, Anukriti; Galusha, Lindsey E; Perez, Jarelys; Nguyen, To-Vy K; Fineza, Camille D; Kachlany, Scott C

2012-05-18

256

Tick capillary feeding for the study of proteins involved in tick-pathogen interactions as potential antigens for the control of tick infestation and pathogen infection  

PubMed Central

Background Ticks represent a significant health risk to animals and humans due to the variety of pathogens they can transmit during feeding. The traditional use of chemicals to control ticks has serious drawbacks, including the selection of acaricide-resistant ticks and environmental contamination with chemical residues. Vaccination with the tick midgut antigen BM86 was shown to be a good alternative for cattle tick control. However, results vary considerably between tick species and geographic location. Therefore, new antigens are required for the development of vaccines controlling both tick infestations and pathogen infection/transmission. Tick proteins involved in tick-pathogen interactions may provide good candidate protective antigens for these vaccines, but appropriate screening procedures are needed to select the best candidates. Methods In this study, we selected proteins involved in tick-Anaplasma (Subolesin and SILK) and tick-Babesia (TROSPA) interactions and used in vitro capillary feeding to characterize their potential as antigens for the control of cattle tick infestations and infection with Anaplasma marginale and Babesia bigemina. Purified rabbit polyclonal antibodies were generated against recombinant SUB, SILK and TROSPA and added to uninfected or infected bovine blood to capillary-feed female Rhipicephalus (Boophilus) microplus ticks. Tick weight, oviposition and pathogen DNA levels were determined in treated and control ticks. Results The specificity of purified rabbit polyclonal antibodies against tick recombinant proteins was confirmed by Western blot and against native proteins in tick cell lines and tick tissues using immunofluorescence. Capillary-fed ticks ingested antibodies added to the blood meal and the effect of these antibodies on tick weight and oviposition was shown. However, no effect was observed on pathogen DNA levels. Conclusions These results highlighted the advantages and some of the disadvantages of in vitro tick capillary feeding for the characterization of candidate tick protective antigens. While an effect on tick weight and oviposition was observed, the effect on pathogen levels was not evident probably due to high tick-to-tick variations among other factors. Nevertheless, these results together with previous results of RNA interference functional studies suggest that these proteins are good candidate vaccine antigens for the control of R. microplus infestations and infection with A. marginale and B. bigemina. PMID:24450836

2014-01-01

257

Fine Specificity of Plasmodium vivax Duffy Binding Protein Binding Engagement of the Duffy Antigen on Human Erythrocytes  

PubMed Central

Plasmodium vivax invasion of human erythrocytes requires interaction of the P. vivax Duffy binding protein (PvDBP) with its host receptor, the Duffy antigen (Fy) on the erythrocyte surface. Consequently, PvDBP is a leading vaccine candidate. The binding domain of PvDBP lies in a cysteine-rich portion of the molecule called region II (PvDBPII). PvDBPII contains three distinct subdomains based upon intramolecular disulfide bonding patterns. Subdomain 2 (SD2) is highly polymorphic and is thought to contain many key residues for binding to Fy, while SD1 and SD3 are comparatively conserved and their role in Fy binding is not well understood. To examine the relative contributions of the different subdomains to binding to Fy and their abilities to elicit strain-transcending binding-inhibitory antibodies, we evaluated recombinant proteins from SD1+2, SD2, SD3, and SD3+, which includes 24 residues of SD2. All of the recombinant subdomains, except for SD2, bound variably to human erythrocytes, with constructs containing SD3 showing the best binding. Antisera raised in laboratory animals against SD3, SD3+, and SD2+3 inhibited the binding of full-length PvDBPII, which is strain transcending, whereas antisera generated to SD1+2 and SD2 failed to generate blocking antibodies. All of the murine monoclonal antibodies generated to full-length PvDBPII that had significant binding-inhibitory activity recognized only SD3. Thus, SD3 binds Fy and elicits blocking antibodies, indicating that it contains residues critical to Fy binding that could be the basis of a strain-transcending candidate vaccine against P. vivax. PMID:22615246

Siddiqui, Asim A.; Xainli, Jia; Schloegel, Jesse; Carias, Lenore; Ntumngia, Francis; Shoham, Menachem; Casey, Joanne L.; Foley, Michael; Adams, John H.

2012-01-01

258

The ESAT-6 Protein of Mycobacterium tuberculosis Interacts with Beta-2-Microglobulin (?2M) Affecting Antigen Presentation Function of Macrophage  

PubMed Central

ESAT-6, an abundantly secreted protein of Mycobacterium tuberculosis (M. tuberculosis) is an important virulence factor, inactivation of which leads to reduced virulence of M. tuberculosis. ESAT-6 alone, or in complex with its chaperone CFP-10 (ESAT-6:CFP-10), is known to modulate host immune responses; however, the detailed mechanisms are not well understood. The structure of ESAT-6 or ESAT-6:CFP-10 complex does not suggest presence of enzymatic or DNA-binding activities. Therefore, we hypothesized that the crucial role played by ESAT-6 in the virulence of mycobacteria could be due to its interaction with some host cellular factors. Using a yeast two-hybrid screening, we identified that ESAT-6 interacts with the host protein beta-2-microglobulin (?2M), which was further confirmed by other assays, like GST pull down, co-immunoprecipitation and surface plasmon resonance. The C-terminal six amino acid residues (90–95) of ESAT-6 were found to be essential for this interaction. ESAT-6, in complex with CFP-10, also interacts with ?2M. We found that ESAT-6/ESAT-6:CFP-10 can enter into the endoplasmic reticulum where it sequesters ?2M to inhibit cell surface expression of MHC-I-?2M complexes, resulting in downregulation of class I-mediated antigen presentation. Interestingly, the ESAT-6:?2M complex could be detected in pleural biopsies of individuals suffering from pleural tuberculosis. Our data highlight a novel mechanism by which M. tuberculosis may undermine the host adaptive immune responses to establish a successful infection. Identification of such novel interactions may help us in designing small molecule inhibitors as well as effective vaccine design against tuberculosis. PMID:25356553

Parveen, Nazia; Jha, Vishwanath; Valluri, Vijaya Lakshmi; Ghosh, Sudip; Mukhopadhyay, Sangita

2014-01-01

259

Bactericidal/permeability-increasing protein (BPI) is an important antigen for anti-neutrophil cytoplasmic autoantibodies (ANCA) in vasculitis.  

PubMed Central

Indirect immunofluorescence (IIF) techniques have shown that ANCA are useful serological markers for some small vessel vasculitides, and ELISA assays, using purified molecules as solid-phase ligand, have helped to identify proteinase 3 (PR3) and myeloperoxidase (MPO) as two of the major ANCA antigens. There remain a substantial number of serum samples, which are positive by IIF, yet recognize neither PR3 nor MPO (double-negative samples). We found, by Western blot analysis of soluble neutrophil granule proteins, that certain of these double-negative samples recognized a 55-kD doublet of which the first eight residues shared N-terminal amino acid sequence homology with BPI, a potent antibiotic towards Gram-negative bacteria. We developed a simple, quick and robust two-step immunobiochemical method to purify BPI. This was then employed to detect anti-BPI autoantibodies by ELISA and Western blot analysis. We tested 100 double-negative samples and 400 consecutive new samples sent for routine ANCA testing in the anti-BPI ELISA. We found that 45 of the 100 double-negative and 44 of the 400 new routine samples recognized BPI. By Western blot analysis 20/20 positive anti-BPI samples blotted the 55-kD protein. Inhibition assays confirmed the specificity of binding. Review of the 89 anti-BPI-positive patients showed a male dominance (M:F ratio 55:34), a mean age of 60.4 years and clinical diagnoses ranging from organ limited vasculitis to widespread systemic vasculitis. Images Fig. 2 Fig. 5 PMID:7813109

Zhao, M H; Jones, S J; Lockwood, C M

1995-01-01

260

Surface-Exposed Antigenic Cleavage Fragments ofNeisseria gonorrhoeae Proteins IAandIB  

Microsoft Academic Search

andproteinase K.Protein IA(PIA) ofstrain 7122(0,serotype 1,serogroupI)was resistant toproteolysis bytolylsulfonyl phenylalanyl chloromethyl ketone-trypsin andalpha-chymotrypsin andonlyslightly affected byproteinase K, aslong asitwasassociated withintact bacteria orisolated outer membranes. Purified PIAhowever wascleaved bythese enzymes,resulting intwotofivefragments. Incontrast, allpreparations ofstrains 5766opaque phenotype (O',serotype 7,serogroupII)and1955(O+,serotype 9b,serogroupIII) were accessible to proteolysis, resulting incleavage fragments ofPIBcompatible tothose described previously by0. Barrera and J.Swanson(Infect. Immun.44:565-568, 1984), M.S.Blakeetal.(Infect. Immun.33:212-222, 1981), and Blake(inG.K.Schoolnik,

STEFAN SCHMITT; GERLINDE LAYH; THOMAS B. BUCHANAN

1986-01-01

261

The Mycobacterium leprae antigen 85 complex gene family: identification of the genes for the 85A, 85C, and related MPT51 proteins.  

PubMed Central

The genes for two novel members (designated 85A and 85C) of the Mycobacterium leprae antigen 85 complex family of proteins and the gene for the closely related M. leprae MPT51 protein were isolated. The complete DNA sequence of the M. leprae 85C gene and partial sequences of the 85A and MPT51 genes are presented. As in M. tuberculosis, the M. leprae 85A, 85C, and previously identified 85B component genes are not closely linked on the genome. However, the MPT51 genes of both species localize close to the respective 85A component genes. Like the 85B component, the M. leprae 85A-MPT51 and 85C antigens are recognized by T cells from healthy contacts and leprosy patients. Images PMID:8359887

Rinke de Wit, T F; Bekelie, S; Osland, A; Wieles, B; Janson, A A; Thole, J E

1993-01-01

262

Antigenic Properties and Diagnostic Potential of Baculovirus-Expressed Infectious Bursal Disease Virus Proteins VPX and VP3  

Microsoft Academic Search

The routine technique for detecting antibodies specific to infectious bursal disease virus (IBDV) is a serological evaluation by enzyme-linked immunosorbent assay (ELISA) with preparations of whole virions as the antigens. To avoid using complete virus in the standard technique, we have developed two new antigens through the expression of the VPX and VP3 genes in insect cells. VPX and especially

JORGE L. MARTINEZ-TORRECUADRADA; BEATRIZ LAZARO; J. F. Rodriguez; J. I. Casal

2000-01-01

263

Prevalence of Kaposi's sarcoma associated herpesvirus infection measured by antibodies to recombinant capsid protein and latent immunofluorescence antigen  

Microsoft Academic Search

Summary Background Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8, may be the infectious cause of KS. Its prevalence in the general population, on the basis of detection of the virus genome, is controversial. To investigate the seroprevalence, we measured antibodies to a recombinant capsid-related (lytic cycle) KSHV antigen and a latent antigen complex. Methods We selected potentially

Guy R Simpson; Thomas F Schulz; Denise Whitby; Pamela M Cook; Chris Boshoff; Lucille Rainbow; Mark R Howard; Shou-Jiang Gao; Roy A Bohenzky; Peter Simmonds; Christine Lee; Annemiek de Ruiter; Angelos Hatzakis; Richard S Tedder; Ian V D Weller; Robin A Weiss; Patrick S Moore

1996-01-01

264

Diversity of ?? T-cell antigens  

PubMed Central

In the last two decades, it has become clear that ?? T cells recognize a diverse array of antigens including self and foreign, large and small, and peptidic and non-peptidic molecules. In this respect, ?? antigens as a whole resemble more the antigens recognized by antibodies than those recognized by ?? T cells. Because of this antigenic diversity, no single mechanism—such as the major histocompatibility complex (MHC) restriction of ?? T cells—is likely to provide a basis for all observed T-cell antigen receptor (TCR)-dependent ?? T-cell responses. Furthermore, available evidence suggests that many individual ?? T cells are poly-specific, probably using different modes of ligand recognition in their responses to unrelated antigens. While posing a unique challenge in the maintenance of self-tolerance, this broad reactivity pattern might enable multiple overlapping uses of ?? T-cell populations, and thus generate a more efficient immune response. PMID:23085946

Born, Willi K; Kemal Aydintug, M; O'Brien, Rebecca L

2013-01-01

265

Immunohistochemical study of seminiferous epithelium in adult bovine testis using monoclonal antibodies against Ki67 protein and proliferating cell nuclear antigen (PCNA)  

Microsoft Academic Search

The distribution pattern of proliferating cell nuclear antigen (PCNA) and Ki-67 protein was studied in adult bovine seminiferous epithelium by means of immunohistochemistry using monoclonal antibodies. Tailoring the methodological protocol for each of the two proliferation markers was a necessary prerequisite for obtaining optimal results in tubular sections and whole-mounts. A-, I- and B-spermatogonia displayed PCNA-positive nuclei, except during meta-,

Karl-Heinz Wrobel; Daniela Bickel; Richard Kujat

1996-01-01

266

Expression, immunolocalization, and serological reactivity of a novel sphingomyelin phosphodiesterase-like protein, an excretory/secretory antigen from Clonorchis sinensis.  

PubMed

Clonorchiasis, caused by Clonorchis sinensis infection, is a zoonotic parasitic disease of hepatobiliary system in which the proteins released by adult are major pathogenetic factors. In this study, we first characterized a putative sphingomyelin phosphodiesterase (CsSMPase) A-like secretory protein, which was highly expressed in the adult worm. The full-length gene was cloned. The putative protein is of relatively low homology comparing with SMPase from other species, and of rich T cell and B cell epitopes, suggesting that it is an antigen of strong antigenicity. The complete coding sequence of the gene was expressed in the Escherichia coli. The recombinant CsSMPase (rCsSMPase) can be recognized by C. sinensis-infected serum, and the protein immunoserum can recognize a specific band in excretory/secretory products (ESPs) of C. sinensis adult by western blotting. Immunolocalization revealed that CsSMPase was not only localized on tegument, ventral sucker of metacercaria, and the intestine of adult but also on the nearby epithelium of bile duct of the infected Sprague-Dawley rats, implying that CsSMPase was mainly secreted and excreted through adult intestine and directly interacted with bile duct epithelium. Although immunized rats evoked high level antibody response, the antigen level was low in clonorchiasis patients. And the sensitivity and specificity of rCsSMPase were 50.0 % (12/24) and 88.4 % (61/69), in sera IgG-ELISA, respectively. It is likely due to the fact that CsSMPase binding to the plasma membrane of biliary epithelium decreases the antigen immune stimulation. PMID:23568580

Huang, Yanwei; Zheng, Youwei; Li, Yuzhe; Yang, Mei; Li, Ting; Zeng, Suxiang; Yu, Xinbing; Huang, Huaiqiu; Hu, Xuchu

2013-06-01

267

Intracellular Delivery of a Protein Antigen with an Endosomal-Releasing Polymer Enhances CD8 T-Cell Production and Prophylactic Vaccine Efficacy  

PubMed Central

Protein-based vaccines have significant potential as infectious disease and anticancer therapeutics, but clinical impact has been limited in some applications by their ability to generate a coordinated cellular immune response. Here, a pH-responsive carrier incorporating poly(propylacrylic acid) (PPAA was evaluated to test whether improved cytosolic delivery of a protein antigen could enhance CD8+ cytotoxic lymphocyte generation and prophylactic tumor vaccine responses. PPAA was directly conjugated to the model ovalbumin antigen via reducible disulfide linkages and was also tested in a particulate formulation after condensation with the cationic poly(dimethylaminoethyl methacrylate) (PDMAEMA). Intracellular trafficking studies revealed that both PPAA-containing formulations were stably internalized compared to control conjugates and evaded exocytotic pathways, leading to increased intracellular accumulation and potential access to the cytosolic MHC-1 antigen presentation pathway. In an EG.7-OVA mouse tumor protection model, both PPAA-containing carriers robustly inhibited tumor growth and led to an approximately 3.5 fold increase in the longevity of tumor free survival relative to controls. Mechanistically this response was attributed to the 8-fold increase in production of ovalbumin-specific CD8+ T-lymphocytes and an 11-fold increase in production of anti-ovalbumin IgG. Significantly, this is one of the first demonstrated examples of in vivo immunotherapeutic efficacy using soluble protein-polymer conjugates. These results suggest that carriers enhancing cytosolic delivery of protein antigens could lead to more robust CD8+ T-cell response and demonstrate the potential of pH-responsive PPAA-based carriers for therapeutic vaccine applications. PMID:21043513

Foster, Suzanne; Duvall, Craig L.; Crownover, Emily F.; Hoffman, Allan S.; Stayton, Patrick S.

2010-01-01

268

Generation of a Baculovirus Recombinant Prostate-Specific Membrane Antigen and Its Use in the Development of a Novel Protein Biochip Quantitative Immunoassay  

Microsoft Academic Search

Prostate-specific membrane antigen (PSMA) is a 100-kDa transmembrane glycoprotein identified by the monoclonal antibody 7E11-C5.3 from the human prostate tumor cell line LNCaP. Because of its significant upregulation in androgen refractory and metastatic prostate cancers, PSMA may be a useful prognostic biomarker and a target for developing novel therapeutic strategies. However, the lack of abundant pure PSMA protein and the

Zhen Xiao; Xi Jiang; Mary Lou Beckett; George L. Wright

2000-01-01

269

Conservation and variability in Archaea: protein antigens with tandem repeats encoded by a cluster of genes with common motifs in Methanosarcina mazei S-6  

Microsoft Academic Search

Three open reading frames, orf492, orf375 and orf783, were identified in a 5.9-kb DNA fragment from the genome of Methanosarcina mazei S-6 that code for proteins recognized by antibodies against cell-surface antigens. The deduced amino-acid (aa) sequences of orfs492 and 375, i.e., ORF492 and ORF375, contain seven and four copies of an approx. 42-aa repeat, respectively. The aa sequence of

Linda E. Mayerhofer; Everly Conway de Macario; Alberto J. L. Macario

1995-01-01

270

Smoking is Unrelated to Female Sexual Function.  

PubMed

Background. Previous research shows that smoking status is unrelated to female sexual difficulties. However, degree of nicotine dependence has not been measured, and the assessment of sexual functioning has not specified penile-vaginal intercourse (henceforth, intercourse), which is more clearly impaired by sexual difficulties than other sexual behaviors. Objectives. To test if smoking status is associated with poorer female sexual function during intercourse, and if nicotine dependence rather than smoking status is related to poorer female sexual function. Methods. During 2012, 129 Portuguese community women reported their smoking status, and completed the Fagerström Test for Nicotine Dependence, the Female Sexual Function Index (FSFI), and an adaptation of the FSFI to assess sexual functioning specifically during intercourse, as well as the desire thereof. Results. Smokers reported higher desire for intercourse and were more likely to have actually engaged in it in the past 4 weeks. Among the coitally active women in the preceding 4 weeks, nicotine dependence correlated with lower desire for intercourse. Smoking status and nicotine dependence were unrelated to arousal, lubrication, orgasm, satisfaction, pain. Conclusions. The findings are consistent with many studies that fail to demonstrate an increased risk of sexual difficulties among female smokers. However, nicotine dependence, rather than smoking status per se, might be associated with lower libido. The results suggest the possibility of an inverse U-shaped relationship between smoking and libido with a moderate use of tobacco being associated with higher sexual desire. PMID:25290661

Costa, Rui Miguel; Peres, Luís

2015-01-28

271

Eimeria tenella heat shock protein 70 enhances protection of recombinant microneme protein MIC2 subunit antigen vaccination against E. tenella challenge.  

PubMed

Heat shock proteins have been reported to stimulate the immune system via innate receptors. Our study found that the novel immunopotentiator, Eimeria tenella (E. tenella) heat shock protein 70 (HSP70), enhanced protective immunity elicited by E. tenella antigen microneme protein 2 (EtMIC2) against avian coccidiosis. It demonstrated that the expression of TLR2 and TLR4 were strongly upregulated in EtHSP70 and EtMIC2 plus EtHSP70 stimulated chicken embryo fibroblasts (CEF) compared with untreated controls and EtMIC2 alone. In addition, the same treatment induced high levels of interleukin (IL)-12 and interferon (IFN)-? that are critical cytokines of innate immunity. In vivo experiments involved using broiler chickens subcutaneously immunized with EtMIC2 alone or EtMIC2 plus EtHSP70 at 7 and 14 days post-hatch, which were then orally challenged with live E. tenella at 7 days following secondary immunization. Body weight gains, cecal lesion scores, fecal oocyst shedding, serum antibody responses against MIC2, and intestinal cytokine transcript levels were assessed as measures of protective immunity. Chickens immunized with EtMIC2 plus EtHSP70 showed increased body weight gains, decreased oocyst shedding, increased serum antibody responses, and high levels of IL-12, IFN-?, and IL-17 compared with the EtMIC2 only or control groups. Moreover, chickens immunized with EtHSP70 alone showed significantly protective effect against E. tenella infection. In summary, this study provides the first evidence of the immunoenhancing activities of EtHSP70 in poultry. PMID:22494937

Zhang, Lei; Ma, Liping; Liu, Renqiang; Zhang, Yunfei; Zhang, Shouping; Hu, Chunmei; Song, Meng; Cai, Jianping; Wang, Ming

2012-09-10

272

Evaluation of an in silico predicted specific and immunogenic antigen from the OmcB protein for the serodiagnosis of Chlamydia trachomatis infections  

PubMed Central

Background The OmcB protein is one of the most immunogenic proteins in C. trachomatis and C. pneumoniae infections. This protein is highly conserved leading to serum cross reactivity between the various chlamydial species. Since previous studies based on recombinant proteins failed to identify a species specific immune response against the OmcB protein, this study evaluated an in silico predicted specific and immunogenic antigen from the OmcB protein for the serodiagnosis of C. trachomatis infections. Results Using the ClustalW and Antigenic programs, we have selected two predicted specific and immunogenic regions in the OmcB protein: the N-terminal (Nt) region containing three epitopes and the C-terminal (Ct) region containing two epitopes with high scores. These regions were cloned into the PinPoint Xa-1 and pGEX-6P-1 expression vectors, incorporating a biotin purification tag and a glutathione-S-transferase tag, respectively. These regions were then expressed in E. coli. Only the pGEX-6P-1 has been found suitable for serological studies as its tag showed less cross reactivity with human sera and was retained for the evaluation of the selected antigens. Only the Ct region of the protein has been found to be well expressed in E. coli and was evaluated for its ability to be recognized by human sera. 384 sera were tested for the presence of IgG antibodies to C. trachomatis by our in house microimmunofluorescence (MIF) and the developed ELISA test. Using the MIF as the reference method, the developed OmcB Ct ELISA has a high specificity (94.3%) but a low sensitivity (23.9). Our results indicate that the use of the sequence alignment tool might be useful for identifying specific regions in an immunodominant antigen. However, the two epitopes, located in the selected Ct region, of the 24 predicted in the full length OmcB protein account for approximately 25% of the serological response detected by MIF, which limits the use of the developed ELISA test when screening C. trachomatis infections. Conclusion The developed ELISA test might be used as a confirmatory test to assess the specificity of serological results found by MIF. PMID:19077181

Frikha-Gargouri, Olfa; Gdoura, Radhouane; Znazen, Abir; Gargouri, Boutheina; Gargouri, Jalel; Rebai, Ahmed; Hammami, Adnene

2008-01-01

273

Negative staining and immunoelectron microscopy of adhesion-deficient mutants of Streptococcus salivarius reveal that the adhesive protein antigens are separate classes of cell surface fibril.  

PubMed Central

The subcellular distribution of the cell wall-associated protein antigens of Streptococcus salivarius HB, which are involved in specific adhesive properties of the cells, was studied. Mutants which had lost the adhesive properties and lacked the antigens at the cell surface were compared with the parent strain. Immunoelectron microscopy of cryosections of cells labeled with affinity-purified, specific antisera and colloidal gold-protein A complexes was used to locate the antigens. Antigen C (AgC), a glycoprotein involved in attachment to host surfaces, was mainly located in the fibrillar layer outside the cell wall. A smaller amount of label was also found throughout the cytoplasmic area in the form of small clusters of gold particles, which suggests a macromolecular association. Mutant HB-7, which lacks the wall-associated AgC, accumulated AgC reactivity intracellularly. Intracellular AgC was often found associated with isolated areas of increased electron density, but sometimes seemed to fill the entire interior of the cell. Antigen B (AgB), a protein responsible for interbacterial coaggregation, was also located in the fibrillar layer, although its distribution differed from that of the wall-associated AgC since AgB was found predominantly in the peripheral areas. A very small amount of label was also found in the cytoplasmic area as discrete gold particles. Mutant HB-V5, which lacks wall-associated AgB, was not labeled in the fibrillar coat, but showed the same weak intracellular label as the parent strain. Immunolabeling with serum against AgD, another wall-associated protein but of unknown function, demonstrated its presence in the fibrillar layer of strain HB. Negatively stained preparations of whole cells of wild-type S. salivarius and mutants that had lost wall-associated AgB or AgC revealed that two classes of short fibrils are carried on the cell surface at the same time. AgB and AgC are probably located on separate classes of short, protease-sensitive fibrils 91 and 72 nm in length, respectively. A third class of only very sparsely distributed short fibrils (63 nm) was observed on mutant HB-V51, which lacks both wall-associated AgB and AgC antigens. The identity of these fibrils and whether they are present on the wild type are not clear. The function of long, protease-resistant fibrils of 178 nm, which are also present on the wild-type strain, remains unknown. Images PMID:2419308

Weerkamp, A H; Handley, P S; Baars, A; Slot, J W

1986-01-01

274

Induction of protective T-helper 1 immune responses against Echinococcus granulosus in mice by a multi-T-cell epitope antigen based on five proteins  

PubMed Central

In this study, we designed an experiment to predict a potential immunodominant T-cell epitope and evaluate the protectivity of this antigen in immunised mice. The T-cell epitopes of the candidate proteins (EgGST, EgA31, Eg95, EgTrp and P14-3-3) were detected using available web-based databases. The synthesised DNA was subcloned into the pET41a+ vector and expressed in Escherichia coli as a fusion to glutathione-S-transferase protein (GST). The resulting chimeric protein was then purified by affinity chromatography. Twenty female C57BL/6 mice were immunised with the antigen emulsified in Freund's adjuvant. Mouse splenocytes were then cultured in Dulbecco's Modified Eagle's Medium in the presence of the antigen. The production of interferon-? was significantly higher in the immunised mice than in the control mice (> 1,300 pg/mL), but interleukin (IL)-10 and IL-4 production was not statistically different between the two groups. In a challenge study in which mice were infected with 500 live protoscolices, a high protectivity level (99.6%) was demonstrated in immunised BALB/C mice compared to the findings in the control groups [GST and adjuvant (Adj) ]. These results demonstrate the successful application of the predicted T-cell epitope in designing a vaccine against Echinococcus granulosus in a mouse model. PMID:23827994

Esmaelizad, Majid; Ahmadian, Gholamreza; Aghaiypour, Khosrow; Shamsara, Mehdi; Paykari, Habibellah; Tebianian, Majid

2013-01-01

275

Crystal structure of Lyme disease antigen outer surface protein A complexed with?an?Fab  

PubMed Central

OspA (outer surface protein A) is an abundant immunogenic lipoprotein of the Lyme disease spirochete Borrelia burgdorferi. The crystal structure of a soluble recombinant form of OspA was solved in a complex with the Fab fragment of mouse monoclonal antibody 184.1 and refined to a resolution of 1.9 ?. OspA has a repetitive antiparallel ? topology with an unusual nonglobular region of “freestanding” sheet connecting globular N- and C-terminal domains. Arrays of residues with alternating charges are a predominant feature of the folding pattern in the nonglobular region. The 184.1 epitope overlaps with a well conserved surface in the N-terminal domain, and a hydrophobic cavity buried in a positively charged cleft in the C-terminal domain is a potential binding site for an unknown ligand. An exposed variable region on the C-terminal domain of OspA is predicted to be an important factor in the worldwide effectiveness of OspA-based vaccines. PMID:9108020

Li, Hong; Dunn, John J.; Luft, Benjamin J.; Lawson, Catherine L.

1997-01-01

276

Synthesis of a putative subtype specific antigenic heptapeptide from Escherichia coli K88 ad protein fimbriae.  

PubMed

The heptapeptide methyl ester Phe-Asn-Glu-Asn-Met-Ala-Tyr-OMe covering the amino acid sequence of the region 213-219 of Escherichia Coli K88 ad protein fimbriae is synthesized using N alpha-t-butyloxycarbonyl-protection and benzyl groups for side-chain-protection. All condensation reactions are performed in 84-97% yield by preactivation of the protected amino acids by dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt), and reaction of the resulting active ester with amine in the presence of 4-methylmorpholine (NMM). A mechanism is proposed for the nitrile formation in the side-chain of activated asparagine, and the suppression of this side-reaction is investigated. The repetitive deprotection is performed in a mixture of trifluoroacetic acid (TFA), phenol and p-cresol to give the TFA salts in virtually quantitatively yields. The final deprotection of the heptapeptide is carried out in a mixture of 25% hydrogen fluoride (HF) and dimethyl sulfide (DMS) in an overall yield of 48%. The serological and conformational properties of the synthetic peptide are under investigation. PMID:2873700

Meldal, M

1986-04-01

277

Identification of Pigment Cell Antigens Defined by Vitiligo Antibodies  

Microsoft Academic Search

Patients with vitiligo have circulating antibodies to pigment cells. To characterize this response further and to identify the antigens defined by vitiligo antibodies, sera of 23 patients with vitiligo and 22 patients with unrelated conditions were analyzed by immunoprecipitation and SDS-PAGE analysis of 125I-labeled cell antigens on pigment and control cells. Antibodies to pigment cell antigens were present in 18

Jian Cui; Ronald Harning; Milagros Henn; Jean-Claude Bystryn

1992-01-01

278

The extracellular domain of the Epstein-Barr virus BZLF2 protein binds the HLA-DR beta chain and inhibits antigen presentation.  

PubMed Central

The Epstein-Barr virus BZLF2 gene encodes a glycoprotein that associates with gH and gL and facilitates the infection of B lymphocytes. In order to determine whether the BZLF2 protein recognizes a B-cell-specific surface antigen, a soluble protein containing the extracellular portion of the BZLF2 protein linked to the Fc portion of human immunoglobulin G1 (BZLF2.Fc) was expressed from mammalian cells. BZLF2.Fc was used in an expression cloning system and found to bind to a beta-chain allele of the HLA-DR locus of the class II major histocompatibility complex (MHC). Analysis of amino- and carboxy-terminal deletion mutants of the BZLF2.Fc protein indicated that the first 90 amino acids of BZLF2.Fc are not required for HLA-DR beta-chain recognition. Site-directed mutagenesis of an HLA-DR beta-chain cDNA and subsequent immunoprecipitation of expressed mutant beta-chain proteins using BZLF2.Fc indicated that the beta1 domain, which participates in the formation of peptide binding pockets, is required for BZLF2.Fc recognition. The addition of BZLF2.Fc to sensitized peripheral blood mononuclear cells in vitro abolished their proliferative response to antigen and inhibited cytokine-dependent cytotoxic T-cell generation in mixed lymphocyte cultures. Flow-cytometric analysis of Akata cells induced to express late Epstein-Barr virus antigens indicated that expression of BZLF2 did not result in reduced surface expression levels of MHC class II. The ability of BZLF2.Fc to bind to the HLA-DR beta chain suggests that the BZLF2 protein may interact with MHC class II on the surfaces of B cells. PMID:8764069

Spriggs, M K; Armitage, R J; Comeau, M R; Strockbine, L; Farrah, T; Macduff, B; Ulrich, D; Alderson, M R; Müllberg, J; Cohen, J I

1996-01-01

279

Simultaneous cytoplasmic and nuclear protein expression of melanoma antigen-A family and NY-ESO-1 cancer-testis antigens represents an independent marker for poor survival in head and neck cancer.  

PubMed

The prognosis of head and neck squamous cell carcinoma (HNSCC) patients remains poor. The identification of high-risk subgroups is needed for the development of custom-tailored therapies. The expression of cancer-testis antigens (CTAs) has been linked to a worse prognosis in other cancer types; however, their prognostic value in HNSCC is unclear because only few patients have been examined and data on CTA protein expression are sparse. A tissue microarray consisting of tumor samples from 453 HNSCC patients was evaluated for the expression of CTA proteins using immunohistochemistry. Frequency of expression and the subcellular expression pattern (nuclear, cytoplasmic, or both) was recorded. Protein expression of melanoma antigen (MAGE)-A family CTA, MAGE-C family CTA and NY-ESO-1 was found in approximately 30, 7 and 4% of tumors, respectively. The subcellular expression pattern in particular had a marked impact on the patients' prognosis. Median overall survival (OS) of patients with (i) simultaneous cytoplasmic and nuclear expression compared to (ii) either cytoplasmic or nuclear expression and (iii) negative patients was 23.0 versus 109.0 versus 102.5 months, for pan-MAGE (p?protein expression of MAGE-A family members or NY-ESO-1 represent a subgroup with an extraordinarily poor survival. The development of immunotherapeutic strategies targeting these CTA may, therefore, be a promising approach to improve the outcome of HNSCC patients. PMID:24482145

Laban, Simon; Atanackovic, Djordje; Luetkens, Tim; Knecht, Rainald; Busch, Chia-Jung; Freytag, Marcus; Spagnoli, Giulio; Ritter, Gerd; Hoffmann, Thomas K; Knuth, Alexander; Sauter, Guido; Wilczak, Waldemar; Blessmann, Marco; Borgmann, Kerstin; Muenscher, Adrian; Clauditz, Till S

2014-09-01

280

Increased proteolysis of diphtheria toxin by human monocytes after heat shock: a subsidiary role for heat-shock protein 70 in antigen processing  

PubMed Central

The expression of heat-shock proteins (hsp) increases after exposure to various stresses including elevated temperatures, oxidative injury, infection and inflammation. As molecular chaperones, hsp have been shown to participate in antigen processing and presentation, in part through increasing the stability and expression of major histocompatibility complex molecules. Heat shock selectively increases human T-cell responses to processed antigen, but does not affect T-cell proliferation induced by non-processed antigens. Here, we have analysed the mechanisms by which stress such as heat shock, and the ensuing hsp over-expression affect the processing of diphtheria toxin (DT) in peripheral blood monocytes. We found that heat shock increased DT proteolysis in endosomes and lysosomes while the activities of the cathepsins B and D, classically involved in DT proteolysis, were decreased. These effects correlated with the heat-shock-mediated increase in hsp 70 expression observed in endosomes and lysosomes. Actinomycin D or blocking anti-hsp 70 antibodies abolished the heat-shock-mediated increase in DT proteolysis. These data indicate that the increased expression of hsp 70 constitutes a subsidiary mechanism that facilitates antigen proteolysis in stressed cells. Confirming these data, presentation by formaldehyde-fixed cells of DT proteolysates that were obtained with endosomes and lysosomes from heat-shocked peripheral blood monocytes showed higher stimulation of T cells than those generated with endosomes and lysosomes from control peripheral blood monocytes. PMID:17116171

Polla, Barbara S; Gabert, Françoise; Peyrusse, Brigitte M-N; Jacquier-Sarlin, Muriel R

2007-01-01

281

Visualization of Pseudomonas aeruginosa O antigens by using a protein A-dextran-colloidal gold conjugate with both immunoglobulin G and immunoglobulin M monoclonal antibodies.  

PubMed Central

Two lipopolysaccharide O-antigen-specific monoclonal antibodies, MA1-8 (an immunoglobulin G1 [IgG1]) and MF15-4 (an IgM), were used to localize the O antigen of the lipopolysaccharide of Pseudomonas aeruginosa PAO1. A protein A-dextran-gold conjugate with an average particle diameter of 12.5 nm was used to label bacterial cells treated with MA1-8, while a second antibody (goat anti-mouse IgM) was required before the same probe could interact with cells treated with the IgM antibody MF15-4. Both antibodies resulted in exclusive labeling of the surface of P. aeruginosa PAO1 but not that of an isogenic O-antigen-lacking rough mutant. When the monoclonal antibodies became attached to the cell surface of P. aeruginosa PAO1, resulting in an even coating, the foldings and other topographic details could not be discerned by negative staining. In thin sections of monoclonal-antibody-treated bacteria, a 20- and a 30- to 40-nm thick amorphous layer was observed around the outside of the outer membrane when MA1-8 (IgG) and MF15-4 (IgM) plus goat anti-mouse IgM antibodies were used, respectively. This amorphous layer presumably resulted from the stabilization of the lipopolysaccharide structure by the monoclonal antibodies which prevented the long O-antigen chains from collapsing owing to dehydration. Images PMID:2440850

Lam, J S; Lam, M Y; MacDonald, L A; Hancock, R E

1987-01-01

282

Adaptor protein-3 is required in dendritic cells for optimal Toll-like receptor signaling from phagosomes and antigen presentation to CD4+ T cells  

PubMed Central

SUMMARY Effective major histocompatibility complex-II (MHC-II) antigen presentation from phagocytosed particles requires phagosome-intrinsic toll-like receptor (TLR) signaling, but the molecular mechanisms underlying TLR delivery to phagosomes and how signaling regulates antigen presentation are incompletely understood. We show a requirement in dendritic cells (DCs) for adaptor protein-3 (AP-3) in efficient TLR recruitment to phagosomes and MHC-II presentation of antigens internalized by phagocytosis but not receptor-mediated endocytosis. DCs from AP-3-deficient pearl mice elicited impaired CD4+ T cell activation and Th1 effector function to particulate antigen in vitro and to recombinant Listeria monocytogenes infection in vivo. Whereas phagolysosome maturation and peptide:MHC-II complex assembly proceeded normally in pearl DCs, peptide:MHC-II export to the cell surface was impeded. This correlated with reduced TLR4 recruitment and proinflammatory signaling from phagosomes by particulate TLR ligands. We propose that AP-3-dependent TLR delivery from endosomes to phagosomes and subsequent signaling mobilize peptide:MHC-II export from intracellular stores. PMID:22560444

Mantegazza, Adriana R.; Guttentag, Susan H.; El-Benna, Jamel; Sasai, Miwa; Iwasaki, Akiko; Shen, Hao; Laufer, Terri M.; Marks, Michael S.

2012-01-01

283

Characterization of a cross-reactive, immunodominant and HLA-promiscuous epitope of Mycobacterium tuberculosis-specific major antigenic protein PPE68.  

PubMed

PPE68 (Rv3873), a major antignic protein encoded by Mycobacteriun tuberculosis-specific genomic region of difference (RD)1, is a strong stimulator of peripheral blood mononuclear cells (PBMCs) obtained from tuberculosis patients and Mycobacterium bovis bacillus Calmette Guerin (BCG)-vaccianted healthy subjects in T helper (Th)1 cell assays, i.e. antigen-induced proliferation and interferon-gamma (IFN-?) secretion. To confirm the antigen-specific recognition of PPE68 by T cells in IFN-? assays, antigen-induced human T-cell lines were established from PBMCs of M. Bovis BCG-vaccinated and HLA-heterogeneous healthy subjects and tested with peptide pools of RD1 proteins. The results showed that PPE68 was recognized by antigen-specific T-cell lines from HLA-heteregeneous subjects. To further identify the immunodominant and HLA-promiscuous Th1-1 cell epitopes present in PPE68, 24 synthetic peptides covering the sequence of PPE68 were indivdually analyzed for HLA-DR binding prediction analysis and tested with PBMCs from M. bovis BCG-vaccinated and HLA-heterogeuous healthy subjects in IFN-? assays. The results identified the peptide P9, i.e. aa 121-VLTATNFFGINTIPIALTEMDYFIR-145, as an immunodominant and HLA-DR promiscuous peptide of PPE68. Furthermore, by using deletion peptides, the immunodominant and HLA-DR promiscuous core sequence was mapped to aa 127-FFGINTIPIA-136. Interestingly, the core sequence is present in several PPE proteins of M. tuberculosis, and conserved in all sequenced strains/species of M. tuberculosis and M. tuberculosis complex, and several other pathogenic mycobacterial species, including M. leprae and M. avium-intracellulalae complex. These results suggest that the peptide aa 121-145 may be exploited as a peptide-based vaccine candidate against tuberculosis and other mycobacterial diseases. PMID:25136958

Mustafa, Abu S

2014-01-01

284

Androgen receptor-associated protein complex binds upstream of the androgen-responsive elements in the promoters of human prostate-specific antigen and kallikrein 2 genes.  

PubMed Central

An increasing number of proteins which bind to hormone-dependent nuclear receptors and mediate their effects on gene expression are being identified. The human prostate-specific antigen (PSA) and kallikrein 2 (KLK2) genes are regulated by the androgen receptor (AR). Using electrophoresis mobility shift assays (EMSA), a common nuclear protein(s) which binds upstream of the androgen-responsive elements (AREs) in the PSA and KLK2 promoters was identified. Binding occurred between bp -539 and -399 and bp -349 and -224 in the PSA and KLK2 promoters respectively, which were shown previously to be necessary for AR-mediated transactivation. Glutathione S-transferase (GST)-AR fusion proteins were constructed to determine whether the AR interacted directly with this protein or protein complex. Specific interactions were observed with AR fusion proteins containing the DNA binding domain. EMSA supershift experiments and GST-AR pull-down experiments followed by Western blotting identified a Fos-related protein(s) of approximately 40 kDa as part of this complex. Competition experiments with a double-stranded oligonucleotide containing an AP-1 binding site demonstrated that DNA binding was not mediated by AP-1. These results indicate that a Fos-containing protein complex distinct from AP-1 binds upstream of the AREs in the PSA and KLK2 promoters, interacts with the AR and may participate in regulation of these two androgen-responsive genes. PMID:9241247

Sun, Z; Pan, J; Balk, S P

1997-01-01

285

The Shc Family Protein Adaptor, Rai, Negatively Regulates T Cell Antigen Receptor Signaling by Inhibiting ZAP-70 Recruitment and Activation  

PubMed Central

Rai/ShcC is a member of the Shc family of protein adaptors expressed with the highest abundance in the central nervous system, where it exerts a protective function by coupling neurotrophic receptors to the PI3K/Akt survival pathway. Rai is also expressed, albeit at lower levels, in other cell types, including T and B lymphocytes. We have previously reported that in these cells Rai attenuates antigen receptor signaling, thereby impairing not only cell proliferation but also, opposite to neurons, cell survival. Here we have addressed the mechanism underlying the inhibitory activity of Rai on TCR signaling. We show that Rai interferes with the TCR signaling cascade one of the earliest steps –recruitment of the initiating kinase ZAP-70 to the phosphorylated subunit of the TCR/CD3 complex, which results in a generalized dampening of the downstream signaling events. The inhibitory activity of Rai is associated to its inducible recruitment to phosphorylated CD3, which occurs in the physiological signaling context of the immune synapse. Rai is moreover found as a pre-assembled complex with ZAP-70 and also constitutively interacts with the regulatory p85 subunit of PI3K, similar to neuronal cells, notwithstanding the opposite biological outcome, i.e. impairment of PI-3K/Akt activation. The data highlight the ability of Rai to establish interactions with the TCR and key signaling mediators which, either directly (e.g. by inhibiting ZAP-70 recruitment to the TCR or sequestering ZAP-70/PI3K in the cytosol) or indirectly (e.g. by promoting the recruitment of effectors responsible for signal extinction) prevent full triggering of the TCR signaling cascade. PMID:22242145

Ferro, Micol; Savino, Maria Teresa; Ortensi, Barbara; Finetti, Francesca; Genovese, Luca; Masi, Giulia; Ulivieri, Cristina; Benati, Daniela; Pelicci, Giuliana; Baldari, Cosima T.

2011-01-01

286

The Shc family protein adaptor, Rai, negatively regulates T cell antigen receptor signaling by inhibiting ZAP-70 recruitment and activation.  

PubMed

Rai/ShcC is a member of the Shc family of protein adaptors expressed with the highest abundance in the central nervous system, where it exerts a protective function by coupling neurotrophic receptors to the PI3K/Akt survival pathway. Rai is also expressed, albeit at lower levels, in other cell types, including T and B lymphocytes. We have previously reported that in these cells Rai attenuates antigen receptor signaling, thereby impairing not only cell proliferation but also, opposite to neurons, cell survival. Here we have addressed the mechanism underlying the inhibitory activity of Rai on TCR signaling. We show that Rai interferes with the TCR signaling cascade one of the earliest steps--recruitment of the initiating kinase ZAP-70 to the phosphorylated subunit of the TCR/CD3 complex, which results in a generalized dampening of the downstream signaling events. The inhibitory activity of Rai is associated to its inducible recruitment to phosphorylated CD3, which occurs in the physiological signaling context of the immune synapse. Rai is moreover found as a pre-assembled complex with ZAP-70 and also constitutively interacts with the regulatory p85 subunit of PI3K, similar to neuronal cells, notwithstanding the opposite biological outcome, i.e. impairment of PI-3K/Akt activation. The data highlight the ability of Rai to establish interactions with the TCR and key signaling mediators which, either directly (e.g. by inhibiting ZAP-70 recruitment to the TCR or sequestering ZAP-70/PI3K in the cytosol) or indirectly (e.g. by promoting the recruitment of effectors responsible for signal extinction) prevent full triggering of the TCR signaling cascade. PMID:22242145

Ferro, Micol; Savino, Maria Teresa; Ortensi, Barbara; Finetti, Francesca; Genovese, Luca; Masi, Giulia; Ulivieri, Cristina; Benati, Daniela; Pelicci, Giuliana; Baldari, Cosima T

2011-01-01

287

IgG Responses to Pneumococcal and Haemophilus Influenzae Protein Antigens Are Not Impaired in Children with a History of Recurrent Acute Otitis Media  

PubMed Central

Background Vaccines including conserved antigens from Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi) have the potential to reduce the burden of acute otitis media. Little is known about the antibody response to such antigens in young children with recurrent acute otitis media, however, it has been suggested antibody production may be impaired in these children. Methods We measured serum IgG levels against 4 pneumococcal (PspA1, PspA 2, CbpA and Ply) and 3 NTHi (P4, P6 and PD) proteins in a cross-sectional study of 172 children under 3 years of age with a history of recurrent acute otitis media (median 7 episodes, requiring ventilation tube insertion) and 63 healthy age-matched controls, using a newly developed multiplex bead assay. Results Children with a history of recurrent acute otitis media had significantly higher geometric mean serum IgG levels against NTHi proteins P4, P6 and PD compared with healthy controls, whereas there was no difference in antibody levels against pneumococcal protein antigens. In both children with and without a history of acute otitis media, antibody levels increased with age and were significantly higher in children colonised with S. pneumoniae or NTHi compared with children that were not colonised. Conclusions Proteins from S. pneumoniae and NTHi induce serum IgG in children with a history of acute otitis media. The mechanisms in which proteins induce immunity and potential protection requires further investigation but the dogma of impaired antibody responses in children with recurrent acute otitis media should be reconsidered. PMID:23152850

Wiertsema, Selma P.; Corscadden, Karli J.; Mowe, Eva N.; Zhang, Guicheng; Vijayasekaran, Shyan; Coates, Harvey L.; Mitchell, Timothy J.; Thomas, Wayne R.; Richmond, Peter C.; Kirkham, Lea-Ann S.

2012-01-01

288

Interaction of Epstein-Barr Virus Nuclear Antigen Leader Protein (EBNA-LP) with HS1-Associated Protein X-1: Implication of Cytoplasmic Function of EBNA-LP  

PubMed Central

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) consists of W1W2 repeats and a unique C-terminal Y1Y2 domain and has been suggested to play an important role in EBV-induced transformation. To identify the cellular factors interacting with EBNA-LP, we performed a yeast two-hybrid screen, using EBNA-LP cDNA containing four W1W2 repeats as bait and an EBV-transformed human peripheral blood lymphocyte cDNA library as the source of cellular genes. Our results were as follows. (i) All three cDNAs in positive yeast colonies were found to encode the same cellular protein, HS1-associated protein X-1 (HAX-1), which is localized mainly in the cytoplasm and has been suggested to be involved in the regulation of B-cell signal transduction and apoptosis. (ii) Mutational analysis of EBNA-LP revealed that the association with HAX-1 is mediated by the W1W2 repeat domain. (iii) A purified chimeric protein consisting of glutathione S-transferase fused to EBNA-LP specifically formed complexes with HAX-1 transiently expressed in COS-7 cells. (iv) When EBNA-LP and HAX-1 were coexpressed in COS-7 cells, EBNA-LP was specifically coimmunoprecipitated with HAX-1. (v) Careful cell fractionation experiments of an EBV-infected lymphoblastoid cell line revealed that EBNA-LP is localized in the cytoplasm as well as in the nucleus. (vi) When EBNA-LP containing four W1W2 repeats was expressed in COS-7 cells, EBNA-LP was detected mainly in the nucleus by immunofluorescence assay. Interestingly, when EBNA-LP containing a single W1W2 repeat was expressed in COS-7 cells, EBNA-LP was localized predominantly in the cytoplasm and was colocalized with HAX-1. These results indicate that EBNA-LP is in fact present and may have a significant function in the cytoplasm, possibly by interacting with and affecting the function of HAX-1. PMID:11024139

Kawaguchi, Yasushi; Nakajima, Kaori; Igarashi, Mie; Morita, Tomoko; Tanaka, Michiko; Suzuki, Mikiko; Yokoyama, Akihiko; Matsuda, Go; Kato, Kentaro; Kanamori, Mikiko; Hirai, Kanji

2000-01-01

289

Effects of bemiparin on airway responses to antigen in sensitized Brown-Norway rats.  

PubMed

Heparins have demonstrated activity in asthma. The effects of bemiparin, a low molecular weight heparin, were examined on antigen-induced responses in sensitized Brown-Norway rats. Inhaled bemiparin (1 mg/ml) reduced the acute bronchospasm produced by aerosol antigen, prevented airway hyperresponsiveness to 5-hydroxytryptamine postantigen exposure, and reduced the eosinophil count (from 0.205+/-0.062 to 0.054+/-0.016 x 10(6) cells/ml in antigen and antigen+bemiparin groups, respectively; P<0.05), eosinophil peroxidase activity, and proteins in the bronchoalveolar lavage fluid (BALF), as well as the transiently augmented mucin Muc5ac expression. Hyperresponsiveness to adenosine was not affected by bemiparin. In similar experiments, inhaled fondaparinux (1 mg/ml) did not affect the antigen-induced responses, while a low-anticoagulant low molecular weight heparin was effective. In conclusion, bemiparin showed beneficial effects in experimental asthma, probably unrelated to its anticoagulant activity, which extends the previous positive findings obtained with other heparins. PMID:15659317

Suchankova, Jana; Mata, Manuel; Cortijo, Julio; Morcillo, Esteban J

2005-01-10

290

An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins  

PubMed Central

T lymphocytes recognize antigens consisting of peptides presented by class I and II major histocompatibility complex (MHC) molecules. The peptides identified so far have been predictable from the amino acid sequences of proteins. We have identified the natural peptide target of a CTL clone that recognizes the tyrosinase gene product on melanoma cells. The peptide results from posttranslational conversion of asparagine to aspartic acid. This change is of central importance for peptide recognition by melanoma-specific T cells, but has no impact on peptide binding to the MHC molecule. This posttranslational modification has not been previously described for any MHC-associated peptide and represents the first demonstration of posttranslational modification of a naturally processed class I-associated peptide. This observation is relevant to the identification and prediction of potential peptide antigens. The most likely mechanism for production of this peptide leads to the suggestion that antigenic peptides can be derived from proteins that are translated into the endoplasmic reticulum. PMID:8627164

1996-01-01

291

Chronic pneumonia in calves after experimental infection with Mycoplasma bovis strain 1067: Characterization of lung pathology, persistence of variable surface protein antigens and local immune response  

PubMed Central

Background Mycoplasma bovis is associated with pneumonia in calves characterized by the development of chronic caseonecrotic lesions with the agent persisting within the lesion. The purposes of this study were to characterize the morphology of lung lesions, examine the presence of M. bovis variable surface protein (Vsp) antigens and study the local immune responses in calves after infection with M. bovis strain 1067. Methods Lung tissue samples from eight calves euthanased three weeks after experimental infection with M. bovis were examined by bacteriology and pathology. Lung lesions were evaluated by immunohistochemical (IHC) staining for wide spectrum cytokeratin and for M. bovis Vsp antigens and pMB67 antigen. IHC identification and quantitative evaluation of CD4+ and CD8+ T lymphocytes and immunoglobulin (IgG1, IgG2, IgM, IgA)-containing plasma cells was performed. Additionally, expression of major histocompatibility complex class II (MHC class II) was studied by IHC. Results Suppurative pneumonic lesions were found in all calves. In two calves with caseonecrotic pneumonia, necrotic foci were surrounded by epithelial cells resembling bronchial or bronchiolar epithelium. In all calves, M. bovis Vsp antigens were constantly present in the cytoplasm of macrophages and were also present extracellularly at the periphery of necrotic foci. There was a considerable increase in numbers of IgG1- and IgG2-positive plasma cells among which IgG1-containing plasma cells clearly predominated. Statistical evaluation of the numbers of CD4+ and CD8+ T cells, however, did not reveal statistically significant differences between inoculated and control calves. In M. bovis infected calves, hyperplasia of bronchus-associated lymphoid tissue (BALT) was characterized by strong MHC class II expression of lymphoid cells, but only few of the macrophages demarcating the caseonecrotic foci were positive for MHC class II. Conclusions The results from this study show that infection of calves with M. bovis results in various lung lesions including caseonecrotic pneumonia originating from bronchioli and bronchi. There is long-term persistence of M. bovis as demonstrated by bacteriology and immunohistochemistry for M. bovis antigens, i.e. Vsp antigens and pMB67. The persistence of the pathogen and its ability to evade the specific immune response may in part result from local downregulation of antigen presenting mechanisms and an ineffective humoral immune response with prevalence of IgG1 antibodies that, compared to IgG2 antibodies, are poor opsonins. PMID:22305416

2012-01-01

292

Purification and properties of M protein extracted from group A streptococci with pepsin: covalent structure of the amino terminal region of type 24 M antigen  

PubMed Central

M protein was extracted from type 24, group A streptococci with pepsin at pH 5.8 and was further purified by ammonium sulfate precipitation, ribonuclease digestion, ion-exchange chromatography, and isoelectric focusing. The purified pepsin extract of M (pep M) protein was shown to be free of nontype-specific immunoreactivity in (a) complement fixation tests with heterologous M antiserum, (b) skin tests in normal adult guinea pigs, and (c) passive hemagglutination tests for the presence of lipoteichoic acid sensitizing or antigenic activity. The pep M24 was highly immunogenic; two of three rabbits developed opsonic antibody titers of 1:256 and the third a titer of 1:32 6 wk after a single injection of 100-pg doses of pep M24 emulsified in complete Freund's adjuvant. The antisera lacked nontype-specific antibodies and produced single precipitin lines in agar gel diffusion tests against crude HC1 extracts of the homologous M protein. Thus, the type-specific antigenic determinant(s) of type 24 M protein appears to be separable from immunotoxic, cross-reactive antigens without loss of immunogenicity in rabbits. The mobility of pep M24 upon electrophoresis in 10 percent sodium dodecyl sulfate pelyacrylamide gel was consistent with an average mol wt of 33,500 daltons. Amino acid analysis demonstrated a predominance of alanine, followed by glutamic acid, lysine, leucine, and aspartic acid. Pep M24 contained an estimated six to seven methionine residues and approximately ten phenylalanine residues per molecule. No other aromatic amino acids were detected. Automatic Edman degradation of pep M24 yielded the sequence of the first 29 amino acids (the amino terminal amino acid being valine) of the amino terminal region of the molecule. The detection of only one new amino acid at each step of Edman degradation confirmed the homogeneity of the purified pep M24. PMID:325168

Beachey, EH; Stollerman, GH; Chiang, EY; Chiang, TM; Seyer, JM; Kang, AH

1977-01-01

293

The rate of nuclear cytoplasmic protein transport is determined by the casein kinase II site flanking the nuclear localization sequence of the SV40 T-antigen.  

PubMed Central

We have previously demonstrated [Rihs, H.-P. and Peters, R. (1989) EMBO J., 8, 1479-1484] that the nuclear transport of recombinant proteins in which short fragments of the SV40 T-antigen are fused to the amino terminus of Escherichia coli beta-galactosidase is dependent on both the nuclear localization sequence (NLS, T-antigen residues 126-132) and a phosphorylation-site-containing sequence (T-antigen residues 111-125). While the NLS determines the specificity, the rate of transport is controlled by the phosphorylation-site-containing sequence. The present study furthers this observation and examines the role of the various phosphorylation sites. Purified, fluorescently labeled recombinant proteins were injected into the cytoplasm of Vero or hepatoma (HTC) cells and the kinetics of nuclear transport measured by laser microfluorimetry. By replacing serine and threonine residues known to be phosphorylated in vivo, we identified the casein kinase II (CK-II) site S111/S112 to be the determining factor in the enhancement of the transport. Either of the residues 111 or 112 was sufficient to elicit the maximum transport enhancement. The other phosphorylation sites (S120, S123, T124) had no influence on the transport rate. Examination of the literature suggested that many proteins harboring a nuclear localization sequence also contain putative CK-II sites at a distance of approximately 10-30 amino acid residues from the NLS. CK-II has been previously implicated in the transmission of growth signals to the nucleus. Our results suggest that CK-II may exert this role by controlling the rate of nuclear protein transport. Images PMID:1848177

Rihs, H P; Jans, D A; Fan, H; Peters, R

1991-01-01

294

Elongation Factor-1? Is a Novel Protein Associated with Host Cell Invasion and a Potential Protective Antigen of Cryptosporidium parvum *  

PubMed Central

The phylum Apicomplexa comprises obligate intracellular parasites that infect vertebrates. All invasive forms of Apicomplexa possess an apical complex, a unique assembly of organelles localized to the anterior end of the cell and involved in host cell invasion. Previously, we generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina sporozoites. This antigen was highly conserved among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium. In the present study, we identified the apical cytoskeletal antigen of Cryptosporidium parvum (C. parvum) and further characterized this antigen in C. parvum to assess its potential as a target molecule against cryptosporidiosis. Indirect immunofluorescence demonstrated that the reactivity of 6D-12-G10 with C. parvum sporozoites was similar to those of anti-?- and anti-?-tubulins antibodies. Immunoelectron microscopy with the 6D-12-G10 mAb detected the antigen both on the sporozoite surface and underneath the inner membrane at the apical region of zoites. The 6D-12-G10 mAb significantly inhibited in vitro host cell invasion by C. parvum. MALDI-TOF/MS and LC-MS/MS analysis of tryptic peptides revealed that the mAb 6D-12-G10 target antigen was elongation factor-1? (EF-1?). These results indicate that C. parvum EF-1? plays an essential role in mediating host cell entry by the parasite and, as such, could be a candidate vaccine antigen against cryptosporidiosis. PMID:24085304

Matsubayashi, Makoto; Teramoto-Kimata, Isao; Uni, Shigehiko; Lillehoj, Hyun S.; Matsuda, Haruo; Furuya, Masaru; Tani, Hiroyuki; Sasai, Kazumi

2013-01-01

295

Purified protein derivative of Mycobacterium tuberculosis and excretory-secretory antigen(s) of Toxocara canis expand in vitro human T cells with stable and opposite (type 1 T helper or type 2 T helper) profile of cytokine production.  

PubMed Central

A large series of T cell clones (TCC) specific for purified protein derivative (PPD) of Mycobacterium tuberculosis (total 60) or Toxocara canis excretory/secretory (TES) antigen (total 69) were established from the peripheral blood of two healthy individuals and analyzed for their profile of cytokine production in response to stimulation with either the specific antigen or the polyclonal activator phorbol myristate acetate plus anti-CD3 antibody. Under both these experimental conditions, the great majority of PPD-specific TCC secreted IL-2 and IFN-gamma but not, or limited amounts of, IL-4 and IL-5. In contrast, most TES-specific TCC secreted IL-4 and IL-5 but not, or limited amounts of, IL-2 and IFN-gamma. PPD-specific TCC that failed to secrete IL-4 and IL-5, and TES-specific TCC that failed to secrete IL-2 and IFN-gamma, were found to lack transcripts for IL-4 and IL-5, or for IL-2 and IFN-gamma, respectively. During the course of the study, over a 6-mo period, the functional phenotype of both TES- and PPD-specific TCC was repeatedly assessed and remained constant. These data demonstrate that T cells with stable Th1 or Th2 functional pattern exist not only in mice but also in humans and suggest that in the course of natural immunization certain infectious agents preferentially expand T cell subsets with stable and definite profile of cytokine production. PMID:1829097

Del Prete, G F; De Carli, M; Mastromauro, C; Biagiotti, R; Macchia, D; Falagiani, P; Ricci, M; Romagnani, S

1991-01-01

296

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

SciTech Connect

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

Tsurimoto, Toshiki; Stillman, B. (Cold Spring Harbor Laboratory, NY (USA))

1990-02-01

297

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

PubMed Central

The proliferating-cell nuclear antigen (PCNA) and the replication factors A and C (RF-A and RF-C) are cellular proteins essential for complete elongation of DNA during synthesis from the simian virus 40 origin of DNA replication in vitro. All three cooperate to stimulate processive DNA synthesis by DNA polymerase delta on a primed single-stranded M13 template DNA and as such can be categorized as DNA polymerase accessory proteins. Biochemical analyses with highly purified RF-C and PCNA have demonstrated functions that are completely analogous to the functions of bacteriophage T4 DNA polymerase accessory proteins. A primer-template-specific DNA binding activity and a DNA-dependent ATPase activity copurified with the multisubunit protein RF-C and are similar to the functions of the phage T4 gene 44/62 protein complex. Furthermore, PCNA stimulated the RF-C ATPase activity and is, therefore, analogous to the phage T4 gene 45 protein, which stimulates the ATPase function of the gene 44/62 protein complex. Indeed, some primary sequence similarities between human PCNA and the phage T4 gene 45 protein could be detected. These results demonstrate a striking conservation of the DNA replication apparatus in human cells and bacteriophage T4. Images PMID:1967833

Tsurimoto, T; Stillman, B

1990-01-01

298

Additions of killed whole cell bacteria preparations to Freund complete adjuvant alter laying hen antibody response to soluble protein antigen.  

PubMed

Passive transfer of antibodies from hen to egg has value to both the producer of commercial polyclonal egg antibody and the producer of hatching eggs. Water-in-oil emulsions are commonly amended with immune stimulants such as Mycobacteria (e.g., Freund complete adjuvant; FCA) to increase antibody production to soluble protein antigens (SPA). Recent discoveries of the mechanisms by which microbial products act as adjuvants led us to hypothesize that additions of killed whole cell bacteria (bacterins) to FCA could improve antibody responses to SPA. All injections used in each experiment were water-in-oil emulsions (50:50) containing 3 mg/mL of phospholipase A(2) (PLA(2)) immunogen. Additionally, all primary control and treatment injections contained heat-killed Mycobacterium butyricum immunogens from FCA. In addition to PLA(2) and FCA, primary treatment injections contained various microbial bacterin immunogens. Hence, the experimental treatment of all experiments was addition of a commercial source of microbial bacterin to FCA for the primary injection only. Booster injections were the same as the primary control injections except Freund incomplete adjuvant replaced FCA. Anti-body titers to PLA(2) in yolk were determined by ELISA. Bacterins tested as additives to FCA were Escherichia coli, Staphylococcus aureus, Streptococcus suis, and Corynebacterium pseudotuberculosis. Escherichia coli bacterin added to FCA decreased egg yolk antibody titer to SPA by 23% in hens of different ages and strains (P < 0.0001). In a second experiment, a 51% decrease in antibody production associated with E. coli bacterin was sustained for several weeks after the primary immunization (P = 0.003). Staphylococcus aureus or Streptococcus suis combined with FCA increased egg yolk antibody 62 and 51%, respectively (P < 0.05), and Corynebacterium pseudotuberculosis had no effect. In conclusion, the addition of bacterin to FCA can influence hen antibody response to SPA as measured in egg yolks. It is hypothesized that the difference in antibody production may be related to the composition of various pathogen associated molecular patterns in the primary injection. PMID:18420981

Trott, D L; Hellestad, E M; Yang, M; Cook, M E

2008-05-01

299

Pandemic influenza vaccine: characterization of A/California/07/2009 (H1N1) recombinant hemagglutinin protein and insights into H1N1 antigen stability  

PubMed Central

Background The recent H1N1 influenza pandemic illustrated the shortcomings of the vaccine manufacturing process. The A/California/07/2009 H1N1 pandemic influenza vaccine or A(H1N1)pdm09 was available late and in short supply as a result of delays in production caused by low yields and poor antigen stability. Recombinant technology offers the opportunity to shorten manufacturing time. A trivalent recombinant hemagglutinin (rHA) vaccine candidate for seasonal influenza produced using the baculovirus expression vector system (BEVS) was shown to be as effective and safe as egg-derived trivalent inactivated vaccine (TIV) in human clinical studies. In this study, we describe the characterization of the A/California/07/2009 rHA protein and compare the H1N1 pandemic rHA to other seasonal rHA proteins. Results Our data show that, like other rHA proteins, purified A/California/07/2009 rHA forms multimeric rosette-like particles of 20–40?nm that are biologically active and immunogenic in mice as assayed by hemagglutination inhibition (HAI) antibody titers. However, proteolytic digest analysis revealed that A/California/07/2009 rHA is more susceptible to proteolytic degradation than rHA proteins derived from other seasonal influenza viruses. We identified a specific proteolytic site conserved across multiple hemagglutinin (HA) proteins that is likely more accessible in A/California/07/2009 HA, possibly as a result of differences in its protein structure, and may contribute to lower antigen stability. Conclusion We conclude that, similar to the recombinant seasonal influenza vaccine, recombinant A(H1N1)pdm09 vaccine is likely to perform comparably to licensed A(H1N1)pdm09 vaccines and could offer manufacturing advantages. PMID:23110350

2012-01-01

300

Evaluation of protective potential of Yersinia pestis outer membrane protein antigens as possible candidates for a new-generation recombinant plague vaccine.  

PubMed

Plague caused by Yersinia pestis manifests itself in bubonic, septicemic, and pneumonic forms. Although the U.S. Food and Drug Administration recently approved levofloxacin, there is no approved human vaccine against plague. The capsular antigen F1 and the low-calcium-response V antigen (LcrV) of Y. pestis represent excellent vaccine candidates; however, the inability of the immune responses to F1 and LcrV to provide protection against Y. pestis F1(-) strains or those which harbor variants of LcrV is a significant concern. Here, we show that the passive transfer of hyperimmune sera from rats infected with the plague bacterium and rescued by levofloxacin protected naive animals against pneumonic plague. Furthermore, 10 to 12 protein bands from wild-type (WT) Y. pestis CO92 reacted with the aforementioned hyperimmune sera upon Western blot analysis. Based on mass spectrometric analysis, four of these proteins were identified as attachment invasion locus (Ail/OmpX), plasminogen-activating protease (Pla), outer membrane protein A (OmpA), and F1. The genes encoding these proteins were cloned, and the recombinant proteins purified from Escherichia coli for immunization purposes before challenging mice and rats with either the F1(-) mutant or WT CO92 in bubonic and pneumonic plague models. Although antibodies to Ail and OmpA protected mice against bubonic plague when challenged with the F1(-) CO92 strain, Pla antibodies were protective against pneumonic plague. In the rat model, antibodies to Ail provided protection only against pneumonic plague after WT CO92 challenge. Together, the addition of Y. pestis outer membrane proteins to a new-generation recombinant vaccine could provide protection against a wide variety of Y. pestis strains. PMID:23239803

Erova, Tatiana E; Rosenzweig, Jason A; Sha, Jian; Suarez, Giovanni; Sierra, Johanna C; Kirtley, Michelle L; van Lier, Christina J; Telepnev, Maxim V; Motin, Vladimir L; Chopra, Ashok K

2013-02-01

301

Combining Viral Vectored and Protein-in-adjuvant Vaccines Against the Blood-stage Malaria Antigen AMA1: Report on a Phase 1a Clinical Trial.  

PubMed

The development of effective vaccines against difficult disease targets will require the identification of new subunit vaccination strategies that can induce and maintain effective immune responses in humans. Here we report on a phase 1a clinical trial using the AMA1 antigen from the blood-stage Plasmodium falciparum malaria parasite delivered either as recombinant protein formulated with Alhydrogel adjuvant with and without CPG 7909, or using recombinant vectored vaccines-chimpanzee adenovirus ChAd63 and the orthopoxvirus MVA. A variety of promising "mixed-modality" regimens were tested. All volunteers were primed with ChAd63, and then subsequently boosted with MVA and/or protein-in-adjuvant using either an 8- or 16-week prime-boost interval. We report on the safety of these regimens, as well as the T cell, B cell, and serum antibody responses. Notably, IgG antibody responses primed by ChAd63 were comparably boosted by AMA1 protein vaccine, irrespective of whether CPG 7909 was included in the Alhydrogel adjuvant. The ability to improve the potency of a relatively weak aluminium-based adjuvant in humans, by previously priming with an adenoviral vaccine vector encoding the same antigen, thus offers a novel vaccination strategy for difficult or neglected disease targets when access to more potent adjuvants is not possible. PMID:25156127

Hodgson, Susanne H; Choudhary, Prateek; Elias, Sean C; Milne, Kathryn H; Rampling, Thomas W; Biswas, Sumi; Poulton, Ian D; Miura, Kazutoyo; Douglas, Alexander D; Alanine, Daniel Gw; Illingworth, Joseph J; de Cassan, Simone C; Zhu, Daming; Nicosia, Alfredo; Long, Carole A; Moyle, Sarah; Berrie, Eleanor; Lawrie, Alison M; Wu, Yimin; Ellis, Ruth D; Hill, Adrian V S; Draper, Simon J

2014-12-01

302

Antigen-induced suppression of the in vitro lymphocyte response to different antigens and mitogens  

PubMed Central

Certain concentrations of antigen stimulated DNA synthesis in sensitized human lymphocytes cultivated in vitro, higher and lower concentrations being less stimulatory. The simultaneous addition of two antigens in low concentrations to the same cells caused an additive response. The decreased response to a high antigen dose did not affect the capacity of the cells to respond to the simultaneous addition of another antigen, as determined at the population level as well as at the cellular level by autoradiography. Presumably specific immunological paralysis was induced by high antigen doses. Addition of low antigen doses for 1–3 days to human sensitized lymphocytes cultivated in vitro resulted in decreased DNA synthesis as a response to the same antigen added in an optimal dose. Suppression of DNA synthesis was not caused by induction of tolerance or antibody suppression, because the cells also failed to respond to an unrelated antigen and to non-specific mitogens, such as PHA and ALS. Most likely the suppressed response after antigen pretreatment represents a phenomenon analogous to antigenic competition, although this term is not appropriate, since there need not be competition between antigens for a detectable effect. No soluble mediators of suppression could be demonstrated in the supernatant of suppressed cultures. PMID:5026855

Möller, Göran; Kashiwagi, Noboru

1972-01-01

303

Seroreactivity to a Large Panel of Field-Derived Plasmodium falciparum Apical Membrane Antigen 1 and Merozoite Surface Protein 1 Variants Reflects Seasonal and Lifetime Acquired Responses to Malaria.  

PubMed

Parasite antigen diversity poses an obstacle to developing an effective malaria vaccine. A protein microarray containing Plasmodium falciparum apical membrane antigen 1 (AMA1, n = 57) and merozoite surface protein 1 19-kD (MSP119, n = 10) variants prevalent at a malaria vaccine testing site in Bandiagara, Mali, was used to assess changes in seroreactivity caused by seasonal and lifetime exposure to malaria. Malian adults had significantly higher magnitude and breadth of seroreactivity to variants of both antigens than did Malian children. Seroreactivity increased over the course of the malaria season in children and adults, but the difference was more dramatic in children. These results help to validate diversity-covering protein microarrays as a promising tool for measuring the breadth of antibody responses to highly variant proteins, and demonstrate the potential of this new tool to help guide the development of malaria vaccines with strain-transcending efficacy. PMID:25294612

Bailey, Jason A; Pablo, Jozelyn; Niangaly, Amadou; Travassos, Mark A; Ouattara, Amed; Coulibaly, Drissa; Laurens, Matthew B; Takala-Harrison, Shannon L; Lyke, Kirsten E; Skinner, Jeff; Berry, Andrea A; Jasinskas, Algis; Nakajima-Sasaki, Rie; Kouriba, Bourema; Thera, Mahamadou A; Felgner, Philip L; Doumbo, Ogobara K; Plowe, Christopher V

2015-01-01

304

Human cytotoxic T cells stimulated by antigen on dendritic cells recognize the N, SH, F, M, 22K, and 1b proteins of respiratory syncytial virus.  

PubMed Central

We examined the human cytotoxic T-cell repertoire of nine adults to 9 of the 10 proteins of respiratory syncytial (RS) virus. Peripheral blood mononuclear cells from normal adults were stimulated with RS virus in vitro. The resulting polyclonal cultures were tested for lysis of B-lymphoblastoid cell lines infected with recombinant vaccinia viruses expressing each of nine individual RS virus proteins. The use of peripheral blood dendritic cells to present antigen gave more easily reproducible results over a shorter culture period than conventional methods. The six RS virus proteins most strongly recognized were the nucleoprotein N (nine of nine donors with greater than 10% above background lysis; P = 0.0004), the surface proteins SH (six of nine donors; P = 0.002) and F (five of nine donors; P = 0.008), the matrix proteins M (five of nine donors; P = 0.004) and 22K (three of nine donors; P = 0.01) and the nonstructural protein 1b (six of nine donors; P = 0.004). There was no significant recognition of the major surface glycoprotein G (two of nine donors), the internal phosphoprotein P (one of nine donors), or the nonstructural protein 1c (one of nine donors). Recognition was major histocompatibility complex class I restricted, but no association between major histocompatibility complex phenotype and protein specificity of T cells was seen. Recognition of F and 22K appeared to be associated with recent infection indicated by increased levels of anti-RS virus immunoglobulin G antibody in serum measured by enzyme-linked immunosorbent assay. Since cytotoxic T-cell recognition of RS virus proteins has been demonstrated to be important in the clearance of virus from infected hosts, the N, M, SH, 1b, F, and 22K proteins should be considered potential vaccine components. Images PMID:1548754

Cherrie, A H; Anderson, K; Wertz, G W; Openshaw, P J

1992-01-01

305

A single amino acid substitution (R441A) in the receptor-binding domain of SARS coronavirus spike protein disrupts the antigenic structure and binding activity  

SciTech Connect

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) has two major functions: interacting with the receptor to mediate virus entry and inducing protective immunity. Coincidently, the receptor-binding domain (RBD, residues 318-510) of SAR-CoV S protein is a major antigenic site to induce neutralizing antibodies. Here, we used RBD-Fc, a fusion protein containing the RBD and human IgG1 Fc, as a model in the studies and found that a single amino acid substitution in the RBD (R441A) could abolish the immunogenicity of RBD to induce neutralizing antibodies in immunized mice and rabbits. With a panel of anti-RBD mAbs as probes, we observed that R441A substitution was able to disrupt the majority of neutralizing epitopes in the RBD, suggesting that this residue is critical for the antigenic structure responsible for inducing protective immune responses. We also demonstrated that the RBD-Fc bearing R441A mutation could not bind to soluble and cell-associated angiotensin-converting enzyme 2 (ACE2), the functional receptor for SARS-CoV and failed to block S protein-mediated pseudovirus entry, indicating that this point mutation also disrupted the receptor-binding motif (RBM) in the RBD. Taken together, these data provide direct evidence to show that a single amino acid residue at key position in the RBD can determine the major function of SARS-CoV S protein and imply for designing SARS vaccines and therapeutics.

He Yuxian [Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10021 (United States)]. E-mail: yhe@nybloodcenter.org; Li Jingjing [Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10021 (United States); Jiang Shibo [Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10021 (United States)

2006-05-26

306

Human cytolytic T cell recognition of Yersinia pestis virulence proteins that target innate immune responses.  

PubMed

Cell contact by the plague bacterium Yersinia pestis initiates the injection of several virulence factors that target biochemical pathways critical for host clearance of bacteria. Despite this impairment of innate immunity, it is unclear whether antigen recognition by T cells is equally affected. We present evidence that human cytolytic T cells respond to Y. pestis virulence proteins presented by infected monocytes and dendritic cells. These T cell antigens consisted of a panel of proteins encoded by pCD1, a 70-kDa plasmid that harbors virulence factors and transport proteins of the cell contact-dependent, type III secretion system. Infected cells retained the ability to process and present tetanus toxoid to T cells, which indicates that responses to unrelated antigens were also maintained. Our results indicate that T cell immunity remains functional during Y. pestis infection, which thus suggests the potential benefits of therapeutic vaccination and strategies that emphasize the inclusion of cytotoxic T lymphocyte responses. PMID:17109349

Saikh, Kamal U; Kissner, Teri L; Dyas, Beverly; Tropea, Joseph E; Waugh, David S; Ulrich, Robert G

2006-12-15

307

The evolutionarily conserved ribosomal protein L23 and the cationic urease beta-subunit of Yersinia enterocolitica O:3 belong to the immunodominant antigens in Yersinia-triggered reactive arthritis: implications for autoimmunity.  

PubMed Central

BACKGROUND: Reactive arthritis (ReA) is a T cell mediated inflammatory process. The immune response is primarily directed against a triggering organism, although autoimmunity has been invoked in long-lasting, antibiotic-resistant disease. Although a variety of different species are known to trigger Reactive arthritis, the clinical manifestations are strikingly similar as well as closely associated to the HLA-B27 (70%). MATERIALS AND METHODS: Various antigenic fractions and single antigens of Yersinia enterocolitica were prepared, and their immunological activity was assessed by proliferation of synovial fluid mononuclear cells from 10 Reactive arthritis patients. The gene encoding one hitherto unknown antigen has been sequenced. Nonapeptides deduced from sequences of the target antigens were tested in an assembly assay. RESULTS: Two immunodominant proteins of Yersinia enterocolitica were found, one being the urease beta-subunit and the other the 50 S ribosomal protein L23. The latter has been sequenced and belongs to the evolutionarily conserved ribosomal proteins with homology to procaryotes and eucaryotes. One nonapeptide derived from the urease beta-subunit was identified as a possible epitope for HLA-B27-restricted cytotoxic T cells by its high affinity. This epitope is also highly conserved. CONCLUSION: Sharing of conserved immunodominant proteins between different disease triggering microorganisms could provide an explanation of the shared clinical picture in Reactive arthritis. Moreover, autoimmunity in Reactive arthritis might be mediated by antigen mimicry between evolutionarily conserved epitopes of ribosomal proteins and their host analogs. Images FIG. 1 FIG. 1 PMID:8790600

Mertz, A. K.; Daser, A.; Skurnik, M.; Wiesmüller, K. H.; Braun, J.; Appel, H.; Batsford, S.; Wu, P.; Distler, A.; Sieper, J.

1994-01-01

308

Characterization of anti-P monoclonal antibodies directed against the ribosomal protein-RNA complex antigen and produced using Murphy Roths large autoimmune-prone mice.  

PubMed

Autoantibodies, including anti-ribosomal P proteins (anti-P), are thought to be produced by an antigen-driven immune response in systemic lupus erythematosus (SLE). To test this hypothesis, we reconstituted the ribosomal antigenic complex in vitro using human P0, phosphorylated P1 and P2 and a 28S rRNA fragment covering the P0 binding site, and immunized Murphy Roths large (MRL)/lrp lupus mice with this complex without any added adjuvant to generate anti-P antibodies. Using hybridoma technology, we subsequently obtained 34 clones, each producing an anti-P monoclonal antibody (mAb) that recognized the conserved C-terminal tail sequence common to all three P proteins. We also obtained two P0-specific monoclonal antibodies, but no antibody specific to P1, P2 or rRNA fragment. Two types of mAbs were found among these anti-P antibodies: one type (e.g. 9D5) reacted more strongly with the phosphorylated P1 and P2 than that with their non-phosphorylated forms, whereas the other type (e.g. 4H11) reacted equally with both phosphorylated and non-phosphorylated forms of P1/P2. Both 9D5 and 4H11 inhibited the ribosome/eukaryotic elongation factor-2 (eEF-2)-coupled guanosine triphosphate (GTP)ase activity. However, preincubation with a synthetic peptide corresponding to the C-terminal sequence common to all three P proteins, but not the peptide that lacked the last three C-terminal amino acids, mostly prevented the mAb-induced inhibition of GTPase activity. Thus, at least two types of anti-P were produced preferentially following the immunization of MRL mice with the reconstituted antigenic complex. Presence of multiple copies of the C-termini, particularly that of the last three C-terminal amino acid residues, in the antigenic complex appears to contribute to the immunogenic stimulus. PMID:25255895

Sato, H; Onozuka, M; Hagiya, A; Hoshino, S; Narita, I; Uchiumi, T

2015-02-01

309

Helper T-cell antigenic site identification in the acquired immunodeficiency syndrome virus gp120 envelope protein and induction of immunity in mice to the native protein using a 16-residue synthetic peptide.  

PubMed Central

Much effort has been devoted to the analysis of antibodies to acquired immunodeficiency syndrome virus antigens, but no studies, to our knowledge, have defined antigenic sites of this virus that elicit T-cell immunity, even though such immunity is important in protection against many other viruses. T cells tend to recognize only a limited number of discrete sites on a protein antigen. Analysis of immunodominant helper T-cell sites has suggested that such sites tend to form amphipathic helices. An algorithm based on this model was used to identify two candidate T-cell sites, env T1 and env T2, in the envelope protein of human T-lymphotropic virus type IIIB that were conserved in other human immunodeficiency virus isolates. Corresponding peptides were synthesized and studied in genetically defined inbred and F1 mice for induction of lymph node proliferation. After immunization with a 426-residue recombinant envelope protein fragment, significant responses to native gp 120, as well as to each peptide, were observed in both F1 combinations studied. Conversely, immunization with env T1 peptide induced T-cell immunity to the native gp 120 envelope protein. The genetics of the response to env T1 peptide were further examined and revealed a significant response in three of four independent major histocompatibility haplotypes tested, an indication of high frequency responsiveness in the population. Identification of helper T-cell sites should facilitate development of a highly immunogenic, carrier-free vaccine that induces T-cell and B-cell immunity. The ability to elicit T-cell immunity to the native viral protein by immunization with a 16-residue peptide suggests that such sites represent potentially important components of an effective vaccine for acquired immunodeficiency syndrome. Images PMID:2438696

Cease, K B; Margalit, H; Cornette, J L; Putney, S D; Robey, W G; Ouyang, C; Streicher, H Z; Fischinger, P J; Gallo, R C; DeLisi, C

1987-01-01

310

The Dok-3/Grb2 Protein Signal Module Attenuates Lyn Kinase-dependent Activation of Syk Kinase in B Cell Antigen Receptor Microclusters*  

PubMed Central

Recruitment of the growth factor receptor-bound protein 2 (Grb2) by the plasma membrane-associated adapter protein downstream of kinase 3 (Dok-3) attenuates signals transduced by the B cell antigen receptor (BCR). Here we describe molecular details of Dok-3/Grb2 signal integration and function, showing that the Lyn-dependent activation of the BCR transducer kinase Syk is attenuated by Dok-3/Grb2 in a site-specific manner. This process is associated with the SH3 domain-dependent translocation of Dok-3/Grb2 complexes into BCR microsignalosomes and augmented phosphorylation of the inhibitory Lyn target SH2 domain-containing inositol 5? phosphatase. Hence, our findings imply that Dok-3/Grb2 modulates the balance between activatory and inhibitory Lyn functions with the aim to adjust BCR signaling efficiency. PMID:23223229

Lösing, Marion; Goldbeck, Ingo; Manno, Birgit; Oellerich, Thomas; Schnyder, Tim; Bohnenberger, Hanibal; Stork, Björn; Urlaub, Henning; Batista, Facundo D.; Wienands, Jürgen; Engelke, Michael

2013-01-01

311

The BSR4 protein is up-regulated in Toxoplasma gondii bradyzoites, however the dominant surface antigen recognised  

E-print Network

The BSR4 protein is up-regulated in Toxoplasma gondii bradyzoites, however the dominant surface ino- sitol-anchored proteins, many of which are stage-specific. Previous transient transfection of BSR-transcriptional regulation of the protein. In this study, we show that BSR4 protein is present in both tachyzoites

Sheridan, Jennifer

312

Alum increases antigen uptake, reduces antigen degradation and sustains antigen presentation by DCs in vitro  

PubMed Central

Aluminium adjuvants (alum) have been the only widely approved adjuvants for use in human vaccines since the 1920s, however, the mechanism of action of these adjuvants remains elusive. Due to increasing demand for novel adjuvants, a clearer understanding of the mechanisms that allow these important agents to affect adaptive immune responses will make a significant contribution to the rational design of future vaccines. Using a novel approach to tracking antigen and antigen presentation, we demonstrate that alum induces higher antigen accumulation and increased antigen presentation by dendritic cells (DCs) in vitro. Antigen accumulation was 100-fold higher and antigen presentation 10-fold higher following alum treatment when compared with soluble protein alone. We also observed that alum causes an initial reduction in presentation compared with soluble antigen, but eventually increases the magnitude and duration of antigen presentation. This was associated with reduced protein degradation in DCs following alum treatment. These studies demonstrate the dynamic alterations in antigen processing and presentation induced by alum that underlie enhanced DC function in response to this adjuvant. PMID:22732235

Ghimire, Tirth R.; Benson, Robert A.; Garside, Paul; Brewer, James M.

2012-01-01

313

Identification of a Meiosis-Specific Protein as a Member of the Class of Cancer\\/Testis Antigens  

Microsoft Academic Search

Little is known about the function of human cancer\\/testis antigens (CTAs), such as MAGE, BAGE, GAGE, HOM-MEL-40, and NY-ESO-1, the expression of which is restricted to human malignancies and testis. When screening a cDNA expression library enriched for testis-specific representative long transcripts for reactivity with high-titered IgG antibodies from the serum of a patient with renal cell carcinoma, one repeatedly

Ozlem Tureci; Ugur Sahin; Carsten Zwick; Michael Koslowski; Gerhard Seitz; Michael Pfreundschuh

1998-01-01

314

The P Domain of Norovirus Capsid Protein Forms Dimer and Binds to Histo-Blood Group Antigen Receptors  

Microsoft Academic Search

Received 28 November 2003\\/Accepted 18 February 2004 Noroviruses (NVs) are the most important pathogen of epidemic nonbacterial gastroenteritis. The recent finding that NVs recognize human histo-blood group antigens (HBGAs) as receptors provided a new approach to study the pathogenesis of NVs. Using computational and site-directed mutagenesis approaches, our inves- tigators previously identified a plausible binding pocket in the P domain

Ming Tan; Rashmi S. Hegde; Xi Jiang

2004-01-01

315

Identification of two novel HSP90 proteins in Babesia orientalis: molecular characterization, and computational analyses of their structure, function, antigenicity and inhibitor interaction  

PubMed Central

Background HSP90 protects the cells from heat stress and facilitates protein maturation and stability. The full genome sequences of piroplasms contain two putative HSP90 proteins, which are yet uncharacterized. To this end, the two putative HSP90 proteins of Babesia orientalis were identified and characterized by molecular and in silico methods. Methods The two putative proteins in B. orientalis genome showing homology with putative HSP90 of other piroplasms were cloned and sequenced. A computational analysis was carried out to predict the antigenic determinants, structure and function of these proteins. The interactions of two HSP90 isoforms with respective inhibitors were also examined through docking analysis. Results The length of BoHSP90-A gene (amplified from gDNA) was 2706 bp with one intron from position 997 to 1299 bp. This gene amplified from cDNA corresponded to full length CDS with an open reading frame (ORF) of 2403 bp encoding a 800 amino acid (AA) polypeptide with a predicted size of 91.02 kDa. The HSP90-B gene was intronless with an ORF of 2349 bp, and predicted polypeptide comprised of 797 AA with a size of 90.59 kDa. The AA sequences of these two proteins of B. orientalis were the most identical to those of B. bovis. The BoHSP90-A and BoHSP90-B were recognized as 90 kDa in the parasite lysate by the rabbit antisera raised against the recombinant BoHSP90 proteins. The anti-B. orientalis buffalo serum reacted with the rBoHSP90s expressed in E. coli, indicating that these proteins might be secreted by the parasite before entry into host cells. The overall structure and functional analyses showed several domains involved in ATPase activity, client protein binding and HSP90 dimerization. Likewise, several HSP90 inhibitors showed binding to ATP binding pockets of BoHSP90-A and BoHSP90-B, as observed through protein structure-ligand interaction analysis. Conclusions The two putative HSP90 proteins in B. orientalis were recognized as 90 kDa. The rBoHSP90-A and rBoHSP90-B were reacted with the B. orientalis infected buffalo serum. The computational structure and functional analyses revealed that these two proteins may have chaperonic activity. The protein structure-ligand interaction analyses indicated that these two proteins had many drug target sites. PMID:24970594

2014-01-01

316

An enhanced antigen-retrieval protocol for immunohistochemical staining of formalin-fixed, paraffin-embedded tissues.  

PubMed

Formalin is the most commonly used fixative for light microscopy because of its preservation of -morphological details. A major adverse effect of formalin fixation is formation of cross-linkages between epitopes (amino acid residues) and unrelated proteins by formaldehyde groups. The great majority of monoclonal and polyclonal antibodies used for immunohistochemical (IHC) staining of formalin-fixed, paraffin-embedded (FFPE) tissues necessitate unmasking antigens for antigen retrieval. There are currently two major antigen-retrieval procedures based on treatment of deparaffinized tissue sections with heat or, less commonly, with enzymatic digestion. The use of various antigen-retrieval solutions and heating sources does not allow standardization of IHC staining and minimalization of interlaboratory discrepancies. We developed a novel modified antigen-retrieval protocol for reversing the effect of -formalin fixation. The key feature of this protocol is treatment of deparaffinized tissue sections at reduced constant heat (97(o)C in a water bath) for 40 min in 25 mM Tris-HCl (pH 8.5), 1 mM EDTA, and 0.05% SDS (Tris-EDTA-SDS) buffer. Sections are then immunostained with primary and secondary antibodies conjugated with polymer-labeled Horse Radish Peroxidase. Compared to conventional antigen-retrieval procedures, this protocol more efficiently reverses the effect of formalin fixation of a wide variety of cellular antigens and in most instances decreases the use of primary antibody by 2-40 times, resulting in cost savings. Moreover, this protocol eliminates the need for using different antigen-retrieval methods in the laboratory, which reduces both time and labor for medical technologists. PMID:21370027

Syrbu, Sergei I; Cohen, Michael B

2011-01-01

317

Virus-like particles from escherichia coli-derived untagged papaya ringspot virus capsid protein purified by immobilized metal affinity chromatography enhance the antibody response against a soluble antigen.  

PubMed

There is a growing interest in using virus-like particles (VLPs) as scaffolds for the presentation of antigens of choice to the immune system. In this work, VLPs from papaya ringspot virus capsid protein expressed in Escherichia coli were evaluated as enhancers of antibody response against a soluble antigen. Interestingly, although the capsid protein lacks a histidine tag, its purification by immobilized metal affinity chromatography was achieved. The formation of VLPs was demonstrated by electron microscopy for the first time for this capsid protein. VLPs were enriched by polyethylene glycol precipitation. Additionally, these VLPs were chemically coupled to green fluorescent protein in order to evaluate them as antigen carriers; however, bioconjugate instability was observed. Nonetheless, the adjuvant effect of these VLPs on BALB/c mice was evaluated, using GFP as antigen, resulting in a significant increase in anti-GFP IgG response, particularly, IgG1 class, demonstrating that the VLPs enhance the immune response against the antigen chosen in this study. PMID:25119647

Guerrero-Rodríguez, Jesús; Manuel-Cabrera, Carlos Alberto; Palomino-Hermosillo, Y Apatzingan; Delgado-Guzmán, Paola Guadalupe; Escoto-Delgadillo, Martha; Silva-Rosales, Laura; Herrera-Rodríguez, Sara Elisa; Sánchez-Hernández, Carla; Gutiérrez-Ortega, Abel

2014-12-01

318

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

PubMed Central

AIM: To investigate the effects of p57kip2, cyclinE protein and proliferating cell nuclear antigen (PCNA) on occurrence and progression of human pancreatic cancer. METHODS: The expression of p57kip2, cyclinE protein and PCNA in tumor tissues and adjacent tissues from 32 patients with pancreatic cancer was detected by SP immunohistochemical technique. RESULTS: The positive expression rate of p57kip2 protein in tumor tissues was 46.9%, which was lower than that in adjacent pancreatic tissues (?2 = 5.317, P<0.05). p57kip2 protein positive expression remarkably correlated with tumor cell differentiation (P<0.05), but not with lymph node metastasis (P>0.05). The positive expression rate of cyclinE protein in tumor tissues was 68.8%, which was higher than that in adjacent pancreatic tissues (?2 = 4.063, P<0.05). CyclinE protein positive expression significantly correlated with tumor cell differentiation and lymph node metastasis (P<0.05). The positive expression rate of PCNA in the tumor tissues was 71.9%, which was higher than that in adjacent pancreatic tissues (?2 = 5.189, P<0.05). PCNA positive expression remarkably correlated with tumor cell differentiation and lymph node metastasis (P<0.05). CONCLUSION: The decreased expression of p57kip2 and/or overexpression of cyclinE protein and PCNA may contribute to the occurrence and progression of pancreatic cancer. p57kip2, cyclinE protein, and PCNA play an important role in occurrence and progression of pancreatic cancer. PMID:16124066

Yue, Hui; Jiang, Hui-Yong

2005-01-01

319

Antigenic homogeneity of male Müllerian gland (MG) secretory proteins of a caecilian amphibian with secretory proteins of the mammalian prostate gland and seminal vesicles: evidence for role of the caecilian MG as a male accessory reproductive gland.  

PubMed

Whereas in all other vertebrates the Müllerian ducts of genetic males are aborted during development, under the influence of Müllerian-inhibiting substance, in the caecilian amphibians they are retained as a pair of functional glands. It has long been speculated that the Müllerian gland might be the male accessory reproductive gland but there has been no direct evidence to this effect. The present study was undertaken to determine whether the caecilian Müllerian gland secretory proteins would bear antigenic similarity to secretory proteins of the prostate gland and/or the seminal vesicles of a mammal. The secretory proteins of the Müllerian gland of Ichthyophis tricolor were evaluated for cross-reactivity with antisera raised against rat ventral prostate and seminal vesicle secretory proteins, adopting SDS-PAGE, two-dimensional electrophoresis and immunoblot techniques. Indeed there was a cross-reaction of five Müllerian gland secretory protein fractions with prostatic protein antiserum and of three with seminal vesicle protein antiserum. A potential homology exists because in mammals the middle group of the prostate primordia is derived from a diverticulum of the Müllerian duct. Thus this study, by providing evidence for expression of prostatic and seminal vesicle proteins in the Müllerian gland, substantiates the point that in caecilians the Müllerian glands are the male accessory reproductive glands. PMID:25160003

Radha, Arumugam; Sree, Sreesha; Faisal, Kunnathodi; Kumar, G Pradeep; Oommen, Oommen V; Akbarsha, Mohammad A

2014-10-01

320

HBc and HBe antigenicity and DNA-binding activity of major core protein P22 in hepatitis B virus core particles isolated from the cytoplasm of human liver cells.  

PubMed Central

Highly purified hepatitis B virus core particles were obtained in large amounts from the cytoplasm of infected human liver cells. This DNA polymerase-negative core preparation had only hepatitis B core antigen-specific antigenicity and showed a surprising stability. Two forms of a single protein of 22,000 molecular weight, P22, were resolved electrophoretically; the slower moving species, P22a, appeared to be a reduced form of the protein, and the faster moving species, P22b, could have represented a conformational isomer containing an intramolecular disulfide bond(s). The immunological properties and DNA-binding activity of the reduced form, P22a, were examined following separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by transfer onto nitrocellulose membranes (Western blotting). We found that the hepatitis B virus C gene protein shared the antigenic site responsible for both hepatitis B core and e antigen reactivity. We also demonstrated that the core protein(s) bound specifically the genomic hepatitis B virus DNA in comparison with a plasmid DNA (pBR322). This last observation was further substantiated by a radioimmunological method. P22a was also found to be phosphorylated in vitro by the endogenous protein kinase activity, copurified with the hepatitis B core antigen particles. These findings suggest that P22 is a multifunctional protein which is incorporated into core particles within the cytoplasm of the host cell before DNA encapsidation. A critical role of this protein in hepatitis B virus assembly is suggested. Images PMID:2578575

Petit, M A; Pillot, J

1985-01-01

321

Expression of Foreign Antigens on the Surface of Escherichia coli by Fusion to the Outer Membrane Protein TraT  

Microsoft Academic Search

The traT gene is one of the F factor transfer genes and encodes an outer membrane protein which is involved in interactions between an Escherichia coli and its surroundings. This protein was altered so as to permit the expression of foreign proteins on the outer membrane of E. coli in this study. A 729-bp DNA fragment, including the leader and

Hsin-Hou Chang; Shih Yi Sheu; Szecheng J. Lo

1999-01-01

322

Oral immunization with recombinant Salmonella typhimurium expressing surface protein antigen A of Streptococcus sobrinus: persistence and induction of humoral responses in rats.  

PubMed Central

Recombinant Salmonella typhimurium has been used as an oral vaccine for various microbial pathogens. Here we report immune responses in Fischer rats orally immunized with a recombinant S. typhimurium strain encoding surface protein antigen A (SpaA) of Streptococcus sobrinus. The attenuated S. typhimurium chi 4072 delta cya delta crp delta asd mutant used in this study contains the Asd+ plasmid pYA2905 expressing a fragment of the SpaA protein. Salmonella cells were cleared from spleens by 7 days and from Peyer's patches by 14 days in rats receiving a single oral immunization of 10(9) CFU of chi 4072. In animals receiving multiple (i.e., days 0 and 7 or days 0, 7, and 21) immunizations, Salmonella cells were cleared from the Peyer's patches by 25 days following the initial immunization. Antigen-specific systemic and mucosal antibody responses were greater in rats receiving multiple immunizations than in those receiving a single immunization. Serum anti-Salmonella activity was potentiated following boosting on day 21. Mucosal immunoglobulin A antibody responses were also greater in rats receiving multiple immunizations than in rats receiving a single immunization. Anti-Salmonella and anti-Streptococcus immunoglobulin A activity persisted longer in rats boosted on day 21 than in rats immunized on days 0 and 7. These data indicate that oral immunization of rats with the recombinant S. typhimurium chi 4072(pYA2905) vaccine induces systemic as well as mucosal antibody responses specific to the Salmonella cells and to the cloned SpaA protein. This is the first report of the use of an attenuated mutant of the murine pathogen S. typhimurium as an oral vaccine in rats. Images PMID:8039885

Redman, T K; Harmon, C C; Michalek, S M

1994-01-01

323

Extending Targeted Immune Depletion to Unrelated Cord Blood Transplantation  

Cancer.gov

In this pilot study, patients with leukemia, lymphoma, multiple myeloma, or certain premalignant blood disorders (such as myelodysplastic syndromes) will undergo targeted immune-depleting chemotherapy followed by unrelated double cord blood transplant.

324

Mechanism design for fractional scheduling on unrelated machines  

E-print Network

Mechanism design for fractional scheduling on unrelated machines George Christodoulou1 , Elias,panni}@mpi-inf.mpg.de 2 Department of Informatics, University of Athens elias@di.uoa.gr Abstract. In this paper, we

Christodoulou, Giorgos

325

Antigenic and immunogenic properties of recombinant hemagglutinin proteins from H1N1 A/Brisbane/59/07 and B/Florida/04/06 when produced in various protein expression systems  

PubMed Central

Antibodies directed against the influenza hemagglutinin (HA) protein largely mediate virus neutralization and confer protection against infection. Consequently, many studies and assays of influenza vaccines are focused on HA-specific immune responses. Recombinant HA (rHA) proteins can be produced in a number of protein expression and cell culture systems. These range from baculovirus infection of insect cell cultures, to transient transfection of plants, to stably transfected human cell lines. Furthermore, the rHA proteins may contain genetic modifications, such as histidine tags or trimerization domains, intended to ease purification or enhance protein stability. However, no systematic study of these different forms of the HA protein have been conducted. It is not clear which, if any, of these different protein expression systems or structural modifications improve or diminish the biological behavior of the proteins as immunogens or antigens in immune assays. Therefore we set out to perform systematic evaluation of rHA produced in different proteins expression systems and with varied modifications. Five rHA proteins based on recent strains of seasonal influenza A and five based on influenza B HA were kindly provided by the Biodefense and Emerging Infections Reagent repository (BEIR). These proteins were evaluated in a combination of biochemical and structural assays, in vitro humoral and cellular immune assays, and in an animal vaccination model. Marked differences in the behavior of the individual proteins was evident suggesting that they are not equal when being used to detect an immune response. They were, nevertheless, similar at eliciting neutralizing antibody responses. PMID:22609035

Santiago, Felix W.; Emo, Kris Lambert; Fitzgerald, Theresa; Treanor, John J.

2012-01-01

326

Twelve Unrelated Translocation Mongols: Cytogenetic, Genetic and Parental Age Data  

Microsoft Academic Search

Cytogenetic analyses of 6 unrelated 13–15\\/21 translocation mongols, 6 unrelated 21–22\\/21 translocation mongols and almost all of their parents and sibs have been carried out. Parents carrying the translocation chromosome were found in 2 of the 13–15\\/21 families, but not in any of the 21–22\\/21 families (one father could not be tested). Mean parental ages for either type of translocation

F. R. Sergovich; H. C. Soltan; D. H. Carr

1964-01-01

327

HLA antigens in Japanese populations.  

PubMed Central

HLA antigens in 841 healthy, unrelated Japanese from nine widely separated geographic localities were studied. The five most common antigens observed in order of decreasing frequency were for the HLA-A locus: HLA-A9, A2, A10, AW32 and A11; and for the HLA-B locus: HLA-'B5' (= HLA-B5+B17), BW40, B12, B14 and B8. The allelic frequency of undetected antigens of the HLA-A locus was .14-.37, and that of the HLA-B locus, .32-.67, indicating that there were serological difficulties in typing for Japanese antigens using antisera from Caucasians. Marked gene frequency clines were observed for HLA-A9 and HLA-A2 from south (Okinawa) to north (Nagoya). Two haplotypes, HLA-A9, B5 and HLA-A10, BW40 were shown to be in linkage disequilibrium in four of the nine subpopulations. PMID:941906

Yasuda, N; Tsuji, K; Aizawa, M; Itakura, K; Inou, T

1976-01-01

328

Recognition of CMV pp65 protein antigen by human CD4 T-cell lines induced with an immunodominant peptide pool.  

PubMed

Cellular immunity against cytomegalovirus (CMV) is essential for recovery from infection and control of viral latency. In immunocompromised hosts, this balance between CMV and cellular immunity is lost. Accordingly, restoration of the CD8 compartment specific for CMV is beneficial for immunocompromised patients. It is clear that CMV-specific CD4 cells provide helper functions facilitating long-term persistence of CD8 cells. Considering the dearth of data on CMV-specific T-helper cells, we investigated the CD4 responses to the immunodominant protein pp65 to define antigenic peptides. Such peptides were pooled and used to generate long-term T-cell lines. The lines were responsive to CMV and pp65. T cells were selected with individual peptides to produce monospecific lines for accurate definition of fine epitope specificity and to confirm human leukocyte antigen HLA-DR restriction. Furthermore, these lines lost alloreactivity, suggesting that they can be generated from the allodonor for adoptive immunoreconstitution of stem cell graft recipients. PMID:15172454

Li Pira, Giuseppina; Bottone, Laura; Ivaldi, Federico; Pelizzoli, Roberta; Bracci, Luisa; Lozzi, Luisa; Scarso, Lucia; Tripodi, Gino; Manca, Fabrizio

2004-05-01

329

Major cytoplasmic membrane protein of Legionella pneumophila, a genus common antigen and member of the hsp 60 family of heat shock proteins, induces protective immunity in a guinea pig model of Legionnaires' disease.  

PubMed

We have examined the capacity of the major cytoplasmic membrane protein (MCMP) of Legionella pneumophila, a genus common antigen and member of the hsp 60 family of heat shock proteins, to induce protective immunity in a guinea pig model of Legionnaires' disease. We purified MCMP to homogeneity from L. pneumophila by buffer extraction, ion-exchange chromatography, and molecular sieve chromatography. Guinea pigs immunized with MCMP developed a strong cell-mediated immune response to the immunogen manifest by marked cutaneous delayed-type hypersensitivity. Guinea pigs immunized with MCMP and then challenged with a lethal aerosol dose of L. pneumophila exhibited a high level of protective immunity. Altogether, in four independent experiments, 55 of 64 (86%) animals immunized three times with 0.6-40 micrograms MCMP including 11 of 11 (100%) animals immunized three times with 40 micrograms MCMP survived aerosol challenge with L. pneumophila compared with 1 of 29 (3%) sham-immunized control animals (P < 0.0001, Cochran-Mantel-Haenszel X2 statistic for pooled data). To our knowledge, MCMP is the first member of the hsp 60 family of proteins shown to induce protective immunity to a microbial pathogen. MCMP has potential as a vaccine against Legionnaires' disease. Since MCMP is a genus common antigen, vaccination with a combination of MCMPs derived from different Legionella species has the potential of inducing protective immunity against all the major Legionella species causing human disease. PMID:8432872

Blander, S J; Horwitz, M A

1993-02-01

330

Expression of foreign antigens on the surface of Escherichia coli by fusion to the outer membrane protein TraT  

Microsoft Academic Search

ThetraT gene is one of the F factor transfer genes and encodes an outer membrane protein which is involved in interactions between anEscherichia coli and its surroundings. This protein was altered so as to permit the expression of foreign proteins on the outer membrane ofE. coli in this study. A 729-bp DNA fragment, including the leader and entire structural gene

Hsin-Hou Chang; Shih Yi Sheu; Szecheng J. Lo

1999-01-01

331

Goodpasture Antigen-binding Protein/Ceramide Transporter Binds to Human Serum Amyloid P-Component and Is Present in Brain Amyloid Plaques*  

PubMed Central

Serum amyloid P component (SAP) is a non-fibrillar glycoprotein belonging to the pentraxin family of the innate immune system. SAP is present in plasma, basement membranes, and amyloid deposits. This study demonstrates, for the first time, that the Goodpasture antigen-binding protein (GPBP) binds to human SAP. GPBP is a nonconventional Ser/Thr kinase for basement membrane type IV collagen. Also GPBP is found in plasma and in the extracellular matrix. In the present study, we demonstrate that GPBP specifically binds SAP in its physiological conformations, pentamers and decamers. The START domain in GPBP is important for this interaction. SAP and GPBP form complexes in blood and partly colocalize in amyloid plaques from Alzheimer disease patients. These data suggest the existence of complexes of SAP and GPBP under physiological and pathological conditions. These complexes are important for understanding basement membrane, blood physiology, and plaque formation in Alzheimer disease. PMID:22396542

Mencarelli, Chiara; Bode, Gerard H.; Losen, Mario; Kulharia, Mahesh; Molenaar, Peter C.; Veerhuis, Robert; Steinbusch, Harry W. M.; De Baets, Marc H.; Nicolaes, Gerry A. F.; Martinez-Martinez, Pilar

2012-01-01

332

Antigenic structure of the complete nonstructural (NS) 2 and 5 proteins of hepatitis C virus (HCV): anti-HCV NS2 and NS5 antibody reactivities in relation to HCV serotype, presence of HCV RNA, and acute HCV infection.  

PubMed Central

Antigenic regions within the nonstructural (NS) 2 and 5 proteins of hepatitis C virus (HCV) were identified and characterized by the use of 127 overlapping synthetic peptides and a serum panel consisting of 167 human serum samples from persons with antibodies to HCV. Initially, 20 anti-HCV-positive serum samples were used to screen the peptides covering the complete NS2 and NS5 proteins. Among the 27 overlapping peptides spanning the NS2 protein of HCV, only the peptide covering residues 960 to 975 was recognized by human sera. Within the 100 peptides covering the NS5 protein, major linear antigenic regions were located at residues 2284 to 2329 within the putative NS5a and at residues 2584 to 2599 and 2944 to 2959 within the putative NS5b. Additional minor linear antigenic regions were also identified within the NS5. The sequence of the antigenic region of the NS2 protein is, unlike most parts of the NS2 protein, highly conserved among the described types of HCV, whereas the sequence of the major antigenic region of NS5 shows variability among HCV types. The recognition of a peptide corresponding to a part of the major region of NS5 was found to be dependent on HCV type. In 129 anti-HCV-positive serum samples, the prevalence of antibodies to the NS2 protein was found to be 23% among HCV RNA-positive sera and 10% among HCV RNA-negative sera. In the same samples, reactivity to the major linear antigenic regions of HCV NS5 was found in 68% of the HCV RNA-positive sera and 67% of the HCV RNA-negative sera. Of 18 serum samples from five patients with acute HCV infections, and who seroconverted with respect to anti-HCV, 4 were found to be reactive to one or more of the 100 NS5 peptides and in three serum samples the NS5 reactivities were found to shorten the time for serodiagnosis of cross-reactive with a region form residues 2584 to 2599 of NS5, which has 67% homology with a six-residue sequence of NS2. In conclusion, in this study we have identified and evaluated the potential use of synthetic peptides corresponding to linear antigenic regions of the NS2 and NS5 proteins. PMID:7496964

Zhang, Z X; Chen, M; Sönnerborg, A; Sällberg, M

1994-01-01

333

DNA elongation by the human DNA polymerase lambda polymerase and terminal transferase activities are differentially coordinated by proliferating cell nuclear antigen and replication protein A.  

PubMed

DNA polymerase lambda contains template-dependent (DNA polymerase) and template-independent (terminal transferase) activities. In this study we enzymologically characterized the terminal transferase activity of polymerase lambda (pol lambda-tdt). Pol lambda-tdt activity was strongly influenced by the nature of the 3'-terminal sequence of the DNA substrate, and it required a single-stranded (ss) DNA 3'-overhang of about 9-12 nucleotides for optimal activity. The strong preference observed for pyrimidine versus purine nucleotide incorporation was found to be due, at least partially, to a steric block imposed by the residue Tyr-505 in the active site of pol lambda. Pol lambda-tdt was found to be able to elongate a 3'-ssDNA end by two alternative mechanisms: first, a template-independent one resulting in addition of 1 or 2 nucleotides, and second, a template-dependent one where a homopolymeric tract as short as 3 nucleotides at the 3'-end could be used as a template to direct DNA polymerization by a looping back mechanism. Furthermore repetitive cycles of DNA synthesis resulted in the expansion of such a short homopolymeric terminal sequence. Most importantly we found that the proliferating cell nuclear antigen was able to selectively block the looping back mechanism while stimulating the single terminal nucleotide addition. Finally replication protein A completely suppressed the transferase activity of pol lambda while stimulating the polymerase activity, suggesting that proliferating cell nuclear antigen and replication protein A can coordinate the polymerase and the terminal transferase activities of pol lambda. PMID:15537631

Maga, Giovanni; Ramadan, Kristijan; Locatelli, Giada A; Shevelev, Igor; Spadari, Silvio; Hübscher, Ulrich

2005-01-21

334

Signaling and transcriptional changes critical for transformation of human cells by simian virus 40 small tumor antigen or protein phosphatase 2A B56gamma knockdown.  

PubMed

One set of genes sufficient for transformation of primary human cells uses the combination of Ha-Ras-V12, the telomerase catalytic subunit hTERT, SV40 large tumor antigen (LT), and SV40 small tumor antigen (ST). Whereas SV40 LT inactivates the retinoblastoma protein and p53, the contribution of ST is poorly understood. The essential helper function of ST requires a functional interaction with protein phosphatase 2A (PP2A). Here we have identified changes in gene expression induced by ST and show that ST mediates these changes through both PP2A-dependent and PP2A-independent mechanisms. Knockdown of PP2A B56gamma subunit can substitute for ST expression to fully transform cells expressing LT, hTERT, and Ras-V12. We also identify those genes affected similarly in two cell lines that have been fully transformed from a common parental line by two alternative mechanisms, namely ST expression or PP2A B56gamma subunit knockdown. ST altered expression of genes involved in proliferation, apoptosis, integrin signaling, development, immune responses, and transcriptional regulation. ST reduced surface expression of MHC class I molecules, consistent with a need for SV40 to evade immune detection. ST expression enabled cell cycle progression in reduced serum and src phosphorylation in anchorage-independent media, whereas B56gamma knockdown required normal serum levels for these phenotypes. Inhibitors of integrin and src signaling prevented anchorage-independent growth of transformed cells, suggesting that integrin and src activation are key ST-mediated events in transformation. Our data support a model in which ST promotes survival through constitutive integrin signaling, src phosphorylation, and nuclear factor kappaB activation, while inhibiting cell-cell adhesion pathways. PMID:15466190

Moreno, Carlos S; Ramachandran, Sumathi; Ashby, Danita G; Laycock, Noelani; Plattner, Courtney A; Chen, Wen; Hahn, William C; Pallas, David C

2004-10-01

335

Discovery of Epstein-Barr virus (EBV)-encoded RNA signal and EBV nuclear antigen leader protein DNA sequence in pet dogs.  

PubMed

The aim of this study was to investigate Epstein-Barr virus (EBV)-related virus infection in pet dogs. The presence of antibodies to EBV antigens and EBV-related DNA was determined by Western blot analysis and PCR, respectively. Among 36 pet dogs examined for serum antibodies, 32 (88.9%) were positive for EBV-specific thymidine kinase, 15 (41.7%) for EBV-encoded DNA-binding protein and 10 (27.8%) for EBV-specific DNA polymerase. A BamHI W fragment sequence encoding part of the EBV nuclear antigen leader protein was detected by PCR in corresponding leukocyte DNA samples. Among 21 dogs tested, 15 (71.4%) were positive for the BamHI W fragment sequence. The specificity of the amplified DNA fragments was confirmed by DNA sequencing. Within the amplified region of the BamHI W fragment (241 bp), DNA sequences detected in 10 dogs had 99.2% (two nucleotide variations), 99.6% (one nucleotide variation) or 100% identity to that of EBV. Furthermore, an EBV-encoded RNA signal was detected by in situ hybridization in dog lymphocytes, as well as in bone-marrow sections, indicating a latent infection with EBV or an EBV-like virus. In conclusion, although the sample size was small, these results showed that a widespread EBV-related gammaherpesvirus could be detected in the peripheral blood and bone marrow of pet dogs. Although no evident zoonotic transmission was detected, further studies are imperative for disclosing the biological significance of this canine EBV-like virus, which may correlate with human disorders. PMID:15784884

Chiou, Shiow-Her; Chow, Kuan-Chih; Yang, Chih-Huan; Chiang, Shu-Fen; Lin, Chun-Hao

2005-04-01

336

Comparative study of Plasmodium falciparum erythrocyte membrane protein 1-DBL? domain variants with respect to antigenic variations and docking interaction analysis with glycosaminoglycans.  

PubMed

The variant surface antigen PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1) encoded by the polymorphic multi-copy var gene family plays an important role in parasite biology and the host-parasite interactions. Sequestration and antigenic variation is an essential component in the survival and pathogenesis of Plasmodium falciparum and contributes to chronic infection. The DBL? domain of PfEMP1 is a potential target for immuno-epidemiological studies and has been visualized as a vaccine candidate against severe malaria. Specific host receptors like heparin, heparan sulphate, blood group A and complement receptor 1 have been reported to bind the DBL? domain. Although heparin has been experimentally shown to disrupt the parasite-host interaction and effectively disrupt rosetting, the binding sites for the DBL? domain and the mechanism behind heparin-mediated rosette inhibition have not been elucidated. In this study, 3D structures and epitopes of the DBL? domain in 3D7 and in two Indian isolates have been predicted and compared. We have carried out docking studies on DBL? domains with human GAG receptors (heparin and heparan sulphate) to predict the strength of association between the protein-ligand interactions. The DBL? domain structures showed extensive diversity and polymorphism in their binding sites. The docking results indicate that heparin binds more effectively with high affinity as compared to heparan sulphate with some common interacting residues. These common residues can play an important role in rosetting and will aid in the designing of inhibitors specific to the interactions between DBL? and heparin or heparan sulphate would be important in malaria treatment. Thus it may lead to the development of novel interference strategies to block red blood cell invasion and provide protection against malaria. PMID:24995459

Agrawal, Megha R; Ozarkar, Aarti D; Gupta, Shipra; Deobagkar, Dileep N; Deobagkar, Deepti D

2014-07-29

337

Use of recombinant antigens expressed in Escherichia coli K-12 to map B-cell and T-cell epitopes on the immunodominant 65-kilodalton protein of Mycobacterium bovis BCG.  

PubMed Central

In gene libraries of Mycobacterium bovis BCG, Mycobacterium tuberculosis, and Mycobacterium leprae, recombinants were frequently encountered that expressed an immunodominant 65-kilodalton (kDa) protein antigen that was shown to react with a high proportion of mycobacterium-reactive human and murine T cells and murine monoclonal antibodies. In this study, recombinant antigens were used to map T-cell and B-cell epitopes on the M. bovis BCG 65-kDa protein that was previously designated MbaA. Four different T-cell-epitope-containing regions (amino acid residues 1 through 16, 17 through 61, 85 through 108, and 235 through 279) were defined that were recognized by seven T-cell clones from patients with tuberculoid leprosy. These regions are distinct from two previously described T-cell epitopes recognized by T cells from a tuberculosis patient. As T-cell clones restricted by different class II determinants were shown to be specific for different regions on the 65-kDa protein, the presented data suggested that the products of different human leukocyte antigen class II loci and alleles present different parts of MbaA to the immune system. B-cell epitopes recognized by 20 monoclonal antibodies were assigned to eight different regions of MbaA. Using 15 of these antibodies, we previously showed that MbaA was antigenically related to a common antigen present in many bacterial species. The dispersed localization of the involved epitopes defined here shows that various different parts of MbaA are indeed conserved. These results show that well-defined recombinant antigens are useful tools for the localization of both B- and T-cell-epitope-containing regions of a protein. Peptides synthesized from the sequences of such regions may then exactly define the epitopes relevant for the development of specific diagnostic tests or of vaccines against mycobacteria. Images PMID:2453469

Thole, J E; van Schooten, W C; Keulen, W J; Hermans, P W; Janson, A A; de Vries, R R; Kolk, A H; van Embden, J D

1988-01-01

338

Expression and characterization of highly antigenic domains of chicken anemia virus viral VP2 and VP3 subunit proteins in a recombinant E. coli for sero-diagnostic applications  

PubMed Central

Background Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. Generally, CAV infection occurs via vertical transmission in young chicks that are less than two weeks old, which are very susceptible to the disease. Therefore, epidemiological investigations of CAV infection and/or the evaluation of the immunization status of chickens is necessary for disease control. Up to the present, systematically assessing viral protein antigenicity and/or determining the immunorelevant domain(s) of viral proteins during serological testing for CAV infection has never been performed. The expression, production and antigenic characterization of CAV viral proteins such as VP1, VP2 and VP3, and their use in the development of diagnostic kit would be useful for CAV infection prevention. Results Three CAV viral proteins VP1, VP2 and VP3 was separately cloned and expressed in recombinant E. coli. The purified recombinant CAV VP1, VP2 and VP3 proteins were then used as antigens in order to evaluate their reactivity against chicken sera using indirect ELISA. The results indicated that VP2 and VP3 show good immunoreactivity with CAV-positive chicken sera, whereas VP1 was found to show less immunoreactivity than VP2 and VP3. To carry out the further antigenic characterization of the immunorelevant domains of the VP2 and VP3 proteins, five recombinant VP2 subunit proteins (VP2-435N, VP2-396N, VP2-345N, VP2-171C and VP2-318C) and three recombinant VP3 subunit proteins (VP3-123N, VP3-246M, VP3-366C), spanning the defined regions of VP2 and VP3 were separately produced by an E. coli expression system. These peptides were then used as antigens in indirect ELISAs against chicken sera. The results of these ELISAs using truncated recombinant VP2 and VP3 subunit proteins as coating antigen showed that VP2-345N, VP2-396N and VP3-246M gave good immunoreactivity with CAV-positive chicken sera compared to the other subunit proteins. Moreover, the VP2-396N and VP2-345 based ELISAs had better sensitivity (97.5%) and excellent specificity (100%) during serodiagnosis testing using a mean plus three standard deviations cut-off. The VP3-246M based ELISA showed a sensitivity of 85% and a specificity of 100% at the same cut-off value. Conclusions This is the first report to systematically assess the antigenic characteristics of CAV viral proteins for sero-diagnosis purposes. Purified recombinant VP2-396N and VP2-345N subunit proteins, which span defined regions of VP2, were demonstrated to have good antigenicity and higher sensitivities than VP3-246M and were able to recognize CAV-positive chicken serum using an ELISA assay. The defined antigenicity potential of these chimeric subunit proteins produced by expression in E. coli seem to have potential and could be useful in the future for the development of the CAV diagnostic tests based on a subunit protein ELISA system. PMID:23937712

2013-01-01

339

Nonpermissive HLA-DPB1 mismatch increases mortality after myeloablative unrelated allogeneic hematopoietic cell transplantation.  

PubMed

We examined current outcomes of unrelated donor allogeneic hematopoietic cell transplantation (HCT) to determine the clinical implications of donor-recipient HLA matching. Adult and pediatric patients who had first undergone myeloablative-unrelated bone marrow or peripheral blood HCT for acute myelogenous leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, and myelodysplastic syndrome between 1999 and 2011 were included. All had high-resolution typing for HLA-A, -B, -C, and -DRB1. Of the total (n = 8003), cases were 8/8 (n = 5449), 7/8 (n = 2071), or 6/8 (n = 483) matched. HLA mismatch (6-7/8) conferred significantly increased risk for grades II to IV and III to IV acute graft vs host disease (GVHD), chronic GVHD, transplant-related mortality (TRM), and overall mortality compared with HLA-matched cases (8/8). Type (allele/antigen) and locus (HLA-A, -B, -C, and -DRB1) of mismatch were not associated with overall mortality. Among 8/8 matched cases, HLA-DPB1 and -DQB1 mismatch resulted in increased acute GVHD, and HLA-DPB1 mismatch had decreased relapse. Nonpermissive HLA-DPB1 allele mismatch was associated with higher TRM compared with permissive HLA-DPB1 mismatch or HLA-DPB1 match and increased overall mortality compared with permissive HLA-DPB1 mismatch in 8/8 (and 10/10) matched cases. Full matching at HLA-A, -B, -C, and -DRB1 is required for optimal unrelated donor HCT survival, and avoidance of nonpermissive HLA-DPB1 mismatches in otherwise HLA-matched pairs is indicated. PMID:25161269

Pidala, Joseph; Lee, Stephanie J; Ahn, Kwang Woo; Spellman, Stephen; Wang, Hai-Lin; Aljurf, Mahmoud; Askar, Medhat; Dehn, Jason; Fernandez Viña, Marcelo; Gratwohl, Alois; Gupta, Vikas; Hanna, Rabi; Horowitz, Mary M; Hurley, Carolyn K; Inamoto, Yoshihiro; Kassim, Adetola A; Nishihori, Taiga; Mueller, Carlheinz; Oudshoorn, Machteld; Petersdorf, Effie W; Prasad, Vinod; Robinson, James; Saber, Wael; Schultz, Kirk R; Shaw, Bronwen; Storek, Jan; Wood, William A; Woolfrey, Ann E; Anasetti, Claudio

2014-10-16

340

Nonpermissive HLA-DPB1 mismatch increases mortality after myeloablative unrelated allogeneic hematopoietic cell transplantation  

PubMed Central

We examined current outcomes of unrelated donor allogeneic hematopoietic cell transplantation (HCT) to determine the clinical implications of donor-recipient HLA matching. Adult and pediatric patients who had first undergone myeloablative-unrelated bone marrow or peripheral blood HCT for acute myelogenous leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, and myelodysplastic syndrome between 1999 and 2011 were included. All had high-resolution typing for HLA-A, -B, -C, and -DRB1. Of the total (n = 8003), cases were 8/8 (n = 5449), 7/8 (n = 2071), or 6/8 (n = 483) matched. HLA mismatch (6-7/8) conferred significantly increased risk for grades II to IV and III to IV acute graft vs host disease (GVHD), chronic GVHD, transplant-related mortality (TRM), and overall mortality compared with HLA-matched cases (8/8). Type (allele/antigen) and locus (HLA-A, -B, -C, and -DRB1) of mismatch were not associated with overall mortality. Among 8/8 matched cases, HLA-DPB1 and -DQB1 mismatch resulted in increased acute GVHD, and HLA-DPB1 mismatch had decreased relapse. Nonpermissive HLA-DPB1 allele mismatch was associated with higher TRM compared with permissive HLA-DPB1 mismatch or HLA-DPB1 match and increased overall mortality compared with permissive HLA-DPB1 mismatch in 8/8 (and 10/10) matched cases. Full matching at HLA-A, -B, -C, and -DRB1 is required for optimal unrelated donor HCT survival, and avoidance of nonpermissive HLA-DPB1 mismatches in otherwise HLA-matched pairs is indicated. PMID:25161269

Lee, Stephanie J.; Ahn, Kwang Woo; Spellman, Stephen; Wang, Hai-Lin; Aljurf, Mahmoud; Askar, Medhat; Dehn, Jason; Fernandez Viña, Marcelo; Gratwohl, Alois; Gupta, Vikas; Hanna, Rabi; Horowitz, Mary M.; Hurley, Carolyn K.; Inamoto, Yoshihiro; Kassim, Adetola A.; Nishihori, Taiga; Mueller, Carlheinz; Oudshoorn, Machteld; Petersdorf, Effie W.; Prasad, Vinod; Robinson, James; Saber, Wael; Schultz, Kirk R.; Shaw, Bronwen; Storek, Jan; Wood, William A.; Woolfrey, Ann E.; Anasetti, Claudio

2014-01-01

341

A novel O-linked glycan modulates Campylobacter jejuni major outer membrane protein-mediated adhesion to human histo-blood group antigens and chicken colonization  

PubMed Central

Campylobacter jejuni is an important cause of human foodborne gastroenteritis; strategies to prevent infection are hampered by a poor understanding of the complex interactions between host and pathogen. Previous work showed that C. jejuni could bind human histo-blood group antigens (BgAgs) in vitro and that BgAgs could inhibit the binding of C. jejuni to human intestinal mucosa ex vivo. Here, the major flagella subunit protein (FlaA) and the major outer membrane protein (MOMP) were identified as BgAg-binding adhesins in C. jejuni NCTC11168. Significantly, the MOMP was shown to be O-glycosylated at Thr268; previously only flagellin proteins were known to be O-glycosylated in C. jejuni. Substitution of MOMP Thr268 led to significantly reduced binding to BgAgs. The O-glycan moiety was characterized as Gal(?1–3)-GalNAc(?1–4)-GalNAc(?1–4)-GalNAc?1-Thr268; modelling suggested that O-glycosylation has a notable effect on the conformation of MOMP and this modulates BgAg-binding capacity. Glycosylation of MOMP at Thr268 promoted cell-to-cell binding, biofilm formation and adhesion to Caco-2 cells, and was required for the optimal colonization of chickens by C. jejuni, confirming the significance of this O-glycosylation in pathogenesis. PMID:24451549

Mahdavi, Jafar; Pirinccioglu, Necmettin; Oldfield, Neil J.; Carlsohn, Elisabet; Stoof, Jeroen; Aslam, Akhmed; Self, Tim; Cawthraw, Shaun A.; Petrovska, Liljana; Colborne, Natalie; Sihlbom, Carina; Borén, Thomas; Wooldridge, Karl G.; Ala'Aldeen, Dlawer A. A.

2014-01-01

342

Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen.  

PubMed Central

HeLa cells contain a serine/threonine protein kinase (DNA-PK) that is strongly activated in vitro by low concentrations of double-stranded DNA (dsDNA). Activation was specific for dsDNA; both natural DNAs and synthetic oligonucleotides functioned as kinase activators. The fact that DNA-PK activity was rapidly inhibited by incubation with dsDNA and ATP suggests that DNA-PK activity also may be regulated by autophosphorylation. During gel filtration, DNA-PK activity behaved as a 350-kDa protein, and highly purified DNA-PK contained a dsDNA-binding, 350-kDa polypeptide that was phosphorylated in a dsDNA-dependent manner. We conclude that this 350-kDa polypeptide is likely to be DNA-PK. Previously we showed that the dsDNA-activated kinase phosphorylates two threonines at the N terminus of hsp90 alpha (S. P. Lees-Miller and C. W. Anderson, J. Biol. Chem. 264:17275-17280, 1989). Here we show that DNA-PK also phosphorylates the simian virus 40 large tumor antigen, the mouse tumor-suppressor protein p53, the human Ku autoantigen, and two unidentified HeLa DNA-associated polypeptides of 52 and 110 kDa. Identification of these and other newly identified DNA-binding substrates suggest that the dsDNA-activated kinase may regulate transcription, DNA replication, or cell growth. Images PMID:2247067

Lees-Miller, S P; Chen, Y R; Anderson, C W

1990-01-01

343

Myosin-cross-reactive antigen (MCRA) protein from Bifidobacterium breve is a FAD-dependent fatty acid hydratase which has a function in stress protection  

PubMed Central

Background The aim of this study was to determine the catalytic activity and physiological role of myosin-cross-reactive antigen (MCRA) from Bifidobacterium breve NCIMB 702258. MCRA from B. breve NCIMB 702258 was cloned, sequenced and expressed in heterologous hosts (Lactococcus and Corynebacterium) and the recombinant proteins assessed for enzymatic activity against fatty acid substrates. Results MCRA catalysed the conversion of palmitoleic, oleic and linoleic acids to the corresponding 10-hydroxy fatty acids, but shorter chain fatty acids were not used as substrates, while the presence of trans-double bonds and double bonds beyond the position C12 abolished hydratase activity. The hydroxy fatty acids produced were not metabolised further. We also found that heterologous Lactococcus and Corynebacterium expressing MCRA accumulated increasing amounts of 10-HOA and 10-HOE in the culture medium. Furthermore, the heterologous cultures exhibited less sensitivity to heat and solvent stresses compared to corresponding controls. Conclusions MCRA protein in B. breve can be classified as a FAD-containing double bond hydratase, within the carbon-oxygen lyase family, which may be catalysing the first step in conjugated linoleic acid (CLA) production, and this protein has an additional function in bacterial stress protection. PMID:21329502

2011-01-01

344

High mobility group (HMG) non-histone chromosomal proteins HMG1 and HMG2 are significant target antigens of perinuclear anti-neutrophil cytoplasmic antibodies in autoimmune hepatitis  

PubMed Central

BACKGROUND—High mobility group (HMG) non-histone chromosomal proteins HMG1 and HMG2 have been identified as novel antigens of perinuclear anti-neutrophil cytoplasmic antibodies (p-ANCAs), and the existence of anti-HMG1 and anti-HMG2 antibodies in a population of patients with ulcerative colitis has been reported.?AIMS—To investigate whether HMG1 and HMG2 are target antigens for p-ANCAs in autoimmune hepatitis (AIH).?PATIENTS—Serum samples from 28 patients with AIH, 44 patients with primary biliary cirrhosis (PBC), 27 patients with chronic hepatitis C, and 23 patients with chronic hepatitis B were tested.?METHODS—ANCAs were detected by routine indirect immunofluorescence (IIF). Anti-HMG1 and anti-HMG2 antibodies were assayed by enzyme linked immunosorbent assay.?RESULTS—p-ANCAs were detected in 89% (25/28) of patients with AIH, 36% (16/44) of patients with PBC, 11% (3/27) of patients with chronic hepatitis C, and 13% (3/23) of patients with chronic hepatitis B. Anti-HMG1 and/or anti-HMG2 antibodies were detected in 89% (25/28) of patients with AIH, 70% (31/44) with PBC, 26% (7/27) with chronic hepatitis C, and 9% (2/23) with chronic hepatitis B. In AIH, anti-HMG1 and/or anti-HMG2 antibodies were detected in 96% (24/25) of p-ANCA positive patients. The p-ANCA staining pattern detected by IIF using sera from patients with AIH disappeared or decreased in titre after preincubation with a mixture of HMG1/HMG2. The presence and titres of those antibodies in AIH correlated significantly with those of p-ANCA, but not with those of anti-nuclear antibody or anti-smooth muscle antibody.?CONCLUSIONS—HMG1 and HMG2 are significant target antigens of p-ANCA in AIH.???Keywords: perinuclear anti-neutrophil cytoplasmic antibodies; chromosomal proteins; high mobility group 1 and 2; autoimmune; hepatitis PMID:10323891

Sobajima, J; Ozaki, S; Uesugi, H; Osakada, F; Inoue, M; Fukuda, Y; Shirakawa, H; Yoshida, M; Rokuhara, A; Imai, H; Kiyosawa, K; Nakao, K

1999-01-01

345

WASp Family Verprolin-homologous Protein-2 (WAVE2) and Wiskott-Aldrich Syndrome Protein (WASp) Engage in Distinct Downstream Signaling Interactions at the T Cell Antigen Receptor Site.  

PubMed

T cell antigen receptor (TCR) engagement has been shown to activate pathways leading to actin cytoskeletal polymerization and reorganization, which are essential for lymphocyte activation and function. Several actin regulatory proteins were implicated in regulating the actin machinery, such as members of the Wiskott-Aldrich syndrome protein (WASp) family. These include WASp and the WASp family verprolin-homologous protein-2 (WAVE2). Although WASp and WAVE2 share several structural features, the precise regulatory mechanisms and potential redundancy between them have not been fully characterized. Specifically, unlike WASp, the dynamic molecular interactions that regulate WAVE2 recruitment to the cell membrane and specifically to the TCR signaling complex are largely unknown. Here, we identify the molecular mechanism that controls the recruitment of WAVE2 in comparison with WASp. Using fluorescence resonance energy transfer (FRET) and novel triple-color FRET (3FRET) technology, we demonstrate how WAVE2 signaling complexes are dynamically regulated during lymphocyte activation in vivo. We show that, similar to WASp, WAVE2 recruitment to the TCR site depends on protein-tyrosine kinase, ZAP-70, and the adaptors LAT, SLP-76, and Nck. However, in contrast to WASp, WAVE2 leaves this signaling complex and migrates peripherally together with vinculin to the membrane leading edge. Our experiments demonstrate that WASp and WAVE2 differ in their dynamics and their associated proteins. Thus, this study reveals the differential mechanisms regulating the function of these cytoskeletal proteins. PMID:25342748

Pauker, Maor H; Reicher, Barak; Joseph, Noah; Wortzel, Inbal; Jakubowicz, Shlomi; Noy, Elad; Perl, Orly; Barda-Saad, Mira

2014-12-12

346

Dominant-negative cAMP-responsive element-binding protein inhibits proliferating cell nuclear antigen and DNA repair, leading to increased cellular radiosensitivity.  

PubMed

Selective inhibition of the epidermal growth factor receptor or mitogen-activated protein kinase (MAPK) results in radiosensitization of cancer cells. One potential mechanism involves cAMP-responsive element-binding protein, which is activated by radiation via the epidermal growth factor receptor/MAPK pathway and which regulates synthesis of proliferating cell nuclear antigen (PCNA), a protein involved in repair of ionizing radiation-induced DNA damage. To test for a role of CREB in cellular radiosensitivity, CHO cells were transfected with plasmids expressing dominant-negative CREB mutants (CR133 or KCREB), and various end-points were measured 48 h later. Basal levels of PCNA-CAT reporter construct activity were reduced by 60 and 40% following expression of CR133 and KCREB, respectively; similar decreases were observed in PCNA protein levels. Pulsed-field gel electrophoresis measurements showed that CR133 inhibited the repair of radiation-induced DNA double-strand breaks, and this effect was reversed by over-expression of PCNA; dominant-negative CREB also significantly inhibited split-dose recovery. Clonogenic assays were used to determine surviving fraction; the dose enhancement ratios for dominant-negative CREB-expressing cells compared with control (vector alone) were 1.5 and 1.3 for CR133 and KCREB, respectively. Importantly, co-transfection of mutant CREB and a construct constitutively expressing PCNA protein restored radiosensitivity of CHO cells back to wild-type levels. Moreover, cells expressing either CREB mutant showed no significant cell cycle redistribution. These data demonstrate that genetic disruption of CREB results in radiosensitization, and that this effect can be explained by a mechanism involving decreased PCNA expression and inhibition of DNA repair. PMID:12734192

Amorino, George P; Mikkelsen, Ross B; Valerie, Kristoffer; Schmidt-Ullrich, Rupert K

2003-08-01

347

HLA-matched unrelated donor hematopoietic cell transplantation after nonmyeloablative conditioning for patients with hematologic malignancies.  

PubMed

A hematopoietic cell transplantation (HCT) approach was developed for elderly or ill patients with hematologic malignancies that employed nonmyeloablative conditioning to avoid common regimen-related toxicities and relied on graft-versus-tumor effects for control of malignancy. Eighty-nine patients, median age 53 years, were given fludarabine (90 mg/m2) and 2 Gy total body irradiation. Marrow (n = 18) or granulocyte colony-stimulating factor (G-CSF)-stimulated peripheral blood mononuclear cells (G-PBMCs; n = 71) were transplanted from unrelated donors matched for human leukocyte antigen A (HLA-A), -B, -C antigens and -DRB1 and -DQB1 alleles. Postgrafting immunosuppression included mycophenolate mofetil and cyclosporine. Donor T-cell chimerism was higher for G-PBMCs compared with marrow recipients. Durable engraftment was observed in 85% of G-PBMCs and 56% of marrow recipients. Cumulative probabilities of grade II, III, and IV acute graft-versus-host disease (GVHD) were 42%, 8%, and 2%, respectively. Nonrelapse mortality at day 100 and at 1 year was 11% and 16%, respectively. One-year overall survivals and progression-free survivals were 52% and 38%, respectively. G-PBMC recipients had improved survival (57% vs 33%) and progression-free survival (44% vs 17%) compared with marrow recipients. HLA-matched unrelated donor HCT after nonmyeloablative conditioning is feasible in patients ineligible for conventional HCT. G-PBMCs conferred higher donor T-cell chimerism, greater durable engraftment, and better progression-free and overall survivals compared with marrow. PMID:12791654

Maris, Michael B; Niederwieser, Dietger; Sandmaier, Brenda M; Storer, Barry; Stuart, Monic; Maloney, David; Petersdorf, Effie; McSweeney, Peter; Pulsipher, Michael; Woolfrey, Ann; Chauncey, Thomas; Agura, Ed; Heimfeld, Shelly; Slattery, John; Hegenbart, Ute; Anasetti, Claudio; Blume, Karl; Storb, Rainer

2003-09-15

348

Partial or total replacement of fish meal by soybean protein on growth, protein utilization, potential estrogenic or antigenic effects, cholesterolemia and flesh quality in rainbow trout, Oncorhynchus mykiss  

Microsoft Academic Search

Groups of rainbow trout (initial body weight 83 ± 1 g) were fed diets (crude protein (CP) 46%; gross energy 21 kJ\\/g DM; crude fat 12%) containing graded levels of either a soyflour (CP 52% DM) or a soy protein concentrate (CP 72% DM) supplemented with L-methionine as partial or total replacement of fish meal protein. A growth trial was

S. J. Kaushik; J. P. Cravedi; J. P. Lalles; J. Sumpter; B. Fauconneau; M. Laroche

1995-01-01

349

The stability and immunogenicity of a protein antigen encapsulated in biodegradable microparticles based on blends of lactide polymers and polyethylene glycol.  

PubMed

Protein-loaded microparticles were produced from blends of poly(ethylene glycol) (PEG) with poly(L-lactide) (PLA) homopolymer or poly(DL-lactide co-glycolide) copolymers (PLG) using a water-in oil-in oil method. The stability of ovalbumin (OVA) associated with microparticles prepared using PEG and 50:50 PLG, 75:25 PLG and PLA, respectively, was analysed by SDS-PAGE and quantified by scanning densitometry following incubation in PBS at 37 degrees C for up to 1 month. Fragmentation and aggregation of OVA was detected with all 3 formulations. The extent of both processes correlated with the degradation rate of the lactide polymer used and decreased in the order PLA < 75:25 PLG < 50:50 PLG. Extensive degradation of the PLG/PEG microparticles also occurred over 4 weeks whereas the use of PLA/PEG blends resulted in a stable microparticle morphology and much reduced fragmentation and aggregation of the associated protein. Following a single sub-cutaneous immunisation, high levels of specific serum IgG antibody were elicited by OVA associated with the PLA/PEG particles. Injection of OVA associated with the 75:25 PLG/PEG microparticles resulted in very low levels of specific antibody. A higher response was induced by the 50:50 PLG/PEG formulation but there was very large inter-animal variation in this group. Antibody levels elicited by all 3 formulations were significantly higher than those elicited by a single injection of soluble OVA. Analysis of antigen specific IgG1 and IgG2a antibody subtype levels also revealed the greater efficacy of the PLA/PEG microparticles as an adjuvant system. The use of PLA/PEG microparticles shows improved protein loading and delivery capacity while maintaining a high level of stability of the associated protein. These results indicate a strong correlation between the stability of microencapsulated antigen and the magnitude of the immune response following sub-cutaneous immunisation. PMID:10075157

Lavelle, E C; Yeh, M K; Coombes, A G; Davis, S S

1999-02-12

350

Cancer Immunotherapy Targeting the High Molecular Weight Melanoma-Associated Antigen Protein Results in a Broad Antitumor Response and Reduction of Pericytes in the Tumor Vasculature  

Microsoft Academic Search

The high molecular weight melanoma-associated antigen (HMW-MAA), also known as melanoma chondroitin sulfate proteoglycan, has been used as a target for the immunotherapy of melanoma. This antigen is expressed on the cell surface and has a restricted distribution in normal tissues. Besides its expression in a broad range of transformed cells, this antigen is also found in pericytes, which are

Paulo Cesar Maciag; Matthew M. Seavey; Zhen-Kun Pan; Soldano Ferrone; Yvonne Paterson

351

Isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein loop libraries  

Microsoft Academic Search

The new antigen receptor (NAR) from nurse sharks consists of an immunoglobulin variable domain attached to five constant domains, and is hypothesised to function as an antigen-binding antibody-like molecule. To determine whether the NAR is present in other species we have isolated a number of new antigen receptor variable domains from the spotted wobbegong shark (Orectolobus maculatus) and compared their

Stewart D Nuttall; Usha V Krishnan; Meghan Hattarki; Ross De Gori; Robert A Irving; Peter J Hudson

2001-01-01

352

Evaluation of on-line high-performance size-exclusion chromatography, differential refractometry, and multi-angle laser light scattering analysis for the monitoring of the oligomeric state of human immunodeficiency virus vaccine protein antigen  

Microsoft Academic Search

Chiron has developed a novel mutant form of the human immunodeficiency virus (HIV) envelop protein, o-gp140, that is currently entering Human Phase 1 clinical trials for testing as a prophylactic HIV vaccine. The o-gp140 protein is oligomeric and the quaternary structure is thought to play an important role in its activity as an antigen. As o-gp140 proceeds through the clinical

John Barackman; Isaias Prado; Chulani Karunatilake; Kenji Furuya

2004-01-01

353

The Aeromonas salmonicida subsp. salmonicida exoproteome: global analysis, moonlighting proteins and putative antigens for vaccination against furunculosis  

PubMed Central

Background Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Despite the identification of several virulence factors the pathogenesis is still poorly understood. We have used high-throughput proteomics to display the differences between in vitro secretome of A. salmonicida wild-type (wt, hypervirulent, JF5054) and T3SS-deficient (isogenic ?ascV, extremely low-virulent, JF2747) strains in exponential (GP) and stationary (SP) phases of growth. Results Among the different experimental conditions we obtained semi-quantitative values for a total of 2136 A. salmonicida proteins. Proteins of specific A. salmonicida species were proportionally less detected than proteins common to the Aeromonas genus or those shared with other Aeromonas species, suggesting that in vitro growth did not induce the expression of these genes. Four detected proteins which are unidentified in the genome of reference strains of A. salmonicida were homologous to components of the conjugative T4SS of A. hydrophila pRA1 plasmid. Polypeptides of three proteins which are specific to the 01-B526 strain were also discovered. In supernatants (SNs), the number of detected proteins was higher in SP (326 for wt vs 329 for mutant) than in GP (275 for wt vs 263 for mutant). In pellets, the number of identified proteins (a total of 1536) was approximately the same between GP and SP. Numerous highly conserved cytoplasmic proteins were present in A. salmonicida SNs (mainly EF-Tu, EF-G, EF-P, EF-Ts, TypA, AlaS, ribosomal proteins, HtpG, DnaK, peptidyl-prolyl cis-trans isomerases, GAPDH, Enolase, FbaA, TpiA, Pgk, TktA, AckA, AcnB, Mdh, AhpC, Tpx, SodB and PNPase), and several evidences support the theory that their extracellular localization was not the result of cell lysis. According to the Cluster of Orthologous Groups classification, 29% of excreted proteins in A. salmonicida SNs were currently poorly characterized. Conclusions In this part of our work we elucidated the whole in vitro exoproteome of hypervirulent A. salmonicida subsp. salmonicida and showed the secretion of several highly conserved cytoplasmic proteins with putative moonlighting functions and roles in virulence. All together, our results offer new information about the pathogenesis of furunculosis and point out potential candidates for vaccine development. PMID:24127837

2013-01-01

354

Use of Recombinant Flagellin Protein as a Tracer Antigen in a Fluorescence Polarization Assay for Diagnosis of Leptospirosis  

Microsoft Academic Search

The objective of the present study was to investigate the usefulness of a recombinant flagellar protein, FlaB, of Leptospira interrogans serovar pomona in the serodiagnosis of leptospirosis by the fluorescence polarization assay (FPA). The recombinant protein FlaB was purified to homogeneity by a combination of nickel-nitriloa- cetic acid agarose chromatography, electrophoresis, and electroelution. Purified FlaB was labeled with fluo- rescein

NASREEN I. BUGHIO; MIN LIN; OM P. SURUJBALLI

1999-01-01

355

Immunohistochemical detection of p53 protein in basal cell skin cancer after microwave-assisted antigen retrieval  

Microsoft Academic Search

p53 is the most frequently altered tumor-suppressor gene in skin cancer. In normal tissues the p53 protein (wild type) has\\u000a a very short half-life and it is not detectable immunohistochemically. In contrast, the mutant p53 protein has an extended\\u000a half-life in tumor cells and can be detected by immunohistochemical methods. p53 is widely used as an indicator of tumor aggression

E. Evke; F. Z. Minbay; S. G. Temel; Z. Kahveci

2009-01-01

356

Immunogenic Salivary Proteins of Triatoma infestans: Development of a Recombinant Antigen for the Detection of Low-Level Infestation of Triatomines  

PubMed Central

Background Triatomines are vectors of Trypanosoma cruzi, the etiological agent of Chagas disease in Latin America. The most effective vector, Triatoma infestans, has been controlled successfully in much of Latin America using insecticide spraying. Though rarely undertaken, surveillance programs are necessary in order to identify new infestations and estimate the intensity of triatomine bug infestations in domestic and peridomestic habitats. Since hosts exposed to triatomines develop immune responses to salivary antigens, these responses can be evaluated for their usefulness as epidemiological markers to detect infestations of T. infestans. Methodology/Principal Findings T. infestans salivary proteins were separated by 2D-gel electrophoresis and tested for their immunogenicity by Western blotting using sera from chickens and guinea pigs experimentally exposed to T. infestans. From five highly immunogenic protein spots, eight salivary proteins were identified by nano liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoLC-ESI-MS/MS) and comparison to the protein sequences of the National Center for Biotechnology Information (NCBI) database and expressed sequence tags of a unidirectionally cloned salivary gland cDNA library from T. infestans combined with the NCBI yeast protein sub-database. The 14.6 kDa salivary protein [gi|149689094] was produced as recombinant protein (rTiSP14.6) in a mammalian cell expression system and recognized by all animal sera. The specificity of rTiSP14.6 was confirmed by the lack of reactivity to anti-mosquito and anti-sand fly saliva antibodies. However, rTiSP14.6 was recognized by sera from chickens exposed to four other triatomine species, Triatoma brasiliensis, T. sordida, Rhodnius prolixus, and Panstrongylus megistus and by sera of chickens from an endemic area of T. infestans and Chagas disease in Bolivia. Conclusions/Significance The recombinant rTiSP14.6 is a suitable and promising epidemiological marker for detecting the presence of small numbers of different species of triatomines and could be developed for use as a new tool in surveillance programs, especially to corroborate vector elimination in Chagas disease vector control campaigns. PMID:19841746

Schwarz, Alexandra; Helling, Stefan; Collin, Nicolas; Teixeira, Clarissa R.; Medrano-Mercado, Nora; Hume, Jen C. C.; Assumpção, Teresa C.; Marcus, Katrin; Stephan, Christian; Meyer, Helmut E.; Ribeiro, José M. C.; Billingsley, Peter F.; Valenzuela, Jesus G.; Sternberg, Jeremy M.; Schaub, Günter A.

2009-01-01

357

Epitope mapping and evaluation of specificity of T-helper sites in four major antigenic peptides of chicken riboflavin carrier protein in outbred rats.  

PubMed

This paper reviews our studies on synthetic peptides spanning the major antigenic determinants of the chicken riboflavin carrier protein (RCP; 219 AA). These determinants are composed of residues 4-24 (YGC), 64-83 (CED), 130-147 (GEN), and 200-219 (HAC) and function as minivaccines in terms of eliciting anti-peptide antibodies which recognize the native protein and are particularly promising contraceptive vaccine candidates. We have used 15-residue synthetic peptides to define short sequences involved in interaction with antibody and with T-cells. We have mapped the boundaries of T-cell epitopes of these peptides in outbred rats by immunizing the animals with each peptide and assaying the popliteal lymph node cell proliferation against a series of overlapping synthetic 15-mers covering the entire length of the individual peptides. The peptides YGC, GEN, and HAC harboured a single T-cell epitope each whereas the peptide CED exhibited bimodal response possessing two epitopes, one at N-terminus and the other at the C-terminus. These studies provide insight into the way in which an immunogen is viewed by the immune system. In addition, preferential T-cell helper function for B cells recognizing unique determinants on the same molecule was demonstrated. This information helps in exploiting synthetic peptides in the construction of designer immunogens which have potential as candidate vaccines. PMID:14575688

Subramanian, Sarada; Andal, S; Karande, Anjali A; Radhakantha Adiga, P

2003-11-01

358

Duffy Antigen Receptor for Chemokine (DARC) Polymorphisms and Its Involvement in Acquisition of Inhibitory Anti-Duffy Binding Protein II (DBPII) Immunity  

PubMed Central

The Plasmodium vivax Duffy binding protein (PvDBP) and its erythrocytic receptor, the Duffy antigen receptor for chemokines (DARC), are involved in the major P. vivax erythrocyte invasion pathway. An open cohort study to analyze DARC genotypes and their relationship to PvDBP immune responses was carried out in 620 volunteers in an agricultural settlement of the Brazilian Amazon. Three cross-sectional surveys were conducted at 6-month intervals, comprising 395, 410, and 407 subjects, respectively. The incidence rates of P. vivax infection was 2.32 malaria episodes per 100 person-months under survey (95% confidence interval [CI] of 1.92-2.80/100 person-month) and, of P. falciparum, 0.04 per 100 person-months (95% CI of 0.007–0.14/100 person-month). The distribution of DARC genotypes was consistent with the heterogeneous ethnic origins of the Amazon population, with a predominance of non-silent DARC alleles: FY*A > FY*B. The 12-month follow-up study demonstrated no association between DARC genotypes and total IgG antibodies as measured by ELISA targeting PvDBP (region II, DBPII or regions II–IV, DBPII-IV). The naturally acquired DBPII specific binding inhibitory antibodies (BIAbs) tended to be more frequent in heterozygous individuals carrying a DARC-silent allele (FY*BES). These results provide evidence that DARC polymorphisms may influence the naturally acquired inhibitory anti-Duffy binding protein II immunity. PMID:24710306

Santos-Alves, Jessica R.; Tang, Michaelis Loren; Sanchez, Bruno A. M.; Sousa, Tais N.; Fontes, Cor J. F.; Nogueira, Paulo A.; Rocha, Roberto S.; Brito, Cristiana F. A.; Adams, John H.; Kano, Flora S.; Carvalho, Luzia H.

2014-01-01

359

Liprin-?4 Is Required for Nickel Induced Receptor Protein Tyrosine Phosphatase-Leukocyte Antigen Related Receptor F (RPTP-LAR) Activity  

PubMed Central

Liprin-?4 was strongly induced following nickel (II) chloride exposure in a variety of cell types including BEAS-2B, A549, BEP2D and BL41 cells. Liprin-?4, a member of the Liprin alpha family, has seven isoforms but only three of these variants were detected in BEAS-2B cells (004, 201 and 202). The level of Liprin-?4 variants 201 and 004 were highly increased in BEAS-2B cells in response to nickel. We showed that Liprin-?4 bound directly to the cytoplasmic region of RPTP-LAR (receptor protein tyrosine phosphatase-leukocyte antigen-related receptor F). The cytoplasmic region of RPTP-LAR contains two phosphatase domains but only the first domain shows activity. The second domain interacts with other proteins. The phosphatase activity was increased both following nickel treatment and also in the presence of nickel ions in cell extracts. Liprin-?4 knock-down lines with decreased expression of Liprin-?4 variants 004 and 201 exhibited greater nickel toxicity compared to controls. The RPTP-LAR phosphatase activity was only slightly increased in a Liprin-?4 knock-down line. Liprin-?4 appeared necessary for the nickel induced tyrosine phosphatase activity. The presence of Liprin-?4 and nickel increased tyrosine phosphatase activity that reduced the global levels of tyrosine phosphorylation in the cell. PMID:21829649

Kiok, Kathrin; Sun, Hong; Clancy, Hailey; Bose, Sutapa; Kluz, Thomas; Wu, Fen; Costa, Max

2011-01-01

360

Oral immunization with recombinant Salmonella typhimurium expressing surface protein antigen A of Streptococcus sobrinus: dose response and induction of protective humoral responses in rats.  

PubMed Central

An attenuated, recombinant Salmonella typhimurium mutant, chi 4072(pYA2905), expressing the surface protein antigen A (SpaA) of Streptococcus sobrinus was investigated for its effectiveness in inducing protective immune responses against S. sobrinus-induced dental caries in an experimental caries model. Fischer rats were orally immunized with either 10(8) or 10(9) CFU of S. typhimurium chi 4072(pYA2905). Persistence of salmonellae in Peyer's patches and spleens and the induction of immune responses were determined. Maximum numbers of salmonellae were recovered from Peyer's patches of rats within the first week of immunization, with higher numbers recovered from rats given 10(9) CFU than from those given 10(8) CFU. Serum anti-Salmonella and anti-SpaA responses increased more rapidly in rats given 10(9) CFU than in rats given 10(8) CFU. The salivary antibody response to SpaA increased with time, but the response varied in the two groups. In a separate study, rats were orally immunized with the recombinant Salmonella mutant and then challenged with cariogenic S. sobrinus 6715. The levels of serum and salivary antibody and caries activity were assessed at the termination of the experiment. Higher levels of salivary immunoglobulin A antibody to SpaA and Salmonella carrier were detected in rats given 10(9) CFU than in those given 10(8) CFU, and these responses were higher than those in nonimmunized controls. Mandibular molars from immunized rats had lower numbers of recoverable streptococci and less extensive carious lesions than those from nonimmunized, control rats. These data indicate that oral immunization with an attenuated recombinant S. typhimurium expressing SpaA of S. sobrinus induces the production of antigen-specific mucosal antibody and confers protection against dental caries. PMID:7729915

Redman, T K; Harmon, C C; Lallone, R L; Michalek, S M

1995-01-01

361

In vitro binding of anthrax protective antigen on bacteriophage T4 capsid surface through Hoc-capsid interactions: A strategy for efficient display of large full-length proteins  

SciTech Connect

An in vitro binding system is described to display large full-length proteins on bacteriophage T4 capsid surface at high density. The phage T4 icosahedral capsid features 155 copies of a nonessential highly antigenic outer capsid protein, Hoc, at the center of each major capsid protein hexon. Gene fusions were engineered to express the 83-kDa protective antigen (PA) from Bacillus anthracis fused to the N-terminus of Hoc and the 130-kDa PA-Hoc protein was expressed in Escherichia coli and purified. The purified PA-Hoc was assembled in vitro on hoc {sup -} phage particles. Binding was specific, stable, and of high affinity. This defined in vitro system allowed manipulation of the copy number of displayed PA and imposed no significant limitation on the size of the displayed antigen. In contrast to in vivo display systems, the in vitro approach allows all the capsid binding sites to be occupied by the 130-kDa PA-Hoc fusion protein. The PA-T4 particles were immunogenic in mice in the absence of an adjuvant, eliciting strong PA-specific antibodies and anthrax lethal toxin neutralizing antibodies. The in vitro display on phage T4 offers a novel platform for potential construction of customized vaccines against anthrax and other infectious diseases.

Shivachandra, Sathish B. [Department of Biology, 103 McCort Ward Hall, Catholic University of America, 620 Michigan Ave., NE, Washington, DC 20064 (United States); Rao, Mangala [Division of Retrovirology, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910 (United States); Janosi, Laszlo [Division of Retrovirology, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910 (United States); Sathaliyawala, Taheri [Department of Biology, 103 McCort Ward Hall, Catholic University of America, 620 Michigan Ave., NE, Washington, DC 20064 (United States); Matyas, Gary R. [Division of Retrovirology, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910 (United States); Alving, Carl R. [Division of Retrovirology, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910 (United States); Leppla, Stephen H. [Bacterial Toxins and Therapeutics Section, National Institute of Allergy and Infectious Diseases, NIH, 30 Convent Dr., Bethesda, MD 20892 (United States); Rao, Venigalla B. [Department of Biology, 103 McCort Ward Hall, Catholic University of America, 620 Michigan Ave., NE, Washington, DC 20064 (United States)]. E-mail: rao@cua.edu

2006-02-05

362

Genetic variation in protein specific antigen detected prostate cancer and the effect of control selection on genetic association studies  

PubMed Central

Background Only a minority of the genetic component of prostate cancer (PrCa) risk has been explained. Some observed associations of single nucleotide polymorphisms (SNPs) with PrCa might arise from associations of these SNPs with circulating prostate specific antigen (PSA) because PSA values are used to select controls. Methods We undertook a genome-wide association study (GWAS) of screen detected PrCa (ProtecT 1146 cases and 1804 controls); meta-analysed the results with those from the previously published UK Genetic Prostate Cancer Study (1854 cases and 1437 controls); investigated associations of SNPs with PrCa using either ‘low’ (PSA <0.5ng/ml) or ‘high’ (PSA ?3ng/ml, biopsy negative) PSA controls; and investigated associations of SNPs with PSA. Results The ProtecT GWAS confirmed previously reported associations of PrCa at 3 loci: 10q11.23, 17q24.3 and 19q13.33. The meta-analysis confirmed associations of PrCa with SNPs near 4 previously identified loci (8q24.21,10q11.23, 17q24.3 and 19q13.33). When comparing PrCa cases with low PSA controls, alleles at genetic markers rs1512268, rs445114, rs10788160, rs11199874, rs17632542, rs266849 and rs2735839 were associated with an increased risk of PrCa, but the effect-estimates were attenuated to the null when using high PSA controls (p for heterogeneity in effect-estimates<0.04). We found a novel inverse association of rs9311171-T with circulating PSA. Conclusions Differences in effect estimates for PrCa observed when comparing low vs. high PSA controls, may be explained by associations of these SNPs with PSA. Impact These findings highlight the need for inferences from genetic studies of PrCa risk to carefully consider the influence of control selection criteria. PMID:24753544

Knipe, Duleeka W; Evans, David M; Kemp, John P.; Eeles, Rosalind; Easton, Douglas F; Kote-Jarai, Zsofia; Al Olama, Ali Amin; Benlloch, Sara; Donovan, Jenny L.; Hamdy, Freddie C.; Neal, David E

2014-01-01

363

The MF6p/FhHDM-1 Major Antigen Secreted by the Trematode Parasite Fasciola hepatica Is a Heme-binding Protein*  

PubMed Central

Blood-feeding parasites have developed biochemical mechanisms to control heme intake and detoxification. Here we show that a major antigen secreted by Fasciola hepatica, previously reported as MF6p, of unknown function (gb|CCA61804.1), and as FhHDM-1, considered to be a helminth defense molecule belonging to the family of cathelicidin-like proteins (gb|ADZ24001.1), is in fact a heme-binding protein. The heme-binding nature of the MF6p/FhHDM-1 protein was revealed in two independent experiments: (i) immunopurification of the secreted protein·heme complexes with mAb MF6 and subsequent analysis by C8 reversed-phase HPLC and MS/MS spectrometry and (ii) analysis of the binding ability of the synthetic protein to hemin in vitro. By immunohistochemistry analysis, we have observed that MF6p/FhHDM-1 is produced by parenchymal cells and transported to other tissues (e.g. vitellaria and testis). Interestingly, MF6p/FhHDM-1 is absent both in the intestinal cells and in the lumen of cecum, but it can be released through the tegumental surface to the external medium, where it binds to free heme molecules regurgitated by the parasite after hemoglobin digestion. Proteins that are close analogs of the Fasciola MF6p/FhHDM-1 are present in other trematodes, including Clonorchis, Opistorchis, Paragonimus, Schistosoma, and Dicrocoelium. Using UV-visible spectroscopy and immunoprecipitation techniques, we observed that synthetic MF6p/FhHDM-1 binds to hemin with 1:1 stoichiometry and an apparent Kd of 1.14 × 10?6 m?1. We also demonstrated that formation of synthetic MF6p/FhHDM-1·hemin complexes inhibited hemin degradation by hydrogen peroxide and hemin peroxidase-like activity in vitro. Our results suggest that MF6p/FhHDM-1 may be involved in heme homeostasis in trematodes. PMID:24280214

Martínez-Sernández, Victoria; Mezo, Mercedes; González-Warleta, Marta; Perteguer, María J.; Muiño, Laura; Guitián, Esteban; Gárate, Teresa; Ubeira, Florencio M.

2014-01-01

364

Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Induces Expression of the Helix-Loop-Helix Protein Id-1 in Human Endothelial Cells  

PubMed Central

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) (also known as human herpesvirus 8) is a gamma-2 herpesvirus believed to be the etiologic agent responsible for KS. The pathogenesis of this potentially life-threatening neoplasm is complex and unclear, and it is currently unknown how KSHV causes KS. Id (named for inhibitor of DNA binding or inhibitor of differentiation) proteins were identified in 1990 and found to be naturally occurring dominant-negative inhibitors of basic helix-loop-helix transcription factors. Id-1, the most well-studied member of this family, has since been shown to play a key role in several biological systems including cellular differentiation, cell cycle regulation, and tumorigenesis. In this report, we demonstrate that Id-1 is expressed at high levels in KS tumor cells both in vitro and in vivo but is expressed at relatively modest levels in endothelial cells (ECs), the likely precursor of the KS tumor cell. Infection of precursor cells with KSHV may be responsible for this enhanced expression, as KSHV infection induced Id-1 27-fold in ECs under our experimental conditions. Furthermore, we demonstrate that the KSHV-encoded latency-associated nuclear antigen (LANA) protein appears to be involved. Expression of LANA in ECs resulted in Id-1 induction that was almost identical to the induction seen with KSHV-infected ECs. These results demonstrate the expression of Id-1 in KS tumor cells and indicate the KSHV LANA protein may be, at least in part, responsible. This may be an important mechanism by which KSHV allows KS tumor cells to escape normal cell cycle regulation and enhances their proliferation. PMID:12719589

Tang, Jun; Gordon, Gabriel M.; Müller, Maike G.; Dahiya, Madhu; Foreman, Kimberly E.

2003-01-01

365

Characterization of an antigenic site that contains a dominant, type-specific neutralization determinant on the envelope protein domain III (ED3) of dengue 2 virus  

SciTech Connect

The surface of the mature dengue virus (DENV) particle consists of 90 envelope (E) protein dimers that mediate both receptor binding and fusion. The E protein ectodomain can be divided into three structural domains designated ED1, ED2, and ED3, of which ED3 contains the critical and dominant virus-specific neutralization sites. In this study the ED3 epitopes recognized by seven, murine, IgG1 DENV-2 type-specific, monoclonal antibodies (MAbs) were determined using site-directed mutagenesis of a recombinant DENV-2 ED3 (rED3) protein. A total of 41 single amino acid substitutions were introduced into the rED3 at 30 different surface accessible residues. The affinity of each MAb with the mutant rED3s was assessed by indirect ELISA and the results indicate that all seven MAbs recognize overlapping epitopes with residues K305 and P384 critical for binding. These residues are conserved among DENV-2 strains and cluster together on the upper lateral face of ED3. A linear relationship was observed between relative occupancy of ED3 on the virion by MAb and neutralization of the majority of virus infectivity ({approx} 90%) for all seven MAbs. Depending on the MAb, it is predicted that between 10% and 50% relative occupancy of ED3 on the virion is necessary for virus neutralization and for all seven MAbs occupancy levels approaching saturation were required for 100% neutralization of virus infectivity. Overall, the conserved antigenic site recognized by all seven MAbs is likely to be a dominant DENV-2 type-specific, neutralization determinant.

Gromowski, Gregory D. [Department of Pathology, Sealy Center for Vaccine Development, Center for Biodefense and Emerging Infectious Diseases, and Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX 77555-0609 (United States); Barrett, Alan D.T. [Department of Pathology, Sealy Center for Vaccine Development, Center for Biodefense and Emerging Infectious Diseases, and Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX 77555-0609 (United States)], E-mail: abarrett@utmb.edu

2007-09-30

366

Cooperative Learning Contingencies: Unrelated versus Related Individual and Group Contingencies  

ERIC Educational Resources Information Center

College students operating under related cooperative contingencies (students had to earn individual credit before being considered for group credit) showed more consistent individual and group improvement on exam performance than students operating under unrelated contingencies (individual credit and group credit were independently determined). A…

Carroll, Erin; Williams, Robert L.; Hautau, Briana

2006-01-01

367

Unrelenting challenges for freaque wave studies in ocean coastal regions  

E-print Network

Unrelenting challenges for freaque wave studies in ocean coastal regions: defining the phenomena P facing. Keywords: ocean waves, freaque waves, the phenomena, freak waves, rogue waves. 1 Introduction The freaque wave has been in existence probably as long as the world's oceans have existed

368

Albright hereditary osteodystrophy and del(2) (q37.3) in four unrelated individuals.  

PubMed

Albright hereditary osteodystrophy (AHO) is a condition with characteristic physical findings (short stature, obesity, round face, brachydactyly) but variable biochemical changes (pseudohypoparathyroidism, pseudopseudohypoparathyroidism). Most patients with AHO have decreased activity of the guanine nucleotide-binding protein (GS protein) that stimulates adenylyl cyclase. The gene encoding the alpha subunit of the GS protein (GNAS1) has been mapped to the long arm of chromosome 20. We describe 4 unrelated individuals with apparent AHO, associated with small terminal deletions of chromosome 2. All 4 patients had normal serum calcium levels consistent with pseudopseudohypoparathyroidism. Del(2) (q37) is the first consistent karyotypic abnormality that has been documented in AHO [Phelan et al., 1993: Am J Hum Genet 53:484]. The finding of the same small terminal deletion in 4 unrelated individuals with a similar phenotype suggests that a gene locus in the 2q37 region is important in the pathogenesis of Albright syndrome. The association of Albright syndrome and the GNAS1 locus on chromosome 20 is well documented. The observation of a second potential disease locus on chromosome 2 may help explain the heterogeneity observed in this disorder. PMID:7573148

Phelan, M C; Rogers, R C; Clarkson, K B; Bowyer, F P; Levine, M A; Estabrooks, L L; Severson, M C; Dobyns, W B

1995-07-31

369

Integrin-associated protein: a 50-kD plasma membrane antigen physically and functionally associated with integrins  

Microsoft Academic Search

Phagocytosis by monocytes or neutrophils can be enhanced by interaction with several proteins or synthetic peptides containing the Arg-Gly-Asp se- quence. Recently we showed that an mAb, B6H12, specifically inhibited this enhancement of neutrophil phagocytosis by inhibiting Arg-Gly-Asp binding to the leukocyte response integrin (Gresham, H. D., J. L. Goodwin, P. M. Allen, D. C. Anderson, and E. J. Brown.

Eric Brown; Lora Hooper; Thang Ho; Hattie Greshamll

1990-01-01

370

Antigenic and Molecular Properties of Type 3 Poliovirus Responsible for an Outbreak of Poliomyelitis in a Vaccinated Population  

Microsoft Academic Search

SUMMARY Virus isolated from an outbreak of poliomyelitis in Finland has been examined serologically and at the molecular level. The causative agent was an antigenically unusual strain of type 3 poliovirus, which was unrelated to the strains used to manufacture either live or killed poliovaccines. It is likely that the antigenic properties of the virus played a part in establishing

D. I. Magrath; D. M. A. Evans; M. Ferguson; G. C. Schild; P. D. Minor; F. Horaud; R. Crainic; M. Stenvik; T. Hovi

1986-01-01

371

Expression of CD95 antigen and Bcl-2 protein in non-Hodgkin's lymphomas and Hodgkin's disease.  

PubMed Central

CD95 (APO-1/Fas) is a member of the superfamily that includes the nerve growth factor and tumor necrosis factor receptors, OX40, CD27, CD30, and CD40. Present on a minority of resting blood lymphocytes, CD95 expression is upregulated on activated T and B lymphocytes and natural killer cells, where binding of the antigen by anti-Fas and anti-APO-1 antibodies has been shown to induce apoptosis. This CD95-mediated apoptosis is at least partially inhibited by expression of the Bcl-2 protooncogene. To evaluate possible roles of CD95 and Bcl-2 in growth regulation of lymphoid neoplasms, we studied by immunohistochemistry the expression of CD95 and Bcl-2 in 67 B- and 5 T-cell lymphomas, and 10 cases of Hodgkin's disease. In all, 29 B and 2 T cell lymphomas, and 9 cases of Hodgkin's disease expressed CD95. Compared with diffuse large B-cell and Burkitt-like lymphomas, lowgrade B-cell lymphomas more frequently expressed CD95 (52% versus 26%; P < .005). None of the B-cell small lymphocytic lymphomas or mantle cell lymphomas expressed CD95, whereas the majority of follicle center lymphomas, extranodal marginal zone B-cell lymphomas, and immunocytomas were CD95+. Of the 29 CD95+ B-cell lymphomas, only 33% of the high-grade group coexpressed Bcl-2, compared with 87% of the low-grade group (P < .04). Two of three peripheral T-cell lymphomas--including one anaplastic large cell lymphoma--expressed CD95. Staining for CD95 was seen in 9 of 10 cases of Hodgkin's disease. The infrequent expression of CD95 in high-grade B-cell lymphomas suggests an association between loss of CD95 expression/function and a more aggressive tumor grade. Whereas frequent coexpression of Bcl-2 with CD95 may protect low-grade B-cell lymphomas against CD95-mediated apoptosis, in the high-grade group such coexpression is infrequent, and other regulators besides Bcl-2 may be involved in modulating the apoptosis signal delivered by CD95. Images Figure 1 Figure 2 PMID:8774139

Nguyen, P. L.; Harris, N. L.; Ritz, J.; Robertson, M. J.

1996-01-01

372

Identification of specific antigenic epitope at N-terminal segment of enterovirus 71 (EV-71) VP1 protein and characterization of its use in recombinant form for early diagnosis of EV-71 infection.  

PubMed

Human enterovirus 71 (EV-71) is the main etiologic agent of hand, foot and mouth disease (HFMD). We sought to identify EV-71 specific antigens and develop serologic assays for acute-phase EV-71 infection. A series of truncated proteins within the N-terminal 100 amino acids (aa) of EV-71 VP1 was expressed in Escherichia coli. Western blot (WB) analysis showed that positions around 11-21 aa contain EV-71-specific antigenic sites, whereas positions 1-5 and 51-100 contain epitopes shared with human coxsackievirus A16 (CV-A16) and human echovirus 6 (E-6). The N-terminal truncated protein of VP1, VP?????, exhibited good stability and was recognized by anti-EV-71 specific rabbit sera. Alignment analysis showed that VP????? is highly conserved among EV-71 strains from different genotypes but was heterologous among other enteroviruses. When the GST-VP????? fusion protein was incorporated as antibody-capture agent in a WB assay and an ELISA for detecting anti-EV-71 IgM in human sera, sensitivities of 91.7% and 77.8% were achieved, respectively, with 100% specificity for both. The characterized EV-71 VP1 protein truncated to positions 6-43 aa has potential as an antigen for detection of anti-EV-71 IgM for early diagnosis of EV-71 infection in a WB format. PMID:24952304

Zhang, Jianhua; Jiang, Bingfu; Xu, Mingjie; Dai, Xing; Purdy, Michael A; Meng, Jihong

2014-08-30

373

Identification of Plasmodium falciparum antigens by antigenic analysis of genomic and proteomic data  

PubMed Central

The recent explosion in genomic sequencing has made available a wealth of data that can now be analyzed to identify protein antigens, potential targets for vaccine development. Here we present, in the context of Plasmodium falciparum, a strategy that rapidly identifies target antigens from large and complex genomes. Sixteen antigenic proteins recognized by volunteers immunized with radiation-attenuated P. falciparum sporozoites, but not by mock immunized controls, were identified. Several of these were more antigenic than previously identified and well characterized P. falciparum-derived protein antigens. The data suggest that immune responses to Plasmodium are dispersed on a relatively large number of parasite antigens. These studies have implications for our understanding of immunodominance and breadth of responses to complex pathogens. PMID:12886016

Doolan, Denise L.; Southwood, Scott; Freilich, Daniel A.; Sidney, John; Graber, Norma L.; Shatney, Lori; Bebris, Lolita; Florens, Laurence; Dobano, Carlota; Witney, Adam A.; Appella, Ettore; Hoffman, Stephen L.; Yates, John R.; Carucci, Daniel J.; Sette, Alessandro

2003-01-01

374

Nasal Immunization with a Fusion Protein Consisting of the Hemagglutinin A Antigenic Region and the Maltose-Binding Protein Elicits CD11c+ CD8+ Dendritic Cells for Induced Long-Term Protective Immunity ?  

PubMed Central

We assessed the efficacy of a fusion protein consisting of the 25-kDa antigenic region of Porphyromonas gingivalis hemagglutinin A and the Escherichia coli maltose-binding protein (25k-hagA-MBP) as a nasal vaccine for the prevention of oral infection with P. gingivalis. Nasal immunization with 25k-hagA-MBP induced high levels of 25k-hagA-specific serum IgG, serum IgA, and salivary IgA antibodies in a Toll-like receptor 4 (TLR4)-dependent manner. These antibody responses were maintained for at least 1 year after immunization. Analysis of cytokine responses showed that nasal administration of 25k-hagA-MBP induced antigen-specific CD4+ T cells producing interleukin 4 (IL-4) and IL-5, but not gamma interferon (IFN-?), in the spleen and cervical lymph nodes (CLNs). Furthermore, increased numbers of CD11c+ CD8?+, but not CD11c+ CD11b+ or CD11c+ B220+, dendritic cells with upregulated expression of CD80, CD86, CD40, and major histocompatibility complex class II (MHC II) molecules were noted in the spleen, CLNs, and nasopharynx-associated lymphoreticular tissues (NALT). Interestingly, when 25k-hagA-MBP or cholera toxin (CT) was given intranasally to enable examination of their presence in neuronal tissues, the amounts of 25k-hagA-MBP were significantly lower than those of CT. Importantly, mice given 25k-hagA-MBP nasally showed a significant reduction in alveolar bone loss caused by oral infection with P. gingivalis, even 1 year after the immunization. These results suggest that 25k-hagA-MBP administered nasally would be an effective and safe mucosal vaccine against P. gingivalis infection and may be an important tool for the prevention of chronic periodontitis in humans. PMID:21115722

Du, Yuan; Hashizume, Tomomi; Kurita-Ochiai, Tomoko; Yuzawa, Satoshi; Abiko, Yoshimitsu; Yamamoto, Masafumi

2011-01-01

375

Kinetics of dengue non-structural protein 1 antigen and IgM and IgA antibodies in capillary blood samples from confirmed dengue patients.  

PubMed

Large-scale epidemiological surveillance of dengue in the field and dengue patient management require simple methods for sample collection, storage, and transportation as well as effective diagnostic tools. We evaluated the kinetics of three biological markers of dengue infection-non-structural protein 1 (NS1) antigen, immunoglobulin M (IgM), and IgA-in sequential capillary blood samples collected from fingertips of confirmed dengue patients. The overall sensitivities and specificities of the tests were 96% and 100%, respectively, for NS1, 58.1% and 100%, respectively, for IgM, and 33% and 100%, respectively, for IgA. During the acute phase of the disease, NS1 was the best marker of dengue infection, with a sensitivity of 98.7%, whereas from day 5, all three markers exhibited relevant levels of sensitivity. This first descriptive study of the kinetics of biological markers of dengue in capillary blood samples confirms the usefulness of this biological compartment for dengue diagnosis and argues for its exploitation in community-level and remote settings. PMID:24470561

Matheus, Séverine; Pham, Thai Binh; Labeau, Bhetty; Huong, Vu Thi Que; Lacoste, Vincent; Deparis, Xavier; Marechal, Vincent

2014-03-01