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1

Chronic bystander infections and immunity to unrelated antigens.  

PubMed

Chronic infections with persistent pathogens such as helminths, mycobacteria, Plasmodium, and hepatitis viruses affect more than a third of the human population and are associated with increased susceptibility to other pathogens as well as reduced vaccine efficacy. Although these observations suggest an impact of chronic infections in modulating immunity to unrelated antigens, little is known regarding the underlying mechanisms. Here, we summarize evidence of the most prevalent infections affecting immunity to unrelated pathogens and vaccines, and discuss potential mechanisms of how different bystander chronic infections might impact immune responses. We suggest that bystander chronic infections affect different stages of host responses and may impact transmission and recognition of other pathogens, innate immune responses, priming and differentiation of adaptive effector responses, as well as the development and maintenance of immunological memory. Further understanding of the immunological effects of coinfection should provide opportunities to enhance vaccine efficacy and control of infectious diseases. PMID:23084915

Stelekati, Erietta; Wherry, E John

2012-10-18

2

Chronic bystander infections and immunity to unrelated antigens  

PubMed Central

Chronic infections with persistent pathogens such as helminths, mycobacteria, Plasmodium and hepatitis viruses affect more than a third of the human population and are associated with increased susceptibility to other pathogens as well as reduced vaccine efficacy. Although these observations suggest an impact of chronic infections in modulating immunity to unrelated antigens, little is known regarding the underlying mechanisms. Here, we summarize evidence of the most prevalent infections affecting immunity to unrelated pathogens and vaccines, and discuss potential mechanisms of how different bystander chronic infections might impact immune responses. We suggest that bystander chronic infections affect different stages of host responses and may impact transmission of other pathogens, recognition and innate immune responses, priming and differentiation of adaptive effector responses, as well as the development and maintenance of immunological memory. Further understanding of the immunological effects of co-infection should provide opportunities to enhance vaccine efficacy and control infectious diseases. PMID:23084915

Stelekati, Erietta; Wherry, E. John

2012-01-01

3

Cell-mediated immune response to unrelated proteins and unspecific inflammation blocked by orally tolerated proteins.  

PubMed

Oral tolerance promotes a generalized decrease in specific immune responsiveness to proteins previously encountered via the oral route. In addition, parenteral immunization with a tolerated protein also triggers a significant reduction in the primary responsiveness to a second unrelated antigen. This is generally explained by 'innocent bystander suppression', suggesting that the transient and episodic effects of inhibitory cytokines released by contact with the tolerated antigen would block responses to the second antigen. In disagreement with this view, we have previously shown that: (i) these inhibitory effects do not require concomitance or contiguity of the injections of the two proteins; (ii) that intravenous or intragastric exposures to the tolerated antigen are not inhibitory; and (iii) that the inhibitory effect, once triggered, persists in the absence of further contact with the tolerated protein, possibly by inhibition of secondary responsiveness (immunological memory). The present work confirms that immunological memory of the second unrelated antigen is hindered by exposure to the tolerated antigen and, in addition, shows that this exposure: (i) inhibits the inflammation triggered by an unrelated antigen through the double effect of inhibiting production of leucocytes in the bone marrow and blocking their migration to inflammed sites; and (ii) significantly blocks footpaw swelling triggered by carrageenan. Taken together, these results conclusively demonstrate that inhibitory effects of parenteral injection of tolerated antigens are much more general than suggested by the 'innocent bystander suppression' hypothesis. PMID:18759750

Ramos, Gustavo C; Rodrigues, Claudiney M; Azevedo, Geraldo M; Pinho, Vanessa; Carvalho, Cláudia R; Vaz, Nelson M

2009-03-01

4

Antigenic Proteins of Borrelia burgdorferi.  

National Technical Information Service (NTIS)

The present invention relates to antigenic Borrelia burgdorferi proteins and their encoding DNA. In particular, the present invention relates to a 39 kilodalton (kDa) Borrelia burgdorferi protein which reacts with Lyme borreliosis serum and a 28 kDa Borre...

W. Simpson, T. Schwan

1990-01-01

5

The identification of tyrosine as a common key residue in unrelated H-2Kd restricted antigenic peptides.  

PubMed

We have compared the activity of several Kd- or Ld-restricted antigenic peptides as competitors in a functional competition assay using cytolytic T lymphocyte (CTL) clones. All of four unrelated Kd-restricted peptides tested could compete with each other but not with the Ld-restricted peptide P91A-. 12-24 (P91A). Moreover, the P91A peptide failed to compete with the four Kd-restricted peptides. In contrast, another Ld-restricted peptide [mouse cytomegalovirus (MCMV) pp89 167-176] could clearly compete with both Kd- and Ld-restricted peptides. The comparison of a series of modified MCMV pp89 peptides suggested that distinct structural features allow the interaction of the peptide with the two different MHC class I molecules. We showed previously that the competitor activity of two different Kd-restricted antigenic peptides was reduced substantially upon Ala substitution of the single Tyr residues present in these peptides. We now show a similar effect for two additional Kd-restricted peptides. Our results thus suggest that Tyr may function as an 'anchor' residue for many antigenic peptides that bind to the Kd molecule. Molecular modeling of the presumed antigen-binding site of the Kd molecule revealed the presence of two deep cavities that may be involved in binding peptide amino acid side chains. A model illustrating one possible interaction of a Tyr-containing peptide with the Kd molecule is presented. PMID:1721832

Maryanski, J L; Romero, P; Van Pel, A; Boon, T; Salemme, F R; Cerottini, J C; Corradin, G

1991-10-01

6

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

7

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

8

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

Microsoft Academic Search

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

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

2009-01-01

9

Hepatitis B Antigen: Antigenic Sites Related to Human Serum Proteins Revealed by Affinity Chromatography  

PubMed Central

Hepatitis B antigen-associated particles, isolated from sera of antigen carriers, were submitted to affinity chromatography on columns of insolubilized antibodies to normal human plasma. The particles adsorbed to the immunosorbent at pH 7.2 and were subsequently eluted at pH 2.2. Exposure of the particles to 8 M urea, 5 M KI, pH 2.2, detergents, organic solvents, or proteolytic enzymes failed to prevent their subsequent adsorption to the immunosorbent. This suggests that antigenic determinants related to human plasma proteins are constituent components of hepatitis B antigen-associated particles. These determinants are distinct from the group-specific (a) and subtype-specific (d or y) sites of the hepatitis B antigen and appear to be related to antigenic specificities on prealbumin, albumin, apolipoproteins C and D, and the ?-chain of immunoglobulin G. PMID:4136767

Neurath, A. Robert; Prince, Alfred M.; Lippin, Arnold

1974-01-01

10

INVITED REVIEW: TARGETING AND EXPRESSION OF ANTIGENIC PROTEINS IN TRANSGENIC  

E-print Network

; hepatitis B surface antigen. Introduction Exploiting plants to produce medicinal products has become a wellINVITED REVIEW: TARGETING AND EXPRESSION OF ANTIGENIC PROTEINS IN TRANSGENIC PLANTS FOR PRODUCTION; editor M. E. Horn) Summary Exploiting plants as biological bioreactors for production and delivery

Korban, Schuyler S.

11

Unconventional antigen retrieval for carbohydrate and protein antigens.  

PubMed

Aldehyde fixation of tissues often adversely affects the reactivity of cellular proteins with antibodies. A most commonly used retrieval technique in immunohistochemistry is high-temperature microwave heating of sections from formaldehyde-fixed and paraffin-embedded tissues. Here we report that pretreatment of paraffin and ultrathin cryosections with N-glycanase F to remove N-glycosidically linked oligosaccharides can result in a dramatic increase in specificity and intensity of immunogold labeling for sugar moieties present on O-glycosidically linked oligosaccharides. This is demonstrated in the immunolocalization of poly alpha2,8 KDN (KDN, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) of megalin in rat kidney. The mechanism of this retrieval procedure is most probably based on the elimination of sterical hindrance by large N-glycosidically linked oligosaccharides. Furthermore, we demonstrate that exposure of ultrathin cryosections to acidic conditions (pH 5.5) at ambient temperature prior to immunogold labeling can result in an increased labeling intensity. This effect was observed for megalin immunoreactive sites in proximal tubular epithelia of rat kidney. It is proposed that the mechanism of this retrieval procedure is based on the depolymerization of methylen and polymethylen bridges introduced by formaldehyde in the acidic milieu. PMID:9860259

Guhl, B; Ziak, M; Roth, J

1998-12-01

12

Extracellular Release of Antigenic Proteins by Helicobacter pylori  

PubMed Central

Screening a Helicobacter pylori genomic library with antisera raised against H. pylori broth culture supernatant resulted in the identification of six antigens: urease, HspB, Lpp20, DnaK, MsrA, and a cysteine-rich 28-kDa protein (designated HcpA). H. pylori antigens may be released into the extracellular space by multiple mechanisms, including specific secretion pathways, autolysis, and membrane vesicle formation. PMID:9596777

Cao, Ping; McClain, Mark S.; Forsyth, Mark H.; Cover, Timothy L.

1998-01-01

13

Extracellular release of antigenic proteins by Helicobacter pylori.  

PubMed

Screening a Helicobacter pylori genomic library with antisera raised against H. pylori broth culture supernatant resulted in the identification of six antigens: urease, HspB, Lpp20, DnaK, MsrA, and a cysteine-rich 28-kDa protein (designated HcpA). H. pylori antigens may be released into the extracellular space by multiple mechanisms, including specific secretion pathways, autolysis, and membrane vesicle formation. PMID:9596777

Cao, P; McClain, M S; Forsyth, M H; Cover, T L

1998-06-01

14

Identification of Antigenic Proteins of the Nosocomial Pathogen Klebsiella pneumoniae  

PubMed Central

The continuous expansion of nosocomial infections around the globe has become a precarious situation. Key challenges include mounting dissemination of multiple resistances to antibiotics, the easy transmission and the growing mortality rates of hospital-acquired bacterial diseases. Thus, new ways to rapidly detect these infections are vital. Consequently, researchers around the globe pursue innovative approaches for point-of-care devices. In many cases the specific interaction of an antigen and a corresponding antibody is pivotal. However, the knowledge about suitable antigens is lacking. The aim of this study was to identify novel antigens as specific diagnostic markers. Additionally, these proteins might be aptly used for the generation of vaccines to improve current treatment options. Hence, a cDNA-based expression library was constructed and screened via microarrays to detect novel antigens of Klebsiella pneumoniae, a prominent agent of nosocomial infections well-known for its extensive antibiotics resistance, especially by extended-spectrum beta-lactamases (ESBL). After screening 1536 clones, 14 previously unknown immunogenic proteins were identified. Subsequently, each protein was expressed in full-length and its immunodominant character examined by ELISA and microarray analyses. Consequently, six proteins were selected for epitope mapping and three thereof possessed linear epitopes. After specificity analysis, homology survey and 3d structural modelling, one epitope sequence GAVVALSTTFA of KPN_00363, an ion channel protein, was identified harboring specificity for K. pneumoniae. The remaining epitopes showed ambiguous results regarding the specificity for K. pneumoniae. The approach adopted herein has been successfully utilized to discover novel antigens of Campylobacter jejuni and Salmonella enterica antigens before. Now, we have transferred this knowledge to the key nosocomial agent, K. pneumoniae. By identifying several novel antigens and their linear epitope sites, we have paved the way for crucial future research and applications including the design of point-of-care devices, vaccine development and serological screenings for a highly relevant nosocomial pathogen. PMID:25333280

Hoppe, Sebastian; Bier, Frank F.; von Nickisch-Rosenegk, Markus

2014-01-01

15

Highly expressed loci are vulnerable to misleading ChIP localization of multiple unrelated proteins  

E-print Network

Chromatin immunoprecipitation (ChIP) is the gold-standard technique for localizing nuclear proteins in the genome. We used ChIP, in combination with deep sequencing (Seq), to study the genome-wide distribution of the Silent ...

Teytelman, Leonid

16

Role of ?-lactamase residues in a common interface for binding the structurally unrelated inhibitory proteins BLIP and BLIP-II.  

PubMed

The ?-lactamase inhibitory proteins (BLIPs) are a model system for examining molecular recognition in protein-protein interactions. BLIP and BLIP-II are structurally unrelated proteins that bind and inhibit TEM-1 ?-lactamase. Both BLIPs share a common binding interface on TEM-1 and make contacts with many of the same TEM-1 surface residues. BLIP-II, however, binds TEM-1 over 150-fold tighter than BLIP despite the fact that it has fewer contact residues and a smaller binding interface. The role of eleven TEM-1 amino acid residues that contact both BLIP and BLIP-II was examined by alanine mutagenesis and determination of the association (k on) and dissociation (k off) rate constants for binding each partner. The substitutions had little impact on association rates and resulted in a wide range of dissociation rates as previously observed for substitutions on the BLIP side of the interface. The substitutions also had less effect on binding affinity for BLIP than BLIP-II. This is consistent with the high affinity and small binding interface of the TEM-1-BLIP-II complex, which predicts per residue contributions should be higher for TEM-1 binding to BLIP-II versus BLIP. Two TEM-1 residues (E104 and M129) were found to be hotspots for binding BLIP while five (L102, Y105, P107, K111, and M129) are hotspots for binding BLIP-II with only M129 as a common hotspot for both. Thus, although the same TEM-1 surface binds to both BLIP and BLIP-II, the distribution of binding energy on the surface is different for the two target proteins, that is, different binding strategies are employed. PMID:24947275

Fryszczyn, Bartlomiej G; Adamski, Carolyn J; Brown, Nicholas G; Rice, Kacie; Huang, Wanzhi; Palzkill, Timothy

2014-09-01

17

Posttranslational protein modifications in antigen recognition and autoimmunity  

Microsoft Academic Search

It is estimated that 50–90% of the proteins in the human body are post-translationally modified. In the proper context, these modifications are necessary for the biological functions of a vast array of proteins and the effector functions of the cells in which they reside. However, it is now clear that some post-translational modifications can create new self antigens (Ags) or

Hester A Doyle; Mark J Mamula

2001-01-01

18

Associations of Urinary Cadmium with Age and Urinary Proteins: Further Evidence of Physiological Variations Unrelated to Metal Accumulation and Toxicity  

PubMed Central

Background: The current risk assessment for environmental cadmium (Cd) largely relies on the assumption that urinary Cd (U-Cd) is a reliable biomarker of the Cd body burden. Recent studies have questioned the validity of this assumption. Objectives: We studied the lifetime trend of U-Cd as a function of diuresis, gender, smoking status, and protein tubular reabsorption. We also analyzed the associations between U-Cd and urinary proteins. Methods: Cd, retinol-binding protein, and albumin were measured in the urine of six cohorts of the general population of Belgium, with a mean age ranging from 5.7 to 88.1 years (n = 1,567). Variations of U-Cd with age were modeled using natural cubic splines. Results: In both genders, U-Cd decreased to a minimum (~ 0.20 ?g/L) at the end of adolescence, then increased until 60–70 years of age (~ 0.60 ?g/L in never-smokers) before leveling off or decreasing. When U-Cd was expressed in micrograms per gram of creatinine, these variations were amplified (minimum, 0.15 µg/g creatinine; maximum, 0.70 µg/g creatinine) and much higher U-Cd values were observed in women. We observed no difference in U-Cd levels between never-smokers and former smokers, and the difference with current smokers did not increase over time. Lifetime curves of U-Cd were higher with increasing urinary retinol-binding protein or albumin, a consequence of the coexcretion of Cd with proteins. Conclusions: At low Cd exposure levels, U-Cd and age are associated through nonlinear and nonmonotonic relationships that appear to be driven mainly by recent Cd intake and physiological variations in the excretion of creatinine and proteins. Citation: Chaumont A, Voisin C, Deumer G, Haufroid V, Annesi-Maesano I, Roels H, Thijs L, Staessen J, Bernard A. 2013. Associations of urinary cadmium with age and urinary proteins: further evidence of physiological variations unrelated to metal accumulation and toxicity. Environ Health Perspect 121:1047–1053;?http://dx.doi.org/10.1289/ehp.1306607 PMID:23774576

Chaumont, Agnes; Voisin, Catherine; Deumer, Gladys; Haufroid, Vincent; Annesi-Maesano, Isabella; Roels, Harry; Thijs, Lutgarde; Staessen, Jan

2013-01-01

19

Conformational heterogeneity in antibody-protein antigen recognition: implications for high affinity protein complex formation.  

PubMed

Specific, high affinity protein-protein interactions lie at the heart of many essential biological processes, including the recognition of an apparently limitless range of foreign proteins by natural antibodies, which has been exploited to develop therapeutic antibodies. To mediate biological processes, high affinity protein complexes need to form on appropriate, relatively rapid timescales, which presents a challenge for the productive engagement of complexes with large and complex contact surfaces (?600-1800 ?(2)). We have obtained comprehensive backbone NMR assignments for two distinct, high affinity antibody fragments (single chain variable and antigen-binding (Fab) fragments), which recognize the structurally diverse cytokines interleukin-1? (IL-1?, ?-sheet) and interleukin-6 (IL-6, ?-helical). NMR studies have revealed that the hearts of the antigen binding sites in both free anti-IL-1? Fab and anti-IL-6 single chain variable exist in multiple conformations, which interconvert on a timescale comparable with the rates of antibody-antigen complex formation. In addition, we have identified a conserved antigen binding-induced change in the orientation of the two variable domains. The observed conformational heterogeneity and slow dynamics at protein antigen binding sites appears to be a conserved feature of many high affinity protein-protein interfaces structurally characterized by NMR, suggesting an essential role in protein complex formation. We propose that this behavior may reflect a soft capture, protein-protein docking mechanism, facilitating formation of high affinity protein complexes on a timescale consistent with biological processes. PMID:24436329

Addis, Philip W; Hall, Catherine J; Bruton, Shaun; Veverka, Vaclav; Wilkinson, Ian C; Muskett, Frederick W; Renshaw, Philip S; Prosser, Christine E; Carrington, Bruce; Lawson, Alastair D G; Griffin, Robert; Taylor, Richard J; Waters, Lorna C; Henry, Alistair J; Carr, Mark D

2014-03-01

20

Detection of new antigenic proteins in Japanese cedar pollen.  

PubMed

In Japan, an increasing number of people suffer from pollenosis, a typical atopic disease. Japanese cedar (Cryptomeria japonica) pollen is the most common allergen that causes pollenosis. Although Cry j 1 and Cry j 2 are the common allergenic proteins contained in the pollen, there is a small population of patients who exhibit positive skin reactions to the pollen extract but are negative for both Cry j 1 and Cry j 2. This suggests that the pollen may contain other antigenic proteins besides these two molecules. In this study, we used sodium dodecyl sulfate-polyacrylamide gel electrophoresis to examine partially purified pollen extract (PPPE) and determine if there were other proteins that reacted to anti-PPPE sera collected from sensitized guinea pigs, mice and rabbits. Subsequently, we detected four bands of proteins with molecular weights around 7, 15, 20 and 31 kDa, which were different from those normally seen for Cry j 1 and Cry j 2. Among these, the 7, 15 and 20 kDa proteins could not be detected when anti-Cry j 1 monoclonal anti-body (mAb) and anti-Cry j 2 mAb were used as antibodies for Western blotting. Therefore, these three proteins may differ from Cry j 1 and Cry j 2 not only in molecular weight but also in antigenicity. In conclusion, three new antigenic proteins that are not identical to Cry j 1 and Cry j 2 have been shown to exist in Japanese cedar pollen. Structural analyses of these proteins may be useful in the development of new therapeutic methods, including specific immunotherapy for pollenosis. PMID:16755010

Matsumura, Daichi; Nabe, Takeshi; Mizutani, Nobuaki; Fujii, Masanori; Kohno, Shigekatsu

2006-06-01

21

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

22

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

23

Multicenter analyses demonstrate significant clinical effects of minor histocompatibility antigens on GvHD and GvL after HLA-matched related and unrelated hematopoietic stem cell transplantation.  

PubMed

The effect of minor H antigen mismatching on the occurrence of graft-versus-host disease (GvHD) and graft-versus-leukemia (GvL) after HLA-matched hematopoietic stem cell transplantation (HSCT) has mainly been demonstrated in single-center studies. Yet, the International Histocompatibility and Immunogenetics Workshops (IHIW) provide a collaborative platform to execute crucial large studies. In collaboration with 20 laboratories of the IHIW, the roles of 10 autosomal and 10 Y chromosome-encoded minor H antigens were investigated on GvHD and relapse incidence in 639 HLA-identical related donor (IRD) and 210 HLA-matched unrelated donor (MUD) HSCT recipients. Donor and recipient DNA samples were genotyped for the minor H antigens HA-1, HA-2, HA-3, HA-8, HB-1, ACC-1, ACC-2, SP110, PANE1, UGT2B17, and HY. The correlations with the primary outcomes GvHD (acute or chronic GvHD), survival, and relapse were statistically analyzed. The results of these multicenter analyses show that none of the HLA class I-restricted HY antigens were found to be associated with any of the primary outcomes. Interestingly, of the HLA class II-restricted HY antigens analyzed, HLA-DQ5 positive recipients showed a significantly increased GvHD-free survival in female-to-male HSCT compared with male-to-female HSCT (P = .013). Yet, analysis of the overall gender effect, thus independent of the known HY antigens, between the gender groups demonstrated an increased GvHD incidence in the female-to-male transplantations (P < .005) and a decreased GvHD-free survival in the female-to-male transplantations (P < .001). Of all autosomally encoded minor H antigens, only mismatching for the broadly expressed minor H antigen HA-8 increased the GvHD incidence in IRD HSCT (Hazard ratio [HR] = 5.28, P < .005), but not in MUD HSCT. Most striking was the influence of hematopoietic restricted minor H antigens on GvL as mismatching for hematopoietic minor H antigens correlated with lower relapse rates (P = .078), higher relapse-free survival (P = .029), and higher overall survival (P = .032) in recipients with GvHD, but not in those without GvHD. In conclusion, the significant GvHD effect of the broadly expressed minor H antigen HA-8 favors matching for HA-8 in IRD, but not in MUD, patient/donor pairs. The GvHD-GvL association demonstrating a significant lower relapse in hematopoietic minor H antigen mismatched patient/donor pairs underlines their clinical applicability for adoptive immunotherapy, enhancing the GvL effect in a GvHD controllable manner. PMID:23756210

Spierings, Eric; Kim, Yeung-Hyen; Hendriks, Matthijs; Borst, Eric; Sergeant, Ruhena; Canossi, Angelica; Oudshoorn, Machteld; Loiseau, Pascale; Dolstra, Harry; Markiewicz, Miroslaw; Leffell, Mary S; Pereira, Noemi; Kircher, Brigitte; Turpeinen, Hannu; Eliaou, Jean-François; Gervais, Thibaut; Laurin, David; Enczmann, Jürgen; Martinetti, Miryam; Thomson, Jackie; Oguz, Fatma; Santarone, Stella; Partanen, Jukka; Siekiera, Urszula; Alessandrino, Emilio Paolo; Kalayoglu, Sevgi; Brand, Ronald; Goulmy, Els

2013-08-01

24

Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest  

Microsoft Academic Search

The INK4a (MTS1, CDKN2) gene encodes an inhibitor (p16INK4a) of the cyclin D-dependent kinases CDK4 and CDK6 that blocks them from phosphorylating the retinoblastoma protein (pRB) and prevents exit from the G1 phase of the cell cycle. Deletions and mutations involving INK4a occur frequently in cancers, implying that p16INK4a, like pRB, suppresses tumor formation. An unrelated protein (p19ARF) arises in

Dawn E. Ouelle; Frédérique Zindy; Richard A. Ashmun; Charles J. Sherr

1995-01-01

25

Comparison of Different Live Vaccine Strategies In Vivo for Delivery of Protein Antigen or Antigen-Encoding DNA and mRNA by Virulence-Attenuated Listeria monocytogenes  

Microsoft Academic Search

Listeria monocytogenes can be used to deliver protein antigens or DNA and mRNA encoding such antigens directly into the cytosol of host cells because of its intracellular lifestyle. In this study, we compare the in vivo efficiencies of activation of antigen-specific CD8 and CD4 T cells when the antigen is secreted by L. monocy- togenes or when antigen-encoding plasmid DNA

Daniela I. M. Loeffler; Christoph U. Schoen; Werner Goebel; Sabine Pilgrim

2006-01-01

26

Absorption of food protein antigen in infants with food protein-induced enterocolitis  

Microsoft Academic Search

Increased gastrointestinal absorption of intact antigen with systemic immunization has been considered a major etiologic factor in the development of food sensitivity. We attempted to test this hypothesis in infants with suspected food protein-induced enter colitis by measuring serum ovalbumin (OVA) concentrations after ingestion of egg white (prior to the performance of good challenges to establish this diagnosis). We first

Geraldine Keating Powell; Phillip J. McDonald; Greggory J. Van Sickle; Randall M. Goldblum

1989-01-01

27

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

28

A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen  

E-print Network

A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective) Anthrax protective antigen (PA) is a 735-aa polypeptide that facili- tates the exit of anthrax lethal as a carrier to deliver antigens into the cytosol for efficient induction of T lymphocyte responses

Lieberman, Judy

29

The Leptospiral Antigen Lp49 is a Two-Domain Protein with Putative Protein Binding Function  

SciTech Connect

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Currently available vaccines have limited effectiveness and therapeutic interventions are complicated by the difficulty in making an early diagnosis of leptospirosis. The genome of Leptospira interrogans was recently sequenced and comparative genomic analysis contributed to the identification of surface antigens, potential candidates for development of new vaccines and serodiagnosis. Lp49 is a membrane-associated protein recognized by antibodies present in sera from early and convalescent phases of leptospirosis patients. Its crystal structure was determined by single-wavelength anomalous diffraction using selenomethionine-labelled crystals and refined at 2.0 Angstroms resolution. Lp49 is composed of two domains and belongs to the all-beta-proteins class. The N-terminal domain folds in an immunoglobulin-like beta-sandwich structure, whereas the C-terminal domain presents a seven-bladed beta-propeller fold. Structural analysis of Lp49 indicates putative protein-protein binding sites, suggesting a role in Leptospira-host interaction. This is the first crystal structure of a leptospiral antigen described to date.

Oliveira Giuseppe,P.; Oliveira Neves, F.; Nascimento, A.; Gomes Guimaraes, B.

2008-01-01

30

Pooled Protein Immunization for Identification of Cell Surface Antigens in Streptococcus sanguinis  

PubMed Central

Background Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species. Methods and Findings We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity. Conclusions The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases. PMID:20668678

Ge, Xiuchun; Kitten, Todd; Munro, Cindy L.; Conrad, Daniel H.; Xu, Ping

2010-01-01

31

DEMONSTRATION OF MULTIPLE ANTIGENIC DETERMINANTS ON 'MYCOPLASMA PNEUMONIAE' ATTACHMENT PROTEIN BY MONOCLONAL ANTIBODIES  

EPA Science Inventory

Distinct multiple antigenic determinants of the attachment protein of Mycoplasma pneumoniae have been identified by limited proteolytic cleavage using specific monoclonal antibodies. Western blots prepared from the gels containing the cleaved fragments were probed with antiserum ...

32

Shift in S-layer protein expression responsible for antigenic variation in Campylobacter fetus.  

PubMed Central

Campylobacter fetus strains possess regular paracrystalline surface layers (S-layers) composed of high-molecular-weight proteins and can change the size and crystalline structure of the predominant protein expressed. Polyclonal antisera demonstrate antigenic cross-reactivity among these proteins but suggest differences in epitopes. Monoclonal antibodies to the 97-kDa S-layer protein of Campylobacter fetus subsp. fetus strain 82-40LP showed three different reactivities. Monoclonal antibody 1D1 recognized 97-kDa S-layer proteins from all C. fetus strains studied; reactivity of monoclonal antibody 6E4 was similar except for epitopes in S-layer proteins from reptile strains and strains with type B lipopolysaccharide. Monoclonal antibody 2E11 only recognized epitopes on S-layer proteins from strains with type A lipopolysaccharide regardless of size. In vitro shift from a 97-kDa S-layer protein to a 127-kDa S-layer protein resulted in different reactivity, indicating that size change was accompanied by antigenic variation. To examine in vivo variation, heifers were genetically challenged with Campylobacter fetus subsp. venerealis strains and the S-layer proteins from sequential isolates were characterized. Analysis with monoclonal antibodies showed that antigenic reactivities of the S-layer proteins were varied, indicating that these proteins represent a system for antigenic variation. Images PMID:7688715

Wang, E; Garcia, M M; Blake, M S; Pei, Z; Blaser, M J

1993-01-01

33

HMGB1-derived peptide acts as adjuvant inducing immune responses to peptide and protein antigen  

PubMed Central

There is a need for new adjuvants that will induce immune responses to subunit vaccines. We show that a short peptide, named Hp91, whose sequence corresponds to an area within the endogenous molecule High mobility group box (HMGB1) protein potentiates cellular immune responses to peptide antigen and cellular and humoral immune responses to protein antigen in vivo. Hp91 promoted the in vivo production of the immunomodulatory cytokines, IFN-?, TNF-?, IL-6, and IL-12 (p70), as well as antigen-specific activation of CD8+ T cells. These results demonstrate the ability of a short immunostimulatory peptide to serve as an adjuvant for subunit vaccines. PMID:20800114

Saenz, R.; da Silva Souza, C.; Huang, C-T; Larsson, M.; Esener, S.; Messmer, D.

2010-01-01

34

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

PubMed Central

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

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

1996-01-01

35

Antigenicity of Recombinant Maltose Binding Protein-Mycobacterium avium subsp. paratuberculosis Fusion Proteins with and without Factor Xa Cleaving  

PubMed Central

Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants. Proteomic studies have shown that M. avium subsp. paratuberculosis expresses certain proteins when exposed to in vitro physiological stress conditions similar to the conditions experienced within a host during natural infection. Such proteins are hypothesized to be expressed in vivo, are recognized by the host immune system, and may be of potential use in the diagnosis of JD. In this study, 50 recombinant maltose binding protein (MBP)-M. avium subsp. paratuberculosis fusion proteins were evaluated using serum samples from sheep infected with M. avium subsp. paratuberculosis, and 29 (58%) were found to be antigenic. Among 50 fusion proteins, 10 were evaluated in MBP fusion and factor Xa-cleaved forms. A total of 31 proteins (62%) were found to be antigenic in either MBP fusion or factor Xa-cleaved forms. Antigenicity after cleavage and removal of the MBP tag was marginally enhanced. PMID:24132604

Begg, Douglas J.; Purdie, Auriol C.; Bannantine, John P.; Whittington, Richard J.

2013-01-01

36

Co-Expression of Lewis y Antigen with Human Epididymis Protein 4 in Ovarian Epithelial Carcinoma  

PubMed Central

Objective The main aims of this study were to explore the molecular structural relationship between Human epididymis protein 4 (HE4) and Lewis y antigen by determining their expression patterns and clinical significance in ovarian epithelial carcinoma. Methods The structural relationship between HE4 and Lewis y antigen was examined using immunoprecipitation and confocal laser scanning microscopy. HE4 and Lewis y were detected in tissues from malignant (53 cases), borderline (27 cases), benign (15 cases) and normal ovarian tissues (15 cases) using immunohistochemical analysis. Results HE4 was present in ovarian cancer, benign tumor tissues, ovarian carcinoma cells, and culture medium, and contained Lewis y antigen. Moreover, expression of Lewis y antigen in HE4 from ovarian cancer was higher than that from benign tumor (P<0.05). HE4 possibly exists as two protein isoforms, both containing Lewis y antigen. Our immunohistochemistry data revealed significantly higher positive expression rates of HE4 in malignant ovarian tissues, compared to benign tumor and normal tissue (P<0.05), similar to Lewis y antigen levels in ovarian cancer (P<0.05). Notably, tissues displaying marked expression of HE4 simultaneously expressed high levels of Lewis y antigen. A linear correlation between the expression patterns of HE4 and Lewis y antigen was evident. Consistently, double-labeling immunofluorescence experiments illustrated co-localization of HE4 and Lewis y antigen within the same area. Conclusions HE4 contains Lewis y antigen. Our results further demonstrate a close correlation between the expression levels of the two antigens, which are significantly high in ovarian cancer. PMID:23894390

Zhuang, Huiyu; Gao, Jian; Hu, Zhenhua; Liu, Juanjuan; Liu, Dawo; Lin, Bei

2013-01-01

37

N-Glycosylation of extracellular matrix protein 1 (ECM1) regulates its secretion, which is unrelated to lipoid proteinosis  

PubMed Central

Extracellular matrix protein 1 (ECM1) is expressed in a wide variety of tissues and plays important roles in extracellular matrix formation. Additionally, ECM1 gene mutations cause lipoid proteinosis (LP), a rare skin condition of genetic origin. However, an effective therapeutic approach of LP is not established. Here, we showed that ECM1 gene mutation observed in LP patients significantly suppresses its secretion. As ECM1 has three putative N-glycosylation sites and most of mutated ECM1 observed in LP patients are defective in these N-glycosylation sites, we investigated the correlation between LP and N-glycosylation of ECM1. We identified that the Asn354 and Asn444 residues in ECM1 were N-glycosylated by mass spectrometry analysis. In addition, an N-linked glycan at Asn354 negatively regulated secretion of ECM1, contrary to LP patient-derived mutants. These results indicate that the defect of N-glycosylation in ECM1 is not involved in the aberration of secretion of LP-derived mutated ECM1. PMID:25379385

Uematsu, Shiho; Goto, Yuki; Suzuki, Takehiro; Sasazawa, Yukiko; Dohmae, Naoshi; Simizu, Siro

2014-01-01

38

Expression of Bcl-2 protein and Fas antigen in non-Hodgkin's lymphomas.  

PubMed Central

Expression of Bcl-2 protein and Fas antigens was analyzed in 12 cases of follicular lymphoma and 32 cases of diffuse lymphoma, including 22 B-cell and 10 T-cell lymphomas. It was shown that 75% of follicular lymphomas had clear expression of both Bcl-2 protein and Fas antigen. Thus, follicular lymphomas may have a growth advantage due to their high expression of Bcl-2 protein, which tended to impede apoptosis mediated by Fas antigen. On the other hand, diffuse lymphomas showed various patterns; 28% were double positive, 16% were only Bcl-2 protein-positive, 28% were only Fas antigen-positive, and 28% were double negative or equivocal. Cytocidal assay of seven leukemia/lymphoma cell lines using anti-human Fas monoclonal antibody revealed that overexpression of Bcl-2 protein tended to impede apoptosis mediated by Fas antigen. However, this inhibitory effect of Bcl-2 protein was incomplete and its effect might be dependent upon cell type. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7519826

Kondo, E.; Yoshino, T.; Yamadori, I.; Matsuo, Y.; Kawasaki, N.; Minowada, J.; Akagi, T.

1994-01-01

39

Computational antigenic epitope prediction by calculating electrostatic desolvation penalties of protein surfaces.  

PubMed

The prediction of antigenic epitopes on the surface of proteins is of great importance for vaccine development and to specifically design recombinant antibodies. Computational methods based on the three-dimensional structure of the protein allow for the detection of noncontinuous epitopes in contrast to methods based on the primary amino-acid sequence only. A method recently developed to predict protein-protein binding sites is presented, and the application to predict putative antigenic epitopes is described in detail. The prediction approach is based on the local perturbation of the electrostatic field at the surface of a protein due to a neutral probe of low dielectric constant that represents an approaching binding partner. The calculated change in electrostatic energy corresponds to an energy penalty of desolvating a protein surface region, and antigenic epitope surface regions tend to be associated with a lower penalty compared to the average protein surface. The protocol to perform the calculations is described and illustrated on an example antigen, the outer surface protein A of Borrelia burgdorferi, a pathogenic organism causing lyme disease. PMID:25048135

Fiorucci, Sébastien; Zacharias, Martin

2014-01-01

40

Protein kinase C delta stimulates antigen presentation by Class II MHC in murine dendritic cells  

Microsoft Academic Search

Maturation of dendritic cells (DCs) regulates protein sorting in endosomal compartments to promote the surface expression of molecules involved in T cell activation. MHC Class II complexes are mobilized to the surface via intracellular effector molecules that remain largely unknown. We here show that protein kinase C (PKC) stimulates Class II antigen surface expression, using knock-in mice that express a

Michael Majewski; Tina O. Bose; Fenna C. M. Sille; Annette M. Pollington; Edda Fiebiger; Marianne Boes

2007-01-01

41

Analysis of spiroplasma proteins: contribution to the taxonomy of group IV spiroplasmas and the characterization of spiroplasma protein antigens.  

PubMed Central

Spiroplasma strains of group IV were compared by two-dimensional protein analyses on polyacrylamide gels. Although considerable diversity was evident, the assemblages studied were less heterogeneous than the known strains of group I. Two electrophoretic techniques were used to identify spiroplasma proteins that had been used to immunize rabbits. These included monoclonal antibodies prepared against Spiroplasma citri. In the first technique, protein antigens were purified by immunoaffinity chromatography, then identified with SDS-PAGE. In the second technique, spiroplasma proteins were first separated by SDS-PAGE, then antigens were identified by antibody binding to blot-transferred proteins. Finally, two-dimensional protein electrophoresis has been used as a source of immunogens to characterize monospecific antibodies against individual S. citri proteins. Images FIG. 1 FIG. 2 PMID:6206657

Mouches, C.; Candresse, T.; McGarrity, G. J.; Bove, J. M.

1983-01-01

42

An analysis of bison erythrocyte antigens and blood proteins  

E-print Network

procedures adopted for bovine blood characterization. However, the information available on the Bos bison species is still limited. Development and use of more tests for red cell antigens and blood enzymes and tests to demonstrate the genotypic... more recent summary of the 12 blood group systems and blood factors described for cattle to date (Rasmusen, 1975 and Caldwell, 1982). Bison Blood Groups It has been shown that various blood typing reagents used in detecting cattle (Bos taurus...

Zamora, Linda Elia

2012-06-07

43

Yersinia outer proteins (YOPS) E, K and N are antigenic but non-protective compared to V antigen, in a murine model of bubonic plague  

Microsoft Academic Search

The pathogenicYersiniaeproduce a range of virulence proteins, encoded by a 70 kb plasmid, which are essential for infection, and also form part of a contact-dependent virulence mechanism. One of these proteins, V antigen, has been shown to confer a high level of protection against parenteral infection withY. pestisin murine models, and is considered to be a protective antigen. In this

Sophie E. C. Leary; Kate F. Griffin; Edouard E. Galyov; Jason Hewer; E. Diane Williamson; Anna Holmström; Åke Forsberg; Richard W. Titball

1999-01-01

44

Baculovirus expression and antigenic characterization of the capsid proteins of three Norwalk-like viruses.  

PubMed

Human caliciviruses (HuCVs) are antigenically diverse. The antigenic relationships among different HuCVs have been difficult to study because HuCVs cannot be passaged in the laboratory. In this study, we describe cloning, sequencing and expression of the viral capsid proteins of three HuCVs that were identified in outbreaks of acute gastroenteritis in Virginia in 1997-1998. Yields of the capsid proteins similar to previously expressed recombinant Norwalk virus were obtained using the baculovirus expression system. Recombinant VA97207 capsid protein (rVA97207) and rVA98387, but not rVA98115, formed virus-like particles (VLPs). All three recombinant capsid antigens detected seroresponses in patients involved in outbreaks of acute gastroenteritis associated with genetically homologous or related HuCVs. The antigenic relationships of the three strains were further characterized using hyperimmune antisera against the three capsid antigens as well as four previously characterized recombinant capsid antigens of Norwalk (rNV), Mexico (rMxV), Hawaii (rHV), and Grimsby viruses (rGrV). VA98387 shared 98% aa identity with GrV; rVA98387 was detected by antisera to GrV. VA98115 shared 87% aa identity with Desert Storm virus and 65% aa identity with prototype Norwalk virus (NV); rVA98115 reacted weakly with NV antisera. VA97207 shared 80% aa identity with Amsterdam and 75% aa identity with Leeds strains and rVA97207 was not detected by any of the heterologous antibodies. In conclusion, VA97207 and VA98115 may belong to CV antigenic types not previously expressed, while VA98387 is a GrV-like virus. Low levels of cross-reactive antibodies were detected between types. Further studies to characterize these antigens and to develop enzyme immune assays (EIAs) for these strains are in progress. PMID:11855626

Jiang, X; Zhong, W M; Farkas, T; Huang, P W; Wilton, N; Barrett, E; Fulton, D; Morrow, R; Matson, D O

2002-01-01

45

The secondary structure of heated whey protein and its hydrolysates antigenicity.  

PubMed

Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) were used to investigate the conformational changes of heated whey protein (WP) and the corresponding changes in the hydrolysates immunoreactivity were determined by competitive enzyme-linked immunosorbent assay (ELISA). Results showed that the contents of alpha- helix and beta-sheet of WP did not decrease much under mild heating conditions and the antigenicity was relatively high; when the heating intensity increased (70 degrees for 25 min or 75 degrees C for 20 min), the content of alpha- helix and beta-sheet decreased to the minimum, so was the antigenicity; However, when the WP was heated at even higher temperature and for a longer time, the beta-sheet associated with protein aggregation begun to increase and the antigenicity increased correspondingly. It was concluded that the conformations of heated WP and the antigenicity of its hydrolysates are related and the optimum structure for decreasing the hydrolysates antigeniity is the least content of alpha-helix and beta-sheet. Establishing the relationship between the WP secondary structure and WP hydrolysates antigenicity is significant to supply the reference for antigenicity reduction by enzymolysis. PMID:22242516

Pang, Zhi-Hua; Zhu, Jun; Wu, Wei-Jing; Wang, Fang; Ren, Fa-Zheng; Zhang, Lu-Da; Guo, Hui-Yuan

2011-11-01

46

Distribution of antigenic and non-antigenic proteins in the organism  

Microsoft Academic Search

Rabbits were injected with ovalbumin, bovine serum globulin, and rabbit serum globulin; the proteins were labeled by traces of radioiodine. Ovalbumin is rapibly eliminated from the blood and deposited in the reticuloendothelial organs where it is concentrated in the cytoplasmic granules. Rabbit serum globulin is present in the blood in higher concentration than in the tissues at all times up

Felix Haurowitz; Charles F. Crampton; Herbert H. Reller

1953-01-01

47

Identification and characterization of the protein antigens of Leptospira interrogans serovar hardjo.  

PubMed Central

We radiolabeled Leptospira proteins with [35S]methionine. Solubilized extracts of radiolabeled L. interrogans serovar hardjo strain hardjoprajitno were analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. We compared the protein profile obtained in this manner to the protein profiles of various [35S]methionine-labeled Leptospira spp. The profiles of the pathogenic L. interrogans strains were very similar but not identical and exhibited no obvious relationship to those of the two nonpathogenic species. We used solubilized, radiolabeled hardjoprajitno extracts and a sensitive radioimmunoprecipitation procedure to identify protein antigens recognized by immunoglobulin G antibodies present in various rabbit anti-hardjo sera. Homologous hyperimmune rabbit serum efficiently precipitated a large subset of proteins, the majority of which were between 30,000 and 66,500 daltons. Radioimmunoprecipitations with sera prepared against each of four recent hardjo isolates cultured from infected cattle produced similar results. Immunoprecipitations done with various radiolabeled Leptospira extracts and anti-hardjoprajitno serum demonstrated that the pathogenic leptospires possessed a number of cross-reactive major and minor protein antigens. By cell fractionation procedures, we found that most of the major protein antigens were present in the outer envelope. These proteins were exposed on the leptospiral cell surface because intact radiolabeled leptospires bound antibodies directed against them. Images PMID:3988343

Nunes-Edwards, P L; Thiermann, A B; Bassford, P J; Stamm, L V

1985-01-01

48

Antigenic and structural differences among six proteins II expressed by a single strain of Neisseria gonorrhoeae.  

PubMed Central

Gonococci express a family of related outer membrane proteins designated protein II (P.II), which undergo both phase and antigenic variation. Six P.II proteins have been identified in strain FA1090. We developed monoclonal antibodies specific for each P.II protein. Using these antibodies as probes, we purified the six different P.II proteins of this strain. Despite the relatedness of the proteins, we could not purify all of them by a single purification scheme. Four P.II proteins were purified by chromatofocusing, and the remaining two proteins were purified by hydrophobic interaction chromatography on phenyl-Sepharose. The N-terminal amino acid sequence of the proteins showed a high degree of sequence conservation. However, there was variability at specific amino acid residues, giving each P.II protein a unique N-terminal amino acid sequence. Thus P.II proteins of one strain differ among themselves not only in antigenic determinants and primary structure, but also in other characteristics affecting their properties in different chromatographic systems. Images PMID:3114142

Barritt, D S; Schwalbe, R S; Klapper, D G; Cannon, J G

1987-01-01

49

Comprehensive Antigen Screening Identifies Moraxella catarrhalis Proteins That Induce Protection in a Mouse Pulmonary Clearance Model  

PubMed Central

Moraxella catarrhalis is one of the three most common causative bacterial pathogens of otitis media, however no effective vaccine against M. catarrhalis has been developed so far. To identify M. catarrhalis vaccine candidate antigens, we used carefully selected sera from children with otitis media and healthy individuals to screen small-fragment genomic libraries that are expressed to display frame-selected peptides on a bacterial cell surface. This ANTIGENome technology led to the identification of 214 antigens, 23 of which were selected by in vitro or in vivo studies for additional characterization. Eight of the 23 candidates were tested in a Moraxella mouse pulmonary clearance model, and 3 of these antigens induced significantly faster bacterial clearance compared to adjuvant or to the previously characterized antigen OmpCD. The most significant protection data were obtained with the antigen MCR_1416 (Msp22), which was further investigated for its biological function by in vitro studies suggesting that Msp22 is a heme binding protein. This study comprises one of the most exhaustive studies to identify potential vaccine candidate antigens against the bacterial pathogen M. catarrhalis. PMID:23671716

Verhaegh, Suzanne J. C.; Niebisch, Axel; Hanner, Markus; Selak, Sanja; Schuler, Wolfgang; Morfeldt, Eva; Hellberg, Christel; Nagy, Eszter; Lundberg, Urban; Hays, John P.; Meinke, Andreas; Henriques-Normark, Birgitta

2013-01-01

50

The circumsporozoite protein is an immunodominant protective antigen in irradiated sporozoites  

E-print Network

into the exo-erythrocytic forms (EEFs). Immunization with irradiated sporozoites (IrSp) leads to robust to circumsporozoite protein (CS) that inhibit sporozoite infectivity, and T cells that destroy the EEFs4 . To study Plasmodium genes are expressed in sporozoites5 and EEFs6 , CS is a dominant protective antigen. Nevertheless

Cai, Long

51

Genetically modified anthrax lethal toxin safely delivers whole HIV protein antigens into the  

E-print Network

Genetically modified anthrax lethal toxin safely delivers whole HIV protein antigens, 2000 (received for review January 24, 2000) Bacillus anthrax lethal toxin can be engineered to deliver cytotoxic T lymphocyte response in HIV-infected donor peripheral blood mononuclear cells in vitro

Lieberman, Judy

52

Antigenic Proteins of 'Borrelia burgdorferi' (Continuation in Part of Serial No. 487,716).  

National Technical Information Service (NTIS)

The invention relates to antigenic proteins specific to Borrelia burgdorferi which have a molecular weight of 28 kDa or 39 kDa as determined bySDS-PAGE and are reactive with Lyme borreliosis serum or fragments thereof and to the corresponing DNA. The prot...

W. J. Simpson, T. G. Schwan

1991-01-01

53

Laminin and Bullous Pemphigoid Antigen Are Distinct Basement Membrane Proteins Synthesized by Epidermal Cells  

Microsoft Academic Search

We sought to determine if laminin, a high molecular weight glycoprotein of basement membrane, is synthesized by epidermal cells and whether it is distinct from bullous pemphigoid (BP) antigen, another high molecular weight-protein of basement membrane. By indirect immunofluorescence we detected laminin in cultures of Pam cells (a mouse keratinocyte cell line) and normal human epidermal cells. To directly demonstrate

John R. Stanley; Pamela Hawley-Nelson; Mina Yaar; George H. Martin; Stephen I. Katz

1982-01-01

54

Human Hepatocellular Carcinoma Cell Lines Secrete the Major Plasma Proteins and Hepatitis B Surface Antigen  

Microsoft Academic Search

Analysis of the cell culture fluid from two new human hepatoma-derived cell lines reveals that 17 of the major human plasma proteins are synthesized and secreted by these cells. One of these cell lines, Hep 3B, also produces the two major polypeptides of the hepatitis B virus surface antigen. When Hep 3B is injected into athymic mice, metastatic hepatocellular carcinomas

Barbara B. Knowles; Chin C. Howe; David P. Aden

1980-01-01

55

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

E-print Network

where these slow-growing pathogenic bacteria have been isolated (Brookset al. 1984; Falkinhamet al. 1980Identification of cytoplasmic membrane protein antigens of Mycobacterium avium, M. intracellulare. intracellulare, and M. scrofulaceum. Can. J. Microbiol. 35: 529-534. The cytoplasmic membrane isolated from

Falkinham, Joseph

56

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.; Gioffre, A.; Peralta, A.; Buddle, B. M.; Cataldi, A.

2009-01-01

57

Polyomavirus small T antigen interacts with yes-associated protein to regulate cell survival and differentiation.  

PubMed

Murine polyomavirus small t antigen (PyST) regulates cell cycle, cell survival, apoptosis, and differentiation and cooperates with middle T antigen (MT) to transform primary cells in vitro and in vivo. Like all polyomavirus T antigens, PyST functions largely via its interactions with host cell proteins. Here, we show that PyST binds both Yes-associated protein 1 (YAP1) and YAP2, integral parts of the Hippo signaling pathway, which is a subject of increasing interest in human cancer. The transcription factor TEAD, which is a known target of YAP, is also found in PyST complexes. PyST enhanced YAP association with protein phosphatase 2A (PP2A), leading to decreased YAP phosphorylation. PyST increased YAP levels by decreasing its degradation. This effect was mediated by a reduction in YAP association with ?-transducin repeat protein (?TRCP), which is known to regulate YAP turnover in a phosphorylation-dependent manner. Genetic analysis has identified PyST mutants defective in YAP binding. These mutants demonstrated that YAP binding is important for PyST to block myoblast differentiation and to synergize with the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) to promote cell death in 3T3-L1 preadipocytes placed under differentiation conditions. In addition to YAP binding, both of these phenotypes require PyST binding to PP2A. Importance: The Hippo/YAP pathway is a highly conserved cascade important for tissue development and homeostasis. Defects in this pathway are increasingly being associated with cancer. Polyomavirus small t antigen is a viral oncogene that cooperates with middle T antigen in transformation. On its own, small t antigen controls cell survival and differentiation. By binding YAP, small t antigen brings it together with protein phosphatase 2A. This work shows how this association of small t antigen with YAP is important for its effects on cell phenotype. It also suggests that PyST can be used to characterize cellular processes that are regulated by YAP. PMID:25122798

Hwang, Justin H; Pores Fernando, Arun T; Faure, Nathalie; Andrabi, Shaida; Hahn, William C; Schaffhausen, Brian S; Roberts, Thomas M

2014-10-01

58

Plasmodium falciparum Merozoite Surface Protein 6 Is a Dimorphic Antigen  

PubMed Central

Merozoite surface protein 1 (MSP1) is a highly polymorphic Plasmodium falciparum merozoite surface protein implicated in the invasion of human erythrocytes during the asexual cycle. It forms a complex with MSP6 and MSP7 on the merozoite surface, and this complex is released from the parasite around the time of erythrocyte invasion. MSP1 and many other merozoite surface proteins contain dimorphic elements in their protein structures, and here we show that MSP6 is also dimorphic. The sequences of eight MSP6 genes indicate that the alleles of each dimorphic form of MSP6 are highly conserved. The smaller 3D7-type MSP6 alleles are detected in parasites from all malarious regions of the world, whereas K1-type MSP6 alleles have only been detected in parasites from mainland Southeast Asia. Cleavage of MSP6, which produces the p36 fragment in 3D7-type MSP6 and associates with MSP1, also occurs in K1-type MSP6 but at a different site in the protein. Anti-3D7 MSP6 antibodies weakly inhibited erythrocyte invasion by homologous 3D7 merozoites but did not inhibit a parasite line expressing the K1-type MSP6 allele. Antibodies from hyperimmune individuals affinity purified on an MSP3 peptide cross-reacted with MSP6; therefore, MSP6 may also be a target of antibody-dependent cellular inhibition. PMID:15039357

Pearce, J. Andrew; Triglia, Tony; Hodder, Anthony N.; Jackson, David C.; Cowman, Alan F.; Anders, Robin F.

2004-01-01

59

ABC proteins in antigen translocation and viral inhibition.  

PubMed

How ABC transporters work is a key issue because of their important roles in multidrug resistance of pathogenic bacteria, reduced efficacy of antitumor drugs, cholesterol metabolism, cell homeostasis and immune response. In the past few years, significant progress has been made in crystallization and structure determination of (mostly) bacterial ABC transporters, as well as in functional studies on ABC systems involved in human pathology. In this review, we use the transporter associated with antigen processing (TAP) to illustrate what is known regarding the mechanism of substrate transport. We also discuss the chemical basis of substrate recognition by TAP and the allosteric cross-talk between the binding of substrate, the release of chemical energy by ATP hydrolysis and cross-membrane translocation. Finally, we detail the role of TAP in a large macromolecular assembly, which optimally loads MHC class I molecules, and the interference with this machinery by TAP-targeted viral factors. Because of structural and probable mechanistic similarities, the understanding of the detailed structure and mechanism of TAP will be applicable to all ABC systems, including those of medical relevance. PMID:20644544

Parcej, David; Tampé, Robert

2010-08-01

60

Immunogenicity of hypothetical highly conserved proteins as novel antigens in Anaplasma marginale.  

PubMed

Anaplasma marginale is a tick-transmitted Gram-negative intraerythrocytic bacterium and the etiological agent of bovine Anaplasmosis. Even though considerable research efforts have been undertaken, Anaplasmosis vaccine development remains a challenging field. Outer-membrane-specific antigens responsible for the ability of more complex immunogens could have a significant role in the protective response. Thus, the identification of outer-membrane antigens represents a major goal in the development of bacterial vaccines. Considering that 40 % of the annotated proteins in A. marginale remain as hypothetical, we selected three candidate antigens, AM1108, AM127, and AM216 based on experimental evidence, in silico structure prediction of ?-barrel outer membrane, and orthology clustering. Sequence alignment and analysis demonstrated a high degree of conservation for the three proteins between the isolates from Argentina compared to the American strains. We confirmed the transcription of the three genes in the intraerythrocytic stage. AM1108 and AM216 recombinant proteins elicited specific T-cell response proliferation and a significant rise in TNF-? and IFN-? transcript levels, respectively. Only AM1108 was able to be recognized by specific antibodies from infected bovines. This study allowed the identification of new candidate components of the outer-membrane fraction of A. marginale. Further studies will be required to analyze their potential as effective antigens for being included in rational vaccine strategies. PMID:24126603

Nuñez, Pablo A; Moretta, Rosalia; Ruybal, Paula; Wilkowsky, Silvina; Farber, Marisa D

2014-03-01

61

Purification, characterization, and localization of a protein antigen shared by thermophilic campylobacters.  

PubMed

A protein antigen with an apparent molecular weight (Mr) of 31,000 was isolated from 0.2 M glycine hydrochloride (pH 2.2) extracts of a typical human fecal isolate, Campylobacter jejuni VC74. The protein was purified to homogeneity on a preparative scale by immunoaffinity chromatography followed by molecular sieving with a Superose 12 column. Isoelectric focusing under nondenaturing conditions indicated a pI of 9.3, and amino acid composition analysis showed that the protein was unusually rich in lysine, containing 14.9 mol% of this basic amino acid. Cysteine and tryptophan were absent. The protein also contained approximately 35% hydrophobic amino acid residues, and N-terminal amino acid analysis showed that 17 of the first 38 residues were hydrophobic. This amino-terminal sequence to residue 22 was virtually identical to that of an antigenically cross-reactive 31,000-Mr protein isolated from another C. jejuni strain belonging to a different heat-labile serogroup. Western blotting (immunoblotting) of glycine extracts of other C. jejuni, Campylobacter coli, and Campylobacter laridis strains belonging to different thermolabile and thermostable serotypes, as well as Campylobacter fetus, with a rabbit polyclonal antiserum raised against the purified C. jejuni VC74 protein showed that all C. jejuni, C. coli, and C. laridis strains tested contained a 31,000-Mr protein with epitopes which were antigenically cross-reactive with the C. jejuni VC74 protein. The antigenically cross-reactive epitopes of this protein were also readily detected by immunodot blot assay of glycine extracts of C. jejuni, C. coli, and C. laridis with monospecific polyclonal antisera to the 31,000-Mr protein, suggesting that this serological test could be a useful addition to those currently employed in the rapid identification of these important pathogens. Slide agglutination reactions, immunofluorescence assay, and immunogold electron microscopy with antisera to purified 31,000-Mr protein and trypsin treatment of whole cells indicated that the cross-reactive epitopes of the 31,000-Mr protein were not exposed on the cell surface. Cell fractionation analysis and immunogold electron microscopy located the protein on the outer surface of the cytoplasmic membrane. This finding suggests that the 31,000-Mr protein is not a good candidate for inclusion in a monovalent subunit Campylobacter vaccine. PMID:2380360

Dubreuil, J D; Kostrzynska, M; Logan, S M; Harris, L A; Austin, J W; Trust, T J

1990-06-01

62

Radioimmunoassay of Mammalian Type-C Viral Proteins: Interspecies Antigenic Reactivities of the Major Internal Polypeptide*  

PubMed Central

Mammalian type-C viruses contain a major internal polypeptide of about 30,000 daltons that is characterized by both intraspecies and interspecies antigenic reactivities. Radioimmunoprecipitation assays were used for measurement of this protein; the assay was based upon interspecies reactivities of the protein. As little as 5 ng of the group-specific antigen of murine leukemia virus can be measured by radioimmunoprecipitation assays, thus providing an approximate 10,000-fold increase in sensitivity over the standard immunodiffusion procedure. The type-C viruses that were recently isolated from a woolly monkey and gibbon ape each have an interspecies type-C antigenic reactivity. The primate viruses, however, could be distinguished from the type-C viruses of murine, rat, hamster, and feline origin that were more highly related to each other. The interspecies reactivity of the 30,000-dalton polypeptide is an immunological marker of the mammalian type-C viruses, since even with this sensitive assay other mammalian viruses with RNA-dependent DNA polymerase activity did not contain the type-C interspecies antigen. Images PMID:4505653

Parks, Wade P.; Scolnick, Edward M.

1972-01-01

63

Identification of early diagnostic antigens from Spirometra erinaceieuropaei sparganum soluble proteins using immunoproteomics.  

PubMed

In order to identify early specific diagnostic antigens of Spirometra erinaceieuropaei (syn. S. erinacei or S. mansoni) sparganum, soluble proteins were analyzed by two-dimensional electrophoresis (2-DE) and western blotting probed with immune sera from infected mice at 14 days post-infection. From a total of approximately 462 proteins spots mainly distributed in pI range of 5-6.6 and with molecular mass of 25-48 kDa, 6 immuno-reactive protein spots with molecular mass of 31.8-38.3 kDa were characterized by MALDI-TOF/TOF-MS. Three proteins were identified as S. erinaceieuropaei cysteine protease, Toxoplasma gondii hypothetical protein and Pecten spp actin, while the remaining were unidentified. The cysteine protease from S. erinaceieuropaei soluble proteins recognized by early infection sera might be developed as diagnostic reagent for early detection of sparganosis. PMID:24974641

Hu, Dan Dan; Cui, Jing; Xiao, Di; Wang, Li; Liu, Li Na; Liu, Ruo Dan; Zhang, Jian Zhong; Wang, Zhong Quan

2014-05-01

64

Elucidating the mechanisms of protein antigen adsorption to the CAF/NAF liposomal vaccine adjuvant systems: effect of charge, fluidity and antigen-to-lipid ratio.  

PubMed

The reverse vaccinology approach has recently resulted in the identification of promising protein antigens, which in combination with appropriate adjuvants can stimulate customized, protective immune responses. Although antigen adsorption to adjuvants influences vaccine efficacy and safety, little is generally known about how antigens and adjuvants interact at the molecular level. The aim of this study was to elucidate the mechanisms of interactions between the equally sized, but oppositely charged model protein antigens ?-lactalbumin and lysozyme, and i) the clinically tested cationic liposomal adjuvant CAF01 composed of cationic dimethyldioctadecylammonium (DDA) bromide and trehalose-6,6'-dibehenate (TDB) or ii) the neutral adjuvant formulation NAF01, where DDA was replaced with zwitterionic distearoylphosphatidylcholine (DSPC). The effect of liposome charge, bilayer rigidity, isoelectric point and antigen-to-lipid ratio was investigated using dynamic light scattering, transmission electron microscopy, differential scanning calorimetry, intrinsic fluorescence and Langmuir monolayers. The net anionic ?-lactalbumin adsorbed onto the cationic liposomes, while there was no measureable attractive interaction with the zwitterionic liposomes. In contrast, the net cationic lysozyme showed very little interaction with either types of liposome. Adsorption of ?-lactalbumin altered its tertiary structure, affected lipid membrane packing below and above the phase transition temperature, and neutralized the liposomal surface charge, resulting in reduced colloidal stability and liposome aggregation. Langmuir studies revealed that ?-lactalbumin was not squeezed out of DDA monolayers upon compression, which suggests additional hydrophobic interactions. Such interactions are thus likely to affect the way vaccine antigens are presented to antigen-presenting cells, and may play an important role for the efficacy of the vaccine-induced immune response. These studies thus exemplify the importance of characterizing the molecular interactions between the vaccine antigen and adjuvant along with immunogenicity and efficacy studies. PMID:24769435

Hamborg, Mette; Rose, Fabrice; Jorgensen, Lene; Bjorklund, Katrine; Pedersen, Helene B; Christensen, Dennis; Foged, Camilla

2014-08-01

65

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

66

Combined Role of the Lewis Antigenic System, Chlamydia Pneumoniae, and C-Reactive Protein in Unstable Angina  

Microsoft Academic Search

OBJECTIVES The goal of this study was to assess the prognostic role of the Lewis antigenic system, Chlamydia pneumoniae (CP) seropositivity (CP), and C-reactive protein (CRP) levels in unstable angina (UA). BACKGROUND The role of CP infection in acute coronary syndromes is contradictory. The Lewis antigenic system, a genetically determined blood group system associated with infections and several disorders, including

Dominick J. Angiolillo; Giovanna Liuzzo; Simona Pelliccioni; Erica De Candia; Raffaele Landolfi; Filippo Crea; Attilio Maseri; Luigi M. Biasucci

2010-01-01

67

Effects of enzymatic hydrolysis of buckwheat protein on antigenicity and allergenicity  

PubMed Central

BACKGROUND/OBJECTIVES Due to its beneficial health effects, use of buckwheat has shown a continuous increase, and concerns regarding the allergic property of buckwheat have also increased. This study was conducted for evaluation of the hydrolytic effects of seven commercial proteases on buckwheat allergens and its allergenicity. MATERIALS/METHODS Extracted buckwheat protein was hydrolyzed by seven proteolytic enzymes at individual optimum temperature and pH for four hours. Analysis was then performed using SDS-PAGE, immunoblotting, and competitive inhibition ELISA (ciELISA) with rabbit antiserum to buckwheat protein, and direct ELISA with pooled serum of 21 buckwheat-sensitive patients. RESULTS Alkaline protease, classified as serine peptidase, was most effective in reducing allergenicity of buckwheat protein. It caused decomposition of the whole buckwheat protein, as shown on SDS-PAGE, and results of immunoblotting showed that the rabbit antiserum to buckwheat protein no longer recognized it as an antigen. Allergenicity showed a decrease of more than 50% when pooled serum of patients was used in ELISA. Two proteolytic enzymes from Aspergillus sp. could not hydrolyze buckwheat allergens effectively, and the allergenicity even appeared to increase. CONCLUSIONS Serine-type peptidases appeared to show a relatively effective reduction of buckwheat allergenicity. However, the antigenicity measured using rabbit antiserum did not correspond to the allergenicity measured using sera from human patients. Production of less allergenic buckwheat protein may be possible using enzymatic hydrolysis. PMID:24944772

Sung, Dong-Eun; Lee, Jeongok; Han, Youngshin; Shon, Dong-Hwa; Ahn, Kangmo

2014-01-01

68

Heterogeneity of CD4-Positive Human T-Cell Clones Which Recognize the Surface Protein Antigen of 'Rickettsia typhi'.  

National Technical Information Service (NTIS)

Immunity to the typhus group of rickettsiae is largely dependent on the effector function of several classes of T lymphocytes, including those which produce gamma interferon. Since the surface protein antigen (SPA) derived from typhus group rickettsiae ha...

F. M. Robbins, M. Carl, R. J. Hartzman, S. Vaidya, W. M. Ching

1989-01-01

69

Protein coated microcrystals formulated with model antigens and modified with calcium phosphate exhibit enhanced phagocytosis and immunogenicity?  

PubMed Central

Protein-coated microcrystals (PCMCs) were investigated as potential vaccine formulations for a range of model antigens. Presentation of antigens as PCMCs increased the antigen-specific IgG responses for all antigens tested, compared to soluble antigens. When compared to conventional aluminium-adjuvanted formulations, PCMCs modified with calcium phosphate (CaP) showed enhanced antigen-specific IgG responses and a decreased antigen-specific IgG1:IgG2a ratio, indicating the induction of a more balanced Th1/Th2 response. The rate of antigen release from CaP PCMCs, in vitro, decreased strongly with increasing CaP loading but their immunogenicity in vivo was not significantly different, suggesting the adjuvanticity was not due to a depot effect. Notably, it was found that CaP modification enhanced the phagocytosis of fluorescent antigen-PCMC particles by J774.2 murine monocyte/macrophage cells compared to soluble antigen or soluble PCMCs. Thus, CaP PCMCs may provide an alternative to conventional aluminium-based acellular vaccines to provide a more balanced Th1/Th2 immune response. PMID:24120484

Jones, Sarah; Asokanathan, Catpagavalli; Kmiec, Dorota; Irvine, June; Fleck, Roland; Xing, Dorothy; Moore, Barry; Parton, Roger; Coote, John

2014-01-01

70

A CD19/Fc fusion protein for detection of anti-CD19 chimeric antigen receptors  

PubMed Central

Background Chimeric Antigen Receptors (CARs) consist of the antigen-recognition portion of a monoclonal antibody fused to an intracellular signaling domain capable of activating T-cells. CARs displayed on the surface of transduced cells perform non-MHC-restricted antigen recognition and activating intracellular signaling pathways for induction of target cytolysis, cytokine secretion and proliferation. Clinical trials are in progress assessing the use of mature T-lymphocytes transduced with CARs targeting CD19 antigen to treat B-lineage malignancies. CD19 is an attractive target for immunotherapy because of its consistent and specific expression in most of the stages of maturation and malignancies of B-lymphocyte origin, but not on hematopoietic stem cells. Antibodies against the extracellular domain of the CAR molecule (anti-Fab, Fc or idiotype) have been used for detection of CAR expression in research and clinical samples by flow cytometry, but may need development for each construct and present significant background in samples from xenograft models. Methods A specific reagent for the detection of anti-CD19 CAR expression was developed, a fusion protein consisting of human CD19 extracellular domains and the Fc region of human IgG1 (CD19sIg). Genes encoding CD19sIg fusion proteins were constructed by fusing either exons 1 to 3 (CD19sIg1-3) or exons 1 to 4 (CD19sIg1-4) of the human CD19 cDNA to a human IgG1Fc fragment. These fusion proteins are intended to work in similar fashion as the MHC Tetramers used for identification of antigen-specific T-cells, and may also have other applications in studies of activation of anti-CD19 CAR bearing cells. The CD19sIg proteins were produced from 293 T cells by stable lentiviral vector transduction and purification from culture medium. Results ELISA assays using several different monoclonal antibodies to CD19 demonstrated dose-related specific binding by the fusion molecule CD19sIg1-4, but no binding by CD19sIg1-3. Conjugation of the CD19sIg1-4 fusion protein to Alexa Fluor 488 allowed specific and sensitive staining of anti-CD19 CAR-bearing cells for flow cytometry assays, detecting as low as 0.5% of CAR-modified primary cells with minimal background staining. Conclusions This fusion molecule is a sensitive reagent for detection of anti-CD19 CAR derived from any monoclonal antibody present in CAR-modified T-cells. PMID:23360526

2013-01-01

71

The application of a protein fraction derived from Treponema Pallidum (reiter strain) as an antigen in the serodiagnosis of syphilis  

Microsoft Academic Search

Summary  A comparison was made between the complement fixation test using a protein fraction derived fromTreponema pallidum (Reiter strain) as an antigen and theTreponema pallidum immobilization test. As appears from the results obtained with 116 syphilitic and 137 presumably non-syphilitic sera the\\u000a complement fixation test with protein antigen showed an excellent sensitivity together with a satisfactory specificity.

J. H. De Bruijn

1957-01-01

72

A Single Amino Acid Substitution Changes Antigenicity of ORF2-Encoded Proteins of Hepatitis E Virus  

PubMed Central

Extensive genomic diversity has been observed among hepatitis E virus (HEV) strains. However, the implication of the genetic heterogeneity on HEV antigenic properties is uncertain. In this study, monoclonal antibodies (Mabs) against truncated ORF2-encoded proteins (aa452–617, designated p166 proteins) derived from HEV strains of Burma (genotype 1a, p166Bur), Pakistan (1b, p166Pak) and Morocco (1c, p166Mor) were raised and used for identification of HEV antigenic diversity. Six Mabs reacted to these 3 p166 proteins as well as p166 proteins constructed from strains derived from Mexico (genotype 2), US (genotype 3) and China (genotype 4), indicating the existence of pan-genotypic epitopes. Two Mabs, 1B5 and 6C7, reacted with p166Bur and p166Mor, but not p166Pak or p166s derived from genotypes 2, 3, and 4, indicating that these 2 Mabs recognized strain-specific HEV epitopes. Both the common and specific epitopes could not be mapped by 23 synthetic peptides spanning the p166Bur sequence, suggesting that they are confirmation-dependent. Comparative sequence analysis showed that p166Bur and p166Mor shared an identical aa sequence along their entire lengths, whereas for p166Pak the aas occupying positions 606 and 614 are different from aas at corresponding positions of p166Bur and p166Mor. Reactivity between 1B5 and p166Bur was abrogated with mutation of p166Bur/A606V, whereas p166Pak acquired the reactivity to 1B5 with mutation of p166Pak/V606A. However, mutations of p166Bur/L614M and P166Pak/M614L did not affect the immunoreactivity. Therefore, the aa occupying position 606 plays a critical role in maintaining the antigenicity of the HEV p166 proteins. PMID:21152284

Liang, Jiu-Hong; Dai, Xing; Dong, Chen; Meng, Ji-Hong

2010-01-01

73

Identification of early diagnostic antigens from major excretory-secretory proteins of Trichinella spiralis muscle larvae using immunoproteomics  

PubMed Central

Background The excretory-secretory (ES) proteins of Trichinella spiralis muscle larvae (ML) come mainly from the excretory granules of the stichosome and the cuticles (membrane proteins), are directly exposed to the host’s immune system, and are the main target antigens, which induce the immune responses. Although the ES proteins are the most commonly used diagnostic antigens for trichinellosis, their main disadvantage are the false negative results during the early stage of infection. The aim of this study was to identify early specific diagnostic antigens from the main components of T. spiralis muscle larval ES proteins. Methods Two-dimensional electrophoresis (2-DE) combined with Western blot were used to screen the early diagnostic antigens from the main components of T. spiralis muscle larval ES proteins. The protein spots recognized by the sera from BALB/c mice infected with T. spiralis at 18 days post-infection (dpi) were identified by MALDI-TOF/TOF-MS and putatively annotated using GO terms obtained from the InterPro databases. Results The ES proteins were analyzed by 2-DE, and more than 33 protein spots were detected with molecular weight varying from 40 to 60 kDa and isoelectric point (pI) from 4 to 7. When probed with the sera from infected mice at 18 dpi, 21 protein spots were recognized and then identified, and they were characterized to correlate with five different proteins of T. spiralis, including two serine proteases, one deoxyribonuclease (DNase) II, and two kinds of trypsin. The five proteins were functionally categorized into molecular function and biological process according to GO hierarchy. Conclusions 2-DE and Western blot combined with MALDI-TOF/TOF-MS were used to screen the diagnostic antigens from the main components of T. spiralis muscle larval ES proteins. The five proteins of T. spiralis identified (two serine proteases, DNase II and two kinds of trypsin) might be the early specific diagnostic antigens of trichinellosis. PMID:24450759

2014-01-01

74

Divergent effects of bacterial lipopolysaccharide on immunity to orally administered protein and particulate antigens in mice.  

PubMed Central

We have investigated whether bacterial lipopolysaccharide (LPS) influences immune responses to dietary protein antigens in experimental animals. Simultaneous intravenous administration of LPS to normal mice fed ovalbumin (OVA) prevented the induction of tolerance for serum IgG antibody responses but did not alter the tolerance of systemic delayed-type hypersensitivity (DTH). In addition, exogenous LPS did not enhance the ability of spleen accessory cells to present OVA to primed T cells. LPS-unresponsive C3H/HeJ mice developed full tolerance of both humoral and cell-mediated immunity after feeding a range of doses of OVA that was equal in degree and persistence to that seen in normal, congenic C3H/HeOla mice and also had normal antigen-presenting cell (APC) activity for OVA. In contrast, C3H/HeJ mice were primed by feeding SRBC instead of developing the systemic tolerance found in normal C3H mice. Our results indicate the complexity of mechanisms that may regulate systemic immunity to orally administered antigens of different forms. Nevertheless, LPS does not modulate DTH responses to fed OVA and does not enhance APC activity, and we conclude that bacterial LPS may be unable to influence hypersensitivity to dietary proteins in man. Images Figure 1 PMID:3488267

Mowat, A M; Thomas, M J; MacKenzie, S; Parrott, D M

1986-01-01

75

Possible Association of GroES and Antigen 85 Proteins with Heat Resistance of Mycobacterium paratuberculosis  

PubMed Central

Conflicting reports on the heat resistance of Mycobacterium paratuberculosis prompted an examination of the effect of culture medium on this property of the organism. M. paratuberculosis was cultured in three types of media (fatty acid-containing medium 7H9-OADC (oleic acid-albumin-dextrose-catalase supplement) and glycerol-containing media WR-GD and 7H9-GD [glycerol-dextrose supplement]) at pH 6.0. M. paratuberculosis grown under these three culture conditions was then tested for heat resistance in distilled water at 65°C. Soluble proteins and mycolic acids of M. paratuberculosis were evaluated by two-dimensional electrophoresis (2-DE) and thin-layer chromatography (TLC), respectively. The type of culture medium used significantly affected the heat resistance of M. paratuberculosis. The decimal reduction times at 65°C (D65°C values; times required to reduce the concentration of bacteria by a factor of 10 at 65°C) for M. paratuberculosis strains grown in 7H9-OADC were significantly higher than those for the organisms grown in WR-GD medium (P < 0.01). When the glycerol-dextrose supplement of WR was substituted for the fatty acid supplement (OADC) in 7H9 medium (resulting in 7H9-GD), the D65°C value was significantly lower than that for the organism grown in 7H9-OADC medium (P = 0.022) but higher than that when it was cultured in WR-GD medium (P = 0.005). Proteomic analysis by 2-DE of soluble proteins extracted from M. paratuberculosis grown without heat stress in the three media (7H9-OADC, 7H9-GD, and WR-GD) revealed that seven proteins were more highly expressed in 7H9-OADC medium than in the other two media. When the seven proteins were subjected to matrix-assisted laser desorption ionization-mass spectrometric analysis, four of the seven protein spots were unidentifiable. The other three proteins were identified as GroES heat shock protein, alpha antigen, and antigen 85 complex B (Ag85B; fibronectin-binding protein). These proteins may be associated with the heat resistance of M. paratuberculosis. Alpha antigen and Ag85B are both trehalose mycolyltransferases involved in mycobacterial cell wall assembly. TLC revealed that 7H9-OADC medium supported production of more trehalose dimycolates and cell wall-bound mycolic acids than did WR-GD medium. The present study shows that in vitro culture conditions significantly affect heat resistance, cell wall synthesis, and protein expression of M. paratuberculosis and emphasize the importance of culture conditions on in vitro and ex vivo studies to estimate heat resistance. PMID:15006794

Sung, Nackmoon; Takayama, Kuni; Collins, Michael T.

2004-01-01

76

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

77

Screening of Predicted Secreted Antigens from Mycobacterium bovis Reveals the Immunodominance of the ESAT-6 Protein Family? †  

PubMed Central

Results of previous studies utilizing bioinformatic approaches in antigen-mining experiments revealed that secreted proteins are among the most frequently recognized antigens from Mycobacterium bovis. Thus, we hypothesized that the analysis of secreted proteins is likely to reveal additional immunogenic antigens that can be used to increase the specificity of diagnostic tests or be suitable vaccination candidates for mycobacterial infections. To test this hypothesis, 382 pools of overlapping peptides spanning 119 M. bovis secreted and potentially secreted proteins were screened for the ability to stimulate a gamma interferon response in vitro using whole blood from tuberculin-positive reactor (TB reactor) cattle. Of the 119 proteins screened, 70 (59%) induced positive responses in the TB reactor animals to various degrees. Strikingly, all but one of the 15 ESAT-6 proteins tested were recognized by at least 30% of the TB reactor animals, with 12 of the 22 most commonly recognized antigens belonging to this protein family. Further analysis of these data demonstrated that there was no significant difference in immunogenicity between the ESAT-6 proteins that were components of potentially intact ESX secretory systems and those corresponding to additional partial esx loci. Importantly for vaccine design, antigenic epitopes in some highly conserved regions shared by numerous ESAT-6 proteins were identified. However, despite this considerable homology, peptide-mapping experiments also revealed that immunodominant peptides were located in regions of amino acid variability. PMID:20086089

Jones, Gareth J.; Gordon, Stephen V.; Hewinson, R. Glyn; Vordermeier, H. Martin

2010-01-01

78

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

79

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

80

Molecular cloning and sequence analysis of the Sta58 major antigen gene of Rickettsia tsutsugamushi: sequence homology and antigenic comparison of Sta58 to the 60-kilodalton family of stress proteins.  

PubMed Central

The scrub typhus 58-kilodalton (kDa) antigen (Sta58) of Rickettsia tsutsugamushi is a major protein antigen often recognized by humans infected with scrub typhus rickettsiae. A 2.9-kilobase HindIII fragment containing a complete sta58 gene was cloned in Escherichia coli and found to express the entire Sta58 antigen and a smaller protein with an apparent molecular mass of 11 kDa (Stp11). DNA sequence analysis of the 2.9-kilobase HindIII fragment revealed two adjacent open reading frames encoding proteins of 11 (Stp11) and 60 (Sta58) kDa. Comparisons of deduced amino acid sequences disclosed a high degree of homology between the R. tsutsugamushi proteins Stp11 and Sta58 and the E. coli proteins GroES and GroEL, respectively, and the family of primordial heat shock proteins designated Hsp10 Hsp60. Although the sequence homology between the Sta58 antigen and the Hsp60 protein family is striking, the Sta58 protein appeared to be antigenically distinct among a sample of other bacterial Hsp60 homologs, including the typhus group of rickettsiae. The antigenic uniqueness of the Sta58 antigen indicates that this protein may be a potentially protective antigen and a useful diagnostic reagent for scrub typhus fever. Images PMID:2108930

Stover, C K; Marana, D P; Dasch, G A; Oaks, E V

1990-01-01

81

An efficient system to generate monoclonal antibodies against membrane-associated proteins by immunisation with antigen-expressing mammalian cells  

PubMed Central

Background The generation of monoclonal antibodies specific for protein antigens usually depends on purified recombinant protein for both immunisation and hybridoma screening. Purification of recombinant protein in sufficient yield and purity is a tedious undertaking and can be demanding especially in the case of membrane proteins. Furthermore, antibodies generated against a purified recombinant protein are frequently incapable of binding to the endogenous protein in its native context. Results We describe a strategy to generate monoclonal antibodies against membrane or membrane-associated proteins that completely bypasses any need for purified recombinant antigen. This approach utilises stably transfected mammalian cells expressing recombinant antigens on their cell surface for immunisation of mice. The transfected cells are also used for measuring seroconversion, hybridoma selection and antibody characterisation. By presenting the antigen in its native conformation for immunisation and hybridoma selection, this procedure promotes the generation of antibodies capable of binding to the endogenous protein. In the present study, we applied this approach successfully for three predicted GPI-anchored proteins of the malaria parasite Plasmodium falciparum. Conclusions The described entirely cell-based technology is a fast and efficient approach for obtaining antibodies reactive with endogenous cell-surface proteins in their native conformation. PMID:21159168

2010-01-01

82

Acute phase protein changes in antigen-induced mono-articular arthritis in rabbits and mice.  

PubMed Central

Acute phase protein levels have been measured during the induction and progression of antigen-induced mono-articular arthritis in rabbits and mice. In rabbits there was a short lived elevation in serum C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) immediately following intra-articular injection which returned to baseline levels 10-12 days after the injection. In BALB/c mice, serum amyloid P-component (SAP) and the third component of complement (C3) were elevated after intra-articular injection, returning towards baseline levels 6 weeks after the injection. The levels of CRP and SAP correlated with the inflammatory changes in the joints during the acute phase of the arthritic response (7 days after intra-articular injection). During the chronic phase the levels of these acute phase proteins bore no relationship to the degree of connective tissue destruction. PMID:2431818

Hunneyball, I M; Spowage, M; Crossley, M J; Rowe, I F; Baltz, M L

1986-01-01

83

Expression of Recombinant Antigenic Proteins from Angiostrongylus cantonensis: A Brief Report  

PubMed Central

Cerebral angiostrongyliasis is an acute inflammation caused by the infection of the nematode Angiostrongylus cantonensis that results in eosinophilic meningitis. The current immunological assay of choice is an immunoblot that detects antibodies to a 31 kDa protein present in crude extracts of the female worm. Recently we have identified diagnostic targets from excretion and secretion products and determined the composition of the 31 kDa antigen after 2-D gel electrophoresis and mass spectrometry. Here we cloned and expressed five proteins in prokaryotic and eukaryotic systems. Recombinant proteins were purified and analysed by Western blot assays and among them 14-3-3, Lec5 and ES7 were recognized by Angiostrongylus-specific serum, although the signal was weak. PMID:23900614

Perelygin, Andrey; Levert, Keith; Lin, Seh-Ching; Lee, Yeuk-Mui; da Silva, Alexandre J; Wilkins, Patricia P; Graeff-Teixeira, Carlos

2013-01-01

84

Plasmodium falciparum Antigen 332 Is a Resident Peripheral Membrane Protein of Maurer's Clefts  

PubMed Central

During the intraerythrocytic development of Plasmodium falciparum, the malaria parasite remodels the host cell cytosol by inducing membranous structures termed Maurer's clefts and inserting parasite proteins into the red blood cell cytoskeleton and plasma membrane. Pf332 is the largest known asexual malaria antigen that is exported into the red blood cell cytosol where it associates with Maurer's clefts. In the current work, we have utilized a set of different biochemical assays to analyze the solubility of the endogenous Pf332 molecule during its trafficking from the endoplasmic reticulum within the parasite to the host cell cytosol. Solubilization studies demonstrate that Pf332 is synthesized and trafficked within the parasite as a peripheral membrane protein, which after export into the host cell cytosol associates with the cytoplasmic side of Maurer's clefts in a peripheral manner. By immunofluorescence microscopy and flow cytometry, we show that Pf332 persists in close association with Maurer's clefts throughout trophozoite maturation and schizogony, and does not become exposed at the host cell surface. Our data also indicate that Pf332 interacts with the host cell cytoskeleton, but only in very mature parasite stages. Thus, the present study describes Pf332 as a resident peripheral membrane protein of Maurer's clefts and suggests that the antigen participates in host cytoskeleton modifications at completion of the intraerythrocytic developmental cycle. PMID:23185236

Nilsson, Sandra; Angeletti, Davide; Wahlgren, Mats; Chen, Qijun; Moll, Kirsten

2012-01-01

85

Plasmodium falciparum antigen 332 is a resident peripheral membrane protein of Maurer's clefts.  

PubMed

During the intraerythrocytic development of Plasmodium falciparum, the malaria parasite remodels the host cell cytosol by inducing membranous structures termed Maurer's clefts and inserting parasite proteins into the red blood cell cytoskeleton and plasma membrane. Pf332 is the largest known asexual malaria antigen that is exported into the red blood cell cytosol where it associates with Maurer's clefts. In the current work, we have utilized a set of different biochemical assays to analyze the solubility of the endogenous Pf332 molecule during its trafficking from the endoplasmic reticulum within the parasite to the host cell cytosol. Solubilization studies demonstrate that Pf332 is synthesized and trafficked within the parasite as a peripheral membrane protein, which after export into the host cell cytosol associates with the cytoplasmic side of Maurer's clefts in a peripheral manner. By immunofluorescence microscopy and flow cytometry, we show that Pf332 persists in close association with Maurer's clefts throughout trophozoite maturation and schizogony, and does not become exposed at the host cell surface. Our data also indicate that Pf332 interacts with the host cell cytoskeleton, but only in very mature parasite stages. Thus, the present study describes Pf332 as a resident peripheral membrane protein of Maurer's clefts and suggests that the antigen participates in host cytoskeleton modifications at completion of the intraerythrocytic developmental cycle. PMID:23185236

Nilsson, Sandra; Angeletti, Davide; Wahlgren, Mats; Chen, Qijun; Moll, Kirsten

2012-01-01

86

Evaluation of Brugia malayi sheath protein (Shp-1) as a diagnostic antigen for human lymphatic filariasis.  

PubMed

Lymphatic filariasis is the second leading cause of permanent long-term disability globally and control of this disease needs efficient diagnostic methods. In this study, abundantly expressing microfilarial sheath protein (Shp-1) from Brugia malayi was characterized as a filarial diagnostic candidate using samples from different clinical population. Monoclonal antibodies were developed against E. coli expressed recombinant Shp-1 in order to assess its efficiency in filarial antigen detection assay system. Endemic Normal (EN, n = 170), asymptomatic microfilaeremics (MF, n = 65), symptomatic chronic pathology (CP, n = 45) and non endemic normal (NEN, n = 10) sera were analyzed by antigen capture enzyme-linked immunosorbent assay. Of the 290 individuals, all MF individuals (both brugian and bancroftian) were positive in this assay followed by CP and EN. When compared with SXP-1 and Og4C3 antigen assays, all assays detected Wb MF correctly, Bm MF was detected by Shp-1 and SXP-1 assays, and only Shp-1 was able to detect EN (12%) and CP (29%). Results showed that this assay may be useful for monitoring prior to mass drug administration. PMID:24389369

Jawaharlal, Jeya Prita Parasurama; Ravishankaran, Rajendran; Shridharan, Radhika Nagamangalam; Lawrence, Ansel Vishal; Karande, Anjali Anoop; Perumal, Kaliraj

2014-03-01

87

Association of HLA-DQ7 antigen with cow milk protein allergy in Italian children.  

PubMed

In this study we investigated the HLA association with cow milk allergy. Thirty-seven Italian children with cow milk allergy and 35 randomly selected age-matched healthy children as control group were included in the study. DNA typing was performed by restriction fragment length polymorphism (RFLP) technique. We show the first statistically significant positive association between the expression of the HLA-DQ7 antigen and cow milk allergy. Several immunological tests (skin prick test, RIA, radioallergosorbent test (RAST) and ELISA) were performed to evaluate the humoral immune responses of DQ7 positive and DQ7 negative allergic patients. Our results show that among the DQ7 positive patients the majority presented a high humoral response. Furthermore, the in vitro proliferative response of patients to the beta-lactoglobulin antigen was performed to evaluate their cell-mediated immune response. We observed that the number of the nonresponders was higher in the DQ7 positive patients when compared to the DQ7 negative patients. Our data indicate an association of HLA-DQ7 antigen with cow milk protein allergy and that the DQ7 positive patients had a prevalence of humoral rather than cellular responses. PMID:9617782

Camponeschi, B; Lucarelli, S; Frediani, T; Barbato, M; Quintieri, F

1997-05-01

88

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

89

Functional Analysis of the Highly Antigenic Outer Capsid Protein, Hoc, a Virus Decoration Protein from T4-like Bacteriophages  

PubMed Central

Summary Bacteriophage T4 is decorated with 155 copies of the highly antigenic outer capsid protein, Hoc. The Hoc molecule (40 kDa) is present at the center of each hexameric capsomer and provides a good platform for surface display of pathogen antigens. Biochemical and modeling studies show that Hoc consists of a string of four domains, three immunoglobulin (Ig)-like and one non-Ig domain at the C-terminus. Biochemical data suggest that the Hoc protein has two functional modules, a capsid binding module containing domains 1 and 4 and a solvent-exposed module containing domains 2 and 3. This model is consistent with the dumbbell-shaped cryo-EM density of Hoc observed in the reconstruction of the T4 capsid. Mutagenesis localized the capsid binding site to the C-terminal 25 amino acids, which are predicted to form two ?-strands flanking a capsid binding loop. Mutations in the loop residues, ESRNG, abolished capsid binding, suggesting that these residues might interact with the major capsid protein, gp23*. With the conserved capsid binding module forming a foothold on the virus and the solvent-exposed module able to adapt to bind to a variety of surfaces, Hoc probably provides survival advantages to the phage, such as increasing the virus concentration near the host, efficient dispersion of the virus, and exposing the tail for more efficient contact with the host cell surface prior to infection. PMID:20497329

Sathaliyawala, Taheri; Islam, Mohammad Z.; Li, Qin; Fokine, Andrei; Rossmann, Michael G.; Rao, Venigalla B.

2010-01-01

90

Simian virus 40 large T antigen stabilizes the TATA-binding protein-TFIIA complex on the TATA element.  

PubMed

Large T antigen (T antigen), the early gene product of simian virus 40 (SV40), is a potent transcriptional activator of both cellular and viral genes. Recently we have shown that T antigen is tightly associated with TFIID and, in this position, performs a TATA-binding protein (TBP)-associated factor (TAF)-like function. Based on this observation, we asked whether T antigen affected steps in preinitiation complex assembly. Using purified components in in vitro complex assembly assays, we found that T antigen specifically enhances the formation of the TBP-TFIIA complex on the TATA element. T antigen accomplishes this by increasing the rate of formation of the TBP-TFIIA complex on the TATA element and by stabilizing the complexes after they are formed on the promoter. In addition, DNA immunoprecipitation experiments indicate that T antigen is associated with the stabilized TBP-TFIIA complexes bound to the DNA. In this regard, it has previously been shown that T antigen interacts with TBP; in the present study, we show that T antigen also interacts with TFIIA in vitro. In testing the ability of T antigen to stabilize the TBP-TFIIA complex, we found that stabilization is highly sensitive to the specific sequence context of the TATA element. Previous studies showed that T antigen could activate simple promoters containing the TATA elements from the hsp70 and c-fos gene promoters but failed to significantly activate similar promoters containing the TATA elements from the promoters of the SV40 early and adenovirus E2a genes. We find that the ability to stabilize the TBP-TFIIA complex on the hsp70 and c-fos TATA elements, and not on the SV40 early and E2A TATA elements, correlates with the ability or inability to activate promoters containing these TATA elements. PMID:9632777

Damania, B; Lieberman, P; Alwine, J C

1998-07-01

91

Immunological Research on Basic Specific and Non-Specific Protein Antigens of the Type Streptococcus Pyogenes (Group a).  

National Technical Information Service (NTIS)

Immunoelectrophoresis and electrophoresis have confirmed that acid-alcohol gamma extracts contained several type specific and non-specific protein antigens which were basic proteins. Their isoelectric point is above 8.5, while that of the non-type specifi...

R. Wahl, J. Goichot, G. Drach

1966-01-01

92

Demonstration of antigenic variation among rabies virus isolates by using monoclonal antibodies to nucleocapsid proteins.  

PubMed Central

Rabies virus isolates from terrestrial animals in six areas of the United States were examined with a panel of monoclonal antibodies to nucleocapsid proteins. Characteristic differences in immunofluorescence reactions permitted the formation of four antigenically distinct reaction groups from the 231 isolates tested. The geographic distribution of these groups corresponded well with separate rabies enzootic areas recognized by surveillance of sylvatic rabies in the United States. Distinctive reaction patterns were also identified for viral proteins from four infected bat species, and identical patterns were found in eight isolated cases of rabies in terrestrial animals. These findings suggest that monoclonal antibodies can be used to study the prevalence, distribution, and transmission of rabies among wildlife species. PMID:2429983

Smith, J S; Reid-Sanden, F L; Roumillat, L F; Trimarchi, C; Clark, K; Baer, G M; Winkler, W G

1986-01-01

93

Inactivation of the alpha C Protein Antigen Gene, bca, by a Novel Shuttle\\/Suicide Vector Results in Attenuation of Virulence and Immunity in Group B Streptococcus  

Microsoft Academic Search

The alpha C protein of group B Streptococcus (GBS) is a major surface-associated antigen. Although its role in the biology and virulence of GBS has not been defined, it is opsonic and capable of eliciting protective immunity. The alpha C protein is widely distributed among clinical isolates and is a potential protein carrier and antigen in conjugate vaccines to prevent

Jing Li; Dennis L. Kasper; Frederick M. Ausubel; Bernard Rosner; James L. Michel

1997-01-01

94

Macrophage inflammatory protein-1 alpha influences eosinophil recruitment in antigen-specific airway inflammation.  

PubMed

Allergic airway inflammation is characterized by peribronchial eosinophil accumulation within the submucosa of the airway of the lung. In the present study we have utilized a model of airway inflammation induced by intratracheal challenge with parasite (Schistosoma mansoni) egg antigen (SEA) in presensitized mice. The recruitment of neutrophils and eosinophils into the airway was found to be maximal at 8 and 48 h post challenge, respectively. Since macrophage inflammatory protein-1 alpha (MIP-1 alpha) has previously been found to be chemotactic for eosinophils, in vitro, we postulated that MIP-1 alpha was involved in the airway inflammation and more specifically in eosinophil recruitment into the airway. Initial studies demonstrated an increase in MIP-1 alpha mRNA expression at 8 h post-SEA challenge, as compared to vehicle-treated control mice. We next demonstrated a significant increase in MIP-1 alpha protein in the lungs of SEA-challenged mice at 8 h compared to control challenged mice, correlating to the mRNA data. Immunohistochemical staining of lungs from SEA-challenged mice demonstrated MIP-1 alpha protein expression in airway epithelial cells, alveolar macrophages and in recruited mononuclear cell populations. Immunolocalization of MIP-1 alpha to cells within the bronchoalveolar lavage fluid demonstrated that macrophages and eosinophils stained positive for the protein. To determine the contribution of MIP-1 alpha expression to eosinophil accumulation, SEA-challenged mice were passively immunized with either neutralizing MIP-1 alpha antibodies or normal rabbit IgG, 3-4 h prior to the intratracheal SEA challenge. These studies demonstrated a > 50% decrease in eosinophil recruitment to the lungs and airway in animals receiving neutralizing MIP-1 alpha antibodies with no effect on early neutrophil recruitment. These results suggest that the production of MIP-1 alpha, induced by an antigen-specific response, plays an important role in recruitment of eosinophils in this airway model of inflammation. PMID:7843237

Lukacs, N W; Strieter, R M; Shaklee, C L; Chensue, S W; Kunkel, S L

1995-01-01

95

Protein antigens of encapsulated Klebsiella pneumoniae surface exposed after growth in the presence of subinhibitory concentrations of cephalosporins.  

PubMed Central

It recently has been reported by us that cephalosporins, at a concentration below that influencing growth rate, reduced the production of enterochelin and capsule formation of iron-depleted Klebsiella pneumoniae. We now report on the antigenicity of the outer membrane components and surface-exposed protein antigens of iron-depleted cells grown in the presence or absence of cephalosporins. All major outer membrane proteins, including iron-regulated membrane proteins, were immunogenic. Encapsulated K. pneumoniae grown in antibiotic-free media had three protein antigens (60, 35.5, and 32.5 kilodaltons) exposed on the surface that were accessible to antibodies. Growth of the same cultures in the presence of subinhibitory concentrations of cephalosporins resulted in the exposure of a greater number of protein antigenic determinants, including iron-regulated membrane proteins, which become readily accessible to antibodies. It was also found that immunoblotting was generally more sensitive than conventional staining of the acrylamide gel with Coomassie blue in the detection of proteins. Images PMID:3914857

Kadurugamuwa, J L; Anwar, H; Brown, M R; Zak, O

1985-01-01

96

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

97

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 (LNI) 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) 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; Elliott, T. F.; Holubec, K.; Baker, T. L.; Love, J. E.

2004-01-01

98

Enhanced discrimination of malignant from benign pancreatic disease by measuring the CA 19-9 antigen on specific protein carriers.  

PubMed

The CA 19-9 assay detects a carbohydrate antigen on multiple protein carriers, some of which may be preferential carriers of the antigen in cancer. We tested the hypothesis that the measurement of the CA 19-9 antigen on individual proteins could improve performance over the standard CA 19-9 assay. We used antibody arrays to measure the levels of the CA 19-9 antigen on multiple proteins in serum or plasma samples from patients with pancreatic adenocarcinoma or pancreatitis. Sample sets from three different institutions were examined, comprising 531 individual samples. The measurement of the CA 19-9 antigen on any individual protein did not improve upon the performance of the standard CA 19-9 assay (82% sensitivity at 75% specificity for early-stage cancer), owing to diversity among patients in their CA 19-9 protein carriers. However, a subset of cancer patients with no elevation in the standard CA 19-9 assay showed elevations of the CA 19-9 antigen specifically on the proteins MUC5AC or MUC16 in all sample sets. By combining measurements of the standard CA 19-9 assay with detection of CA 19-9 on MUC5AC and MUC16, the sensitivity of cancer detection was improved relative to CA 19-9 alone in each sample set, achieving 67-80% sensitivity at 98% specificity. This finding demonstrates the value of measuring glycans on specific proteins for improving biomarker performance. Diagnostic tests with improved sensitivity for detecting pancreatic cancer could have important applications for improving the treatment and management of patients suffering from this disease. PMID:22220206

Yue, Tingting; Maupin, Kevin A; Fallon, Brian; Li, Lin; Partyka, Katie; Anderson, Michelle A; Brenner, Dean E; Kaul, Karen; Zeh, Herbert; Moser, A James; Simeone, Diane M; Feng, Ziding; Brand, Randall E; Haab, Brian B

2011-01-01

99

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

PubMed Central

Group A rotaviruses are the major etiologic agents of acute gastroenteritis worldwide in children and young animals. Among its structural proteins, VP6 is the most immunogenic and is highly conserved within this group. Lactococcus lactis is a food-grade, Gram-positive, and nonpathogenic lactic acid bacteria that has already been explored as a mucosal delivery system of heterologous antigens. In this work, the nisin-controlled expression system was used to display the VP6 protein at the cell surface of L. lactis. Conditions for optimal gene expression were established by testing different nisin concentrations, cell density at induction, and incubation times after induction. Cytoplasmic and cell wall protein extracts were analyzed by Western blot and surface expression was confirmed by flow cytometry. Both analysis provided evidence that VP6 was efficiently expressed and displayed on the cell surface of L. lactis. Furthermore, the humoral response of mice immunized with recombinant L. lactis was evaluated and the displayed recombinant VP6 protein proved to be immunogenic. In conclusion, this is the first report of displaying VP6 protein on the surface of L. lactis to induce a specific immune response against rotavirus. These results provide the basis for further evaluation of this VP6-displaying L. lactis as a mucosal delivery vector in a mouse model of rotavirus infection. PMID:23984337

Esteban, L. E.; Temprana, C. F.; Arguelles, M. H.; Glikmann, G.; Castello, A. A.

2013-01-01

100

Adsorption of multimeric T cell antigens on carbon nanotubes: effect on protein structure and antigen-specific T cell stimulation.  

PubMed

Antigen-specific activation of cytotoxic T cells can be enhanced up to three-fold more than soluble controls when using functionalized bundled carbon nanotube substrates ((b) CNTs). To overcome the denaturing effects of direct adsorption on (b) CNTs, a simple but robust method is demonstrated to stabilize the T cell stimulus on carbon nanotube substrates through non-covalent attachment of the linker neutravidin. PMID:23090793

Fadel, Tarek R; Li, Nan; Shah, Smith; Look, Michael; Pfefferle, Lisa D; Haller, Gary L; Justesen, Sune; Wilson, Corey J; Fahmy, Tarek M

2013-03-11

101

EBNA-5, an Epstein-Barr Virus-Encoded Nuclear Antigen, Binds to the Retinoblastoma and p53 Proteins  

Microsoft Academic Search

Epstein-Barr virus (EBV) immortalized human lymphoblastoid cell lines express six virally encoded nuclear proteins, designated EBV nuclear antigens 1-6 (EBNA-1-6). We show that the EBNA-5 protein (alternatively designated EBNA-LP) that is required for B-cell transformation can form a molecular complex with the retinoblastoma (RB) and p53 tumor suppressor proteins. Using EBNA-5 deletion mutants, we have found that a 66-amino acid-long

Laszlo Szekely; Galina Selivanova; Kristinn P. Magnusson; George Klein; Klas G. Wiman

1993-01-01

102

Antigenic and phylogenetic studies on a variant Newcastle disease virus using anti?fusion protein monoclonal antibodies and partial sequencing of the fusion protein gene  

Microsoft Academic Search

A virulent Newcastle disease virus (NDV) isolate, 34\\/90, reported to show considerable antigenic diversity from more classical strains of NDV, including vaccine strains, was evaluated phylogenetically and for the presence of neutralizing epitopes on the fusion protein. Comparison of a 309 nucleotide sequence of the fusion protein gene of 34\\/90 with other viruses confirmed the diversity of this virus, placing

M. S. Collins; Sally Franklin; I. Strong; G. Meulemans; D. J. Alexander

1998-01-01

103

Improved reactivity of hepatitis C virus core protein epitopes in a conformational antigen-presenting system.  

PubMed Central

Recent studies have identified several epitopes in the N-terminal portion of the nucleocapsid protein which are predominantly recognized by sera of patients infected with hepatitis C virus (HCV). The characterization of the sequences recognized by theses antibodies and the evaluation of their reactivities have been performed mainly with synthetic peptides. However, synthetic peptides are notoriously unreliable as antigens when the immune response is directed against conformational epitopes. In order to improve the detection of antibody responses in HCV-infected patients, we have evaluated the reactivities of three immunodominant regions of the HCV core protein (residues 1 to 20, 21 to 40, and 32 to 46) displayed in a conformation-specific manner on the surface of the Flock House virus (FHV) capsid protein. The results obtained with these proteins in the analysis of 94 serum samples positive by anti-HCV enzyme-linked immunosorbent assay where then compared with those obtained with the corresponding synthetic peptides. The sequence most reactive both with the peptide and with the FHV protein was the region from residues 1 to 20, confirming the low conformational requirements for the display of these residues. On the other hand, the already reported conformational nature of residues 32 to 46 is in keeping with its observed high reactivity when displayed by the FHV recombinant protein and with the low reactivity displayed by its corresponding synthetic peptide. Finally, the high reactivity observed for the chimeric protein displaying the region from residues 21 to 40, as opposed to the results obtained with the synthetic peptide, also suggests that this sequence contains one or more conformational epitopes whose structures cannot be mimicked correctly with synthetic peptides. PMID:9067642

Buratti, E; Di Michele, M; Song, P; Monti-Bragadin, C; Scodeller, E A; Baralle, F E; Tisminetzky, S G

1997-01-01

104

Effect of ultraviolet B radiation on S-100 protein antigen in epidermal Langerhans cells  

SciTech Connect

Ultraviolet B (UVB) radiation has been shown to induce significant alterations in both function and surface antigen expression of epidermal Langerhans cells (ELC). In this study the effect of UVB radiation on ELC marker S-100 protein antigen (S-100 Ag) which is present in the nucleus and cytoplasm of human ELC was investigated. A total of 34 sites on 31 volunteers were exposed to 3 MED (minimal erythema dose) of UVB and biopsied at various times up to 7 days after irradiation. Skin from 9 noninjured and 7 slice-wounded subjects served as controls. The avidin-biotin-peroxidase staining technique was used to identify S-100 Ag in sections of formalin-fixed, paraffin-embedded tissue, and the numbers of stained suprabasal dendritic cells were then counted over a 200 basal cell length of interfollicular epidermis. Noninjured skin had 3.56 +/- 3.01 cells, whereas slice-wounded skin had elevated numbers (greater than 10.0 cells) at 1, 24, and 48 h after injury. Following UVB irradiation, a significant (p less than 0.001) increase in antigen-positive cells (14 +/- 3.46) was found at 1 h; this number declined to just below normal at 12 h, but by 48 h returned to and remained at preinjury levels. In contrast to previous observations of the depletion of ELC surface markers by UVB radiation, the authors demonstrate here that the numbers of S-100 Ag-positive ELC actually increase following comparable doses of radiation. Since this increase occurs so rapidly following both UVB irradiation and slice injury, S-100 Ag may be synthesized or unmasked within the ELC as a response to wounding of the epidermis.

Schneider, S.A.; Fukuyama, K.; Maceira, J.; Epstein, W.L.

1985-02-01

105

Comparative studies on antigenicity and allergenicity of native and denatured egg white proteins.  

PubMed

The binding activities of IgG and IgE antibodies from egg-allergic patients to physically or chemically treated egg white proteins were examined and compared with those of rabbit anti-egg white IgG antibodies. The sera from eight patients and four rabbit antibodies were used in this study. The binding activities of human IgG antibody to partially denatured ovotransferrin (Tf), ovalbumin (OA), and lysozyme (Lys) forms were increased, whereas carboxymethylation (RCM) and heat treatment caused a dramatic decrease in the antigenicity of Tf and ovomucoid (OVM). Tf and OVM were major immunogenic antigens for the rabbit IgG response. Urea also caused Tf to exhibit greater rabbit IgG binding activity. In contrast, human and rabbit antibodies did not react with ovomucin. Partially denatured Tf and Lys also induced strong IgE binding activities. The allergenicity of Tf, OVM, and Lys was decreased by RCM, whereas OA retained its binding capacity. These results suggested that anti-OA IgE recognizes more sequential epitopes and that anti-OVM and Lys antibodies recognize both conformational and sequential epitopes. Tf and OVM were dominant allergens for the IgE antibodies of anaphylaxis patients, whereas IgE from atopic patients bound more strongly with OA and OVM. PMID:11958641

Mine, Yoshinori; Zhang, Jie Wei

2002-04-24

106

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

107

Characterization of antigenic domains and epitopes in the ORF3 protein of a Chinese isolate of avian hepatitis E virus.  

PubMed

Avian hepatitis E virus (HEV) is an emerging virus associated with the big liver and spleen disease or hepatitis-splenomegaly syndrome in chickens and subclinical infections by the virus are also common. The complete genome of avian HEV contains three open-reading frames (ORFs) in which ORF2 protein is part of virus particles and thus contains primary epitopes. Antigenic epitopes of avian HEV ORF2 protein have been described but those associated with the ORF3 have not. To analyze the antigenic domains and epitopes in the ORF3 protein of a Chinese isolate of avian HEV (CaHEV), we generated a series of antigens comprised of the complete ORF3 and also five truncated overlapping ORF3 peptides. The antibodies used in this study were mouse antisera and monoclonal antibodies against ORF3, positive chicken sera from Specific Pathogen Free chickens experimentally infected with CaHEV and clinical chicken sera. Using these antigens and antibodies, we identified three antigenic domains at amino acids (aa) 1-28, 55-74 and 75-88 in which aa 75-88 was a dominant domain. The dominant domain contained at least two major epitopes since field chickens infected with avian HEV produced antibodies against the domain and epitopes. These results provide useful information for future development of immunoassays for the diagnosis of avian HEV infection. PMID:24021883

Zhao, Qin; Sun, Ya-ni; Hu, Shou-bin; Wang, Xin-jie; Xiao, Yi-hong; Hsu, Walter H; Xiao, Shu-qi; Wang, Cheng-bao; Mu, Yang; Hiscox, Julian A; Zhou, En-Min

2013-12-27

108

Identification of a Nonstructural DNA-Binding Protein (DBP) as an Antigen with Diagnostic Potential for Human Adenovirus  

PubMed Central

Background Human adenoviruses (HAdVs) have been implicated as important agents in a wide range of human illnesses. To date, 58 distinct HAdV serotypes have been identified and can be grouped into six species. For the immunological diagnosis of adenoviruses, the hexon protein, a structural protein, has been used. The potential of other HAdV proteins has not been fully addressed. Methodology/Principal Findings In this study, a nonstructural antigenic protein, the DNA binding protein (DBP) of human adenovirus 5 and 35 (Ad5, Ad35) - was identified using immunoproteomic technology. The expression of Ad5 and Ad35 DBP in insect cells could be detected by rhesus monkey serum antibodies and healthy adult human serum positive for Ad5 and Ad35. Recombinant DBPs elicited high titer antibodies in mice. Their conserved domain displayed immunological cross-reactions with heterologous DBP antibodies in Western blot assays. DBP-IgM ELISA showed higher sensitivity adenovirus IgM detection than the commercial Adenovirus IgM Human ELISA Kit. A Western blot method developed based on Ad5 DBP was highly consistent with (?2?=? 44.9, P<0.01) the Western blot assay for the hexon protein in the detection of IgG, but proved even more sensitive. Conclusions/Significance The HAdV nonstructural protein DBP is an antigenic protein that could serve as an alternative common antigen for adenovirus diagnosis. PMID:23516396

Zhou, Hongli; Wu, Chao; Paranhos-Baccala, Glaucia; Vernet, Guy; Jin, Qi; Wang, Jianwei; Hung, Tao

2013-01-01

109

Effects of vancomycin on platelets, plasma proteins and hepatitis B surface antigen.  

PubMed

The antibiotic vancomycin shares many similarities with ristocetin, an agent noted for its effects on platelets and plasma fibrinogen. Vancomycin did not aggregate platelets as ristocetin, but platelets were incorporated into precipitates induced by vancomycin. Fibrinogen and factor VIII were precipitated from plasma at low concentrations of vancomycin. The precipitated fibrinogen remained clottable. Hepatitis B surface antigen was selectively precipitated from serum and could be recovered from the precipitate. Rabbits receiving bolus intravenous injections of high doses of vancomycin developed hypofibrinogenemia and thrombocytopenia within minutes and often went on to die. Studies with 125I-vancomycin revealed little stable binding of the antibiotic to platelets or fibrinogen. A relationship is suggested between the potent protein precipitating effects and phlebitis at the infusion site commonly associated with vancomycin therapy. PMID:1188837

Coller, B S; Lundberg, W B; Gralnick, H R

1975-09-30

110

Polyoma middle tumor antigen interacts with SHC protein via the NPTY (Asn-Pro-Thr-Tyr) motif in middle tumor antigen.  

PubMed Central

Polyomavirus middle tumor antigen (MT) transforms a large number of cell types by binding to and modulating the activities of cellular proteins. Previous genetic analysis defined in MT an independent motif, NPTY (Asn-Pro-Thr-Tyr), required for transformation. This report demonstrates that NPTY is required for interaction between MT and SHC protein, a Src homology 2 (SH2)-containing protooncogene product implicated in activating Ras via association with GRB2 protein. SHC is phosphorylated on tyrosine and associates with GRB2 in MT-transformed cells. These effects require an intact NPTY motif in MT. SHC immunoprecipitates from MT-transformed cells possess kinase activity that phosphorylates not only SHC and MT but also the 85-kDa subunit of phosphatidylinositol 3-kinase. This result suggests that a complex exists that contains, at a minimum, MT, Src family tyrosine kinases, phosphatidylinositol 3-kinase, and SHC. Images PMID:8022784

Campbell, K S; Ogris, E; Burke, B; Su, W; Auger, K R; Druker, B J; Schaffhausen, B S; Roberts, T M; Pallas, D C

1994-01-01

111

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

112

Identification of antigenic proteins associated with trichloroethylene-induced autoimmune disease by serological proteome analysis  

SciTech Connect

Although many studies indicated that trichloroethylene (TCE) could induce autoimmune diseases and some protein adducts were detected, the proteins were not identified and mechanisms remain unknown. To screen and identify autoantigens which might be involved in TCE-induced autoimmune diseases, three groups of sera were collected from healthy donors (I), patients suffering from TCE-induced exfoliative dermatitis (ED) (II), and the healed ones (III). Serological proteome analysis (SERPA) was performed with total proteins of TCE-treated L-02 liver cells as antigen sources and immunoglobins of the above sera as probes. Highly immunogenic spots (2-fold or above increase compared with group I) in group II and III were submitted to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem mass spectrometry sequencing. Western blot analysis was followed using commercial antibodies and individual serum. Six proteins were identified. Among them, Enoyl Coenzyme A hydratase peroxisoma 1 and lactate dehydrogenase B only showed stronger immunogenicity for group II sera, while Purine nucleoside phosphorylase, ribosomal protein P0 and proteasome activator subunit1 isoform1 also showed stronger immunogenicity for group III sera. Noteworthy, NM23 reacted only with group II sera. Western blot analysis of NM23 expression indicated that all of the individual serum of group II showed immune activity, which confirmed the validity of SERPA result. These findings revealed that there exist autoantibodies in group II and III sera. Besides, autoantibodies of the two stages of disease course were different. These autoantigens might serve as biomarkers to elucidate mechanisms underlying TCE toxicity and are helpful for diagnosis, therapy and prognosis of TCE-induced autoimmune diseases.

Liu Jianjun; Xing Xiumei; Huang Haiyan; Jiang Yingzhi; He Haowei; Xu Xinyun; Yuan Jianhui; Zhou Li; Yang Linqing [Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, No. 21, Rd 1st Tianbei, 518020 Shenzhen (China); Zhuang Zhixiong, E-mail: bio-research@hotmail.co [Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, No. 21, Rd 1st Tianbei, 518020 Shenzhen (China)

2009-11-01

113

Localization and Fine Mapping of Antigenic Sites on the Nucleocapsid Protein N of Porcine Reproductive and Respiratory Syndrome Virus with Monoclonal Antibodies  

Microsoft Academic Search

The purpose of this study was to analyze the antigenic structure of the nucleocapsid protein N of the Lelystad virus isolate of porcine reproductive and respiratory syndrome virus (PRRSV) and to identify antigenic differences between this prototype European isolate and other North American isolates. To do this, we generated a panel of monoclonal antibodies (mAbs) directed against the N protein

J. J. M. Meulenberg; A. P. van Nieuwstadt; A. van Essen-Zandbergen; J. N. A. Bos-de Ruijter; J. P. M. Langeveld; R. H. Meloen

1998-01-01

114

Proline-rich cell surface antigens of horseshoe crab hemocytes are substrates for protein cross-linking with a clotting protein coagulin.  

PubMed

Monoclonal antibodies were raised against hemocytes of the horseshoe crab Tachypleus tridentatus. All of the antibodies obtained reacted with the same protein bands on SDS-PAGE of hemocyte lysate. Flow cytometry and biotinylation of surface substances on the hemocytes indicated that the antigens are major peripheral proteins of hemocytes. The antigens were purified from hemocyte lysate and were good substrates for the horseshoe crab hemocyte transglutaminase (HcTGase). Transglutaminases play an important role during the final stage of blood coagulation in mammals and crustaceans. Although HcTGase did not intermolecularly cross-link a clottable protein coagulogen or its proteolytic product coagulin, HcTGase promoted the cross-linking of coagulin with the surface antigens, resulting in the formation of a stable polymer. We determined the nucleotide sequences for two isoproteins of the antigens. The two proteins containing 271 and 284 residues (66% identity) were composed of tandem repeats of proline-rich segments. We named them proxins-1 and -2 after proline-rich proteins for protein cross-linking. Proxins may form a stable physical barrier against invading pathogens in cooperation with hemolymph coagulation at injured sites. PMID:12189150

Osaki, Tsukasa; Okino, Nozomu; Tokunaga, Fuminori; Iwanaga, Sadaaki; Kawabata, Shun-Ichiro

2002-10-18

115

Molecular characterization of a 35-kilodalton protein of Borrelia burgdorferi, an antigen of diagnostic importance in early Lyme disease.  

PubMed Central

Antibodies against a 35-kDa antigen of Borrelia burgdorferi are detectable in the serum of about half of patients with early Lyme disease. The gene encoding this antigen was isolated from a genomic library of B. burgdorferi B31 (low passage), and full-length expression of the recombinant gene product was achieved in Escherichia coli. Antiserum raised against the recombinant protein was reactive with a B. burgdorferi protein of the same molecular size as the diagnostic 35-kDa antigen cited in an earlier study of criteria for the sero-diagnosis of early Lyme disease. Also, the recombinant protein was reactive with serum from patients with early Lyme disease who were seropositive for the 35-kDa antigen. DNA sequence analysis of the gene indicated an open reading frame of 909 bp encoding a protein with a calculated molecular mass of 34.3 kDa. This gene did not possess the usual initiation codon ATG but rather probably used a TTG codon. The deduced amino acid sequence of the N terminus exhibited a motif similar to that for signal peptides of lipoproteins. Southern blotting revealed a chromosomal location for this gene; and it was specific for B. burgdorferi, B. afzellii, and B. garinii but not for B. hermsii, B. coriaciae, or B. turicatae. PMID:8968885

Gilmore, R D; Kappel, K J; Johnson, B J

1997-01-01

116

Antigenic Comparison of Envelope Protein E between Japanese Encephalitis Virus and Some Other Flaviviruses Using Monoclonal Antibodies  

Microsoft Academic Search

SUMMARY The antigenic relationships between Japanese encephalitis (JE) virus and several other flaviviruses have been investigated using anti-JE virus monoclonal antibodies (MAbs). Seventeen MAbs directed against envelope protein E of JE virus were characterized and divided into eight MAb groups based on reactivity patterns in haemagglutination inhibition test, neutralization (N) test, ELISA and competitive binding assay with JE virus. The

JUNKO KIMURA-KURODA; KOTARO YASUI

1986-01-01

117

Protein Array Profiling of Tic Patient Sera Reveals a Broad Range and Enhanced Immune Response against Group A Streptococcus Antigens  

PubMed Central

The human pathogen Group A Streptococcus (Streptococcus pyogenes, GAS) is widely recognized as a major cause of common pharyngitis as well as of severe invasive diseases and non-suppurative sequelae associated with the existence of GAS antigens eliciting host autoantibodies. It has been proposed that a subset of paediatric disorders characterized by tics and obsessive-compulsive symptoms would exacerbate in association with relapses of GAS-associated pharyngitis. This hypothesis is however still controversial. In the attempt to shed light on the contribution of GAS infections to the onset of neuropsychiatric or behavioral disorders affecting as many as 3% of children and adolescents, we tested the antibody response of tic patient sera to a representative panel of GAS antigens. In particular, 102 recombinant proteins were spotted on nitrocellulose-coated glass slides and probed against 61 sera collected from young patients with typical tic neuropsychiatric symptoms but with no overt GAS infection. Sera from 35 children with neither tic disorder nor overt GAS infection were also analyzed. The protein recognition patterns of these two sera groups were compared with those obtained using 239 sera from children with GAS-associated pharyngitis. This comparative analysis identified 25 antigens recognized by sera of the three patient groups and 21 antigens recognized by tic and pharyngitis sera, but poorly or not recognized by sera from children without tic. Interestingly, these antigens appeared to be, in quantitative terms, more immunogenic in tic than in pharyngitis patients. Additionally, a third group of antigens appeared to be preferentially and specifically recognized by tic sera. These findings provide the first evidence that tic patient sera exhibit immunological profiles typical of individuals who elicited a broad, specific and strong immune response against GAS. This may be relevant in the context of one of the hypothesis proposing that GAS antigen-dependent induction of autoantibodies in susceptible individuals may be involved the occurrence of tic disorders. PMID:19623252

Bombaci, Mauro; Grifantini, Renata; Mora, Marirosa; Reguzzi, Valerio; Petracca, Roberto; Meoni, Eva; Balloni, Sergio; Zingaretti, Chiara; Falugi, Fabiana; Manetti, Andrea G. O.; Margarit, Immaculada; Musser, James M.; Cardona, Francesco; Orefici, Graziella; Grandi, Guido; Bensi, Giuliano

2009-01-01

118

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

119

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

120

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

121

Heligmosomoides bakeri antigen rescues CD4-positive T cells from glucocorticoid-induced apoptosis by Bcl-2 protein expression.  

PubMed

Heligmosomoides bakeri infection in mice is associated with a dominant CD4(+) T-cell response and with the activity of natural Treg cells with CD4(+) CD25(+) phenotype. The polarization of Th2 T-cell phenotype and the increase in the CD4(+) CD25(+) T cell population are regulated by glucocorticoids that induce apoptosis in CD4(+) CD25(-) T cells and inhibit apoptosis in CD4(+) CD25(+) T cells. However, exposure of mice to H. bakeri antigen induces a high glucocorticoid concentration in serum and a reduction in the number of CD4-positive; CD4(+) CD25(-) and CD4(+) CD25(+) apoptotic T cells in mesenteric lymph node cells. In this study to evaluate the in vitro effect of the anti-apoptotic property of H. bakeri antigen on T cells, apoptosis of these cells was induced by glucocorticoids-dexamethasone (Dex). Excretory-secretory (ES) antigen of the nematode prevented Dex-induced apoptosis in CD4-positive T cells with CD4(+) CD25(-) and CD4(+) CD25(High) phenotype by Bcl-2 protein expression. Contrary to the effect on CD4-positive T cells, survival of CD8(+) T cells was not connected with expression of Bcl-2 protein. This suggest that H. bakeri antigen modulates CD4-positive T cell sensitivity to glucocorticoid-induced apoptosis by induction of Bcl-2 protein. PMID:21306399

Donskow, K; Drela, N; Doligalska, M

2011-03-01

122

Identification of an antigenic domain in the N-terminal region of avian hepatitis E virus (HEV) capsid protein that is not common to swine and human HEVs.  

PubMed

The antigenic domains located in the C-terminal 268 amino acid residues of avian hepatitis E virus (HEV) capsid protein have been characterized. This region shares common epitopes with swine and human HEVs. However, epitopes in the N-terminal 338 amino acid residues have never been reported. In this study, an antigenic domain located between amino acids 23 and 85 was identified by indirect ELISA using the truncated recombinant capsid proteins as coating antigens and anti-avian HEV chicken sera as primary antibodies. In addition, this domain did not react with anti-swine and human HEV sera. These results indicated that the N-terminal 338 amino acid residues of avian HEV capsid protein do not share common epitopes with swine and human HEVs. This finding is important for our understanding of the antigenicity of the avian HEV capsid protein. Furthermore, it has important implications in the selection of viral antigens for serological diagnosis. PMID:25209807

Wang, Lizhen; Sun, Yani; Du, Taofeng; Wang, Chengbao; Xiao, Shuqi; Mu, Yang; Zhang, Gaiping; Liu, Lihong; Widén, Frederik; Hsu, Walter H; Zhao, Qin; Zhou, En-Min

2014-12-01

123

Structure of the C-terminal domain of AspA (antigen I/II-family) protein from Streptococcus pyogenes.  

PubMed

The pathogenic bacteria Streptococcus pyogenes can cause an array of diseases in humans, including moderate infections such as pharyngitis (strep throat) as well as life threatening conditions such as necrotizing fasciitis and puerperal fever. The antigen I/II family proteins are cell wall anchored adhesin proteins found on the surfaces of most oral streptococci and are involved in host colonization and biofilm formation. In the present study we have determined the crystal structure of the C2-3-domain of the antigen I/II type protein AspA from S. pyogenes M type 28. The structure was solved to 1.8 Å resolution and shows that the C2-3-domain is comprised of two structurally similar DEv-IgG motifs, designated C2 and C3, both containing a stabilizing covalent isopeptide bond. Furthermore a metal binding site is identified, containing a bound calcium ion. Despite relatively low sequence identity, interestingly, the overall structure shares high similarity to the C2-3-domains of antigen I/II proteins from Streptococcus gordonii and Streptococcus mutans, although certain parts of the structure exhibit distinct features. In summary this work constitutes the first step in the full structure determination of the AspA protein from S. pyogenes. PMID:24918040

Hall, Michael; Nylander, Sa; Jenkinson, Howard F; Persson, Karina

2014-01-01

124

Structure of the C-terminal domain of AspA (antigen I/II-family) protein from Streptococcus pyogenes  

PubMed Central

The pathogenic bacteria Streptococcus pyogenes can cause an array of diseases in humans, including moderate infections such as pharyngitis (strep throat) as well as life threatening conditions such as necrotizing fasciitis and puerperal fever. The antigen I/II family proteins are cell wall anchored adhesin proteins found on the surfaces of most oral streptococci and are involved in host colonization and biofilm formation. In the present study we have determined the crystal structure of the C2–3-domain of the antigen I/II type protein AspA from S. pyogenes M type 28. The structure was solved to 1.8 Å resolution and shows that the C2–3-domain is comprised of two structurally similar DEv-IgG motifs, designated C2 and C3, both containing a stabilizing covalent isopeptide bond. Furthermore a metal binding site is identified, containing a bound calcium ion. Despite relatively low sequence identity, interestingly, the overall structure shares high similarity to the C2–3-domains of antigen I/II proteins from Streptococcus gordonii and Streptococcus mutans, although certain parts of the structure exhibit distinct features. In summary this work constitutes the first step in the full structure determination of the AspA protein from S. pyogenes. PMID:24918040

Hall, Michael; Nylander, ?sa; Jenkinson, Howard F.; Persson, Karina

2014-01-01

125

Safety and immunogenicity of the tetravalent protein-conjugated meningococcal vaccine (MCV4) in recipients of related and unrelated allogeneic hematopoietic stem cell transplantation.  

PubMed

Given the high morbidity and mortality associated with meningococcal disease, in 2007 the Advisory Committee of Immunization Practices recommended immunization of all children ages 11-18 with a protein-conjugated meningococcal vaccine. There are limited data on the immunogenicity of this vaccine after allogeneic hematopoietic stem cell transplantation (allo-HCT). Since 2007, we have immunized 48 patients with the MCV4 vaccine. Two vaccinated patients who lacked follow-up titers were excluded from this analysis. Stem cells were derived from an HLA-identical sibling (n = 17) or an alternative donor (n = 29). The median time to vaccination was 2.34 years after allo-HCT. Only 7 patients responded to all 4 serogroups, and 16 patients responded to none of the serogroups. The response to serogroups A, C, Y, and W-135 was 52%, 30%, 46%, and 33%, respectively. The ability to respond to 2 or more serogroups was not affected by age, diagnosis, time to vaccination, or history of graft-versus-host disease. Receipt of a T cell-depleted graft was associated with a poorer response (P = .044). Eight of 16 patients who received a second MCV4 vaccination responded to all 4 serogroups. This retrospective study suggests that response to a single MCV4 vaccination is poor after allo-HCT. Administration of a 2-dose series, as currently recommended for patients with asplenia, complement deficiency, and HIV infection, should be evaluated in this patient population. PMID:21820392

Mahler, Michelle B; Taur, Ying; Jean, Raymond; Kernan, Nancy A; Prockop, Susan E; Small, Trudy N

2012-01-01

126

ATM Protein Physically and Functionally Interacts with Proliferating Cell Nuclear Antigen to Regulate DNA Synthesis*  

PubMed Central

Ataxia telangiectasia (A-T) is a pleiotropic disease, with a characteristic hypersensitivity to ionizing radiation that is caused by biallelic mutations in A-T mutated (ATM), a gene encoding a protein kinase critical for the induction of cellular responses to DNA damage, particularly to DNA double strand breaks. A long known characteristic of A-T cells is their ability to synthesize DNA even in the presence of ionizing radiation-induced DNA damage, a phenomenon termed radioresistant DNA synthesis. We previously reported that ATM kinase inhibition, but not ATM protein disruption, blocks sister chromatid exchange following DNA damage. We now show that ATM kinase inhibition, but not ATM protein disruption, also inhibits DNA synthesis. Investigating a potential physical interaction of ATM with the DNA replication machinery, we found that ATM co-precipitates with proliferating cell nuclear antigen (PCNA) from cellular extracts. Using bacterially purified ATM truncation mutants and in vitro translated PCNA, we showed that the interaction is direct and mediated by the C terminus of ATM. Indeed, a 20-amino acid region close to the kinase domain is sufficient for strong binding to PCNA. This binding is specific to ATM, because the homologous regions of other PIKK members, including the closely related kinase A-T and Rad3-related (ATR), did not bind PCNA. ATM was found to bind two regions in PCNA. To examine the functional significance of the interaction between ATM and PCNA, we tested the ability of ATM to stimulate DNA synthesis by DNA polymerase ?, which is implicated in both DNA replication and DNA repair processes. ATM was observed to stimulate DNA polymerase activity in a PCNA-dependent manner. PMID:22362778

Gamper, Armin M.; Choi, Serah; Matsumoto, Yoshihiro; Banerjee, Dibyendu; Tomkinson, Alan E.; Bakkenist, Christopher J.

2012-01-01

127

The regulated long-term delivery of therapeutic proteins by using antigen-specific B lymphocytes  

Microsoft Academic Search

Memory lymphocytes are important mediators of the immune response. These cells are long-lived and undergo clonal expansion upon reexposure to specific antigen, differentiating into effector cells that secrete Ig or cytokines while maintaining a residual pool of memory T and B lymphocytes. Here, the ability of antigen-specific lymphocytes to undergo repeated cycles of antigen-driven clonal expansion and contraction is exploited

Katalin Takács; Camille Du Roure; Stephen Nabarro; Niall Dillon; John H. McVey; Zoe Webster; Angus MacNeil; István Bartók; Christopher Higgins; David Gray; Matthias Merkenschlager; Amanda G. Fisher

2004-01-01

128

HIGH PREVALENCE OF ANTITHROMBIN III, PROTEIN C AND PROTEIN S DEFICIENCY, BUT NO FACTOR V LEIDEN MUTATION IN VENOUS THROMBOPHILIC CHINESE PATIENTS IN TAIWAN  

Microsoft Academic Search

We studied the prevalence of antithrombin III (AT III), protein C (PC) and protein S (PS) deficiencies and factor V Leiden mutation in thrombophilia in Taiwan. Eighty-five consecutive and unrelated patients with otherwise unexplained venous thrombophilia were studied. Both antigen and activity of inhibitors were determined using commercial kits (Stago), activated PC sensitivity ratio (APC SR) by Coatest (Chromogenix), and

Ming-Ching Shen; Jen-Shiou Lin; Woei Tsay

1997-01-01

129

The Wzz (Cld) Protein in Escherichia coli: Amino Acid Sequence Variation Determines O-Antigen Chain Length Specificity  

PubMed Central

The O antigen is a polymer with a repeated unit. The chain length in most Escherichia coli strains has a modal value of 10 to 18 O units, but other strains have higher or lower modal values. wzz (cld/rol) mutants have a random chain length distribution, showing that the modal distribution is determined by the Wzz protein. Cloned wzz genes from E. coli strains with short (7 to 16), intermediate (10 to 18), and long (16 to 25) modal chain lengths were transferred to a model system, and their effects on O111 antigen were studied. The O111 chain length closely resembled that of the parent strains. We present data based on the construction of chimeric wzz genes and site-directed mutagenesis of the wzz gene to show that the modal value of O-antigen chain length of E. coli O1, O2, O7, and O157 strains can be changed by specific amino acid substitutions in wzz. It is concluded that the O-antigen chain length heterogeneity in E. coli strains is the result of amino acid sequence variation of the Wzz protein. PMID:9573151

Franco, Agustin V.; Liu, Dan; Reeves, Peter R.

1998-01-01

130

Repression of the Drosophila proliferating-cell nuclear antigen gene promoter by zerknuellt protein  

SciTech Connect

A 631-bp fragment containing the 5{prime}-flanking region of the Drosophila melanogaster proliferating-cell nuclear antigen (PCNA) gene was placed upstream of the chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in Drosophila Kc cells transfected with this plasmid and a set of 5{prime}-deletion derivatives revealed that the promoter function resided within a 192-bp region. Cotransfection with a zerknuellt (zen)-expressing plasmid specifically repressed CAT expression. However, cotransfection with expression plasmids for a nonfunctional zen mutation, even skipped, or bicoid showed no significant effect on CAT expression. RNase protection analysis revealed that the repression by zen was at the transcription step. The target sequence of zen was mapped within the 34-bp region of the PCNA gene promoter, even though it lacked zen protein-binding sites. Transgenic flies carrying the PCNA gene regulatory region fused with lacZ were established. These results indicate that zen indirectly represses PCNA gene expression, probably by regulating the expression of some transcription factor(s) that binds to the PCNA gene promoter.

Yamaguchi, Masamitsu; Hirose, Fumiko; Nishida, Yasuyoshi; Matsukage, Akio (Aichi Cancer Center Research Inst., Nagoya (Japan))

1991-10-01

131

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

132

Latex-protein complexes from an acute phase recombinant antigen of Toxoplasma gondii for the diagnosis of recently acquired toxoplasmosis.  

PubMed

The synthesis and characterization of latex-protein complexes (LPC), from the acute phase recombinant antigen P35 (P35Ag) of Toxoplasma gondii and "core-shell" carboxylated or polystyrene (PS) latexes (of different sizes and charge densities) are considered, with the aim of producing immunoagglutination reagents able to detect recently acquired toxoplasmosis. Physical adsorption (PA) and chemical coupling (CC) of P35Ag onto latex particles at different pH were investigated. Greater amounts of adsorbed protein were obtained on PS latexes than on carboxylated latexes, indicating that hydrophobic forces govern the interactions between the protein and the particle surface. In the CC experiments, the highest amount of bound protein was obtained at pH 6, near the isoelectric point of the protein (IP=6.27). At this pH, it decreased both the repulsion between particle surface and protein, and the repulsion between neighboring molecules. The LPC were characterized and the antigenicity of the P35Ag protein coupled on the particles surface was evaluated by Enzyme-Linked ImmunoSorbent Assay (ELISA). Results from ELISA showed that the P35Ag coupled to the latex particles surface was not affected during the particles sensitization by PA and CC and the produced LPC were able to recognize specific anti-P35Ag antibodies present in the acute phase of the disease. PMID:24905682

Peretti, Leandro E; Gonzalez, Verónica D G; Marcipar, Iván S; Gugliotta, Luis M

2014-08-01

133

Antibodies to the protein core of the small cell lung cancer workshop antigen cluster-w4 and to the leucocyte workshop antigen CD24 recognize the same short protein sequence leucine-alanine-proline.  

PubMed Central

We recently described the identity of the small cell lung cancer (SCLC) cluster-w4 antigen and the human B cell differentiation marker CD24, a glycosylphosphatidylinositol (GPI)-anchored, highly glycosylated surface molecule of only 31-35 amino acids [15]. The specificities of three anti-cluster-w4 and of eleven anti-CD24 MoAbs have been investigated with respect to their binding capacity to the protein core of cluster-w4/CD24 antigen. Four overlapping peptides spanning this protein core were synthesized. MoAbs shown to bind to two overlapping peptides by antibody binding inhibition using the cluster-w4/CD24-positive SCLC cell line SW2 and by direct peptide binding detected in an ELISA were investigated in more detail. To determine the exact epitopes recognized by these MoAbs, an epitope mapping assay using peptides synthesized onto polyethylene pins was established. The three anti-cluster-w4 MoAbs SWA11, SWA21 and SWA22 and the anti-CD24 MoAbs OKB2 and ALB9 recognized the same short leucine-alanine-proline (LAP) sequence in an area without potential glycosylation sites close to the GPI anchor of the protein core of the cluster-w4/CD24 antigen. PMID:7688677

Weber, E; Lehmann, H P; Beck-Sickinger, A G; Wawrzynczak, E J; Waibel, R; Folkers, G; Stahel, R A

1993-01-01

134

Uncoupling of induced protein processing from maturation in dendritic cells exposed to a highly antigenic preparation from a helminth parasite  

PubMed Central

Toll-like receptor (TLR) ligands induce dendritic cell (DC) maturation. During this process cells initiate proteolytic degradation of internalized protein antigens into peptides that complex with MHC II, and simultaneously increase expression of costimulatory molecules and of cytokines such as IL-6, IL-12 and IL-23. In these ways, TLR-activated DCs are able to activate naïve Th cells and initiate Th1 and Th17 responses, and TLR-ligands thus serve as adjuvants for these types of responses. In contrast, products from helminth parasites generally do not activate DCs, and act as adjuvants for Th2 response induction. We have explored the underlying basis for this form of adjuvanticity. We show that exposure of DCs to soluble antigens from the eggs of the helminth parasite Schistosoma mansoni (SEA) leads to the induction of proteolysis of internalized antigen. This occurs in the absence of significant induction of costimulatory molecule expression or production of proinflammatory cytokines. SEA-induced antigen processing occurs independently of MyD88 or Trif, but is significantly attenuated by inhibition of p38, but not ERK, signaling. In DCs exposed to SEA, ligation of CD40 provides a necessary second signal that stimulates costimulatory molecule expression, allowing DCs to mature into capable antigen presenting cells. Collectively, the data demonstrate the existence of a MyD88/Trif-independent, p38-dependent pathway of antigen processing in DCs, which is uncoupled from conventional DC maturation and is associated with induction of Th2 type immune responses. PMID:19017945

Marshall, Fraser A.; Pearce, Edward J.

2010-01-01

135

A Cloned MajorSchistosoma mansoniEgg Antigen with Homologies to Small Heat Shock Proteins Elicits Th1 Responsiveness  

Microsoft Academic Search

Inschistosomiasismansoni,solubleeggantigensoftheworminducechronicT-cell-mediatedgranulomatous tissue responses. Since thefirst preparation of crude soluble egg antigen extract, a dearth of highly purified antigenshashamperedtheidentificationofgranulomainducermolecules.Herewereportthatacloned38-kDa egg polypeptide (r38) with homologies to small heat shock proteins is a strong immunogen. The recombinant and the sodium dodecyl sulfate-polyacrylamide gel electrophoresis separated and eluted native 38-kDa (p38) polypeptides, used in microgram amounts and unaided by adjuvant, sensitized mice for a

YINLONG CAI; JANE G. LANGLEY; DAVID I. SMITH; ANDDOV L. BOROS

1996-01-01

136

Novel immunoassay for carcinoembryonic antigen based on protein A-conjugated immunosensor chip by surface plasmon resonance and cyclic voltammetry  

Microsoft Academic Search

In this study, an immunosensor chip utilizing surface plasmon resonance (SPR) and cyclic voltammetry (CV) was fabricated for\\u000a detecting carcinoembryonic antigen (CEA). Specifically, we applied in parallel an SPR instrument and a CV device to monitor\\u000a the assembly of carcinoembryonic antibody (anti-CEA) on a protein A-conjugated surface and the subsequent ligand reaction.\\u000a The immunosensor chips were constructed by various concentrations

Dian-Ping Tang; Ruo Yuan; Ya-Qin Chai

2006-01-01

137

Antibody response to polyhistidine-tagged peptide and protein antigens attached to liposomes via lipid-linked nitrilotriacetic acid in mice.  

PubMed

Particulate delivery systems enhance antibody responses to subunit antigens. However, covalent attachment of protein antigens can disrupt protein structure and mask critical epitopes, altering the antibody response to the antigen. In this report, we evaluate noncovalent metal chelation via nitrilotriacetic acid (NTA) as a nondestructive method to attach peptide and protein antigens to liposomes. Two model antigens, ovalbumin (OVA) and a peptide derived from the membrane-proximal region of HIV-1 gp41 (N-MPR), were polyhistidinylated and attached to liposomes via monovalent NTA (mono-NTA; K(D) [equilibrium dissociation constant], ?10 ?M), trivalent NTA (tris-NTA; K(D), ?1 nM), or a covalent linkage. Attachment of N-MPR, but not OVA, to liposomes via an NTA lipid elicited stronger antibody responses in BALB/c mice than a formulation in which unassociated antigen was simply admixed with control liposomes lacking NTA. However, the tris-NTA linkage did not increase antibody responses to either N-MPR or OVA compared to the level for the mono-NTA linkage, despite the greater liposomal association of the antigen. For both antigens, covalently attaching them to a lipid elicited significantly stronger antibody responses than NTA-anchored antigens (OVA titer, 3.4 × 10(6) versus 1.4 × 10(6) to 1.6 × 10(6) [P < 0.001]; N-MPR titer, 4.4 × 10(4) versus 5.5 × 10(2) to 7.6 × 10(2) [P < 0.003]). The data indicate that NTA linkages may increase antibody titers to weak antigens such as N-MPR, but NTA-mediated attachment remains inferior to covalent conjugation. Moreover, enhancements in antigen-liposome affinity do not result in increased antibody titers. Thus, additional improvements of NTA-mediated conjugation technology are necessary to achieve an effective, nondestructive method for increasing the humoral response to antigens in particulate vaccines. PMID:21159923

Watson, Douglas S; Platt, Virginia M; Cao, Limin; Venditto, Vincent J; Szoka, Francis C

2011-02-01

138

In vivo neutralization of eosinophil-derived major basic protein inhibits antigen-induced bronchial hyperreactivity in sensitized guinea pigs.  

PubMed

This study examines the effect of purified rabbit antiguinea pig eosinophil-derived major basic protein (MBP) Ig on antigen-induced bronchial hyperreactivity to inhaled acetylcholine in aerosol-sensitized guinea pigs. Ovalbumin inhalation by sensitized guinea pigs induced a rise in the numbers of eosinophils and in the levels of MBP in the bronchoalveolar lavage fluid, which peaked at 24 h and resolved at 72 h. Antigen-challenged animals exhibited bronchial hyperreactivity to inhale acetylcholine at 72 h, but not at 6 or 24 h. The intranasal administration of 200 microliter of purified rabbit anti-guinea pig MBP Ig, at 2.5 mg/ml, but not of the control preimmune rabbit Ig, 1 h before and 5 h after ovalbumin inhalation suppressed bronchial hyperreactivity to acetylcholine at 72 h without affecting the number of eosinophils accumulating in the bronchoalveolar lavage fluid. These findings indicate that antigen challenge in sensitized guinea pigs is followed by early eosinophil infiltration and activation within the airways and by late bronchial hyperreactivity. Neutralization of endogenously secreted MBP by a specific antiserum prevented antigen-induced bronchial hyperreactivity, suggesting that eosinophil degranulation plays an important role in the alterations of bronchopulmonary function in the guinea pig. PMID:8613536

Lefort, J; Nahori, M A; Ruffie, C; Vargaftig, B B; Pretolani, M

1996-02-15

139

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

PubMed Central

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

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

2013-01-01

140

Rheumatoid arthritis synovial membrane contains a 62,000-molecular-weight protein that shares an antigenic epitope with the Epstein-Barr virus-encoded associated nuclear antigen.  

PubMed Central

A monoclonal antibody, selected for reactivity with the Epstein-Barr virus (EBV)-encoded antigen EBNA-1, exhibited strong reactivity with the synovial lining cells in joint biopsies from 10 of 12 patients with rheumatoid arthritis (RA) and adherent cells eluted from these tissues. No staining of RA synovial membrane frozen tissue sections or eluted synovial-lining cells was obtained with monoclonal antibodies directed against other EBV-encoded antigens (anti-p160, anti-gp200/350) or with monoclonal antibodies directed against antigens encoded by cytomegalovirus, herpes simplex viruses, or human T cell leukemia virus type I. Among 12 osteoarthritis and normal synovial biopsies only rare reactive cells were noted. Characterization of the antigen(s) in RA synovium by the Western immunoblotting technique revealed a 62,000-molecular-weight (mol-wt) protein, in contrast to the 70,000-85,000-mol-wt EBNA-1 antigen found in EBV-transformed cells. The structural basis for the cross-reactivity of the RA synovial membrane 62,000-mol-wt protein and the EBNA-1 antigen appears to reside in the glycine-alanine rich region of these molecules. A rabbit antibody directed against a synthetic peptide (IR3-VI-2) derived from the glycine-alanine-rich region of EBNA-1 reacted with the 70,000-85,000-mol-wt EBNA-1 antigen in EBV-infected cells and with the 62,000-mol-wt molecule in RA synovial membrane extracts. Since strong antibody responses to EBNA-1 are known to exist in RA patients, these results suggest that immune responses to a cross-reactive antigen may play a role in the pathogenesis of RA. Images PMID:2422209

Fox, R; Sportsman, R; Rhodes, G; Luka, J; Pearson, G; Vaughan, J

1986-01-01

141

Immunogenicity of a Fusion Protein Comprising Coli Surface Antigen 3 and Labile B Subunit of Enterotoxigenic Escherichia coli  

PubMed Central

Background: Enterotoxigenic Escherichia coli (ETEC) strains are the major causes of diarrheal disease in humans and animals. Colonization factors and enterotoxins are the major virulence factors in ETEC pathogenesis. For the broad-spectrum protection against ETEC, one could focus on colonization factors and non-toxic heat labile as a vaccine candidate. Methods: A fusion protein is composed of a major fimbrial subunit of coli surface antigen 3, and the heat-labile B subunit (LTB) was constructed as a chimeric immunogen. For optimum level expression of protein, the gene was synthesized with codon bias of E. coli. Also, recombinant protein was expressed in E. coli BL21DE3. ELISA and Western tests were carried out for determination of antigen and specificity of antibody raised against recombinant protein in animals. The anti-toxicity and anti-adherence properties of the immune sera against ETEC were also evaluated. Results: Immunological analyses showed the production of high titer of specific antibody in immunized mice. The built-in LTB retains native toxin properties which were approved by GM1 binding assay. Pre-treatment of the ETEC cells with anti-sera significantly decreased their adhesion to Caco-2 cells. Conclusion: The results indicated the efficacy of the recombinant chimeric protein as an effective immunogen inducing strong humoral response. The designated chimer would be an interesting prototype for a vaccine and worthy of further investigation. PMID:25326019

Alerasol, Masoome; Mousavi Gargari, Seyed Latif; Nazarian, Shahram; Bagheri, Samane

2014-01-01

142

A surface receptor specific for human IgA on group B streptococci possessing the Ibc protein antigen  

PubMed Central

A number of group B streptococcal strains of various serotypes, Ia, Ib, Ic, II, and III were examined for their ability to bind human IgG and IgA. No strains of group B streptococci were found to bind IgG, but many strains possessing the Ibc protein antigen(s) were found to bind a significant amount of IgA. The extent of IgA binding correlated with the amount of a 130,000 mol wt, detergent-extractable protein, and reactivity with the Ic typing sera. Using nitrocellulose blots, it was found that the 130,000 mol wt protein bound human IgA. A method was developed to purify the protein while retaining its ability to bind human IgA. Using solid phase radioimmunoassays, it was determined that the protein bound to the Fc region of monomeric or polymeric IgA and that it failed to bind IgM or any IgG isotype. PMID:6387034

1984-01-01

143

Immune responses of a chimaeric protein vaccine containing Mycoplasma hyopneumoniae antigens and LTB against experimental M. hyopneumoniae infection in pigs.  

PubMed

A recombinant chimaeric protein containing three Mycoplasma hyopneumoniae antigens (C-terminal portion of P97, heat shock protein P42, and NrdF) fused to an adjuvant, the B subunit of heat-labile enterotoxin of Escherichia coli (LTB), was used to immunize pigs against enzootic pneumonia. The systemic and local immune responses, as well as the efficacy of the chimaeric protein in inducing protection against experimental M. hyopneumoniae infection were evaluated. In total, 60 male piglets, purchased from a M. hyopneumoniae-free herd, at 4 weeks of age were randomly allocated to six different experimental groups of 10 animals each: recombinant chimaeric protein by intramuscular (IM) (1) or intranasal (IN) (2) administration, commercial bacterin by IM administration (3), and the adjuvant LTB by IM (4, control group A) or IN (5, control group B) administration. All groups were immunized at 24 and 38 days of age and challenged at 52 days of age. The sixth group that was not challenged was used as the negative control (IN [n=5] or IM [n=5] administration of the LTB adjuvant). Compared with the non-challenged group, administration of the chimaeric protein induced significant (P<0.05) IgG and IgA responses against all individual antigens present in the chimaera, but it could not confer a significant protection against M. hyopneumoniae infection in pigs. This lack of effectiveness points towards the need for further studies to improve the efficacy of this subunit-based vaccine approach. PMID:24909331

Marchioro, Silvana B; Sácristan, Rubén Del Pozo; Michiels, Annelies; Haesebrouck, Freddy; Conceição, Fabricio R; Dellagostin, Odir A; Maes, Dominiek

2014-08-01

144

Genetic, Structural, and Antigenic Analyses of Glycan Diversity in the O-Linked Protein Glycosylation Systems of Human Neisseria Species? †  

PubMed Central

Bacterial capsular polysaccharides and lipopolysaccharides are well-established ligands of innate and adaptive immune effectors and often exhibit structural and antigenic variability. Although many surface-localized glycoproteins have been identified in bacterial pathogens and symbionts, it not clear if and how selection impacts associated glycoform structure. Here, a systematic approach was devised to correlate gene repertoire with protein-associated glycoform structure in Neisseria species important to human health and disease. By manipulating the protein glycosylation (pgl) gene content and assessing the glycan structure by mass spectrometry and reactivity with monoclonal antibodies, it was established that protein-associated glycans are antigenically variable and that at least nine distinct glycoforms can be expressed in vitro. These studies also revealed that in addition to Neisseria gonorrhoeae strain N400, one other gonococcal strain and isolates of Neisseria meningitidis and Neisseria lactamica exhibit broad-spectrum O-linked protein glycosylation. Although a strong correlation between pgl gene content, glycoform expression, and serological profile was observed, there were significant exceptions, particularly with regard to levels of microheterogeneity. This work provides a technological platform for molecular serotyping of neisserial protein glycans and for elucidating pgl gene evolution. PMID:20363948

B?rud, Bente; Aas, Finn Erik; Vik, Ashild; Winther-Larsen, Hanne C.; Egge-Jacobsen, Wolfgang; Koomey, Michael

2010-01-01

145

Heat shock cognate protein 70 encodes antigenic epitopes recognised by HLA-B4601-restricted cytotoxic T lymphocytes from cancer patients  

Microsoft Academic Search

Heat shock cognate protein 70 (HSC70), a highly conserved protein and a member of the family of molecular chaperones, has the ability to induce cytotoxic T lymphocyte (CTL) responses through binding and carrying antigenic peptides. We demonstrated in this study that the HSC70 gene encodes two antigenic peptides recognised by HLA-B46-restricted and tumour-reactive CTLs established from tumour-infiltrating lymphocytes of a

K Azuma; S Shichijo; H Takedatsu; N Komatsu; H Sawamizu; K Itoh

2003-01-01

146

Characterization of an excreted/secreted antigen form of 14-3-3 protein in Toxoplasma gondii tachyzoites.  

PubMed

The 14-3-3 protein was shown to be present into the parasitophorous vacuole of Toxoplasma gondii-infected human monocyte cells and in the excreted/secreted antigens (ESA). The ESA 14-3-3 protein migrates electrophoretically as the cytosol and the main membranous 14-3-3 isoforms. The excretion/secretion of 14-3-3 was not sensitive to cycloheximide, a protein synthesis inhibitor, even at a concentration which inhibited the production of 14-3-3 inside the tachyzoites. Recombinant 14-3-3/GST protein was used to test the presence of 14-3-3 antibodies in different human sera. A positive immunoreactivity was observed with sera corresponding to acute toxoplasmosis and a possible involvement of 14-3-3 in host immunity is discussed. PMID:15109715

Assossou, Olga; Besson, Françoise; Rouault, Jean-Pierre; Persat, Florence; Ferrandiz, Josette; Mayençon, Martine; Peyron, François; Picot, Stèphane

2004-05-01

147

Isolation and characterization of monoclonal antibodies specific for antigen P1, a major surface protein of mutans streptococci.  

PubMed Central

A panel of 15 murine monoclonal antibodies (MAbs; 14 immunoglobulin G1, 1 immunoglobulin G2a) directed against antigen P1, a major surface protein of mutans streptococci, was prepared. All of these MAbs reacted by the enzyme-linked immunosorbent assay with solubilized wall material from Streptococcus mutans Ingbritt 175 (a serotype c strain which retains significant amounts of P1 in its cell wall), culture supernatant fluid from Ingbritt 162 (a strain which excretes large amounts of P1 into the culture medium), and purified P1. By Western immunoblotting, these MAbs were observed to react with a high-molecular-weight polypeptide which comigrated with antigen P1. None of these MAbs cross-reacted with human heart tissue or with various eucaryotic proteins. When whole cells of various strains of mutans streptococci were screened against the panel of MAbs, the strongest reactivities were noted with strains of serotype c and e S. mutans, while a serotype f strain of S. mutans, along with S. sobrinus and S. cricetus strains, reacted somewhat more weakly. S. rattus strains were completely negative. Results obtained with bacterial culture supernatants were qualitatively similar. The surface localization of antigen P1 was confirmed by electron microscopy with an indirect immunogold technique. In sectioned S. mutans cells, labeling appeared to be associated with a fibrillar "fuzzy coat" layer, which was far more prominent on cells of Ingbritt 175 than on those of Ingbritt 162. Images PMID:3312011

Ayakawa, G Y; Boushell, L W; Crowley, P J; Erdos, G W; McArthur, W P; Bleiweis, A S

1987-01-01

148

Immunization with FSH? fusion protein antigen prevents bone loss in a rat ovariectomy-induced osteoporosis model  

SciTech Connect

Highlights: •A GST-FSH fusion protein was successfully expressed in E. coli. •Immunization with GST-FSH antigen can raise high-titer anti-FSH polyclonal sera. •Anti-FSH polyclonal sera can neutralize osteoclastogenic effect of FSH in vitro. •FSH immunization can prevent bone loss in a rat osteoporosis model. -- Abstract: Osteoporosis, a metabolic bone disease, threatens postmenopausal women globally. Hormone replacement therapy (HTR), especially estrogen replacement therapy (ERT), is used widely in the clinic because it has been generally accepted that postmenopausal osteoporosis is caused by estrogen deficiency. However, hypogonadal ? and ? estrogen receptor null mice were only mildly osteopenic, and mice with either receptor deleted had normal bone mass, indicating that estrogen may not be the only mediator that induces osteoporosis. Recently, follicle-stimulating hormone (FSH), the serum concentration of which increases from the very beginning of menopause, has been found to play a key role in postmenopausal osteoporosis by promoting osteoclastogenesis. In this article, we confirmed that exogenous FSH can enhance osteoclast differentiation in vitro and that this effect can be neutralized by either an anti-FSH monoclonal antibody or anti-FSH polyclonal sera raised by immunizing animals with a recombinant GST-FSH? fusion protein antigen. Moreover, immunizing ovariectomized rats with the GST-FSH? antigen does significantly prevent trabecular bone loss and thereby enhance the bone strength, indicating that a FSH-based vaccine may be a promising therapeutic strategy to slow down bone loss in postmenopausal women.

Geng, Wenxin; Yan, Xingrong; Du, Huicong; Cui, Jihong; Li, Liwen, E-mail: liven@nwu.edu.cn; Chen, Fulin, E-mail: chenfl@nwu.edu.cn

2013-05-03

149

Quantitative analysis of cell surface-associated SV40 large T antigen using a newly developed 3H-protein A binding assay.  

PubMed

We have established a sensitive assay for the quantitative determination of large T antigen determinants on the surface of living simian virus 40 (SV40)-transformed cells (mKSA). Cells in suspension culture were incubated with monoclonal antibodies specific for large T antigen (KT3, directed against the carboxyterminus of large T antigen, and PAb 108, directed against an aminoterminal determinant on large T antigen). After incubation with secondary antibody (rabbit anti-mouse IgG), followed by incubation with 3H-protein A, the cells were sequentially extracted first with the nonionic detergent NP-40, followed by ultrasonication and extraction with the zwitterionic detergent Empigen BB. NP-40 solubilized large T antigen associated with NP-40-soluble constituents of the plasma membrane, whereas Empigen BB solubilized the plasma membrane lamina-associated subclass of large T antigen (U. Klockmann and W. Deppert, 1983, EMBO J., 7, 1151-1157). The amount of cell surface-bound 3H-protein A in the NP-40 and Empigen BB extracts was determined by liquid scintillation counting. In agreement with earlier reports, cell surface large T antigen was mainly found in association with the plasma membrane lamina (PML). Since the specific activity of 3H-protein A was known, it was possible to calculate the number of surface-bound 3H-protein A molecules, and thus to estimate the average number of surface-exposed amino- and carboxyterminal determinants of large T antigen per cell. KT3 recognized about 450-900 carboxyterminal determinants, while PAb 108 bound to about 1200-2400 aminoterminal determinants on the surface of a single mKSA cell. The cellular protein p53 also was detected on the surface of mKSA cells and was found to be present in amounts comparable to cell surface large T antigen. PMID:2471353

Rinke, Y; Deppert, W

1989-06-01

150

Varicellovirus UL49.5 Proteins Differentially Affect the Function of the Transporter Associated with Antigen Processing, TAP  

PubMed Central

Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing (TAP) plays an essential role in MHC class I–restricted antigen presentation, as TAP imports peptides into the ER, where peptide loading of MHC class I molecules takes place. In this study, the UL49.5 proteins of the varicelloviruses bovine herpesvirus 1 (BHV-1), pseudorabies virus (PRV), and equine herpesvirus 1 and 4 (EHV-1 and EHV-4) are characterized as members of a novel class of viral immune evasion proteins. These UL49.5 proteins interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides through inhibition of TAP. BHV-1, PRV, and EHV-1 recombinant viruses lacking UL49.5 no longer interfere with peptide transport. Combined with the observation that the individually expressed UL49.5 proteins block TAP as well, these data indicate that UL49.5 is the viral factor that is both necessary and sufficient to abolish TAP function during productive infection by these viruses. The mechanisms through which the UL49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 block TAP exhibit surprising diversity. BHV-1 UL49.5 targets TAP for proteasomal degradation, whereas EHV-1 and EHV-4 UL49.5 interfere with the binding of ATP to TAP. In contrast, TAP stability and ATP recruitment are not affected by PRV UL49.5, although it has the capacity to arrest the peptide transporter in a translocation-incompetent state, a property shared with the BHV-1 and EHV-1 UL49.5. Taken together, these results classify the UL49.5 gene products of BHV-1, PRV, EHV-1, and EHV-4 as members of a novel family of viral immune evasion proteins, inhibiting TAP through a variety of mechanisms. PMID:18516302

Lipinska, Andrea D.; Wang, Ying; Quinten, Edwin; Reits, Eric A.; Koch, Joachim; Loch, Sandra; Rezende, Marisa Marcondes; Daus, Franz; Bienkowska-Szewczyk, Krystyna; Osterrieder, Nikolaus; Mettenleiter, Thomas C.; Heemskerk, Mirjam H. M.; Tampe, Robert; Neefjes, Jacques J.; Chowdhury, Shafiqul I.; Ressing, Maaike E.; Rijsewijk, Frans A. M.; Wiertz, Emmanuel J. H. J.

2008-01-01

151

The influence of monogalactosyldiacylglycerols from different marine macrophytes on immunogenicity and conformation of protein antigen of tubular immunostimulating complex.  

PubMed

The tubular immunostimulating complex (TI-complex) is a novel nanoparticulate antigen delivery system consisting of cholesterol, triterpene glycoside cucumarioside A(2)-2, and glycolipid monogalactosyldiacylglycerol (MGDG) isolated from marine macrophytes. MGDG is crucial for the formation of a lipid matrix for the protein antigen incorporated in TI-complexes. Fatty acid composition and the physical state of this glycolipid depend on the taxonomic position of marine macrophytes. Therefore, the aim of the present work was to study the capacity of MGDGs, isolated from five species of marine macrophytes, to influence conformation and to enhance immunogenicity of porin from Yersinia pseudotuberculosis (YOmpF) as a model antigen of subunit vaccine based on TI-complexes. The trimeric porin was chosen for these experiments, because it was approximately two times more immunogenic than monomeric porin incorporated in TI-complexes. Immunization of mice with YOmpF within TI-complexes, comprised of different MGDGs, revealed a dependence of the immunostimulating effect of TI-complexes on the microvicosity of this glycolipid. TI-complexes comprising MGDGs from Sargassum pallidum and Ulva fenestrata with medium microviscosity induced maximal levels of anti-porin antibodies (four times higher when compared with those induced by pure porin). The adjuvant effect of TI-complexes based on other MGDGs varied by 2.8, 2.3 and 1.3 times for TI-complexes comprised of MGDGs from Zostera marina, Ahnfeltia tobuchiensis, and Laminaria japonica, respectively. MGDGs are also able to influence cytokine mechanisms of immunological regulation. DSC and spectroscopic studies showed that maximal immunostimulating effect of TI-complexes correlated with a moderate stabilizing influence of MGDGs from S. pallidum and U. fenestrata on the conformation of porin. The results obtained suggest lipid "nanofluidics" as a novel strategy for optimizing the immune response to protein antigens within lipid particulate systems. PMID:22269933

Sanina, Nina M; Kostetsky, Eduard Y; Shnyrov, Valery L; Tsybulsky, Alexander V; Novikova, Olga D; Portniagina, Olga Y; Vorobieva, Natalia S; Mazeika, Andrey N; Bogdanov, Mikhail V

2012-04-01

152

Surrogate Tumor Antigen Vaccination Induces Tumor-Specific Immunity and the Rejection of Spontaneous Metastases  

Microsoft Academic Search

The nonimmunogenic 4T1 murine mammary carcinoma model and a model surrogate tumor antigen (sTA) were employed to explore the possibility of inducing tumor-specific immunity through active immunization in the absence of defined tumor-associated antigens. Immunization of naive mice with protein-based sTA resulted in protection from s.c. challenge, with 4T1 modified to express the sTA (4T1.sTA), or from a sTA-expressing unrelated

Jennifer D. Lewis; Michael H. Shearer; Ronald C. Kennedy; Robert K. Bright

2005-01-01

153

Characterization of the membrane attack complex inhibitory protein CD59 antigen on human amniotic cells and in amniotic fluid.  

PubMed Central

A functional complement system and the potential for its activation are present in human amniotic fluid. We have recently demonstrated that CD59 antigen is present and functionally active on human amniotic epithelial cells (HAEC). We have now further examined the role of this protein on HAEC and have also demonstrated its presence in amniotic fluid (AF). CD59 Ag on HAEC is similar in size to the erythrocyte protein and is anchored via glycosyl phosphatidylinositol. The AF protein retained the capacity to incorporate into target cells and protect against lysis by complement. These data suggest that HAEC secrete into AF a form of CD59 Ag which retains inhibitory activity and which may be important in protection of the foetus from maternal complement in utero. Images Figure 1 Figure 3 Figure 4 PMID:1383132

Rooney, I A; Morgan, B P

1992-01-01

154

The Immune Response to a Vesicular Stomatitis Virus Vaccine Vector Is Independent of Particulate Antigen Secretion and Protein Turnover Rate  

PubMed Central

Vesicular stomatitis virus (VSV) is a highly cytopathic virus being developed as a vaccine vector due to its ability to induce strong protective T cell and antibody responses after a single dose. However, little is known regarding the mechanisms underlying the potent immune responses elicited by VSV. We previously generated a VSV vector expressing the hepatitis B virus middle envelope surface glycoprotein (MS) that induces strong MS-specific T cell and antibody responses in mice. After synthesis in the cytoplasm, the MS protein translocates to the endoplasmic reticulum, where it forms subviral particles that are secreted from the cell. To better understand the contributions of secreted and intracellular protein to the VSV-induced immune response, we produced a vector expressing a secretion-deficient MS mutant (MSC69A) and compared the immunogenicity of this vector to that of the wild-type VSV-MS vector in mice. As expected, the MSC69A protein was not secreted from VSV-infected cells and displayed enhanced proteasome-mediated degradation. Surprisingly, despite these differences in intracellular protein processing, the T cell and antibody responses generated to MSC69A were comparable to those elicited by virus expressing wild-type MS protein. Therefore, when it is expressed from VSV, the immune responses to MS are independent of particulate antigen secretion and the turnover rate of cytoplasmic protein. These results are consistent with a model in which the immune responses to VSV are strongly influenced by the replication cycle of the vector and demonstrate that characteristics of the vector have the capacity to affect vaccine efficacy more than do the properties of the antigen itself. PMID:22345454

Cobleigh, Melissa A.; Bradfield, Clinton; Liu, Yuanjie; Mehta, Anand

2012-01-01

155

Antigenic and immunogenic properties of truncated VP28 protein of white spot syndrome virus in Procambarus clarkii.  

PubMed

Previous studies identify VP28 envelope protein of white spot syndrome virus (WSSV) as its main antigenic protein. Although implicated in viral infectivity, its functional role remains unclear. In the current study, we described the production of polyclonal antibodies to recombinant truncated VP28 proteins including deleted N-terminal (rVP28?N), C-terminal (rVP28?C) and middle (rVP28?M). In antigenicity assays, antibodies developed from VP28 truncations lacking the N-terminal or middle regions showed significantly lowered neutralization of WSSV in crayfish, Procambarus clarkii. Further immunogenicity analysis showed reduced relative percent survival (RPS) in crayfish vaccinating with these truncations before challenge with WSSV. These results indicated that N-terminal (residues 1-27) and middle region (residues 35-95) were essential to maintain the neutralizing linear epitopes of VP28 and responsible in eliciting immune response. Thus, it is most likely that these regions are exposed on VP28, and will be useful for rational design of effective vaccines targeting VP28 of WSSV. PMID:23178263

Du, Hua-Hua; Hou, Chong-Lin; Wu, Xiao-Guo; Xie, Rong-hui; Wang, Yi-Zhen

2013-01-01

156

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

Velasquez, Norma P.; Camargo, Rocio E.; Uzcanga, Graciela L.; Bubis, Jose

2014-01-01

157

Heat treatment improves antigen-specific T cell activation after protein delivery by several but not all yeast genera.  

PubMed

A central prerequisite in using yeast as antigen carrier in vaccination is its efficient interaction with cellular components of the innate immune system, mainly mediated by cell surface structures. Here, we investigated the distribution of major yeast cell wall components such as mannan, ?-glucan and chitin of four different and likewise biotechnologically relevant yeasts (Saccharomyces, Pichia, Kluyveromyces and Schizosaccharomyces) and analyzed the influence of heat-treatment on ?-1,3-glucan exposure at the outer yeast cell surface as well as the amount of yeast induced reactive oxygen species (ROS) production by antigen presenting cells (APC) in human blood. We found that yeasts significantly differ in the distribution of their cell wall components and that heat-treatment affected both, cell wall composition and yeast-induced ROS production by human APCs. We further show that heat-treatment modulates the activation of antigen specific memory T cells after yeast-mediated protein delivery in different ways and thus provide additional support of using yeast as vehicle for the development of novel T cell vaccines. PMID:24674665

Bazan, Silvia Boschi; Breinig, Tanja; Schmitt, Manfred J; Breinig, Frank

2014-05-01

158

SV40 LARGE T ANTIGEN INTERACTS WITH THE MITOTIC CHECKPOINT PROTEIN BUB1  

Microsoft Academic Search

INTRODUCTION. Simian virus 40 (SV40) is a member of the papovavirus family of DNA tumour viruses. The viral oncoprotein SV40 Large T antigen (LT) is sufficient for immortalization of many rodent cell types, and can also transform these cells at a low frequency (1). It carries out these functions mainly by association with host factors. Some of the critical activities

Rowena Lock; Marina Cotsiki; Ole Gjoerup; Alan Entwistle; Tom Roberts; Erica Golemis; Parmjit Jat

159

Goodpasture Antigen-binding Protein and Its Spliced Variant, Ceramide Transfer Protein, Have Different Functions in the Modulation of Apoptosis during Zebrafish Development*S?  

PubMed Central

Human Goodpasture antigen-binding protein (GPBP) is an atypical protein kinase that phosphorylates the Goodpasture auto-antigen, the ?3 chain of collagen IV. The COL4A3BP gene is alternatively spliced producing two protein isoforms: GPBP and GPBP?26. The latter lacks a serine-rich domain composed of 26 amino acid residues. Both isoforms also function as ceramide transfer proteins (CERT). Here, we explored the function of Gpbp and Gpbp?26/CERT during embryogenesis in zebrafish. We cloned both splice variants of the zebrafish gene and found that they are differentially expressed during development. We used antisense oligonucleotide-mediated loss-of-function and synthetic mRNA-based gain-of-function approaches. Our results show that the loss-of-function phenotype is linked to cell death, evident primarily in the muscle of the somites, extensive loss of myelinated tracks, and brain edema. These results indicate that disruption of the nonvesicular ceramide transport is detrimental to normal embryonic development of somites and brain because of increased apoptosis. Moreover, this phenotype is mediated by Gpbp but not Gpbp?26/CERT, suggesting that Gpbp is an important factor for normal skeletal muscle and brain development. PMID:18424781

Granero-Molto, Froilan; Sarmah, Swapnalee; O'Rear, Lynda; Spagnoli, Anna; Abrahamson, Dale; Saus, Juan; Hudson, Billy G.; Knapik, Ela W.

2008-01-01

160

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; Narvanen, Ale; Stenman, Ulf-Hakan; Koistinen, Hannu

2014-01-01

161

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

162

Does Binding of Complement Factor H to the Meningococcal Vaccine Antigen, Factor H Binding Protein, Decrease Protective Serum Antibody Responses?  

PubMed Central

Factor H binding protein (fHbp) is a principal antigen in a multicomponent meningococcal vaccine recently licensed in Europe for prevention of serogroup B diseases. The protein recruits the complement downregulator, factor H (fH), to the bacterial surface, which enables the organism to resist complement-mediated bacteriolysis. Binding is specific for human fH. In preclinical studies, mice and rabbits immunized with fHbp vaccines developed serum bactericidal antibody responses, which in humans predict protection against developing meningococcal disease. These studies, however, were in animals whose fH did not bind to the vaccine antigen. Here we review the immunogenicity of fHbp vaccines in human fH transgenic mice. The data suggest that animals with high serum human fH concentrations have impaired protective antibody responses. Further, mutant fHbp vaccines with single amino acid substitutions that decrease fH binding are superior immunogens, possibly by unmasking epitopes in the fH binding site that are important for eliciting serum bactericidal antibody responses. Humans immunized with fHbp vaccines develop serum bactericidal antibody, but achieving broad coverage in infants required incorporation of additional antigens, including outer membrane vesicles, which increased rates of fever and local reactions at the injection site. The experimental results in transgenic mice predict that fHbp immunogenicity can be improved in humans by using mutant fHbp vaccines with decreased fH binding. These results have important public health implications for developing improved fHbp vaccines for control of serogroup B meningococcal disease and for development of vaccines against other microbes that bind host molecules. PMID:23740919

Ram, Sanjay; Beernink, Peter T.

2013-01-01

163

Evidence for an inhibitory feedback loop regulating simian virus 40 large T-antigen fusion protein nuclear transport.  

PubMed Central

Nuclear protein import is central to eukaryotic cell function. It is dependent on ATP, temperature and cytosolic factors, and requires specific targeting sequences called nuclear localization signals (NLSs). Nuclear import kinetics was studied in vitro using digitonin-permeabilized cells of the HTC rat hepatoma cell line and a fluorescently labelled beta-galactosidase fusion protein carrying amino acids 111-135 of the simian virus 40 large T-antigen (T-ag), including the NLS. Nuclear accumulation was rapid, reaching steady-state after about 80 min at 37 degrees C (t1/2 at about 17 min). Surprisingly, maximal nuclear concentration was found to be directly proportional to the concentration of the cytosolic extract and of cytoplasmic T-ag protein. Neither preincubation of cells for 1 h at 37 degrees C before the addition of T-ag protein nor the addition of fresh transport medium after 1 h and continuation of the incubation for another hour affected the maximal nuclear concentration. If cells were allowed to accumulate T-ag protein for 1 h before the addition of fresh transport medium containing different concentrations of T-ag protein and incubated for a further hour, the maximal nuclear concentration did not change unless the concentration of T-ag protein in the second transport mixture exceeded that in the first, in which case the nuclear concentration increased. Nuclear import of T-ag thus appeared (i) to be strictly unidirectional over 2 h at 37 degrees C and (ii) to be regulated by an inhibitory feedback loop, whereby the cytosolic concentration of protein appears to determine directly the precise end point of nuclear accumulation. This study represents the first characterization of this previously undescribed mechanism of regulation of nuclear protein import. PMID:8670127

Seydel, U; Jans, D A

1996-01-01

164

Liver cytosolic 1 antigen-antibody system in type 2 autoimmune hepatitis and hepatitis C virus infection  

Microsoft Academic Search

Within the multiform liver\\/kidney microsomal (LKM) family, a subgroup of sera that reacts with a liver cytosolic (LC) protein has been isolated and the new antigen-antibody system is called LC1. Unlike LKM antibody type 1 (anti-LKM1), anti-LC1 is said to be unrelated to hepatitis C virus (HCV) infection and has therefore been proposed as a marker of 'true' autoimmune hepatitis

M Lenzi; P Manotti; L Muratori; M Cataleta; G Ballardini; F Cassani; F B Bianchi

1995-01-01

165

GILT modulates CD4+ T cell tolerance to the melanocyte differentiation antigen tyrosinase-related protein 1  

PubMed Central

Gamma-interferon-inducible lysosomal thiol reductase (GILT) facilitates MHC class II-restricted processing though endocytic reduction of protein disulfide bonds and is necessary for efficient class II-restricted processing of melanocyte differentiation antigen, tyrosinase-related protein 1 (TRP1). Using class II-restricted, TRP1-specific T cell repector transgenic mice, we identify a novel role for GILT in the maintenance of tolerance to TRP1. TRP1-specific thymocytes are centrally deleted in the presence of GILT and TRP1. In contrast, CD4 single positive thymocytes and peripheral T cells develop in the absence of GILT or TRP1, demonstrating that GILT is required for negative selection of TRP1-specific thymocytes. Although TRP1-specific T cells escape thymic deletion in the absence of GILT, they are tolerant to TRP1 and do not induce vilitigo. TRP1-specific T cells that develop in the absence of GILT have diminished IL-2 and IFN-? production. Furthermore, GILT-deficient mice have a four-fold increase in the percentage of TRP1-specific regulatory T cells compared to TRP1-deficient mice, and depletion of regulatory T cells partially restores the ability of GILT-deficient TRP1-specific CD4+ T cells to induce vitiligo. Thus, GILT plays a critical role in regulating CD4+ T cell tolerance to an endogenous skin-restricted antigen relevant to controlling autoimmunity and generating effective immunotherapy for melanoma. PMID:21833020

Rausch, Matthew P.; Hastings, Karen Taraszka

2011-01-01

166

Identification and characterization of a Trypanosoma congolense 46 kDa protein as a candidate serodiagnostic antigen.  

PubMed

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

2014-06-01

167

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

168

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...

169

CGM1a Antigen of Neutrophils, a Receptor of Gonococcal Opacity Proteins  

Microsoft Academic Search

Neisseria gonorrhoeae (GC) or Escherichia coli expressing phase-variable opacity (Opa) protein (Opa+ GC or Opa+ GC or Opa+ E. coli) adhere to human neutrophils and stimulate phagocytosis, whereas their counterparts not expressing Opa protein (Opa- GC or Opa- E. coli) E. coli) adhere to human neutrophils and stimulate phagocytosis, whereas their counterparts not expressing Opa protein Opa+ GC or E.

Tie Chen; Emil C. Gotschlich

1996-01-01

170

Recombinant dengue virus type 2 envelope\\/hepatitis B surface antigen hybrid protein expressed in Pichia pastoris can function as a bivalent immunogen  

Microsoft Academic Search

A truncated version of the dengue virus type 2 envelope protein (Den2E) encoding the first 395 amino acid (aa) residues, and Den2E fused in-frame with the full-length 226-aa hepatitis B surface antigen (Den2E-HBsAg) protein were expressed in the methylotrophic yeast, Pichia pastoris. Both the recombinant proteins showed evidence of the capacity to form high molecular weight aggregates. Electron microscopic analysis

Himani Bisht; Dipti A Chugh; Manoj Raje; S Swaminathan; Navin Khanna

2002-01-01

171

Amino Acid Sequence and Antigenicity of the Amino-terminus of the 168 kDa Adherence Protein of Mycoplasma pneumoniae  

Microsoft Academic Search

The amino-terminal end of the 168 kDa adherence protein from the membrane of Mycoplasma pneumoniae was sequenced up to 12 amino acids. A synthetic peptide containing nine amino acids of this sequence was used to study the antigenicity of the amino-terminus of the 168 kDa protein and the involvement of the homologous sequence of the protein in the adherence process.

E. JACOBS; K. FUCHTE; W. BREDT

1987-01-01

172

Free intraglomerular malarial antigens.  

PubMed Central

Infection with Plasmodium berghei leads to a rapidly lethal disease in different strains of mice. Nude athymic mice are not able to produce circulating antibodies (IgG or IgM) against plasmodial antigens. Nevertheless, plasmodium-related antigens can be detected in the glomeruli of nude mice, in relation to the rising parasitaemia. This deposition is unrelated to the deposition of other immunoreactants (IgG, IgM or C3). The presence of the latter as well as the circulating auto-antibodies did not correlate with the intercurrent infection and control and experimental animals behaved likewise. These results indicate an intraglomerular localization of free malarial antigens. It is suggested that this may represent a basic mechanism for in situ formation of immune complexes in immunocompetent mice. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:3896290

Pakasa, M.; Van Damme, B.; Desmet, V. J.

1985-01-01

173

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

174

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

175

Mapping 28 erythrocyte antigen, plasma protein and enzyme polymorphisms using an efficient genomic scan of the porcine genome.  

PubMed

One hundred and fifty-four microsatellite markers were selected for genomic scanning of the porcine genome and were grouped into amplification sets to reduce the cost and labour required. Thirty amplification sets had two markers (duplex), 20 sets had three markers (triplex) and five sets had four markers (quadruplex) while 14 markers were analysed separately. The selection criteria for microsatellites were: ease of scoring, level of polymorphism, genetic location and ability to be genotyped in a multiplexed polymerase chain reaction (PCR). The selected microsatellites were chosen to span the entire genome flanked by the porcine linkage map with intervals between adjacent markers of 15-20 cM where possible. The utility of this set of markers was demonstrated by linkage analyses with loci controlling blood plasma protein and red cell enzyme polymorphisms (n = 13), erythrocyte antigens (n = 15), the S blood group, coat colour and ryanodine receptor from 174 backcross Meishan-White Composite pigs. These loci displayed various forms of inheritance and most (24 loci) have been placed in linkage groups. Significant two-point linkages (lod > 3.0) were detected for each polymorphic marker. These results provide the first linkage assignments for phosphoglucomutase (PGM2) and erythrocyte antigen F (EAF) to SSC8; and serum amylase (AMY) and erythrocyte antigen I (EAI) to SSC18. All of the remaining polymorphic loci (n = 24) mapped to previously identified regions confirming earlier results. Most of the markers used in this study should be useful in resource populations of various breed crosses as the number of alleles detected in a multibreed reference population was one of the selection criteria. PMID:9363592

Rohrer, G A; Vögeli, P; Stranzinger, G; Alexander, L J; Beattie, C W

1997-10-01

176

Deduced amino acid sequences at the fusion protein cleavage site of Newcastle disease viruses showing variation in antigenicity and pathogenicity.  

PubMed

The amino acid sequence at the F2/F1 cleavage site of the F0 fusion protein of 17 strains of Newcastle disease virus (NDV) was deduced from sequencing a 32 nucleotide area of the genome by reverse transcription and polymerase chain reaction (PCR) techniques. With the addition of sequences at the same area previously published for 9 other viruses comparisons were made of a total of 26 NDV strains and isolates (11 of low virulence, 15 of high virulence or mesogenic) covering ten antigenic groups determined by reactions with monoclonal antibodies. All the virulent viruses and the mesogenic strain Komarov showed the amino acid sequence 112R/K-R-Q-K/R-R116 for the C-terminus of the F2 protein and phenylalanine (F) at the N-terminus of the F1 protein, residue 117. The mesogenic isolate of the antigenic variant NDV responsible for the recent panzootic in racing pigeons, often termed "pigeon paramyxovirus type 1", examined in this study had the sequence 112G-R-Q-K-R-F117. The deduced amino acid sequence in the corresponding region of all viruses of low virulence was 112G/E-K/R-Q-G/E-R-L117. The virulent virus, PMV-1/chicken/Ireland/34/90 (34/90), which had a close antigenic relationship to a group of avirulent viruses, three of which were examined in the present study as representatives of the monoclonal antibody group H, showed between 4-6 nucleotide differences from these viruses in the 32 nucleotide region studied. These resulted in differences in the deduced amino acid sequence at residue 112 E-->K, 115 E-->K and 117-->F, giving 34/90 a typical virulent virus motif at the cleavage site. Despite the extremely small portion of the genome studied there were several areas which appeared characteristic for 34/90 and the three group H viruses of low virulence, which suggests that they may have arisen from the same gene pool. PMID:8435046

Collins, M S; Bashiruddin, J B; Alexander, D J

1993-01-01

177

B-Cell Responses to Pregnancy-Restricted and -Unrestricted Plasmodium falciparum Erythrocyte Membrane Protein 1 Antigens in Ghanaian Women Naturally Exposed to Malaria Parasites  

PubMed Central

Protective immunity to Plasmodium falciparum malaria acquired after natural exposure is largely antibody mediated. IgG-specific P. falciparum EMP1 (PfEMP1) proteins on the infected erythrocyte surface are particularly important. The transient antibody responses and the slowly acquired protective immunity probably reflect the clonal antigenic variation and allelic polymorphism of PfEMP1. However, it is likely that other immune-evasive mechanisms are also involved, such as interference with formation and maintenance of immunological memory. We measured PfEMP1-specific antibody levels by enzyme-linked immunosorbent assay (ELISA) and memory B-cell frequencies by enzyme-linked immunosorbent spot (ELISPOT) assay in a cohort of P. falciparum-exposed nonpregnant Ghanaian women. The antigens used were a VAR2CSA-type PfEMP1 (IT4VAR04) with expression restricted to parasites infecting the placenta, as well as two commonly recognized PfEMP1 proteins (HB3VAR06 and IT4VAR60) implicated in rosetting and not pregnancy restricted. This enabled, for the first time, a direct comparison in the same individuals of immune responses specific for a clinically important parasite antigen expressed only during well-defined periods (pregnancy) to responses specific for comparable antigens expressed independent of pregnancy. Our data indicate that PfEMP1-specific B-cell memory is adequately acquired even when antigen exposure is infrequent (e.g., VAR2CSA-type PfEMP1). Furthermore, immunological memory specific for VAR2CSA-type PfEMP1 can be maintained for many years without antigen reexposure and after circulating antigen-specific IgG has disappeared. The study provides evidence that natural exposure to P. falciparum leads to formation of durable B-cell immunity to clinically important PfEMP1 antigens. This has encouraging implications for current efforts to develop PfEMP1-based vaccines. PMID:24566620

Ampomah, Paulina; Stevenson, Liz; Ofori, Michael F.; Barfod, Lea

2014-01-01

178

Transformation of human T-cell clones by Herpesvirus saimiri: intact antigen recognition by autonomously growing myelin basic protein-specific T cells.  

PubMed Central

Herpesvirus saimiri has recently been shown to immortalize human T cells. It was unknown, however, whether Herpesvirus saimiri transformation affects T-cell receptor (TCR) expression and signal transduction. In the present study, we have transformed CD4+ human T-cell clones specific for human myelin basic protein. The transformed T cells were grown in interleukin 2 and divided in the absence of antigen and antigen-presenting cells. They retained the membrane phenotype of activated T cells and secreted the cytokines interferon gamma and lymphotoxin, but interleukin 4 was not detected. Further, the transformed T cells continued to express the original TCR as demonstrated by TCR variable-region-V beta-specific monoclonal antibodies and TCR sequencing. Antigen-specific recognition and signal transduction by the TCR were demonstrated by myelin-basic-protein-induced HLA-DR-restricted secretion of interferon gamma and lymphotoxin and by myelin-basic-protein-specific proliferation. Antigen specificity and reactivity have been maintained for > 1 year after transformation. Transformation with Herpesvirus saimiri now allows the production of virtually unlimited numbers of (auto)antigen-specific T cells expressing functional TCR and a stable membrane phenotype. This technology will facilitate studies of the pathogenesis of putative autoimmune diseases, such as multiple sclerosis, and may be of help in TCR-targeted immunotherapy. PMID:7504291

Weber, F; Meinl, E; Drexler, K; Czlonkowska, A; Huber, S; Fickenscher, H; Muller-Fleckenstein, I; Fleckenstein, B; Wekerle, H; Hohlfeld, R

1993-01-01

179

Vesicular Stomatitis Virus Matrix Protein Impairs CD1d-Mediated Antigen Presentation through Activation of the p38 MAPK Pathway?  

PubMed Central

Natural killer T (NKT) cells are unique T lymphocytes that recognize CD1d-bound lipid antigens and play an important role in both innate and acquired immune responses against infectious diseases and tumors. We have already shown that a vesicular stomatitis virus (VSV) infection results in the rapid inhibition of murine CD1d-mediated antigen presentation to NKT cells. In the present study, it was found that the VSV matrix (VSV-M) protein is an important element in this decrease in antigen presentation postinfection. The VSV-M protein altered the intracellular distribution of murine CD1d molecules, resulting in qualitative (but not quantitative) changes in cell surface CD1d expression. The M protein was distributed throughout the infected cell, and it was found to activate the mitogen-activated protein kinase (MAPK) p38 very early postinfection. Infection of CD1d+ cells with a temperature-sensitive VSV-M mutant at the nonpermissive temperature both substantially reversed the inhibition of antigen presentation by CD1d and delayed the activation of p38. Thus, the VSV-M protein plays an important role in permitting the virus to evade important components of the innate immune response by regulating specific MAPK pathways. PMID:18815300

Renukaradhya, Gourapura J.; Khan, Masood A.; Shaji, Daniel; Brutkiewicz, Randy R.

2008-01-01

180

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

181

A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+ T lymphocytes  

Microsoft Academic Search

Summary Herpes simplex virus (HSV) infection of human fibro- blasts rapidly renders the cells resistant to lysis by HSV-specific CDS+ cytotoxic T lymphocytes (CTLs), which normally recognize cell surface major histocom- patibillty complex (MHC) class I proteins presenting viral peptides. Within 3 hr of infection with HSV, MHC class I protein complexes are retained in the endoplas- mic reticulum (ER)\\/cis

Ian A. York; Cindy Roop; David W. Andrews; Stanley R. Riddell; Frank L. Graham; David C. Johnson

1994-01-01

182

In vivo protein biotinylation for identification of organ-specific antigens accessible from the vasculature  

Microsoft Academic Search

We describe a new methodology, based on terminal perfusion of rodents with a reactive ester derivative of biotin that enables the covalent modification of proteins readily accessible from the bloodstream. Biotinylated proteins from total organ extracts can be purified on streptavidin resin in the presence of strong detergents, digested on the resin and subjected to liquid chromatography–tandem mass spectrometry for

Jascha-N Rybak; Anna Ettorre; Brigitte Kaissling; Raffaella Giavazzi; Giuliano Elia; Dario Neri

2005-01-01

183

Epstein–Barr virus nuclear antigen 3A protein regulates CDKN2B transcription via interaction with MIZ-1  

PubMed Central

The Epstein–Barr virus (EBV) nuclear antigen 3 family of protein is critical for the EBV-induced primary B-cell growth transformation process. Using a yeast two-hybrid screen we identified 22 novel cellular partners of the EBNA3s. Most importantly, among the newly identified partners, five are known to play direct and important roles in transcriptional regulation. Of these, the Myc-interacting zinc finger protein-1 (MIZ-1) is a transcription factor initially characterized as a binding partner of MYC. MIZ-1 activates the transcription of a number of target genes including the cell cycle inhibitor CDKN2B. Focusing on the EBNA3A/MIZ-1 interaction we demonstrate that binding occurs in EBV-infected cells expressing both proteins at endogenous physiological levels and that in the presence of EBNA3A, a significant fraction of MIZ-1 translocates from the cytoplasm to the nucleus. Moreover, we show that a trimeric complex composed of a MIZ-1 recognition DNA element, MIZ-1 and EBNA3A can be formed, and that interaction of MIZ-1 with nucleophosmin (NPM), one of its coactivator, is prevented by EBNA3A. Finally, we show that, in the presence of EBNA3A, expression of the MIZ-1 target gene, CDKN2B, is downregulated and repressive H3K27 marks are established on its promoter region suggesting that EBNA3A directly counteracts the growth inhibitory action of MIZ-1. PMID:25092922

Bazot, Quentin; Deschamps, Thibaut; Tafforeau, Lionel; Siouda, Maha; Leblanc, Pascal; Harth-Hertle, Marie L.; Rabourdin-Combe, Chantal; Lotteau, Vincent; Kempkes, Bettina; Tommasino, Massimo; Gruffat, Henri; Manet, Evelyne

2014-01-01

184

Amino-terminal regions of polyomavirus middle T antigen are required for interactions with protein phosphatase 2A.  

PubMed Central

Polyomavirus middle T antigen (MT) is the major transforming protein of the virus. It functions through interactions with a number of cellular proteins involved in cell proliferation. MT forms complexes with protein phosphatase 2A (PP2A), pp60c-src, phosphatidylinositol 3-kinase, and Shc. We introduced both deletion and point mutations into three regions of MT and examined their ability to associate with PP2A and pp60c-src. The first 25 amino acid residues of MT are required for association with PP2A and pp60c-src. Amino acids 105 to 111, comprising the sequence Cys-Arg-Met-Pro-Leu-Thr-Cys, is also required for complex formation between MT and PP2A. However, the sequence Asp-Lys-Gly-Gly (amino acids 44 to 47), also found in the B subunit of PP2A, is dispensable for complex formation between MT and PP2A. We find a strict correlation between the ability of MT to associate with PP2A and the ability of MT to associate with pp60c-src. One mutant, L5E, associates with a phosphatase other than PP2A, pp60c-src, and phosphatidylinositol 3-kinase in a manner similar to that of wild-type MT yet is reduced in its transforming ability on NIH 3T3 cells. PMID:7538175

Glenn, G M; Eckhart, W

1995-01-01

185

Substrate binding protein SBP2 of a putative ABC transporter as a novel vaccine antigen of Moraxella catarrhalis.  

PubMed

Moraxella catarrhalis is a common respiratory tract pathogen that causes otitis media in children and infections in adults with chronic obstructive pulmonary disease. Since the introduction of the pneumococcal conjugate vaccines with/without protein D of nontypeable Haemophilus influenzae, M. catarrhalis has become a high-priority pathogen in otitis media. For the development of antibacterial vaccines and therapies, substrate binding proteins of ATP-binding cassette transporters are important targets. In this study, we identified and characterized a substrate binding protein, SBP2, of M. catarrhalis. Among 30 clinical isolates tested, the sbp2 gene sequence was highly conserved. In 2 different analyses (whole-cell enzyme-linked immunosorbent assay and flow cytometry), polyclonal antibodies raised to recombinant SBP2 demonstrated that SBP2 expresses epitopes on the bacterial surface of the wild type but not the sbp2 mutant. Mice immunized with recombinant SBP2 showed significantly enhanced clearance of M. catarrhalis from the lung compared to that in the control group at both 25-?g and 50-?g doses (P < 0.001). We conclude that SBP2 is a novel, attractive candidate as a vaccine antigen against M. catarrhalis. PMID:24914218

Otsuka, Taketo; Kirkham, Charmaine; Johnson, Antoinette; Jones, Megan M; Murphy, Timothy F

2014-08-01

186

The Tn antigen-specific lectin from ground ivy is an insecticidal protein with an unusual physiology.  

PubMed

Leaves of ground ivy (Glechoma hederacea) contain a lectin (called Gleheda) that is structurally and evolutionary related to the classical legume lectins. Screening of a population of wild plants revealed that Gleheda accounts for more than one-third of the total leaf protein in some clones, whereas it cannot be detected in other clones growing in the same environment. Gleheda is predominantly expressed in the leaves where it accumulates during early leaf maturation. The lectin is not uniformly distributed over the leaves but exhibits a unique localization pattern characterized by an almost exclusive confinement to a single layer of palisade parenchyma cells. Insect feeding trials demonstrated that Gleheda is a potent insecticidal protein for larvae of the Colorado potato beetle (Leptinotarsa decemlineata). Because Gleheda is not cytotoxic, it is suggested that the insecticidal activity is linked to the carbohydrate-binding specificity of the lectin, which as could be demonstrated by agglutination assays with different types of polyagglutinable human erythrocytes is specifically directed against the Tn antigen structure (N-acetylgalactosamine O-linked to serine or threonine residues of proteins). PMID:12857814

Wang, Weifang; Hause, Bettina; Peumans, Willy J; Smagghe, Guy; Mackie, Anne; Fraser, Robin; van Damme, Els J M

2003-07-01

187

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

188

Baculovirus expression of non-structural protein NS2 and core protein VP7 of African horsesickness virus serotype 3 and their use as antigens in an indirect ELISA.  

PubMed

Non-structural protein NS2 and core protein VP7 of African horsesickness virus serotype 3 (AHSV3) were expressed in Spodoptera frugiperda cells by recombinant baculoviruses containing the relevant genes. These proteins were purified and analysed by polyacrylamide gel electrophoresis and Western blot. NS2 and VP7 were used separately as antigens in an indirect ELISA for the detection of AHSV antibodies. Both antigens cross-reacted with hyperimmune guinea-pig antisera to infected cell lysates of all nine known AHSV serotypes and to antisera obtained from horses immunized with attenuated virus of seven AHSV serotypes. PMID:7989441

Bremer, C W; du Plessis, D H; van Dijk, A A

1994-07-01

189

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

190

T-cell Intracellular Antigen (TIA)-Proteins Deficiency in Murine Embryonic Fibroblasts Alters Cell Cycle Progression and Induces Autophagy  

PubMed Central

Mice lacking either T-cell intracellular antigen 1 (TIA1) or TIA1 related/like protein (TIAR/TIAL1) show high rates of embryonic lethality, suggesting a relevant role for these proteins during embryonic development. However, intrinsic molecular and cellular consequences of either TIA1 or TIAR deficiency remain poorly defined. By using genome-wide expression profiling approach, we demonstrate that either TIA1 or TIAR inactivation broadly alter normal development-associated signalling pathways in murine embryonic fibroblasts (MEF). Indeed, these analyses highlighted alterations of cytokine-cytokine and ECM-receptor interactions and Wnt, MAPK, TGF-beta dependent signalling pathways. Consistent with these results, TIA1 and TIAR knockout (KO) MEF show reduced rates of cell proliferation, cell cycle progression delay and increased cell size. Furthermore, TIA-proteins deficiency also caused metabolic deficiencies, increased ROS levels and DNA damage, promoting a gentle rise of cell death. Concomitantly, high rates of autophagy were detected in both TIA1 and TIAR KO MEF with induction of the formation of autophagosomes, as evidenced by the up-regulation of the LC3B protein, and autolysosomes, measured by colocalization of LC3B and LAMP1, as a survival mechanism attempt. Taken together, these observations support that TIA proteins orchestrate a transcriptome programme to activate specific developmental decisions. This program is likely to contribute to mouse physiology starting at early stages of the embryonic development. TIA1/TIAR might function as cell sensors to maintain homeostasis and promote adaptation/survival responses to developmental stress. PMID:24086455

Sanchez-Jimenez, Carmen; Izquierdo, Jose M.

2013-01-01

191

Conserved epitope on several human vitamin K-dependent proteins: location of the antigenic site and influence of metal ions on antibody binding  

SciTech Connect

A murine monoclonal antibody (designated H-11) produced by injecting mice with purified human protein C was found to bind several human vitamin K-dependent proteins. Using a solid-phase competitive radioimmunoassay with antibody immobilized onto microtiter plates, binding of /sup 125/I-labeled protein C to the antibody was inhibited by increasing amounts of protein C, prothrombin, and Factors X and VII over a concentration range of 1 x 10/sup -8/ to 1 x 10/sup -6/ M. Chemical treatment of prothrombin with a variety of agents did not destroy the antigenic site recognized by the antibody as measured by immunoblotting of prothrombin or prothrombin derivative immobilized onto nitrocellulose. Immunoblotting of purified vitamin K-dependent polypeptides with the monoclonal antibody following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose indicated that the antigenic site was found on the light chains of protein C and Factor X. The exact location of the antigenic determinant for antibody H-11 was established using synthetic peptides. Comparison of protein sequences of bovine and human vitamin K-dependent proteins suggests that the sequence Phe-Leu-Glu-Glu-Xaa-Arg/Lys is required for antibody binding. Increasing concentrations of Ca/sup 2 +/, Mg/sup 2 +/, or Mn/sup 2 +/ partially inhibited binding of /sup 125/I-protein C to the antibody in a solid-phase assay system with half-maximal binding observed at divalent metal ion concentrations of 2, 4, and 0.6 mM, respectively. The antigenic site thus recognized by monoclonal antibody H-11 is located at the amino-terminal region in the highly conserved ..gamma..-carboxyglutamic acid-containing domains of several, but not all, vitamin K-dependent proteins.

Church, W.R.; Messier, T.; Howard, P.R.; Amiral, J.; Meyer, D.; Mann, K.G.

1988-05-05

192

A mycobacterial 65-kD heat shock protein induces antigen-specific suppression of adjuvant arthritis, but is not itself arthritogenic  

PubMed Central

A recombinant (r)65-kD protein from Mycobacterium leprae, at levels far in excess of those present in whole mycobacteria, was unable to induce arthritis. Even when combined with a synthetic adjuvant, CP20961, to mimic the peptidoglycan adjuvant component of the mycobacterial cell wall, the r65-kD protein failed to induce arthritis. Pretreatment with as little as 1 microgram r65-kD protein protected rats against arthritis induced by M. tuberculosis, but this r65-kD protein was markedly less able to protect against arthritis induced by the synthetic adjuvant, CP20961, or type II collagen. The r65-kD protein appears, therefore, to produce an antigen-specific protection against arthritis induced by bacterial cell walls containing the 65-kD protein. Such protection can be overcome, however, by arthritogenic T lymphocytes, suggesting that protection occurs by preventing clonal proliferation of autoreactive T lymphocytes that are induced by the adjuvant properties of mycobacterial cell walls. How the r65-kD protein abrogates this particular adjuvant activity, and the nature of the arthritogenic self antigen(s), remain to be elucidated. PMID:2104920

1990-01-01

193

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

E-print Network

and outer bacterial membranes and a needle which extends the into extracellular space. With the increasing prevalence of drug resistant bacterial strains, vaccines represent one of the most promising strategies to combat these diseases. Proteins located...

Markham, Aaron Paul

2009-07-06

194

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

195

The Haemophilus influenzae HtrA Protein Is a Protective Antigen  

Microsoft Academic Search

The htrA gene from two strains of nontypeable Haemophilus influenzae has been cloned and sequenced, and the encoded approximately 46-kDa HtrA proteins were found to be highly conserved. H. influenzae HtrA has approximately 55% identity with the Escherichia coli and Salmonella typhimurium HtrA stress response proteins, and expression of the H. influenzae htrA gene was inducible by high temperature. Recombinant

SHEENA M. LOOSMORE; YAN-PING YANG; RAY OOMEN; JEAN M. SHORTREED; DEBBIE C. COLEMAN; MICHEL H. KLEIN

1998-01-01

196

Reverse Line Blot Assay for Direct Identification of Seven Streptococcus agalactiae Major Surface Protein Antigen Genes  

Microsoft Academic Search

We developed a multiplex PCR-based reverse line blot hybridization assay (mPCR\\/RLB) to detect the genes encoding members of the family of variable surface-localized proteins of Streptococcus agalactiae (group B streptococcus (GBS)), namely, Bca (C), Rib, Epsilon (Epsilon\\/Alp1\\/Alp5), Alp2, Alp3, and Alp4, and the immunoglobulin A binding protein, Bac (C). We used the assay to identify these genes in a collection

Zuotao Zhao; Fanrong Kong; Gwendolyn L. Gilbert

2006-01-01

197

P80, the HinT interacting membrane protein, is a secreted antigen of Mycoplasma hominis  

Microsoft Academic Search

BACKGROUND: Mycoplasmas are cell wall-less bacteria which encode a minimal set of proteins. In Mycoplasma hominis, the genes encoding the surface-localized membrane complex P60\\/P80 are in an operon with a gene encoding a cytoplasmic, nucleotide-binding protein with a characteristic Histidine triad motif (HinT). HinT is found in both procaryotes and eukaryotes and known to hydrolyze adenosine nucleotides in eukaryotes. Immuno-precipitation

Miriam Hopfe; Ricarda Hoffmann; Birgit Henrich

2004-01-01

198

Biophysical Analysis of the Endoplasmic Reticulum-Resident Chaperone/Heat Shock Protein gp96/GRP94 and Its Complex with Peptide Antigen  

E-print Network

Biophysical Analysis of the Endoplasmic Reticulum-Resident Chaperone/Heat Shock Protein gp96/GRP94) of the endoplasmic reticulum (ER) and cytosol participate, in some cases, in the presenta- tion of peptide antigens develop specific protective immunity against cancers from which the HSPs were originally isolated

Chait, Brian T.

199

Pretreatment with antibody to eosinophil major basic protein prevents hyperresponsiveness by protecting neuronal M2 muscarinic receptors in antigen-challenged guinea pigs.  

PubMed

In antigen-challenged guinea pigs there is recruitment of eosinophils into the lungs and to airway nerves, decreased function of inhibitory M2 muscarinic autoreceptors on parasympathetic nerves in the lungs, and airway hyperresponsiveness. A rabbit antibody to guinea pig eosinophil major basic protein was used to determine whether M2 muscarinic receptor dysfunction, and the subsequent hyperresponsiveness, are due to antagonism of the M2 receptor by eosinophil major basic protein. Guinea pigs were sensitized, challenged with ovalbumin and hyperresponsiveness, and M2 receptor function tested 24 h later with the muscarinic agonist pilocarpine. Antigen-challenged guinea pigs were hyperresponsive to electrical stimulation of the vagus nerves compared with controls. Likewise, loss of M2 receptor function was demonstrated since the agonist pilocarpine inhibited vagally-induced bronchoconstriction in control but not challenged animals. Pretreatment with rabbit antibody to guinea pig eosinophil major basic protein prevented hyperresponsiveness, and protected M2 receptor function in the antigen-challenged animals without inhibiting eosinophil accumulation in the lungs or around the nerves. Thus, hyperresponsiveness is a result of inhibition of neuronal M2 muscarinic receptor function by eosinophil major basic protein in antigen-challenged guinea pigs. PMID:9410903

Evans, C M; Fryer, A D; Jacoby, D B; Gleich, G J; Costello, R W

1997-11-01

200

Chick myotendinous antigen. II. A novel extracellular glycoprotein complex consisting of large disulfide-linked subunits  

PubMed Central

This report describes the biochemical characterization of a novel extracellular matrix component, " myotendinous antigen," which appears early in chick limb morphogenesis at sites connecting developing muscle fibers, tendons, and bone ( Chiquet , M., and D. Fambrough , 1984; J. Cell Biol., 98:1926-1936). This extracellular matrix antigen is a major component of the secretory proteins released into the medium by fibroblast and muscle cultures; the soluble form is characterized here. This form of myotendinous antigen is a large glycoprotein complex consisting of several disulfide linked subunits (Mr approximately 150,000-240,000). The differently sized antigen subunits are related, since they yielded very similar proteolytic cleavage patterns. M1 antibody can bind to the denatured subunits. The antigen subunits, as well as a Mr approximately 80,000 pepsin-resistant antigenic domain derived from them, are resistant to bacterial collagenase. Despite possessing subunits similar in size to fibronectin, myotendinous antigen appears to be both structurally and antigenically unrelated to fibronectin or to other known extracellular matrix components. About seven times more M1 antigen per cell nucleus was released into the medium in fibroblast as compared to muscle cultures. In muscle conditioned medium, myotendinous antigen is noncovalently complexed to very high molecular weight material that could be heavily labeled by [3H]glucosamine and [35S]sulfate. This material is sensitive to chondroitinase ABC and hence appears to contain sulfated glycosaminoglycans. We speculate that myotendinous antigen might interact with proteoglycans on the surface of muscle fibers, thereby acting as a link to tendons. PMID:6202699

1984-01-01

201

Melanomas with concordant loss of multiple melanocytic differentiation proteins: immune escape that may be overcome by targeting unique or undefined antigens.  

PubMed

Melanoma-reactive HLA-A x 0201-restricted cytotoxic T lymphocyte (CTL) lines generated in vitro lyse autologous and HLA-matched allogeneic melanoma cells and recognize multiple shared peptide antigens from tyrosinase, MART-1, and Pme117/gp100. However, a subset of melanomas fail to be lysed by these T cells. In the present report, four different HLA-A x 0201+ melanoma cell lines not lysed by melanoma-reactive allogeneic CTL have been evaluated in detail. All four are deficient in expression of the melanocytic differentiation proteins (MDP) tyrosinase, Pme117/gp100, gp75/ trp-1, and MART-1/Melan-A. This concordant loss of multiple MDP explains their resistance to lysis by melanoma-reactive allogeneic CTL and confirms that a subset of melanomas may be resistant to tumor vaccines directed against multiple MDP-derived epitopes. All four melanoma lines expressed normal levels of HLAA x 0201, and all were susceptible to lysis by xenoreactive-peptide-dependent HLA-A x 0201-specific CTL clones, indicating that none had identifiable defects in antigen-processing pathways. Despite the lack of shared MDP-derived antigens, one of these MDP-negative melanomas, DM331, stimulated an effective autologous CTL response in vitro, which was restricted to autologous tumor reactivity. MHC-associated peptides isolated by immunoaffinity chromatography from HLA-A1 and HLA-A2 molecules of DM331 tumor cells included at least three peptide epitopes recognized by DM331 CTL and restricted by HLA-A1 or by HLA-A x 0201. Recognition of these CTL epitopes cannot be explained by defined, shared melanoma antigens; instead, unique or undefined antigens must be responsible for the autologous-cell-specific anti-melanoma response. These findings suggest that immunotherapy directed against shared melanoma antigens should be supplemented with immunotherapy directed against unique antigens or other undefined antigens, especially in patients whose tumors do not express MDP. PMID:10752474

Slingluff, C L; Colella, T A; Thompson, L; Graham, D D; Skipper, J C; Caldwell, J; Brinckerhoff, L; Kittlesen, D J; Deacon, D H; Oei, C; Harthun, N L; Huczko, E L; Hunt, D F; Darrow, T L; Engelhard, V H

2000-03-01

202

Expression and Purification of Dengue Virus Type 2 Envelope Protein as a Fusion with Hepatitis B Surface Antigen in Pichia pastoris  

Microsoft Academic Search

The methylotrophic yeast, Pichia pastoris, has been used as a host to express the envelope protein (Den2E) of dengue type 2 virus (NGC strain) as a chimera with hepatitis B surface antigen (HBsAg): a protein known to self assemble into virus-like particles (VLPs) and to be efficiently expressed in P. pastoris. The Den2E gene used in this study is a

Himani Bisht; Dipti A. Chugh; Sathyamangalam Swaminathan; Navin Khanna

2001-01-01

203

A single amino acid substitution at N-terminal region of coat protein of turnip mosaic virus alters antigenicity and aphid transmissibility  

Microsoft Academic Search

Summary The antigenic activity of the N-terminal region of coat protein of turnip mosaic virus (TuMV) aphid transmissible strain 1 and non-transmissible strain 31 was examined by using a panel of monoclonal antibodies (MAbs) raised against the two virus strains as well as antisera raised against several synthetic peptides from the N-terminal region of the protein. The reactivity of these

S. Kantrong; H. Saunal; J. P. Briand; N. Sako

1995-01-01

204

Polyarginine-Mediated Protein Delivery to Dendritic Cells Presents Antigen More Efficiently onto MHC Class I and Class II and Elicits Superior Antitumor Immunity  

Microsoft Academic Search

Protein transduction domains (PTDs) have been used increasingly to deliver reagents to a variety of cell types in vitro and in vivo. We have previously shown that HIV TAT-PTD-containing whole protein antigens (Ags)-transduced dendritic cells (DCs) stimulated Ag-specific CD8+ and CD4+ T cells. Although the cytotoxic T lymphocytes (CTL) activity generated was sufficient to prevent engraftment of mice with Ag-expressing

Hiroshi Mitsui; Takashi Inozume; Reiko Kitamura; Naotaka Shibagaki; Shinji Shimada

2006-01-01

205

Identification of classical swine fever virus protein E2 as a target for cytotoxic T cells by using mRNA-transfected antigen-presenting cells  

Microsoft Academic Search

Vaccination of pigs against Classical swine fever virus (CSFV) by using live-virus vaccines induces early protection before detectable humoral immune responses. Immunological analyses indicate that this is associated with T-cell activation, underlining the importance of targeting cytotoxic T-lymphocyte (CTL) responses for vaccine improvement. Antigen-presenting cells (APCs) transfected with mRNA encoding structural protein E2 or non-structural viral proteins NS3¿NS4A were used

M. Ceppi; M. G. M. de Bruin; T. Seuberlich; C. Balmelli; S. Pascolo; J. D. Tratschin; N. Ruggli; D. Wienhold; A. Summerfield

2005-01-01

206

Early Diagnosis of Scrub Typhus with a Rapid Flow Assay Using Recombinant Major Outer Membrane Protein Antigen (r56) of Orientia tsutsugamushi  

Microsoft Academic Search

The variable 56-kDa major outer membrane protein of Orientia tsutsugamushi is the immunodominant an- tigen in human scrub typhus infections. We developed a rapid immunochromatographic flow assay (RFA) for the detection of immunoglobulin M (IgM) and IgG antibodies to O. tsutsugamushi. The RFA employs a trun- cated recombinant 56-kDa protein from the Karp strain as the antigen. The performance of

W.-M. Ching; D. Rowland; Z. Zhang; A. L. Bourgeois; D. Kelly; G. A. Dasch; P. L. Devine

2001-01-01

207

Targeting hepatitis B virus antigens to dendritic cells by heat shock protein to improve DNA vaccine potency  

PubMed Central

AIM: To investigate a novel DNA vaccination based upon expression of the HBV e antigen fused to a heat shock protein (HSP) as a strategy to enhance DNA vaccine potency. METHODS: A pCMV-HBeAg-HSP DNA vaccine and a control DNA vaccine were generated. Mice were immunized with these different construct. Immune responses were measured 2 wk after a second immunization by a T cell response assay, CTL cytotoxicity assay, and an antibody assay in C57BL/6 and BALB/c mice. CT26-HBeAg tumor cell challenge test in vivo was performed in BALB/c mice to monitor anti-tumor immune responses. RESULTS: In the mice immunized with pCMV-HBe-HSP DNA, superior CTL activity to target HBV-positive target cells was observed in comparison with mice immunized with pCMV-HBeAg (44% ± 5% vs 30% ± 6% in E: T > 50:1, P < 0.05). ELISPOT assays showed a stronger T-cell response from mice immunized with pCMV-HBe-HSP than that from pCMV-HBeAg immunized animals when stimulated either with MHC classIor class II epitopes derived from HBeAg (74% ± 9% vs 31% ± 6%, P < 0.01). ELISA assays revealed an enhanced HBeAg antibody response from mice immunized with pCMV-HBe-HSP than from those immunized with pCMV-HBeAg. The lowest tumor incidence and the slowest tumor growth were observed in mice immunized with pCMV-HBe-HSP when challenged with CT26-HBeAg. CONCLUSION: The results of this study demonstrate a broad enhancement of antigen-specific CD4+ helper, CD8+ cytotoxic T-cell, and B-cell responses by a novel DNA vaccination strategy. They also proved a stronger antigen-specific immune memory, which may be superior to currently described HBV DNA vaccination strategies for the treatment of chronic HBV infection. PMID:17990356

Gu, Qin-Long; Huang, Xue; Ren, Wen-Hong; Shen, Lei; Liu, Bing-Ya; Chen, Si-Yi

2007-01-01

208

Protein-peptide affinity determination using an H/D exchange dilution strategy: Application to antigen-antibody interactions  

PubMed Central

A new methodology using hydrogen/deuterium amide exchange (HDX) to determine the binding affinity of protein-peptide interactions is reported. The method, based on our previously established approach, PLIMSTEX (Protein Ligand Interaction by Mass Spectrometry, Titration and H/D Exchange) [J. Am. Chem. Soc. 2003, 125, 5252–5253], makes use of a dilution strategy (dPLIMSTEX) for HDX, using the mass of the peptide ligand as readout. We employed dPLIMSTEX to study the interaction of calcium-saturated calmodulin with the opioid peptide ?-endorphin as a model system; the affinity results are in good agreement with those from traditional PLIMSTEX and with literature values obtained by using other methods. We show that the dPLIMSTEX method is feasible to quantify an antigen-antibody interaction involving a 3-nitrotyrosine modified peptide in complex with a monoclonal anti-nitrotyrosine antibody. A dissociation constant in the low nanomolar range, was determined, and a binding stoichiometry of antibody/peptide of 1:2 was confirmed. In addition, we determined that the epitope in the binding interface contains a minimum of five amino acids. The dPLIMSTEX approach is a sensitive and powerful tool for the quantitative determination of peptide affinities with antibodies, complementary to conventional immuno-analytical techniques. PMID:20444623

Tu, Tingting; Dragusanu, Mihaela; Petre, Brindusa-Alina; Rempel, Don L.; Przybylski, Michael; Gross, Michael L.

2010-01-01

209

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

210

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

211

Phosphoregulation of the RNA-binding Protein Hu Antigen R (HuR) by Cdk5 Affects Centrosome Function*  

PubMed Central

Hu antigen R (HuR) is an mRNA-binding protein belonging to the ELAV family. It is highly expressed in cancer and involved in cell survival and proliferation. The impact of post-translational regulation of HuR and resulting cellular effects are poorly understood. In the current report, we describe a direct interaction between HuR and Cdk5 in glioma. We determined that Cdk5 specifically phosphorylates HuR at the serine 202 residue in the unique hinge region. The molecular consequences of this interaction are an altered HuR ability to bind, stabilize, and promote translation of mRNAs. At the cellular level, the anomalous HuR phosphorylation at this site evokes robust defects in centrosome duplication and cohesion as well as arrest of cell cycle progression. Subcellular fractionation and immunofluorescence technique confirm a direct integration of HuR and Cdk5 with centrosomes. We propose that HuR stores mRNA in the centrosome and that HuR phosphorylation by Cdk5 controls de novo protein synthesis in near proximity to centrosomes and, thus, impacts centrosome function. PMID:22829587

Filippova, Natalia; Yang, Xiuhua; King, Peter; Nabors, L. Burt

2012-01-01

212

The association of Duffy binding protein region II polymorphisms and its antigenicity in Plasmodium vivax isolates from Thailand.  

PubMed

Plasmodium vivax Duffy binding protein II (DBPII) plays an important role in reticulocyte invasion and is a potential vaccine candidate against vivax malaria. However, polymorphisms in DBPII are a challenge for the successful design of a broadly protective vaccine. In this study, the genetic diversity of DBPII among Thai isolates was analyzed from Plasmodium vivax-infected blood samples and polymorphism characters were defined with the MEGA4 program. Sequence analysis identified 12 variant residues that are common among Thai DBPII haplotypes with variant residues L333F, L424I, W437R and I503K having the highest frequency. Variant residue D384K occurs in combination with either E385K or K386N/Q. Additionally, variant residue L424I occurs in conjunction with W437R in most Thai DBPII alleles and these variants frequently occur in combination with the I503K variant. The polymorphic patterns of Thai isolates were defined into 9 haplotypes (Thai DBL-1, -2, -3, etc.…). Thai DBL-2, -5, -6 haplotypes are the most common DBPII variants in Thai residents. To study the association of these Thai DBPII polymorphisms with antigenic character, the functional inhibition of anti-DBPII monoclonal antibodies against a panel of Thai DBL variants was characterized by an in vitro erythrocyte binding inhibition assay. The functional inhibition of anti-DBPII monoclonal antibodies 3C9, 2D10 and 2C6 against Thai variants was significantly different, suggesting that polymorphisms of Thai DBPII variants alter the antigenic character of the target epitopes. In contrast, anti-DBPII monoclonal antibody 2H2 inhibited all Thai DBPII variants equally well. Our results suggest that the immune efficacy of a DBPII vaccine will depend on the specificity of the anti-DBPII antibodies induced and that it is preferable to optimize responses to conserved epitopes for broadly neutralizing protection against P. vivax. PMID:25108177

Chootong, Patchanee; McHenry, Amy M; Ntumngia, Francis B; Sattabongkot, Jetsumon; Adams, John H

2014-12-01

213

Antigenic Mapping of the Recombinant Norwalk Virus Capsid Protein Using Monoclonal Antibodies  

Microsoft Academic Search

Norwalk virus (NV) is the prototype strain of a group of noncultivatable caliciviruses that infect humans and cause outbreaks of epidemic acute nonbacterial gastroenteritis. The NV virion is composed of 180 copies of a single structural protein that, when expressed in insect cells infected with a recombinant baculovirus, assembles into empty recombinant Norwalk virus-like particles (rNV VLPs) which are morphologically

MICHELE E. HARDY; TOMOYUKI N. TANAKA; NORITOSHI KITAMOTO; LAURA J. WHITE; JUDITH M. BALL; XI JIANG; MARY K. ESTES

1996-01-01

214

Existence of uncoupling protein-2 antigen in isolated mitochondria from various tissues  

E-print Network

-3 (UCP3) were raised by operating the blotted proteins into the spleen of minipigs. The antisera,4,15]. The ex- istence of UCP2 mRNA in spleen, macrophages, thymus, bone marrow [1] and Kupfer cells [16] has

Garlid, Keith

215

Antigenicity of Fusion Proteins from Sarcoma-associated Chromosomal Translocations1  

Microsoft Academic Search

Synovial sarcoma (SS), clear cell sarcoma (CCS), and desmoplastic small round cell tumor (DSRCT) are soft-tissue malignancies occurring primarily in adolescents and young adults. These tumors contain specific chromosomal translocations that fuse the 5 region of one gene with the 3 region of another, resulting in the formation of characteristic fusion proteins. These translocations are unique to tumor cells and

B. Scott Worley; Leon T. van den Broeke; Theresa J. Goletz; C. David Pendleton; Emily M. Daschbach; Elaine K. Thomas; Francesco M. Marincola; Lee J. Helman; Jay A. Berzofsky

2001-01-01

216

Preparation and diagnostic utility of a hemagglutination inhibition test antigen derived from the baculovirus-expressed hemagglutinin-neuraminidase protein gene of Newcastle disease virus.  

PubMed

A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 2(13) per 25 ?L, which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4°C. The rHN-based HI assay specifically detected NDV antibodies, but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%, respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays. PMID:23820164

Choi, Kang-Seuk; Kye, Soo-Jeong; Jeon, Woo-Jin; Park, Mi-Ja; Kim, Saeromi; Seul, Hee-Jung; Kwon, Jun-Hun

2013-01-01

217

Preparation and diagnostic utility of a hemagglutination inhibition test antigen derived from the baculovirus-expressed hemagglutinin-neuraminidase protein gene of Newcastle disease virus  

PubMed Central

A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 213 per 25 µL, which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4?. The rHN-based HI assay specifically detected NDV antibodies, but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%, respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays. PMID:23820164

Kye, Soo-Jeong; Jeon, Woo-Jin; Park, Mi-Ja; Kim, Saeromi; Seul, Hee-Jung; Kwon, Jun-Hun

2013-01-01

218

Disruption of the association of 73 kDa heat shock cognate protein with transporters associated with antigen processing (TAP) decreases TAP-dependent translocation of antigenic peptides into the endoplasmic reticulum.  

PubMed

Major histocompatibility complex class I-bound antigenic peptides generated in the cytosol are translocated into the ER by TAP. In the present study, the physical association of HSC73 with TAP in human lymphoblastoid T1 cells was demonstrated. The dissociation was induced in the presence of 10 mM ATP, indicating that the ADP-binding form of HSC73 might be associated with TAP. We found that HSC73-binding immunosuppressant, MeDSG disrupted the HSC73-TAP association, whereas it did not affect the binding of HSC73 to a substrate protein. MHC class I expression on the cell surface was also downregulated. Then, the effect of MeDSG on the TAP-mediated ER translocation was examined using two homologous model peptides, NGT-Bw4 and NGT-Bw6, which had distinct binding affinity to HSC73. Although high-affinity peptide NGT-Bw4 was translocated by TAP, low-affinity peptide NGT-Bw6 was not. The TAP-dependent translocation of NGT-Bw4 was abolished in the presence of MeDSG. Decreased presentation on the cell surface was shown for the human leukocyte antigen (HLA)-A31-restricted natural antigenic peptide F4.2, which had high affinity to HSC73, in the presence of MeDSG. It was indicated that disruption of the HSC73-TAP association resulted in inhibition of TAP-dependent translocation of HSC73-bound peptides. Our findings highlighted an important role of HSC73 for feeding antigenic peptides to TAP, and suggested a possibility that a synthetic polyamine might inhibit the function of HSC73, thereby suppressing MHC class I-restricted presentation of HSC73-bound antigenic peptides. PMID:18380807

Kamiguchi, Kenjiro; Torigoe, Toshihiko; Fujiwara, Osamutaro; Ohshima, Shin; Hirohashi, Yoshihiko; Sahara, Hiroeki; Hirai, Itaru; Kohgo, Yutaka; Sato, Noriyuki

2008-02-01

219

Sequence and Antigenic Variability of the Helicobacter mustelae Surface Ring Protein Hsr  

PubMed Central

We have identified an array of more than 500 repetitive sequences flanking the hsr gene, which encodes the major surface protein of the ferret pathogen Helicobacter mustelae. The repeats show identity exclusively to the amino-terminal half of Hsr. Analysis of Hsr from three strains indicated variability of exposed epitopes. Characterization of an hsr mutant showed that Hsr is not an adhesin. PMID:11292773

Forester, Natasha; Lumsden, John S.; O'Croinin, Tadhg; O'Toole, Paul W.

2001-01-01

220

Piloting of exogenous antigen into cross-presentation pathway by heat shock proteins  

Microsoft Academic Search

Recent evidences have been indicating that heat shock proteins (HSPs) play an important role as a “danger signal” in the extracellular\\u000a milieu on behalf of immune surveillance. Above all, Hsp70, gp96 and Hsp90 have been shown to elicit intriguing efficient CTL\\u000a responses by so called “cross-presentation” with yet entirely unknown mechanism. Here, we discuss that the immunologic roles\\u000a of HSPs,

Yasuaki Tamura; Goro Kutomi; Jun Oura; Toshihiko Torigoe; Noriyuki Sato

221

Cell-free production of trimeric influenza hemagglutinin head domain proteins as vaccine antigens.  

PubMed

In order to effectively combat pandemic influenza threats, there is a need for more rapid and robust vaccine production methods. In this article, we demonstrate E. coli-based cell-free protein synthesis (CFPS) as a method to rapidly produce domains from the protein hemagglutinin (HA), which is present on the surface of the influenza virus. The portion of the HA coding sequence for the "head" domain from the 2009 pandemic H1N1 strain was first optimized for E. coli expression. The protein domain was then produced in CFPS reactions and purified in soluble form first as a monomer and then as a trimer by a C-terminal addition of the T4 bacteriophage foldon domain. Production of soluble trimeric HA head domain was enhanced by introducing stabilizing amino acid mutations to the construct in order to avoid aggregation. Trimerization was verified using size exclusion HPLC, and the stabilized HA head domain trimer was more effectively recognized by antibodies from pandemic H1N1 influenza vaccine recipients than was the monomer and also bound to sialic acids more strongly, indicating that the trimers are correctly formed and could be potentially effective as vaccines. PMID:22729608

Welsh, John P; Lu, Yuan; He, Xiao-Song; Greenberg, Harry B; Swartz, James R

2012-12-01

222

Aggregation of recombinant human botulinum protein antigen serotype C in varying solution conditions: implications of conformational stability for aggregation kinetics.  

PubMed

Solution conditions greatly affect the aggregation rate of a protein. Elucidating these influences provides insight into the critical factors governing aggregation. In this study, recombinant human botulinum protein antigen serotype C [rBoNTC (H(c))] was employed as a model protein. rBoNTC (H(c)) aggregated irreversibly during incubation at 42°C. The aggregation rate was studied as a function of solution conditions, including varying the pH from 3.5 to 8.0 and with or without 150 mM NaCl, 7.5% (w/v) trehalose, and 0.5 M urea. Some solution conditions retarded rBoNTC (H(c)) aggregation, whereas others accelerated aggregation, particularly acidic pH and addition of NaCl or urea. To better understand the mechanism by which these solution conditions influenced aggregation rates, the structure of rBoNTC (H(c)) was characterized using circular dichroism, fluorescence, and ultraviolet absorbance spectroscopies. Conformational stability was assessed from equilibrium urea-induced unfolding studies and by using differential scanning calorimetry (DSC). The activation energy of the aggregation reaction (E(a)) was estimated from an analysis of the heating-rate dependence of the thermal transition observed during DSC heating scans. Overall, for rBoNTC (H(c)), an inverse correlation was found between conformational stability and aggregation rate, as well as between the kinetic barrier to unfolding (i.e., E(a)) and aggregation rate. PMID:20862672

Bai, Shujun; Manning, Mark Cornell; Randolph, Theodore W; Carpenter, John F

2011-03-01

223

Task unrelated thought whilst encoding information  

Microsoft Academic Search

Task unrelated thought (TUT) refers to thought directed away from the current situation, for example a daydream. Three experiments were conducted on healthy participants, with two broad aims. First, to contrast distributed and encapsulated views of cognition by comparing the encoding of categorical and random lists of words (Experiments One and Two). Second, to examine the consequences of experiencing TUT

Jonathan M. Smallwood; Simona F. Baracaia; Michelle Lowe; Marc Obonsawinb

2003-01-01

224

Anthrax Lethal Toxin Impairs CD1d-Mediated Antigen Presentation by Targeting the Extracellular Signal-Related Kinase 1/2 Mitogen-Activated Protein Kinase Pathway?  

PubMed Central

Lethal toxin (LT) is a critical virulence factor of Bacillus anthracis and an important means by which this bacterium evades the host's immune system. In this study, we demonstrate that CD1d-expressing cells treated with LT have reduced CD1d-mediated antigen presentation. We earlier showed an important role for the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 (ERK1/2) in the regulation of CD1d-mediated antigen presentation, and we report here that LT impairs antigen presentation by CD1d in an ERK1/2-dependent manner. Similarly, LT and the ERK1/2 pathway-specific inhibitor U0126 caused a decrease in major histocompatibility complex (MHC) class II-mediated antigen presentation. Confocal microscopy analyses revealed altered intracellular distribution of CD1d and LAMP-1 in LT-treated cells, similar to the case for ERK1/2-inhibited cells. These results suggest that Bacillus anthracis has the ability to evade the host's innate immune system by reducing CD1d-mediated antigen presentation through targeting the ERK1/2 pathway. PMID:20194602

Khan, Masood A.; Gallo, Richard M.; Brutkiewicz, Randy R.

2010-01-01

225

Conformation study of HA(306-318) antigenic peptide of the haemagglutinin influenza virus protein  

NASA Astrophysics Data System (ADS)

Several HLA-DR alleles present the immunodominant HA(306-318) peptide of haemagglutinin of the influenza virus to T cells. NMR data of the peptide in various water solutions exclude any ?-helix or turn conformations. Circular dichroism and Fourier transform infrared spectroscopies indicate an estimated ?-extended structure in water of 31% and 28%, respectively, with spectra shape similar to the ones observed for ?-sheet containing proteins. The H/D amide exchange suggests a stable length-dependent interchain hydrogen-bonding. The partially ?-extended conformation of HA(306-318) in solution might be close to the one found in HA(306-318)-HLA-DR1 complex. These results suggest different interconverting extended conformations of HA(306-318), depending on the microenvironment of the solution medium. This flexibility emphasizes the ability of some peptides to fit more easily the binding site of several HLA-DR molecules. Similar results were obtained on the HIV P25(263-277) peptide which has been previously shown to be a good DR1 binder. From a vibrational point of view, infrared Amide I frequencies of secondary structures in peptides were ascertained. As previously demonstrated for proteins in solution, Fourier transform infrared and circular dichroism spectroscopies appear to be valuable tools for conformational properties of peptides. Their use may contribute to the detection of peptide conformation-binding relationship which has to be further tested by biochemical and biological studies.

Bertrand, A.; Brito, R. M.; Alix, A. J. P.; Lancelin, J. M.; Carvalho, R. A.; Geraldes, C. F. G. C.; Lakhdar-Ghazal, F.

2006-11-01

226

Tumor-produced secreted form of binding of immunoglobulin protein elicits antigen-specific tumor immunity.  

PubMed

Binding of immunoglobulin protein (BiP) is a major molecular chaperone localized in endoplasmic reticulum (ER). It has been demonstrated to interact with nascent Ig. However, contrary to other ER-resident heat shock proteins such as gp96, calreticulin, and ORP150, it is not clear whether tumor-derived BiP plays a role in inducing antitumor immunity. In this study, we show that the tumor-derived secreted form of BiP is capable of inducing antitumor CD8(+) T cell responses. We constructed an ER-retention signal KDEL-deleted mutant of BiP cDNA and transfected it to tumor cells, which resulted in continuous secretion of tumor-derived BiP into the extracellular milieu. We show that this secreted BiP is taken up by bone marrow-derived dendritic cells, and thereafter BiP-associated Ag peptide is cross-presented in association with MHC class I molecules, resulting in elicitation of an Ag-specific CD8(+) T cell response and antitumor effect. This strategy to boost antitumor immune responses shows that a tumor could be its own cellular vaccine via gene modification of the secretion of the tumor Ag-BiP complex. PMID:21339366

Tamura, Yasuaki; Hirohashi, Yoshihiko; Kutomi, Goro; Nakanishi, Katsuya; Kamiguchi, Kenjirou; Torigoe, Toshihiko; Sato, Noriyuki

2011-04-01

227

Development of a nucleic acid lateral flow strip for detection of hepatitis C virus (HCV) core antigen.  

PubMed

The object of this study was to develop a simple, rapid, specific, and highly sensitive method to detect HCV core antigen. A nucleic acid aptamer was designed with the high specificity and sensitivity in a nucleic acid lateral flow strip to compete with HCV core antigen and DNA probes. The lower detection limit of the test strip was calculated to be 10 pg/mL with the scanner and 100 pg/mL with naked eyes. Results showed that there were no cross-interactions with other proteins such as HCV NS3, E1/E2 antigens, HIV p24 antigens, or BSA proteins (HCV unrelated protein). When the viral load exceeded 10(4) copies/mL, the positive coincidence rates of ELISA and strip detection, when compared with the HCV RNA assay, were 98.44% and 97.28%, respectively. The results indicated that the ELISA detection and strip assay were in good agreement with the measured value. The results indicated that a nucleic acid lateral flow strip was a simple, rapid, specific, highly sensitive, and cost-effective field-based method for detecting HCV core antigen. The strip assay is an acceptable alternative to diagnose HCV core antigen and to investigate its epidemiology in clinical laboratories lacking specialized equipment and skills. PMID:23448141

Wang, Chunfeng; Zhang, Lianfeng; Shen, Xuanmei

2013-01-01

228

Cross-priming of minor histocompatibility antigen-specific cytotoxic T cells upon immunization with the heat shock protein gp96  

PubMed Central

Vaccination of mice with heat shock proteins isolated from tumor cells induces immunity to subsequent challenge with those tumor cells the heat shock protein was isolated from but not with other tumor cells (Udono, H., and P.K. Srivastava. 1994. J. Immunol. 152:5398-5403). The specificity of this immune response is caused by tumor-derived peptides bound to the heat shock proteins (Udono., H., and P.K. Srivastava. 1993. J. Exp. Med. 178:1391-1396). Our experiments show that a single immunization with the heat shock protein gp96 isolated from beta- galactosidase (beta-gal) expressing P815 cells (of DBA/2 origin) induces cytotoxic T lymphocytes (CTLs) specific for beta-gal, in addition to minor H antigens expressed by these cells. CTLs can be induced in mice that are major histocompatibility complex (MHC) identical to the gp96 donor cells (H-2d) as well as in mice with a different MHC (H-2b). Thus gp96 is able to induce "cross priming" (Matzinger, P., and M.J. Bevan. 1977. Cell. Immunol. 33:92-100), indicating that gp96-associated peptides are not limited to the MHC class I ligands of the gp96 donor cell. Our data confirm the notion that samples of all cellular antigens presentable by MHC class I molecules are represented by peptides associated with gp96 molecules of that cell, even if the fitting MHC molecule is not expressed. In addition, we extend previous reports on the in vivo immunogenicity of peptides associated gp96 molecules to two new groups of antigens, minor H antigens, and proteins expressed in the cytosol. PMID:7650492

1995-01-01

229

High dose antigen treatment with a peptide epitope of myelin basic protein modulates T cells in multiple sclerosis patients.  

PubMed

One of the auto-antigens aberrantly targeted in Multiple sclerosis is myelin basic protein (MBP). In this study, chronic progressive multiple sclerosis (CPMS) patients receiving the experimental drug MBP8298, on a compassionate care trial, were examined before and after high dose peptide treatment for their circulating regulatory T-cell numbers and their responses to the common mitogens, phytohemagglutinin and poke-weed mitogen. Peripheral blood mononuclear cells (PBMCs) isolated from these patients before treatment displayed anergy upon stimulation with phytohemagglutinin; measured through reduced proliferation, IFN-? and IL-17A secretion in an in vitro cell culture system. 6 Weeks and 6months after treatment their PBMCs displayed a reversal of anergy with phytohemagglutinin stimulation. There was also a marked increase in their CD4(+)CD25(+hi)FoxP3(+) T-cells regulatory T-cells. These results suggest that high dose MBP8298 treatment has a profound effect on the circulating T-cells of CPMS patients, capable of reversing peripheral anergy and establishing T regulation. PMID:23246830

Loo, Eric W; Krantz, Mark J; Agrawal, Babita

2012-11-01

230

[Stress-induced changes of hypothalamic structure cell responses to antigen injection (LPS) (revealed by c-Fos protein expression)].  

PubMed

Stress stimuli are known to influence the intensity if immune response. To elucidate the role of central regulating structures in this changes, analysis of activation level of hypothalamic neurons (revealed by quantity of c-Fos-positive cells) was carried out in rats within 2 hours after intravenous LPS injection and after this--impact associated with electric pain stimulation (EPS). The investigation was carried out in 52 male Wistar rats, 200-250 g. The c-Fos protein expression was analyzed with immunohistochemical method. The increase of c-Fos-positive cells number in 2 hours after LPS injection was observed in AFTN, PVH, LHA, VMH, DMH and PH. After electrical pain stimulation, the quantity of c-Fos-positive cells increased in the same structures. Combined application of electric pain stimulation and LPS injection results in diminished activation level in AHN, PVH, LHA and VMH as compared with typical response to single LPS injection without EPS. The EPS suppresses intensity of the immune response induced by injection of LPS (revealed by local hemolysis method with calculation of antibody-forming cells quantity (%) in the rat spleen). Thus the activation level changes of hypothalamic structures (AHN, PVH, LHA, PH) correlate with development of stress-induced immunosuppression, i. e. morphofunctional description of hypothalamic structures activation as revealed by pattern of activated cell alterations in hypothalamic structures during realization of stress-induced changes of immune system responses to antigen injection. PMID:17385422

Gavrilov, Iu V; Perekrest, S V; Novikova, N S; Korneva, E A

2006-11-01

231

Characterization of Neisseria meningitidis Isolates That Do Not Express the Virulence Factor and Vaccine Antigen Factor H Binding Protein ? †  

PubMed Central

Neisseria meningitidis remains a leading cause of bacterial sepsis and meningitis. Complement is a key component of natural immunity against this important human pathogen, which has evolved multiple mechanisms to evade complement-mediated lysis. One approach adopted by the meningococcus is to recruit a human negative regulator of the complement system, factor H (fH), to its surface via a lipoprotein, factor H binding protein (fHbp). Additionally, fHbp is a key antigen in vaccines currently being evaluated in clinical trials. Here we characterize strains of N. meningitidis from several distinct clonal complexes which do not express fHbp; all strains were recovered from patients with disseminated meningococcal disease. We demonstrate that these strains have either a frameshift mutation in the fHbp open reading frame or have entirely lost fHbp and some flanking sequences. No fH binding was detected to other ligands among the fHbp-negative strains. The implications of these findings for meningococcal pathogenesis and prevention are discussed. PMID:21508163

Lucidarme, Jay; Tan, Lionel; Exley, Rachel M.; Findlow, Jamie; Borrow, Ray; Tang, Christoph M.

2011-01-01

232

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

233

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

PubMed

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

Sreejit, Gopalkrishna; Ahmed, Asma; Parveen, Nazia; Jha, Vishwanath; Valluri, Vijaya Lakshmi; Ghosh, Sudip; Mukhopadhyay, Sangita

2014-10-01

234

Glyceraldehyde-3-phosphate dehydrogenase, an immunogenic Streptococcus equi ssp. zooepidemicus adhesion protein and protective antigen.  

PubMed

Streptococcus equi ssp. zooepidemicus (Streptococcus zooepidemicus, SEZ) is an important pathogen associated with opportunistic infections of a wide range of species, including pigs and humans. The absence of a suitable vaccine makes it difficult to control SEZ infection. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been previously identified as an immunogenic protein using immunoproteomic techniques. In the present study, we confirmed that the sequence of GAPDH was highly conserved with other Streptococcus spp. The purified recombinant GAPDH could elicit a significant humoral antibody response in mice and confer significant protection against challenge with a lethal dose of SEZ. GAPDH could adhere to the Hep-2 cells, confirmed by flow cytometry, and inhibit adherence of SEZ to Hep-2 cells in an adherence inhibition assay. In addition, real-time PCR demonstrated that GAPDH was induced in vivo following infection of mice with SEZ. These suggest that GAPDH could play an important role in the pathogenesis of SEZ infection and could be a target for vaccination against SEZ. PMID:23568215

Fu, Qiang; Wei, Zigong; Liu, Xiaohong; Xiao, Pingping; Lu, Zhaohui; Chen, Yaosheng

2013-04-01

235

Intranasal Immunization with the Cholera Toxin B Subunit-Pneumococcal Surface Antigen A Fusion Protein Induces Protection against Colonization with Streptococcus pneumoniae and Has Negligible Impact on the Nasopharyngeal and Oral Microbiota of Mice  

Microsoft Academic Search

One of the candidate proteins for a mucosal vaccine antigen against Streptococcus pneumoniae is PsaA (pneumococcal surface antigen A). Vaccines targeting mucosal immunity may raise concerns as to possible alterations in the normal microbiota, especially in the case of PsaA, which was shown to have homologs with elevated sequence identity in other viridans group streptococci. In this work, we demonstrate

F. C. Pimenta; E. N. Miyaji; A. P. M. Areas; M. L. S. Oliveira; A. L. S. S. de Andrade; P. L. Ho; S. K. Hollingshead; L. C. C. Leite

2006-01-01

236

Application of encoded library technology (ELT) to a protein-protein interaction target: discovery of a potent class of integrin lymphocyte function-associated antigen 1 (LFA-1) antagonists.  

PubMed

The inhibition of protein-protein interactions remains a challenge for traditional small molecule drug discovery. Here we describe the use of DNA-encoded library technology for the discovery of small molecules that are potent inhibitors of the interaction between lymphocyte function-associated antigen 1 and its ligand intercellular adhesion molecule 1. A DNA-encoded library with a potential complexity of 4.1 billion compounds was exposed to the I-domain of the target protein and the bound ligands were affinity selected, yielding an enriched small-molecule hit family. Compounds representing this family were synthesized without their DNA encoding moiety and found to inhibit the lymphocyte function-associated antigen 1/intercellular adhesion molecule-1 interaction with submicromolar potency in both ELISA and cell adhesion assays. Re-synthesized compounds conjugated to DNA or a fluorophore were demonstrated to bind to cells expressing the target protein. PMID:24593905

Kollmann, Christopher S; Bai, Xiaopeng; Tsai, Ching-Hsuan; Yang, Hongfang; Lind, Kenneth E; Skinner, Steven R; Zhu, Zhengrong; Israel, David I; Cuozzo, John W; Morgan, Barry A; Yuki, Koichi; Xie, Can; Springer, Timothy A; Shimaoka, Motomu; Evindar, Ghotas

2014-04-01

237

Comparisons of voided urine cytology, nuclear matrix protein-22 and bladder tumor associated antigen tests for bladder cancer of geriatric male patients in Taiwan, China  

Microsoft Academic Search

Aim:To compare the results of bladder tumor associated antigen (BTA TRAK), nuclear matrix protein 22 (NMP 22) and voided urine cytology (VUC) in detecting bladder cancer.Methods:A total of 135 elderly male and 50 healthy volunteers enrolled in this study were classified into three groups: (i) 93 patients with bladder cancer; (ii) 42 patients with urinary benign conditions; and (iii) 50

Ke-Hung Tsui; Shao-Ming Chen; Ta-Ming Wang; Horng-Heng Juang; Chien-Lun Chen; Guang-Huan Sun; Phei-Lang Chang

2007-01-01

238

Transformation of Human T-Cell Clones by Herpesvirus Saimiri: Intact Antigen Recognition by Autonomously Growing Myelin Basic Protein-Specific T Cells  

Microsoft Academic Search

Herpesvirus saimiri has recently been shown to immortalize human T cells. It was unknown, however, whether Herpesvirus saimiri transformation affects T-cell receptor (TCR) expression and signal transduction. In the present study, we have transformed CD4^+ human T-cell clones specific for human myelin basic protein. The transformed T cells were grown in interleukin 2 and divided in the absence of antigen

Frank Weber; Edgar Meinl; Klaus Drexler; Anna Czlonkowska; Susanne Huber; Helmut Fickenscher; Ingrid Muller-Fleckenstein; Berhard Fleckenstein; Hartmut Wekerle; Reinhard Hohlfeld

1993-01-01

239

The preparation of protein A-gold complexes with 3 nm and 15 nm gold particles and their use in labelling multiple antigens on ultra-thin sections  

Microsoft Academic Search

Summary  The preparation of a protein A-gold complex (pAg3) using 3 nm gold particles and its application for labelling of intracellular antigens on thin sections is reported. The 3 nm gold particle is the smallest metal particle currently available for cytochemistry and permits a higher resolution of the pAg technique. Furthermore, it can be used in double labelling experiments in conjunction

Jurgen Roth

1982-01-01

240

Inhibition of Ubiquitin\\/Proteasome-Dependent Protein Degradation by the Gly-Ala Repeat Domain of the Epstein-Barr Virus Nuclear Antigen 1  

Microsoft Academic Search

The Epstein-Barr virus (EBV) encoded nuclear antigen (EBNA) 1 is expressed in latently infected B lymphocytes that persist for life in healthy virus carriers and is the only viral protein regularly detected in all EBV associated malignancies. The Gly-Ala repeat domain of EBNA1 was shown to inhibit in cis the presentation of major histocompatibility complex (MHC) class I restricted cytotoxic

Jelena Levitskaya; Anatoly Sharipo; Ainars Leonchiks; Aaron Ciechanover; Maria G. Masucci

1997-01-01

241

Evaluation of a Multiplex PCR-Based Reverse Line Blot-Hybridization Assay for Identification of Serotype and Surface Protein Antigens of Streptococcus agalactiae  

Microsoft Academic Search

Recently, we developed separate multiplex PCR-based re- verse line blot-hybridization (mPCR\\/RLB) assays to identify molecular serotypes (MS) (2) and protein antigen gene profiles (PGPs) (9) of Streptococcus agalactiae (group B streptococcus (GBS)). The aim of this study was to develop an assay that would identify MS and PGPs simultaneously and could poten- tially replace the use of antisera for typing.

Xianyu Zeng; Fanrong Kong; Julie Morgan; Gwendolyn L. Gilbert

2006-01-01

242

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

Microsoft Academic Search

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\\u000a in tissues of experimentally infected goats. The goats were experimentally infected with a virulent PPR virus, which was previously\\u000a isolated from a severe natural disease outbreak in gazelles, during 2002 in

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

2009-01-01

243

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

244

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

245

Biophysical and formulation studies of the Schistosoma mansoni TSP-2 extracellular domain recombinant protein, a lead vaccine candidate antigen for intestinal schistosomiasis  

PubMed Central

A candidate vaccine to prevent human schistosomiasis is under development. The vaccine is comprised of a recombinant 9 kDa antigen protein corresponding to the large extracellular domain of a tetraspanin surface antigen protein of Schistosoma mansoni, Sm-TSP-2. Here, we describe the biophysical profile of the purified, recombinant Sm-TSP-2 produced in the yeast PichiaPink™, which in preclinical studies in mice was shown to be an effective vaccine against intestinal schistosomiasis. Biophysical techniques including circular dichroism, intrinsic and extrinsic fluorescence and light scattering were employed to generate an empirical phase diagram, a color based map of the physical stability of the vaccine antigen over a wide range of temperatures and pH. From these studies a pH range of 6.0–8.0 was determined to be optimal for maintaining the stability and conformation of the protein at temperatures up to 25 °C. Sorbitol, sucrose and trehalose were selected as excipients that prevented physical degradation during storage. The studies described here provide guidance for maximizing the stability of soluble recombinant Sm-TSP-2 in preparation of its further development as a vaccine. PMID:23880663

Cheng, Weiqiang; Curti, Elena; Rezende, Wanderson C; Kwityn, Clifford; Zhan, Bin; Gillespie, Portia; Plieskatt, Jordan; Joshi, Sangeeta B.; Volkin, David B.; Hotez, Peter J; Middaugh, C Russell; Bottazzi, Maria Elena

2013-01-01

246

Proteomics integrated with Escherichia coli vector-based vaccines and antigen microarrays reveals the immunogenicity of a surface sialidase-like protein of Propionibacterium acnes  

PubMed Central

Proteomics is a powerful tool for the identification of proteins, which provides a basis for rational vaccine design. However, it is still a highly technical and time-consuming task to examine a protein’s immunogenicity utilizing traditional approaches. Here, we present a platform for effectively evaluating protein immunogenicity and antibody detection. A tetanus toxin C fragment (Tet-c) was used as a representative antigen to establish this platform. A cell wall-anchoring sialidase-like protein (SLP) of Propionibacterium acnes was utilized to assess the efficacy of this platform. We constructed an Escherichia coli vector-based vaccine by overexpressing Tet-c or SLP in E. coli and utilized an intact particle of E. coli itself as a vaccine (E. coli Tet-c or SLP vector). After ultraviolet (UV) irradiation, the E. coli vector-based vaccines were administered intranasally into imprinting control region mice without adding exogenous adjuvants. For antibody detection, we fabricated antigen microarrays by printing with purified recombinant proteins including Tet-c and SLP. Our results demonstrated that detectable antibodies were elicited in mice 6 weeks after intranasal administration of UV-irradiated E. coli vector-based vaccines. The antibody production of Tet-c and SLP was significantly elevated after boosting. Notably, the platform with main benefits of using E. coli itself as a vaccine carrier provides a critical template for applied proteomics aimed at screening novel vaccine targets. In addition, the novel immunogenic SLP potentially serves as an antigen candidate for the development of vaccines targeting P. acnes-associated diseases. PMID:21136919

Huang, Cheng-Po; Liu, Yu-Tsueng; Nakatsuji, Teruaki; Shi, Yang; Gallo, Richard R.; Lin, Shwu-Bin; Huang, Chun-Ming

2011-01-01

247

Analysis of the Borrelia burgdorferi GeHo fla gene and antigenic characterization of its gene product.  

PubMed Central

The fla gene of Borrelia burgdorferi GeHo was analyzed and expressed in Escherichia coli. The structural gene encodes a flagellar protein of 336 amino acids. Comparative sequence analysis of the amino acid sequence revealed a high degree of sequence conservation with flagellins from both phylogenetically related and unrelated bacteria. The antigenic properties of the B. burgdorferi Fla protein were studied by synthesizing overlapping octapeptides, which were screened by using a battery of different monoclonal and polyclonal antibodies from various species directed against native and denatured flagellar proteins. No single species-independent immunodominant epitope could be located. However, immunoreactive oligopeptides clustered within the variable middle region (N-180 to I-260). This region could constitute a candidate antigen for more specific and sensitive serodiagnosis of Lyme borreliosis. Images PMID:1704884

Gassmann, G S; Jacobs, E; Deutzmann, R; Gobel, U B

1991-01-01

248

Fibronectin-Binding Antigen 85 and the 10-Kilodalton GroES- Related Heat Shock Protein Are the Predominant TH-1 Response Inducers in Leprosy Contacts  

Microsoft Academic Search

Peripheral blood mononuclear cells from 27 healthy leprosy contacts were analyzed for lymphoproliferation and TH-1 cytokine secretion (interleukin-2 and gamma interferon) in response to heat shock proteins with molecular masses of 65, 18, and 10 kDa fromMycobacterium lepraeand the 30-32-kDa antigen 85 (Ag 85) from MycobacteriumbovisBCG.Cellsfrom18and19of19lepromin-positivecontactsproliferatedorproducedTH-1 cytokines in response to theM. leprae10-kDa protein and to Ag 85, respectively. Limiting-dilution analysis

P. LAUNOIS; M. NIANG; J. L. CARTEL; I. MANE; A. DROWART

249

The rat leukocyte antigen MRC OX-44 is a member of a new family of cell surface proteins which appear to be involved in growth regulation.  

PubMed Central

Moloney murine leukemia virus (MoMuLV)-induced rat T-cell lymphomas express discrete 1.8-, 2.2-, and 4-kb mRNA transcripts hybridizing under conditions of reduced stringency to a probe derived from a region upstream of the first exon of the Tpl-1/Ets-1 gene. Screening a cDNA library from one rat T-cell lymphoma with this genomic probe yielded 15 cDNA clones which were derived from 10 different genes. One of these genes, defined by the cDNA clone pRcT7a, was expressed as a 1.8-kb mRNA transcript in spleen and thymus but not in other normal rat tissues. Expression of the gene defined by the pRcT7a cDNA clone in a series of MoMuLV-induced rat T-cell lymphomas showed a perfect correlation with the expression of the rat leukocyte antigen MRC OX-44. Because of this observation, the pRcT7a clone was sequenced and it was shown to identify a gene coding for a 219-amino-acid protein. The homology between pRcT7a and the Tpl-1 probe used for its detection mapped within the 3' untranslated region of the pRcT7a cDNA clone. The pRcT7a protein, which exhibits four putative transmembrane regions and three putative glycosylation sites, contains a region which is nearly identical in sequence to a peptide derived from the rat leukocyte antigen MRC OX-44. This finding suggested that the pRcT7a cDNA clone defines the gene coding for OX-44. To confirm this finding, a pRcT7a construct in the retrovirus vector pZipNeo was introduced into the OX-44- T-cell lymphoma line 2788. Immunostaining with the MRC OX-44 monoclonal antibody followed by flow cytometry revealed that following gene transfer, the 2788 cells became OX-44+. Sequence comparisons revealed that pRcT7a/MRC OX-44 is a member of a family of genes which includes the melanoma-specific antigen ME491; the human leukocyte antigen CD37; the protein TAPA-1, which is expressed on the surface of human T cells and appears to be involved in growth regulation; the human gastrointestinal tumor antigen CO-029; and the Schistosoma mansoni-associated antigen Sm23. Images PMID:2017181

Bellacosa, A; Lazo, P A; Bear, S E; Tsichlis, P N

1991-01-01

250

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

251

Immunoproteomics: The Key to Discovery of New Vaccine Antigens Against Bacterial Respiratory Infections  

PubMed Central

The increase in antibiotic resistance and the shortage of new antimicrobials to prevent difficult bacterial infections underlines the importance of prophylactic therapies to prevent infection by bacterial pathogens. Vaccination has reduced the incidence of many serious diseases, including respiratory bacterial infections. However, there are many pathogens for which no vaccine is available and some vaccines are not effective among all age groups or among immunocompromised individuals. Immunoproteomics is a powerful technique which has been used to identify potential vaccine candidates to protect against pathogenic bacteria. The combination of proteomics with the detection of immunoreactive antigens using serum highlights immunogenic proteins that are expressed during infection. This is particularly useful when patient serum is used as the antigens that promote a humoral response during human infection are identified. This review outlines examples of vaccine candidates that have been identified using immunoproteomics and have successfully protected animals against challenge when tested in immunisation studies. Many immunoreactive proteins are common to several unrelated pathogens, however some of these are not always protective in animal immunisation and challenge studies. Furthermore, examples of well-established immunogens, including Bordetella pertussis antigen FHA were not detected in immunoproteomics studies, indicating that this technology may underrepresent the immunoreactive proteins in a pathogen. Although only one step in the pathway towards an efficacious approved vaccine, immunoproteomics is an important technology in the identification of novel vaccine antigens. PMID:23305366

Dennehy, Ruth; McClean, Siobhan

2012-01-01

252

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

253

Weather adjustment using seemingly unrelated regression  

SciTech Connect

Seemingly unrelated regression (SUR) is a system estimation technique that accounts for time-contemporaneous correlation between individual equations within a system of equations. SUR is suited to weather adjustment estimations when the estimation is: (1) composed of a system of equations and (2) the system of equations represents either different weather stations, different sales sectors or a combination of different weather stations and different sales sectors. SUR utilizes the cross-equation error values to develop more accurate estimates of the system coefficients than are obtained using ordinary least-squares (OLS) estimation. SUR estimates can be generated using a variety of statistical software packages including MicroTSP and SAS.

Noll, T.A. [Idaho Power Company, Boise, ID (United States)

1995-05-01

254

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

255

Use of Viral Lysate Antigen Combined with Recombinant Protein in Western Immunoblot Assay as Confirmatory Test for Serodiagnosis of Severe Acute Respiratory Syndrome  

PubMed Central

A Western immunoblot assay for confirmatory serodiagnosis of severe acute respiratory syndrome (SARS) was developed utilizing viral lysate antigens combined with a recombinant nucleocapsid protein, GST-N (glutathione S-transferase-nucleocapsid) of the SARS coronavirus (SARS-CoV). The viral lysate antigens were separated by electrophoresis and transblotted onto nitrocellulose membranes. The resultant membrane was subsequently added with the GST-N recombinant protein at a specific location. The positions of bands corresponding to some of the structural proteins immobilized on the membrane were then located and verified with mouse or rabbit antisera specific to the respective proteins. The Western immunoblot assay was able to detect antibodies to SARS-CoV in all 40 serum specimens from SARS patients and differentiate the SARS-positive samples from those of the healthy donor or non-SARS patient controls (150 samples) when set criteria were followed. In addition, when the immunoblot was used to test samples considered falsely positive by an in-house-developed SARS-specific enzyme-linked immunosorbent assay, band patterns different from those with samples from SARS patients were obtained. PMID:15539520

Guan, Ming; Chen, Hsiao Ying; Tan, Phuay Heng; Shen, Shuo; Goh, Phuay-Yee; Tan, Yee-Joo; Pang, Peow Hoon; Lu, Yang; Fong, Priscilla Yiquan; Chin, Daria

2004-01-01

256

Facilitation of cell adhesion by immobilized dengue viral nonstructural protein 1 (NS1): arginine-glycine-aspartic acid structural mimicry within the dengue viral NS1 antigen.  

PubMed

Dengue virus infection causes life-threatening hemorrhagic fever. Increasing evidence implies that dengue viral nonstructural protein 1 (NS1) exhibits a tendency to elicit potentially hazardous autoantibodies, which show a wide spectrum of specificity against extracellular matrix and platelet antigens. How NS1 elicits autoantibodies remains unclear. To address the hypothesis that NS1 and matrix proteins may have structural and functional similarity, cell-matrix and cell-NS1 interactions were evaluated using a cell-adhesion assay. The present study showed that dengue NS1 immobilized on coverslips resulted in more cell adhesion than did the control proteins. This cell adhesion was inhibited by peptides containing arginine-glycine-aspartic acid (RGD), a motif important for integrin-mediated cell adhesion. In addition, anti-NS1 antibodies blocked RGD-mediated cell adhesion. Although there is no RGD motif in the NS1 protein sequence, these data indicate that RGD structural mimicry exists within the NS1 antigen. PMID:12198607

Chang, Hsin-Hou; Shyu, Huey-Fen; Wang, Yo-Ming; Sun, Der-Shan; Shyu, Rong-Hwa; Tang, Shiao-Shek; Huang, Yao-Shine

2002-09-15

257

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

258

Sensitization of MHC class I-restricted T cells to exogenous proteins: evidence for an alternative class I-restricted antigen presentation pathway.  

PubMed Central

Immunization with exogenous proteins usually fails to immunize CD8+ T cells in vivo. Here we report that chicken ovalbumin (OVA) denatured by heat or sodium dodecyl sulphate (SDS) effectively induced CD8+ cytolytic T cells in vivo. The cytolytic T-lymphocyte (CTL) population generated recognized syngeneic target cells pulsed with the immunodominant OVA peptide (257-264) or transfected with the OVA protein-encoding gene. To analyse the mechanisms of how denatured OVA enters the class I-restricted pathway of antigen presentation, we took advantage of the fact that denatured OVA sensitizes target cells in vitro for lysis by OVA-specific CTL. We found that neither inhibition of protein synthesis (by cycloheximide) nor blocking of transport via the Golgi apparatus (by brefeldin A) interfered with the class I-restricted presentation of denatured OVA in vitro. In addition, transporter associated with antigen presentation (TAP)-dependent transport into the endoplasmic reticulum (ER) was not required for effective presentation, as TAP-deficient cells (RMA-S) could be sensitized effectively by denatured OVA for recognition by class I-restricted CTL. In contrast, class I-restricted presentation of denatured OVA was sensitive to lysosomotropic agents (NH4Cl, vinblastine and leupeptin), indicating that endosomal-like compartments are involved in the presentation of denatured OVA. Sensitization was inhibited at low temperature, yet took place in the presence of sucrose and in the absence of K+, indicating that denatured OVA enters the cell via fluid-phase endocytosis. Hence the results provide further evidence for an alternative class I-restricted pathway of antigen presentation for exogenous proteins. As that pathway seems to be effective in vivo, it offers a new and effective way of vaccination of CD8+ CTL. PMID:7490131

Martinez-Kinader, B; Lipford, G B; Wagner, H; Heeg, K

1995-01-01

259

Murine aortic smooth muscle cells acquire, though fail to present exogenous protein antigens on major histocompatibility complex class II molecules.  

PubMed

In the present study aortic murine smooth muscle cell (SMC) antigen presentation capacity was evaluated using the E?-GFP/Y-Ae system to visualize antigen uptake through a GFP tag and tracking of E? peptide/MHCII presentation using the Y-Ae Ab. Stimulation with IFN-? (100?ng/mL) for 72?h caused a significant (P < 0.01) increase in the percentage of MHC class II positive SMCs, compared with unstimulated cells. Treatment with E?-GFP (100??g/mL) for 48?h induced a significant (P < 0.05) increase in the percentage of GFP positive SMCs while it did not affect the percentage of Y-Ae positive cells, being indicative of antigen uptake without its presentation in the context of MHC class II. After IFN-?-stimulation, ovalbumin- (OVA, 1?mg/mL) or OVA323-339 peptide-(0.5??g/mL) treated SMCs failed to induce OT-II CD4(+) T cell activation/proliferation; this was also accompanied by a lack of expression of key costimulatory molecules (OX40L, CD40, CD70, and CD86) on SMCs. Finally, OVA-treated SMCs failed to induce DO11.10-GFP hybridoma activation, a process independent of costimulation. Our results demonstrate that while murine primary aortic SMCs express MHC class II and can acquire exogenous antigens, they fail to activate T cells through a failure in antigen presentation and a lack of costimulatory molecule expression. PMID:25136640

Maddaluno, Marcella; MacRitchie, Neil; Grassia, Gianluca; Ialenti, Armando; Butcher, John P; Garside, Paul; Brewer, James M; Maffia, Pasquale

2014-01-01

260

Murine Aortic Smooth Muscle Cells Acquire, Though Fail to Present Exogenous Protein Antigens on Major Histocompatibility Complex Class II Molecules  

PubMed Central

In the present study aortic murine smooth muscle cell (SMC) antigen presentation capacity was evaluated using the E?-GFP/Y-Ae system to visualize antigen uptake through a GFP tag and tracking of E? peptide/MHCII presentation using the Y-Ae Ab. Stimulation with IFN-? (100?ng/mL) for 72?h caused a significant (P < 0.01) increase in the percentage of MHC class II positive SMCs, compared with unstimulated cells. Treatment with E?-GFP (100??g/mL) for 48?h induced a significant (P < 0.05) increase in the percentage of GFP positive SMCs while it did not affect the percentage of Y-Ae positive cells, being indicative of antigen uptake without its presentation in the context of MHC class II. After IFN-?-stimulation, ovalbumin- (OVA, 1?mg/mL) or OVA323–339 peptide-(0.5??g/mL) treated SMCs failed to induce OT-II CD4+ T cell activation/proliferation; this was also accompanied by a lack of expression of key costimulatory molecules (OX40L, CD40, CD70, and CD86) on SMCs. Finally, OVA-treated SMCs failed to induce DO11.10-GFP hybridoma activation, a process independent of costimulation. Our results demonstrate that while murine primary aortic SMCs express MHC class II and can acquire exogenous antigens, they fail to activate T cells through a failure in antigen presentation and a lack of costimulatory molecule expression. PMID:25136640

Maddaluno, Marcella; MacRitchie, Neil; Grassia, Gianluca; Ialenti, Armando; Butcher, John P.; Garside, Paul; Brewer, James M.; Maffia, Pasquale

2014-01-01

261

gp330 associates with a 44-kDa protein in the rat kidney to form the Heymann nephritis antigenic complex.  

PubMed Central

Using antibodies isolated from glomeruli of nephritic rats we have previously identified a 330-kDa cell surface glycoprotein (gp330) as a major pathogenic antigen of Heymann nephritis (HN), an experimental model of human membranous glomerulonephritis. Recently, we have isolated a cDNA clone, C14, encoding a polypeptide that contains a pathogenic epitope of HN responsible for the initiation of the disease. Subsequently, another protein, alpha 2-macroglobulin receptor-associated protein (alpha 2-MRAP), which is a subunit of the receptor for human alpha 2-macroglobulin/low density lipoprotein receptor-related protein (LRP), was shown to possess a high degree of sequence homology to the C14 protein (C14p). In this report, we have investigated the relationship between gp330, C14p, and alpha 2-MRAP. Immunoprecipitation studies demonstrate that gp330 forms a heterodimeric association with a 44-kDa polypeptide that is stable to detergent extraction and long-term centrifugation. Further, immunoblotting analysis on the purified complex indicates that the 44-kDa associated protein shares immunological identity to C14p and alpha 2-MRAP. In addition, antibodies eluted from glomeruli of HN rats and antibodies to a C14 fusion protein immunoprecipitated gp330 and the 44-kDa protein, demonstrating that the epitopes responsible for the initial events of HN are accessible within the complex. Based on these data, three models are proposed to explain how pathogenic epitopes in the gp330-44-kDa, HN antigenic complex may be presented at the cell surface and initiate the onset of HN. Images PMID:1495959

Orlando, R A; Kerjaschki, D; Kurihara, H; Biemesderfer, D; Farquhar, M G

1992-01-01

262

Computational design of protein antigens that interact with the CDR H3 loop of HIV broadly neutralizing antibody 2F5.  

PubMed

Rational design of proteins with novel binding specificities and increased affinity is one of the major goals of computational protein design. Epitope-scaffolds are a new class of antigens engineered by transplanting viral epitopes of predefined structure to protein scaffolds, or by building protein scaffolds around such epitopes. Epitope-scaffolds are of interest as vaccine components to attempt to elicit neutralizing antibodies targeting the specified epitope. In this study we developed a new computational protocol, MultiGraft Interface, that transplants epitopes but also designs additional scaffold features outside the epitope to enhance antibody-binding specificity and potentially influence the specificity of elicited antibodies. We employed MultiGraft Interface to engineer novel epitope-scaffolds that display the known epitope of human immunodeficiency virus 1 (HIV-1) neutralizing antibody 2F5 and that also interact with the functionally important CDR H3 antibody loop. MultiGraft Interface generated an epitope-scaffold that bound 2F5 with subnanomolar affinity (K(D)?=?400 pM) and that interacted with the antibody CDR H3 loop through computationally designed contacts. Substantial structural modifications were necessary to engineer this antigen, with the 2F5 epitope replacing a helix in the native scaffold and with 15% of the native scaffold sequence being modified in the design stage. This epitope-scaffold represents a successful example of rational protein backbone engineering and protein-protein interface design and could prove useful in the field of HIV vaccine design. MultiGraft Interface can be generally applied to engineer novel binding partners with altered specificity and optimized affinity. PMID:25043744

Azoitei, M L; Ban, Y A; Kalyuzhny, O; Guenaga, J; Schroeter, A; Porter, J; Wyatt, R; Schief, William R

2014-10-01

263

Comparison of serum human epididymis protein 4 and carbohydrate antigen 125 as markers in ovarian cancer: A meta-analysis  

PubMed Central

Ovarian cancer (OC) is the third most common type of gynecological cancer. Measurements of human epididymis protein 4 (HE4) levels have been suggested for improving the specificity of the laboratory identification of OC. For this meta-analysis, the Medline, Embase and Cochrane databases were searched to identify relevant studies. All the included studies for diagnostic performance were combined with sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratios (DORs) with 95% confidence intervals (CIs), summary receiver operating characteristic (SROC) curves and areas under the SROC curves (AUC). A total of 25 studies including 4,729 patients were identified as eligible for inclusion in the final analysis. The pooled sensitivities and respective 95% CIs for HE4 and carbohydrate antigen 125 (CA125) were 0.74 (0.72–0.76) and 0.74 (0.72–0.76), respectively. The pooled specificities and respective 95% CIs for HE4 and CA125 were 0.90 (0.89–0.91) and 0.83 (0.81–0.84), respectively. The summary DORs and 95% CIs for HE4 and CA125 were 43.35 (29.13–64.51) and 17.06 (10.97–26.51), respectively and the AUCs for HE4 and CA125 were 0.8915 and 0.8538, respectively. In total, 9 studies investigated the diagnostic accuracy of HE4 combined with CA125 for the diagnosis of OC. The pooled sensitivity and 95% CIs of HE4, CA125 and HE4+CA125 in this subgroup were 0.71 (0.67–0.75), 0.74 (0.69–0.78) and 0.90 (0.87–0.92), respectively; the pooled specificity and 95% CIs of HE4, CA125 and HE4+CA125 were 0.92 (0.90–0.94), 0.73 (0.69–0.76) and 0.85 (0.82–0.87), respectively. The diagnostic accuracy of HE4 in distinguishing OC from other benign gynecological diseases was found to be to be superior to that of CA125 and the combination of HE4 and CA125 may enhance the diagnostic sensitivity. PMID:24940495

ZHEN, SHUAI; BIAN, LI-HONG; CHANG, LI-LI; GAO, XIN

2014-01-01

264

[c-FOS protein expression in cells of the variuos hypothalamic structures after electric pain stimulation and injections of antigens].  

PubMed

Hypothalamic structures become activated after stimul. Alteration of c-Fos-positive cells quantity in different hypothalamic structures after electric pain stimulation (EPS), intravenous (iv) injection of antigens (lioppolysaccharide (LPS) and bovine serum albumir (BSA)) was detected with immunohistochemical method. EPS and iv injection of antigens (LPS and BSA) result in c-Fos-positive cells quantity increase in all observed hypothalamic structures. The highest activation level was in AHN and PH after EPS and in AHN, PVH, LHA-28, and PH after iv LPS injection. Comparative analysis of results showed, that c-Fos-positive cells quantity increase after EPS in AHN, PVH, LHA and PH was more significant than after iv injection of antigens (LPS and BSA). LPS injection results in more pronounced cell activation in AHN, PVH, LHA-28 and DMH (according to quantity of c-Fos-positive cells), than BSA injection. PMID:17216716

Gavrilov, Iu V; Perekrest, S V; Novikova, N S

2006-10-01

265

A fluorescent multiplexed bead-based immunoassay (FMIA) for quantitation of IgG against Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis protein antigens.  

PubMed

Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis are pathogens commonly associated with infectious diseases in childhood. This study aimed to develop a fluorescent multiplexed bead-based immunoassay (FMIA) using recombinant proteins for the quantitation of serum IgG antibodies against these bacteria. Eight pneumococcal proteins (Ply, CbpA, PspA1, PspA2, PcpA, PhtD, SP1732-3 and SP2216-1), 3 proteins of H. influenzae (NTHi Protein D, NTHi0371-1, NTHi0830), and 5 proteins of M. catarrhalis (MC Omp CD, MC_RH4_2506, MC_RH4_1701, MC_RH4_3729-1, MC_RH4_4730) were used to develop the FMIA. Optimal coupling concentrations for each protein, comparison of singleplex and multiplex assays, specificity, reproducibility, and correlation to ELISA for six pneumococcal antigens were determined for validation. FMIA was then used to analyze acute and convalescent paired serum samples of 50 children with non-severe pneumonia. The coupling concentrations varied for different antigens, ranging from 1.6 to 32?g of protein/million beads. Correlation between singleplexed and multiplexed assays was excellent, with R?0.987. The FMIA was specific, reaching >92% homologous inhibition for all specificities; heterologous inhibition ?20% was found only in six cases. The assay was repeatable, with averages of intra-assay variation ?10.5%, day-to-day variation ?9.7% and variation between technicians ?9.1%. Comparison with ELISA for pneumococcal antigens demonstrated good correlation with R ranging from 0.854 (PspA2) to 0.976 (PcpA). The samples from children showed a wide range of antibody concentrations and increases in convalescent samples. In conclusion, the FMIA was sensitive, specific, and repeatable, using small amounts of recombinant proteins and sera to detect antibodies against S. pneumoniae, H. influenzae and M. catarrhalis. The methodology would be suitable for studies investigating etiological diagnosis and in experimental vaccine studies. PMID:24530690

Andrade, Dafne C; Borges, Igor C; Laitinen, Hanna; Ekström, Nina; Adrian, Peter V; Meinke, Andreas; Barral, Aldina; Nascimento-Carvalho, Cristiana M; Käyhty, Helena

2014-03-01

266

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

267

Targeting of Nasal Mucosa-Associated Antigen-Presenting Cells In Vivo with an Outer Membrane Protein A Derived from Klebsiella pneumoniae  

PubMed Central

Administration of vaccines by the nasal route has recently proven to be one of the most efficient ways for inducing both mucosal and systemic antibody responses in experimental animals. Our results demonstrate that P40, a well-defined outer membrane protein A from Klebsiella pneumoniae, is indeed a carrier molecule suitable for nasal immunization. Using fragments from the respiratory syncytial virus subgroup A (RSV-A) G protein as antigen models, it has been shown that P40 is able to induce both systemic and mucosal immunity when fused or coupled to a protein or a peptide and administered intranasally (i.n.) to naive or K. pneumoniae-primed mice. Confocal analyses of nasal mucosa-associated lymphoid tissue after i.n. instillation of P40 showed that this molecule is able to cross the nasal epithelium and target CD11c-positive cells likely to be murine dendritic cells or macrophages. More importantly, this targeting of antigen-presenting cells following i.n. immunization with a subunit of the RSV-A molecule in the absence of any mucosal adjuvant results in both upper and lower respiratory tract protection against RSV-A infection. PMID:11553588

Goetsch, Liliane; Gonzalez, Alexandra; Plotnicky-Gilquin, Helene; Haeuw, Jean Francois; Aubry, Jean Pierre; Beck, Alain; Bonnefoy, Jean Yves; Corvaia, Nathalie

2001-01-01

268

Energy filtering transmission electron microscopy immunocytochemistry and antigen retrieval of surface layer proteins from Tannerella forsythensis using microwave or autoclave heating with citraconic anhydride.  

PubMed

Tannerella forsythensis (Bacteroides forsythus), an anaerobic Gram-negative species of bacteria that plays a role in the progression of periodontal disease, has a unique bacterial protein profile. It is characterized by two unique protein bands with molecular weights of more than 200 kDa. It also is known to have a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane. We examined the relationship between high molecular weight proteins and the S-layer using electron microscopic immunolabeling with chemical fixation and an antigen retrieval procedure consisting of heating in a microwave oven or autoclave with citraconic anhydride. Immunogold particles were localized clearly at the outermost cell surface. We also used energy-filtering transmission electron microscopy (EFTEM) to visualize 3, 3'-diaminobenzidine tetrahydrochloride (DAB) reaction products after microwave antigen retrieval with 1% citraconic anhydride. The three-window method for electron spectroscopic images (ESI) of nitrogen by the EFTEM reflected the presence of moieties demonstrated by the DAB reaction with horseradish peroxidase (HRP)-conjugated secondary antibodies instead of immunogold particles. The mapping patterns of net nitrogen were restricted to the outermost cell surface. PMID:22984898

Moriguchi, K; Mitamura, Y; Iwami, J; Hasegawa, Y; Higuchi, N; Murakami, Y; Maeda, H; Yoshimura, F; Nakamura, H; Ohno, N

2012-11-01

269

The BH3-only proteins Bim and Puma cooperate to impose deletional T-cell tolerance to organ-specific antigens  

PubMed Central

Summary Although the pro-apoptotic BH3-only protein, Bim, is required for deletion of autoreactive thymocytes, Bim-deficient mice do not succumb to extensive organ-specific autoimmune disease. To determine whether other BH3-only proteins safeguard tolerance in the absence of Bim, we screened mice lacking Bim alongside other BH3-only proteins. Most strains showed no additional defects, however, mice deficient for both Puma and Bim spontaneously developed autoimmunity in multiple organs and their T-cells could transfer organ-specific autoimmunity. Puma/Bim double-deficient mice had a striking accumulation of mature single positive thymocytes, suggesting a further defect in thymic deletion was the basis for disease. Transgenic mouse models of thymocyte deletion to peripheral neo-antigens confirmed that the loss of Bim and Puma allowed increased numbers of autoreactive thymocytes to escape deletion. Our data show that Puma cooperates with Bim to impose a thymic deletion checkpoint to peripheral self-antigens and cement the notion that defects in apoptosis alone are sufficient to cause autoimmune disease. PMID:22960223

Gray, Daniel H. D.; Kupresanin, Fiona; Berzins, Stuart P.; Herold, Marco J.; O'Reilly, Lorraine A.; Bouillet, Philippe; Strasser, Andreas

2012-01-01

270

Cloning and characterization of the antigenic membrane protein (Amp) gene and in situ detection of Amp from malformed flowers infected with Japanese hydrangea phyllody phytoplasma.  

PubMed

A Japanese hydrangea phyllody (JHP) disease found throughout Japan causes economic damage to the horticultural industry. JHP phytoplasma-infected Japanese hydrangea plants show several disease symptoms involved in floral malformations, such as virescence, phyllody and proliferation. Here, we cloned and characterized the antigenic membrane protein (Amp) gene homolog from the JHP phytoplasma (JHP-amp), expressed the JHP-Amp protein in Escherichia coli cells, and then obtained an antibody against JHP-Amp. The antibody against JHP-Amp had no cross-reactions with the antibody against the Amp protein from a closely related onion yellows phytoplasma. This serologic specificity is probably due to the high diversity of the hydrophilic domains in the Amp proteins. The in situ detection of the JHP-Amp protein revealed that the JHP phytoplasma was localized to the phloem tissues in the malformed flower. This study shows that the JHP-Amp protein is indeed a membrane protein, which is expressed at detectable level in the JHP phytoplasma-infected hydrangea. PMID:18943252

Arashida, R; Kakizawa, S; Ishii, Y; Hoshi, A; Jung, H-Y; Kagiwada, S; Yamaji, Y; Oshima, K; Namba, S

2008-07-01

271

[Comparison of various immune surface labeling methods for scanning electron microscopy with the example of a surface antigen protein of the yeast Candida albicans].  

PubMed

The labeling of immunocomplexes for scanning electron microscopy (SEM) is a fairly new technique, and the various procedures, that have been proposed, have not yet been compared. Such comparative evaluation was performed with Candida protease as a target antigen. This secretory enzyme of the opportunistic yeast Candida albicans can be localized on the surface of fungal blastopores and mycelia, both after growth in proteinaceous medium and upon infection of murine peritoneal macrophages. The presence of the protease antigen was confirmed by immunofluorescence and by immunoperoxidase-light microscopy. The decoration of protease - anti protease complexes for SEM was attempted with colloids derived from the immunoperoxidase reaction, by the immunogold technique, and by antibodies linked to beads of synthetic polymers (polystyrene, polymethacrylate, polyacrolein). In addition, inactivated Staphylococcus aureus was used, which binds to antibodies through its protein-A. The high resolution by SEM of surface structures was matched only by the colloid based decoration techniques. All conjugates with beads suffered from inconsistent binding, which did not correspond with the distribution of the surface antigen. The comparatively best result with beads was obtained with polystyrene (Latex). Colloid based techniques in addition allow for critical point drying, which cannot be applied to synthetic beads in the usual manner. PMID:3901566

Borg, M

1985-01-01

272

A blocking ELISA for detection of antibody to a subgroup-reactive epitope of African horsesickness viral protein 7 (VP7) using a novel gamma-irradiated antigen.  

PubMed

A novel gamma irradiated inactivated cell culture derived African horsesickness viral (AHSV) antigen was used in a blocking ELISA (B-ELISA) for detecting antibody to a subgroup-reactive epitope of AHSV. A monoclonal antibody (MAB), class IgM, against an epitope on African horsesickness (AHS) viral protein 7 (VP7) was developed in BALBc mice and used in the B-ELISA. The MAB, designated F9H, was blocked by 69 serums from equidae with antibody to AHS, but its binding activity was not appreciably affected by 301 serums that did not contain antibodies to AHS virus. An ELISA protocol using a blocking format is described. PMID:8784514

House, J A; Stott, J L; Blanchard, M T; LaRocco, M; Llewellyn, M E

1996-07-23

273

A Trypanosoma cruzi membrane protein shares an epitope with a lymphocyte activation antigen and induces crossreactive antibodies  

PubMed Central

Chagas' disease results from the infection of the protozoan parasite Trypanosoma cruzi and affects several million people in South America. Several alterations of the immune response have been described in this disease, such as severe immunosuppression of both cellular and humoral responses and massive polyclonal stimulation with the generation of autoantibodies crossreacting with host cells and tissues. We have obtained monoclonal antibodies (mAbs) from T. cruzi-infected mice that recognized a 50/55-kD antigen (GP50/55) on the T. cruzi membrane, but not in other parasites of the family Trypanosomatidae. One of these GP50/55-specific mAbs (C10) crossreacts with a 28-kD antigen (p28) expressed on the membrane of greater than 85% of activated mouse T and B lymphocytes, after in vitro activation with concanavalin A, Salmonella typhosa lipopolysaccharide, phorbol dibutyrate ester, or antigen, and on several murine T and B lymphocyte cell lines. Human T and B lymphocytes also express upon activation with phytohemagglutinin or Staphylococcus aureus Cowan I (SAC) a similar antigen recognized by mAb C10, although in a lower proportion of cells (30-40%). Furthermore, this mAb was able to suppress mouse and human T and B cell proliferation to any of those stimuli. In addition, sera from chagasic patients and T. cruzi-infected mice, but not from control patients or littermates, contain antibodies that recognize a similar p28 antigen on B lymphocytes. Furthermore, the immunoglobulin fractions of some chagasic sera also suppress the proliferation of human T lymphocytes. These results suggest a possible pathological role of autoantibodies as an alternative mechanism for T. cruzi-associated immunosuppression. PMID:1375261

1992-01-01

274

Anti-Thy-1 Antibody-mediated Complement-dependent Cytotoxicity is Regulated by the Distribution of Antigen, Antibody and Membrane Complement Regulatory Proteins in Rats  

PubMed Central

Some therapeutic antibodies as anticancer agents exert their effects through the host immune system, but the factors that predict their cytotoxicity, including complement-dependent cytotoxicity (CDC), are unclear. In the present study, we attempted to elucidate some of these factors in a preclinical model. CDC-related mesangiolysis caused by administration of the anti-Thy-1.1 antibody can be studied in the rat anti-Thy-1 glomerulonephritis model, so the model was used in this study. Three animals each were sacrificed at 0.5, 1, 8, 24 and 48 hours after i.v. administration of the anti-Thy-1.1 antibody at 1mg/kg. The distribution of the Thy-1.1 antigen and 2 membrane complement regulatory proteins (mCRPs), Crry and CD55, in three non-treated animals and the distribution of the injected antibody and C3 in the model was studied by immunohistochemistry. In the mesangial cells of the kidney, both expression of the antigen and distribution of the antibody with C3 deposition were observed with weak expression of mCRPs. There was also antigen and antibody distribution in the medullary cells of the adrenal gland and in the lymphocytes of the thymus but no C3 deposition, which was thought to be related to high expression of mCRPs. The antigen was observed in several other organs and tissues without distribution of the antibody. Cell death was only observed in the mesangial cells. These results clearly demonstrate that activation of CDC is regulated by several factors, such as distribution of the target molecule, antibody distribution and the balance among the molecules of the CDC cascade and mCRPs. PMID:23723567

Kato, Chie; Kato, Atsuhiko; Adachi, Kenji; Fujii, Etsuko; Isobe, Kaori; Matsushita, Tomochika; Watanabe, Takeshi; Suzuki, Masami

2013-01-01

275

Generation of MHC class I-restricted cytotoxic T lymphocytes by expression of a viral protein in muscle cells: antigen presentation by non-muscle cells.  

PubMed Central

Expression of reporter genes in muscle cells has been achieved by intramuscular (i.m.) injection of plasmid DNA expression vectors. We previously demonstrated that this technique is an effective means of immunization to elicit both antibodies capable of conferring homologous protection and cell-mediated immunity leading to cross-strain protection against influenza virus challenge in mice. These results suggested that expression of viral proteins by muscle cells can result in the generation of cellular immune responses, including cytotoxic T lymphocytes (CTL). However, because DNA has the potential to be internalized and expressed by other cell types, we sought to determine whether or not induction of CTL required synthesis of antigen in non-muscle cells and if not whether transfer of antigen to antigen-presenting cells from muscle cells may be involved. In the present study we demonstrate that transplantation of nucleoprotein (NP)-transfected myoblasts into syngeneic mice led to the generation of NP-specific antibodies and CTL and cross-strain protective immunity against a lethal challenge with influenza virus. Furthermore transplantation of NP-expressing myoblasts (H-2k) intraperitoneally into F1 hybrid mice (H-2d x H-2k) elicited NPCTL restricted by the MHC haplotype of both parental strains. These results indicate that NP expression by muscle cells after transplantation was sufficient to generate protective cell-mediated immunity and that induction of the CTL response was mediated at least in part, by transfer of antigen from the transplanted muscle cells to a host cell. PMID:8911141

Ulmer, J B; Deck, R R; Dewitt, C M; Donnhly, J I; Liu, M A

1996-01-01

276

Wheat ribosome-inactivating proteins: Seed and leaf forms with different specificities and cofactor requirements  

Microsoft Academic Search

Distinct forms of ribosome-inactivating proteins were purified from wheat (Triticum aestivum L.) germ and leaves and termed tritin-S and tritin-L, respectively. These differ in size and charge and are antigenically unrelated. They are both RNA N-glycosidases which act on 26S rRNA in native yeast (Saccharomyces cerevisiae) ribosomes by the removal of A3024 located in a universally conserved sequence in domain

Andrea J. Massiah; Martin R. Hartley

1995-01-01

277

Recombinant dengue virus type 2 envelope/hepatitis B surface antigen hybrid protein expressed in Pichia pastoris can function as a bivalent immunogen.  

PubMed

A truncated version of the dengue virus type 2 envelope protein (Den2E) encoding the first 395 amino acid (aa) residues, and Den2E fused in-frame with the full-length 226-aa hepatitis B surface antigen (Den2E-HBsAg) protein were expressed in the methylotrophic yeast, Pichia pastoris. Both the recombinant proteins showed evidence of the capacity to form high molecular weight aggregates. Electron microscopic analysis of the purified proteins showed that while Den2E displayed an amorphous morphology, Den2E-HBsAg existed as well-structured virus-like particles (VLPs). Using immuno-gold electron microscopy, these VLPs were demonstrated to contain both components of the Den2E-HBsAg hybrid protein. Seroanalysis showed that the hybrid VLPs could function in vivo as bivalent immunogens, which could elicit immune responses directed against both components of the hybrid protein, as evidenced by ELISA, immunoprecipitation and immunofluorescence data. PMID:12270598

Bisht, Himani; Chugh, Dipti A; Raje, Manoj; Swaminathan, S Swaminathan; Khanna, Navin

2002-10-23

278

Use of In Vivo-Induced Antigen Technology for Identification of Escherichia coli O157:H7 Proteins Expressed during Human Infection  

PubMed Central

Using in vivo-induced antigen technology (IVIAT), a modified immunoscreening technique that circumvents the need for animal models, we directly identified immunogenic Escherichia coli O157:H7 (O157) proteins expressed either specifically during human infection but not during growth under standard laboratory conditions or at significantly higher levels in vivo than in vitro. IVIAT identified 223 O157 proteins expressed during human infection, several of which were unique to this study. These in vivo-induced (ivi) proteins, encoded by ivi genes, mapped to the backbone, O islands (OIs), and pO157. Lack of in vitro expression of O157-specific ivi proteins was confirmed by proteomic analysis of a mid-exponential-phase culture of E. coli O157 grown in LB broth. Because ivi proteins are expressed in response to specific cues during infection and might help pathogens adapt to and counter hostile in vivo environments, those identified in this study are potential targets for drug and vaccine development. Also, such proteins may be exploited as markers of O157 infection in stool specimens. PMID:15845468

John, Manohar; Kudva, Indira T.; Griffin, Robert W.; Dodson, Allen W.; McManus, Bethany; Krastins, Bryan; Sarracino, David; Progulske-Fox, Ann; Hillman, Jeffrey D.; Handfield, Martin; Tarr, Phillip I.; Calderwood, Stephen B.

2005-01-01

279

Solution structure, membrane interactions, and protein binding partners of the tetraspanin Sm-TSP-2, a vaccine antigen from the human blood fluke Schistosoma mansoni.  

PubMed

The tetraspanins (TSPs) are a family of integral membrane proteins that are ubiquitously expressed at the surface of eukaryotic cells. TSPs mediate a range of processes at the surface of the plasma membrane by providing a scaffold for the assembly of protein complexes known as tetraspanin-enriched microdomains (TEMs). We report here the structure of the surface-exposed EC2 domain from Sm-TSP-2, a TSP from Schistosoma mansoni and one of the better prospects for the development of a vaccine against schistosomiasis. This is the first solution structure of this domain, and our investigations of its interactions with lipid micelles provide a general model for interactions between TSPs, membranes, and other proteins. Using chemical cross-linking, eight potential protein constituents of Sm-TSP-2-mediated TEMs were also identified. These include proteins important for membrane maintenance and repair, providing further evidence for the functional role of Sm-TSP-2- and Sm-TSP-2-mediated TEMs. The identification of calpain, Sm29, and fructose-bisphosphate aldolase, themselves potential vaccine antigens, suggests that the Sm-TSP-2-mediated TEMs could be disrupted via multiple targets. The identification of further Sm-TSP-2-mediated TEM proteins increases the available candidates for multiplex vaccines and/or novel drugs targeting TEMs in the schistosome tegument. PMID:24429291

Jia, Xinying; Schulte, Leigh; Loukas, Alex; Pickering, Darren; Pearson, Mark; Mobli, Mehdi; Jones, Alun; Rosengren, Karl J; Daly, Norelle L; Gobert, Geoffrey N; Jones, Malcolm K; Craik, David J; Mulvenna, Jason

2014-03-01

280

Molecular analysis of region 1 of the Escherichia coli K5 antigen gene cluster: a region encoding proteins involved in cell surface expression of capsular polysaccharide.  

PubMed Central

The nucleotide sequence of region 1 of the K5 antigen gene cluster of Escherichia coli was determined. This region is postulated to encode functions which, at least in part, participate in translocation of polysaccharide across the periplasmic space and onto the cell surface. Analysis of the nucleotide sequence revealed five genes that encode proteins with predicted molecular masses of 75.7, 60.5, 44, 43, and 27 kDa. The 27-kDa protein was 70.7% homologous to the CMP-2-keto-3-deoxyoctulosonic acid synthetase enzyme encoded by the E. coli kdsB gene, indicating the presence of a structural gene for a similar enzyme within the region 1 operon. The 43-kDa protein was homologous to both the Ctrb and BexC proteins encoded by the Neisseria meningitidis and Haemophilus influenzae capsule gene clusters, respectively, indicating common stages in the expression of capsules in these gram-negative bacteria. However, no homology was detected between the 75.7, 60.5-, and 44-kDa proteins and any of the proteins so far described for the H. influenzae and N. meningitidis capsule gene clusters. Images PMID:8397187

Pazzani, C; Rosenow, C; Boulnois, G J; Bronner, D; Jann, K; Roberts, I S

1993-01-01

281

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

282

An Influenza Virus M2 Protein Specific Chimeric Antigen Receptor Modulates Influenza A/WSN/33 H1N1 Infection In Vivo  

PubMed Central

A potential target for the development of universal vaccine strategies against Influenza A is the M2 protein – a membrane protein with a highly conserved extracellular domain. In this study we developed engineered T-cell receptors, by fusing M2-specific antibody sequences with T-cell receptor transmembrane and signaling domains to target influenza infected cells. When expressed on T-cells, these novel T-cell receptors (chimeric antigen receptors - CARs) are able to recognize specific antigens on the surface of target cells via an MHC-independent mechanism. Using an existing monoclonal antibody (14C2) specific for the M2 ectodomain (M2e), we generated an M2-specific CAR. We tested the specificity of this M2 CAR in vitro by measuring the activation of T-cells in response to M2-specific peptides or M2-expressing cell lines. Both Jurkat T-cells and peripheral blood mononuclear cells expressing the M2-specific CAR responded to specific antigen stimulation by upregulating NFAT and producing ?-interferon. To test whether the M2-specific CAR are effective at recognizing influenza infected cells in vivo we used an established BALB/c murine infection model. At day 4 post-infection, when M2 CAR expressing splenocytes could be detected in the lung, the Influenza A/WSN/33 virus titre was around 50% of that in control mice. Although the lung virus titre later increased in the treated group, virus was cleared in both groups of mice by day 8. The results provide support for the development of M2e as a target for cell mediated immunotherapy. PMID:23493233

Talbot, Simon J; Blair, Natalie F; McGill, Niolette; Ligertwood, Yvonne; Dutia, Bernadette M; Johannessen, Ingo

2013-01-01

283

Effect of cholinomimetics and adrenomimetics on proliferation of mouse B lymphocytes during primary immune response to protein antigen  

SciTech Connect

The aim of this investigation was to study the effect of neurotransmitters on proliferation of B lymphocytes induced by specific antigen. Experiments were carried out on female mice. To estimate proliferative activity, lymphocytes enriched with B cells were incubated in medium 199 for 2 h at 37 degrees C in a dose of 2.10/sup 6/-5.10/sup 6/ cells with 2 microCi of /sup 3/H-(methyl)-thymidine. The effect of acetylcholine on incorporation of /sup 3/H-thymidine into B lymphocytes of mice immunized with different doses of antigen during culture is shown. Discordance of effects of adrenalin and acetylcholine on incorporation of /sup 3/H-thymidine into B lymphocytes of mice immunized with different doses of ovalbumin is also shown.

Ado, A.D.; Dontsov, V.I.; Gol'dshtein, M.M.

1985-12-01

284

Protein Interactions Targeting the Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus to Cell Chromosomes  

Microsoft Academic Search

Maintenance of Kaposi's sarcoma-associated herpesvirus (KSHV) latent infection depends on the viral episomes in the nucleus being distributed to daughter cells following cell division. The latency-associated nuclear antigen (LANA) is constitutively expressed in all KSHV-infected cells. LANA binds sequences in the terminal repeat regions of the KSHV genome and tethers the viral episomes to chromosomes. To better understand the mechanism

Anita Krithivas; Masahiro Fujimuro; Magdalena Weidner; David B. Young; S. Diane Hayward

2002-01-01

285

Molecular cloning of the common acute lymphoblastic leukemia antigen (CALLA) identifies a type II integral membrane protein  

Microsoft Academic Search

Common acute lymphoblastic leukemia antigen (CALLA) is a 100-kDa cell-surface glycoprotein expressed on most acute lymphoblastic leukemias and certain other immature lymphoid malignancies and on normal lymphoid progenitors. The latter are either uncommitted to B- or T-cell lineage or committed to only the earliest stages of B- or T-lymphocyte maturation. To elucidate the primary structure of CALLA, the authors purified

M. A. Shipp; N. E. Richardson; P. H. Sayre; N. R. Brown; E. L. Masteller; L. K. Clayton; J. Ritz; E. L. Reinherz

1988-01-01

286

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

287

Immunohistochemical study of intermediate filament proteins on routinely processed, celloidin-embedded human temporal bone sections by using a new technique for antigen retrieval.  

PubMed

Although immunohistochemical studies of intermediate filament proteins have been carried out on temporal bone sections by using modified fixation/embedding techniques to preserve antigenicity, there have been no light microscopic studies concerning immunohistochemical staining on routinely formalin-fixed celloidin-embedded human temporal bone sections. A method for immunostaining routinely processed celloidin-embedded tissues would be extremely valuable in that it would permit study of the extensive collections of formalin-celloidin temporal bone specimens that exist in major centers of otopathologic research. Recently, we have developed a new technique which can be used to retrieve the antigenicity masked by formalin fixation and decalcification. This method requires immersing slides for 30 min at room temperature in a solution of saturated sodium hydroxide in methanol before immunostaining. Using this method, 45 celloidin-embedded human temporal bone sections were stained with monoclonal antibodies to keratin, vimentin, neurofilament, glial fibrillary acidic protein and desmin as primary antibodies using a sensitive streptavidin-biotin procedure. The results obtained by using this technique are at least equivalent to those obtained with modified fixatives, cryosections or immuno-electron microscopy. This new method may provide a useful approach for studying routinely processed, celloidin-embedded human temporal bone sections and open a new field in immuno-otopathology. PMID:7680182

Shi, S R; Tandon, A K; Haussmann, R R; Kalra, K L; Taylor, C R

1993-01-01

288

Expression of p53 protein and ki-67 antigen in oral premalignant lesions and oral squamous cell carcinomas: An immunohistochemical study  

PubMed Central

Aim: To study expression of p53 protein and ki-67 antigen in normal, non-dysplastic, dysplastic, premalignant and malignant lesions of the oral mucosa. Materials and Methods: The standard immunohistochemical method along with MIB-1 and DO-7; DAKO antibodies was used to study the expression of p53 and ki-67 in paraffin-embedded tissue specimens. Results: All samples studied showed positive staining for p53 and ki-67. Only one case each from leukoplakia and oral squamous cell carcinoma (OSCC) groups showed negative staining for ki-67. The staining was confined to basal layer in most of the cases except OSCC in which it was seen in all layers. The intensity of staining was moderate to intense. The percentage of p53-positive cells in normal mucosa was 15-25% which was increased to 95% in malignant mucosa. Statistical analysis revealed that the expression of p53 and ki-67 increases as normal oral mucosa becomes dysplastic and undergoes malignant transformation. Conclusion: These results emphasize the potential use of p53 protein and ki-67 antigen as markers of malignant transformation and carcinogenesis in oral premalignant lesions, conditions and OSCC, respectively; and in future they may serve as prognostic tools in the early detection of malignant transformation in oral premalignant lesions and conditions. PMID:22442608

Humayun, S.; Prasad, V. Ram

2011-01-01

289

Down-regulation of class I HLA antigens and of the Epstein-Barr virus-encoded latent membrane protein in Burkitt lymphoma lines.  

PubMed Central

Epstein-Barr virus (EBV)-carrying Burkitt lymphoma (BL) cells are relatively or completely resistant to the lytic effect of major histocompatibility complex class I HLA antigen-restricted cytotoxic T lymphocytes (CTLs) generated by stimulating lymphocytes of EBV-seropositive donors with the autologous EBV-transformed lymphoblastoid cell line (LCL). We previously found that EBV-negative and EBV-carrying BL lines derived from HLA-A11-positive donors were not only resistant to lysis by the HLA-A11-restricted CTL generated by stimulation with the autologous LCL, but also to HLA-A11-specific CTL derived from lymphocytes of an EBV-seronegative donor stimulated with an allogeneic LCL. Using the same and additional cell lines, we now show that the CTL resistance of the BL lines is probably due to a selective down-regulation of HLA-A11. We also show that the EBV-encoded latent membrane protein is expressed at a lower level in the EBV-carrying BL lines than in EBV-transformed LCLs. Only one of eight in vitro EBV-converted BL lines that shifted to a more LCL-like growth pattern expressed LMP at a high level. This line also reexpressed the HLA-A11 antigen that was undetectable in its EBV-negative progenitor. Our findings suggest that the typical BL cell phenotype is associated with low expression of both proteins. Images PMID:3037521

Masucci, M G; Torsteindottir, S; Colombani, J; Brautbar, C; Klein, E; Klein, G

1987-01-01

290

A pre-clinical model of double versus single unit unrelated cord blood transplantation  

PubMed Central

Cord blood transplantation (CBT) with units containing total nucleated cell (TNC) dose >2.5×107/kg is associated with improved engraftment and decreased transplant-related mortality. For many adults no single cord blood units are available that meet the cell dose requirements. We developed a dog model of CBT to evaluate approaches to overcome the problem of low cell dose cord blood units. This study primarily compared double- versus single-unit CBT. Unrelated dogs were bred and cord blood units were harvested. We identified unrelated recipients that were dog leukocyte antigen (DLA)-88 (class I) and DLA-DRB1 (class II) allele-matched with cryopreserved units. Each unit contained ? 1.7×107 TNC/kg. Recipients were given 9.2 Gy total body irradiation and DLA-matched unrelated cord blood with post-grafting cyclosporine and mycophenolate mofetil. After double-unit CBT, 5 dogs engrafted and 4 survived long term with one dominant engrafting unit and prompt immune reconstitution. In contrast, 0 of 5 dogs given single-unit CBT survived beyond 105 days (p=0.03, log-rank test); neutrophil and platelet recovery was delayed (both p=0.005) and recipients developed fatal infections. This new large animal model showed that outcomes were improved after double-unit compared to single-unit CBT. After double-unit CBT, the non-engrafted unit facilitates engraftment of the dominant unit. PMID:20304085

Georges, George E.; Lesnikov, Vladimir; Baran, Szczepan W.; Aragon, Anna; Lesnikova, Marina; Jordan, Robert; Yang, Ya-Ju Laura; Yunusov, Murad Y.; Zellmer, Eustacia; Heimfeld, Shelly; Venkataraman, Gopalakrishnan M.; Harkey, Michael A.; Graves, Scott S.; Storb, Rainer; Storer, Barry E.; Nash, Richard A.

2010-01-01

291

Phase 3 study comparing methotrexate and tacrolimus with methotrexate and cyclosporine for prophylaxis of acute graft-versus-host disease after marrow transplantation from unrelated donors  

Microsoft Academic Search

After the transplantation of unmodified marrow from human leukocyte antigen- matched unrelated donors receiving cy- closporine (CSP) and methotrexate (MTX), the incidence of acute graft-versus-host disease (GVHD) is greater than 75%. Ta- crolimus is a macrolide compound that, in previous preclinical and clinical stud- ies, was effective in combination with MTX for the prevention of acute GVHD. Between March 1995

Richard A. Nash; Joseph H. Antin; Chatchada Karanes; Joseph W. Fay; Belinda R. Avalos; Andrew M. Yeager; Donna Przepiorka; Stella Davies; Finn B. Petersen; Pamela Bartels; Donald Buell; William Fitzsimmons; Claudio Anasetti; Rainer Storb; Voravit Ratanatharathorn

2000-01-01

292

The Predictive Value of HLA-DR Matching and Cytokine Gene Polymorphisms in Renal Allograft Acute Rejection: A Living-unrelated Donor (LURD) Study  

Microsoft Academic Search

Background: In addition to Human Leukocyte Antigens (HLA) compatibility, gene polymorphisms in cytokines might also be important in the quality of allogeneic im- mune response. Objective: To evaluate the influence of HLA-DR matching and a number of cytokine gene polymorphisms on acute rejection after living-unrelated donor (LURD) kidney transplantation. Methods: A total of 42 renal transplants per- formed at Hashemi

Nader Tajik; Tohid Kazemi; Aliakbar Delbandi; Ahad Ghods; Alireza Salek Moghaddam

293

Major membrane surface proteins of Mycoplasma hyopneumoniae selectively modified by covalently bound lipid  

SciTech Connect

Surface protein antigens of Mycoplasma hyopneumoniae were identified by direct antibody-surface binding or by radioimmunoprecipitation of surface /sup 125/I-labeled proteins with a series of monoclonal antibodies (MAbs). Radioimmunoprecipitation of TX-114-phase proteins from cells labeled with (/sup 35/S) methionine, /sup 14/C-amino acids, or (/sup 3/H) palmitic acid showed that proteins p65, p50, and p44 were abundant and (with one other hydrophobic protein, p60) were selectively labeled with lipid. Alkaline hydroxylamine treatment of labeled proteins indicated linkage of lipids by amide or stable O-linked ester bonds. Proteins p65, p50, and p44 were highly immunogenic in the natural host as measured by immunoblots of TX-114-phase proteins with antisera from swine inoculated with whole organisms. These proteins were antigenically and structurally unrelated, since hyperimmune mouse antibodies to individual gel-purified proteins were monospecific and gave distinct proteolytic epitope maps. Intraspecies size variants of one surface antigen of M. hyopneumoniae were revealed by a MAb to p70 (defined in strain J, ATCC 25934), which recognized a large p73 component on strain VPP11 (ATCC 25617). In addition, MAb to internal, aqueous-phase protein p82 of strain J failed to bind an analogous antigen in strain VPP11.

Wise K.S.; Kim, M.F.

1987-12-01

294

Solubilization of the Epstein-Barr virus-determined nuclear antigen and its characterization as a DNA-binding protein.  

PubMed Central

The Eptstein-Barr virus (EBV)-determined nuclear antigen (EBNA) was solubilized from isolate nuclei of two EBV-transformed cell lines- Raji and AW-Ramos, by high-salt treatment. Its DNA-binding properties were studied by DNA-cellulose chromatography and a 51Cr release complement fixation assay. EBNA binds to both double-stranded and single-stranded calf thymus DNA, showing a higher affinity to double-stranded DNA. There was no detectable difference in the DNA binding of EBNA prepared from Raji and AW-Ramos cells. PMID:192908

Luka, J; Siegert, W; Klein, G

1977-01-01

295

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

296

ON THE CONFIGURATION-LP FOR SCHEDULING ON UNRELATED MACHINES  

E-print Network

ON THE CONFIGURATION-LP FOR SCHEDULING ON UNRELATED MACHINES JOSÃ? VERSCHAE AND ANDREAS WIESE Abstract. One of the most important open problems in machine scheduling is the problem of scheduling a set of jobs on unrelated machines to minimize the makespan. The best known approximation algorithm

Nabben, Reinhard

297

Dynamic nature of the association of large tumor antigen and p53 cellular protein with the surfaces of simian virus 40-transformed cells.  

PubMed Central

A molecular complex of simian virus 40 large tumor antigen (T-Ag) and p53 cellular protein is present on the surface of simian virus 40-transformed mouse cells. The stability of the association of the two proteins with the cell surface was characterized. Cells were either surface iodinated by the lactoperoxidase technique or metabolically labeled with [35S]methionine, and surface antigens were detected by differential immunoprecipitation with specific antibodies immediately after labeling or after incubation at 37 degrees C. A rapid, concomitant disappearance of T-Ag and p53 from the cell surface was observed. The half-life of iodinated surface T-Ag was less than 30 min, whereas that of [35S]methionine-labeled surface T-Ag was 1 to 2 h. Although T-Ag and p53 were rapidly lost, both were also rapidly replaced on the cell surface, since newly exposed molecules could be detected when cells were reiodinated after a 2-h chase period. Control experiments established that the loss of the surface molecules was not induced by the iodination reaction. The appearance of surface T-Ag was prevented when cellular protein synthesis was inhibited with cycloheximide. The disappearance and replacement of T-Ag and p53 appeared to be energy-independent processes, as neither was inhibited by sodium azide or 2,4-dinitrophenol. Incubation of iodinated cells at 4 degrees C did block the loss of T-Ag and p53. These observations suggest that T-Ag and p53 are coordinately turned over in the plasma membrane. The nature of the association of the T-Ag-p53 complex with the cell surface can best be described as highly dynamic. Images PMID:6690721

Santos, M; Butel, J S

1984-01-01

298

The Nucleocapsid Protein of Rift Valley Fever Virus Is a Potent Human CD8+ T Cell Antigen and Elicits Memory Responses  

PubMed Central

There is no licensed human vaccine currently available for Rift Valley Fever Virus (RVFV), a Category A high priority pathogen and a serious zoonotic threat. While neutralizing antibodies targeting the viral glycoproteins are protective, they appear late in the course of infection, and may not be induced in time to prevent a natural or bioterrorism-induced outbreak. Here we examined the immunogenicity of RVFV nucleocapsid (N) protein as a CD8+ T cell antigen with the potential for inducing rapid protection after vaccination. HLA-A*0201 (A2)-restricted epitopic determinants were identified with N-specific CD8+ T cells from eight healthy donors that were primed with dendritic cells transduced to express N, and subsequently expanded in vitro by weekly re-stimulations with monocytes pulsed with 59 15mer overlapping peptides (OLPs) across N. Two immunodominant epitopes, VT9 (VLSEWLPVT, N121–129) and IL9 (ILDAHSLYL, N165–173), were defined. VT9- and IL9-specific CD8+ T cells identified by tetramer staining were cytotoxic and polyfunctional, characteristics deemed important for viral control in vivo. These peptides induced specific CD8+ T cell responses in A2-transgenic mice, and more importantly, potent N-specific CD8+ T cell reactivities, including VT9- and IL9-specific ones, were mounted by mice after a booster vaccination with the live attenuated RVF MP-12. Our data suggest that the RVFV N protein is a potent human T cell immunogen capable of eliciting broad, immunodominant CD8+ T cell responses that are potentially protective. Understanding the immune responses to the nucleocapsid is central to the design of an effective RVFV vaccine irrespective of whether this viral protein is effective as a stand-alone immunogen or only in combination with other RVFV antigens. PMID:23527138

Xu, Weidong; Watts, Douglas M.; Costanzo, Margaret C.; Tang, Xiaolei; Venegas, Leon A.; Jiao, Feng; Sette, Alessandro; Sidney, John; Sewell, Andrew K.; Wooldridge, Linda; Makino, Shinji; Morrill, John C.; Peters, Clarence J.; Kan-Mitchell, June

2013-01-01

299

Fibronectin-binding antigen 85 and the 10-kilodalton GroES-related heat shock protein are the predominant TH-1 response inducers in leprosy contacts.  

PubMed

Peripheral blood mononuclear cells from 27 healthy leprosy contacts were analyzed for lymphoproliferation and TH-1 cytokine secretion (interleukin-2 and gamma interferon) in response to heat shock proteins with molecular masses of 65, 18, and 10 kDa from Mycobacterium leprae and the 30-32-kDa antigen 85 (Ag 85) from Mycobacterium bovis BCG. Cells from 18 and 19 of 19 lepromin-positive contacts proliferated or produced TH-1 cytokines in response to the M. leprae 10-kDa protein and to Ag 85, respectively. Limiting-dilution analysis for two lepromin-positive contacts indicated that about one-third of M. leprae-reactive T cells displayed specificity to the M. leprae 10-kDa protein and Ag 85. The M. leprae 65- and 18-kDa proteins were less potent TH-1 response inducers: gamma interferon and interleukin-2 could be measured in 14 and 19 lepromin-positive contacts, respectively. In contrast, very low or undetectable proliferative and cytokine responses were found for 8 lepromin-negative contacts. Our data demonstrate that the fibronectin-binding Ag 85 and the 10-kDa GroES homolog are powerful mycobacterial TH-1 response inducers in the vast majority of lepromin-positive contacts and suggest that they might be valuable candidates for a future subunit vaccine. PMID:7806388

Launois, P; N'Diaye, M N; Cartel, J L; Mane, I; Drowart, A; Van Vooren, J P; Sarthou, J L; Huygen, K

1995-01-01

300

Complex Alternative Cytoplasmic Protein Isoforms of the Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen 1 Generated through Noncanonical Translation Initiation  

PubMed Central

Kaposi's sarcoma-associated herpesvirus (KSHV) latency associated-nuclear antigen 1 (LANA1) protein is constitutively expressed in all KSHV-infected cells, as well as in all forms of KSHV-associated malignancies. LANA1 is a multifunctional KSHV oncoprotein containing multiple repeat sequences that is important for viral episome maintenance and the regulation of cellular and viral gene expression. We characterize here multiple LANA1 isoforms and show that ?50% of LANA1 is naturally generated as N-terminally truncated shoulder proteins that are detected on SDS-PAGE as faster-migrating shoulder bands designated LANA1S. Higher-molecular-weight LANA1S isoforms initiate downstream at noncanonical sites within the N-terminal region, whereas lower-molecular-weight LANA1S isoforms initiate downstream within the central repeat 1 domain. LANA1S proteins lack an N-terminal nuclear localization signal motif, and some isoforms differ from full-length, canonical LANA1 by localizing to perinuclear and cytoplasmic sites. Although LANA1 has until now been assumed to be solely active in the nucleus, this finding indicates that this major KSHV oncoprotein may have cytoplasmic activities as well. KSHV overcomes its limited genetic coding capacity by generating alternatively initiated protein isoforms that may have distinct biological functions. PMID:23255808

Toptan, Tuna; Fonseca, Lidia; Kwun, Hyun Jin; Chang, Yuan

2013-01-01

301

Antigenic differences in nuclear proteins of normal liver and hepatoma. Identification of a nuclear protein present in hepatocytes but absent in hepatoma cells  

Microsoft Academic Search

Two main types of proteins are associated with chromatin. The histones are a well characterized group of basic proteins, which seem to be involved in the general organization of the chromatin. The nonhistone proteins (NHP) ~ are a much more heterogeneous group of proteins and are believed to be involved in the regulation of gene expression (1, 2). If gene

ERKKI RUOSLAHTI; EVA ENGVALL; HANNU JALANKO; DAVID E. COMINGS

1977-01-01

302

Proteins Antigenically Related toMethyl-Accepting Chemotaxis Proteins ofEscherichia coli Detected inaWide RangeofBacterial Species  

Microsoft Academic Search

Thefourmethyl-accepting chemotaxis proteins ofEscherichia coli, often called transducers, aretransmem- branereceptor proteins thatexhibit substantial identity amongthesequences oftheir cytoplasmic domains. Thus, antiserum raised tooneofthese proteins recognizes theothers andmight beexpected torecognize related proteins inother bacteria. We usedantiserum raised tothetransducer Trginimmunoblot experiments to probe awiderange ofbacterial species forthepresence ofantigenically related proteins. Suchproteins were detected inover20different species, representing 6ofthe11eubacterial phyla defined byanalysis ofrRNA sequences aswellasonearchaebacterial

GERALD L. HAZELBAUER

1993-01-01

303

Immune Response to Cryptococcus neoformans Soluble Polysaccharide I. Serological Assay for Antigen and Antibody  

PubMed Central

Chromium chloride was used as a coupling agent for the conjugation of purified cryptococcal polysaccharide to sheep erythrocytes. Sensitized erythrocytes were used in a passive hemagglutination (PHA) assay for antibody to cryptococcal polysaccharide and a passive hemagglutination inhibition (PHI) assay for antigen. The PHA assay was more sensitive than complement fixation, agglutination, or precipitation tests for antibody. The PHI assay could detect submicrogram quantities of soluble polysaccharide. Antigen or antibody could be detected in serum or spinal fluid from seven of eight patients with cryptococcosis. Tests for antigen or antibody were negative with sera from patients with histoplasmosis, blastomycosis, coccidioidomycosis, aspergillosis, or allescheriosis. A low frequency (3%) of positive reactors for antibody was found among sera from normal persons and from persons with unrelated diseases; whereas, all tests for antigen were negative. The assay showed a high degree of sensitivity for immunoglobulins of the immunoglobulin M class; however, cryptococcal antibody of the immunoglobulin G class was also detected. The immunological specificity of the polysaccharide preparation was due to carbohydrate rather than to protein associated with the polysaccharide. PMID:4570986

Kozel, Thomas R.; Cazin, John

1972-01-01

304

Secretion of parasite-specific immunoglobulin G by purified blood B lymphocytes from immune individuals after in vitro stimulation with recombinant Plasmodium falciparum merozoite surface protein-119 antigen  

PubMed Central

The C-terminal 19 000 MW fragment of merozoite surface protein-1 (MSP119) is one of the most promising candidate antigens for a malaria vaccine. Baculovirus recombinant Plasmodium falciparum MSP119 has been used to define conditions for the in vitro production of specific antibodies by purified human blood B cells in a culture system where T-cell signals were provided by the engagement of CD40 molecules and exogenous cytokines. MSP119 preferentially induced surface immunoglobulin G (IgG) -positive (s?+) B lymphocytes from P. falciparum-immune donors to differentiate and produce antigen-specific IgG. In contrast, naïve B cells or cells from non-immune donors could not be induced to secrete parasite-specific IgG in vitro. Although IgG secretion was obtained in the absence of exogenous cytokines, it was dependent on B-cell-derived interleukin-10 (IL-10) and/or B-cell factor(s) under the control of IL-10, since IgG levels were significantly decreased in the presence of neutralizing anti-IL-10 antibodies. These results demonstrate at the cellular level that a single malaria vaccine candidate polypeptide can direct parasite-specific antibody production mediated by the secretion of potentiating factors. PMID:10447733

Garraud, O; Diouf, A; Holm, I; Nguer, C M; Spiegel, A; Perraut, R; Longacre, S

1999-01-01

305

Development of hepatitis B virus capsids into a whole-chain protein antigen display platform: New particulate Lyme disease vaccines  

Microsoft Academic Search

The immunogenicity of peptides and small protein fragments can be considerably enhanced by their presentation on particulate carriers such as capsid-like particles (CLPs) from hepatitis B virus (HBV). HBV CLPs are icosahedral nanoparticles formed by 90 or 120 core protein dimers. Insertions into the immunodominant c\\/e1 B cell epitope, a surface-exposed loop on the HBV capsid protein, are especially immunogenic.

Michael Nassal; Claudia Skamel; Maren Vogel; Peter A. Kratz; Thomas Stehle; Reinhard Wallich; Markus M. Simon

2008-01-01

306

Characterization and modification of the carboxy-terminal sequences of bluetongue virus type 10 NS1 protein in relation to tubule formation and location of an antigenic epitope in the vicinity of the carboxy terminus of the protein.  

PubMed Central

Bluetongue virus produces large numbers of tubules during infection. The tubules are formed from a 552-amino-acid, 64-kDa NS1 protein encoded by the viral double-stranded RNA segment M6. A series of deletion and extension mutants of bluetongue virus serotype 10 NS1 has been generated and expressed in insect cells in order to identify the carboxy-terminal components of the protein which are important for tubule formation. The mutants AcCT5 and AcCT10, lacking 5 and 10 of the carboxy-terminal residues, respectively, were prepared. By analyzing their abilities to form tubules, it was shown that AcCT5 was capable of this function whereas AcCT10 was not, indicating that the last five amino acids are not strongly involved in NS1 tubule formation. Extension mutants including foreign antigenic sequences involving up to 16 amino acids added to the C terminus of NS1 were shown to form tubules, although an extension of 19 amino acids inhibited tubule formation. Analysis of a panel of monoclonal antibodies has established that an NS1 antigenic site is located near the carboxy terminus of the protein. It appears to be exposed on the surface of tubules. The opportunities to develop new vaccines using recombinant NS1 to deliver foreign epitopes are discussed. PMID:7535866

Monastyrskaya, K; Gould, E A; Roy, P

1995-01-01

307

RALP1 Is a Rhoptry Neck Erythrocyte-Binding Protein of Plasmodium falciparum Merozoites and a Potential Blood-Stage Vaccine Candidate Antigen  

PubMed Central

Erythrocyte invasion by merozoites is an obligatory stage of Plasmodium infection and is essential to disease progression. Proteins in the apical organelles of merozoites mediate the invasion of erythrocytes and are potential malaria vaccine candidates. Rhoptry-associated, leucine zipper-like protein 1 (RALP1) of Plasmodium falciparum was previously found to be specifically expressed in schizont stages and localized to the rhoptries of merozoites by immunofluorescence assay (IFA). Also, RALP1 has been refractory to gene knockout attempts, suggesting that it is essential for blood-stage parasite survival. These characteristics suggest that RALP1 can be a potential blood-stage vaccine candidate antigen, and here we assessed its potential in this regard. Antibodies were raised against recombinant RALP1 proteins synthesized by using the wheat germ cell-free system. Immunoelectron microscopy demonstrated for the first time that RALP1 is a rhoptry neck protein of merozoites. Moreover, our IFA data showed that RALP1 translocates from the rhoptry neck to the moving junction during merozoite invasion. Growth and invasion inhibition assays revealed that anti-RALP1 antibodies inhibit the invasion of erythrocytes by merozoites. The findings that RALP1 possesses an erythrocyte-binding epitope in the C-terminal region and that anti-RALP1 antibodies disrupt tight-junction formation, are evidence that RALP1 plays an important role during merozoite invasion of erythrocytes. In addition, human sera collected from areas in Thailand and Mali where malaria is endemic recognized this protein. Overall, our findings indicate that RALP1 is a rhoptry neck erythrocyte-binding protein and that it qualifies as a potential blood-stage vaccine candidate. PMID:24002067

Ito, Daisuke; Hasegawa, Tomoyuki; Miura, Kazutoyo; Yamasaki, Tsutomu; Arumugam, Thangavelu U.; Thongkukiatkul, Amporn; Takeo, Satoru; Takashima, Eizo; Sattabongkot, Jetsumon; Han, Eun-Taek; Long, Carole A.; Torii, Motomi

2013-01-01

308

Expression and purification of Dengue virus type 2 envelope protein as a fusion with hepatitis B surface antigen in Pichia pastoris.  

PubMed

The methylotrophic yeast, Pichia pastoris, has been used as a host to express the envelope protein (Den2E) of dengue type 2 virus (NGC strain) as a chimera with hepatitis B surface antigen (HBsAg): a protein known to self assemble into virus-like particles (VLPs) and to be efficiently expressed in P. pastoris. The Den2E gene used in this study is a truncated version encoding the first 395 amino acid (aa) residues of the mature Den2E protein; the HBsAg gene encodes the full length 226 aa HBsAg protein. Two in-frame gene fusions were constructed for intracellular expression in P. pastoris. The first one contains the HBsAg gene as the 5' partner and the Den2E gene as the 3'partner (HBsAg-Den2E). In the second one, the relative positions of the two partners of the gene fusion were reversed to create the hybrid Den2E-HBsAg gene. These fusion genes were integrated into the genome of P. pastoris under the control of the methanol-inducible alcohol oxidase (AOX1) promoter. Of the two fusions, the Den2E-HBsAg gene was expressed at higher levels in P. pastoris based on Northern analysis. The hybrid protein ( approximately 68 kDa) expressed by this clone was purified to near homogeneity using a combination of acid precipitation, hydrophobic interaction, and immunoaffinity chromatographic steps. Final purification achieved was approximately 1400-fold with a yield of approximately 26%. The chimeric protein was found to possess the ability to assemble into high molecular weight aggregates (akin to HBsAg particles). The recombinant fusion protein eluted close to the void volume of a Sepharose CL-4B column indicating its macromolecular nature. On a CsCl density gradient the recombinant fusion protein sedimented to a position very similar to that of HBsAg VLPs. The hybrid protein is recognized by the two neutralizing monoclonals against the two components of the chimeric protein. PMID:11570849

Bisht, H; Chugh, D A; Swaminathan, S; Khanna, N

2001-10-01

309

Solution Structure of the Factor H-binding Protein, a Survival Factor and Protective Antigen of Neisseria meningitidis*S?  

PubMed Central

Factor H-binding protein is a 27-kDa lipoprotein of Neisseria meningitidis discovered while screening the bacterial genome for vaccine candidates. In addition to being an important component of a vaccine against meningococcus in late stage of development, the protein is essential for pathogenesis because it allows the bacterium to survive and grow in human blood by binding the human complement factor H. We recently reported the solution structure of the C-terminal domain of factor H-binding protein, which contains the immunodominant epitopes. In the present study, we report the structure of the full-length molecule, determined by nuclear magnetic resonance spectroscopy. The protein is composed of two independent barrels connected by a short link. Mapping the residues recognized by monoclonal antibodies with bactericidal or factor H binding inhibition properties allowed us to predict the sites involved in the function of the protein. The structure therefore provides the basis for designing improved vaccine molecules. PMID:19196709

Cantini, Francesca; Veggi, Daniele; Dragonetti, Sara; Savino, Silvana; Scarselli, Maria; Romagnoli, Giacomo; Pizza, Mariagrazia; Banci, Lucia; Rappuoli, Rino

2009-01-01

310

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

311

Improvement of protein immobilization for the elaboration of tumor-associated antigen microarrays: application to the sensitive and specific detection of tumor markers from breast cancer sera.  

PubMed

There is an urgent need to identify relevant tumor markers showing high sensitivity and specificity for early diagnosis and prognosis of breast cancer. Protein microarrays have demonstrated to be cost-effective, high through-put and powerful tools for screening and identifying tumor markers with only minute samples. Autoantibodies directed against tumor-associated antigens (TAAs) were shown to be relevant tumor markers. However, due to the variability of immune response from one individual to another and depending on the type of cancer, detection of only one type of anti-TAA autoantibody is not sufficient to give a reliable and precise diagnosis. It is necessary to use a set of several TAAs for determining specific autoimmune profiles. Therefore, combining various TAAs on different surfaces could improve sensitivity and specificity for anti-TAA autoantibody detection. Herein a panel of 10 proteins, including well-known tumor-associated antigens (TAAs) and potential new biomarkers of breast cancer, were immobilized onto microstructured microarray under optimized conditions (spotting pH buffer, surface chemistry, blocking procedure), in order to determine an autoimmune signature of breast cancer. Sera from 29 breast cancer patients and 28 healthy donors were screened in sandwich immunoassays on the miniaturized system to detect the eventual presence of anti-TAAs autoantibodies. Results indicated that the detection level of each anti-TAA autoantibody in a given serum sample was strongly dependant on the surface chemistry. Combining five TAAs (p53, Hsp60, Hsp70, Her2-Fc, NY-ESO-1) on two different surface chemistries (NHS and APDMES) allowed the significant detection of more than 82% breast cancer sera. PMID:23017679

Yang, Zhugen; Chevolot, Yann; Géhin, Thomas; Solassol, Jérôme; Mange, Alain; Souteyrand, Eliane; Laurenceau, Emmanuelle

2013-02-15

312

Nuclear Relocalization of the Pre-mRNA Splicing Factor PSF during Apoptosis Involves Hyperphosphorylation, Masking of Antigenic Epitopes, and Changes in Protein Interactions  

PubMed Central

The spatial nuclear organization of regulatory proteins often reflects their functional state. PSF, a factor essential for pre-mRNA splicing, is visualized by the B92 mAb as discrete nuclear foci, which disappeared during apoptosis. Because this mode of cell death entails protein degradation, it was considered that PSF, which like other splicing factors is sensitive to proteolysis, might be degraded. Nonetheless, during the apoptotic process, PSF remained intact and was N-terminally hyperphosphorylated on serine and threonine residues. Retarded gel migration profiles suggested differential phosphorylation of the molecule in mitosis vs. apoptosis and under-phosphorylation during blockage of cells at G1/S. Experiments with the use of recombinant GFP-tagged PSF provided evidence that in the course of apoptosis the antigenic epitopes of PSF are masked and that PSF reorganizes into globular nuclear structures. In apoptotic cells, PSF dissociated from PTB and bound new partners, including the U1–70K and SR proteins and therefore may acquire new functions. PMID:11514619

Shav-Tal, Yaron; Cohen, Michal; Lapter, Smadar; Dye, Billy; Patton, James G.; Vandekerckhove, Joel; Zipori, Dov

2001-01-01

313

A novel O-linked glycan modulates Campylobacter jejuni major outer membrane protein-mediated adhesion to human histo-blood group antigens and chicken colonization.  

PubMed

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 Thr(268); previously only flagellin proteins were known to be O-glycosylated in C. jejuni. Substitution of MOMP Thr(268) 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-Thr(268); modelling suggested that O-glycosylation has a notable effect on the conformation of MOMP and this modulates BgAg-binding capacity. Glycosylation of MOMP at Thr(268) 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

314

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

315

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

316

Early Diagnosis of Scrub Typhus with a Rapid Flow Assay Using Recombinant Major Outer Membrane Protein Antigen (r56) of Orientia tsutsugamushi  

PubMed Central

The variable 56-kDa major outer membrane protein of Orientia tsutsugamushi is the immunodominant antigen in human scrub typhus infections. We developed a rapid immunochromatographic flow assay (RFA) for the detection of immunoglobulin M (IgM) and IgG antibodies to O. tsutsugamushi. The RFA employs a truncated recombinant 56-kDa protein from the Karp strain as the antigen. The performance of the RFA was evaluated with a panel of 321 sera (serial bleedings of 85 individuals suspected of scrub typhus) which were collected in the Pescadore Islands, Taiwan, from 1976 to 1977. Among these 85 individuals, IgM tests were negative for 7 cases by both RFA and indirect fluorescence assay (IFA) using Karp whole-cell antigen. In 29 cases specific responses were detected by the RFA earlier than by IFA, 44 cases had the same detection time, and 5 cases were detected earlier by IFA than by RFA. For IgG responses, 4 individuals were negative with both methods, 37 cases exhibited earlier detection by RFA than IFA, 42 cases were detected at the same time, and 2 cases were detected earlier by IFA than by RFA. The sensitivities of RFA detection of antibody in sera from confirmed cases were 74 and 86% for IgM and IgG, respectively. When IgM and IgG results were combined, the sensitivity was 89%. A panel of 78 individual sera collected from patients with no evidence of scrub typhus was used to evaluate the specificity of the RFA. The specificities of the RFA were 99% for IgM and 97% for IgG. The sensitivities of IFA were 53 and 73% for IgM and IgG, respectively, and were 78% when the results of IgM and IgG were combined. The RFA test was significantly better than the IFA test for the early detection of antibody to scrub typhus in primary infections, while both tests were equally sensitive with reinfected individuals. PMID:11238230

Ching, W.-M.; Rowland, D.; Zhang, Z.; Bourgeois, A. L.; Kelly, D.; Dasch, G. A.; Devine, P. L.

2001-01-01

317

Early diagnosis of scrub typhus with a rapid flow assay using recombinant major outer membrane protein antigen (r56) of Orientia tsutsugamushi.  

PubMed

The variable 56-kDa major outer membrane protein of Orientia tsutsugamushi is the immunodominant antigen in human scrub typhus infections. We developed a rapid immunochromatographic flow assay (RFA) for the detection of immunoglobulin M (IgM) and IgG antibodies to O. tsutsugamushi. The RFA employs a truncated recombinant 56-kDa protein from the Karp strain as the antigen. The performance of the RFA was evaluated with a panel of 321 sera (serial bleedings of 85 individuals suspected of scrub typhus) which were collected in the Pescadore Islands, Taiwan, from 1976 to 1977. Among these 85 individuals, IgM tests were negative for 7 cases by both RFA and indirect fluorescence assay (IFA) using Karp whole-cell antigen. In 29 cases specific responses were detected by the RFA earlier than by IFA, 44 cases had the same detection time, and 5 cases were detected earlier by IFA than by RFA. For IgG responses, 4 individuals were negative with both methods, 37 cases exhibited earlier detection by RFA than IFA, 42 cases were detected at the same time, and 2 cases were detected earlier by IFA than by RFA. The sensitivities of RFA detection of antibody in sera from confirmed cases were 74 and 86% for IgM and IgG, respectively. When IgM and IgG results were combined, the sensitivity was 89%. A panel of 78 individual sera collected from patients with no evidence of scrub typhus was used to evaluate the specificity of the RFA. The specificities of the RFA were 99% for IgM and 97% for IgG. The sensitivities of IFA were 53 and 73% for IgM and IgG, respectively, and were 78% when the results of IgM and IgG were combined. The RFA test was significantly better than the IFA test for the early detection of antibody to scrub typhus in primary infections, while both tests were equally sensitive with reinfected individuals. PMID:11238230

Ching, W M; Rowland, D; Zhang, Z; Bourgeois, A L; Kelly, D; Dasch, G A; Devine, P L

2001-03-01

318

Characterisation of novel linear antigen epitopes on North American-type porcine reproductive and respiratory syndrome virus M protein.  

PubMed

The M protein, encoded by the porcine reproductive and respiratory syndrome virus (PRRSV) ORF6 gene, is considered to be one of the most conserved PRRSV proteins. In recent decades, highly specific monoclonal antibodies (Mabs) have been exploited to provide reliable diagnoses for many diseases. In this study, two different Mab clones targeting the linear epitopes on the PRRSV M protein were generated and characterized. Both Mabs showed binding activity against the native PRRSV virion and recombinant M protein when analyzed by immunofluorescence assay (IFA) and Western blot. The targeted epitope of each Mab was mapped by serial truncation of the M protein to generate overlapping fragments. Fine epitope mapping was then performed using a panel of expressed polypeptides. The polypeptide sequences of the two epitopes recognized by Mabs 1C8 and 3F7 were (3)SSLD(6) and (155)VLGGRKAVK(163), respectively, with the former being a newly identified epitope on the M protein. In both cases, these two epitopes were finely mapped for the first time. Alignments of Mab epitope sequences revealed that the two epitopes on the M protein were highly conserved between the North American-type strains. These Mabs, along with their mapped epitopes, are useful for the development of diagnostic and research tools, including immunofluorescence, ELISA and Western blot. PMID:25037720

Wang, Qian; Chen, Jiazeng; Peng, Jinmei; An, Tongqing; Leng, Chaoliang; Sun, Yan; Guo, Xin; Ge, Xinna; Tian, Zhijun; Yang, Hanchun

2014-11-01

319

Safety of treatment with DLA-identical or unrelated mesenchymal stromal cells in DLA-identical canine bone marrow transplantation  

PubMed Central

Background: Although in vitro and in vivo experiments have suggested that mesenchymal stromal cells (MSC) may have important immunomodulatory functions in allogeneic hematopoietic cell transplantation (HCT), results from clinical studies have been inconsistent. In the current study we investigate the safety of dog leukocyte antigen (DLA) identical or third party unrelated MSC in DLA-identical HCT. Results: There were no differences between treatment groups in depth of granulocyte or platelet nadirs, time to granulocyte or platelet engraftment, rate of acute GVHD or rejection. All dogs tolerated the MSC infusion well, although 2 dogs treated with unrelated MSC were euthanized on day 9 due to complications unrelated to the MSC infusion. While no formation of ectopic tissue was observed, GFP positive signals in bone marrow, spleen or liver were detected at time of necropsy in 75% and 50% of dogs treated with DLA-identical or unrelated MSC, respectively. Discussion: Treatment with DLA-identical or unrelated MSC in high dose DLA-identical HCT is safe, and provides a large animal HCT model in which to investigate immunological mechanisms and optimal treatment strategies for future human trials. Methods: Fourteen dogs were treated with 920 cGy total body irradiation (TBI) followed by transplantation of marrow from DLA-identical littermates and immunosuppression with cyclosporine. Prior to infusion of marrow, dogs received infusions of DLA-identical MSC from the marrow donor (n = 4), unrelated MSC (n = 4), or culture medium (n = 6), within 1 h of TBI. MSC obtained from relevant donors were ex-vivo expanded and transduced with GFP-retrovirus before infusion. PMID:23723082

Kornblit, Brian; Leisenring, Wendy M.; Santos, Erlinda B.; Storb, Rainer; Sandmaier, Brenda M.

2013-01-01

320

An antigen chimera of poliovirus induces antibodies against human papillomavirus type 16.  

PubMed Central

It has been established that the surface of poliovirus type 1 can be extensively modified to incorporate antigenic domains from other poliovirus serotypes and from unrelated viruses. The fact that the modified (chimeric) viruses exhibit dual antigenicity and immunogenicity led us to explore the possibility of using the Sabin vaccine strain of poliovirus type 1 as a vector for the presentation of antigenic domains from human papillomavirus type 16 (HPV-16), a virus associated with the development of cervical carcinoma. We report here the construction and characterization of a chimeric poliovirus containing a 16-residue sequence derived from the major capsid protein (L1) of HPV-16. This virus chimera stimulated the production in rabbits of antibodies which recognized the HPV-16-derived peptide and an L1 fusion protein synthesized in Escherichia coli and detected HPV-16 in human biopsy material by immunoperoxidase staining. The possibility that poliovirus-HPV chimeras could be used as vaccines against HPV-16 is discussed. Images PMID:2154604

Jenkins, O; Cason, J; Burke, K L; Lunney, D; Gillen, A; Patel, D; McCance, D J; Almond, J W

1990-01-01

321

Heat shock proteins in juvenile idiopathic arthritis: Keys for understanding remitting arthritis and candidate antigens for immune therapy  

Microsoft Academic Search

Juvenile idiopathic arthritis (JIA) is in a majority of the cases of self-limiting, and sometimes even a self-remitting, disease.\\u000a A growing amount of data suggests that active T cell regulation determines, at least partly, the clinical outcome of JIA.\\u000a In experimental models of arthritis, a group of highly conserved microbial proteins, heat shock proteins (hsps), can be used\\u000a to effectively

Berent Prakken; Wietse Kuis; Willem van Eden; Salvatore Albani

2002-01-01

322

Association of Simian Virus 40 Vp1 with 70-Kilodalton Heat Shock Proteins and Viral Tumor Antigens  

Microsoft Academic Search

Proper folding of newly synthesized viral proteins in the cytoplasm is a prerequisite for the formation of infectious virions. The major capsid protein Vp1 of simian virus 40 forms a series of disulfide-linked inter- mediates during folding and capsid formation. In addition, we report here that Vp1 is associated with cellular chaperones (HSP70) and a cochaperone (Hsp40) which can be

Peggy P. Li; Noriko Itoh; Marika Watanabe; Yunfan Shi; Peony Liu; Hui-Jung Yang; Harumi Kasamatsu

2009-01-01

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

Antigenic regions within the hepatitis C virus envelope 1 and non-structural proteins: identification of an IgG3-restricted recognition site with the envelope 1 protein.  

PubMed Central

Antibody binding to antigenic regions of hepatitis C virus (HCV) envelope 1 (E1; residues 183-380, E2/non-structural (NS) 1 (residues 380-437), NS1 (residues 643-690), and NS4 (1684-1751) proteins were assayed for 50 sera with antibodies to HCV (anti-HCV) and for 46 sera without anti-HCV. Thirty-four peptides, 18 residues long with an eight-amino acid overlap within each HCV region, were synthesized and tested with all 96 sera. Within the E region 183-380, the major binding site was located to residues 203-220, and was recognized by eight sera. Within the E2/NS1 region 380-437, the peptide covering residues 410-427 was recognized by two sera, and within the NS1 region 643-690, peptides covering residues 663-690 were recognized by four sera. Within the NS4 region 1684-1751, 27 sera were reactive to one or more of the NS4 peptides, and 21 out of these were reactive with peptide 1694-1711. One part of the major binding site could be located to residues 1701-1704, with the sequence Leu-Tyr-Arg-Glu. The IgG1, IgG3 and IgG4 subclasses were reactive with the five antigenic regions of HCV core, residues 1-18, 11-28, 21-38, 51-68 and 101-118. Reactivity to the major envelope site consisted almost exclusively of IgG3, and reactivity to the major site of NS4 consisted only of IgG1. Thus, a non-restricted IgG response to linear HCV-encoded binding sites was found to the core protein, whereas IgG subclass-restricted linear binding sites were found within the E1 protein, and within the NS4 protein. PMID:7680297

Sallberg, M; Ruden, U; Wahren, B; Magnius, L O

1993-01-01

325

Isolation and biochemical and molecular analyses of a species-specific protein antigen from the gastric pathogen Helicobacter pylori.  

PubMed

A protein of Mr 26,000 which was present in large quantities in extracts of cells of Helicobacter pylori was purified to homogeneity by ammonium sulfate precipitation followed by gel filtration and reversed-phase chromatography or anion-exchange chromatography. The protein appeared to be associated with the soluble fraction of the cell, and antibodies raised against the protein were reactive with whole-cell lysates of a variety of H. pylori strains in a simple immunodot blot assay. This reaction was species specific. Protein sequence determination of the amino terminus and internal cyanogen bromide fragments and amino acid composition analysis were performed. An oligonucleotide derived from these data was used to clone a fragment encoding most of the coding sequence. Expression in Escherichia coli was dependent on vector promoters. The DNA sequence of the fragment was determined. DNA probes derived from the cloned fragment hybridized to genomic DNA of all H. pylori strains tested, but not to DNAs of Helicobacter mustelae, Wolinella succinogenes, various Campylobacter species, and a panel of gram-negative enteric bacteria. The apparent uniqueness of this protein may be exploited for the development of species-specific diagnostics for this gastric pathogen. PMID:1987145

O'Toole, P W; Logan, S M; Kostrzynska, M; Wadström, T; Trust, T J

1991-01-01

326

Effective vaccination of cattle using the virion G protein of bovine ephemeral fever virus as an antigen.  

PubMed

In a series of experiments, the envelope glycoprotein (G protein) of bovine ephemeral fever virus (BEFV) induced immunity against challenge with virulent virus. Protection correlated with the level of specific serum antibodies to G protein measured by a blocking ELISA test and with the level of neutralizing antibody. The optimum vaccination regimen consisted of two injections given 21 days apart at a dose rate of 0.32 microgram per cow of purified G protein emulsified in the adjuvant Quil A. This schedule conferred immunity for the duration of the preliminary experiment (46 days). Immunity to severe disease, but not to infection, remained for at least 12 months after vaccination, although BEFV could not be reisolated from vaccinated cattle following challenge. Unvaccinated cattle used as controls exhibited typical signs of clinical ephemeral fever and BEFV was recovered from all control animals following challenge. PMID:7975863

Uren, M F; Walker, P J; Zakrzewski, H; St George, T D; Byrne, K A

1994-07-01

327

Internalization of a Bacillus anthracis protective antigen-c-Myc fusion protein mediated by cell surface anti-c-Myc antibodies.  

PubMed Central

BACKGROUND: Anthrax toxin, secreted by Bacillus anthracis, consists of protective antigen (PA) and either lethal factor (LF) or edema factor (EF). PA, the receptor-binding component of the toxin, translocates LF or EF into the cytosol, where the latter proteins exert their toxic effects. We hypothesized that anthrax toxin fusion proteins could be used to kill virus-infected cells and tumor cells, if PA could be redirected to unique receptors found only on these cells. MATERIALS AND METHODS: To test this hypothesis in a model system, amino acids 410-419 of the human p62(c-myc) epitope were fused to the C-terminus of PA to redirect PA to the c-Myc-specific hybridoma cell line 9E10. RESULTS: The PA-c-Myc fusion protein killed both mouse macrophages and 9E10 hybridoma cells when administered with LF or an LF fusion protein (FP59), respectively. Similar results were obtained with PA, which suggests that PA-c-Myc used the endogenous PA receptor to enter the cells. By blocking the endogenous PA receptors on 9E10 cells with the competitive inhibitor PA SNKEDeltaFF, the PA-c-Myc was directed to an alternate receptor, i.e., the anti-c-Myc antibodies presented on the cell surface. The c-Myc IgG were proven to act as receptors because the addition of a synthetic peptide containing the c-Myc epitope along with PA SNKEDeltaFF further reduced the toxicity of PA-c-Myc + FP59. CONCLUSION: This study shows that PA can be redirected to alternate receptors by adding novel epitopes to the C-terminus of PA, enabling the creation of cell-directed toxins for therapeutic purposes. PMID:9508786

Varughese, M.; Chi, A.; Teixeira, A. V.; Nicholls, P. J.; Keith, J. M.; Leppla, S. H.

1998-01-01

328

Cooperative action of cellular proteins YB-1 and Pur alpha with the tumor antigen of the human JC polyomavirus determines their interaction with the viral lytic control element.  

PubMed Central

Human JC polyomavirus (JCV) is the etiologic agent of the neurodegenerative disease progressive mulifocal leukoencephalopathy. By using JCV as a model, we investigated the role of the viral early protein tumor antigen (TAg) in the binding of two cellular proteins, Pura alpha and YB-1, to JCV regulatory sequences. Results from band-shift assays with purified YB-1, Pur alpha, and TAg indicated that efficient binding of Pur alpha, a strong activator of early gene transcription, to a single-stranded target sequence corresponding to the viral lytic control element, is diminished in the presence of the late gene activator YB-1, which recognizes the opposite strand of the Pur alpha binding site. Of particular interest was the ability of Pur alpha and TAg to enhance binding of YB-1 to DNA molecules without being associated with this complex. Binding studies using a mutant peptide encompassing the N terminus of YB-1 indicated that the C terminus of YB-1 is important for its DNA binding activity. The ability of Pur alpha and TAg to increase binding of YB-1 to DNA is independent of the YB-1 C terminus. Similarly, results from band-shift assays using Pur alpha variants indicated that two distinct regions of this protein contribute either to its ability to bind DNA or to its ability to enhance YB-1 DNA binding activity. Based on the interaction of Pur alpha, YB-1, and TAg, and their binding to DNA, a model is proposed for the role of these proteins in transcription of viral early and late genes during the lytic cycle. Images Fig. 1 Fig. 2 Fig. 3 Fig. 5 PMID:7862639

Chen, N N; Chang, C F; Gallia, G L; Kerr, D A; Johnson, E M; Krachmarov, C P; Barr, S M; Frisque, R J; Bollag, B; Khalili, K

1995-01-01

329

Identification of classical swine fever virus protein E2 as a target for cytotoxic T cells by using mRNA-transfected antigen-presenting cells.  

PubMed

Vaccination of pigs against Classical swine fever virus (CSFV) by using live-virus vaccines induces early protection before detectable humoral immune responses. Immunological analyses indicate that this is associated with T-cell activation, underlining the importance of targeting cytotoxic T-lymphocyte (CTL) responses for vaccine improvement. Antigen-presenting cells (APCs) transfected with mRNA encoding structural protein E2 or non-structural viral proteins NS3-NS4A were used to identify viral genes encoding CTL epitopes. Monocyte-derived dendritic cells (DCs) and fibrocytes served as the APCs. In vitro translation of the mRNA and microscopic analysis of transfected cells demonstrated that E2 and NS3-NS4A could be identified. APCs transfected with either of the mRNA molecules restimulated CSFV-specific T cells to produce gamma interferon and specific cytotoxic activity against CSFV-infected target cells. The presence of CTL epitopes on E2 was confirmed by using d/d-haplotype MAX cells expressing E2 constitutively as target cells in d/d-haplotype CTL assays. A potent CTL activity against E2 was detected early (1-3 weeks) after CSFV challenge. This work corroborates the existence of CTL epitopes within the non-structural protein domain NS3-NS4A of CSFV. Furthermore, epitopes on the E2 protein can also now be classified as targets for CTLs, having important implications for vaccine design, especially subunit vaccines. As for the use of mRNA-transfected APCs, this represents a simple and efficient method to identify viral genes encoding CTL epitopes in outbred populations. PMID:16099911

Ceppi, M; de Bruin, M G M; Seuberlich, T; Balmelli, C; Pascolo, S; Ruggli, N; Wienhold, D; Tratschin, J D; McCullough, K C; Summerfield, A

2005-09-01

330

Antigen-Specific T-Cell Responses in Humans after Intranasal Immunization with a Meningococcal Serogroup B Outer Membrane Vesicle Vaccine  

PubMed Central

We have studied the ability of the Norwegian group B meningococcal outer membrane vesicle (OMV) vaccine, when administered intranasally without adjuvant, to induce T-cell responses in humans. A group of 12 vaccinees was immunized with four doses of OMVs (250 ?g of protein/dose) at weekly intervals, and a single booster dose was given 5 months later. In vitro T-cell proliferation in response to the OMV vaccine, purified PorA (class 1) protein, PorB (class 3) protein, and one unrelated control antigen (Mycobacterium bovis BCG) was measured by [3H]thymidine incorporation into peripheral blood mononuclear cells obtained from the vaccinees before and after the immunizations. The nasal OMV immunizations induced antigen-specific T-cell responses in the majority of the vaccinees when tested against OMVs (7 of 12) and the PorA antigen (11 of 12). None of the vaccinees showed a vaccine-induced T-cell response to the PorB antigen after the initial four doses. Although some individuals responded to all the vaccine antigens after the booster dose, this response was not significant when the vaccinees were analyzed as a group. We have also demonstrated that the PorA antigen-specific T-cell responses correlated with anti-OMV immunoglobulin A (IgA) levels in nasal secretions, with anti-OMV IgG levels in serum, and with serum bactericidal activity. In conclusion, we have shown that it is possible to induce antigen-specific T-cell responses in humans by intranasal administration of a meningococcal OMV vaccine without adjuvant. PMID:9916109

Oftung, Fredrik; Naess, Lisbeth Meyer; Wetzler, Lee M.; Korsvold, Gro Ellen; Aase, Audun; H?iby, E. Arne; Dalseg, Rolf; Holst, Johan; Michaelsen, Terje E.; Haneberg, Bj?rn

1999-01-01

331

Diagnostic Value of Proteins of Three Borrelia Species (Borrelia burgdorferi Sensu Lato) and Implications for Development and Use of Recombinant Antigens for Serodiagnosis of Lyme Borreliosis in Europe  

PubMed Central

More and more assays for the serodiagnosis of Lyme borreliosis (LB) are based on recombinant antigens. However, so far, there is no consensus as to which are the most specific and sensitive proteins and how they should be used in combination to obtain tests with the best discrimination abilities. The present study was preceded by a detailed analysis of Western blots (WB) using whole-cell lysates of Borrelia burgdorferi sensu stricto strain PKa2, B. afzelii PKo, and B. garinii PBi (U. Hauser, G. Lehnert, R. Lobentanzer, and B. Wilske, J. Clin. Microbiol. 35:1433–1444, 1997). For the present work, the data bank from that study, containing information about the reactivities of 330 sera (from patients at different stages of LB [n = 189]; control group, n = 141), was reused. The specificities and sensitivities of various combinations of proteins from different strains were calculated for different interpretation criteria. For immunoglobulin G (IgG) WB, the recommended combination of antigens available to date as recombinant proteins included p83/100 of PKa2, p83/100 of PKo, p39 of PKo, p39 of PBi, and OspC of PBi (interpretation criterion, at least one reactive band required for a positive WB; specificity, 96.5%; sensitivity, 56.1%). The further addition of p58 of PKo, p17 of PKo, or p14 of PKo was most favorable in terms of both a considerable gain of sensitivity and little loss of specificity. IgG Western blotting with a whole-cell lysate of strain PKo might be improved by the addition of OspC of PBi. For IgG WB, the best combination, out of all bands, was p83/100, p58, p39, p30, and p21 of all three strains and OspC of PBi, p17b of PBi, p56 of PKa2, p43 of PKo, p17 of PKo, and p14 of PKo (interpretation criterion, at least two reactive bands required for a positive WB; specificity, 97.2%; sensitivity, 61.4%). An interpretation criterion of at least two reactive bands is more reliable than one of only one reactive band. For IgM WB, the best combination was OspC of PKo, OspC of PBi, p39 of all three strains, p17 of PKo, and strong reactions with p41 of all three strains (interpretation criterion, at least one reactive band required; specificity, 97.9%; sensitivity, 47.0%). PMID:9665948

Hauser, Ulrike; Lehnert, Gisela; Wilske, Bettina

1998-01-01

332

Purification and characterization of protein disulphide-isomerase from the unicellular green alga Chlamydomonas reinhardii. A 120 kDa dimer antigenically distinct from the vertebrate enzyme.  

PubMed Central

Protein disulphide-isomerase (PDI) has been isolated from the unicellular green alga Chlamydomonas reinhardii and purified by (NH4)2SO4 precipitation, gel filtration and DEAE-Sephacel, hydroxyapatite and f.p.l.c. chromatography. The active algal enzyme is a 120 kDa dimer with a subunit molecular mass of 60 kDa when determined by SDS/PAGE. Although similar in size to the previously isolated vertebrate PDIs, the algal enzyme is antigenically distinct, polyclonal antibodies against the algal PDI showing no cross-reactivity with the vertebrate enzyme on immunoblots, and vice versa. The anti-(algal PDI) antiserum did not inhibit algal PDI activity, and C. reinhardii PDI could be immobilized on anti-PDI-Protein A-Sepharose in active form. In contrast with the situation in vertebrates, where PDI functions as a subunit of prolyl 4-hydroxylase, the C. reinhardii PDI is not associated with the algal prolyl 4-hydroxylase. Images Fig. 1. Fig. 4. PMID:2344363

Kaska, D D; Kivirikko, K I; Myllylä, R

1990-01-01

333

Production, characterization, and application of monoclonal antibodies specific to recombinant (E2) structural protein in antigen-capture ELISA for clinical diagnosis of Chikungunya virus.  

PubMed

The resurgence of Chikungunya (CHIK) virus in the form of an explosive, unprecedented epidemic with high virulence and unusual numbers of fatalities has created an immense public health concern in recent years. In the absence of an effective vaccine and specific antiviral therapy, early accurate diagnosis is essential for the best patient management. The present study describes the production and characterization of high-affinity and selective monoclonal antibodies (Mabs) against recombinant E2 protein (rE2) of the CHIK virus. The reactivity of Mabs for rE2 protein was demonstrated using ELISA. The specificity of the generated Mabs for rE2 was demonstrated by Western blot and indirect immunofluorescence. The application of this CHIK virus E2-specific monoclonal antibody in early clinical diagnosis was demonstrated by various analytical methods, such as immunoblotting, indirect immunofluorescence assay (IFA), and antigen-capture ELISA (AC-ELISA), for the detection as well as the identification of the novel ECSA genotypes of CHIK virus. These findings suggest that the high-affinity E2-specific monoclonal antibodies reported in this study will be useful for early clinical diagnosis and epidemiological studies of CHIK virus in developing countries. PMID:22420756

Kumar, Jyoti S; Khan, Mohsin; Gupta, Garima; Bhoopati, Manna; Lakshmana Rao, P V; Parida, Manmohan

2012-04-01

334

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

335

The NS1 protein of influenza A virus suppresses interferon-regulated activation of antigen-presentation and immune-proteasome pathways.  

PubMed

The NS1 protein of influenza virus counters host antiviral defences primarily by antagonizing the type I interferon (IFN) response. Both the N-terminal dsRNA-binding domain and the C-terminal effector domain are required for optimal suppression of host responses during infection. To better understand the regulatory role of the NS1 effector domain, we used an NS1-truncated mutant virus derived from human H1N1 influenza isolate A/Texas/36/91 (Tx/91) and assessed global transcriptional profiles from two independent human lung cell-culture models. Relative to the wild-type Tx/91-induced gene expression, the NS1 mutant virus induced enhanced expression of innate immune genes, specifically NF-?B signalling-pathway genes and IFN-? and -? target genes. We queried an experimentally derived IFN gene set to gauge the proportion of IFN-responsive genes that are suppressed specifically by NS1. We show that the C-terminally truncated NS1 mutant virus is less efficient at suppressing IFN-regulated gene expression associated with activation of antigen-presentation and immune-proteasome pathways. This is the first report integrating genomic analysis from two independent human culture systems, including primary lung cells, using genetically similar H1N1 influenza viruses that differ only in the length of the NS1 protein. PMID:21593271

Tisoncik, Jennifer R; Billharz, Rosalind; Burmakina, Svetlana; Belisle, Sarah E; Proll, Sean C; Korth, Marcus J; García-Sastre, Adolfo; Garcíia-Sastre, Adolfo; Katze, Michael G

2011-09-01

336

Expression of the Grb2-related protein of the lymphoid system in B cell subsets enhances B cell antigen receptor signaling through mitogen-activated protein kinase pathways.  

PubMed

Adapter proteins play a critical role in regulating signals triggered by Ag receptor cross-linking. These small molecules link receptor proximal events with downstream signaling pathways. In this study, we explore the expression and function of the Grb2-related protein of the lymphoid system (GrpL)/Grb2-related adaptor downstream of Shc adapter protein in human B cells. GrpL is expressed in naive B cells and is down-regulated following B cell Ag receptor ligation. By contrast, germinal center and memory B cells express little or no GrpL. Using human B cell lines, we detected constitutive interactions between GrpL and B cell linker protein, Src homology (SH)2 domain-containing leukocyte protein of 76 kDa, hemopoietic progenitor kinase 1, and c-Cbl. The N-terminal SH3 domain of GrpL binds c-Cbl while the C-terminal SH3 domain binds B cell linker protein and SH2 domain-containing leukocyte protein of 76 kDa. Exogenous expression of GrpL in a GrpL-negative B cell line leads to enhanced Ag receptor-induced extracellular signal-related kinase and p38 mitogen-activated protein kinase phosphorylation. Thus, GrpL expression in human B cell subsets appears to regulate Ag receptor-mediated signaling events. PMID:12496419

Yankee, Thomas M; Solow, Sasha A; Draves, Kevin D; Clark, Edward A

2003-01-01

337

The ribosomal protein P0 of soybean (Glycine max L. Merr.) has antigenic cross-reactivity to soybean seed lectin  

Microsoft Academic Search

Soybean (Glycine max L. Merr.) mutants lacking the ability to produce the lectin normally found in soybean seeds (SBL) are designated Le-. A protein of higher molecular weight that cross-reacts with antibodies raised to SBL was found at nearly equivalent levels in roots, hypocotyls, and leaves, and at lower levels in cotyledons and dry seeds of both Le+ and Le-

Keith L. Wycoff; Pieternel van Rhijn; Ann M. Hirsch

1997-01-01

338

The Tn Antigen-Specific Lectin from Ground Ivy Is an Insecticidal Protein with an Unusual Physiology  

Microsoft Academic Search

Leaves of ground ivy (Glechoma hederacea) contain a lectin (called Gleheda) that is structurally and evolutionary related to the classical legume lectins. Screening of a population of wild plants revealed that Gleheda accounts for more than one-third of the total leaf protein in some clones, whereas it cannot be detected in other clones growing in the same environment. Gleheda is

Weifang Wang; Bettina Hause; Willy J. Peumans; Guy Smagghe; Anne Mackie; Robin Fraser; Els J. M. Van Damme

2003-01-01

339

Epstein-Barr virus nuclear antigen leader protein localizes to promoters and enhancers with cell transcription factors and EBNA2  

PubMed Central

Epstein–Barr virus (EBV) nuclear antigens EBNALP (LP) and EBNA2 (E2) are coexpressed in EBV-infected B lymphocytes and are critical for lymphoblastoid cell line outgrowth. LP removes NCOR and RBPJ repressive complexes from promoters, enhancers, and matrix-associated deacetylase bodies, whereas E2 activates transcription from distal enhancers. LP ChIP-seq analyses identified 19,224 LP sites of which ?50% were ±2 kb of a transcriptional start site. LP sites were enriched for B-cell transcription factors (TFs), YY1, SP1, PAX5, BATF, IRF4, ETS1, RAD21, PU.1, CTCF, RBPJ, ZNF143, SMC3, NF?B, TBLR, and EBF. E2 sites were also highly enriched for LP-associated cell TFs and were more highly occupied by RBPJ and EBF. LP sites were highly marked by H3K4me3, H3K27ac, H2Az, H3K9ac, RNAPII, and P300, indicative of activated transcription. LP sites were 29% colocalized with E2 (LP/E2). LP/E2 sites were more similar to LP than to E2 sites in associated cell TFs, RNAPII, P300, and histone H3K4me3, H3K9ac, H3K27ac, and H2Az occupancy, and were more highly transcribed than LP or E2 sites. Gene affected by CTCF and LP cooccupancy were more highly expressed than genes affected by CTCF alone. LP was at myc enhancers and promoters and of MYC regulated ccnd2, 23 med complex components, and MYC regulated cell survival genes, igf2r and bcl2. These data implicate LP and associated TFs and DNA looping factors CTCF, RAD21, SMC3, and YY1/INO80 chromatin-remodeling complexes in repressor depletion and gene activation necessary for lymphoblastoid cell line growth and survival. PMID:24167291

Portal, Daniel; Zhou, Hufeng; Zhao, Bo; Kharchenko, Peter V.; Lowry, Elizabeth; Wong, Limsoon; Quackenbush, John; Holloway, Dustin; Jiang, Sizun; Lu, Yong; Kieff, Elliott

2013-01-01

340

Low-Dose Adenovirus Vaccine Encoding Chimeric Hepatitis B Virus Surface Antigen-Human Papillomavirus Type 16 E7 Proteins Induces Enhanced E7-Specific Antibody and Cytotoxic T-Cell Responses  

PubMed Central

Induction of effective immune responses may help prevent cancer progression. Tumor-specific antigens, such as those of human papillomaviruses involved in cervical cancer, are targets with limited intrinsic immunogenicity. Here we show that immunization with low doses (106 infectious units/dose) of a recombinant human adenovirus type 5 encoding a fusion of the E7 oncoprotein of human papillomavirus type 16 to the carboxyl terminus of the surface antigen of hepatitis B virus (HBsAg) induces remarkable E7-specific humoral and cellular immune responses. The HBsAg/E7 fusion protein assembled efficiently into virus-like particles, which stimulated antibody responses against both carrier and foreign antigens, and evoked antigen-specific kill of an indicator cell population in vivo. Antibody and T-cell responses were significantly higher than those induced by a control adenovirus vector expressing wild-type E7. Such responses were not affected by preexisting immunity against either HBsAg or adenovirus. These data demonstrate that the presence of E7 on HBsAg particles does not interfere with particle secretion, as it occurs with bigger proteins fused to the C terminus of HBsAg, and results in enhancement of CD8+-mediated T-cell responses to E7. Thus, fusion to HBsAg is a convenient strategy for developing cervical cancer therapeutic vaccines, since it enhances the immunogenicity of E7 while turning it into an innocuous secreted fusion protein. PMID:16188983

Baez-Astua, Andres; Herraez-Hernandez, Elsa; Garbi, Natalio; Pasolli, Hilda A.; Juarez, Victoria; zur Hausen, Harald; Cid-Arregui, Angel

2005-01-01

341

Characterization of PbPga1, an Antigenic GPI-Protein in the Pathogenic Fungus Paracoccidioides brasiliensis  

PubMed Central

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), one of the most prevalent mycosis in Latin America. P. brasiliensis cell wall components interact with host cells and influence the pathogenesis of PCM. Cell wall components, such as glycosylphosphatidylinositol (GPI)-proteins play a critical role in cell adhesion and host tissue invasion. Although the importance of GPI-proteins in the pathogenesis of other medically important fungi is recognized, little is known about their function in P. brasiliensis cells and PCM pathogenesis. We cloned the PbPga1 gene that codifies for a predicted GPI-anchored glycoprotein from the dimorphic pathogenic fungus P. brasiliensis. PbPga1 is conserved in Eurotiomycetes fungi and encodes for a protein with potential glycosylation sites in a serine/threonine-rich region, a signal peptide and a putative glycosylphosphatidylinositol attachment signal sequence. Specific chicken anti-rPbPga1 antibody localized PbPga1 on the yeast cell surface at the septum between the mother cell and the bud with stronger staining of the bud. The exposure of murine peritoneal macrophages to rPbPga1 induces TNF-? release and nitric oxide (NO) production by macrophages. Furthermore, the presence of O-glycosylation sites was demonstrated by ?-elimination under ammonium hydroxide treatment of rPbPga1. Finally, sera from PCM patients recognized rPbPga1 by Western blotting indicating the presence of specific antibodies against rPbPga1. In conclusion, our findings suggest that the PbPga1gene codifies for a cell surface glycoprotein, probably attached to a GPI-anchor, which may play a role in P. brasiliensis cell wall morphogenesis and infection. The induction of inflammatory mediators released by rPbPga1 and the reactivity of PCM patient sera toward rPbPga1 imply that the protein favors the innate mechanisms of defense and induces humoral immunity during P. brasiliensis infection. PMID:23024763

Valim, Clarissa X. R.; Basso, Luiz Roberto; dos Reis Almeida, Fausto B.; Reis, Thaila Fernanda; Damasio, Andre Ricardo Lima; Arruda, Luisa Karla; Martinez, Roberto; Roque-Barreira, Maria Cristina; Oliver, Constance; Jamur, Maria Celia; Coelho, Paulo Sergio Rodrigues

2012-01-01

342

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

343

Phosphorylation of the Invariant Chain by Protein Kinase C Regulates MHC Class II Trafficking to Antigen-Processing Compartments  

Microsoft Academic Search

The invariant chain (Ii) plays a critical role in the transport of newly synthesized class II molecules to endosomal Ag-processing compartments. Of the two major isoforms of human Ii, only Ii-p35 is phosphorylated in vivo, and inhibiting Ii phosphorylation inhibits the trafficking of newly synthesized class II molecules to Ag-processing compartments. We now report that a member of the protein

Howard A. Anderson; Daniel T. Bergstralh; Tatsuyoshi Kawamura; Andrew Blauvelt; Paul A. Roche

344

Selection of Glutamate-Rich Protein Long Synthetic Peptides for Vaccine Development: Antigenicity and Relationship with Clinical Protection and Immunogenicity  

Microsoft Academic Search

Antibodies against three long synthetic peptides (LSPs) derived from the glutamate-rich protein (GLURP) of Plasmodium falciparum were analyzed in three cohorts from Liberia, Ghana, and Senegal. Two overlapping LSPs, LR67 and LR68, are derived from the relatively conserved N-terminal nonrepeat region (R0), and the third, LR70, is derived from the R2 repeat region. A high prevalence of antibody responses to

MICHAEL THEISEN; DANIEL DODOO; AISSATOU TOURE-BALDE; SOE SOE; GIAMPIETRO CORRADIN; KWADWO K. KORAM; JØRGEN A. L. KURTZHALS; LARS HVIID; THOR THEANDER; BARTHOLOMEW AKANMORI; MOHAMADOU NDIAYE; PIERRE DRUILHE

2001-01-01

345

Importance of M-Protein C Terminus as Substrate Antigen for Serodetection of Equine Arteritis Virus Infection  

Microsoft Academic Search

Equine arteritis virus (EAV), an enveloped positive-stranded RNA virus, is the prototype of the arterivirus group. In a previous paper (A. Kheyar, S. Martin, G. St.-Laurent, P. J. Timoney, W. H. McCollum, and D. Archambault, Clin. Diagn. Lab. Immunol. 4:648-652, 1997), we have shown that the unglycosylated membrane (M) protein, which is composed of 162 amino acids (aa), is a

Célia Jeronimo; Denis Archambault

2002-01-01

346

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

347

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

348

HLA-A2 presents shared tumor-associated antigens derived from endogenous proteins in ovarian cancer.  

PubMed

Tumor-associated lymphocytes (TAL) from the malignant ascites and tumor-infiltrating lymphocytes (TIL) from the solid tumor were isolated from six consecutive untreated ovarian cancer patients. Tumor-specific CTL were generated from both TAL and TIL using solid phase anti-CD3, low dose IL-2 (50 IU/ml), and repeated tumor stimulation. The specificity of TAL and TIL was tested in standard cytotoxicity assays using autologous tumor, several allogeneic ovarian tumors, and the NK-sensitive cell line, K562. Anti-HLA-A-B-C mAb, W6/32, was used to demonstrate that these tumor-specific TAL and TIL were HLA class I-restricted. The ability of the ascitic and solid tumor to present Ag by HLA class I was assessed using Brefeldin A, a fungal metabolite that blocks the endogenous Ag-processing pathway in the viral model. Brefeldin A significantly inhibited tumor-specific cytotoxicity as well as HLA class I expression on the cell surface, suggesting an endogenous source of tumor-associated Ag. Despite previous reports of antigenic heterogeneity in ovarian cancer, shared tumor-associated Ag were shown to exist in this disease as demonstrated by significant allogeneic recognition of HLA-A2-matched patients as opposed to unmatched controls. Specifically, CTL from HLA-A2+ patients lysed HLA-A2+ allogeneic targets significantly better than HLA-A2- allogeneic or HLA-A2+ melanoma targets. There was no such difference with HLA-A2- effectors. Furthermore, HLA-A2 was confirmed to be a major restriction element in ovarian cancer by the blocking of HLA-A2+ effectors against both autologous and allogeneic HLA-A2+ targets with the anti-HLA-A2 mAb, BB7.2. These findings verify a similar lymphocyte/tumor interaction as has been documented in melanoma, suggesting a common mechanism of recognition of these human tumors by lymphocytes. PMID:8228240

Peoples, G E; Goedegebuure, P S; Andrews, J V; Schoof, D D; Eberlein, T J

1993-11-15

349

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 Vina, 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

350

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

351

Self-assembly of in vitro-translated human papillomavirus type 16 L1 capsid protein into virus-like particles and antigenic reactivity of the protein.  

PubMed Central

The human papillomavirus type 16 (HPV-16) L1 capsid protein is the major component of the HPV virion. We prepared L1 protein of HPV-16 in a cell-free system. The L1 gene was cloned in an expression plasmid and transcribed and translated in vitro in a rabbit reticulocyte lysate. The expressed protein had the molecular mass (55 kDa) expected for the L1 protein, and it assembled into virus-like particles that closely resembled papillomavirus virions. The protein retained conformational epitopes, as evidenced by its reactivity with monoclonal antibodies which recognize only intact viral particles. In radioimmunoprecipitation assays with sera from college women grouped by their genital tract HPV DNA status, high reactivity was found in 68% of HPV-16 DNA-positive women, in 23% of women with other HPVs, and in 19% of HPV-negative women. In comparison, none of the sera of children were reactive. The results of the radioimmunoprecipitation assays showed a significant correlation with results obtained with the same sera in an enzyme-linked immunosorbent assay with virus-like particles produced in baculovirus (chi-square test for linear trend, P = 0.0023). Although the amounts of L1 protein obtained are small, the ability to produce virus-like particles by in vitro translation may be useful in the study of virus assembly, virus binding, and the immunological response to HPV infection. PMID:8914767

Iyengar, S; Shah, K V; Kotloff, K L; Ghim, S J; Viscidi, R P

1996-01-01

352

Specific glycoprotein antigens on the surface of insect and mammalian stages of Trypanosoma cruzi.  

PubMed Central

Two major surface antigens on Trypanosoma cruzi, the causative agent of Chagas disease, have been described [Nogueira, N., Chaplan, S., Tydings, J., Unkeless, J. & Cohn, Z. (1981) J. Exp. Med. 153, 629-639]. One, a Mr 75,000 glycoprotein (GP), is specific for the culture forms (insect-host stages) of the organisms--epimastigotes and metacyclic trypomastigotes. The other, a Mr 90,000 GP, was found in vertebrate-host stages of the organisms--bloodstream-form trypomastigotes. We now report that these two major surface antigens of T. cruzi seem to be unrelated proteins, as judged by tryptic and chymotryptic peptide analysis. Antibodies were raised in rabbits against epimastigote or trypomastigote proteins which had been immunoprecipitated with human antisera. These trypomastigote and epimastigote protein antisera reacted only with the homologous immunogen, as determined by immunoprecipitation of surface-labeled organisms and by immunofluorescence. The Mr 75,000 GP is detected only in cultured (insect stage) epimastigotes and metacyclic trypomastigotes. The Mr 90,000 GP is only present in bloodstream-form trypomastigotes, amastigotes, and trypomastigotes obtained from infected muscle cells in vitro. Therefore, the insect and vertebrate stages of this species display distinctive surface GPs that can be identified by surface labeling and immunoprecipitation techniques in six strains of T. cruzi (Y, CL, Peru, Colombiana, SF-12, and SF-21) isolated from widely different areas of South America. Images PMID:6175966

Nogueira, N; Unkeless, J; Cohn, Z

1982-01-01

353

Opa Proteins of Pathogenic Neisseriae Initiate Src Kinase-Dependent or Lipid Raft-Mediated Uptake via Distinct Human Carcinoembryonic Antigen-Related Cell Adhesion Molecule Isoforms? †  

PubMed Central

Several pathogenic bacteria exploit human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) for adhesion to and invasion into their host cells. CEACAM isoforms have characteristic expression patterns on epithelial, endothelial, or hematopoietic cells, providing bacteria with distinct sets of receptors on particular tissues. For example, while CEACAM1 and CEACAM6 have a wide tissue distribution, CEACAM3, CEACAM4, and CEACAM8 are uniquely expressed on primary human granulocytes, whereas CEA and CEACAM7 are limited to epithelia. By reconstitution of a CEACAM-deficient cell line with individual CEACAMs, we have analyzed the requirements for CEACAM-mediated internalization of Neisseria gonorrhoeae. Our results point to two mechanistically different uptake pathways triggered by either epithelial CEACAMs (CEACAM1, CEA, and CEACAM6) or the granulocyte-specific CEACAM3. In particular, CEACAM3-mediated uptake critically depends on Src family protein tyrosine kinase (PTK) activity, and CEACAM3 associates with the SH2 domains of several Src PTKs. In contrast, epithelial CEACAMs require the integrity of cholesterol-rich membrane microdomains and are affected by cholesterol depletion, whereas CEACAM3-mediated uptake by transfected cells or the opsonin-independent phagocytosis by human granulocytes is not altered in the presence of cholesterol chelators. These results allow the subdivision of all human CEACAMs known to be utilized as pathogen receptors into functional groups and point to important consequences for bacterial engagement of distinct CEACAM isoforms. PMID:17517873

Schmitter, Tim; Pils, Stefan; Weibel, Stephanie; Agerer, Franziska; Peterson, Lisa; Buntru, Alexander; Kopp, Kathrin; Hauck, Christof R.

2007-01-01

354

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

355

Serodiagnosis of equine infectious anemia by agar gel immunodiffusion and ELISA using a recombinant p26 viral protein expressed in Escherichia coli as antigen.  

PubMed

We used a p26 recombinant protein (p26r) from equine infectious-anemia virus (EIAV) expressed in Escherichia coli as antigen to standardize an agar-gel immunodiffusion (AGIDp26r) test and an indirect ELISA (ELISAp26r) for the detection of antibodies against EIAV in 720 equine sera from Brazil. We evaluated the tests's relative diagnostic sensitivities (relSe) and relative diagnostic specificities (relSp) against a commercial AGID kit (Idexx, USA). We used three sera panels: panel A--196 AGID-negative sera from an AIE non-endemic controlled area; panel B--194 AGID-negative sera from an AIE endemic area and panel C--330 AGID-positive sera from an AIE endemic area. ELISAp26r cut-off value was defined with TG-ROC using sera from panels A and C. AGIDp26r showed an agreement of 100% with the commercial kit. When applied to sera from panels A and C, ELISAp26r showed an agreement of 100% with the kit, but, although relSe was 100% for panel C, the ELISAp26r had relSp of 93.3%. PMID:17109980

Piza, Adriana S Toledo; Pereira, Alessandra Rael; Terreran, Maria Thereza; Mozzer, Otto; Tanuri, Amílcar; Brandão, Paulo Eduardo; Richtzenhain, Leonardo José

2007-03-17

356

Induction of the nuclear I{kappa}B protein I{kappa}B-{zeta} upon stimulation of B cell antigen receptor  

SciTech Connect

The nuclear I{kappa}B protein I{kappa}B-{zeta} is barely detectable in resting cells and is induced in macrophages and fibroblasts following stimulation of innate immunity via Toll-like receptors. The induced I{kappa}B-{zeta} associates with nuclear factor (NF)-{kappa}B in the nucleus and plays crucial roles in its transcriptional regulation. Here, we examined the induction of I{kappa}B-{zeta} in B lymphocytes, one of the major players in adaptive immunity. Upon crosslinking of the surface immunoglobulin complex, I{kappa}B-{zeta} mRNA was robustly induced in murine B-lymphoma cell line A20 cells. While the crosslinking activated NF-{kappa}B and induced its target gene, I{kappa}B-{alpha}, co-crosslinking of Fc{gamma} receptor IIB to the surface immunoglobulin complex inhibited NF-{kappa}B activation and the induction of I{kappa}B-{zeta} and I{kappa}B-{alpha}, suggesting critical roles for NF-{kappa}B in the induction. These results indicate that I{kappa}B-{zeta} is also induced by stimulation of B cell antigen receptor, suggesting that I{kappa}B-{zeta} is involved in the regulation of adaptive immune responses.

Hijioka, Kuniaki [Department of Molecular and Cellular Biochemistry, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan); Matsuo, Susumu [Department of Molecular and Cellular Biochemistry, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan); Eto-Kimura, Akiko [Department of Molecular and Cellular Biochemistry, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan); Takeshige, Koichiro [Department of Molecular and Cellular Biochemistry, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan); Muta, Tatsushi [Department of Molecular and Cellular Biochemistry, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan) and Laboratory of Cell Recognition and Response, Graduate School of Life Sciences, Tohoku University, Sendai 980-8578 (Japan)]. E-mail: tmuta@biology.tohoku.ac.jp

2007-05-04

357

Ku Antigen-DNA Conformation Determines the Activation of DNA-Dependent Protein Kinase and DNA Sequence-Directed Repression of Mouse Mammary Tumor Virus Transcription  

PubMed Central

Mouse mammary tumor virus (MMTV) transcription is repressed by DNA-dependent protein kinase (DNA-PK) through a DNA sequence element, NRE1, in the viral long terminal repeat that is a sequence-specific DNA binding site for the Ku antigen subunit of the kinase. While Ku is an essential component of the active kinase, how the catalytic subunit of DNA-PK (DNA-PKcs) is regulated through its association with Ku is only beginning to be understood. We report that activation of DNA-PKcs and the repression of MMTV transcription from NRE1 are dependent upon Ku conformation, the manipulation of DNA structure by Ku, and the contact of Ku80 with DNA. Truncation of one copy of the overlapping direct repeat that comprises NRE1 abrogated the repression of MMTV transcription by Ku–DNA-PKcs. Remarkably, the truncated element was recognized by Ku–DNA-PKcs with affinity similar to that of the full-length element but was unable to promote the activation of DNA-PKcs. Analysis of Ku–DNA-PKcs interactions with DNA ends, double- and single-stranded forms of NRE1, and the truncated NRE1 element revealed striking differences in Ku conformation that differentially affected the recruitment of DNA-PKcs and the activation of kinase activity. PMID:10330147

Giffin, Ward; Gong, Wenrong; Schild-Poulter, Caroline; Hache, Robert J. G.

1999-01-01

358

Selective deletion of antigen-specific CD8+ T cells by MHC class I tetramers coupled to the type I ribosome-inactivating protein saporin  

PubMed Central

CD8+ cytotoxic T lymphocytes (CTLs) are important effector cells responsible for tissue destruction in several autoimmune and allograft-related diseases. To discover if pathogenic T cells could be selectively deleted, we investigated the ability of a toxin coupled to major histocompatibility complex (MHC) class I tetramers to kill antigen-specific CD8+ T cells. H2-Db tetramers were assembled using streptavidin conjugated to the ribosome-inactivating protein (RIP) saporin (SAP). These tetramers inhibited ribosome activity in vitro, retained the T-cell receptor (TCR)–binding specificity of their nontoxic counterparts, and were internalized by 100% of target cells, leading to cell death in 72 hours. Cytotoxicity was dependent on the tetramer dose and avidity for the T cell. A single injection of the SAP-coupled tetramer eliminated more than 75% of cognate, but not control, T cells. This work demonstrates the therapeutic potential of cytotoxic tetramers to selectively eradicate pathogenic clonotypes while leaving overall T-cell immunity intact. PMID:17179221

Barnes, Carie; Woolard, Matthew D.; Johnson, Michael D. L.; Cullen, John M.; Collins, Edward J.; Frelinger, Jeffrey A.

2007-01-01

359

Bacterial ClpB heat-shock protein, an antigen-mimetic of the anorexigenic peptide ?-MSH, at the origin of eating disorders.  

PubMed

The molecular mechanisms at the origin of eating disorders (EDs), including anorexia nervosa (AN), bulimia and binge-eating disorder (BED), are currently unknown. Previous data indicated that immunoglobulins (Igs) or autoantibodies (auto-Abs) reactive with ?-melanocyte-stimulating hormone (?-MSH) are involved in regulation of feeding and emotion; however, the origin of such auto-Abs is unknown. Here, using proteomics, we identified ClpB heat-shock disaggregation chaperone protein of commensal gut bacteria Escherichia coli as a conformational antigen mimetic of ?-MSH. We show that ClpB-immunized mice produce anti-ClpB IgG crossreactive with ?-MSH, influencing food intake, body weight, anxiety and melanocortin receptor 4 signaling. Furthermore, chronic intragastric delivery of E. coli in mice decreased food intake and stimulated formation of ClpB- and ?-MSH-reactive antibodies, while ClpB-deficient E. coli did not affect food intake or antibody levels. Finally, we show that plasma levels of anti-ClpB IgG crossreactive with ?-MSH are increased in patients with AN, bulimia and BED, and that the ED Inventory-2 scores in ED patients correlate with anti-ClpB IgG and IgM, which is similar to our previous findings for ?-MSH auto-Abs. In conclusion, this work shows that the bacterial ClpB protein, which is present in several commensal and pathogenic microorganisms, can be responsible for the production of auto-Abs crossreactive with ?-MSH, associated with altered feeding and emotion in humans with ED. Our data suggest that ClpB-expressing gut microorganisms might be involved in the etiology of EDs. PMID:25290265

Tennoune, N; Chan, P; Breton, J; Legrand, R; Chabane, Y N; Akkermann, K; Järv, A; Ouelaa, W; Takagi, K; Ghouzali, I; Francois, M; Lucas, N; Bole-Feysot, C; Pestel-Caron, M; do Rego, J-C; Vaudry, D; Harro, J; Dé, E; Déchelotte, P; Fetissov, S O

2014-01-01

360

Molecular cloning and expression of a unique receptor-like protein-tyrosine-phosphatase in the leucocyte-common-antigen-related phosphate family.  

PubMed Central

Protein-tyrosine-phosphatases (PTPases) have been implicated in the regulation of certain tyrosine kinase growth factor receptors in that they dephosphorylate the activated (autophosphorylated) form of the receptors. In order to identify PTPases that potentially act on receptor targets in liver, we used the human leucocyte common antigen-related PTPase (LAR) cDNA [Streuli, Krueger, Hall, Schlossman and Saito (1988) J. Exp. Med. 168, 1523-1530] and isolated two closely related transmembrane PTPase homologues from a rat hepatic cDNA library. Both PTPases had large extracellular domains that contained three immunoglobulin-like repeats and eight type-III fibronectin repeats. Both enzymes had tandem homologous PTPase domains following a single hydrophobic transmembrane domain. One sequence encoded the rat homologue of LAR. The second PTPase, designated LAR-PTP2, had 79 and 90% identity with rat LAR in the respective cytoplasmic PTPase domains, with only 57% sequence similarity in the extracellular domain. The catalytic domains of LAR and LAR-PTP2 prepared by bacterial expression were active in dephosphorylating a variety of phosphotyrosyl substrates but did not hydrolyse phosphoserine or phosphothreonine residues of labelled casein. Both enzymes exhibited rapid turnover numbers of 4-7 s-1 for myelin basic protein and 78-150 s-1 for derivatized lysozyme. LAR and LAR-PTP2 displayed similar PTPase activity towards the simultaneous dephosphorylation of receptors of intact insulin and epidermal growth factor from liver membranes. These data indicate that there is a family of LAR-related PTPases that may regulate the phosphorylation state of receptor tyrosine kinases in liver and other tissues. Images Figure 1 Figure 4 Figure 5 Figure 6 PMID:8068021

Zhang, W R; Hashimoto, N; Ahmad, F; Ding, W; Goldstein, B J

1994-01-01

361

Amyotrophic Lateral Sclerosis-linked Mutant SOD1 Sequesters Hu Antigen R (HuR) and TIA-1-related Protein (TIAR)  

PubMed Central

Down-regulation of vascular endothelial growth factor (VEGF) in the mouse leads to progressive and selective degeneration of motor neurons similar to amyotrophic lateral sclerosis (ALS). In mice expressing ALS-associated mutant superoxide dismutase 1 (SOD1), VEGF mRNA expression in the spinal cord declines significantly prior to the onset of clinical manifestations. In vitro models suggest that dysregulation of VEGF mRNA stability contributes to that decline. Here, we show that the major RNA stabilizer, Hu Antigen R (HuR), and TIA-1-related protein (TIAR) colocalize with mutant SOD1 in mouse spinal cord extracts and cultured glioma cells. The colocalization was markedly reduced or abolished by RNase treatment. Immunoanalysis of transfected cells indicated that colocalization occurred in insoluble aggregates and inclusions. RNA immunoprecipitation showed a significant loss of VEGF mRNA binding to HuR and TIAR in mutant SOD1 cells, and there was marked depletion of HuR from polysomes. Ectopic expression of HuR in mutant SOD1 cells more than doubled the mRNA half-life of VEGF and significantly increased expression to that of wild-type SOD1 control. Cellular effects produced by mutant SOD1, including impaired mitochondrial function and oxidative stress-induced apoptosis, were reversed by HuR in a gene dose-dependent pattern. In summary, our findings indicate that mutant SOD1 impairs post-transcriptional regulation by sequestering key regulatory RNA-binding proteins. The rescue effect of HuR suggests that this impairment, whether related to VEGF or other potential mRNA targets, contributes to cytotoxicity in ALS. PMID:19805546

Lu, Liang; Wang, Shuying; Zheng, Lei; Li, Xuelin; Suswam, Esther A.; Zhang, Xiaowen; Wheeler, Crystal G.; Nabors, L. B.; Filippova, Natalia; King, Peter H.

2009-01-01

362

The serodominant secreted effector protein of Salmonella, SseB, is a strong CD4 antigen containing an immunodominant epitope presented by diverse HLA class II alleles.  

PubMed

Detailed characterization of the protective T-cell response in salmonellosis is a pressing unmet need in light of the global burden of human Salmonella infections and the likely contribution of CD4 T cells to immunity against this intracellular infection. In previous studies screening patient sera against antigen arrays, SseB was noteworthy as a serodominant target of adaptive immunity, inducing significantly raised antibody responses in HIV-seronegative compared with seropositive patients. SseB is a secreted protein, part of the Espa superfamily, localized to the bacterial surface and forming part of the translocon of the type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2. We demonstrate here that SseB is also a target of CD4 T-cell immunity, generating a substantial response after experimental infection in human volunteers, with around 0·1% of the peripheral repertoire responding to it. HLA-DR/peptide binding studies indicate that this protein encompasses a number of peptides with ability to bind to several different HLA-DR alleles. Of these, peptide 11 (p11) was shown in priming of both HLA-DR1 and HLA-DR4 transgenic mice to contain an immunodominant CD4 epitope. Analysis of responses in human donors showed immunity focused on p11 and another epitope in peptide 2. The high frequency of SseB-reactive CD4 T cells and the broad applicability to diverse HLA genotypes coupled with previous observations of serodominance and protective vaccination in mouse challenge experiments, make SseB a plausible candidate for next-generation Salmonella vaccines. PMID:24891088

Reynolds, Catherine J; Jones, Claire; Blohmke, Christoph J; Darton, Thomas C; Goudet, Amelie; Sergeant, Ruhena; Maillere, Bernard; Pollard, Andrew J; Altmann, Daniel M; Boyton, Rosemary J

2014-11-01

363

A431 cell variants lacking the blood group A antigen display increased high affinity epidermal growth factor-receptor number, protein-tyrosine kinase activity, and receptor turnover  

PubMed Central

The epidermal growth factor receptor (EGF-R) of human A431 cells bears an antigenic determinant that is closely related to the human blood group A carbohydrate structure. Labeling studies with blood group A reactive anti-EGF-R monoclonal antibodies and various lectins revealed that A431 cultures are heterogeneous with respect to blood group A expression. We have isolated clonal variants of these cells that either express (A431A+ cells) or completely lack (A431A- cells) the blood group A specific N-acetyl-D-galactosamine (GalNAc) residue. We show that this difference is due to the absence of a UDP-GalNAc:Gal transferase activity in A431A- cells. Subsequently, we have compared EGF-R functioning in these cell lines. Scatchard analysis of EGF- binding shows that in A431A- cells 6.3% of the EGF-R belongs to a high affinity subclass (Kd = 0.4 nM) while in A431A+ this subclass represents only 3.2% of the total receptor pool. The elevated level of high affinity receptors in A431A- cells is accompanied by a parallel increase in receptor protein- tyrosine kinase activity. In membrane preparations of A431A- cells, receptor autophosphorylation as well as phosphorylation of a tyrosine-containing peptide substrate is 2-3-fold higher as compared with A431A+ cells. In intact A431A-cells, the difference in receptor activity is measured as a 2-3-fold elevated level of receptor phosphorylation and a 2-3-fold higher abundance of phosphotyrosine in total cellular protein in A431A- cells. In addition, [35S]methionine pulse-chase experiments showed a ligand-independent increase in turnover of EGF-R in A431A- cells: the receptor's half life in these cells is 10 h as compared with 17 h in A431A+ cells. Our results suggest a possible involvement of GalNAc residue(s) in determining EGF-R affinity, protein-tyrosine kinase activity and turnover in A431 cells. Furthermore, our results indicate that high affinity EGF-R are the biologically active species with respect to protein-tyrosine kinase activity. PMID:2458364

1988-01-01

364

Improved Immunodiagnosis of Cystic Hydatid Disease by Using a Synthetic Peptide with Higher Diagnostic Value Than That of Its Parent Protein, Echinococcus granulosus Antigen B  

Microsoft Academic Search

The assays are used for the diagnosis of hydatid disease are still imperfect. The reported diagnostic sensitivity and specificity vary greatly depending on the panel of sera used, the laboratory conducting the assay, and, more critically, the antigen used. To contribute to its standardization, we have recently ranked the diagnostic performances of the major parasite antigens and the available synthetic

GUALBERTO GONZALEZ-SAPIENZA; CARMEN LORENZO; ALBERTO NIETO

2000-01-01

365

Effects of chemotherapy on antibody levels directed against PGL-I and 85A and 85B protein antigens in lepromatous patients.  

PubMed

IgG antibodies against antigens 85A and 85B from Mycobacterium bovis BCG, IgM antibodies against phenolic glycolipid-I (PGL-I) and circulating PGL-I antigen were measured in the serum of 11 patients with lepromatous leprosy receiving multidrug therapy (MDT). Before treatment, 6 patients were reactive to antigen 85A, 10 patients to antigen 85B, and 11 patients to PGL-I; circulating PGL-I was detected in the sera of all of them. After 2 years of MDT PGL-I antigen could no longer be detected in all of the patients, except for two who were not compliant with treatment. IgG antibodies directed against the 85A and 85B antigens and IgM antibodies against the PGL-I antigen also decreased significantly during treatment but more slowly. The determination of circulating PGL-I antigen remains the most appropriate tool for monitoring lepromatous leprosy under MDT. PMID:8326178

Drowart, A; Chanteau, S; Huygen, K; De Cock, M; Cartel, J L; De Bruyn, J; Launois, P; Yernault, J C; Van Vooren, J P

1993-03-01

366

Control of tick infestations and pathogen prevalence in cattle and sheep farms vaccinated with the recombinant Subolesin-Major Surface Protein 1a chimeric antigen  

PubMed Central

Background Despite the use of chemical acaricides, tick infestations continue to affect animal health and production worldwide. Tick vaccines have been proposed as a cost-effective and environmentally friendly alternative for tick control. Vaccination with the candidate tick protective antigen, Subolesin (SUB), has been shown experimentally to be effective in controlling vector infestations and pathogen infection. Furthermore, Escherichia coli membranes containing the chimeric antigen composed of SUB fused to Anaplasma marginale Major Surface Protein 1a (MSP1a) (SUB-MSP1a) were produced using a simple low-cost process and proved to be effective for the control of cattle tick, Rhipicephalus (Boophilus) microplus and R. annulatus infestations in pen trials. In this research, field trials were conducted to characterize the effect of vaccination with SUB-MSP1a on tick infestations and the prevalence of tick-borne pathogens in a randomized controlled prospective study. Methods Two cattle and two sheep farms with similar geographical locations and production characteristics were randomly assigned to control and vaccinated groups. Ticks were collected, counted, weighed and classified and the prevalence of tick-borne pathogens at the DNA and serological levels were followed for one year prior to and 9 months after vaccination. Results Both cattle and sheep developed antibodies against SUB in response to vaccination. The main effect of the vaccine in cattle was the 8-fold reduction in the percent of infested animals while vaccination in sheep reduced tick infestations by 63%. Female tick weight was 32-55% lower in ticks collected from both vaccinated cattle and sheep when compared to controls. The seroprevalence of Babesia bigemina was lower by 30% in vaccinated cattle, suggesting a possible role for the vaccine in decreasing the prevalence of this tick-borne pathogen. The effect of the vaccine in reducing the frequency of one A. marginale msp4 genotype probably reflected the reduction in the prevalence of a tick-transmitted strain as a result of the reduction in the percent of tick-infested cattle. Conclusions These data provide evidence of the dual effect of a SUB-based vaccine for controlling tick infestations and pathogen infection/transmission and provide additional support for the use of the SUB-MSP1a vaccine for tick control in cattle and sheep. PMID:24398155

2014-01-01

367

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

368

In vitro selection and evolution of functional proteins by using ribosome display  

PubMed Central

We report here a system with which a correctly folded complete protein and its encoding mRNA both remain attached to the ribosome and can be enriched for the ligand-binding properties of the native protein. We have selected a single-chain fragment (scFv) of an antibody 108-fold by five cycles of transcription, translation, antigen-affinity selection, and PCR. The selected scFv fragments all mutated in vitro by acquiring up to four unrelated amino acid exchanges over the five generations, but they remained fully compatible with antigen binding. Libraries of native folded proteins can now be screened and made to evolve in a cell-free system without any transformation or constraints imposed by the host cell. PMID:9144168

Hanes, Jozef; Pluckthun, Andreas

1997-01-01

369

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

370

Circulatory antigen of Heymann nephritis. III. Presence of the 70-kD circulatory protein in the immune deposits of Heymann nephritis.  

PubMed Central

An antigen of 70 kD size has been isolated previously from normal rat serum which has immunological cross-reactivity to the Heymann nephritis antigen, F x 1A. Its role in the pathogenesis of Heymann's nephritis was unknown. In this investigation we tested for the presence of 70-kD circulatory antigen in the glomerular immune deposits of Heymann's nephritis. Further, its presence was correlated with severity of disease. It was observed that the presence of the 70-kD antigen strongly correlated with the existence of electron-dense deposits in the lamina rara externa (LRE) of the glomerular capillary wall and with pathologic proteinuria. Temporally, the presence of the 70-kD antigen in the immune deposits was followed by large electron-dense deposits, enhanced complement activity and proteinuria. The data suggest that in the growing immune complex lattice in the LRE, the 70-kD circulatory antigen by virtue of its small size, mobility and antigen cross-reactivity facilitates cross linking and coalescence of immune complexes, resulting in electron-dense immune deposits (EDD) formation which initiates complement activation and consequent proteinuria. PMID:1893629

Singh, A K; Schwartz, M M

1991-01-01

371

Characterisation of Sarcoptes scabiei antigens.  

PubMed

In pig herds, the status of Sarcoptes scabiei infections is routinely monitored by serodiagnosis. Crude antigen for ELISA is usually prepared from S. scabiei var. canis or other variations and may lead to variations in the outcome of different tests, making assay standardisation difficult. This study was performed to investigate the antigen profiles of S. scabiei, including differences between hydrophilic and more hydrophobic protein fractions, by Western blotting with sera from pigs with defined infection status. Potential cross-reactivity among S. scabiei (var. canis, suis and bovis), Dermatophagoides farinae and Tyrophagus putrescentiae was also analysed. Hydrophobic S. scabiei antigens were detectable in the range of 40-50 kDa, whilst the hydrophilic fraction showed no specific antigenicity. In the hydrophobic fractions of D. farinae and T. putrescentiae, two major protein fractions in a similar size range could be identified, but no cross-reactivity with Sarcoptes-positive sera was detectable. However, examination of the hydrophilic fractions revealed cross-reactivity between Sarcoptes-positive sera and both the house dust mite and the storage mite in the range of 115 and 28/38 kDa. Specific bands in the same range (42 and 48 kDa) could be detected in blots from hydrophobic fractions of all three tested variations of S. scabiei (var. canis, bovis and suis). These results show that there are considerable differences in mange antibody reactivity, including reactions with proteins from free-living mites, which may interfere with tests based on hydrophilic antigens. Further refinement of antigen and the use of specific hydrophobic proteins could improve ELISA performance and standardisation. PMID:20865427

Hejduk, Gloria; Hofstätter, Katja; Löwenstein, Michael; Peschke, Roman; Miller, Ingrid; Joachim, Anja

2011-02-01

372

Dynamic appearance of antigenic epitopes effective for viral neutralization during membrane fusion initiated by interactions between HIV-1 envelope proteins and CD4/CXCR4.  

PubMed

HIV-1 entry into cells is mediated by interactions between the envelope (Env) gp120 and gp41 proteins with CD4 and chemokine receptors via an intermediate called the viral fusion complex (vFC). Here, mAbs were used to find the dynamic changes in expression of antigenic epitopes during vFC formation. A CD4-specific mAb (R275) and anti-vFC mAbs, designated F12-1, F13-6 and F18-4 that recognize the epitopes only appeared by the co-culture of env-transfected 293FT and CD4-transfected 293 cells, were developed by immunizing ganp-gene transgenic mice with an vFC-like structure formed by the same co-culture. The epitopes recognized by the mAbs appeared at different time points during vFC formation: F18-4 appeared first, followed by F13-6, and finally F12-1. The anti-vFC mAbs had little effect on vFC formation or virus neutralization; however, interestingly F12-1 and F18-4 increased exposure of the OKT4-epitope on the domain 3 in the extracellular region of CD4. R275, which recognizes the epitope closely associated with the OKT4-determinant on the domain 3, showed the marked inhibition of vFC formation and viral neutralization activity. The Ab binding to the epitopes appeared during viral membrane fusion might reinforce the appearance of the target epitopes for effective neutralization activity. PMID:22226668

Toda, Teppei; Kuwahara, Kazuhiko; Kondo, Naoyuki; Matsuda, Zene; Maeda, Yosuke; Maeda, Kazuhiko; Sakaguchi, Nobuo

2012-09-01

373

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

PubMed Central

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

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

2009-01-01

374

Inhibition of mitogen-activated protein kinase pathway can induce upregulation of human leukocyte antigen class I without PD-L1-upregulation in contrast to interferon-? treatment.  

PubMed

Recently, we reported that human leukocyte antigen (HLA) class I expression is predominantly regulated by the mitogen-activated protein kinase (MAPK) pathway as one of the oncogenic regulations of HLA class I expression. In the present study, we examined mechanisms of how HLA class I and PD-L1 are regulated by MAPK inhibitors and interferon-? (IFN-?). Furthermore, we evaluated the expression of major signal transduction molecules by Western blot and anti-tumor CTL activity by a cytotoxic assay when HLA class I and PD-L1 were modulated by MAPK inhibitors and/or IFN-?. As a result, we confirmed, as a more general phenomenon, that the inhibition of MAPK could upregulate HLA class I expression in a panel of human solid tumors (n = 26). Of note, we showed that MAPK inhibitors act on the upregulation of HLA class I expression through a different pathway from IFN-?; there was an additive effect in the upregulation of HLA class I when treated with the combination of MAPK inhibitors and IFN-?, and there was no overlapping activation of JAK2/STAT1 and Erk1/2 molecules when treated with either IFN-? or MAPK inhibitors. Furthermore, we showed that IFN-?-treatment impaired the tumor-specific CTL activity due to the upregulation of PD-L1 in spite of the upregulation of HLA class I, while MAPK inhibitors can augment the tumor-specific CTL activity due to the upregulated HLA class I without PD-L1 alterations. In conclusion, in addition to the original anti-proliferative activity, MAPK inhibitors may work toward the enhancement of T-cell-mediated anti-tumor immunity through the upregulation of HLA class I without the upregulation of PD-L1. PMID:25154680

Mimura, Kousaku; Kua, Ley-Fang; Shiraishi, Kensuke; Kee Siang, Lim; Shabbir, Asim; Komachi, Mayumi; Suzuki, Yoshiyuki; Nakano, Takashi; Yong, Wei-Peng; So, Jimmy; Kono, Koji

2014-10-01