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1

Epitopes shared by unrelated antigens of Borrelia burgdorferi.  

PubMed Central

An immunoglobulin M kappa-chain murine monoclonal antibody (CAB) reacted in a Western blot (immunoblot) with approximately 30 polypeptides from a whole-cell lysate of several American and European Borrelia burgdorferi strains. The reactive antigen with the highest M(r) was measured at 93 kDa (p93) and had an NH2-terminal sequence identical to the one previously reported for this antigen. The lowest reactive antigen had an M(r) of 16,000. All antigens recognized by CAB had isoelectric points within a narrow acidic range, between 5.4 and 6.2. Thus, the objective of this study was to determine whether the broad reactivity of CAB could be due to degradation of the antigen with the highest M(r), since such spontaneous degradation of p93 has already been reported, and to determine whether CAB could recognize shared epitopes in different antigens. Treatment of B. burgdorferi with protease inhibitors did not result in changes in CAB reactivity, indicating that if such degradation existed, it was most likely not due to the action of endogenous proteases. Likewise, protease treatment of intact organisms and recovery of the antigens in the insoluble fraction of a Triton X-114 partition indicated that they were internal and thus less likely to be degraded by experimental procedures. Amino-terminal sequences of other reactive polypeptides showed one approximately 72-kDa polypeptide to be identical to the DnaK homolog of B. burgdorferi. Two other antigens at approximately 49 and 47 kDa were blocked to Edman degradation. Finally, one sequenced polypeptide with a molecular mass of approximately 38.5 kDa had a strong identity with glyceraldehyde-3-phosphate dehydrogenase of other bacteria and vertebrates. Thus, while it cannot be ruled out that some of the CAB reactivity may be due to fragmentation of p93, there is strong evidence to indicate the presence of a shared epitope in at least three, possibly five, unrelated antigens of B. burgdorferi. A linear epitope within amino acid residues 357 to 371 of p93 was identified. Evidence is presented for a discontinuous epitope in the carboxy-terminal region of the DnaK homolog, which bears strong amino acid identity with the p93 epitope. The conserved amino acid sequences necessary for these shared epitopes indicate possible genetic and/or functional relatedness among these various antigens. Images PMID:7509314

Anda, P; Backenson, P B; Coleman, J L; Benach, J L

1994-01-01

2

THE EMERGENCE OF ANTIBODIES WITH EITHER IDENTICAL OR UNRELATED INDIVIDUAL ANTIGENIC SPECIFICITY DURING REPEATED IMMUNIZATIONS WITH STREPTOCOCCAL VACCINES  

PubMed Central

Electrophoretically monodisperse antibody components in rabbit antisera to the carbohydrates of the Groups A and C streptococci have been examined for their individual antigenic specificity. In these antibody components which were isolated by preparative electrophoresis, individual antigenic specificity was confined to the specific antibody and was absent in the nonantibody ?-globulin. Radioprecipitation experiments and the use of immune absorbent columns constructed from goat anti-antisera, which had been absorbed with fraction II, revealed that all the specific antibody in an electrophoretically monodisperse component was reactive with the homologous anti-antibody. Antibodies with either identical or distinct individual antigenic specificities may occur in the same rabbit with repeated immunizations. Antibodies with identical antigenic specificity had identical electrophoretic mobility, whereas antibodies with unrelated antigenic specificities had distinct electrophoretic mobilities. In the interval between immunizations, if antibody to the carbohydrate antigen was absent, there was no detectable antibody with individual antigenic specificity. PMID:4192569

Eichmann, Klaus; Braun, Dietmar G.; Feizi, Ten; Krause, Richard M.

1970-01-01

3

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

4

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

5

Identification of protein antigens of group B streptococci, with special reference to the Ibc antigens  

PubMed Central

The protein antigens of prototypes of five types of group B streptococcal strains were extracted with HCl or Triton X-100, separated by sodium dodecyl sulfate polyacrylamide electrophoresis, transferred to nitrocellulose, and examined by immunochemical staining. The Ibc proteins are shown to consist of at least two distinct protein antigens and their breakdown products. One antigen, the "beta" antigen, exists primarily as a 130,000 mol wt protein that is also able to bind human IgA. The "alpha" antigen, which has no known function, appears as a number of proteins of various molecular weights from 20,000 to 120,000. Another set of antigens, the R protein antigens of type III strains, has been identified as a group of acid-labile proteins varying in molecular weight from 100,000 to 130,000. In addition, two previously undescribed antigens have been found that are common to all five group B types. PMID:6387035

1984-01-01

6

Antigenic analysis of Campylobacter flagellar protein and other proteins.  

PubMed

Outer membrane proteins of Campylobacter jejuni and other campylobacter species were analyzed for their antigenic potentials by immunoblotting. Polypeptides were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred electrophoretically, and reacted with rabbit antisera to C. jejuni. Each Campylobacter species analyzed demonstrated a unique outer membrane protein antigenic profile; interspecies antigen sharing was observed to be compatible with the degree of DNA relatedness between the species. The most highly conserved outer membrane protein antigen was the flagellum (molecular weight, 62,000). An aflagellate mutant was found to be untypable with the heat-labile system, in contrast to its parental isolate. The immunogenic potentials of C. jejuni proteins were examined by immunoblot analysis of sera from infected humans. Sera of convalescent patients, reacted with their homologous C. jejuni isolates, recognized a variety of campylobacter proteins. The most consistent immunogen in human infection was the flagellar protein. Patient sera assayed by the immunoblot technique were easily distinguished from control sera, which did not recognize specific campylobacter antigens. These findings suggest that the campylobacter flagellar protein is an essential determinant of the heat-labile antigen typing scheme and is the dominant immunogen recognized during C. jejuni infections in humans. PMID:2578478

Wenman, W M; Chai, J; Louie, T J; Goudreau, C; Lior, H; Newell, D G; Pearson, A D; Taylor, D E

1985-01-01

7

The Stability and Formation of Native Proteins from Unfolded Monomers Is Increased through Interactions with Unrelated Proteins  

PubMed Central

The intracellular concentration of protein may be as high as 400 mg per ml; thus it seems inevitable that within the cell, numerous protein-protein contacts are constantly occurring. A basic biochemical principle states that the equilibrium of an association reaction can be shifted by ligand binding. This indicates that if within the cell many protein-protein interactions are indeed taking place, some fundamental characteristics of proteins would necessarily differ from those observed in traditional biochemical systems. Accordingly, we measured the effect of eight different proteins on the formation of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) from guanidinium chloride unfolded monomers. The eight proteins at concentrations of micrograms per ml induced an important increase on active dimer formation. Studies on the mechanism of this phenomenon showed that the proteins stabilize the dimeric structure of TbTIM, and that this is the driving force that promotes the formation of active dimers. Similar data were obtained with TIM from three other species. The heat changes that occur when TbTIM is mixed with lysozyme were determined by isothermal titration calorimetry; the results provided direct evidence of the weak interaction between apparently unrelated proteins. The data, therefore, are strongly suggestive that the numerous protein-protein interactions that occur in the intracellular space are an additional control factor in the formation and stability of proteins. PMID:17551578

Rodríguez-Almazán, Claudia; Torner, Francisco J.; Costas, Miguel; Pérez-Montfort, Ruy; de Gómez-Puyou, Marieta Tuena; Puyou, Armando Gómez

2007-01-01

8

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

PubMed

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 information regulator (Sir) complex in Saccharomyces cerevisiae. We analyzed ChIP-Seq peaks of the Sir2, Sir3, and Sir4 silencing proteins and discovered 238 unexpected euchromatic loci that exhibited enrichment of all three. Surprisingly, published ChIP-Seq datasets for the Ste12 transcription factor and the centromeric Cse4 protein indicated that these proteins were also enriched in the same euchromatic regions with the high Sir protein levels. The 238 loci, termed "hyper-ChIPable", were in highly expressed regions with strong polymerase II and polymerase III enrichment signals, and the correlation between transcription level and ChIP enrichment was not limited to these 238 loci but extended genome-wide. The apparent enrichment of various proteins at hyper-ChIPable loci was not a consequence of artifacts associated with deep sequencing methods, as confirmed by ChIP-quantitative PCR. The localization of unrelated proteins, including the entire silencing complex, to the most highly transcribed genes was highly suggestive of a technical issue with the immunoprecipitations. ChIP-Seq on chromatin immunoprecipitated with a nuclear-localized GFP reproduced the above enrichment in an expression-dependent manner: induction of the GAL genes resulted in an increased ChIP signal of the GFP protein at these loci, with presumably no biological relevance. Whereas ChIP is a broadly valuable technique, some published conclusions based upon ChIP procedures may merit reevaluation in light of these findings. PMID:24173036

Teytelman, Leonid; Thurtle, Deborah M; Rine, Jasper; van Oudenaarden, Alexander

2013-11-12

9

Probability of Finding Marrow Unrelated Donor (MUD) for an Indian patient in a Multi-national Human Leukocyte Antigen (HLA) Registry.  

PubMed

With an increase in the number of transplants happening globally, hematopoietic stem cells (HSC) transplantation from matched unrelated donor (MUD) has begun. The increasing trend of MUD transplants across countries has been largely facilitated with the conspicuous growth of volunteer HSC donor noted in the last decade i.e. 8 million HSC donors in 2002 to more than 22 million in 2013 registered in 71 member registries of the Bone Marrow Donor Worldwide (BMDW). Some populations of the world are still very poorly represented in these registries. Since, the chances of successful engraftment and disease free survival are directly proportional to the HLA compatibility between the recipient and the prospective donor, the diversity of the HLA system at the antigenic and allelic level and the heterogeneity of HLA data of the registered donors has a bearing on the probability of finding a volunteer unrelated HSC donor for patients from such populations. In the present study 126 patients were identified suffering from hematological diseases requiring MUD transplant. Their HLA typing was performed and search was done using BMDW database. The search results for these Indian patients in the multinational registry as well as in the Indian Registries were analyzed using mean, range, standard deviation and finally evaluated in terms of probability for finding matched donor (MUD). Total Asian population is only 11 % in the BMDW making it difficult to find a MUD for an Asian patient. The current study supports this, experimentally; revealing that the probability of finding an allele match for an Indian patient in the multinational Human Leukocyte Antigen (HLA) registries is 16 % and a dismal 0.008 % in the Indian registries (donors in Indian registries is just 33,678 as compared to 22.5 million in BMDW). This greatly, emphasizes on enhancing the number of Indian donors in Indian and multi-national registries. PMID:25825557

Tiwari, Aseem K; Bhati-Kushwaha, Himakshi; Kukreja, Pooja; Mishra, Vikash C; Tyagi, Neetu; Sharma, Ashish; Raina, Vimarsh

2015-06-01

10

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

11

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

Microsoft Academic Search

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

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

2008-01-01

12

Isoform I (mdr3) is the major form of P-glycoprotein expressed in mouse brain capillaries. Evidence for cross-reactivity of antibody C219 with an unrelated protein.  

PubMed Central

P-glycoprotein (P-gp) is expressed in various non-cancerous tissues such as the endothelial cells of the blood-brain barrier. We used several monoclonal antibodies (mAbs) and isoform-specific polyclonal antibodies to establish which P-gp isoforms are expressed in isolated mouse brain capillaries. P-gp class I isoform was detected in capillaries with a Western immunoblotting procedure using a specific antiserum. No immunoreactivity was observed with either class II- or class III-specific antisera. Immunoreactivity was observed with mAb C219. However, this antibody detected two distinct immunoreactive proteins (155 and 190 kDa) in the isolated brain capillaries. These two proteins comigrated as a broad band when the samples were submitted to heat prior to gel electrophoresis. The glycoprotein nature of these two antigens was evaluated by their sensitivity to N-glycanase treatment. Following this treatment, the size of the proteins was reduced from 190 and 155 kDa to 180 and 120 kDa, respectively. Triton X-114 phase-partitioning studies showed that the 190 kDa immunoreactive protein was poorly solubilized by Triton X-114, while the 155 kDa protein was partitioned in the detergent-rich phase. In labelling experiments, only the 155 kDa protein was photolabelled with [125I]iodoarylazidoprazosin. These results show that a 190 kDa protein detected by antibody C219 is an antigen unrelated to the three P-gp isoforms presently known. Cross-reactivity of C219 with an unrelated protein emphasizes the fact that more than one antibody should be used in the assessment of P-gp expression in cell lines and tissues. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7848274

Jetté, L; Pouliot, J F; Murphy, G F; Béliveau, R

1995-01-01

13

Regulation of beta-lactamase activity by remote binding of heme: functional coupling of unrelated proteins through domain insertion.  

PubMed

Coupling the activities of normally disparate proteins into one functional unit has significant potential in terms of constructing novel switching components for synthetic biology or as biosensors. It also provides a means of investigating the basis behind transmission of conformation events between remote sites that is integral to many biological processes, including allostery. Here we describe how the structures and functions of two normally unlinked proteins, namely, the heme binding capability of cytochrome b(562) and the antibiotic degrading beta-lactamase activity of TEM, have been coupled using a directed evolution domain insertion approach. The important small biomolecule heme directly modulates in vivo and in vitro the beta-lactamase activity of selected integral fusion proteins. The presence of heme decreased the concentration of ampicillin tolerated by Escherichia coli and the level of in vitro hydrolysis of nitrocefin by up to 2 orders of magnitude. Variants with the largest switching magnitudes contained insertions at second-shell sites that abut key catalytic residues. Spectrophotometry confirmed that heme bound to the integral fusion proteins in a manner similar to that of cytochrome b(562). Circular dichroism suggested that only subtle structural changes rather than gross folding-unfolding events were responsible for modulating beta-lactamase activity, and size exclusion chromatography confirmed that the integral fusion proteins remained monomeric in both the apo and holo forms. Thus, by sampling a variety of insertion positions and linker sequences, we are able to couple the functions of two unrelated proteins by domain insertion. PMID:20602528

Edwards, Wayne R; Williams, Abigail J; Morris, Josephine L; Baldwin, Amy J; Allemann, Rudolf K; Jones, D Dafydd

2010-08-10

14

Prediction of protein antigenic determinants from amino acid sequences  

SciTech Connect

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

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

1981-06-01

15

Selecting unrelated donors  

PubMed Central

TEASER You are selecting an unrelated donor for a 40 year old man with AML in CR2. Which donor would you select? FULL CASE You are selecting an unrelated donor for a 40 year old man with AML in second complete remission with high-risk cytogenetics. He is CMV seronegative with blood type A?. He is an only child. The search coordinators have identified the following options: OptionAge/sexHLA matching (9/10 matched donors)CMV statusBlood typeYour ResponseDonor 150 FSingle allele mismatch at HLA-ACMV ?A+24%Donor 230 MSingle allele mismatch at HLA-CCMV +O+22%Donor 330 MSingle antigen mismatch at HLA-DQCMV +A+54% PMID:19203734

2015-01-01

16

Complexes of polyoma virus medium T antigen and cellular proteins.  

PubMed

Antibodies against synthetic peptides corresponding to the carboxyl-terminal six amino acids, Lys-Arg-Ser-Arg-His-Phe (KF), and an internal region, Glu-Glu-Glu-Glu-Tyr-Met-Pro-Met-Glu (EE), of polyoma virus medium T antigen were used successively to purify medium T antigen by affinity chromatography. Medium T antigen from cell extracts was first bound to anti-KF antibodies and released from the immune complex with excess KF peptide; then it was bound to anti-EE antibodies and released with excess EE peptide. Two proteins, pp60c-src and a new protein of approximately equal to 61,000 Da (61-kDa protein), were copurified because they formed complexes with medium T antigen. The 61-kDa protein-medium T antigen complex was detected in extracts from wild-type-infected and transformed cells but not from cells infected with NG59 virus, which has a mutation in the medium T gene and is transformation defective. Instead, NG59 medium T antigen formed a complex with another cellular protein of approximately equal to 72,000 Da. PMID:2415976

Grussenmeyer, T; Scheidtmann, K H; Hutchinson, M A; Eckhart, W; Walter, G

1985-12-01

17

The Role of Heat Shock Proteins in Antigen Cross Presentation  

PubMed Central

Heat shock proteins (HSPs) are molecular chaperones that bind tumor antigens and mediate their uptake into antigen presenting cells. HSP–antigen complexes are then directed toward either the MHC class I pathway through antigen cross presentation or the conventional class II pathway, leading to activation of T cell subsets. Uptake of HSP-chaperoned polypeptides can involve both receptor-mediated and receptor-independent routes, and mechanisms of antigen sorting between the Class I and II pathways after uptake are currently under investigation. The processes involved in internalization of HSP–antigen complexes differ somewhat from the mechanisms previously determined for (unchaperoned) particulate and free soluble antigens. A number of studies show that HSP-facilitated antigen cross presentation requires uptake of the complexes by scavenger receptors (SR) followed by processing in the proteasome, and loading onto MHC class I molecules. In this review we have examined the roles of HSPs and SR in antigen uptake, sorting, processing, cell signaling, and activation of innate and adaptive immunity. PMID:22566944

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

2012-01-01

18

Changes in structural and antigenic properties of proteins by radiation  

NASA Astrophysics Data System (ADS)

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

Kume, Tamikazu; Matsuda, Tsukasa

1995-08-01

19

Conformational Dynamics and Antigenicity in the Disordered Malaria Antigen Merozoite Surface Protein 2  

PubMed Central

Merozoite surface protein 2 (MSP2) of Plasmodium falciparum is an abundant, intrinsically disordered protein that is GPI-anchored to the surface of the invasive blood stage of the malaria parasite. Recombinant MSP2 has been trialled as a component of a malaria vaccine, and is one of several disordered proteins that are candidates for inclusion in vaccines for malaria and other diseases. Nonetheless, little is known about the implications of protein disorder for the development of an effective antibody response. We have therefore undertaken a detailed analysis of the conformational dynamics of the two allelic forms of MSP2 (3D7 and FC27) using NMR spectroscopy. Chemical shifts and NMR relaxation data indicate that conformational and dynamic properties of the N- and C-terminal conserved regions in the two forms of MSP2 are essentially identical, but significant variation exists between and within the central variable regions. We observe a strong relationship between the conformational dynamics and the antigenicity of MSP2, as assessed with antisera to recombinant MSP2. Regions of increased conformational order in MSP2, including those in the conserved regions, are more strongly antigenic, while the most flexible regions are minimally antigenic. This suggests that modifications that increase conformational order may offer a means to tune the antigenicity of MSP2 and other disordered antigens, with implications for vaccine design. PMID:25742002

Andrew, Dean; Krishnarjuna, Bankala; Nová?ek, Ji?í; Žídek, Lukáš; Sklená?, Vladimír; Richards, Jack S.; Beeson, James G.; Anders, Robin F.; Norton, Raymond S.

2015-01-01

20

Sequence conservation in the chagasin family suggests a common trend in cysteine proteinase binding by unrelated protein inhibitors.  

PubMed

The recently described inhibitor of cysteine proteinases from Trypanosoma cruzi, chagasin, was found to have close homologs in several eukaryotes, bacteria and archaea, the first protein inhibitors of cysteine proteases in prokaryotes. These previously uncharacterized 110-130 residue-long proteins share a well-conserved sequence motif that corresponds to two adjacent beta-strands and the short loop connecting them. Chagasin-like proteins also have other conserved, mostly aromatic, residues, and share the same predicted secondary structure. These proteins adopt an all-beta fold with eight predicted beta-strands of the immunoglobulin type. The phylogenetic distribution of the chagasins generally correlates with the presence of papain-like cysteine proteases. Previous studies have uncovered similar trends in cysteine proteinase binding by two unrelated inhibitors, stefin and p41, that belong to the cystatin and thyroglobulin families, respectively. A hypothetical model of chagasin-cruzipain interaction suggests that chagasin may dock to the cruzipain active site in a similar manner with the conserved NPTTG motif of chagasin forming a loop that is similar to the wedge structures formed at the active sites of papain and cathepsin L by stefin and p41. PMID:12142451

Rigden, Daniel J; Mosolov, Vladimir V; Galperin, Michael Y

2002-08-01

21

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

22

Outcomes of patients with myeloid malignancies treated with allogeneic hematopoietic stem cell transplantation from matched unrelated donors compared with one human leukocyte antigen mismatched related donors using HLA typing at 10 loci.  

PubMed

Most candidates for hematopoietic stem cell transplantation (HSCT) lack a human leukocyte antigen (HLA)-identical sibling donor. Some patients may have a related donor with whom they are mismatched at 1 antigen/allele. It is not known whether such a match is preferable to a matched unrelated donor (MUD). We evaluated the outcomes (survival, relapse, nonrelapse mortality [NRM]) of all 28 patients with a single HLA antigen/allele mismatch identified through high-resolution HLA typing at HLA-A, -B, -C, -DRB1, and -DQB1, and all 318 patients with myeloid malignancies who received transplants from a 10/10 MUD treated during the same period of time at a single institution. Overall, outcomes for patients treated from a 1-antigen/allele mismatch related donor were significantly worse than from a MUD, primarily because of increased NRM. Overall survival (OS) rates at 3 years for 1-antigen/allele mismatched related donor and MUD transplant recipients were 19% and 45% (P = .007), and NRM rates were 40% and 26% (P = .05), respectively. Patients with class I mismatches appeared to have poorer OS than did patients with class II mismatches. A higher incidence of graft rejection was identified in the mismatched related donor group (P = .02). These results indicate that transplant outcomes are better with a MUD than with a 1 antigen/allele-mismatched related donor. PMID:20969970

Ciurea, Stefan O; Saliba, Rima M; Rondon, Gabriela; Patah, Poliana A; Aung, Fleur; Cano, Pedro; Andersson, Borje S; Kebriaei, Partow; Popat, Uday; Fernandez-Vina, Marcelo; Champlin, Richard E; de Lima, Marcos

2011-06-01

23

Linking Two Seemingly Unrelated Diseases, Cancer and Acute Respiratory Distress Syndrome, Through a Dictyostelium Secreted Protein  

E-print Network

adenosine (86). The cytoplasmic domain of membrane DPPIV interacts with CD45, a membrane-linked tyrosine phosphatase, and initiates signaling to downstream effectors (87). DPPIV also interacts with the mannose 6-phosphate receptor, a partnership... that results in receptor internalization following T-cell activation (87). Interactions between DPPIV and the extracellular matrix protein molecules fibronectin and collagen are thought to increase cell migration (87). Additionally, soluble DPPIV (s...

Herlihy, Sarah E

2014-06-05

24

Identification of antigenic proteins of the nosocomial pathogen Klebsiella pneumoniae.  

PubMed

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

25

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

26

Presentation of peptide antigens by mouse CD1 requires endosomal localization and protein antigen processing  

PubMed Central

Mouse CD1(mCD1) molecules have been reported to present two types of antigens: peptides or proteins and the glycolipid ?-galactosylceramide. Here, we demonstrate that a protein antigen, chicken ovalbumin (Ova), must be processed to generate peptides presented by mCD1 to CD8+ T cells. The processing and mCD1-mediated presentation of chicken Ova depend on endosomal localization because inhibitors of endosomal acidification and endosomal recycling pathways block T cell reactivity. Furthermore, a cytoplasmic tail mutant of mCD1, which disrupts endosomal localization, has a greatly reduced capacity to present Ova to mCD1 restricted cells. Newly synthesized mCD1 molecules, however, are not required for Ova presentation, suggesting that molecules recycling from the cell surface are needed. Because of these data showing that mCD1 trafficks to endosomes, where it can bind peptides derived from exogenous proteins, we conclude that peptide antigen presentation by mCD1 is likely to be a naturally occurring phenomenon. In competition assays, ?-galactosylceramide did not inhibit Ova presentation, and presentation of the glycolipid was not inhibited by excess Ova or the peptide epitope derived from it. This suggests that, although both lipid and peptide presentation may occur naturally, mCD1 may interact differently with these two types of antigens. PMID:9826697

Tangri, Shabnam; Brossay, Laurent; Burdin, Nicolas; Lee, Delphine J.; Corr, Maripat; Kronenberg, Mitchell

1998-01-01

27

Chromatographic Separation and Antigenic Analysis of Proteins of the Oncornaviruses  

PubMed Central

Gel filtration of avian tumor virus proteins in 6 m guanidine hydrochloride clearly resolved seven major protein species. The antigenic activity of these proteins was recovered in good yield after removal of the denaturing solvent, permitting a correlation of specific polypeptides with the principal antigens of the virion. Two of the proteins, of molecular weights 70,000 and 32,000, contain carbohydrate and are situated on the viral membrane, as shown by their being accessible in the intact virus to specific antibodies. Four proteins, with molecular weights (in guanidine) of 27,000, 19,000, 15,000, and 12,000, have different group-specific (gs) antigens and are enclosed within the viral membrane. The smallest protein, with a molecular weight of 10,000, has not previously been described; it is not detectable with antisera and possesses a mobility identical to that of one gs protein when subjected to electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate. Of the proteins lacking carbohydrate, three are present in the virion in a molecular ratio of 2:2:1, and the two others, present in almost equal amount, are rich in lysine and arginine. Images PMID:4332144

Fleissner, Erwin

1971-01-01

28

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

29

Comparative Analyses of the Proteins and Antigens of Five Herpesviruses  

Microsoft Academic Search

SUMMARY The proteins of five herpesviruses - herpes simplex types I and 2 (HSV-I and HSV-2), bovine mammillitis virus (BMV), pseudorabies virus (PRV) and equine abortion virus (EAV) - have been compared by polyacrylamide gel electrophoresis of purified virus and by antigenic analysis using virus neutralization and agar-gel immunodiffusion tests. Although there were general similarities in the number, size range

R. A. Killington; JANE YEO; R. W. Honess; D. H. Watson; BARBARA E. DUNCAN; I. W. Halliburton; JENNIFER MUMFORD

1977-01-01

30

Prediction of protein antigenic determinants from amino acid sequences  

Microsoft Academic Search

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

T. P. Hopp; K. R. Woods

1981-01-01

31

Cell surface protein antigen from Wolinella recta ATCC 33238T.  

PubMed Central

A high-molecular-weight (approximately 150,000) protein was selectively isolated by acid extraction from the cell surface of Wolinella recta and purified by negative adsorption on DEAE-cellulose and gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that this protein was found in W. recta but not in other Wolinella species, such as W. curva and W. succinogenes. Sera from patients with periodontitis reacted strongly with this protein antigen, whereas sera from healthy donors showed little or no reactivity, as determined by immunoblotting analysis. In serum, titers of immunoglobulin G antibodies to the protein antigen were significantly higher in patients with periodontitis than in periodontally healthy donors, as detected by an enzyme-linked immunosorbent assay. Images PMID:2753998

Kokeguchi, S; Kato, K; Kurihara, H; Murayama, Y

1989-01-01

32

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

PubMed

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

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

2013-01-01

33

Antigenicity and Immunogenicity of Plasmodium vivax Merozoite Surface Protein-3  

PubMed Central

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

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

2013-01-01

34

Genes for the major protein antigens of Mycobacterium tuberculosis: the etiologic agents of tuberculosis and leprosy share an immunodominant antigen.  

PubMed Central

Mycobacterium tuberculosis genes encoding immunologically relevant proteins were isolated by systematically screening a lambda gt11 recombinant DNA expression library with a collection of murine monoclonal antibodies directed against protein antigens of this pathogen. These antibodies, previously characterized by a World Health Organization workshop on monoclonal antibodies against mycobacteria, were used to isolate DNA sequences encoding five major protein antigens of this pathogen. To evaluate the extent of crossreactivity between these M. tuberculosis antigens and antigens of Mycobacterium leprae, recombinant antigens were probed with monoclonal antibodies directed against the protein antigens of these bacilli. One of the antigens, a 65-kDa protein, has determinants common to M. tuberculosis and M. leprae. We find not only that this antigen is recognized by mouse monoclonal antibodies but that it is the major protein recognized by anti-M. tuberculosis rabbit sera. The 65-kDa proteins of M. tuberculosis and M. leprae appear to play a role in the humoral and cell-mediated immune response to these pathogens. Images PMID:3104901

Husson, R N; Young, R A

1987-01-01

35

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

36

Cloning, expression, and antigenicity of 14 proteins from Campylobacter jejuni.  

PubMed

Fourteen Campylobacter jejuni genes--porA, cadF, omp18, dnaK, flaC, peb1, peb2, peb3, peb4, ahpC, groEL, tuF, hipO, and Cj0069--were cloned and expressed in Escherichia coli BL21. The recombinant proteins were purified on histidine (His) and glutathione S-transferase (GST) trap columns using the ÄKTA Explorer 100 System. Recombinant proteins were visualized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The antigenicities of these recombinant proteins were assessed by Western blotting and enzyme-linked immunosorbent assays with anti-C. jejuni immune rabbit sera. Four recombinant proteins, including rGST-PorA, rHis-CadF, rGST-GroEL, and rGST-TuF, demonstrated reactions with both anti-serum and preimmune serum, while rHis-DnaK, rGST-FlaC, rGST-PEB2, rGST-PEB3, rGST-PEB4, and rGST-HipO showed variable antigenicity characteristics to the anti-sera derived from different C. jejuni strains. rHis-Omp18, rHis-PEB1, and rGST-AhpC demonstrated universal and specific antigenities with the entire anti-sera panel tested in this present study, while recombinant rGST-Cj0069 and rHis-DnaK did not react with any of the anti-C. jejuni sera tested. In conclusion, rGST-AhpC may be useful as a potential serodiagnostic antigen for C. jejuni infection. PMID:22779748

Zhang, Maojun; Meng, Fanliang; Cao, Fangfang; Qiao, Bo; Liu, Guodong; Liu, Hongying; Zhou, Yizhuang; Dong, Haiyan; Gu, Yixin; Xiao, Di; Zhang, Yongchan; Zhang, Jianzhong

2012-08-01

37

Interactions between polyomavirus medium T antigen and three cellular proteins of 88, 61, and 37 kilodaltons.  

PubMed

Affinity-purified medium T antigen of wild-type polyomavirus and dl8, a transforming mutant with a deletion in the medium T gene, is associated with three cellular proteins with apparent molecular weights of 88,000 (88K protein), 61,000 (61K protein), and 37,000 (37K protein). Medium T antigen encoded by the nontransforming hrt mutants fails to associate with these proteins, whereas medium T antigen of the nontransforming mutant dl1015 is able to do so. Medium T antigen of the nontransforming mutant dl23 binds to the 61K and 37K proteins; however, binding to the 88K protein is uncertain. The pattern of complex formation between these proteins and medium T antigen resembles that of pp60c-src and medium T antigen. The binding of medium T antigen to the 88K, 61K, and 37K proteins, as well as to pp60c-src, might represent a necessary but insufficient step in transformation. By mixing extracts from infected and uninfected cells, complex formation between medium T antigen and the 88K, 61K, and 37K proteins can be demonstrated in vitro. Pulse-chase experiments indicated that in vivo the association between medium T antigen and the 61K and 37K proteins is a slow process. The latter two proteins are probably bound to each other in uninfected cells. On two-dimensional gels of whole-cell extract, the 61K protein comigrated with a minor protein with an isoelectric point of 5.2. The 61K protein was neither phosphorylated nor glycosylated. Polyomavirus tumor serum precipitated the 61K and 37K proteins independently of medium T antigen. Therefore, the 61K protein or the 37K protein or both have the properties of a cellular tumor antigen. PMID:2824823

Grussenmeyer, T; Carbone-Wiley, A; Scheidtmann, K H; Walter, G

1987-12-01

38

Interactions between polyomavirus medium T antigen and three cellular proteins of 88, 61, and 37 kilodaltons.  

PubMed Central

Affinity-purified medium T antigen of wild-type polyomavirus and dl8, a transforming mutant with a deletion in the medium T gene, is associated with three cellular proteins with apparent molecular weights of 88,000 (88K protein), 61,000 (61K protein), and 37,000 (37K protein). Medium T antigen encoded by the nontransforming hrt mutants fails to associate with these proteins, whereas medium T antigen of the nontransforming mutant dl1015 is able to do so. Medium T antigen of the nontransforming mutant dl23 binds to the 61K and 37K proteins; however, binding to the 88K protein is uncertain. The pattern of complex formation between these proteins and medium T antigen resembles that of pp60c-src and medium T antigen. The binding of medium T antigen to the 88K, 61K, and 37K proteins, as well as to pp60c-src, might represent a necessary but insufficient step in transformation. By mixing extracts from infected and uninfected cells, complex formation between medium T antigen and the 88K, 61K, and 37K proteins can be demonstrated in vitro. Pulse-chase experiments indicated that in vivo the association between medium T antigen and the 61K and 37K proteins is a slow process. The latter two proteins are probably bound to each other in uninfected cells. On two-dimensional gels of whole-cell extract, the 61K protein comigrated with a minor protein with an isoelectric point of 5.2. The 61K protein was neither phosphorylated nor glycosylated. Polyomavirus tumor serum precipitated the 61K and 37K proteins independently of medium T antigen. Therefore, the 61K protein or the 37K protein or both have the properties of a cellular tumor antigen. Images PMID:2824823

Grussenmeyer, T; Carbone-Wiley, A; Scheidtmann, K H; Walter, G

1987-01-01

39

Goodpasture Antigen-binding Protein (GPBP) Directs Myofibril Formation  

PubMed Central

Goodpasture antigen-binding protein-1 (GPBP-1) is an exportable non-conventional Ser/Thr kinase that regulates glomerular basement membrane collagen organization. Here we provide evidence that GPBP-1 accumulates in the cytoplasm of differentiating mouse myoblasts prior to myosin synthesis. Myoblasts deficient in GPBP-1 display defective myofibril formation, whereas myofibrils assemble with enhanced efficiency in those overexpressing GPBP-1. We also show that GPBP-1 targets the previously unidentified GIP130 (GPBP-interacting protein of 130 kDa), which binds to myosin and promotes its myofibrillar assembly. This report reveals that GPBP-1 directs myofibril formation, an observation that expands its reported role in supramolecular organization of structural proteins to the intracellular compartment. PMID:21832087

Revert-Ros, Francisco; López-Pascual, Ernesto; Granero-Moltó, Froilán; Macías, Jesús; Breyer, Richard; Zent, Roy; Hudson, Billy G.; Saadeddin, Anas; Revert, Fernando; Blasco, Raül; Navarro, Carmen; Burks, Deborah; Saus, Juan

2011-01-01

40

Similar transplantation outcomes for acute myeloid leukemia and myelodysplastic syndrome patients with haploidentical versus 10/10 human leukocyte antigen-matched unrelated and related donors.  

PubMed

Allogeneic stem cell transplantation for patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) has been performed primarily with an HLA-matched donor. Outcomes of haploidentical transplantation have recently improved, and a comparison between donor sources in a uniform cohort of patients has not been performed. We evaluated outcomes of 227 patients with AML/MDS treated with melphalan-based conditioning. Donors were matched related (MRD) (n = 87, 38%), matched unrelated (MUD) (n = 108, 48%), or haploidentical (n = 32, 14%). No significant differences were found between haploidentical and MUD transplantation outcomes; however, there was a trend for improved outcomes in the MRD group, with 3-year progression-free survival for patients in remission of 57%, 45%, and 41% for MRD, MUD, and haploidentical recipients, respectively (P = .417). Recovery of T cell subsets was similar for all groups. These results suggest that haploidentical donors can safely extend transplantation for AML/MDS patients without an HLA-matched donor. Prospective studies comparing haploidentical and MUD transplantation are warranted. PMID:25263628

Di Stasi, Antonio; Milton, Denái R; Poon, L M; Hamdi, Amir; Rondon, Gabriela; Chen, Julianne; Pingali, Sai R; Konopleva, Marina; Kongtim, Piyanuch; Alousi, Amin; Qazilbash, Muzaffar H; Ahmed, Sairah; Bashir, Qaiser; Al-atrash, Gheath; Oran, Betul; Hosing, Chitra M; Kebriaei, Partow; Popat, Uday; Shpall, Elizabeth J; Lee, Dean A; de Lima, Marcos; Rezvani, Katayoun; Khouri, Issa F; Champlin, Richard E; Ciurea, Stefan O

2014-12-01

41

Stress proteins and the immune response to mycobacteria — Antigens as virulence factors?  

Microsoft Academic Search

The immune response to mycobacterial infection includes pathogenic as well as protective activities. It is possible that different types of immune responses are associated with recognition of different antigenic determinants. Amongst the antigens which are prominent in antibody and T cell recognition of mycobacteria, we have identified members of highly conserved stress protein families. Mapping of antigenic determinants on stress

D B Young; A Mehlert; V Bal; P Mendez-Samperio; J Ivanyi; J R Lamb

1988-01-01

42

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

43

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

PubMed Central

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

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

2008-01-01

44

Large T antigens of many polyomaviruses are able to form complexes with the retinoblastoma protein.  

PubMed

Stable protein complexes between the large T antigens of mouse, monkey, baboon, or human polyomaviruses and the retinoblastoma protein were detected by an in vitro coimmunoprecipitation assay. All of the large T antigens tested were able to bind to both human and mouse retinoblastoma polypeptides, showing that these interactions have been conserved during evolution. PMID:2154613

Dyson, N; Bernards, R; Friend, S H; Gooding, L R; Hassell, J A; Major, E O; Pipas, J M; Vandyke, T; Harlow, E

1990-03-01

45

A major allogenic leukocyte antigen in the agnathan hagfish  

PubMed Central

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

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

2013-01-01

46

Towards Preserving the Immunogenicity of Protein Antigens Carried by Nanoparticles While Avoiding the Cold Chain  

PubMed Central

Nanoparticles are an attractive vaccine carrier with potent adjuvant activity. Data from our previous studies showed that immunization of mice with lecithin/glyceryl monostearate-based nanoparticles with protein antigens conjugated onto their surface induced a strong, quick, and long-lasting antigen-specific immune response. In the present study, we evaluated the feasibility of preserving the immunogenicity of protein antigens carried by nanoparticles without refrigeration using these antigen-conjugated nanoparticles as a model. The nanoparticles were lyophilized, and the immunogenicity of the antigens was evaluated in a mouse model using bovine serum albumin or the Bacillus anthracis protective antigen protein as model antigens. With proper excipients, the nanoparticles can be lyophilized while maintaining the immunogenicity of the antigens. Moreover, the immunogenicity of the model antigen conjugated onto the nanoparticles was undamaged after a relatively extended period of storage at room temperature or under accelerated conditions (37°C) when the nanoparticles were lyophilized with 5% mannitol plus 1% polyvinylpyrrolidone. To our knowledge, the present study represents an early attempt to preserve the immunogenicity of the protein antigens carried by nanoparticles without refrigeration. PMID:20416366

Sloat, Brian R.; Sandoval, Michael A.; Cui, Zhengrong

2010-01-01

47

Characterization of the interaction of polyomavirus middle T antigen with type 2A protein phosphatase.  

PubMed Central

Two cellular proteins of 36 and 63 kDa which bind the small T and middle T antigens of polyomavirus recently have been identified as the catalytic and regulatory subunits of the phosphoserine/threonine-specific type 2A protein phosphatase (PP2A). We report here the presence of phosphoseryl phosphatase activity associated with polyomavirus small T and middle T antigens in immunoprecipitates prepared from virus-infected and transformed cells. Phosphatase activity was also found associated with middle T-antigen mutants, some of which had been defined previously to associate with 36- and 63-kDa cellular proteins. Middle T-antigen-associated phosphatase activity was sensitive to okadaic acid and microcystin-LR, inhibitors of PP2A, and insensitive to inhibitor 1 or 2, orthovanadate, or EDTA. Using antiserum specific for the catalytic subunit of PP2A, we found that unlike the majority of PP2A, middle T-antigen-bound PP2A was membrane associated. However, no gross change in the amount, activity, or localization of PP2A could be attributed to middle T-antigen expression in transformed cells. Anti-PP2A antibodies coprecipitated a 63-kDa protein from normal cells and in addition coprecipitated middle T antigen, 60- and 61-kDa proteins (identified as src family members), and an 81-kDa protein from middle T-antigen-transformed cells. Furthermore, we detected protein kinase activity in PP2A immunoprecipitates and protein phosphatase activity in src immune complexes from extracts of middle T-antigen-transformed, but not normal, cells. These results reinforce the notion that at least a portion of middle T antigen bridges a protein kinase with a protein phosphatase. Images PMID:1371166

Ulug, E T; Cartwright, A J; Courtneidge, S A

1992-01-01

48

Complexes of Polyoma Virus Medium T Antigen and Cellular Proteins  

Microsoft Academic Search

Antibodies against synthetic peptides corresponding to the carboxyl-terminal six amino acids, Lys-Arg-Ser-Arg-His-Phe (KF), and an internal region, Glu-Glu-Glu-Glu-Tyr-Met-Pro-Met-Glu (EE), of polyoma virus medium T antigen were used successively to purify medium T antigen by affinity chromatography. Medium T antigen from cell extracts was first bound to anti-KF antibodies and released from the immune complex with excess KF peptide; then it

Thomas Grussenmeyer; Karl Heinz Scheidtmann; Mary Anne Hutchinson; Walter Eckhart; Gernot Walter

1985-01-01

49

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

50

Passive immunity to yersiniae mediated by anti-recombinant V antigen and protein A-V antigen fusion peptide.  

PubMed Central

LcrV (V antigen), a known unstable 37.3-kDa monomeric peptide encoded on the ca. 70-kb Lcr plasmid of Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica, has been implicated as a regulator of the low-calcium response, virulence factor, and protective antigen. In this study, lcrV of Y. pestis was cloned into protease-deficient Escherichia coli BL21. The resulting recombinant V antigen underwent marked degradation from the C-terminal end during purification, yielding major peptides of 36, 35, 34, and 32 to 29 kDa. Rabbit gamma globulin raised against this mixture of cleavage products provided significant protection against 10 minimum lethal doses of Y. pestis (P < 0.01) and Y. pseudotuberculosis (P < 0.02). To both stabilize V antigen and facilitate its purification, plasmid pPAV13 was constructed so as to encode a fusion of lcrV and the structural gene for protein A (i.e., all but the first 67 N-terminal amino acids of V antigen plus the signal sequence and immunoglobulin G-binding domains but not the cell wall-associated region of protein A). The resulting fusion peptide, termed PAV, could be purified to homogeneity in one step by immunoglobulin G affinity chromatography and was stable thereafter. Rabbit polyclonal gamma globulin directed against PAV provided excellent passive immunity against 10 minimum lethal doses of Y. pestis (P < 0.005) and Y. pseudotuberculosis (P < 0.005) but was ineffective against Y. enterocolitica. Protection failed after absorption with excess PAV, cloned whole V antigen, or a large (31.5-kDa) truncated derivative of the latter but was retained (P < 0.005) upon similar absorption with a smaller (19.3-kDa) truncated variant, indicating that at least one protective epitope resides internally between amino acids 168 and 275. Images PMID:7927675

Motin, V L; Nakajima, R; Smirnov, G B; Brubaker, R R

1994-01-01

51

Identification of major antigenic proteins of Edwardsiella tarda recognized by Japanese flounder antibody.  

PubMed

Edwardsiella tarda is a fish pathogen that causes systemic infections in fresh water and marine fish. Determining the antigenic proteins is important for the development of an immunodiagnostic tests and a vaccine for effective infection control in fish. In the current study, antigens were detected by immunoblotting and affinity column chromatography using a Japanese flounder (Paralichthys olivaceus) antibody produced by experimental infection with E. tarda. GroEL, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), outer membrane protein A, filament protein, 30S ribosomal protein S6, 50S ribosomal protein L9, cold shock protein, and carbon storage protein were identified as antigens of E. tarda through biochemical analyses of the molecular weights, isoelectric points, and N-terminal amino-acid sequences. These proteins can be easily detected in flounder infected with E. tarda and are potential diagnostic markers. PMID:19564499

Sakai, Takamitsu; Matsuyama, Tomomasa; Nishioka, Toyohiro; Nakayasu, Chihaya; Kamaishi, Takashi; Yamaguchi, Kenichi; Iida, Takaji

2009-07-01

52

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 ...

53

A model for the hepatitis B virus core protein: prediction of antigenic sites and relationship to RNA virus capsid proteins.  

PubMed Central

The sequences of the core proteins from several serotypes of human hepatitis B virus and related mammalian and avian hepadnaviruses are aligned with the vp3 capsid protein of mengo virus, a picornavirus. The homology indicates an eight-stranded antiparallel beta-barrel fold for the hepatitis protein, as observed in the tertiary structure of the picornavirus protein. The locations of known antigenic sites and other modifications are consistent with this structure for the core protein. The predicted folding suggests additional exposed antigenic sites and supports an evolutionary relationship between this family of enveloped DNA viruses and enveloped and non-enveloped RNA viruses. PMID:2456212

Argos, P; Fuller, S D

1988-01-01

54

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

Microsoft Academic Search

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

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

1984-01-01

55

PostTranslational Modifications of Proteins: Implications for Aging, Antigen Recognition, and Autoimmunity  

Microsoft Academic Search

Proteins are complex organic molecules susceptible to numerous post-translational modifications occurring spontaneously during aging or as a consequence of physiologic or pathologic processes. Antigenicity and interactions of proteins with components of the immune system may be profoundly affected by post-translational modifications. Thus, modified self-antigens may be absent (not-tolerated) during early T-cell selection and trigger reactions by the immune system as

Paul A. C. Cloos; Stephan Christgau

2004-01-01

56

Evaluation of Anaplasma marginale major surface protein 3 (MSP3) as a diagnostic test antigen.  

PubMed Central

An immunodominant surface protein, major surface protein 3 (MSP3), has been proposed as an antigen suitable for use in the diagnosis of bovine anaplasmosis. We further characterized MSP3 to examine its potential as a test antigen for the serological diagnosis of carrier cattle. The specificity of this antigen in detecting infected cattle as well as the conservation of MSP3 between strains of Anaplasma marginale was evaluated by using immunoblots of A. marginale proteins separated by one- and two-dimensional polyacrylamide gel electrophoreses. Immune sera from animals infected with Anaplasma ovis, Ehrlichia risticii, and Ehrlichia ewingii reacted with the MSP3 antigen of A. marginale. One-dimensional gel electrophoresis of A. marginale proteins demonstrated size polymorphism of MSP3 between different geographic isolates. Two-dimensional gel electrophoresis revealed at least three different antigens migrating at the 86-kDa molecular size, and sera from animals infected with different strains of A. marginale reacted with different 86-kDa antigens. These results indicate that MSP3 may share cross-reactive epitopes with antigens found in A. ovis and some Ehrlichia spp. In addition, MSP3 is not conserved between different isolates of A. marginale, and at least in the isolate from Florida, what was previously identified as MSP3 is actually a group of three or more 86-kDa antigens with different isoelectric points. The cross-reactivity of MSP3 with some Ehrlichia spp., the variability of MSP3 between isolates, and the multiple 86-kDa antigens recognized by various sera suggest that MSP3 is not a suitable candidate for use as a recombinant test antigen. PMID:8788999

Alleman, A R; Barbet, A F

1996-01-01

57

Antigenic proteins of Flavobacterium psychrophilum recognized by ayu Plecoglossus altivelis antisera.  

PubMed

Flavobacterium psychrophilum is the causative agent of bacterial coldwater disease (BCWD) in ayu Plecoglossus altivelis altivelis and is responsible for substantial economic losses in ayu culture in Japan. To develop effective vaccines for the disease, we identified antigenic proteins of F. psychrophilum using immunoglobulin from ayu that had recovered from BCWD. The whole protein extracted from the bacterium was separated using 2-dimensional polyacrylamide gel electrophoresis and was transferred to a polyvinylidene fluoride membrane. Subsequently, antigenic proteins of the bacterium were detected using western blotting and ayu antisera against F. psychrophilum. Each protein spot showing antigenicity was subjected to tandem mass spectrometry (MS/MS) analysis using a MALDI-QIT-TOF mass spectrometer. Protein identification based on the MS/MS data was performed using the genome database for F. psychrophilum JIP02/86, and the subcellular localization for each identified protein was predicted with web-based tools (LipoP and PSORTb). In total, 62 antigenic proteins were identified: of these, 46 were putative cytoplasmic proteins (e.g. elongation factor Tu and heat shock protein 90). The remaining 21 proteins were identified as putative membrane-bound or secreted proteins and are potential vaccine candidates. These proteins include OmpA, Omp 121, M13 family metallopeptidase, and M48 family metalloprotease. PMID:24553416

Kato, Goshi; Sakai, Takamitsu; Suzuki, Kyuma; Yamaguchi, Kenichi; Takano, Tomokazu; Matsuyama, Tomomasa; Nakayasu, Chihaya

2014-02-19

58

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

PubMed

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 1 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; Souza, C da Silva; Huang, C-T; Larsson, M; Esener, S; Messmer, D

2010-11-01

59

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

60

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

61

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

62

Production of extensive chickpea (Cicer arietinum L.) protein hydrolysates with reduced antigenic activity.  

PubMed

Chickpea protein isolate was used as starting material for the production of hypoallergenic protein hydrolysates. Western blotting of the protein isolate showed that IgE in sensitized patient sera strongly bound to the basic polypeptidic chains and recognized the acidic ones of 11S globulin. During the hydrolysis process by the individual and/or sequential action of endo- and exoproteases, a high reduction of antigenic activity was observed. Results suggest that the presence of intact or partially hydrolyzed basic polypeptide chains of 11S globulin are responsible for the formation of IgE complexes in protein hydrolysates obtained by exoprotease treatment; however, the digestion of these polypeptide chains by individual action of endoprotease caused a high loss of antigenic activity. The most effective reduction of antigenicity, >90%, was observed in extensive hydrolyzed chickpea proteins obtained by sequential treatment with endo- and exopeptidases. This chickpea protein hydrolysate could be useful for the elaboration of specialized hypoallergenic food products. PMID:10552721

Clemente, A; Vioque, J; Sanchez-Vioque, R; Pedroche, J; Millán, F

1999-09-01

63

An analysis of bison erythrocyte antigens and blood proteins  

E-print Network

hemolysis, agglutination of the erythrocytes and allergic reactions. Karl Landsteiner (Zmijewski, 1978) in 1901, was the first to point out that there were immunologic differences among red blood cell antigens. He found that humans of blood group A... that there existed a simple genetic relationship between the presence and absence of particular red cell antigens. Since Landsteiner (Land- stei ner, 1936) recognised s1mi lar differences in the blood cells of man, many attempts have been made to demonstrate...

Zamora, Linda Elia

1983-01-01

64

Detection and antigenicity of chlamydial proteins that bind eukaryotic cell membrane proteins.  

PubMed

Chlamydia psittaci proteins capable of binding eukaryotic cell membranes were identified and antigenically characterized. Cell membrane proteins (CMP) of noninfected cells were labeled with biotin (B-CMP), then were extracted with 1% Triton X-100. Nitrocellulose membrane strips containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of chlamydial elementary bodies (EB) were reacted with the B-CMP extract, followed by addition of streptavidin-conjugated horse radish peroxidase. Among the various strains of chlamydiae examined, a protein of approximately 16 to 18 kDa consistently bound B-CMP. A second larger protein, ranging in molecular mass from 24 to 32 kDa, also bound B-CMP. Immunoblotting techniques were used to analyze the reactions of antisera from immunized and experimentally infected animals to these proteins. A rabbit polyclonal antiserum produced against the 18-kDa adhesin of a serovar-1 strain of C psittaci (B577) reacted strongly with 18-kDa proteins of all C psittaci strains, but weakly with that of C trachomatis. Mouse antisera raised against the serovar-2 (FC-Stra) 28-kDa protein reacted only with proteins of the homologous serovar. Sera from experimentally infected animals did not react with the C trachomatis 18-kDa adhesion protein, but did react in 2 patterns with related and nonrelated C psittaci isolates. Two rabbits inoculated with infective serovar-1 EB and 1 rabbit inoculated with a serovar-2 strain reacted specifically with the 18-kDa proteins of their homologous serovars. In contrast, 2 other rabbits inoculated with the same serovar-2 strain produced antisera that reacted with all C psittaci 18-kDa proteins, as did serum from a similarly inoculated bull.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1378251

Baghian, A; Schnorr, K L

1992-06-01

65

Murine T-cell response to native and recombinant protein antigens of Rickettsia tsutsugamushi.  

PubMed Central

A polyclonal T-cell line with TH1 characteristics was used to assess the murine cellular immune response to native and recombinant Rickettsia tsutsugamushi antigens. Proliferation of this T-cell line was observed in response to numerous native antigen fractions, which indicates that the murine T-helper-cell response is directed at multiple scrub typhus antigens with no apparent antigenic immunodominance. Subsequent analysis of recombinant R. tsutsugamushi antigens made it possible to identify a 47-kDa scrub typhus antigen (Sta47) that was stimulatory for the polyclonal T-cell line. Recombinant clones encoding 56-, 58-, and 110-kDa antigens (Sta56, Sta58, and Sta110, respectively) were unable to induce proliferation of this T-cell line. DNA sequence analysis of the cloned rickettsial insert encoding the Sta47 protein revealed the presence of four open reading frames potentially encoding proteins of 47, 30, 18, and 13 kDa. Analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated and eluted fractions of lysates from the recombinant HB101(pRTS47B4.3) demonstrated that the fractions containing the 47-kDa protein as well as those containing proteins less than 18 kDa were stimulatory. Selected synthetic amphipathic peptides derived from the Sta47 antigen sequence identified a 20-amino-acid peptide that gave a 10-fold increase in T-cell proliferation over a control malarial peptide of similar length. Recognition of the 47-kDa antigen by a T-cell line with TH1 characteristics implicates this protein as one of potential importance in protection studies and future vaccine development. Images PMID:8478055

Hickman, C J; Stover, C K; Joseph, S W; Oaks, E V

1993-01-01

66

Co-administration of non-carrier nanoparticles boosts antigen immune response without requiring protein conjugation.  

PubMed

Nanotechnology promises a revolution in medicine including through new vaccine approaches. The use of nanoparticles in vaccination has, to date, focused on attaching antigen directly to or within nanoparticle structures to enhance antigen uptake by immune cells. Here we question whether antigen incorporation with the nanoparticle is actually necessary to boost vaccine effectiveness. We show that the immunogenicity of a sub-unit protein antigen was significantly boosted by formulation with silica nanoparticles even without specific conjugation of antigen to the nanoparticle. We further show that this effect was observed only for virus-sized nanoparticles (50 nm) but not for larger (1,000 nm) particles, demonstrating a pronounced effect of nanoparticle size. This non-attachment approach has potential to radically simplify the development and application of nanoparticle-based formulations, leading to safer and simpler nanoparticle applications in vaccine development. PMID:24793947

Wibowo, Nani; Chuan, Yap P; Seth, Arjun; Cordoba, Yoann; Lua, Linda H L; Middelberg, Anton P J

2014-06-17

67

Lumazine synthase protein cage nanoparticles as antigen delivery nanoplatforms for dendritic cell-based vaccine development  

PubMed Central

Purpose Protein cages are promising nanoplatform candidates for efficient delivery systems due to their homogenous size and structure with high biocompatibility and biodegradability. In this study, we investigate the potential of lumazine synthase protein cage as an antigen delivery system to dendritic cells (DCs), which induce antigen-specific T cell proliferation. Materials and Methods Ovalbumin (OVA) peptides OT-1 (SIINFEKL) and OT-2 (ISQAVHAAHAEINEAGR) were genetically inserted to lumazine synthase and each protein cage was over-expressed in Escherichia coli as a soluble protein. The efficiency of antigen delivery and the resulting antigen-specific T cell proliferation by DCs was examined in vitro as well as in vivo. Results We successfully generated and characterized OVA peptides carrying lumazine synthase protein cages. The OT-1 and OT-2 peptides carried by lumazine synthases were efficiently delivered and processed by DCs in vitro as well as in vivo, and induced proliferation of OT-1-specific CD8+T cells and OT-2-specific CD4+T cells. Conclusion Our data demonstrate the potential of lumazine synthase protein cage being used as a novel antigen delivery system for DC-based vaccine development in future clinical applications. PMID:25003097

2014-01-01

68

Proteins and antigens of Pasteurella multocida serotype 1 from fowl cholera.  

PubMed

The protein profiles of Pasteurella multocida serotype 1 isolates and the response of chickens to serotype 1 antigens were investigated using SDS-PAGE. Patterns obtained with Coomassie blue staining of soluble protein extracts were similar. The major difference between isolates was the position of one of the major proteins in the 34-38 kDa region. When chickens were experimentally infected with a clinical isolate of P. multocida serotype 1 various proteins were recognised by immunoblotting, including one with a relative molecular weight of 34 kDa; however, no reactions were observed in the region where LPS is known to migrate. When these infection sera were used in an EIA with purified LPS obtained from Heddleston serotype 1 type strain (X-73) they reacted strongly. Serum used for serotyping isolates in the gel diffusion precipitin test recognised many antigens in common with sera from infected birds, but some antigens were specific to typing sera. PMID:1829560

Ireland, L; Adler, B; Milner, A R

1991-04-01

69

Overexpression and Enzymatic Assessment of Antigenic Fragments of Hyaluronidase Recombinant Protein From Streptococcus pyogenes  

PubMed Central

Background: Hyaluronidase catalyzes the hydrolysis of hyaluronan polymers to N-acetyl-D-glucosamine and D-glucuronic acid. This enzyme is a dimer of identical subunits. Hyaluronidase has different pharmaceutical and medical applications. Previously, we produced a recombinant hyaluronidase antigenic fragment of Streptococcus pyogenes. Objectives: This study aimed to improve the protein production and purity of hyaluronidase recombinant protein from S. pyogenes. In addition, the enzymatic activity of this protein was investigated. Materials and Methods: The expression of hyaluronidase antigenic fragments was optimized using IPTG concentration, time of induction, temperature, culture, and absorbance of 0.6-0.8-1 at 600 nm. Afterwards, the expressed proteins were purified and the enzymatic activity was assessed by turbid metric method. Results: Data indicated that maximum protein is produced in OD = 0.8, 0.5 mM Isopropyl ?-D-1-thiogalactopyranoside (IPTG), 37ºC, NB 1.5x, without glucose, incubated for overnight. The enzymatic activity of the recombinant protein was similar to the commercial form of hyaluronidase. Conclusions: The results showed that an antigenic fragment of the recombinant hyaluronidase protein from S. pyogenes has a considerable enzymatic activity. It can be suggested to use it for medical purposes. In addition, applications of bioinformatics software would facilitate the production of a smaller protein with same antigenic properties and enzymatic activity. PMID:25789122

Sadoogh Abbasian, Shabnam; Ghaznavi Rad, Ehsanollah; Akbari, Neda; Zolfaghari, Mohammad Reza; pakzad, Iraj; Abtahi, Hamid

2014-01-01

70

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

71

Enzymatic Hydrolysis of Heated Whey: Iron-Binding Ability of Peptides and Antigenic Protein Fractions  

Microsoft Academic Search

This study evaluated the influence of various en- zymes on the hydrolysis of whey protein concentrate (WPC) to reduce its antigenic fractions and to quantify the peptides having iron-binding ability in its hydroly- sates. Heated (for 10 min at 100°C) WPC (2% protein solution) was incubated with 2% each of Alcalase, Fla- vourzyme, papain, and trypsin for 30, 60, 90,

S. B. Kim; I. S. Seo; M. A. Khan; K. S. Ki; W. S. Lee; H. J. Lee; H. S. Shin; H. S. Kim

2007-01-01

72

Targeting and expression of antigenic proteins in transgenic plants for production of edible oral vaccines  

Microsoft Academic Search

Summary  Exploiting plants as biological bioreactors for production and delivery of edible oral subunit vaccines is a promising application\\u000a of biotechnology. Efforts to enhance expression levels of transgenes coding for antigenic proteins by exploiting promoters,\\u000a targeting sequences, and enhancer elements have produced rather low quantities of the antigen in plant tissues, but enough\\u000a to induce immune responses in feeding studies. This

Schuyler S. Korban

2002-01-01

73

Protease digestion of hepatitis A virus: disparate effects on capsid proteins, antigenicity, and infectivity.  

PubMed Central

High concentrations of either trypsin or chymotrypsin caused nearly complete cleavage of capsid protein VP2 of hepatitis A virus but did not significantly reduce the infectivity, thermostability, or antigenicity of the virus. Chymotrypsin also had a lesser effect on VP1. These findings indicate the presence of a protease-accessible VP2 surface site which neither contributes significantly to the dominant antigenic site nor plays a role in the attachment of the virus to putative cell receptors. Images PMID:1654460

Lemon, S M; Amphlett, E; Sangar, D

1991-01-01

74

Recombinant early secreted antigen target 6 protein as a skin test antigen for the specific detection of Mycobacterium tuberculosis infection  

PubMed Central

Although the delayed-type hypersensitivity skin test reaction to tuberculin purified protein derivative (PPD) is used worldwide for tuberculosis (TB) detection, it is incapable of distinguishing Mycobacterium tuberculosis (MTB) infection from bacille Calmette–Guérin (BCG) vaccination or infection with non-tuberculous Mycobacteria. As a result, there is an urgent need for a more specific diagnostic tool for TB. This study reports the skin reactions of guinea pigs and human volunteers to recombinant early secreted antigen target 6 (rESAT6), a secretory protein found only in MTB, M. bovis and few other mycobacterial species. These volunteers had varying histories of BCG vaccination and exposure to MTB, allowing us to determine the specificity of their response to TB exposure. Our results show that 1·0 ?g of the purified MTB rESAT6 antigen elicited a positive skin response in both animals and humans exposed to MTB, as well as in animals exposed to M. bovis and M. marinum, all species of Mycobacteria that contain the gene for early secreted antigen target 6 (ESAT6). ESAT6 appears to be more specific to MTB infection than PPD, as demonstrated by the fact that we saw no skin responses in the BCG-vaccinated volunteers, nor in the guinea pigs sensitized with BCG vaccine, or with Mycobacteria that do not contain the gene encoding ESAT6. We believe that this is the first report of the use of a rESAT6 protein in a skin test in human volunteers, and that these data support its use in the specific detection of MTB infection. PMID:18321349

Wu, X; Zhang, L; Zhang, J; Zhang, C; Zhu, L; Shi, Y

2008-01-01

75

Protection of Mice with a Tuberculosis Subunit Vaccine Based on a Fusion Protein of Antigen 85B and ESAT-6  

Microsoft Academic Search

In this study, we investigated the potential of a tuberculosis subunit vaccine based on fusion proteins of the immunodominant antigens ESAT-6 and antigen 85B. When the fusion proteins were administered to mice in the adjuvant combination dimethyl dioctadecylammonium bromide-monophosphoryl lipid A, a strong dose- dependent immune response was induced to both single components as well as to the fusion proteins.

ANJA WEINREICH OLSEN; LAURENS A. H. VAN PINXTEREN; LIMEI MENG OKKELS; PETER BIRK RASMUSSEN; PETER ANDERSEN

2001-01-01

76

Strong antibody responses induced by protein antigens conjugated onto the surface of lecithin-based nanoparticles.  

PubMed

An accumulation of research over the years has demonstrated the utility of nanoparticles as antigen carriers with adjuvant activity. Herein we defined the adjuvanticity of a novel lecithin-based nanoparticle engineered from emulsions. The nanoparticles were spheres of around 200nm. Model protein antigens, bovine serum albumin (BSA) or Bacillus anthracis protective antigen (PA) protein, were covalently conjugated onto the nanoparticles. Mice immunized with the BSA-conjugated nanoparticles developed strong anti-BSA antibody responses comparable to that induced by BSA adjuvanted with incomplete Freund's adjuvant and 6.5-fold stronger than that induced by BSA adsorbed onto aluminum hydroxide. Immunization of mice with the PA-conjugated nanoparticles elicited a quick, strong, and durable anti-PA antibody response that afforded protection of the mice against a lethal dose of anthrax lethal toxin challenge. The potent adjuvanticity of the nanoparticles was likely due to their ability to move the antigens into local draining lymph nodes, to enhance the uptake of the antigens by antigen-presenting cells (APCs), and to activate APCs. This novel nanoparticle system has the potential to serve as a universal protein-based vaccine carrier capable of inducing strong immune responses. PMID:19729045

Sloat, Brian R; Sandoval, Michael A; Hau, Andrew M; He, Yongqun; Cui, Zhengrong

2010-01-01

77

Proteomic Screening of Antigenic Proteins from the Hard Tick, Haemaphysalis longicornis (Acari: Ixodidae)  

PubMed Central

Proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. For detection of antigens from Haemaphysalis longicornis, 1-dimensional electrophoresis (1-DE) quantitative immunoblotting technique combined with 2-dimensional electrophoresis (2-DE) immunoblotting was used for whole body proteins from unfed and partially fed female ticks. Reactivity bands and 2-DE immunoblotting were performed following 2-DE electrophoresis to identify protein spots. The proteome of the partially fed female had a larger number of lower molecular weight proteins than that of the unfed female tick. The total number of detected spots was 818 for unfed and 670 for partially fed female ticks. The 2-DE immunoblotting identified 10 antigenic spots from unfed females and 8 antigenic spots from partially fed females. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) of relevant spots identified calreticulin, putative secreted WC salivary protein, and a conserved hypothetical protein from the National Center for Biotechnology Information and Swiss Prot protein sequence databases. These findings indicate that most of the whole body components of these ticks are non-immunogenic. The data reported here will provide guidance in the identification of antigenic proteins to prevent infestation and diseases transmitted by H. longicornis. PMID:25748713

Kim, Young-Ha; slam, Mohammad Saiful; You, Myung-Jo

2015-01-01

78

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

79

Homologous cellular proteins associated with simian virus 40 small T antigen and polyomavirus medium T antigen.  

PubMed Central

The simian virus 40 small T-associated 56,000-Mr (56K) and 32K cellular proteins were shown to be closely related to the polyomavirus medium T-associated 61K and 37K cellular proteins as demonstrated by two-dimensional polyacrylamide gel electrophoresis and V8 protease peptide mapping. Images PMID:3184277

Walter, G; Carbone-Wiley, A; Joshi, B; Rundell, K

1988-01-01

80

A recombinant hybrid protein as antigen for an anti-blood stage malaria vaccine.  

PubMed

Based on investigations on several blood stage antigens from Plasmodium falciparum we have expressed a hybrid protein in E. coli containing 262 amino acids of the serine-stretch protein SERP and 189 amino acids of the histidine alanine rich protein HRPII. Antibodies raised against the hybrid protein by immunization of rabbits and Aotus monkeys react with both corresponding schizont polypeptides. Two Aotus monkeys immunized with the SERP/HRPII hybrid protein showed only low parasitemias after challenge infection with P. falciparum, compared to the control group. The result suggests that hybrid proteins of this type may be the basis for the development of a malaria vaccine. PMID:2049032

Knapp, B; Hundt, E; Enders, B; Küpper, H A

1991-02-01

81

Polymer-metal complexes of protein antigens--new highly effective immunogens.  

PubMed

The mechanism of interaction of the copolymers N-vinylpyrrolidone-acrylic acid and N-vinylpyrrolidone-maleic anhydride with bovine serum albumin, influenza virus total surface antigen (haemagglutinin and neuraminidase), and the BCG protein fraction in the presence of divalent copper ions was investigated. Novel water-soluble triple polymer-metal complexes of the above protein antigens were formed. These complexes showed high immunogenicity and conferred high levels of immunological protection. Study of the replication of pathogenic influenza A virus in animal lungs showed that, in mice immunised with the triple complex containing surface glycoprotein influenza virus A antigens, reproduction of the homologous virus was sharply inhibited, and immunisation of B mice, exhibiting pronounced T-cell deficiency, with complexes containing the BCG protein fraction ensured development of a high level of protection with respect to BCG infection. PMID:2103830

Mustafaev, M I; Norimov, A Sh

1990-03-01

82

Antigenicity in sheep of synthetic peptides derived from stress-regulated Mycobacterium avium subsp. paratuberculosis proteins and comparison with recombinant protein and complex native antigens.  

PubMed

Serum antibody enzyme-linked immunosorbent assay is the most commonly used test for diagnosis of Mycobacterium avium subsp. paratuberculosis infection in ruminants. However, the assay requires serum preabsorption with Mycobacterium phlei proteins to reduce cross reactions potentially contributed by the exposure of livestock to environmental mycobacteria. To trial the discovery of novel antigens which do not require serum absorption, synthetic MAP-specific peptides were selected based on in silico research to identify putative B cell epitopes. Four peptides from previously identified stress-regulated proteins were synthesized and evaluated using enzyme linked immunosorbent assay to detect Mycobacterium avium subsp. paratuberculosis specific antibodies in sheep. Two peptides were from hypothetical MAP proteins (MAP3567 and MAP1168c) and two were from proteins with known function (MAP2698c, an acyl-acyl carrier protein desaturase-DesA2 and MAP2487c a carbonic anhydrase). The ability of each peptide to discriminate between unexposed and MAP exposed (infected and vaccinated) animals was similar to that of the parent recombinant MAP antigen, with area under receiver operating curve values of 0.86-0.93. Assays run with a combination of two peptides showed slightly higher reactivity than those of individual peptides. Peptides evaluated in this study had diagnostic potential similar to corresponding recombinant proteins but not superior to a complex native MAP antigen or a commercial assay. Further study is required to investigate other peptides for their diagnostic potential, and this may be simpler and cheaper than subunit protein-based research. PMID:23815825

Gurung, Ratna B; Begg, Douglas J; Purdie, Auriol C; Whittington, Richard J

2014-03-15

83

Peptic and Tryptic Hydrolysis of Native and Heated Whey Protein to Reduce Its Antigenicity  

Microsoft Academic Search

This study examined the effects of enzymes on the production and antigenicity of native and heated whey protein concentrate (WPC) hydrolysates. Native and heated (10 min at 100°C) WPC (2% protein solution) were incubated at 50°C for 30, 60, 90, and 120 min with0.1, 0.5,and1% pepsinand thenwith0.1, 0.5,and 1% trypsin on a protein-equivalent basis. A greater degree of hydrolysis was

S. B. Kim; K. S. Ki; M. A. Khan; W. S. Lee; H. J. Lee; B. S. Ahn; H. S. Kim

2007-01-01

84

Rapid Expression of Mycobacterium avium subsp. paratuberculosis Recombinant Proteins for Antigen Discovery?  

PubMed Central

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease, a chronic granulomatous enteritis of ruminants and other species. Detection of infection in animals is hampered by the lack of sensitive and specific diagnostic assays. We describe here an approach that utilizes translationally active PCR fragments for the rapid in vitro transcription and translation of recombinant proteins for antigen discovery in M. avium subsp. paratuberculosis. The investigations showed that the MAP1272c protein selectively reacts with sera from Johne's disease-positive cattle and represents an antigen of potential utility in M. avium subsp. paratuberculosis immunodiagnostics. PMID:17079432

Li, Lingling; Munir, Shirin; Bannantine, John P.; Sreevatsan, Srinand; Kanjilal, Sagarika; Kapur, Vivek

2007-01-01

85

The endothelial protein PLVAP in lymphatics controls the entry of lymphocytes and antigens into lymph nodes.  

PubMed

In the lymphatic sinuses of draining lymph nodes, soluble lymph-borne antigens enter the reticular conduits in a size-selective manner and lymphocytes transmigrate to the parenchyma. The molecular mechanisms that control these processes are unknown. Here we unexpectedly found that PLVAP, a prototypic endothelial protein of blood vessels, was synthesized in the sinus-lining lymphatic endothelial cells covering the distal conduits. In PLVAP-deficient mice, both small antigens and large antigens entered the conduit system, and the transmigration of lymphocytes through the sinus floor was augmented. Mechanistically, the filtering function of the lymphatic sinus endothelium was dependent on diaphragms formed by PLVAP fibrils in transendothelial channels. Thus, in the lymphatic sinus, PLVAP forms a physical sieve that regulates the parenchymal entry of lymphocytes and soluble antigens. PMID:25665101

Rantakari, Pia; Auvinen, Kaisa; Jäppinen, Norma; Kapraali, Maria; Valtonen, Joona; Karikoski, Marika; Gerke, Heidi; Iftakhar-E-Khuda, Imtiaz; Keuschnigg, Johannes; Umemoto, Eiji; Tohya, Kazuo; Miyasaka, Masayuki; Elima, Kati; Jalkanen, Sirpa; Salmi, Marko

2015-04-01

86

Identification of a 68-kilodalton protective protein antigen from Bordetella bronchiseptica.  

PubMed Central

A 68-kilodalton (kd) outer membrane protein antigen of Bordetella bronchiseptica has been identified by using monoclonal antibodies that recognized two nonoverlapping determinants. Antibody BB05 also reacted with homologous proteins from Bordetella pertussis and Bordetella parapertussis but not with another 12 organisms from various bacterial genera. Passive injection of BB05 antibody protected mice from aerosol infection with B. bronchiseptica as shown by reduced mortality and reduced pathology of turbinate bones. The 68-kd B. bronchiseptica antigen was purified by BB05-based affinity chromatography and evaluated for its potency to immunize mice actively against either intraperitoneal or aerosol challenge with B. bronchiseptica. Immunization with the 68-kd antigen in incomplete Freund adjuvant significantly reduced the levels of mortality in intraperitoneally challenged mice. In the aerosol infection model, injection of the 68-kd antigen with complete or incomplete Freund adjuvant or saponin reduced the bacterial counts in the lungs of infected mice. These results suggest that the 68-kd protein may represent a potential "protective" antigen of B. bronchiseptica. Images PMID:3972452

Montaraz, J A; Novotny, P; Ivanyi, J

1985-01-01

87

Western blot analysis of antibody response to pneumococcal protein antigens in a murine model of pneumonia.  

PubMed Central

To detect new antigen candidates for serological tests, we studied the antibody response to pneumococcal protein antigens in mice infected intratracheally with various Streptococcus pneumoniae strains. Sera were tested by Western blotting against whole-cell protein extracts. Mice developed a detectable immunoglobulin G-type response against a small number of polypeptides. The antibody response was strain dependent: sera from individuals infected with the same strain gave similar banding patterns on immunoblots. The banding patterns varied with the strain used for infection. However, a band at 36 to 38 kDa was recognized by all reactive sera. This band appeared to correspond to a polypeptide that was antigenically well conserved among the different S. pneumoniae serotypes. An antibody response to this antigen developed in mice irrespective of the capsular type, the virulence, and the susceptibility to penicillin G of the infecting strain. Thus, this 36- to 38-kDa protein antigen may be of value for the development of a serological test for humans. PMID:9384307

Mouneimne, H; Juvin, M; Beretti, J L; Azoulay-Dupuis, E; Vallee, E; Geslin, P; Petitpretz, P; Berche, P; Gaillard, J L

1997-01-01

88

Enhanced Discrimination of Malignant from Benign Pancreatic Disease by Measuring the CA 19-9 Antigen on Specific Protein Carriers  

Microsoft Academic Search

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

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

2011-01-01

89

Sperm protein 17 is an oncofetal antigen: a lesson from a murine model.  

PubMed

Sperm protein 17 (Sp17) was originally identified in the flagellum of spermatozoa and subsequently included in the subfamily of tumor-associated antigens known as cancer-testes antigens (CTA). Sp17 has been associated with the motility and migratory capacity in tumor cells, representing a link between gene expression patterns in germinal and tumor cells of different histological origins. Here we review the relevance of Sp17 expression in the mouse embryo and cancerous tissues, and present additional data demonstrating Sp17 complex expression pattern in this murine model. The expression of Sp17 in embryonic as well as adult neoplastic cells, but not normal tissues, suggests this protein should be considered an "oncofetal antigen." Further investigations are necessary to elucidate the mechanisms and functional significance of Sp17 aberrant expression in human adult cells and its implication in the pathobiology of cancer. PMID:24811209

Arnaboldi, F; Menon, A; Menegola, E; Di Renzo, F; Mirandola, L; Grizzi, F; Figueroa, J A; Cobos, E; Jenkins, M; Barajon, I; Chiriva-Internati, Maurizio

2014-10-01

90

Association of simian virus 40 small-t antigen with the 61-kilodalton component of a cellular protein complex.  

PubMed Central

Two cellular proteins, 61 and 37 kDa, are found in association with the simian virus 40 (SV40) small-t antigen. Fractionation in standard chromatography systems showed that these proteins were associated with one another in uninfected cells, suggesting that the small-t antigen may bind the complex as a whole and not each individual protein independently. In the presence of N-ethylmaleimide, the 37-kDA protein was selectively released from immune complexes, leaving the small-t antigen and 61-kDa protein in association. This result suggests that the small-t antigen may bind only the 61-kDa protein and that the 37-kDa protein may be associated with immune complexes by virtue of its association with the 61-kDa cellular protein. Images PMID:2170691

Joshi, B; Rundell, K

1990-01-01

91

Effects of roasting, blanching, autoclaving, and microwave heating on antigenicity of almond (Prunus dulcis L.) proteins.  

PubMed

Whole, unprocessed Nonpareil almonds were subjected to a variety of heat processing methods that included roasting (280, 300, and 320 degrees F for 20 and 30 min each; and 335 and 350 degrees F for 8, 10, and 12 min each), autoclaving (121 degrees C, 15 psi, for 5, 10, 15, 20, 25, and 30 min), blanching (100 degrees C for 1, 2, 3, 4, 5, and 10 min), and microwave heating (1, 2, and 3 min). Proteins were extracted from defatted almond flour in borate saline buffer, and immunoreactivity of the soluble proteins (normalized to 1 mg protein/mL for all samples) was determined using enzyme linked immunosorbent assay (ELISA). Antigenic stability of the almond major protein (amandin) in the heat-processed samples was determined by competitive inhibition ELISA using rabbit polyclonal antibodies raised against amandin. Processed samples were also assessed for heat stability of total antigenic proteins by sandwich ELISA using goat and rabbit polyclonal antibodies raised against unprocessed Nonpareil almond total protein extract. ELISA assays and Western blotting experiments that used both rabbit polyclonal antibodies and human IgE from pooled sera indicated antigenic stability of almond proteins when compared with that of the unprocessed counterpart. PMID:12033826

Venkatachalam, M; Teuber, S S; Roux, K H; Sathe, S K

2002-06-01

92

Association of protein phosphatase 2A with polyoma virus medium tumor antigen.  

PubMed Central

The polyoma virus medium and small tumor antigens, as well as simian virus 40 small tumor antigen, form specific complexes with two cellular proteins designated 61- and 37-kDa proteins. In this report, we demonstrate that the 61- and 37-kDa proteins correspond to the A and C subunits, respectively, of the serine- and threonine-specific protein phosphatase 2A (PP2A). On the one hand, antibodies raised against the 61-kDa protein reacted specifically with the purified A subunit of PP2A. Furthermore, the amino acid sequences of seven tryptic peptides from the A subunit were almost identical to sequences of the 61-kDa protein as deduced from the corresponding cDNA sequence. On the other hand, antibodies against the purified C subunit (catalytic subunit) of PP2A reacted specifically with the medium tumor antigen-associated 37-kDa protein. These data suggest a role of PP2A in cell transformation by polyoma virus and simian virus 40. Images PMID:2157202

Walter, G; Ruediger, R; Slaughter, C; Mumby, M

1990-01-01

93

Kinetics and extent of protein tyrosine kinase activation in individual T cells upon antigenic stimulation  

PubMed Central

Using human CD4+ T-cell clones and peptide-pulsed antigen-presenting cells (APC) we measured, at the single cell level, different steps in the T-cell activation cascade. Simultaneous analysis of T-cell antigen receptor (TCR) down-regulation and interferon-? (IFN-?) production shows that both the level of TCR occupancy and the amount of IFN-? produced by single T cells increase in an antigen dose-dependent fashion. Conversely, commitment of T cells to IFN-? production does not occur as soon as a defined number of TCR have been engaged, but requires the same duration of sustained signalling at low as well as at high antigen concentrations. Measurement of phosphotyrosine levels by flow cytometry reveals that, upon conjugation with APC, individual T cells undergo an antigen dose-dependent activation of protein tyrosine kinases (PTK), which parallels the level of TCR occupancy. In antigen-stimulated T cells the increased phosphotyrosine staining is localized in the area of contact with APC, as shown by confocal microscopy. PTK activation is sustained for at least 2 hr after conjugation, and is required to maintain a sustained increase in intracellular Ca2+ concentration. Our results show, for the first time, a direct correlation between the level of TCR occupancy and the activation of PTK in individual T cells and offer an explanation for how the number of triggered TCR can be ‘counted’ and integrated in a corresponding biological response. PMID:10447744

Müller, S; Demotz, S; Bulliard, C; Valitutti, S

1999-01-01

94

Kinetics and extent of protein tyrosine kinase activation in individual T cells upon antigenic stimulation.  

PubMed

Using human CD4+ T-cell clones and peptide-pulsed antigen-presenting cells (APC) we measured, at the single cell level, different steps in the T-cell activation cascade. Simultaneous analysis of T-cell antigen receptor (TCR) down-regulation and interferon-gamma (IFN-gamma) production shows that both the level of TCR occupancy and the amount of IFN-gamma produced by single T cells increase in an antigen dose-dependent fashion. Conversely, commitment of T cells to IFN-gamma production does not occur as soon as a defined number of TCR have been engaged, but requires the same duration of sustained signalling at low as well as at high antigen concentrations. Measurement of phosphotyrosine levels by flow cytometry reveals that, upon conjugation with APC, individual T cells undergo an antigen dose-dependent activation of protein tyrosine kinases (PTK), which parallels the level of TCR occupancy. In antigen-stimulated T cells the increased phosphotyrosine staining is localized in the area of contact with APC, as shown by confocal microscopy. PTK activation is sustained for at least 2 hr after conjugation, and is required to maintain a sustained increase in intracellular Ca2+ concentration. Our results show, for the first time, a direct correlation between the level of TCR occupancy and the activation of PTK in individual T cells and offer an explanation for how the number of triggered TCR can be 'counted' and integrated in a corresponding biological response. PMID:10447744

Müller, S; Demotz, S; Bulliard, C; Valitutti, S

1999-06-01

95

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; Schüler, Wolfgang; Morfeldt, Eva; Hellberg, Christel; Nagy, Eszter; Lundberg, Urban; Hays, John P.; Meinke, Andreas; Henriques-Normark, Birgitta

2013-01-01

96

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

97

Serum IGF-binding protein-6 and prostate specific antigen in breast cancer  

Microsoft Academic Search

Objective: Recent studies have demonstrated the presence of the IGF-binding proteins (IGFBPs) and prostate specific antigen (PSA), an IGFBP protease, in human breast tissue. We sought to investigate the differences in serum IGFs, IGFBP-1, -3 and -6, and PSA between patients with surgically proven breast cancer and patients with benign breast disease. Design and Methods: Concentrations of IGFs, IGFBP-1, -3

Karmal K Kaulsay; Kok-Onn Lee

1999-01-01

98

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 compartment of mammalian cells. The engineered anthrax toxin vaccine appears unlikely to induce an antibody

Lieberman, Judy

99

SEPPA: a computational server for spatial epitope prediction of protein antigens  

Microsoft Academic Search

In recent years, a lot of efforts have been made in conformational epitope prediction as antigen proteins usually bind antibodies with an assembly of sequentially discontinuous and structurally com- pact surface residues. Currently, only a few meth- ods for spatial epitope prediction are available with focus on single residue propensity scales or continual segments clustering. In the method of SEPPA,

Jing Sun; Di Wu; Tianlei Xu; Xiaojing Wang; Xiaolian Xu; Lin Tao; Y. X. Li; Zhi-wei Cao

2009-01-01

100

Entamoeba histolytica antigenic protein detected in pus aspirates from patients with amoebic liver abscess.  

PubMed

Entamoeba histolytica is a causative agent of amoebic liver abscess (ALA) and is endemic in many underdeveloped countries. We investigated antigenic E. histolytica proteins in liver abscess aspirates using proteomics approach. Pus samples were first tested by real-time PCR to confirm the presence of E. histolytica DNA and the corresponding serum samples tested for E. histolytica-specific IgG by a commercial ELISA. Proteins were extracted from three and one pool(s) of pus samples from ALA and PLA (pyogenic liver abscess) patients respectively, followed by analysis using isoelectric focussing, SDS-PAGE and Western blot. Unpurified pooled serum samples from infected hamsters and pooled human amoebic-specific IgG were used as primary antibodies. The antigenic protein band was excised from the gel, digested and analysed by MALDI-TOF/TOF and LC-MS/MS. The results using both primary antibodies showed an antigenic protein band of ?14kDa. Based on the mass spectrum analysis, putative tyrosine kinase is the most probable identification of the antigenic band. PMID:23680184

Othman, Nurulhasanah; Mohamed, Zeehaida; Yahya, Maya Mazuwin; Leow, Voon Meng; Lim, Boon Huat; Noordin, Rahmah

2013-08-01

101

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

PubMed

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

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

1983-01-01

102

Cloning, expression and antigenic analysis of heat shock protein A gene of human Helicobacter pylori  

Microsoft Academic Search

AIM: To construct a recombinant vector containing gene encoding heat shock protein A with a Mr of 13 000 from human Helicobacter pylori (H pylori) and express it in E. coli BL21, and to explore the antigenicity. METHODS: The target gene was amplified from H pylori chromosome by PCR, and then inserted into the prokaryotic expression vector pET32a (+) digested

Zheng Jiang; Dan Pu; Ai-Long Huang; Xiao-Hong Tao; Pi-Long Wang

2003-01-01

103

Effects of adsorption to aluminum salt adjuvants on the structure and stability of model protein antigens.  

PubMed

The effect of adsorption onto aluminum salt adjuvants on the structure and stability of three model protein antigens was studied using fluorescence and Fourier transform infrared spectroscopies, as well as isothermal titration and differential scanning calorimetric techniques. Lysozyme was preferentially adsorbed to aluminum phosphate (Adju-Phos), whereas ovalbumin and bovine serum albumin were better adsorbed to aluminum hydroxide (Alhydrogel). A linearized Langmuir adsorption isotherm was used to obtain information regarding the binding interactions between proteins and adjuvants. Binding energetics and stoichiometry data obtained from isothermal titration calorimetry measurements were complex. Based on the spectroscopic and differential scanning calorimetry studies, the structure of all three proteins, when adsorbed to the surface of an aluminum salt, was altered in such a way as to render the proteins less thermally stable. Besides the pharmaceutical significance of this destabilization, we consider the possibility that this phenomenon may facilitate the presentation of antigens and thus contribute to the adjuvant activity of the aluminum salts. PMID:15684430

Jones, LaToya S; Peek, Laura J; Power, Jonathan; Markham, Aaron; Yazzie, Brian; Middaugh, C Russell

2005-04-01

104

Size and Antigenic Comparisons among the Structural Proteins of Selected Autonomous Parvoviruses  

Microsoft Academic Search

SUMMARY The size and antigenic relationships among structural proteins (VPs) of canine parvovirus (CPV), feline parvovirus (FPV), porcine parvovirus (PPV), minute virus of mice (MVM) and bovine parvovirus (BPV) were determined by SDS-PAGE of radiolabelled, purified virus and immunoprecipitated viral proteins. Mature virions of CPV, FPV, PPV and MVM were composed of three VPs designated VP1, VP2 and VP3. The

W. L. Mengeling; J. F. Ridpath; A. C. Vorwald

1988-01-01

105

Antigenic analysis of the major outer membrane protein of Haemophilus somnus with monoclonal antibodies.  

PubMed Central

The major outer membrane protein of Haemophilus somnus possesses at least five distinct epitopes. Three surface-exposed epitopes on the major outer membrane protein include a conserved epitope with potential for development of a vaccine and a diagnostic test and two variable epitopes responsible for antigenic differences among strains; the remaining two epitopes are well preserved among strains but not exposed on the cell surface. Images PMID:8478121

Tagawa, Y; Haritani, M; Ishikawa, H; Yuasa, N

1993-01-01

106

Identification, characterization and antigenicity of the Plasmodium vivax rhoptry neck protein 1 (PvRON1)  

PubMed Central

Background Plasmodium vivax malaria remains a major health problem in tropical and sub-tropical regions worldwide. Several rhoptry proteins which are important for interaction with and/or invasion of red blood cells, such as PfRONs, Pf92, Pf38, Pf12 and Pf34, have been described during the last few years and are being considered as potential anti-malarial vaccine candidates. This study describes the identification and characterization of the P. vivax rhoptry neck protein 1 (PvRON1) and examine its antigenicity in natural P. vivax infections. Methods The PvRON1 encoding gene, which is homologous to that encoding the P. falciparum apical sushi protein (ASP) according to the plasmoDB database, was selected as our study target. The pvron1 gene transcription was evaluated by RT-PCR using RNA obtained from the P. vivax VCG-1 strain. Two peptides derived from the deduced P. vivax Sal-I PvRON1 sequence were synthesized and inoculated in rabbits for obtaining anti-PvRON1 antibodies which were used to confirm the protein expression in VCG-1 strain schizonts along with its association with detergent-resistant microdomains (DRMs) by Western blot, and its localization by immunofluorescence assays. The antigenicity of the PvRON1 protein was assessed using human sera from individuals previously exposed to P. vivax malaria by ELISA. Results In the P. vivax VCG-1 strain, RON1 is a 764 amino acid-long protein. In silico analysis has revealed that PvRON1 shares essential characteristics with different antigens involved in invasion, such as the presence of a secretory signal, a GPI-anchor sequence and a putative sushi domain. The PvRON1 protein is expressed in parasite's schizont stage, localized in rhoptry necks and it is associated with DRMs. Recombinant protein recognition by human sera indicates that this antigen can trigger an immune response during a natural infection with P. vivax. Conclusions This study shows the identification and characterization of the P. vivax rhoptry neck protein 1 in the VCG-1 strain. Taking into account that PvRON1 shares several important characteristics with other Plasmodium antigens that play a functional role during RBC invasion and, as shown here, it is antigenic, it could be considered as a good vaccine candidate. Further studies aimed at assessing its immunogenicity and protection-inducing ability in the Aotus monkey model are thus recommended. PMID:22024312

2011-01-01

107

The ability of heat-killed Mycobacterium vaccae to stimulate a cytotoxic T-cell response to an unrelated protein is associated with a 65 kilodalton heat-shock protein  

PubMed Central

Exogenous antigens are generally presented by Class II major histocompatibility (MHC) molecules. When administered with an adjuvant, however, they are capable of inducing a CD8+ T-cell response where antigen recognition is associated with Class I MHC. Accordingly, immunization with soluble ovalbumin (OVA) alone does not activate CD8+ cytotoxic T cells (CTL) but when given in complete Freund's adjuvant (CFA), or in formulations of a number of novel adjuvants, an OVA-specific CD8+ CTL response can be detected. We show in this report that immunization with soluble OVA mixed with heat-killed Mycobacterium vaccae, but not with other common pathogenic and saprophytic mycobacteria, can activate OVA-specific CD8+ CTL. An OVA-specific CTL response is detected when mice are immunized by either the intraperitoneal or intranasal route and their spleen cells are re-stimulated in vitro. Adjuvant activity of heat-killed M. vaccae is present in M. vaccae culture filtrate, in soluble protein components of whole M. vaccae and in the 65 kDa heat-shock protein (hsp) of M. vaccae. Mycobacterium vaccae has previously been shown to have no adverse side-effects in humans. The current results suggest that M. vaccae may be useful as an adjuvant for vaccines and other immunotherapies where CD8+ CTL responses to exogenous proteins are crucial. PMID:11260328

Skinner, M A; Prestidge, R; Yuan, S; Strabala, T J; Tan, P L J

2001-01-01

108

[Hyperprolactinemia unrelated to prolactinoma].  

PubMed

Hyperprolactinemia, defined as prolactin levels above the upper limit of normal range, is the most frequent hypothalamus-pituitary dysfunction. Clinical symptoms of hyperprolactinemia in women include oligomenorrhea, infertility, and galactorrhea, while in men the condition may lead to hypogonadism, decreased libido, erectile dysfunction, infertility, gynecomastia, and, in rare instances, galactorrhea. In many patients, hyperprolactinemia results from the presence of prolactinoma, which is considered as the most common hormone-secreting pituitary tumors. However, transient or long-term hyperprolactinemia may also develop during different physiological situations or due to several diseases. It is also a frequent but often neglected side effect of many drugs, particularly of antipsychotics. Finally, hyperprolactinemia may be secondary to the predominance of high molecular mass circulating prolactin forms that have been postulated to represent complexes of prolactin and anti-prolactin immunoglobulins (macroprolactinemia). The cause of hyperprolactinemia determines its treatment. In this paper, we review the causes of hyperprolactinemia unrelated to prolactinoma, providing a differential diagnosis of this condition. PMID:25764785

Krysiak, Robert; Okopie?, Bogus?aw

2014-01-01

109

Original article Permeability of milk protein antigens across the intestinal  

E-print Network

-Lactogtobutin (!-Lg), a-Lactalbumin (a-La) and p-casein (!-cas). Pepsin-trypsin hydrolysis and permeability in isolated rabbit ileum in Ussing chamber were suited by ELISA and radiolabelled-protein measurement. Pepsin â-Lactalbumine (a-La), et la #-caséine (/3-cas) du lait de vache. L hydrolyse successive pepsine

Paris-Sud XI, Université de

110

Extracellular Release of Antigenic Proteins by Helicobacter pylori  

Microsoft Academic Search

Helicobacter pylori is a gram-negative bacterium that colo- nizes the gastric mucosa of humans. The mechanisms by which H. pylori elicits an inflammatory response and persists for de- cades in the gastric mucosa remain incompletely understood (4, 14). Secreted proteins play an important role in the patho- genesis of many bacterial infections (20), and therefore, in this study we sought

PING CAO; MARK S. MCCLAIN; MARK H. FORSYTH

1998-01-01

111

Transcutaneous immunization with hydrophilic recombinant gp100 protein induces antigen-specific cellular immune response  

PubMed Central

The objective of this study was to evaluate the potential of transcutaneous immunization with tumor antigen to induce cell-mediated immunity. For this purpose, hydrophilic recombinant gp100 protein (HR-gp100) was topically applied on human intact skin in vitro, and used as a vaccine in a mouse model. We demonstrate that HR-gp100 permeates into human skin, and is processed and presented by human dendritic cells. In a mouse model, an HR-gp100-based vaccine triggered antigen-specific T cell responses, as shown by proliferation assays, ELISA and intracellular staining for IFN-?. Transcutaneous antigen delivery may provide a safe, simple and effective method to elicit cell mediated immunity. PMID:20947070

Eisenberg, Galit; Machlenkin, Arthur; Frankenburg, Shoshana; Mansura, Adva; Pitcovski, Jacob; Yefenof, Eitan; Peretz, Tamar; Lotem, Michal

2010-01-01

112

Isolation and Characterization of Antibody Fragments Selective for Specific Protein Morphologies from Nanogram Antigen Samples  

PubMed Central

We developed atomic force microscope (AFM) based protocols that enable isolation and characterization of antibody based reagents that selectively bind target protein variants using low nanogram amounts or less of unpurified starting material. We isolated single chain antibody fragments (scFvs) that specifically recognize an oligomeric amyloid-beta (A?) species correlated with Alzheimer’s disease (AD) using only a few nanograms of an enriched but not purified sample obtained from human AD brain tissue. We employed several subtractive panning steps to remove all phage binding non-desired antigens and then employed a single positive panning step using minimal antigen. We also used AFM to characterize the specificity of the isolated clones, again using minimal material, selecting the C6 scFv based on expression levels. We show that C6 selectively binds cell and brain derived oligomeric A?. The protocols described are readily adapted to isolating antibody based reagents against other antigenic targets with limited availability. PMID:23359572

Kasturirangan, Srinath; Reasoner, Tim; Schulz, Philip; Boddapati, Shanta; Emadi, Sharareh; Valla, Jon; Sierks, Michael R.

2013-01-01

113

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

PubMed Central

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

2014-01-01

114

Systematic analysis of natural antibody responses to P. falciparum merozoite antigens by protein arrays.  

PubMed

With the completion of the functional genome, merozoite proteome and stage-specific transcriptomes of the intraerythrocytic developmental cycle of Plasmoidum falciparum, the development of new vaccine candidates targeting Plasmodium merozoites is now possible. Here we report using protein array technology to detect antibody responses to Plasmodium merozoite proteins by screening the serum of Plasmodium-exposed individuals. A total of 138 genes encoding P. falciparum merozoite proteins were cloned using the In-Fusion cloning method and expressed using a wheat germ cell-free system (WGCF). These proteins were then screened with serum from Plasmodium-exposed individuals and unexposed subjects using protein arrays. A total of 30 highly immunoreactive merozoite antigens were identified (21.7% of 138 target proteins), including 10 well-characterized blood-stage vaccine candidates for P. falciparum. In addition, we report for the first time 7 proteins (MSP3.5, MRSP2, ETRAMP11.2, ETRAMP14.1 and RALP1, and two hypothetical proteins PFA0210c and PF14_0572) as being immunologically reactive. These novel Plasmodium merozoite antigens may be potential vaccine candidates for blood-stage malaria, and warrant further study. PMID:23201113

Fan, Yan-Ting; Wang, Yue; Ju, Chuan; Zhang, Ting; Xu, Bin; Hu, Wei; Chen, Jun-Hu

2013-01-14

115

Specific nongluten proteins of wheat are novel target antigens in celiac disease humoral response.  

PubMed

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

Huebener, Sina; Tanaka, Charlene K; Uhde, Melanie; Zone, John J; Vensel, William H; Kasarda, Donald D; Beams, Leilani; Briani, Chiara; Green, Peter H R; Altenbach, Susan B; Alaedini, Armin

2015-01-01

116

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

PubMed Central

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

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

2014-01-01

117

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

SciTech Connect

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

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

1982-06-01

118

Application of Protein Microarrays for Multiplexed Detection of Antibodies to Tumor Antigens in Breast Cancer  

PubMed Central

There is strong preclinical evidence that cancer, including breast cancer, undergoes immune surveillance. This continual monitoring, by both the innate and the adaptive immune systems, recognizes changes in protein expression, mutation, folding, glycosylation, and degradation. Local immune responses to tumor antigens are amplified in draining lymph nodes, and then enter the systemic circulation. The antibody response to tumor antigens, such as p53 protein, are robust, stable, and easily detected in serum, may exist in greater concentrations than their cognate antigens, and are potential highly specific biomarkers for cancer. However, antibodies have limited sensitivities as single analytes, and differences in protein purification and assay characteristics have limited their clinical application. For example, p53 autoantibodies in the sera are highly specific for cancer patients, but are only detected in the sera of 10-20% of patients with breast cancer. Detection of p53 autoantibodies is dependent on tumor burden, p53 mutation, rapidly decreases with effective therapy, but is relatively independent of breast cancer subtype. Although antibodies to hundreds of other tumor antigens have been identified in the sera of breast cancer patients, very little is known about the specificity and clinical impact of the antibody immune repertoire to breast cancer. Recent advances in proteomic technologies have the potential for rapid identification of immune response signatures for breast cancer diagnosis and monitoring. We have adapted programmable protein microarrays for the specific detection of autoantibodies in breast cancer. Here, we present the first demonstration of the application of programmable protein microarray ELISAs for the rapid identification of breast cancer autoantibodies. PMID:18311903

Anderson, Karen S.; Ramachandran, Niroshan; Wong, Jessica; Raphael, Jacob V.; Hainsworth, Eugenie; Demirkan, Gokhan; Cramer, Daniel; Aronzon, Diana; Hodi, F. Stephen; Harris, Lyndsay; Logvinenko, Tanya; LaBaer, Joshua

2012-01-01

119

Research Article www.ijptonline.com ANTIGEN PROTEIN FROM XYLELLA FASTIDIOSA: NEW PARADIGM OF SYNTHETIC VACCINE DEVELOPMENT  

E-print Network

Received on 14-07-2010 Accepted on 29-07-2010 Xylella fastidiosa is a pathogenic bacterium that infects plants, causing a variety of diseases in over 100 plant species. Peptide fragments of antigen protein can be used to select nonamers for use in rational vaccine design and to increase the understanding of roles of the immune system in pathogenic diseases. Analysis shows MHC class II binding peptides of antigen protein from Xylella fastidiosa are important determinant for protection of host form infection. In this assay, we used PSSM and SVM algorithms for antigen design and predicted the binding affinity of antigen protein having 790 amino acids, which shows 782 nonamers. Binding ability prediction of antigen peptides to major histocompatibility complex (MHC) class I & II molecules is important in vaccine development from Xylella fastidiosa.

Gomase V. S; Chitlange N. R

120

INVITED REVIEW: TARGETING AND EXPRESSION OF ANTIGENIC PROTEINS IN TRANSGENIC PLANTS FOR PRODUCTION OF EDIBLE ORAL VACCINES  

Microsoft Academic Search

Summary Exploiting plants as biological bioreactors for production and delivery of edible oral subunit vaccines is a promising application of biotechnology. Efforts to enhance expression levels of transgenes coding for antigenic proteins by exploiting promoters, targeting sequences, and enhancer elements have produced rather low quantities of the antigen in plant tissues, but enough to induce immune responses in feeding studies.

SCHUYLER S. KORBAN

121

Effect of Attenuated Salmonella enterica Serovar Typhimurium Expressing a Streptococcus mutans Antigen on Secondary Responses to the Cloned Protein  

Microsoft Academic Search

Attenuated Salmonella enterica serovar Typhimurium has been used for targeted delivery of recombinant antigens to gut- and nose-associated lymphoid tissues. Contradictory reports have described the effect of preexisting immunity to the antigen delivery vehicle. We decided to examine this discrepancy by studying the effect of immunizing mice by the intranasal (i.n.) route with Salmonella expressing an insoluble protein and to

CHRISTINA JESPERSGAARD; PING ZHANG; GEORGE HAJISHENGALLIS; MICHAEL W. RUSSELL; SUZANNE M. MICHALEK

2001-01-01

122

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

Microsoft Academic Search

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

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

1988-01-01

123

Plant heat shock protein 70 as carrier for immunization against a plant-expressed reporter antigen  

Microsoft Academic Search

Mammalian Heat Shock Proteins (HSP), have potent immune-stimulatory properties due to the natural capability to associate\\u000a with polypeptides and bind receptors on antigen presenting cells. The present study was aimed to explore whether plant HSP,\\u000a and in particular HSP70, share similar properties. We wanted in particular to evaluate if HSP70 extracted in association to\\u000a naturally bound polypeptides from plant tissues

Giampaolo Buriani; Camillo Mancini; Eugenio Benvenuto; Selene Baschieri

2011-01-01

124

Application of an acid proteinase from Monascus purpureus to reduce antigenicity of bovine milk whey protein  

Microsoft Academic Search

An acid proteinase from Monascus purpureus No. 3403, MpuAP, was previously purified and some characterized in our laboratory (Agric Biol Chem 48:1637–1639, 1984). However, further information about this enzyme is lacking. In this study, we investigated MpuAP’s comprehensive substrate\\u000a specificity, storage stability, and prospects for reducing antigenicity of whey proteins for application in the food industry.\\u000a MpuAP hydrolyzed primarily five

P. L. Nilantha Lakshman; Shinjiro Tachibana; Hirohide Toyama; Toki Taira; Toshihiko Suganuma; Worapot Suntornsuk; Masaaki Yasuda

125

?-NAP, a cerebellar degeneration antigen, is a neuron-specific vesicle coat protein  

Microsoft Academic Search

We have identified a target antigen in autoimmune cerebellar degeneration, ?-NAP, that is closely related to the ?-adaptin and ?-COP coat proteins. ?-NAP is a non-clathrin-associated phosphoprotein expressed exclusively in neurons, from E12 through adulthood. ?-NAP is present in the neuronal soma and nerve terminal as soluble and membrane-bound pools and is associated with a discrete set of nerve-terminal vesicles.

Lori S Newman; Matthew O McKeever; Hirotaka J Okano; Robert B Darnell

1995-01-01

126

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

127

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

SciTech Connect

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

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

1981-07-01

128

Recombinant Bacillus anthracis spore proteins enhance protection of mice primed with suboptimal amounts of protective antigen  

PubMed Central

Inactivated Bacillus anthracis spores given with protective antigen (PA) contribute to immunity against anthrax in several animal models. Antiserum raised against whole irradiated B. anthracis spores has been shown to have anti-germination and opsonic activities in vitro. Based on these observations, we hypothesized that surface-exposed spore proteins might serve as supplemental components of a PA-based anthrax vaccine. The protective anti-spore serum was tested for reactivity with recombinant forms of 30 proteins known, or believed to be, present within the B. anthracis exosporium. Eleven of those proteins were reactive with this antiserum, and, subsequently a subset of this group was used to generate rabbit polyclonal antibodies. These sera were evaluated for recognition of the immunogens on intact spores generated from Sterne strain, as well as from an isogenic mutant lacking the spore surface protein Bacillus collagen-like antigen (BclA). The data were consistent with the notion that the antigens in question were located beneath BclA on the basal surface of the exosporium. A/J mice immunized with either the here-to-for hypothetical protein p5303 or the structural protein BxpB, each in combination with subprotective levels of PA, showed enhanced protection against subcutaneous spore challenge. While neither anti-BxpB or anti-p5303 antibodies reduced the rate of spore germination in vitro, both caused increased uptake and lead to a higher rate of destruction by phagocytic cells. We conclude that by facilitating more efficient phagocytic clearance of spores, antibodies against individual exosporium components can contribute to protection against B. anthracis infection. PMID:18657585

Cybulski, Robert J.; Sanz, Patrick; McDaniel, Dennis; Darnell, Steve; Bull, Robert L.; O’Brien, Alison D.

2008-01-01

129

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

130

Cellular proteins that associate with the middle and small T antigens of polyomavirus.  

PubMed Central

We have used two-dimensional gel electrophoresis to analyze in more detail the cellular proteins which associate with the middle and small tumor antigens (MT and ST, respectively) of polyomavirus. Proteins with molecular masses of 27, 29, 36, 51, 61, 63, and 85 kilodaltons (kDa) that specifically coimmunoprecipitated with MT were identified on these gels. The 36-, 51-, 61-, 63-, and 85-kDa proteins are probably the same as the proteins of similar sizes previously reported by a number of groups, whereas the 27- and 29-kDa proteins represent proteins that are heretofore undescribed. The 27- and 29-kDa proteins were abundant cellular proteins, whereas the others were minor cellular constituents. The association of each of these proteins with MT was sensitive to one or more mutations in MT that rendered it transformation defective. The association of the 85-kDa protein was the most sensitive indicator of the transformation competence of MT mutants. In addition, the 85-kDa protein was the only associated protein whose association with MT changed consistently in parallel with MT-associated phosphatidylinositol kinase activity. Furthermore, the fraction of the 85-kDa protein which was found associated with the MT complex contained 15 to 20% of its phosphate content on tyrosine. The 36- and 63-kDa proteins complexed with both polyomavirus MT and ST and comigrated on two-dimensional gels with two simian virus 40 ST-associated proteins originally described by Rundell and coworkers (K. Rundell, E. O. Major, and M. Lampert, J. Virol. 37:1090-1093, 1981). None of the other MT-associated proteins associated significantly with ST. Images PMID:2845116

Pallas, D C; Cherington, V; Morgan, W; DeAnda, J; Kaplan, D; Schaffhausen, B; Roberts, T M

1988-01-01

131

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

Microsoft Academic Search

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

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

132

Cancer associated aberrant protein O-glycosylation can modify antigen processing and immune response.  

PubMed

Aberrant glycosylation of mucins and other extracellular proteins is an important event in carcinogenesis and the resulting cancer associated glycans have been suggested as targets in cancer immunotherapy. We assessed the role of O-linked GalNAc glycosylation on antigen uptake, processing, and presentation on MHC class I and II molecules. The effect of GalNAc O-glycosylation was monitored with a model system based on ovalbumin (OVA)-MUC1 fusion peptides (+/- glycosylation) loaded onto dendritic cells co-cultured with IL-2 secreting OVA peptide-specific T cell hybridomas. To evaluate the in vivo response to a cancer related tumor antigen, Balb/c or B6.Cg(CB)-Tg(HLA-A/H2-D)2Enge/J (HLA-A2 transgenic) mice were immunized with a non-glycosylated or GalNAc-glycosylated MUC1 derived peptide followed by comparison of T cell proliferation, IFN-? release, and antibody induction. GalNAc-glycosylation promoted presentation of OVA-MUC1 fusion peptides by MHC class II molecules and the MUC1 antigen elicited specific Ab production and T cell proliferation in both Balb/c and HLA-A2 transgenic mice. In contrast, GalNAc-glycosylation inhibited the presentation of OVA-MUC1 fusion peptides by MHC class I and abolished MUC1 specific CD8+ T cell responses in HLA-A2 transgenic mice. GalNAc glycosylation of MUC1 antigen therefore facilitates uptake, MHC class II presentation, and antibody response but might block the antigen presentation to CD8+ T cells. PMID:23189185

Madsen, Caroline B; Petersen, Cecilie; Lavrsen, Kirstine; Harndahl, Mikkel; Buus, Søren; Clausen, Henrik; Pedersen, Anders E; Wandall, Hans H

2012-01-01

133

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

134

Antigen heterogeneity among isolates of Mycoplasma bovis is generated by high-frequency variation of diverse membrane surface proteins.  

PubMed

The protein and antigen profiles of 11 isolates of Mycoplasma bovis were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis of whole organisms. The isolates examined included the type strain PG45 and 10 other filter-cloned strains or purified isolates both from animals without clinical signs and from clinical cases of bovine mastitis, arthritis, or pneumonia. While the overall protein patterns visualized by silver staining were very similar, marked differences in the antigen banding profiles were detected by rabbit antiserum prepared against whole organisms from one of the strains analyzed. This antigenic heterogeneity was shown to be independent of the geographical origin, the type of clinical disease, and the site of isolation and was also observed among serial isolates from a single animal. Antigen profiles were further monitored throughout sequentially subcloned populations of the PG45 strain. This clonal analysis revealed a high-frequency variation in the expression levels of several prominent antigens. All of these variable antigens were defined by detergent-phase fractionation with Triton X-114 as amphiphilic integral membrane proteins. A subset of different-sized membrane proteins was identified by a monoclonal antibody raised against a PG45 subclone expressing a 63- and a 46-kDa variant antigen within that set. The selective susceptibility of these proteins to trypsin treatment of intact organisms and their ability to bind the monoclonal antibody in colony immunoblots demonstrated that they were exposed on the cell surface. In addition, their preferential recognition by serum antibodies from individual cattle with naturally induced M. bovis mastitis or arthritis confirmed that they were major immunogens of this organism. These studies establish that the apparent antigenic heterogeneity among M. bovis isolates reported here does not represent stable phenotypic strain differences generated from accumulated mutational events but reflects distinct expression patterns of diverse, highly variable membrane surface proteins. PMID:7927789

Rosengarten, R; Behrens, A; Stetefeld, A; Heller, M; Ahrens, M; Sachse, K; Yogev, D; Kirchhoff, H

1994-11-01

135

High-affinity reactions between antigen-specific T-cell receptors and peptides associated with allogeneic and syngeneic major histocompatibility complex class I proteins.  

PubMed

We report here that the intrinsic affinities of the antigen-specific T-cell receptors (TCR) of two unrelated CD8+ T-cell clones for their respective peptide-major histocompatibility complex (MHC) ligands are higher than the values generally thought to prevail for TCR. The TCR of one clone (2C) binds an allogeneic class I MHC protein (Ld) in association with an alpha-ketoglutarate dehydrogenase nonapeptide (QLSPFPFDL, termed QL9) with an intrinsic affinity (intrinsic equilibrium association constant) of 1-2 x 10(7) M-1. The TCR of the other clone (4G3) binds a syngeneic class I MHC protein (Kb) in association with an ovalbumin octapeptide (SIINFEKL, termed pOV8) with an intrinsic affinity of 1.5 x 10(6) M-1. A comparison of the two clones, combined with current views of T-cell repertoire selection in the thymus, leads us to propose that TCR affinities are generally likely to be higher for allogeneic MHC-peptide complexes than for syngeneic MHC-peptide complexes. PMID:7972089

Sykulev, Y; Brunmark, A; Tsomides, T J; Kageyama, S; Jackson, M; Peterson, P A; Eisen, H N

1994-11-22

136

Prostate-specific antigen and gross cystic disease fluid protein-15 are co-expressed in androgen receptor-positive breast tumours.  

PubMed Central

Androgens regulate breast cancer cell proliferation via androgen receptor (AR)-mediated mechanisms. To investigate further the androgen-responsiveness of human breast tumours, we examined the immunohistochemical expression of the AR and two androgen-regulated proteins, prostate-specific antigen (PSA) and gross cystic disease fluid protein-15 (GCDFP-15), in 72 primary breast tumours. AR immunoreactivity was present in the nuclei of breast tumour cells and was correlated with oestrogen receptor (ER; P < 0.05) and progesterone receptor (PR; P < 0.01) status. PSA and GCDFP-15 immunoreactivity was present in the cytoplasm of tumour cells but not the adjacent stromal cells. AR-positive cells were present in 85% (61/72) of breast tumours, and 98% (43/44) of PSA-positive and 92% (44/48) of GCDFP-15-positive tumours were also positive for AR. Positive immunoreactivity for both PSA and GCDFP-15 in breast tumours was highly dependent on AR status (odds ratios of 24.0 and 4.5 respectively), but unrelated to age, ER and PR status and axillary lymph node involvement. PSA immunoreactivity was more frequently observed in moderate and well-differentiated tumours and was significantly (P < 0.001) associated with GCDFP-15 immunoreactivity. In conclusion, PSA and GCDFP-15 immunoreactivity was dependent on the presence of AR, but not ER or PR in primary breast tumours. Images Figure 1 PMID:9703283

Hall, R. E.; Clements, J. A.; Birrell, S. N.; Tilley, W. D.

1998-01-01

137

Nonclassically secreted proteins as possible antigens for vaccine development: a reverse vaccinology approach.  

PubMed

Reverse vaccinology strategies have already been applied to a variety of microorganisms and have contributed significantly to vaccine development. However, most of the studies focused on an individual organism or on proteins with signature sequence motifs commonly found in known secreted proteins from bacteria. In this work, we applied a reverse vaccinology strategy based on conservation, virulence, and nonclassically surface exposure criterions to identify potential antigens in two microorganisms with significant degree of genomic plasticity among isolates (Streptococcus pneumoniae and Leptospira spp.), which imposes a major limitation to the production of a multistrain component vaccine. PSORTb 3.0.2 was run to predict the subcellular localization of the proteins. OrthoMCL was run to identify groups of the most conserved proteins between strains. Virulence prediction was done for the most conserved proteins, and SecretomeP was run to predict the nonclassically secreted proteins among the potential virulence factors. Based on the above criteria, we identified 37 proteins conserved between 16 genomes of S. pneumoniae and 12 proteins conserved between 5 leptospiral genomes as potential vaccine candidates. PMID:25672322

de Alvarenga Mudadu, Mauricio; Carvalho, Viviane; Leclercq, Sophie Yvette

2015-04-01

138

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

139

Label-Free Electrochemical Diagnosis of Viral Antigens with Genetically Engineered Fusion Protein  

PubMed Central

We have developed a simple electrochemical biosensing strategy for the label-free diagnosis of hepatitis B virus (HBV) on a gold electrode surface. Gold-binding polypeptide (GBP) fused with single-chain antibody (ScFv) against HBV surface antigen (HBsAg), in forms of genetically engineered protein, was utilized. This GBP-ScFv fusion protein can directly bind onto the gold substrate with the strong binding affinity between the GBP and the gold surface, while the recognition site orients toward the sample for target binding at the same time. Furthermore, this one-step immobilization strategy greatly simplifies a fabrication process without any chemical modification as well as maintaining activity of biological recognition elements. This system allows specific immobilization of proteins and sensitive detection of targets, which were verified by surface plasmon resonance analysis and successfully applied to electrochemical cyclic voltammetry and impedance spectroscopy upto 0.14 ng/mL HBsAg. PMID:23112590

Heo, Nam Su; Zheng, Shun; Yang, MinHo; Lee, Seok Jae; Lee, Sang Yup; Kim, Hwa-Jung; Park, Jung Youn; Lee, Chang-Soo; Park, Tae Jung

2012-01-01

140

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

PubMed

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

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

2015-04-01

141

Structural and antigenic identification of the ORF12 protein (alpha TIF) of equine herpesvirus 1.  

PubMed

The equine herpesvirus 1 (EHV-1) homolog of the herpes simplex virus type 1 (HSV-1) tegument phosphoprotein, alpha TIF (Vmw65; VP16), was identified previously as the product of open reading frame 12 (ORF12) and shown to transactivate immediate early (IE) gene promoters. However, a specific virion protein corresponding to the ORF12 product has not been identified definitively. In the present study the ORF12 protein, designated ETIF, was identified as a 60-kDa virion component on the basis of protein fingerprint analyses in which the limited proteolysis profiles of the major 60-kDa in vitro transcription/ translation product of an ORF12 expression vector (pT7-12) were compared to those of purified virion proteins of similar size. ETIF was localized to the viral tegument in Western blot assays of EHV-1 virions and subvirion fractions using polyclonal antiserum and monoclonal antibodies generated against a glutathione-S-transferase-ETIF fusion protein. Northern and Western blot analyses of EHV-1-infected cell lysates prepared under various metabolic blocks indicated that ORF12 is expressed as a late gene, and cross reaction of polyclonal anti-GST-ETIF with a 63.5-kDa HSV-1 protein species suggested that ETIF and HSV-1 alpha TIF are antigenically related. Last, DNA band shift assays used to assess ETIF-specific complex formation indicated that ETIF participates in an infected cell protein complex with the EHV-1 IE promoter TAATGARAT motif. PMID:9143293

Lewis, J B; Thompson, Y G; Feng, X; Holden, V R; O'Callaghan, D; Caughman, G B

1997-04-14

142

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

143

Salmonella enterica Serotype Typhimurium Fimbrial Proteins Serve as Antigens during Infection of Mice  

Microsoft Academic Search

The Salmonella enterica serotype Typhimurium genome contains 13 operons with homology to fimbrial gene sequences. Here we investigated the role of 11 serotype Typhimurium fimbrial proteins, including FimA, AgfA (CsgA), BcfA, StbA, SthA, LpfA, PefA, StdA, StcA, StiA, and StfA, as antigens during the infection of genetically resistant mice (CBA). Upon the growth of serotype Typhimurium in standard laboratory broth

Andrea Humphries; Sandra DeRidder; Andreas J. Baumler

2005-01-01

144

Correlation between molecular size of the surface array protein and morphology and antigenicity of the Campylobacter fetus S layer.  

PubMed

The correlation between the molecular size of the surface layer protein (S protein) and both structure and antigenicity of the Campylobacter fetus surface layer (S layer) was investigated in several clinical strains and their spontaneous variants which produce S proteins of molecular weights (MW) different from those of the parents. Only three molecular sizes of the S proteins were observed (98, 127, and 149 kDa) in the parental and variant strains. Immunologically, the 98-kDa protein and the 149-kDa protein but not the 127-kDa protein were cross-reactive. Freeze-etching analysis showed that the 98-kDa S protein formed a hexagonal arrangement with a 24-nm center-to-center space and that the S proteins with larger MW (127 or 149 kDa) formed tetragonal ones with an 8-nm center-to-center space. Thus, the MW changes of the S proteins seen in the variant strains were associated with both morphological and antigenic changes in S layer. These observations support the hypothesis that the pattern and antigenicity of the C. fetus S layer is determined by the particular type of S protein. Furthermore, the presence of the two different S layer patterns on a single bacterial cell indicates that multiple S proteins can be produced and expressed in a single cell. PMID:2037362

Fujimoto, S; Takade, A; Amako, K; Blaser, M J

1991-06-01

145

Merozoite Surface Antigen 2 Proteins of Babesia bovis Vaccine Breakthrough Isolates Contain a Unique Hypervariable Region Composed of Degenerate Repeats  

Microsoft Academic Search

The merozoite surface antigen 2 (MSA-2) proteins of Babesia bovis are members of the variable merozoite surface antigen (VMSA) family that have been implicated in erythrocyte invasion and are important targets for antibody-mediated blocking of invasion. Extensive sequence variation in another VMSA member, MSA-1, has been shown in all vaccine breakthrough isolates. To test the hypothesis that the msa-2 genes

Shawn J. Berens; Kelly A. Brayton; John B. Molloy; Russell E. Bock; Ala E. Lew; Terry F. McElwain

2005-01-01

146

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

Microsoft Academic Search

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

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

2000-01-01

147

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

Microsoft Academic Search

Cell-mediated immune responses are critical for protective immunity to mycobacterial infections. Recent progress in defining mycobacterial antigens has determined that region of difference 1 (RD1) gene products induce strong T-cell responses, particularly the early secretory antigenic target 6-kDa (ESAT-6) protein and culture filtrate protein 10 (CFP10). However, comprehensive analysis of the immune response towards these antigens is incompletely characterized. To

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

2005-01-01

148

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

Microsoft Academic Search

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

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

2004-01-01

149

Effects of pH, temperature and enzyme-to-substrate ratio on the antigenicity of whey protein hydrolysates prepared by Alcalase  

Microsoft Academic Search

Response surface methodology was used to study the effects of pH (7.0–11.0), temperature (30–60°C), and enzyme-to-substrate ratio (4000–8000unitsg?1protein) on the residual antigenicity of whey protein concentrate (WPC, 77.5% protein) hydrolysates obtained with Alcalase. It was shown that enzymatic hydrolysis with Alcalase could reduce the antigenicity of WPC for ?-lactalbumin (?-LA) and ?-lactoglobulin (?-LG) effectively and the reduction of antigenicity could

Hai Zheng; Xiaoqin Shen; Guanhao Bu; Yongkang Luo

2008-01-01

150

Spherical Body Protein 4 Is a New Serological Antigen for Global Detection of Babesia bovis Infection in Cattle ?  

PubMed Central

Five Babesia bovis recombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), C-terminal rhoptry-associated protein 1 (BbRAP-1/CT), truncated thrombospondin-related anonymous protein (BbTRAP-T), spherical body protein 1 (BbSBP-1), and spherical body protein 4 (BbSBP-4), were evaluated as diagnostic antigens to detect the infection in cattle. The recombinant proteins were highly antigenic when tested with experimentally B. bovis-infected bovine serum in Western blot analysis. Furthermore, five antisera that had been raised against each of the recombinant proteins reacted specifically with the corresponding authentic protein, as determined in Western blot analysis. Next, enzyme-linked immunosorbent assays (ELISAs) using these recombinant proteins were evaluated for diagnostic use, and the sensitivity and specificity of each protein were demonstrated with a series of serum samples from experimentally B. bovis-infected cattle. Furthermore, a total of 669 field serum samples collected from cattle in regions of B. bovis endemicity in seven countries were tested with the ELISAs, and the results were compared to those of an indirect fluorescent antibody test (IFAT), as a reference. Among five recombinant antigens, recombinant BbSBP-4 (rBbSBP-4) had the highest concordance rate (85.3%) and kappa value (0.705), indicating its reliability in the detection of specific antibodies to B. bovis in cattle, even in different geographical regions. Overall, we have successfully developed an ELISA based on rBbSBP-4 as a new serological antigen for a practical and sensitive test which will be applicable for epidemiologic survey and control programs in the future. PMID:21123520

Terkawi, Mohamad Alaa; Huyen, Nguyen Xuan; Wibowo, Putut Eko; Seuseu, Faasoa Junior; Aboulaila, Mahmoud; Ueno, Akio; Goo, Youn-Kyoung; Yokoyama, Naoaki; Xuan, Xuenan; Igarashi, Ikuo

2011-01-01

151

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

PubMed Central

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

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

2013-01-01

152

Molecular modeling of the human sperm associated antigen 11 B (SPAG11B) proteins.  

PubMed

Antimicrobial proteins and peptides are ubiquitous in nature with diverse structural and biological properties. Among them, the human beta-defensins are known to contribute to the innate immune response. Besides the defensins, a number of defensin-like proteins and peptides are expressed in many organ systems including the male reproductive system. Some of the protein isoforms encoded by the sperm associated antigen 11B (SPAG11) gene in humans are beta-defensin-like and exhibit structure dependent and salt tolerant antimicrobial activity, besides contributing to sperm maturation. Though some of the functional roles of these proteins are reported, the structural and molecular features that contribute to their antimicrobial activity is not yet reported. In this study, using in silico tools, we report the three dimensional structure of the human SPAG11B proteins and their C-terminal peptides. web-based hydropathy, amphipathicity, and topology (WHAT) analyses and grand average of hydropathy (GRAVY) indices show that these proteins and peptides are amphipathic and highly hydrophilic. Self-optimized prediction method with alignment (SOPMA) analyses and circular dichroism data suggest that the secondary structure of these proteins and peptides primarily contain beta-sheet and random coil structure and alpha-helix to a lesser extent. Ramachandran plots show that majority of the amino acids in these proteins and peptides fall in the permissible regions, thus indicating stable structures. The secondary structure of SPAG11B isoforms and their peptides were not perturbed with increasing NaCl concentration (0-300?mM) and at different pH (3, 7, and 10), thus reinforcing our previously reported observation that their antimicrobial activity is salt tolerant. To the best of our knowledge, for the first time, results of our study provide vital information on the structural features of SPAG11B protein isoforms and their contribution to antimicrobial activity. PMID:25573789

Narmadha, Ganapathy; Yenugu, Suresh

2015-04-01

153

Structural characteristics of immunogenic liver-stage antigens derived from P. falciparum malarial proteins.  

PubMed

A fully effective antimalarial vaccine must contain multiple proteins from the different development stages of Plasmodium falciparum parasites involved in host-cell invasion or their biologically active fragments. It must therefore include sporozoite molecules able to induce protective immunity by blocking the parasite's access to hepatic cells, and/or proteins involved in the development of this stage, amongst which are included the Liver Stage Antigen-1 (LSA-1) and the Sporozoite and Liver Stage Antigen (SALSA). Our studies have focused on the search for an association between the structure of high activity binding peptides (HABPs), including both conserved native and their modified analogues, and their ability to bind to the MHC Class II HLA-DR molecules during formation of the MHCII-peptide-TCR complex leading to inducing the appropriate immune response. These studies are part of a logical and rational strategy for developing multi-stage, multi-component, minimal subunit-based vaccines, mainly against the P. falciparum malaria. PMID:19422790

Cifuentes, Gladys; Vanegas, Magnolia; Martinez, Nora Lorena; Pirajan, Camilo; Patarroyo, Manuel Elkin

2009-07-10

154

Analysis of feline calicivirus capsid protein genes: identification of variable antigenic determinant regions of the protein  

Microsoft Academic Search

Three isolates of feline calicivirus (FCV) designated NADC, KCD and CFI\\/68 were compared for bio- chemical, serological and genetic variation within the capsid protein gene. The M r of the capsid protein from purified virions was approximately 66 000 for the NADC virus isolate, which differed slightly from the relative mobilities of the purified capsid proteins of the KCD and

Bruce S. Seal; Jufia F. Ridpath; William L. Mengeling

1993-01-01

155

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

156

Echinococcus granulosus antigen B: a Hydrophobic Ligand Binding Protein at the host-parasite interface.  

PubMed

Lipids are mainly solubilized by various families of lipid binding proteins which participate in their transport between tissues as well as cell compartments. Among these families, Hydrophobic Ligand Binding Proteins (HLBPs) deserve special consideration since they comprise intracellular and extracellular members, are able to bind a variety of fatty acids, retinoids and some sterols, and are present exclusively in cestodes. Since these parasites have lost catabolic and biosynthetic pathways for fatty acids and cholesterol, HLBPs are likely relevant for lipid uptake and transportation between parasite and host cells. Echinococcus granulosus antigen B (EgAgB) is a lipoprotein belonging to the HLBP family, which is very abundant in the larval stage of this parasite. Herein, we review the literature on EgAgB composition, structural organization and biological properties, and propose an integrated scenario in which this parasite HLBP contributes to adaptation to mammalian hosts by meeting both metabolic and immunomodulatory parasite demands. PMID:25451555

Silva-Álvarez, Valeria; Folle, Ana Maite; Ramos, Ana Lía; Zamarreño, Fernando; Costabel, Marcelo D; García-Zepeda, Eduardo; Salinas, Gustavo; Córsico, Betina; Ferreira, Ana María

2015-02-01

157

Identification of the outer membrane proteins of Campylobacter pyloridis and antigenic cross-reactivity between C. pyloridis and C. jejuni.  

PubMed

The outer membrane and surface exposed proteins of four strains of the gastric Campylobacter-like organism Campylobacter pyloridis were identified by SDS-PAGE of Sarkosyl-insoluble membranous material and 125I-surface-labelled whole bacteria. Although constant outer membrane proteins (molecular mass 61, 54 and 31 kDa) were observed in these strains, several variable 125I-labelled surface proteins were detected. C. pyloridis does not appear to express a single surface-exposed major outer membrane protein like that of C. jejuni and C. coli. Putative flagella proteins were identified from isolated flagella and acid-extractable surface material and by immunoblotting with anti-flagella antibodies. Several major protein antigens were observed by immunoblotting with anti-C. pyloridis antisera. At least two of these antigens cross-reacted with anti-C. jejuni antiserum. This cross-reaction appears to be caused primarily by flagellar antigens. However, one major protein antigen (61 kDa) was not cross-reactive with C. jejuni and may, therefore, be useful in serological tests for the specific diagnosis of C. pyloridis infections. PMID:3309141

Newell, D G

1987-01-01

158

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

159

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

160

Qualitative and quantitative assessment of meningococcal antigens to evaluate the potential strain coverage of protein-based vaccines  

PubMed Central

A unique multicomponent vaccine against serogroup B meningococci incorporates the novel genome-derived proteins fHbp, NHBA, and NadA that may vary in sequence and level of expression. Measuring the effectiveness of such vaccines, using the accepted correlate of protection against invasive meningococcal disease, could require performing the serum bactericidal assay (SBA) against many diverse strains for each geographic region. This approach is impractical, especially for infants, where serum volumes are very limited. To address this, we developed the meningococcal antigen typing system (MATS) by combining a unique vaccine antigen-specific ELISA, which detects qualitative and quantitative differences in antigens, with PorA genotyping information. The ELISA correlates with killing of strains by SBA and measures both immunologic cross-reactivity and quantity of the antigens NHBA, NadA, and fHbp. We found that strains exceeding a threshold value in the ELISA for any of the three vaccine antigens had ?80% probability of being killed by immune serum in the SBA. Strains positive for two or more antigens had a 96% probability of being killed. Inclusion of multiple different antigens in the vaccine improves breadth of coverage and prevents loss of coverage if one antigen mutates or is lost. The finding that a simple and high-throughput assay correlates with bactericidal activity is a milestone in meningococcal vaccine development. This assay allows typing of large panels of strains and prediction of coverage of protein-based meningococcal vaccines. Similar assays may be used for protein-based vaccines against other bacteria. PMID:20962280

Donnelly, John; Medini, Duccio; Boccadifuoco, Giuseppe; Biolchi, Alessia; Ward, Joel; Frasch, Carl; Moxon, E. Richard; Stella, Maria; Comanducci, Maurizio; Bambini, Stefania; Muzzi, Alessandro; Andrews, William; Chen, Jie; Santos, George; Santini, Laura; Boucher, Philip; Serruto, Davide; Pizza, Mariagrazia; Rappuoli, Rino; Giuliani, Marzia Monica

2010-01-01

161

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.; Argüelles, M. H.; Glikmann, G.; Castello, A. A.

2013-01-01

162

Identification of a New Antigen Epitope in the Nuclear Localization Signal Region of Porcine Circovirus Type 2 Capsid Protein  

Microsoft Academic Search

Objective: Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic syndrome in pigs. The capsid (Cap) protein encoded by ORF2 is the main structural protein involved in the host immune protective response to PCV2. It is therefore important to map the antigenic epitopes of the PCV2 Cap protein. Methods: In this study, 5 monoclonal antibodies (mAbs) against

Longjun Guo; Yuehua Lu; Liping Huang; Yanwu Wei; Changming Liu

2011-01-01

163

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

PubMed

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

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

2014-09-01

164

MAP1272c Encodes an NlpC/P60 Protein, an Antigen Detected in Cattle with Johne's Disease  

PubMed Central

The protein encoded by MAP1272c has been shown to be an antigen of Mycobacterium avium subsp. paratuberculosis that contains an NlpC/P60 superfamily domain found in lipoproteins or integral membrane proteins. Proteins containing this domain have diverse enzymatic functions that include peptidases, amidases, and acetyltransferases. The NlpC protein was examined in comparison to over 100 recombinant proteins and showed the strongest antigenicity when analyzed with sera from cattle with Johne's disease. To further localize the immunogenicity of NlpC, recombinant proteins representing defined regions were expressed and evaluated with sera from cattle with Johne's disease. The region from amino acids 74 to 279 was shown to be the most immunogenic. This fragment was also evaluated against a commercially available enzyme-linked immunosorbent assay (ELISA). Two monoclonal antibodies were produced in mice immunized with the full-length protein, and each recognized a distinct epitope. These antibodies cross-reacted with proteins from other mycobacterial species and demonstrated variable sizes of the proteins expressed from these subspecies. Both antibodies were further analyzed, and their interaction with MAP1272c and MAP1204 was characterized by a solution-based, luminescent binding assay. These tools provide additional means to study a strong antigen of M. avium subsp. paratuberculosis. PMID:22593240

Lingle, Cari K.; Stabel, Judith R.; Ramyar, Kasra X.; Garcia, Brandon L.; Raeber, Alex J.; Schacher, Pascal; Kapur, Vivek; Geisbrecht, Brian V.

2012-01-01

165

Antigenic structure of the central conserved region of protein G of bovine respiratory syncytial virus.  

PubMed Central

Epitopes were resolved at the amino acid level for nine monoclonal antibodies (MAbs) directed against the central conserved region of protein G of bovine respiratory syncytial virus (BRSV-G). Peptide binding studies showed which amino acids in the epitope contributed to antibody binding. The details of the epitopes were compared with the high-resolution structure of a synthetic peptide corresponding to the central conserved region of BRSV-G, and this indicated which face of the central conserved region is the antigenic structure. The major linear epitope of the central conserved region of BRSV-G is located at the tip of the loop, overlapping a relatively flat surface formed by a double disulfide-bonded cystine noose. At least one, but possibly two sulfur atoms of a disulfide bridge that line the conserved pocket at the center of the flat surface, is a major contributor to antibody binding. Some of the residue positions in the epitope have mutated during the evolution of RSV-G, which suggests that the virus escaped antibody recognition with these mutations. Mutations that occur at positions 177 and 180 may have only a local effect on the antigenic surface, without influencing the structure of the backbone, whereas mutations at positions 183 and 184 will probably have major structural consequences. The study explains the antigenic, structural, and functional importance of each residue in the cystine noose which provides information for peptide vaccine design. Additionally, analysis of the epitopes demonstrated that two point mutations at positions 180 and 205 define the preliminary classification of BRSV subgroups. PMID:9094683

Langedijk, J P; Meloen, R H; Taylor, G; Furze, J M; van Oirschot, J T

1997-01-01

166

A novel mechanism for regulating the activity of proliferating cell nuclear antigen by a small protein.  

PubMed

Proliferating cell nuclear antigen (PCNA) forms a trimeric ring that associates with and influences the activity of many proteins participating in DNA metabolic processes and cell cycle progression. Previously, an uncharacterized small protein, encoded by TK0808 in the archaeon Thermococcus kodakarensis, was shown to stably interact with PCNA in vivo. Here, we show that this protein, designated Thermococcales inhibitor of PCNA (TIP), binds to PCNA in vitro and inhibits PCNA-dependent activities likely by preventing PCNA trimerization. Using hydrogen/deuterium exchange mass spectrometry and site-directed mutagenesis, the interacting regions of PCNA and TIP were identified. Most proteins bind to PCNA via a PCNA-interacting peptide (PIP) motif that interacts with the inter domain connecting loop (IDCL) on PCNA. TIP, however, lacks any known PCNA-interacting motif, suggesting a new mechanism for PCNA binding and regulation of PCNA-dependent activities, which may support the development of a new subclass of therapeutic biomolecules for inhibiting PCNA. PMID:24728986

Li, Zhuo; Huang, Richard Y-C; Yopp, Daniel C; Hileman, Travis H; Santangelo, Thomas J; Hurwitz, Jerard; Hudgens, Jeffrey W; Kelman, Zvi

2014-05-01

167

Genetic and antigenic characterization of Babesia bovis merozoite spherical body protein Bb-1.  

PubMed

A Babesia bovis merozoite protein, Bb-1, was localized by immunoelectron microscopy to an apical organelle known as the spherical body. This unique structure appears to be analogous to dense granules of other apicomplexan protozoa. Similar to previously described dense granule proteins of Plasmodium spp., Bb-1 is secreted during or just after invasion of host erythrocytes and becomes associated with the cytoplasmic face of the infected cell. The amino terminal sequence of Bb-1 contains a predicted signal peptide and is similar to the amino terminus of another spherical body protein (BvVA1/225) which is also translocated to the erythrocyte membrane. Importantly, these two spherical body proteins are the major components of a protective fraction of B. bovis antigen. There is marked conservation of Bb-1 amino acid sequences and B-lymphocyte epitopes among geographic strains. However, a divergent Bb-1 allele (Bv80) in Australia strains encodes six regions of amino acid polymorphism, including a region of tetrapeptide repeats in the C-terminal half of the polypeptide. Two of the polymorphic regions map to previously defined Th1 epitopes on Bb-1. PMID:7770080

Hines, S A; Palmer, G H; Brown, W C; McElwain, T F; Suarez, C E; Vidotto, O; Rice-Ficht, A C

1995-02-01

168

A novel mechanism for regulating the activity of proliferating cell nuclear antigen by a small protein  

PubMed Central

Proliferating cell nuclear antigen (PCNA) forms a trimeric ring that associates with and influences the activity of many proteins participating in DNA metabolic processes and cell cycle progression. Previously, an uncharacterized small protein, encoded by TK0808 in the archaeon Thermococcus kodakarensis, was shown to stably interact with PCNA in vivo. Here, we show that this protein, designated Thermococcales inhibitor of PCNA (TIP), binds to PCNA in vitro and inhibits PCNA-dependent activities likely by preventing PCNA trimerization. Using hydrogen/deuterium exchange mass spectrometry and site-directed mutagenesis, the interacting regions of PCNA and TIP were identified. Most proteins bind to PCNA via a PCNA-interacting peptide (PIP) motif that interacts with the inter domain connecting loop (IDCL) on PCNA. TIP, however, lacks any known PCNA-interacting motif, suggesting a new mechanism for PCNA binding and regulation of PCNA-dependent activities, which may support the development of a new subclass of therapeutic biomolecules for inhibiting PCNA. PMID:24728986

Li, Zhuo; Huang, Richard Y.-C.; Yopp, Daniel C.; Hileman, Travis H.; Santangelo, Thomas J.; Hurwitz, Jerard; Hudgens, Jeffrey W.; Kelman, Zvi

2014-01-01

169

Cooperative serum bactericidal activity between human antibodies to meningococcal factor H binding protein and neisserial heparin binding antigen.  

PubMed

A meningococcal group B vaccine containing multiple protein antigens including factor H binding protein (fHbp) and Neisserial heparin binding antigen (NHba) is in clinical development. The ability of antibodies against individual antigens to interact and augment protective immunity is unknown. We assayed human complement-mediated bactericidal activity (SBA) in stored sera from six immunized adults before and after depletion of antibodies to fHbp and/or NHba. All six subjects developed ? 4-fold increases in SBA titer against a test strain with fHbp in the variant 1 group with an amino acid sequence that matched the vaccine antigen (GMT <1:4 baseline, to 1:139 after 3 doses of vaccine). By adsorption 88 to >95% of the SBA was directed against fHbp. Four subjects developed ? 4-fold increases in SBA titer against a test strain with a heterologous fHbp variant 2 antigen and a homologous NHba amino acid sequence that matched the vaccine antigen (GMT <1:4 baseline, to 1:45). SBA was directed primarily against NHba in one subject, against fHbp in a second, while depletion of either anti-NHba or anti-fHbp antibody removed the majority of SBA in sera from two subjects. In all four subjects, depletion of both anti-fHbp and anti-NHba antibodies removed more SBA than depletion of either antibody individually. Mixing a mouse non-bactericidal anti-fHbp variant 1 antiserum with a mouse anti-NHba antiserum also augmented the anti-NHba SBA titer against this test strain. For meningococcal vaccines that target relatively sparsely exposed antigens such fHbp or NHba, non-bactericidal antibodies against individual antigens can cooperate and elicit SBA. PMID:21241734

Vu, David M; Wong, Tracy T; Granoff, Dan M

2011-02-24

170

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

171

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

172

Molecular cloning and sequence of cDNA encoding polyoma medium tumor antigen-associated 61-kDa protein.  

PubMed Central

Polyoma virus medium tumor antigen forms specific complexes with several cellular proteins; among these is a protein of approximately 61 kDa. With antibodies directed against medium tumor antigen, the 61-kDa protein was purified from human 293 cells that were infected with a hybrid adenovirus and overexpressed medium tumor antigen. The purified 61-kDa protein was partially digested with protease V8, and one of the protease V8 fragments was isolated and partially sequenced. The amino acid sequence information was used to design mixed oligonucleotide probes for screening a cDNA library from human placenta. A clone was isolated that hybridized with two separate probes; the clone contained an insert with an open reading frame for 589 amino acids. By in vitro translation of the transcript from this insert, a protein was generated that had the same size and yielded the same pattern of protease V8 fragments as the original 61-kDa protein. Its amino acid sequence reveals 15 repeats, the majority of which are 39 amino acids long. This protein bears no resemblance to proteins in the data bank that was searched. Images PMID:2554323

Walter, G; Ferre, F; Espiritu, O; Carbone-Wiley, A

1989-01-01

173

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

SciTech Connect

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

Liu, Cheng-Der; Cheng, Chi-Ping; Fang, Jia-Shih; Chen, Ling-Chih [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China)] [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China); Zhao, Bo; Kieff, Elliott [Department of Medicine and Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women’s Hospital and Harvard Medical School, 181 Longwood Ave., Boston 02115, MA (United States)] [Department of Medicine and Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women’s Hospital and Harvard Medical School, 181 Longwood Ave., Boston 02115, MA (United States); Peng, Chih-Wen, E-mail: pengcw@mail.tcu.edu.tw [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China)] [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China)

2013-01-18

174

Antigenic characterization of recombinant hemagglutinin proteins derived from different avian influenza virus subtypes.  

PubMed

Since the advent of highly pathogenic variants of avian influenza virus (HPAIV), the main focus of avian influenza research has been the characterization and detection of HPAIV hemagglutinin (HA) from H5 and H7 subtypes. However, due to the high mutation and reassortation rate of influenza viruses, in theory any influenza strain may acquire increased pathogenicity irrespective of its subtype. A comprehensive antigenic characterization of influenza viruses encompassing all 16 HA and 9 neuraminidase subtypes will provide information useful for the design of differential diagnostic tools, and possibly, vaccines. We have expressed recombinant HA proteins from 3 different influenza virus HA subtypes in the baculovirus system. These proteins were used to generate polyclonal rabbit antisera, which were subsequently employed in epitope scanning analysis using peptide libraries spanning the entire HA. Here, we report the identification and characterization of linear, HA subtype-specific as well as inter subtype-conserved epitopes along the HA proteins. Selected subtype-specific epitopes were shown to be suitable for the differentiation of anti-HA antibodies in an ELISA. PMID:20140098

Mueller, Matthias; Renzullo, Sandra; Brooks, Roxann; Ruggli, Nicolas; Hofmann, Martin A

2010-01-01

175

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

PubMed Central

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

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

2013-01-01

176

Human immunodeficiency virus (HIV) antigens: structure and serology of multivalent human mucin MUC1-HIV V3 chimeric proteins.  

PubMed Central

Molecular modeling and two-dimensional NMR techniques enable us to identify structural features in the third variable region (V3) loop of the human immunodeficiency virus (HIV) surface glycoprotein gp120, in particular the principal neutralizing determinant (PND), that remain conserved despite the sequence variation. The conserved structure of the PND is a solvent-accessible protruding motif or a knob, structurally isomorphous with the immunodominant knobs in the tandem repeat protein of human mucin 1 (MUC1) (a tumor antigen for breast, pancreatic, and ovarian cancer). We have replaced the mucin antigenic knobs by the PND knobs of the HIV MN isolate in a set of chimeric human MUC1/HIV V3 antigens. This produced multivalent HIV antigens in which PNDs are located at regular intervals and separated by extended mucin spacers. In this article we show by two-dimensional NMR spectroscopy that the multivalent antigens preserve the PNDs in their native structure. We also demonstrate by ELISA that the antigens correctly present the PNDs for binding to monoclonal antibodies or polyclonal antisera from HIV-infected patients. Images Fig. 2 Fig. 3 PMID:7816840

Fontenot, J D; Gatewood, J M; Mariappan, S V; Pau, C P; Parekh, B S; George, J R; Gupta, G

1995-01-01

177

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

PubMed

We describe here a single plasmid DNA immunogen representing the broadest antigen repertoire among HIV vaccine candidates. This pDNA was "ANTIGENeered" for the regulated expression of thirteen complete and two non-functional HIV protein antigens. These proteins self assemble into complex virus-like particles (VLP(+)). Multiple irreversible safety features were introduced by genetic modifications including the complete impairment of integration, reverse transcription, the function of Nef and the 3'LTR. Epitope analysis predicted that the pDNA-derived protein repertoire can potentially present over 3000 T cell epitopes. However, the expressed antigens have different potential to induce HIV-specific CD4(+) and CD8(+) T cells supporting our hypothesis that HIV vaccines should contain all possible regulatory and structural proteins. This immunogen is the active pharmaceutical ingredient of DermaVir, a therapeutic vaccine product candidate that recently successfully completed Phase II clinical trials and meets the safety, immunogenicity and cost requirements of an HIV vaccine. PMID:21109034

Somogyi, Eszter; Xu, Jianqing; Gudics, Agnes; Tóth, József; Kovács, Attila L; Lori, Franco; Lisziewicz, Julianna

2011-01-17

178

Generation of antigen specific CD8 + cytotoxic T cells following immunization with soluble protein formulated with novel glycoside adjuvants  

Microsoft Academic Search

Presentation of peptide on MHC class I molecules is essential to elicit cytolytic T cell (CTL) activity. Such peptides are a result of the cytosolic, or class I, antigen processing pathway. Due to the segregation of the class I and the exogenous processing pathway, soluble protein cannot enter the class I pathway and is thus incapable of inducing CTL. However

N. A Sheikh; P Rajananthanan; G. S Attard; W. J. W Morrow

1999-01-01

179

Blood-group antigen recognition by a solute-binding protein from a serotype 3 strain of Streptococcus pneumoniae  

PubMed Central

Streptococcus pneumoniae is a common bacterial pathogen that is well-known for its ability to cause acute respiratory disease (pneumonia), ear infections, and other serious illnesses. This Gram-positive bacterium relies on its carbohydrate metabolizing capabilities for full virulence in its host; however, the range of glycan targets that it can attack is presently not fully appreciated. S. pneumoniae is known to have a fucose utilization operon that in the TIGR4 strain plays a role in its virulence. Here we identify a second type of fucose utilization operon that is present in a subset of S. pneumoniae strains, including the serotype 3 strain SP3-BS71. This operon contains a transporter with a solute-binding protein, FcsSBP (Fucose Solute Binding Protein), that interacts tightly (Ka ~1 × 106 M?1) and specifically with soluble A- and B-antigen trisaccharides but displays no selectivity between these two sugars. The structure of the FcsSBP in complex with the A-trisaccharide antigen, determined to 2.35 Å, reveals its mode of binding to the reducing end of this sugar thus highlighting this protein’s requirement for soluble blood-group antigen ligands. Overall, this report exposes a heretofore unknown capability of certain S. pneumoniae strains to transport and potentially metabolize the histo-blood group antigen carbohydrates of its host. PMID:19285508

Higgins, Melanie A.; Abbott, D. Wade; Boulanger, Martin J.; Boraston, Alisdair B.

2010-01-01

180

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

181

Identification of antigenic Edwardsiella tarda surface proteins and their role in pathogenesis.  

PubMed

Edwardsiella tarda causes an infectious fish disease called edwardsiellosis. Several outer membrane proteins (OMPs) are associated with virulence factors and are attractive as vaccine candidates. In this study, 4 immuno-reactive OMPs of E. tarda were detected using anti-sera from flounder infected with E. tarda. Using matrix-assisted laser desorption/ionization mass spectrometry analyses, 2 of the 4 OMPs were identified as OmpA and murein lipoprotein (Lpp), which are highly conserved surface proteins in gram-negative bacteria. For further characterization of these surface proteins, we generated ompA- and lpp-inactivated mutants by insertion of a kanamycin cassette in the corresponding genes, and named these mutants E. tarda CK99 and CK164, respectively. As expected, immuno-reactive OmpA and Lpp proteins were absent in E. tarda CK99 and CK164, respectively, confirming that OmpA and Lpp are antigenic surface proteins. Interestingly, the LD(50) value of E. tarda CK164 in fish (2.0 × 10(8) colony-forming unit [CFU]/fish) was greater than that of the parental strain (3.0 × 10(7) CFU/fish). The LD(50) of E. tarda CK99 did not differ from that of its parental strain. After administering attenuated E. tarda CK164 to fish, we monitored the E. tarda-specific immune response profile. We observed that the E. tarda-specific serum IgM titer increased in a time-dependent manner, and was much higher than the value observed after the administration of a heat-killed E. tarda control. Moreover, fish vaccinated with E. tarda CK164 were 100% protected when challenged by CK41, a pathogenic strain. Our results suggest that E. tarda CK164 can potentially be used for developing an effective live attenuated vaccine for edwardsiellosis that can be applied in the aquaculture industry. PMID:23231854

Yu, Jong Earn; Yoo, Ah Young; Choi, Kyoung-Hwa; Cha, Jaeho; Kwak, Inseok; Kang, Ho Young

2013-02-01

182

FlaA, a putative flagellar outer sheath protein, is not an immunodominant antigen associated with Lyme disease.  

PubMed Central

FlaA was recently found to be associated with flagellar filaments of Borrelia burgdorferi. We tested whether antibodies to this protein are a good indicator of infection, as antibodies to FlaA proteins in other spirochetal infections show an increase in titer. Although overproduction of intact FlaA was highly toxic to Escherichia coli, truncated proteins which lacked the N-terminal signal sequence could be successfully overexpressed. Immunoblotting with sera from mammalian hosts infected with B. burgdorferi indicated that FlaA is not an immunodominant antigen in Lyme disease. However, sera from two patients reacted with both recombinant and native FlaA protein, suggesting that B. burgdorferi FlaA was antigenic and expressed in vivo. PMID:9199479

Ge, Y; Charon, N W

1997-01-01

183

Discovery of GAMA, a Plasmodium falciparum merozoite micronemal protein, as a novel blood-stage vaccine candidate antigen.  

PubMed

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

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

2011-11-01

184

Recovery and Altered Neutralization Specificities of Chimeric Viruses Containing Capsid Protein Domain Exchanges from Antigenically Distinct Strains of Feline Calicivirus  

PubMed Central

Feline calicivirus (FCV) strains can show significant antigenic variation when tested for cross-reactivity with antisera produced against other FCV strains. Previous work has demonstrated the presence of hypervariable amino acid sequences in the capsid protein of FCV (designated regions C and E) that were postulated to constitute the major antigenic determinants of the virus. To examine the involvement of hypervariable sequences in determining the antigenic phenotype, the nucleotide sequences encoding the E regions from three antigenically distinct parental FCV strains (CFI, KCD, and NADC) were exchanged for the equivalent sequences in an FCV Urbana strain infectious cDNA clone. Two of the three constructs were recovered as viable, chimeric viruses. In six additional constructs, of which three were recovered as viable virus, the E region from the parental viruses was divided into left (N-terminal) and right (C-terminal) halves and engineered into the infectious clone. A final viable construct contained the C, D, and E regions of the NADC parental strain. Recovered chimeric viruses showed considerable antigenic variation from the parental viruses when tested against parental hyperimmune serum. No domain exchange was able to confer complete recognition by parental antiserum with the exception of the KCD E region exchange, which was neutralized at a near-homologous titer with KCD antiserum. These data demonstrate that it is possible to recover engineered chimeric FCV strains that possess altered antigenic characteristics. Furthermore, the E hypervariable region of the capsid protein appears to play a major role in the formation of the antigenic structure of the virion where conformational epitopes may be more important than linear in viral neutralization. PMID:10627517

Neill, John D.; Sosnovtsev, Stanislav V.; Green, Kim Y.

2000-01-01

185

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

PubMed Central

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

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

2014-01-01

186

Development of a sandwich Dot-ELISA for detecting bovine viral diarrhea virus antigen with E2 recombinant protein  

Microsoft Academic Search

The IgG antibodies of rabbit anti-E2 protein of the bovine viral diarrhea virus were prepared by a general method from high\\u000a efficiency serum immunized by E2 recombinant protein antigen expressed in E. coli prokaryotic expression system and were labeled to make enzyme-labeled antibody with the method of NaIO4. A sandwich Dot enzyme-linked immunosorbent assay (Dot-ELISA) for the detection of BVDV

Yuelan Zhao; Yuzhu Zuo; Lei Zhang; Jinghui Fan; Hanchun Yang; Jianhua Qin

2009-01-01

187

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

PubMed

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

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

2015-01-01

188

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

PubMed Central

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

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

2015-01-01

189

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

190

Conservation of antigen components from two recombinant hybrid proteins protective against malaria.  

PubMed

Recently, we have shown that two hybrid proteins carrying partial sequences of the blood-stage antigens SERP, HRPII, and MSAI from Plasmodium falciparum confer protective immunity on Aotus monkeys against an experimental parasite infection (B. Knapp, E. Hundt, B. Enders, and H. A. Küpper, Infect. Immun. 60:2397-2401, 1992). The malarial components of the hybrid proteins consist of amino acid residues 630 to 892 of SERP, amino acid residues 146 to 260 of MSAI, and the 189 C-terminal residues of HRPII. We have studied the diversity of these protein regions in field isolates of P. falciparum. Genomic DNA was extracted from the blood of six donors from two different areas where malaria is endemic. The gene regions of SERP and MSAI coding for the corresponding sequences of the protective hybrid proteins and the exon II region of the HRPII gene were amplified by polymerase chain reaction and sequenced. All three regions were found to be highly conserved. In the 262-amino-acid fragment of SERP, one single conservative amino acid substitution was found. The exon II region of HRPII showed only a slight variability in number and arrangement of the repeat units. The 115-amino-acid fragment of MSAI which is located within an N-terminal region known to be conserved among different parasite strains was shown to be the most variable among the vaccine components: amino acid substitutions were found in 14 different positions of this MSAI region when both laboratory strains and field isolates were compared. PMID:8432609

Knapp, B; Nau, U; Hundt, E

1993-03-01

191

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

PubMed

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

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

2014-11-01

192

SEPPA: a computational server for spatial epitope prediction of protein antigens  

PubMed Central

In recent years, a lot of efforts have been made in conformational epitope prediction as antigen proteins usually bind antibodies with an assembly of sequentially discontinuous and structurally compact surface residues. Currently, only a few methods for spatial epitope prediction are available with focus on single residue propensity scales or continual segments clustering. In the method of SEPPA, a concept of ‘unit patch of residue triangle’ was introduced to better describe the local spatial context in protein surface. Besides that, SEPPA incorporated clustering coefficient to describe the spatial compactness of surface residues. Validated by independent testing datasets, SEPPA gave an average AUC value over 0.742 and produced a successful pick-up rate of 96.64%. Comparing with peers, SEPPA shows significant improvement over other popular methods like CEP, DiscoTope and BEpro. In addition, the threshold scores for certain accuracy, sensitivity and specificity are provided online to give the confidence level of the spatial epitope identification. The web server can be accessed at http://lifecenter.sgst.cn/seppa/index.php. Batch query is supported. PMID:19465377

Sun, Jing; Wu, Di; Xu, Tianlei; Wang, Xiaojing; Xu, Xiaolian; Tao, Lin; Li, Y. X.; Cao, Z. W.

2009-01-01

193

The interaction among protein C inhibitor, prostate-specific antigen, and the semenogelin system.  

PubMed

The kallikrein-like serine protease, prostate-specific antigen (PSA), is mixed in human seminal plasma with its protein substrates semenogelin (Sg) -I, Sg-II, and protein C inhibitor (PCI), which are produced in seminal vesicles. In the seminal plasma, PSA degrades Sg-I, and Sg-II, which are major components in insoluble coagula, and PCI inhibits PSA by forming a PSA-PCI complex. Digestion of seminal coagula with PSA releases PCI and PSA-PCI complex from the coagula into a soluble phase, suggesting the presence of active PCI within the coagula. PCI forms a ternary complex with PSA and Sg-II in the seminal plasma. The binding of Sg-II to PSA and PCI is influenced by pH, ionic strength, heparin, negatively charged dextran sulfate, divalent cations, and particularly by Zn 2 +. These observations suggest that binding of PCI to Sg in seminal vesicles regulates the PSA-catalyzed degradation of Sg in seminal plasma; the complex formation among PCI, PSA, and Sg is modulated by several factors in seminal plasma. PMID:17253189

Suzuki, Koji; Kise, Hideaki; Nishioka, Junji; Hayashi, Tatsuya

2007-02-01

194

Formation of potential titanium antigens based on protein binding to titanium dioxide nanoparticles  

PubMed Central

Degradation products of titanium implants include free ions, organo-metallic complexes, and particles, ranging from nano to macro sizes. The biological effects, especially of nanoparticles, is yet unknown. The main objective of this study was to develop Ti-protein antigens in physiological solutions that can be used in testing of cellular responses. For this purpose, 0.1% TiO2 nanoparticles less than 100 nm were mixed with human serum albumin (HSA), 0.1% and 1%, in cell culture medium (DMEM, pH 7.2). The Ti concentrations in the resulting solutions were analyzed by inductively coupled plasma mass spectrometry. The stability of the nanoparticles in suspension was analyzed by UV-vis spectrophotometer and Dynamic Light Scattering. The concentration of Ti in suspension was dependent on the presence and concentration of HSA. Albumin prevented high aggregation rate of TiO2 nanoparticles in cell culture medium. It is shown that nano TiO2-protein stable aggregates can be produced under physiological conditions at high concentrations, and are candidates for use in cellular tests. PMID:18488417

Vamanu, Carmen Irina; Høl, Paul Johan; Allouni, Zouhir Ekeland; Elsayed, Said; Gjerdet, Nils Roar

2008-01-01

195

Serological diagnosis of Chagas' disease: a potential confirmatory assay using preserved protein antigens of Trypanosoma cruzi.  

PubMed Central

The diagnosis of Chagas' disease relies mostly on data provided by immunologic tests, but inconclusive results often require elucidation, especially in blood banks. When six different types of Trypanosoma cruzi epimastigote antigens were studied by an immunoblotting assay (IBA), a preserved protein antigen (Ag PP) was found to present the most interesting immunochemical features because of its high reactivity with anti-T. cruzi antibodies. Thus, the IBA with Ag PP (PP IBA) was assessed with panels of coded and noncoded serum samples prepared in different laboratories, including the Brazilian Reference Laboratory for Chagas' Disease. It was found that serum samples from patients proved (clinically, eletrocardiographically, serologically, and epidemiologically) to have Chagas' disease consistently recognized 12 bands (140, 100, 85, 78, 59, 57, 46, 35, 27, 23, 20, and 18 kDa) of Ag PP. In contrast, sera from nonchagasic patients, including patients with mucocutaneous leishmaniasis, were negative or reacted weakly, and one serum sample did not have more than five different bands. These bands were 78, 57, 46, 35, 27, 23, 20, or 18 kDa. A criterion was adopted to interpret the results obtained in the PP IBA. The criterion considered positive a serum sample recognizing all 12 bands and considered negative a serum sample that did not recognize any of the bands except the eight nonspecific bands mentioned above. The PP IBA indicated maximum sensitivity and specificity as well as high positive and negative predictive values. The data demonstrate that the PP IBA discriminates chagasic from nonchagasic infections and seems to be applicable as a confirmatory assay for elucidating inconclusive results obtained by standard serology. PMID:9196203

Mendes, R P; Hoshino-Shimizu, S; Moura da Silva, A M; Mota, I; Heredia, R A; Luquetti, A O; Leser, P G

1997-01-01

196

Monoclonal antibody studies of the antigenic determinants of human plasma retinol-binding protein.  

PubMed

A battery of monoclonal antibodies (MoAbs) against human retinol-binding protein (RBP) was produced to obtain useful probes for the study of the antigenic determinants of RBP. The 12 antibodies all reacted with human RBP by immunoblotting. Based on antibody cross-competition radioimmunoassays, four distinct and different groups of antibodies were identified: group I, 1A4 and 2F4; group II, 1G10, 5C5, 6F4, and 7G3; group III, 5H6, 6C7, 10G5, and 14E3; and group IV, 5H9 and 13A1. Information about the epitopes of RBP recognized by these MoAbs was obtained by testing the reactivity of each antibody with human, rabbit, and rat RBPs by immunoblotting. Group I and group IV antibodies reacted to a similar extent with human, rabbit, and rat RBPs. Group II antibodies reacted strongly with human and rabbit RBPs, but reacted very weakly with rat RBP. Group III antibodies reacted strongly with human RBP, but did not react with rabbit or rat RBP. Thus, the epitopes for group I and group IV antibodies appear to be regions of the RBP molecule that are conserved across the three species, whereas group III antibodies recognized only human RBP. In a preliminary study, the reactivity of each antibody with purified cyanogen bromide fragments of RBP was tested by slot immunoblotting. None of the MoAbs reacted with any of the cyanogen bromide fragments. This study shows that MoAbs specific for at least four different regions of the RBP molecule can be produced; hence, RBP contains at least four major antigenic domains. PMID:15539214

Davila-Bloom, M E; Blaner, W S; Goodman, D S

1990-05-01

197

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

198

Mass Spectrometry of Human Leukocyte Antigen Class I Peptidomes Reveals Strong Effects of Protein Abundance and Turnover on Antigen Presentation*  

PubMed Central

HLA class I molecules reflect the health state of cells to cytotoxic T cells by presenting a repertoire of endogenously derived peptides. However, the extent to which the proteome shapes the peptidome is still largely unknown. Here we present a high-throughput mass-spectrometry-based workflow that allows stringent and accurate identification of thousands of such peptides and direct determination of binding motifs. Applying the workflow to seven cancer cell lines and primary cells, yielded more than 22,000 unique HLA peptides across different allelic binding specificities. By computing a score representing the HLA-I sampling density, we show a strong link between protein abundance and HLA-presentation (p < 0.0001). When analyzing overpresented proteins – those with at least fivefold higher density score than expected for their abundance – we noticed that they are degraded almost 3 h faster than similar but nonpresented proteins (top 20% abundance class; median half-life 20.8h versus 23.6h, p < 0.0001). This validates protein degradation as an important factor for HLA presentation. Ribosomal, mitochondrial respiratory chain, and nucleosomal proteins are particularly well presented. Taking a set of proteins associated with cancer, we compared the predicted immunogenicity of previously validated T-cell epitopes with other peptides from these proteins in our data set. The validated epitopes indeed tend to have higher immunogenic scores than the other detected HLA peptides. Remarkably, we identified five mutated peptides from a human colon cancer cell line, which have very recently been predicted to be HLA-I binders. Altogether, we demonstrate the usefulness of combining MS-analysis with immunogenesis prediction for identifying, ranking, and selecting peptides for therapeutic use. PMID:25576301

Bassani-Sternberg, Michal; Pletscher-Frankild, Sune; Jensen, Lars Juhl; Mann, Matthias

2015-01-01

199

Dephosphorylation of JC virus agnoprotein by protein phosphatase 2A: Inhibition by small t antigen  

PubMed Central

Previous studies have demonstrated that the JC virus (JCV) late regulatory protein agnoprotein is phosphorylated by the serine/threonine-specific protein kinase-C (PKC) and mutants of this protein at the PKC phosphorylation sites exhibit defects in the viral replication cycle. We have now investigated whether agnoprotein phosphorylation is regulated by PP2A, a serine/threonine-specific protein phosphatase and whether JCV small t antigen (Sm t-Ag) is involved in this regulation. Protein–protein interaction studies demonstrated that PP2A associates with agnoprotein and dephosphorylates it at PKC-specific sites. Sm t-Ag was also found to interact with PP2A and this interaction inhibited the dephosphorylation of agnoprotein by PP2A. The interaction domains of Sm t-Ag and agnoprotein with PP2A were mapped, as were the interaction domains of Sm t-Ag with agnoprotein. The middle portion of Sm t-Ag (aa 82–124) was found to be critical for the interaction with both agnoprotein and PP2A and the N-terminal region of agnoprotein for interaction with Sm t-Ag. To further understand the role of Sm t-Ag in JCV regulation, a stop codon was introduced at Ser90 immediately after splice donor site of the JCV early gene and the functional consequences of this mutation were investigated. The ability of this mutant virus to replicate was substantially reduced compared to WT. Next, the functional significance of PP2A in JCV replication was examined by siRNA targeting. Downregulation of PP2A caused a significant reduction in the level of JCV replication. Moreover, the impact of Sm t-Ag on agnoprotein phosphorylation was investigated by creating a double mutant of JCV, where Sm t-Ag stop codon mutant was combined with an agnoprotein triple phosphorylation mutant (Ser7, Ser11 and Thr21 to Ala). Results showed that double mutant behaves much like the triple phosphorylation mutant of agnoprotein during viral replication cycle, which suggests that agnoprotein might be an important target of Sm t-Ag with respect to the regulation of its phosphorylation. Collectively, these results suggest that there is an interplay between agnoprotein, Sm t-Ag and PP2A with respect to the regulation of JCV life cycle and this could be important for the progression of the JCV-induced disease, PML. PMID:18353419

Sariyer, Ilker K.; Khalili, Kamel; Safak, Mahmut

2009-01-01

200

Plant heat shock protein 90 as carrier-adjuvant for immunization against a reporter antigen.  

PubMed

Here, we evaluated the modulation of the immune response induced by Hsp90 of Nicotiana benthamiana (NbHsp90.3) against the Maltose Binding Protein (MBP) as a reporter antigen. Equimolar quantities of recombinant proteins were administered in mice as follows: MBP alone (MBP group), a mixture of MBP and rNbHsp90.3 (MBP+rNbHsp90.3 group) and the fusion of MBP to rNbHsp90.3 (MBP-rNbHsp90.3 group). The covalent linkage between NbHsp90.3 and MBP to bring a fusion protein was essential to induce the strong specific antibody response with predominance of IgG2a. Eighty-four days after the first immunization, splenocyte proliferation from MBP-rNbHsp90.3-immunized mice was consistently higher than that from MBP and MBP+rNbHsp90.3 groups. In addition, splenocytes from MBP-rNbHsp90.3 immunized mice produced higher levels of IFN-? than controls. Finally, both formulations with rNbHsp90.3 significantly enhanced the MHC class I expression levels, but only rNbHsp90.3 covalent bound to MBP induced a specific cellular immune response against MBP measured as increased percentage of CD8(+) T cells. Taken together, these results suggest that plant HSP90s could be incorporated as adjuvants in vaccines that require the generation of a Th1 response along with a CD8 cytotoxic cell response to confer immunity. PMID:24120680

Corigliano, Mariana G; Fenoy, Ignacio; Sander, Valeria; Maglioco, Andrea; Goldman, Alejandra; Clemente, Marina

2013-12-01

201

Dendritic cells transduced with protein antigens induce cytotoxic lymphocytes and elicit antitumor immunity.  

PubMed

Dendritic cell (DC)-based vaccines are being developed for treatment of patients with cancer, in part because DC are potent inducers of CD8(+) CTL. DC MHC class I:antigenic peptide complexes that are required for CTL elicitation are most often generated by incubating DC with peptides or by transfecting (or transducing) DC with cDNAs (or viral vectors) that encode protein Ags. The former approach is feasible when MHC class I Ags and relevant peptides are known. The latter approach may be hampered by inefficient DC transfection (transduction) and/or difficulties associated with preparation and use of viral vectors. Herein we demonstrate that a bacterial recombinant model tumor-associated Ag (OVA) that contains the HIV TAT protein transduction domain (PTD) was readily engineered and purified, efficiently transduced murine lymphocytes and DC, and was processed by proteasomes for MHC class I-restricted presentation to CTL. In addition, PTD-containing rOVA was processed and presented by DC to CD4 T cells as efficiently as native OVA or rOVA lacking the PTD. PTD-OVA-transduced DC induced CTL in vivo in a Th cell-independent fashion and vaccinated against OVA-expressing tumors. In contrast, rOVA lacking the PTD did not enter the DC MHC class I presentation pathway and DC treated with this protein did not prime OVA-specific CTL in vivo. Treatment of mice harboring clinically apparent OVA-expressing tumors with PTD-OVA-transduced DC resulted in tumor regression in some animals. This straightforward vaccination strategy may translate into DC-based treatments for patients with cancer and other serious diseases. PMID:11859130

Shibagaki, Naotaka; Udey, Mark C

2002-03-01

202

Mannosylated Mucin-Type Immunoglobulin Fusion Proteins Enhance Antigen-Specific Antibody and T Lymphocyte Responses  

PubMed Central

Targeting antigens to antigen-presenting cells (APC) improve their immunogenicity and capacity to induce Th1 responses and cytotoxic T lymphocytes (CTL). We have generated a mucin-type immunoglobulin fusion protein (PSGL-1/mIgG2b), which upon expression in the yeast Pichia pastoris became multivalently substituted with O-linked oligomannose structures and bound the macrophage mannose receptor (MMR) and dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) with high affinity in vitro. Here, its effects on the humoral and cellular anti-ovalbumin (OVA) responses in C57BL/6 mice are presented. OVA antibody class and subclass responses were determined by ELISA, the generation of anti-OVA CTLs was assessed in 51Cr release assays using in vitro-stimulated immune spleen cells from the different groups of mice as effector cells and OVA peptide-fed RMA-S cells as targets, and evaluation of the type of Th cell response was done by IFN-?, IL-2, IL-4 and IL-5 ELISpot assays. Immunizations with the OVA ? mannosylated PSGL-1/mIgG2b conjugate, especially when combined with the AbISCO®-100 adjuvant, lead to faster, stronger and broader (with regard to IgG subclass) OVA IgG responses, a stronger OVA-specific CTL response and stronger Th1 and Th2 responses than if OVA was used alone or together with AbISCO®-100. Also non-covalent mixing of mannosylated PSGL-1/mIgG2b, OVA and AbISCO®-100 lead to relatively stronger humoral and cellular responses. The O-glycan oligomannoses were necessary because PSGL-1/mIgG2b with mono- and disialyl core 1 structures did not have this effect. Mannosylated mucin-type fusion proteins can be used as versatile APC-targeting molecules for vaccines and as such enhance both humoral and cellular immune responses. PMID:23071675

Johansson, Tomas; Nilsson, Anki; Chatzissavidou, Nathalie; Sjöblom, Magnus; Rova, Ulrika; Holgersson, Jan

2012-01-01

203

A 230-kD Basic Protein Is the Major Bullous Pemphigoid Antigen  

Microsoft Academic Search

Recent studies have demonstrated that bullous pemphigoid (BP) antigen, in extracts of cultured human keratinocytes and normal human epidermis, is a 230-kD polypeptide. However, other recent studies have suggested that there is heterogeneity of EP antigen at the molecular level. To demonstrate that this 230-kD polypeptide is the major BP antigen and to further characterize it, we tested multiple BP

Stephan Mueller; Vera Klaus-Kovtun; John R. Stanley

1989-01-01

204

Evaluation of Mycobacterium tuberculosis Early Secreted Antigenic Target 6 Recombinant Protein as a Diagnostic Marker in Skin Test  

PubMed Central

Objectives Tuberculosis (TB) is the leading infectious disease in the developing world. Delayed-type hypersensitivity skin test diagnoses TB using tuberculin purified protein derivative (PPD), but this test is incapable of distinguishing Mycobacterium tuberculosis (MTB) infection from bacillus Calmette–Guérin (BCG) vaccination or an infection caused by nontuberculous mycobacteria (NTM). This study was performed to evaluate the use of recombinant early secretory antigenic target 6 (rESAT-6), a secretory protein found only in MTB, Mycobacterium bovis, and few other mycobacterial species, as a skin marker for MTB in guinea pigs. Methods We prepared recombinant MTB ESAT-6 and evaluated its use as a specific antigen for MTB in guinea pigs. Results Our results show that the purified MTB rESAT-6 antigen is capable of inducing a positive reaction only in guinea pigs sensitized to MTB. No such reaction was observed in the animals sensitized to M. bovis, BCG vaccination, or NTM (Mycobacterium avium). Conclusion Our study results confirm that the ESAT-6 antigen is more specific to MTB infection than PPD and could be used in more specific skin tests for detection of MTB in large animals and in humans. PMID:25737829

Moradi, Jale; Mosavari, Nader; Ebrahimi, Mahmoud; Arefpajohi, Reza; Tebianian, Majid

2014-01-01

205

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

PubMed

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

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

2010-07-01

206

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

207

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

208

Elastin, a novel extracellular matrix protein adhering to mycobacterial antigen 85 complex.  

PubMed

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 (K(D) = 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

2013-02-01

209

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

PubMed Central

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

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

2011-01-01

210

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

E-print Network

and Its Complex with Peptide Antigen Nora A. Linderoth, Martha N. Simon,§ Natalia A. Rodionova, Martine ReceiVed October 4, 2000 ABSTRACT: Animals vaccinated with heat shock protein (HSP)-peptide complexes. This autologous specific immunity has been demonstrated using a number of HSP-peptide antigen complexes

Chait, Brian T.

211

Human CD8+ herpes simplex virus-specific cytotoxic T-lymphocyte clones recognize diverse virion protein antigens.  

PubMed Central

The role of the HLA class I-restricted, CD8+, herpes simplex virus (HSV)-specific cytotoxic T lymphocytes (CTL) in the control of human HSV infections is controversial because previous reports suggest that a substantial portion of the antigen-specific lytic response is mediated by CD4+ cells. To address this question directly, we isolated HSV-specific CD8+ CTL clones from a patient with recurrent genital herpes. These CTL were cloned by coculturing responder peripheral blood mononuclear cells (PBMC) with phytohemagglutinin-stimulated PBMC that had been infected with live HSV-2 and then irradiated prior to the addition of responder cells. After 1 week, CTL were cloned by limiting dilution using phytohemagglutinin stimulation and allogeneic feeder PBMC. Seven clones were isolated; all seven clones were CD8+ CD4- CD3+ DRbright, six lysed only HSV-2-infected targets, and one lysed both HSV-1- and HSV-2-infected targets. Antigen presentation was restricted by two to three different HLA class I loci. To determine the antigens recognized by these HSV-specific CTL, target cells were infected with HSV in the presence of acyclovir, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, or cycloheximide in a series of drug block/release protocols to limit the repertoire of viral gene expression to select transcriptional classes. Five of the clones exhibited a different pattern of cytotoxicity, suggesting that each recognized a distinct HSV antigen. One of the clones appears to be directed against an immediate-early antigen; six of the clones recognize virion proteins. Five of these clones recognized internal virion proteins that could be introduced into target cells by HSV infection in the absence of virus gene expression. Antigen specificity was further tested by using vaccinia virus vectors that express glycoproteins gD2 and gB2 or the tegument protein VP16. One clone lysed vaccinia virus/gD2-infected target cells; the remaining clones did not recognize any of these gene products. The diversity of the CD8+ response from a single individual indicated that several different antigens are recognized when presented in the context of a variety of class I HLA alleles, a pattern that markedly differs from that described for another human herpesvirus, cytomegalovirus. Images PMID:1310769

Tigges, M A; Koelle, D; Hartog, K; Sekulovich, R E; Corey, L; Burke, R L

1992-01-01

212

Simultaneous Purification of Murine Mammary Tumor Virus Structural Proteins: Analysis of Antigenic Reactivities of Native gp34 by Radioimmunocompetition Assays  

PubMed Central

All the structural proteins (gp47, gp34, p27, p23, p16, and p12) of the murine mammary tumor virus (MuMTV) were simultaneously purified utilizing alkylagarose chromatography as the initial fractionation step. Least-hydrophobic MuMTV polypeptides (p23, p16) and the slightly hydrophobic p27 were separated from moderately hydrophobic proteins gp47 and p12 by passage through octylimino (C8)-agarose; the gp47 and p12 could be removed from the matrix by elution with ethylene glycol, whereas the most hydrophobic MuMTV protein, gp34, was eluted using nonionic detergent together with ethylene glycol. Subsequent purification steps involved ion-exchange or gel filtration chromatography. The resulting protein preparations appeared near-homogeneous on analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Recoveries of MuMTV proteins, based on their approximate individual contribution to total virus protein, ranged from about 20% for gp47 to greater than 100% for the minor structural component p23, the major phosphoprotein of MuMTV. Antiserum against purified C3H MuMTV gp34, together with purified, radioiodinated gp34, was used to develop a radioimmunoassay which showed that from 13 to 14% of total MuMTV protein by weight is gp34. Using this assay system, the group-specific antigenic reactivity of gp34 was also demonstrated. When solubilized preparations of C3H, RIII, and GR MuMTV's were used as competing antigens in gp34 radioimmunoassays with anti-C3H MuMTV serum, both group- and type-specific differences in antigenic reactivity were found. Images PMID:90171

Marcus, Stuart L.; Kopelman, Rebecca; Sarkar, Nurul H.

1979-01-01

213

Identification and partial characterization of a novel bipartite protein antigen associated with the outer membrane of Escherichia coli.  

PubMed Central

A study by crossed immunoelectrophoresis performed in conjunction with precipitate excision and polypeptide analysis identified a new antigen complex in the envelope of Escherichia coli ML308-225. This antigen corresponds to antigen 43 in the crossed immunoelectrophoresis profile of membrane vesicles (P. Owen and H. R. Kaback, Proc. Natl. Acad. Sci. USA 75:3148-3152, 1978). Immunoprecipitation experiments conducted with specific antiserum revealed that the complex was expressed on the cell surface and that it contained, in equal stoichiometry, two chemically distinct polypeptides termed alpha and beta (Mrs of 60,000 and 53,000, respectively). The beta polypeptide was heat modifiable, displaying an apparent Mr of 37,000 when solubilized at temperatures below 70 degrees C. Analysis of fractions obtained following cell disruption, isopycnic centrifugation, and detergent extraction indicated that both alpha and beta polypeptides were components of the outer membrane. The two polypeptides were not linked by disulfide bonds, and neither was peptidoglycan associated. The complex contained no detectable lipopolysaccharide, enzyme activity, fatty acyl groups, or other cofactors. Neither correlated with E. coli proteins of similar molecular weight which had previously been shown to be associated with the outer membrane. Antibodies were raised to individual alpha and beta polypeptides. Each of these sera was shown to be subunit specific when tested against denatured membrane proteins. In contrast, each immunoglobulin preparation coprecipitated both alpha and beta polypeptides when tested against undenatured proteins derived from Triton X-100-treated membranes. The results reveal the presence of a novel bipartite protein antigen in the outer membrane of E. coli. Images PMID:3301815

Owen, P; Caffrey, P; Josefsson, L G

1987-01-01

214

The Babesia bovis Merozoite Surface Antigen 2 Locus Contains Four Tandemly Arranged and Expressed Genes Encoding Immunologically Distinct Proteins  

Microsoft Academic Search

Members of the variable merozoite surface antigen (vmsa) gene family of Babesia bovis encode membrane proteins involved in erythrocyte invasion. In this study, we have identified and sequenced the complete 8.3-kb genomic locus containing msa-2, a member of the vmsa family, in the biologically cloned Mexico Mo7 strain. Four tandemly arranged copies of msa-2-related genes were found in the locus.

Monica Florin-Christensen; Carlos E. Suarez; Stephen A. Hines; Guy H. Palmer; Wendy C. Brown; Terry F. McElwain

2002-01-01

215

CLONING AND EXPRESSION OF AN ANTIGENIC DOMAIN OF A MAJOR SURFACE PROTEIN (NCP43) OF Neospora caninum  

Microsoft Academic Search

LIMA JUNIOR, M.S.DA C.; ANDREOTTI, R.; CAETANO, A.R.; PAIVA, F.; MATOS, M. DE F.C. (Cloning and expression of an antigenic domain of a major surface protein (Nc-p43) of Neospora caninum.) Clonagem e expressão de um domínio antigênico da proteína majoritária de superfície (Nc-p43) de Neospora caninum. Revista Brasileira de Parasitologia Veterinária, v. 16, n. 2, p. 61-66, 2007. Embrapa Gado

MANOEL S. DA; C. LIMA; RENATO ANDREOTTI; ALEXANDRE R. CAETANO; FERNANDO PAIVA; MARIA DE FÁTIMA C. MATOS

216

Immunohistochemical analysis of the cell cycle-associated antigens Ki67 and retinoblastoma protein in parathyroid carcinomas and adenomas  

Microsoft Academic Search

The morphologic distinction between parathyroid carcinoma and adenoma can be a difficult diagnostic problem. We analyzed nuclear\\u000a immunoreactivity for the cell cycle-associated antigen Ki-67 with monoclonal antibody (MAb) MIB-1 and for retinoblastoma (RB)\\u000a protein with two polyclonal antisera in 24 parathyroid carcinomas and 35 adenomas, which were formalin fixed and paraffin\\u000a embedded to determine if these antibodies could assist in

Ricardo V. Lloyd; J. Aidan Carney; Jorge A. Ferreiro; Long Jin; Geoffrey B. Thompson; Jon A. van Heerden; Clive S. Grant; Peter C. Wollan

1995-01-01

217

Comparison of antigenic proteins from Lactococcus garvieae KG (?) and KG (+) strains that are recognized by olive flounder ( Paralichthys olivaceus) antibodies  

Microsoft Academic Search

Lactococcus garvieae is an important etiological agent of lactococcosis in various fish species including olive flounder (Paralichthys olivaceus). In this study, proteomic and immunoproteomic analyses were employed to compare the antigenic profiles of strains KG9408, MS93003, and NSS9310 strains of L. garvieae. Proteomic analysis using two-dimensional gel electrophoresis (2-DE) revealed differences in five protein spots among the different L. garvieae

Gee-Wook Shin; Seong-Won Nho; Seong-Bin Park; Ho-Bin Jang; In-Seok Cha; Mi-Ae Ha; Young-Rim Kim; Rishikesh S. Dalvi; Seong-Joon Joh; Tae-Sung Jung

2009-01-01

218

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

PubMed Central

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

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

2008-01-01

219

Local and systemic immune responses induced by a recombinant chimeric protein containing Mycoplasma hyopneumoniae antigens fused to the B subunit of Escherichia coli heat-labile enterotoxin LTB.  

PubMed

A multi-antigen chimera composed of three antigens of Mycoplasma hyopneumoniae (R1, P42, and NrdF) and the mucosal adjuvant Escherichia coli heat-labile enterotoxin B subunit (LTB) was constructed, and its antigenic and immunogenic properties were evaluated in mice and pigs. In addition, we compared the effect of the fusion and co-administration of these proteins in mice. Antibodies against each subunit recognized the chimeric protein. Intranasal and intramuscular immunization of mice with the chimeric protein significantly increased IgG and IgA levels in the serum and tracheobronchial lavages, respectively, against some of the antigens present in the chimeric. Swine immunized with the chimeric protein developed an immune response against all M. hyopneumoniae antigens present in the fusion with a statistically significant difference (P<0.05). The adjuvant rLTB enhanced the immune response in both fused and co-administered antigens; however, better results were obtained with the chimeric protein. This multi-antigen is a promising vaccine candidate that may help control M. hyopneumoniae infection. PMID:25091529

Marchioro, Silvana Beutinger; Fisch, Andressa; Gomes, Charles K; Jorge, Sérgio; Galli, Vanessa; Haesebrouck, Freddy; Maes, Dominiek; Dellagostin, Odir; Conceição, Fabricio R

2014-09-17

220

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

PubMed Central

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

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

2005-01-01

221

The Major Cicatricial Pemphigoid Antigen Is a 180-kD Protein that Shows Immunologic Cross-Reactivities with the Bullous Pemphigoid Antigen  

Microsoft Academic Search

Recent studies have shown that sera from patients with cicatricial pemphigoid (CP) contained autoantibodies against epidermal antigens of molecular weight 230 kD and\\/or 180 kD by immunoblotting, similar to those recognized by bullous pemphigoid (BP) sera. Previous immunoprecipitation studies have shown that BP sera only precipitated the 230-kD antigen. To characterize the CP antigen(s) we tested 10 CP sera, 10

Philippe Bernard; Catherine Prost; Nathalie Durepaire; Nicole Basset-Seguin; Liliane Didierjean; Jean-Hilaire Saurat

1992-01-01

222

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

223

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

PubMed Central

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

1996-01-01

224

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

225

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

Børud, Bente; Aas, Finn Erik; Vik, Åshild; Winther-Larsen, Hanne C.; Egge-Jacobsen, Wolfgang; Koomey, Michael

2010-01-01

226

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

PubMed

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

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

2013-10-01

227

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

PubMed Central

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

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

2012-01-01

228

Peptide Inhibitors of the Malaria Surface Protein, Apical Membrane Antigen 1: Identification of Key Binding Residues  

PubMed Central

Apical membrane antigen 1 (AMA1) is essential for malaria parasite invasion of erythrocytes and is therefore an attractive target for drug development. Peptides that bind AMA1 have been identified from random peptide libraries expressed on the surface of phage. Of these, R1, which binds to a hydrophobic ligand binding site on AMA1, was a particularly potent inhibitor of parasite invasion of erythrocytes in vitro. The solution structure of R1 contains a turn-like conformation between residues 5–10. Here the importance of residues in this turn-like structure for binding to AMA1 was examined by site-directed mutagenesis and NMR spectroscopy. The peptide was expressed as a fusion protein following replacement of Met16 by Leu in order to accommodate cyanogen bromide cleavage. This modified peptide (R2) displayed the same affinity for AMA1 as R1, showing that the identity of the side chain at position 16 was not critical for binding. Substitution of Phe5, Pro7, Leu8, and Phe9 with alanine led to significant (7.5- to > 350-fold) decreases in affinity for AMA1. Comparison of backbone amide and C?H chemical shifts for these R2 analogues with corresponding values for R2 showed no significant changes, with the exception of R2(P7A), where slightly larger differences were observed, particularly for residues flanking position 7. The absence of significant changes in the secondary chemical shifts suggests that these mutations had little effect on the solution conformation of R2. The identification of a non-polar region of these peptides containing residues essential for AMA1 binding establishes a basis for the design of anti-malarial drugs based on R1 mimetics. PMID:21213258

Lee, Erinna F.; Yao, Shenggen; Sabo, Jennifer K.; Fairlie, W. Douglas; Stevenson, Rachel A.; Harris, Karen S.; Anders, Robin F.; Foley, Michael; Norton, Raymond S.

2011-01-01

229

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

230

Identification of an immunodominant antigenic site involving the capsid protein VP3 of hepatitis A virus.  

PubMed Central

Hepatitis A virus, an hepatotropic picornavirus, is a common cause of acute hepatitis in man for which there is no available vaccine. Competitive binding studies carried out in solid phase suggest that neutralizing monoclonal antibodies to hepatitis A virus recognize a limited number of epitopes on the capsid surface, although the polypeptide locations of these epitopes are not well defined. Neutralization-escape mutants, selected for resistance to monoclonal antibodies, demonstrate broad cross-resistance to other monoclonal antibodies. Sequencing of virion RNA from several of these mutants demonstrated that replacement of aspartic acid residue 70 of capsid protein VP3 (residue 3070) with histidine or alanine confers resistance to neutralization by monoclonal antibody K2-4F2 and prevents binding of this antibody and other antibodies with similar solid-phase competition profiles. These results indicate that residue 3070 contributes to an immunodominant antigenic site. Mutation at residue 102 of VP1 (residue 1102) confers partial resistance against antibody B5-B3 and several other antibodies but does not prevent antibody attachment. Both VP3 and VP1 sites align closely in the linear peptide sequences with sites of neutralization-escape mutations in poliovirus and human rhinovirus, suggesting conservation of structure among these diverse picornaviruses. However, because partial neutralization resistance to several monoclonal antibodies (2D2, 3E1, and B5-B3) was associated with mutation at either residue 3070 or residue 1102, these sites appear more closely related functionally in hepatitis A virus than in these other picornaviruses. PMID:2460866

Ping, L H; Jansen, R W; Stapleton, J T; Cohen, J I; Lemon, S M

1988-01-01

231

Antigenic domains of the streptococcal Pep M5 protein. Localization of epitopes crossreactive with type 6 M protein and identification of a hypervariable region of the M molecule  

PubMed Central

Pep M5, the pepsin-derived N-terminal half of the group A streptococcal type 5 M protein exhibits immunologic crossreaction with type 6 M protein, localizing some of the M6-crossreactive epitope(s) within this segment of the M5 protein. Based on the amino acid sequence of the Pep M5 protein, two structurally distinct domains have been recognized within its coiled-coil structure. We have now found that peptides derived from both the structurally distinct domains of the Pep M5 protein contain antigenic epitopes. Furthermore, only the peptides from the C-terminal domain of the Pep M5 protein crossreacted with rabbit anti-M6 sera, whereas those from the N-terminal domain did not. Consistent with this, sequence analyses of the arginyl peptides of the Pep M6 protein, the pepsin-derived N-terminal half of the M6 protein, revealed extensive homology of some of these peptides with regions within the C-terminal domain of the Pep M5 molecule. While an arginyl peptide of the Pep M6 protein exhibits 84% homology with region 150-186 of the Pep M5 protein, the C-terminal hexadecapeptide of the Pep M6 protein is virtually identical with the corresponding region of the Pep M5 protein. These results are suggestive of conformational similarities in the region around the pepsin-susceptible site within the M5 and M6 proteins. In addition, one or more epitopes of the M5 protein that are crossreactive with the M6 protein may be placed close to the pepsin- susceptible site of the M5 protein. Previous studies have suggested the N-terminal half of the M proteins to be the variable part of the molecule among the different M protein serotypes. The present results suggest that the N-terminal quarter of the M protein may represent the hypervariable domain of the M molecule. PMID:2416864

1986-01-01

232

Antigen sequence typing of outer membrane protein (fetA) gene of Neisseria meningitidis serogroup A from Delhi & adjoining areas  

PubMed Central

Background & objectives: Meningitis caused by Neisseria meningitidis is a fatal disease. Meningococcal meningitis is an endemic disease in Delhi and irregular pattern of outbreaks has been reported in India. All these outbreaks were associated with serogroup A. Detailed molecular characterization of N. meningitidis is required for the management of this fatal disease. In this study, we characterized antigenic diversity of surface exposed outer membrane protein (OMP) FetA antigen of N. meningitidis serogroup A isolates obtained from cases of invasive meningococcal meningitis in Delhi, India. Methods: Eight isolates of N. meningitidis were collected from cerebrospinal fluid during October 2008 to May 2011 from occasional cases of meningococcal meningitis. Seven isolates were from outbreaks of meningococcal meningitis in 2005-2006 in Delhi and its adjoining areas. These were subjected to molecular typing of fetA gene, an outer membrane protein gene. Results: All 15 N. meningitides isolates studied were serogroup A. This surface exposed porin is putatively under immune pressure. Hence as a part of molecular characterization, genotyping was carried out to find out the diversity in outer membrane protein (FetA) gene among the circulating isolates of N. meningitidis. All 15 isolates proved to be of the same existing allele type of FetA variable region (VR) when matched with global database. The allele found was F3-1 for all the isolates. Interpretation & conclusions: There was no diversity reported in the outer membrane protein FetA in the present study and hence this protein appeared to be a stable molecule. More studies on molecular characterization of FetA antigen are required from different serogroups circulating in different parts of the world. PMID:25758575

Dwivedi, S.; Das, B.K.; Kapil, A.; Sood, S.; Chaudhry, R.; Gupta, S.; Deb, M.; Nair, D.; Aneja, S.

2014-01-01

233

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

Microsoft Academic Search

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

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

1987-01-01

234

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

PubMed

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

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

2005-07-01

235

The latency-associated nuclear antigen, a multifunctional protein central to Kaposi’s sarcoma-associated herpesvirus latency  

PubMed Central

Latency-associated nuclear antigen (LANA) is encoded by the Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) open reading frame 73. LANA is expressed during latent KSHV infection of cells, including tumor cells, such as primary effusion lymphoma, KS and multicentric Castleman’s disease. Latently infected cells have multiple extrachromosomal copies of covalently closed circular KSHV genomes (episomes) that are stably maintained in proliferating cells. LANA’s best characterized function is that of mediating episome persistence. It does so by binding terminal repeat sequences to the chromosomal matrix, thus ensuring episome replication with each cell division and efficient DNA segregation to daughter nuclei after mitosis. To achieve these functions, LANA associates with different host cell proteins, including chromatin-associated proteins and proteins involved in DNA replication. In addition to episome maintenance, LANA has transcriptional regulatory effects and affects cell growth. LANA exerts these functions through interactions with different cell proteins. PMID:22122438

Ballestas, Mary E; Kaye, Kenneth M

2013-01-01

236

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

237

A subgroup-specific antigenic site in the G protein of respiratory syncytial virus forms a disulfide-bonded loop.  

PubMed Central

An antigenic site (represented by 15 amino acids, residues 174 to 188, designated peptide 12) of the large glycoprotein G of respiratory syncytial virus was demonstrated to be subgroup specific in peptide enzyme-linked immunosorbent assay tests with murine monoclonal antibodies and human postinfection sera. The role of individual amino acids in this subgroup-specific site was determined by use of single-amino-acid-deletion sets of peptides. When monoclonal antibodies were reacted with the deletion sets, a broad amino acid dependence of 11 or 12 residues, Cys-176 (Ile-175 in subgroup B) to Cys-186, was found. Human postinfection sera exhibited a narrower reaction profile (for subgroup A, Cys-182 to Trp-183; for subgroup B, Cys-176 to Lys-183). Reduction of peptides on microtiter plates by treatment with dithiothreitol completely destroyed their antigenic activity in tests with monoclonal antibodies and human postinfection sera of subgroup B. A variant of peptide 12 containing all four cysteines of the G protein (represented by 16 amino acids, residues 172 to 187, designated peptide 12var) also was subgroup specific. We concluded that the activity of the antigenic site in tests with monoclonal antibodies for subgroups A and B appears to depend on intrapeptide disulfide bonds. Reactions with postinfection sera of subgroup B also may depend on a disulfide bond. In contrast, postinfection sera of subgroup A appeared to have the capacity to identify a subgroup-specific site in a linear form of the selected 15-amino-acid-long peptide. Treatment of peptides with dithiothreitol had no effect on their antigenic activity in tests with human postinfection sera of subgroup A. These findings have relevance for molecular engineering of peptide antigens for use in respiratory syncytial virus subgroup-specific site-directed serology. PMID:1697913

Akerlind-Stopner, B; Utter, G; Mufson, M A; Orvell, C; Lerner, R A; Norrby, E

1990-01-01

238

The BTB-kelch Protein KLHL6 Is Involved in B-Lymphocyte Antigen Receptor Signaling and Germinal Center Formation†  

PubMed Central

BTB-kelch proteins can elicit diverse biological functions but very little is known about the physiological role of these proteins in vivo. Kelch-like protein 6 (KLHL6) is a BTB-kelch protein with a lymphoid tissue-restricted expression pattern. In the B-lymphocyte lineage, KLHL6 is expressed throughout ontogeny, and KLHL6 expression is strongly upregulated in germinal center (GC) B cells. To analyze the role of KLHL6 in vivo, we have generated mouse mutants of KLHL6. Development of pro- and pre-B cells was normal but numbers of subsequent stages, transitional 1 and 2, and mature B cells were reduced in KLHL6-deficient mice. The antigen-dependent GC reaction was blunted (smaller GCs, reduced B-cell expansion, and reduced memory antibody response) in the absence of KLHL6. Comparison of mutants with global loss of KLHL6 to mutants lacking KLHL6 specifically in B cells demonstrated a B-cell-intrinsic requirement for KLHL6 expression. Finally, B-cell antigen receptor (BCR) cross-linking was less sensitive in KLHL6-deficient B cells compared to wild-type B cells as measured by proliferation, Ca2+ response, and activation of phospholipase C?2. Our results strongly point to a role for KLHL6 in BCR signal transduction and formation of the full germinal center response. PMID:16166635

Kroll, Jens; Shi, Xiaozhong; Caprioli, Arianna; Liu, Hong-Hsing; Waskow, Claudia; Lin, Keng-Mean; Miyazaki, Toru; Rodewald, Hans-Reimer; Sato, Thomas N.

2005-01-01

239

Characterization of the Babesia gibsoni 12-kDa protein as a potential antigen for the serodiagnosis.  

PubMed

A novel gene, BgP12, encoding a 12-kDa protein was identified from Babesia gibsoni. The full-length cDNA of BgP12 contains an open reading frame of 378 bp, corresponding to 126 amino acid (aa) residues consisting of a putative 26 aa signal peptide and a 100 aa mature protein. The recombinant BgP12 (rBgP12) lacking the N-terminal signal peptide was expressed in Escherichia coli as a soluble glutathione S-transferase (GST) fusion protein (rBgP12) that produced an anti-rBgP12 serum in mice after immunization. Using this anti-rBgP12 serum, a native 12-kDa protein in B. gibsoni was recognized by Western blot analysis. Immunofluorescent antibody tests (IFAT) revealed that BgP12 was mainly seen during the ring stage of B. gibsoni trophozoite. An indirect enzyme-linked immunosorbent assay (ELISA) using the rBgP12 detected specific antibodies in the sequential sera of a dog experimentally infected with B. gibsoni beginning 10 days post-infection to 442 days post-infection, even when the dog became chronically infected and showed a low level of parasitemia. Moreover, the antigen did not show cross-reaction with antibodies to the closely related apicomplexan parasites, indicating that the rBgP12 might be an immunodominant antigen for B. gibsoni infection that could be used as a diagnostic antigen for B. gibsoni infection with high specificity and sensitivity. PMID:19000776

Goo, Youn-Kyoung; Jia, Honglin; Aboge, G Oluga; Terkawi, M Alaa; Lee, Eung-goo; Yamagishi, Junya; Nishikawa, Yoshifumi; Jang, Hyung-Kwan; Sunaga, Fujiko; Namikawa, Kazuhiko; Fujisaki, Kozo; Xuan, Xuenan

2009-03-01

240

Comparison of colorimetric assays with quantitative amino acid analysis for protein quantification of Generalized Modules for Membrane Antigens (GMMA).  

PubMed

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

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

2015-01-01

241

Unrelated donor BMT recipients given pretransplant low-dose antithymocyte globulin have outcomes equivalent to matched sibling BMT: a matched pair analysis  

Microsoft Academic Search

Fifty-seven patients receiving unrelated donor (UD) BMT were matched for disease and stage with 57 recipients of genotypically matched related donor (MRD) BMT. All UD recipients were matched serologically for A and B and by high resolution for DR and DQ antigens. All patients received CsA and ‘short course’ methotrexate with folinic acid. Unrelated donor BMT patients also received thymoglobulin

P Duggan; K Booth; A Chaudhry; D Stewart; JD Ruether; S Glück; D Morris; CB Brown; B Herbut; M Coppes; R Anderson; J Wolff; M Egeler; S Desai; AR Turner; L Larratt; E Gyonyor; JA Russell

2002-01-01

242

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

PubMed Central

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

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

2013-01-01

243

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

PubMed

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

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

2011-01-14

244

Epstein-Barr Viral BNLF2a Protein Hijacks the Tail-anchored Protein Insertion Machinery to Block Antigen Processing by the Transport Complex TAP*  

PubMed Central

Virus-infected cells are eliminated by cytotoxic T lymphocytes, which recognize viral epitopes displayed on major histocompatibility complex class I molecules at the cell surface. Herpesviruses have evolved sophisticated strategies to escape this immune surveillance. During the lytic phase of EBV infection, the viral factor BNLF2a interferes with antigen processing by preventing peptide loading of major histocompatibility complex class I molecules. Here we reveal details of the inhibition mechanism of this EBV protein. We demonstrate that BNLF2a acts as a tail-anchored protein, exploiting the mammalian Asna-1/WRB (Get3/Get1) machinery for posttranslational insertion into the endoplasmic reticulum membrane, where it subsequently blocks antigen translocation by the transporter associated with antigen processing (TAP). BNLF2a binds directly to the core TAP complex arresting the ATP-binding cassette transporter in a transport-incompetent conformation. The inhibition mechanism of EBV BNLF2a is distinct and mutually exclusive of other viral TAP inhibitors. PMID:21984826

Wycisk, Agnes I.; Lin, Jiacheng; Loch, Sandra; Hobohm, Kathleen; Funke, Jessica; Wieneke, Ralph; Koch, Joachim; Skach, William R.; Mayerhofer, Peter U.; Tampé, Robert

2011-01-01

245

Epstein-Barr viral BNLF2a protein hijacks the tail-anchored protein insertion machinery to block antigen processing by the transport complex TAP.  

PubMed

Virus-infected cells are eliminated by cytotoxic T lymphocytes, which recognize viral epitopes displayed on major histocompatibility complex class I molecules at the cell surface. Herpesviruses have evolved sophisticated strategies to escape this immune surveillance. During the lytic phase of EBV infection, the viral factor BNLF2a interferes with antigen processing by preventing peptide loading of major histocompatibility complex class I molecules. Here we reveal details of the inhibition mechanism of this EBV protein. We demonstrate that BNLF2a acts as a tail-anchored protein, exploiting the mammalian Asna-1/WRB (Get3/Get1) machinery for posttranslational insertion into the endoplasmic reticulum membrane, where it subsequently blocks antigen translocation by the transporter associated with antigen processing (TAP). BNLF2a binds directly to the core TAP complex arresting the ATP-binding cassette transporter in a transport-incompetent conformation. The inhibition mechanism of EBV BNLF2a is distinct and mutually exclusive of other viral TAP inhibitors. PMID:21984826

Wycisk, Agnes I; Lin, Jiacheng; Loch, Sandra; Hobohm, Kathleen; Funke, Jessica; Wieneke, Ralph; Koch, Joachim; Skach, William R; Mayerhofer, Peter U; Tampé, Robert

2011-12-01

246

Studies of Plasmodium falciparum rhoptry-associated membrane antigen (RAMA) protein peptides specifically binding to human RBC.  

PubMed

Plasmodium falciparum rhoptry-associated membrane antigen (RAMA) peptides used in normal red blood cell (RBC) binding assays revealed that peptides 33426 (79NINILSSVHRKGRILYDSF97) and 33460 (777HKKREKSISPHSYQKVSTKVQ797) bound with high activity, presenting nanomolar affinity constants. Such high binding activity peptides (HABPs) displayed helicoid and random coil structures as determined by circular dichroism. HABPs inhibited P. falciparumin vitro invasion of normal RBC by up to 61% (depending on concentration), suggesting that some RAMA protein regions could be involved in P. falciparum invasion of RBC. The nature and localisation of receptors on RBC surface responsible for HABP binding were studied using enzyme-treated erythrocytes and structural analysis. PMID:18191882

Pinzón, Carlos Giovanni; Curtidor, Hernando; Bermúdez, Adriana; Forero, Martha; Vanegas, Magnolia; Rodríguez, Jorge; Patarroyo, Manuel E

2008-02-01

247

Protection against Plasmodium chabaudi Malaria Induced by Immunization with Apical Membrane Antigen 1 and Merozoite Surface Protein 1 in the Absence of Gamma Interferon or Interleukin4  

Microsoft Academic Search

Strategies to optimize formulations of multisubunit malaria vaccines require a basic knowledge of under- lying protective immune mechanisms induced by each vaccine component. In the present study, we evaluated the contribution of antibody-mediated and cell-mediated immune mechanisms to the protection induced by immunization with two blood-stage malaria vaccine candidate antigens, apical membrane antigen 1 (AMA-1) and merozoite surface protein 1

James M. Burns; P. R. Flaherty; P. Nanavati; W. P. Weidanz

2004-01-01

248

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

PubMed Central

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

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

2014-01-01

249

Detergent pretreatment of solid phase globular proteins in ELISA`s. Enhanced antigenicity and subsequent sensitivity. Final report, September 1989-September 1991  

SciTech Connect

Methods for pretreatment and rejuvenation of preimmobilized globular proteins used in immunodiagnostics were investigated using reagents routinely used in ELISA`s. Rabbit and goat gamma globulins, functioning as antigens, and antibodies on non-covalent, and covalent solid surfaces, were monitored for detergent mediated desorption, denaturation, non-specific binding and altered antigenicity. The results from fourteen commercially supplied polyvinyl- and polystyrene-derivatized microtiter plates coated with antibody or antigenic lgG were compared with commercial microtiter diagnostic plates with preimmobilized lgG. Wash solutions had no effect on immobilized gamma globulins when the solid phase protein functioned as an antibody on covalent or noncovalent surfaces. In addition to tween 20 removing up to 50% of noncovalently bound protein additional binding sites are apparently exposed on solid phase antigens, evident by an increase in signal, which cannot be explained by nonspecific binding. However, no increase in signal was evident when antigen was preimmobilized covalently. The role of between 20 and other reagent components in ELISA-based assays are explored. The screening of noncovalent preimmobilized antigen coated surfaces prior to use for deteraent mediated enhancement is suggested.

Blanchard, G.C.; Bouhmadouche, M.; Williamson, M.L.

1994-10-01

250

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

251

The human immunodeficiency virus antigen Nef forms protein bodies in leaves of transgenic tobacco when fused to zeolin  

PubMed Central

Protein bodies (PB) are stable polymers naturally formed by certain seed storage proteins within the endoplasmic reticulum (ER). The human immunodeficiency virus negative factor (Nef) protein, a potential antigen for the development of an anti-viral vaccine, is highly unstable when introduced into the plant secretory pathway, probably because of folding defects in the ER environment. The aim of this study was to promote the formation of Nef-containing PB in tobacco (Nicotiana tabacum) leaves by fusing the Nef sequence to the N-terminal domains of the maize storage protein ?-zein or to the chimeric protein zeolin (which efficiently forms PB and is composed of the vacuolar storage protein phaseolin fused to the N-terminal domains of ?-zein). Protein blots and pulse–chase indicate that fusions between Nef and the same ?-zein domains present in zeolin are degraded by ER quality control. Consistently, a mutated zeolin, in which wild-type phaseolin was substituted with a defective version known to be degraded by ER quality control, is unstable in plant cells. Fusion of Nef to the entire zeolin sequence instead allows the formation of PB detectable by electron microscopy and subcellular fractionation, leading to zeolin–Nef accumulation higher than 1% of total soluble protein, consistently reproduced in independent transgenic plants. It is concluded that zeolin, but not its ?-zein portion, has a positive dominant effect over ER quality control degradation. These results provide insights into the requirements for PB formation and avoidance of quality-control degradation, and indicate a strategy for enhancing foreign protein accumulation in plants. PMID:18540021

de Virgilio, Maddalena; Bellucci, Michele; Mainieri, Davide; Rossi, Marika; Benvenuto, Eugenio; Arcioni, Sergio; Vitale, Alessandro

2008-01-01

252

The Major Antigenic Membrane Protein of “Candidatus Phytoplasma asteris” Selectively Interacts with ATP Synthase and Actin of Leafhopper Vectors  

PubMed Central

Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of “Candidatus Phytoplasma asteris”, the chrysanthemum yellows phytoplasmas (CYP) strain, and three others as non-vectors. Interactions between a labelled (recombinant) CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the ? and ? subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity. PMID:21799902

Galetto, Luciana; Bosco, Domenico; Balestrini, Raffaella; Genre, Andrea; Fletcher, Jacqueline; Marzachì, Cristina

2011-01-01

253

Concomitant progressive multifocal leucoencephalopathy and primary central nervous system lymphoma expressing JC virus oncogenic protein, large T antigen  

PubMed Central

This report describes the concomitant occurrence of the JC virus (JCV) induced demyelinating disease progressive multifocal leucoencephalopathy (PML) and a primary central nervous system lymphoma (PCNS-L) in a patient with AIDS. Postmortem neuropathological examination revealed characteristic features of PML including multiple lesions of demyelination, enlarged oligodendrocytes with hyperchromatic nuclei (many containing eosinophilic intranuclear inclusions), and enlarged astrocytes with bizarre hyperchromatic nuclei. Immunohistochemical analysis demonstrated the expression of the JCV capsid protein VP-1 in the nuclei of infected oligodendrocytes and astrocytes. The PCNS-L lesion located in the basal ganglia was highly cellular, distributed perivascularly, and consisted of large atypical plasmacytoid lymphocytes. Immunohistochemical examination of this neoplasm identified it to be of B cell origin. Moreover, expression of the JCV oncogenic protein, T antigen, was detected in the nuclei of the neoplastic lymphocytes. This study provides the first evidence for a possible association between JCV and PCNS-L. PMID:11577180

Gallia, G L; Del Valle, L; Laine, C; Curtis, M; Khalili, K

2001-01-01

254

Analysis of immune responses directed toward a recombinant early secretory antigenic target six-kilodalton protein-culture filtrate protein 10 fusion protein in Mycobacterium bovis-infected cattle.  

PubMed

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

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

2005-10-01

255

Identification of regions in polyomavirus middle T and small t antigens important for association with protein phosphatase 2A.  

PubMed Central

Two subunits of protein phosphatase 2A (PP2A) have been shown previously to bind to the small t and middle T antigens (ST and MT, respectively) of polyomavirus. To determine sequences important for binding of PP2A to ST and MT, we first constructed a series of ST mutants in regions known to be important for biological activity of ST and MT. Several mutations in two small regions just amino terminal to the Cys-X-Cys-X-X-Cys motifs of ST and MT abolished PP2A binding to ST in vitro. Parallel mutations were constructed in MT to investigate the role of PP2A binding in the function of polyomavirus MT. Wild-type and mutant MT proteins were stably expressed in NIH 3T3 cells and analyzed (i) for their ability to induce transformation and (ii) for associated cellular proteins and corresponding enzymatic activities previously described as associating with wild-type MT. A number of the mutant MTs were found to be defective in binding of PP2A as assayed by coimmunoprecipitation. In contrast, a deletion of the highly conserved stretch of amino acids 42 to 47 (His-Pro-Asp-Lys-Gly-Gly) in the ST-MT-large T antigen common region did not affect PP2A binding to MT. MT mutants defective for PP2A binding were also defective in transformation, providing further evidence that association with PP2A is important for the ability of MT to transform cells. All mutants which were impaired for PP2A binding were similarly or more dramatically impaired for associated protein and lipid kinase activities, supporting the possibility that PP2A binding is necessary for the formation and/or stability of an MT-pp60c-src complex. PMID:7538174

Campbell, K S; Auger, K R; Hemmings, B A; Roberts, T M; Pallas, D C

1995-01-01

256

Induction of Antigen-Specific Class I-Restricted Cytotoxic T Cells by Soluble Proteins in vivo  

Microsoft Academic Search

Cytotoxic T lymphocytes (CTL) are induced specifically against viral and tumor antigens presented by major histocompatibility complex class I molecules on the surface of infected or transformed cells. Intracellularly synthesized antigens are processed and associated with class I antigens within cells before presentation on the cell surface. Because of this special requirement for CTL induction, exogenous soluble antigens do not,

Syamal Raychaudhuri; Michelle Tonks; Frank Carbone; Thomas Ryskamp; W. John W. Morrow; Nabil Hanna

1992-01-01

257

The cellular proteins which can associate specifically with polyomavirus middle T antigen in human 293 cells include the major human 70-kilodalton heat shock proteins.  

PubMed Central

We compared the proteins which associate with middle T antigen (MT) of polyomavirus in human cells infected with Ad5(pymT), a recombinant adenovirus which directs the overexpression of MT, with the MT-associated proteins (MTAPs) previously identified in murine fibroblasts expressing MT. MTAPs of 27, 29, 36, and 63 kilodaltons (kDa) appeared to be fairly well conserved between the two species, as judged by comigration on two-dimensional gels. Several 61-kDa MTAP species detected in MT immunoprecipitates from both cell sources also comigrated on these gels. However, no protein comigrating precisely with the murine 85-kDa MTAP could be detected in the human cells. Furthermore, two proteins of 72 and 74 kDa associated with wild-type MT in the infected human cells but not in murine fibroblasts expressing MT. It had been previously reported for murine cells that the 70-kDa heat shock protein associates with a particular mutant MT but not with wild-type MT (G. Walter, A. Carbone, and W.J. Welch, J. Virol. 61:405-410, 1987). By the criteria of comigration on two-dimensional gels, tryptic peptide mapping, and immunoblotting, we showed that the 72- and 74-kDa proteins that associate with wild-type MT in human cells are the major human 70-kDa heat shock proteins. Images PMID:2795710

Pallas, D C; Morgan, W; Roberts, T M

1989-01-01

258

Particle-based transcutaneous administration of HIV-1 p24 protein to human skin explants and targeting of epidermal antigen presenting cells.  

PubMed

Transcutaneous immunization is a promising vaccination strategy for the treatment of infectious diseases and cancer. In this study, we investigate the combination of cyanoacrylate skin surface stripping (CSSS) and particle-based antigen delivery to target the HIV-1 p24 protein to skin antigen presenting cells (APC). The CSSS treatment pre-activates skin APC and opens hair follicles, where protein-loaded particles accumulate and allow for sustained delivery of the loaded antigen to perifollicular APC. We found that poly-lactic acid (PLA) and polystyrene (PS) particles targeted the adsorbed HIV-1 p24 protein to the hair follicles. Small amounts of PS and PLA particles were found to translocate to the epidermis and be internalized by skin cells, whereas most of the particles aggregated in the hair follicle canal, where they released the loaded antigen. The p24 protein diffused to the epidermis and dermis and was detected in skin cells, especially in Langerhans cells and dermal dendritic cells. Furthermore, the combination of CSSS and particle-based delivery resulted in activation and maturation of Langerhans cells (HLA-DR, CD80 and CD83). We conclude that particle-based antigen delivery across partially disrupted skin barrier is a feasible and effective approach to needle-free transcutaneous vaccination. PMID:24384300

Rancan, Fiorenza; Amselgruber, Sarah; Hadam, Sabrina; Munier, Sevérine; Pavot, Vincent; Verrier, Bernard; Hackbarth, Steffen; Combadiere, Behazine; Blume-Peytavi, Ulrike; Vogt, Annika

2014-02-28

259

An Enterococcus faecium Secreted Antigen, SagA, Exhibits Broad-Spectrum Binding to Extracellular Matrix Proteins and Appears Essential for E. faecium Growth  

Microsoft Academic Search

A gene encoding a major secreted antigen, SagA, was identified in Enterococcus faecium by screening an E. faecium genomic expression library with sera from patients with E. faecium-associated endocarditis. Recom- binant SagA protein showed broad-spectrum binding to extracellular matrix (ECM) proteins, including fibrinogen, collagen type I, collagen type IV, fibronectin, and laminin. A fibrinogen-binding protein, purified from culture supernatants of

Fang Teng; Magdalena Kawalec; George M. Weinstock; Waleria Hryniewicz; Barbara E. Murray

2003-01-01

260

Screening of 71 P. multocida Proteins for Protective Efficacy in a Fowl Cholera Infection Model and Characterization of the Protective Antigen PlpE  

Microsoft Academic Search

BackgroundThere is a strong need for a recombinant subunit vaccine against fowl cholera. We used a reverse vaccinology approach to identify putative secreted or cell surface associated P. multocida proteins that may represent potential vaccine candidate antigens.Principal FindingsA high-throughput cloning and expression protocol was used to express and purify 71 recombinant proteins for vaccine trials. Of the 71 proteins tested,

Tamás Hatfaludi; Keith Al-Hasani; Lan Gong; John D. Boyce; Mark Ford; Ian W. Wilkie; Noelene Quinsey; Michelle A. Dunstone; David E. Hoke; Ben Adler

2012-01-01

261

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

262

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...

263

Structural and Antigenic Properties of Merozoite Surface Protein 4 of Plasmodium falciparum  

Microsoft Academic Search

Merozoite surface protein 4 (MSP4) of Plasmodium falciparum is a glycosylphosphatidylinositol-anchored integral membrane protein of 272 residues that possesses a single epidermal growth factor (EGF)-like domain near the carboxyl terminus. We have expressed both full-length MSP4 and a number of fragments in Esche- richia coli and have used these recombinant proteins to raise experimental antisera. All recombinant proteins elicited specific

LINA WANG; CASILDA G. BLACK; VIKKI M. MARSHALL; ROSS L. COPPEL

1999-01-01

264

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

E-print Network

Abstract Antibodies against Escherichia coli-expressed uncou- pling protein-2 (UCP2) and uncoupling protein reacted more intensively with the recombinant UCP2 and UCP3 than with uncoupling protein-1 (UCP1) isolated from brown adipose tissue. Moreover, anti- UCP2 and cross-reacting anti-UCP3 antibodies identified

Garlid, Keith

265

The interaction of C-reactive protein and serum amyloid P component with nuclear antigens  

Microsoft Academic Search

The pentraxins are a family of proteins characterized by cyclic pentameric structure, calcium-dependent ligand binding and sequence homology. The two main representatives of this family are the serum proteins, C-reactive protein (CRP) and serum amyloid P component (SAP). In man CRP is an acute phase reactant which increases up to 1000 fold during the acute phase response whereas SAP is

Terry W. Clos

1996-01-01

266

Rapid "tea-bag" peptide synthesis using 9-fluorenylmethoxycarbonyl (Fmoc) protected amino acids applied for antigenic mapping of viral proteins.  

PubMed

The role of individual amino acids in binding human and macaque antibodies were determined in the human immunodeficiency virus type 1 (HIV-1) gp41, residues 594-613, and for human antibodies in the hepatitis B (HB) virus core/e antigens (HBc/eAg), residues 121-140. Decapeptides with 9 amino acids (aa) overlap were synthesised using a rapid method for simultaneous multiple peptide synthesis with 9-fluorenylmethoxycarbonyl (Fmoc) protection for the alpha-amino group of the aas. One coupling cycle including washing steps was performed within 60-90 min. The crude products were analysed by reversed-phase HPLC and PD-mass spectrometry. With the 11 decapeptides covering residues 594-613 of HIV-1 gp41, the sequences SGKLI at aa 599-603 was found to be the main recognition site for 19 human anti-HIV positive sera. Two macaques repeatedly immunized with a peptide covering aa 594-613 of gp41, preferentially recognised the sequence CTTAVPW at residues 604-610 after 1-2 months of immunisation. One macaque also recognised the sequence CSGKLI, with sera sampled greater than 10 months after start of immunisation. Out of 9 human sera from patients with chronic HB, and reactive to a peptide covering residues 121-140 of HBc/eAg, 8 were found to recognise the sequence TPPA at residues 128-131, with an individual variation within residues 125-133 in regard to N- and C-terminal ends of the recognised antigenic site. Thus, human recognition of this antigenic site overlaps the reported T- and the B-cell recognition site found in mice. We believe that this simple and rapid approach to obtain large numbers of immunologically active peptides can be useful for most laboratories interested in the immunological characterisation of proteins. PMID:1720419

Sällberg, M; Rudén, U; Magnius, L O; Norrby, E; Wahren, B

1991-09-01

267

Oxidation by Neutrophils-Derived HOCl Increases Immunogenicity of Proteins by Converting Them into Ligands of Several Endocytic Receptors Involved in Antigen Uptake by Dendritic Cells and Macrophages  

PubMed Central

The initiation of adaptive immune responses to protein antigens has to be preceded by their uptake by antigen presenting cells and intracellular proteolytic processing. Paradoxically, endocytic receptors involved in antigen uptake do not bind the majority of proteins, which may be the main reason why purified proteins stimulate at most weak immune responses. A shared feature of different types of adjuvants, capable of boosting immunogenicity of protein vaccines, is their ability to induce acute inflammation, characterized by early influx of activated neutrophils. Neutrophils are also rapidly recruited to sites of tissue injury or infection. These cells are the source of potent oxidants, including hypochlorous acid (HOCl), causing oxidation of proteins present in inflammatory foci. We demonstrate that oxidation of proteins by endogenous, neutrophils-derived HOCl increases their immunogenicity. Upon oxidation, different, randomly chosen simple proteins (yeast alcohol dehydrogenase, human and bovine serum albumin) and glycoproteins (human apo-transferrin, ovalbumin) gain the ability to bind with high affinity to several endocytic receptors on antigen presenting cells, which seems to be the major mechanism of their increased immunogenicity. The mannose receptor (CD206), scavenger receptors A (CD204) and CD36 were responsible for the uptake and presentation of HOCl-modified proteins by murine dendritic cells and macrophages. Other scavenger receptors, SREC-I and LOX-1, as well as RAGE were also able to bind HOCl-modified proteins, but they did not contribute significantly to these ligands uptake by dendritic cells because they were either not expressed or exhibited preference for more heavily oxidised proteins. Our results indicate that oxidation by neutrophils-derived HOCl may be a physiological mechanism of conferring immunogenicity on proteins which in their native forms do not bind to endocytic receptors. This mechanism might enable the immune system to detect infections caused by pathogens not recognized by pattern recognition receptors. PMID:25849867

Biedro?, Rafa?; Konopi?ski, Maciej K.; Marcinkiewicz, Janusz; Józefowski, Szczepan

2015-01-01

268

Adult Schistosoma mansoni worms positively modulate soluble egg antigen-induced inflammatory hepatic granuloma formation in vivo. Stereological analysis and immunophenotyping of extracellular matrix proteins, adhesion molecules, and chemokines.  

PubMed Central

Synchronized liver granulomas were induced by injecting Sepharose beads to which SEA soluble egg antigen (SEA) or the concanavalin A binding fraction of SEA had been coupled into a mesenteric vein in naive, single-sex (35 days) and bisexually (28 days) Schistosoma mansoni-infected and Plasmodium berghei-immunized mice. Stereological analysis revealed that peak granuloma formation was already reached 8 days after injection in single-sex infected mice compared with 16 days in naive animals. No difference in granuloma formation between naive and P. berghei-immunized animals and between unisexually and bisexually S. mansoni-infected mice was observed. This suggests that the positive immunomodulatory effect on the granulomogenesis is worm specific and not likely to be due to arousal of the immune system by unrelated factors, nor is it influenced by the gender or degree of maturation of female worms. At all stages in time, the concanavalin A binding-fraction-induced granulomas reached only 65 to 70% of the volume of SEA-induced granulomas. Immunophenotyping of extracellular matrix proteins around deposited heads revealed that fibronectin was the dominant extracellular matrix protein and that also type I and IV collagen and laminin were deposited. Temporal analysis of the expression of the adhesion molecules ICAM-1, LFA-1, VLA-4, and VLA-6 was performed. Morphological evidence is presented for the role of adhesion molecules in the initiation and maintenance of hepatic granuloma formation. The chemokine monocyte chemoattractant protein-1 was expressed in the granuloma and in hepatic artery branches. From these data, it is concluded that adult S. mansoni worms positively modulate schistosomal hepatic granuloma formation in vivo. Adhesion molecules and chemokines play important roles in schistosomal granuloma formation. Images Figure 1 Figure 2 Figure 3 PMID:9176396

Jacobs, W.; Bogers, J.; Deelder, A.; Wéry, M.; Van Marck, E.

1997-01-01

269

Structural Analysis of the Synthetic Duffy Binding Protein (DBP) Antigen DEKnull Relevant for Plasmodium vivax Malaria Vaccine Design  

PubMed Central

The Plasmodium vivax vaccine candidate Duffy Binding Protein (DBP) is a protein necessary for P. vivax invasion of reticulocytes. The polymorphic nature of DBP induces strain-specific immune responses that pose unique challenges for vaccine development. DEKnull is a synthetic DBP based antigen that has been engineered through mutation to enhance induction of blocking inhibitory antibodies. We determined the x-ray crystal structure of DEKnull to identify if any conformational changes had occurred upon mutation. Computational and experimental analyses assessed immunogenicity differences between DBP and DEKnull epitopes. Functional binding assays with monoclonal antibodies were used to interrogate the available epitopes in DEKnull. We demonstrate that DEKnull is structurally similar to the parental Sal1 DBP. The DEKnull mutations do not cause peptide backbone shifts within the polymorphic loop, or at either the DBP dimerization interface or DARC receptor binding pockets, two important structurally conserved protective epitope motifs. All B-cell epitopes, except for the mutated DEK motif, are conserved between DEKnull and DBP. The DEKnull protein retains binding to conformationally dependent inhibitory antibodies. DEKnull is an iterative improvement of DBP as a vaccine candidate. DEKnull has reduced immunogenicity to polymorphic regions responsible for strain-specific immunity while retaining conserved protein folds necessary for induction of strain-transcending blocking inhibitory antibodies. PMID:25793371

Chen, Edwin; Salinas, Nichole D.; Ntumngia, Francis B.; Adams, John H.; Tolia, Niraj H.

2015-01-01

270

Structural Analysis of the Synthetic Duffy Binding Protein (DBP) Antigen DEKnull Relevant for Plasmodium vivax Malaria Vaccine Design.  

PubMed

The Plasmodium vivax vaccine candidate Duffy Binding Protein (DBP) is a protein necessary for P. vivax invasion of reticulocytes. The polymorphic nature of DBP induces strain-specific immune responses that pose unique challenges for vaccine development. DEKnull is a synthetic DBP based antigen that has been engineered through mutation to enhance induction of blocking inhibitory antibodies. We determined the x-ray crystal structure of DEKnull to identify if any conformational changes had occurred upon mutation. Computational and experimental analyses assessed immunogenicity differences between DBP and DEKnull epitopes. Functional binding assays with monoclonal antibodies were used to interrogate the available epitopes in DEKnull. We demonstrate that DEKnull is structurally similar to the parental Sal1 DBP. The DEKnull mutations do not cause peptide backbone shifts within the polymorphic loop, or at either the DBP dimerization interface or DARC receptor binding pockets, two important structurally conserved protective epitope motifs. All B-cell epitopes, except for the mutated DEK motif, are conserved between DEKnull and DBP. The DEKnull protein retains binding to conformationally dependent inhibitory antibodies. DEKnull is an iterative improvement of DBP as a vaccine candidate. DEKnull has reduced immunogenicity to polymorphic regions responsible for strain-specific immunity while retaining conserved protein folds necessary for induction of strain-transcending blocking inhibitory antibodies. PMID:25793371

Chen, Edwin; Salinas, Nichole D; Ntumngia, Francis B; Adams, John H; Tolia, Niraj H

2015-03-01

271

Cultured human Langerhans' cells are superior to fresh cells at presenting native HIV-1 protein antigens to specific CD4+ T-cell lines.  

PubMed Central

Cultured Langerhans' cells (CLC) exhibit enhanced antigen-presenting function compared to freshly isolated LC (FLC), but they are commonly believed to be inefficient at processing intact proteins. In this study, FLC and CLC from normal, human immunodeficiency virus (HIV) seronegative volunteers were compared for their ability to present the HIV-1 envelope glycoprotein gp120 or reverse transcriptase (p66) antigens to autologous, specific CD4+ T cell lines. Epidermal cell suspensions enriched for LC were prepared from suction blister roofs. FLC stimulated T cells at lower antigen concentrations compared to unfractionated peripheral blood mononuclear cells (PBMC). CLC were more potent on a per cell basis than FLC, PBMC or adherent monocytes at presenting native gp120, native p66 or immunogenic peptides. CLC were also more efficient than FLC or PBMC in terms of the amount of antigen required for T-cell activation. Chloroquine and leupeptin inhibited presentation of intact p66, but not of an immunodominant peptide, by FLC or CLC, thus indicating that both cells utilize antigen-processing mechanisms that are based on intracellular acidification and protease activity. Incubation of CLC with monoclonal antibodies against HLA-DR, CD11b, CD18, CD50, CD54, CD58 or CD80, but not anti-major histocompatibility complex class I (MHC-I), inhibited antigen-specific T-cell proliferation to varying degrees. We conclude that human CLC retain the ability to process and present protein antigens potently to CD4+ T cells. Thus, CLC have the capacity to participate actively in the generation and maintenance of T-helper cell immunity to viral antigens during HIV-1 infection. PMID:8698396

Girolomoni, G; Valle, M T; Zacchi, V; Costa, M G; Giannetti, A; Manca, F

1996-01-01

272

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

273

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

274

Pseudomonas aeruginosa Cif Protein Enhances the Ubiquitination and Proteasomal Degradation of the Transporter Associated with Antigen Processing (TAP) and Reduces Major Histocompatibility Complex (MHC) Class I Antigen Presentation*  

PubMed Central

Cif (PA2934), a bacterial virulence factor secreted in outer membrane vesicles by Pseudomonas aeruginosa, increases the ubiquitination and lysosomal degradation of some, but not all, plasma membrane ATP-binding cassette transporters (ABC), including the cystic fibrosis transmembrane conductance regulator and P-glycoprotein. The goal of this study was to determine whether Cif enhances the ubiquitination and degradation of the transporter associated with antigen processing (TAP1 and TAP2), members of the ABC transporter family that play an essential role in antigen presentation and intracellular pathogen clearance. Cif selectively increased the amount of ubiquitinated TAP1 and increased its degradation in the proteasome of human airway epithelial cells. This effect of Cif was mediated by reducing USP10 deubiquitinating activity, resulting in increased polyubiquitination and proteasomal degradation of TAP1. The reduction in TAP1 abundance decreased peptide antigen translocation into the endoplasmic reticulum, an effect that resulted in reduced antigen available to MHC class I molecules for presentation at the plasma membrane of airway epithelial cells and recognition by CD8+ T cells. Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation. PMID:24247241

Bomberger, Jennifer M.; Ely, Kenneth H.; Bangia, Naveen; Ye, Siying; Green, Kathy A.; Green, William R.; Enelow, Richard I.; Stanton, Bruce A.

2014-01-01

275

Molecular model of the A subunit of protein phosphatase 2A: interaction with other subunits and tumor antigens.  

PubMed Central

Protein phosphatase 2A consists of three subunits, the catalytic subunit (C) and two regulatory subunits (A and B). The A subunit has a rod-like shape and consists of 15 nonidentical repeats. It binds the catalytic subunit through repeats 11 to 15 at the C terminus and the tumor antigens encoded by small DNA tumor viruses through overlapping but distinct regions at N-terminal repeats 2 to 8. A model of the A subunit was developed on the basis of the fact that uncharged or hydrophobic amino acids are conserved at eight defined positions within each repeat. Helical wheel projections suggested that each repeat can be arranged as two interacting amphipathic helixes connected by a short loop. Mutational analysis of the A subunit revealed that the proposed loops are important for binding of tumor antigens, the B subunit, and the C subunit. Native gel analysis of mutant A subunits synthesized in vitro demonstrated that the binding region for the B subunit, previously thought to include repeats 2 to 8, covers repeats 1 to 10 and that the B and C subunits cooperate in binding to the A subunit. Images PMID:8254721

Ruediger, R; Hentz, M; Fait, J; Mumby, M; Walter, G

1994-01-01

276

Permeation of antigen protein-conjugated nanoparticles and live bacteria through microneedle-treated mouse skin  

PubMed Central

Background: The present study was designed to evaluate the extent to which pretreatment with microneedles can enhance skin permeation of nanoparticles in vitro and in vivo. Permeation of live bacteria, which are physically nanoparticles or microparticles, through mouse skin pretreated with microneedles was also studied to evaluate the potential risk of microbial infection. Methods and results: It was found that pretreatment of mouse skin with microneedles allowed permeation of solid lipid nanoparticles, size 230 nm, with ovalbumin conjugated on their surface. Transcutaneous immunization in a mouse skin area pretreated with microneedles with ovalbumin nanoparticles induced a stronger antiovalbumin antibody response than using ovalbumin alone. The dose of ovalbumin antigen determined whether microneedle-mediated transcutaneous immunization with ovalbumin nanoparticles induced a stronger immune response than subcutaneous injection of the same ovalbumin nanoparticles. Microneedle treatment permitted skin permeation of live Escherichia coli, but the extent of the permeation was not greater than that enabled by hypodermic injection. Conclusion: Transcutaneous immunization on a microneedle-treated skin area with antigens carried by nanoparticles can potentially induce a strong immune response, and the risk of bacterial infection associated with microneedle treatment is no greater than that with a hypodermic injection. PMID:21753877

Kumar, Amit; Li, Xinran; Sandoval, Michael A; Rodriguez, B Leticia; Sloat, Brian R; Cui, Zhengrong

2011-01-01

277

Pharmaceutical proteins in plants. A strategic genetic engineering approach for the production of tuberculosis antigens.  

PubMed

Tuberculosis (TB) is a re-emerging disease that is considered a major human health priority as well as an important disease of livestock. TB is also a zoonosis, and Mycobacterium tuberculosis and M. bovis, the human and bovine causative agents, respectively, are very closely related. Protection against TB is essentially achieved through vaccination with the Bacille Calmetle-Guerin (BCG) strain of M. bovis. Protection is, however, incomplete, and novel improved vaccines are currently under investigation. Production of protective antigens in transgenic plants, or "pharming," is a promising emerging approach, and a zoonosis-like TB is a good model for investigating the potential of this approach. Pharma-Planta, a European Commission-funded project and consortium, was set up to address this topic, within which a component is aimed at assessing the production efficacy and stability of the TB antigens in different compartments of the plant cell. This article is meant to introduce this promising approach for veterinary medicine by describing the ongoing project and its specific genetic engineering strategy. PMID:19120228

Frutos, Roger; Denise, Hubert; Vivares, Christian; Neuhaus, Jean-Marc; Vitale, Sandro; Pedrazzini, Emmanuela; Ma, Julian; Dix, Phil; Gray, John; Pezzotti, Mario; Conrad, Udo; Robinson, David

2008-12-01

278

Simian virus 40 small tumor antigen inhibits dephosphorylation of protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation.  

PubMed Central

We report that the small tumor (small-t) antigen of simian virus 40 (SV40) forms complexes with nuclear protein phosphatase 2A (PP2A) and regulates the phosphorylation and transcriptional transactivation function of the cyclic AMP (cAMP)-regulatory element binding protein (CREB). PP2A coimmunoprecipitated with small t from nuclear extracts from HepG2 cells expressing small t or from rat liver nuclear extracts to which recombinant small t was added. Protein phosphatase 1 was not detected in small-t immunoprecipitates. In HepG2 cells expressing small t, dibutyryl-cAMP (Bt2cAMP) stimulated the phosphorylation of CREB 65-fold, whereas CREB phosphorylation was stimulated only 5- to 8-fold by Bt2cAMP in cells not expressing small t. Small t also inhibited the dephosphorylation of cAMP-dependent protein kinase (PKA)-phosphorylated CREB in rat liver nuclear extracts. In cells expressing small t, Bt2cAMP-stimulated transcription from the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter was enhanced over the level of transcription from the PEPCK promoter in cells not expressing small t. Small t also enhanced Bt2cAMP-stimulated transcription from a Gal4-responsive promoter in cells expressing a chimeric protein containing the Gal4 DNA-binding domain linked to the CREB transactivation domain. However, small t did not stimulate transcription either from a 5' deletion mutant of the PEPCK promoter that is not able to bind CREB or from the Gal4-responsive promoter in the absence of the Gal4-CREB protein. These data suggest that small t enhances Bt2cAMP-stimulated gene transcription by inhibiting the dephosphorylation of PKA-phosphorylated CREB by nuclear PP2A. These findings support previous observations that nuclear PP2A is the primary phosphatase that dephosphorylates PKA-phosphorylated CREB. Images PMID:8065321

Wheat, W H; Roesler, W J; Klemm, D J

1994-01-01

279

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

PubMed

Cryptococcus neoformans var. neoformans is an important fungal pathogen. The capsule is a well established virulence factor and a target site for diagnostic tests. The CPL1 gene is required for capsular formation and virulence. The protein product Cpl1 has been proposed to be a secreted protein, but the characteristics of this protein have not been reported. Here we sought to characterize Cpl1. Phylogenetic analysis showed that the Cpl1 of C. neoformans var. neoformans and the Cpl1 orthologs identified in C. neoformans var. grubii and C. gattii formed a distinct cluster among related fungi; while the putative ortholog found in Trichosporon asahii was distantly related to the Cryptococcus cluster. We expressed Cpl1 abundantly as a secreted His-tagged protein in Pichia pastoris. The protein was used to immunize guinea pigs and rabbits for high titer mono-specific polyclonal antibody that was shown to be highly specific against the cell wall of C. neoformans var. neoformans and did not cross react with C. gattii, T. asahii, Aspergillus spp., Candida spp. and Penicillium spp. Using the anti-Cpl1 antibody, we detected Cpl1 protein in the fresh culture supernatant of C. neoformans var. neoformans and we showed by immunostaining that the Cpl1 protein was located on the surface. The Cpl1 protein is a specific surface protein of C. neoformans var. neoformans. PMID:25261494

Cai, Jian-Piao; Liu, Ling-Li; To, Kelvin K W; Lau, Candy C Y; Woo, Patrick C Y; Lau, Susanna K P; Guo, Yong-Hui; Ngan, Antonio H Y; Che, Xiao-Yan; Yuen, Kwok-Yung

2015-01-01

280

Unrelated Business Income Tax (UBIT) Policy  

E-print Network

Unrelated Business Income Tax (UBIT) Policy Responsible Administrative Unit: Finance with all income tax regulations of the United States and State of Colorado. As a public institution of higher education in of the State of Colorado, Mines is considered "tax-exempt" from federal and state

281

Genetic and antigenic characterization of Babesia bovis merozoite spherical body protein Bb1  

Microsoft Academic Search

A Babesia bovis merozoite protein, Bb-1, was localized by immunoelectron microscopy to an apical organelle known as the spherical body. This unique structure appears to be analogous to dense granules of other apicomplexan protozoa. Similar to previously described dense granule proteins of Plasmodium spp., Bb-1 is secreted during or just after invasion of host erythrocytes and becomes associated with the

Stephen A. Hines; Guy H. Palmer; Wendy C. Brown; Terry F. McElwain; Carlos E. Suarez; Odillon Vidotto; Allison C. Rice-Ficht

1995-01-01

282

Assessing the Serodiagnostic Potential of 35 Mycobacterium tuberculosis Proteins and Identification of Four Novel Serological Antigens  

Microsoft Academic Search

Improved diagnostic reagents are needed for the detection of Mycobacterium tuberculosis infections, and the development of a serodiagnostic test would complement presently available diagnostic methods. The aim of the present study was to identify novel serological targets for use for the future serodiagnosis of tuberculosis (TB). We cloned and expressed 35 M. tuberculosis proteins as recombinant proteins in Escherichia coli

Karin Weldingh; Ida Rosenkrands; Limei Meng Okkels; T. Mark; Peter Andersen

2005-01-01

283

The Tn Antigen-Specific Lectin from Ground Ivy Is an Insecticidal Protein with an Unusual Physiology1  

PubMed Central

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.

2003-01-01

284

Substrate Binding Protein SBP2 of a Putative ABC Transporter as a Novel Vaccine Antigen of Moraxella catarrhalis  

PubMed Central

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.

2014-01-01

285

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

PubMed Central

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 to homogeneity, obtained the NH2-terminal sequence from both the intact protein and derived tryptic and V8 protease peptides and isolated CALLA cDNAs from a Nalm-6 cell line lambda gt10 library using redundant oligonucleotide probes. The CALLA cDNA sequence predicts a 750-amino acid integral membrane protein with a single 24-amino acid hydrophobic segment that could function as both a transmembrane region and a signal peptide. The COOH-terminal 700 amino acids, including six potential N-linked glycosylation sites compose the extracellular protein segment, whereas the 25 NH2-terminal amino acids remaining after cleavage of the initiation methionine form the cytoplasmic tail. CALLA+ cells contain CALLA transcripts of 2.7 to 5.7 kilobases with the major 5.7- and 3.7-kilobase mRNAs being preferentially expressed in specific cell types. Images PMID:2968607

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

1988-01-01

286

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

287

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

PubMed

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 against Escherichia coli. When the PspA nanoparticle formulations were administered subcutaneously to mice they 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 D; Briles, David E; Wannemuehler, Michael J; Narasimhan, Balaji

2013-09-01

288

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

289

Expression of P32 protein of goatpox virus in Pichia pastoris and its potential use as a diagnostic antigen in ELISA.  

PubMed

The present study was undertaken to express goatpox virus (GTPV) P32 protein in Pichia pastoris and evaluate its potential use as a diagnostic antigen in ELISA. The amplified P32 gene of GTPV was cloned into pPICZalphaA vector and characterized by PCR, restriction enzyme digestion and sequencing. The characterized linear recombinant plasmids were transformed in Pichia host GSII5 strain by electroporation and the zeocin resistant Pichia transformant containing P32 gene was selected and confirmed by PCR. The expression of P32 protein in Pichia was induced with 0.5% methanol at 30 degrees C. The optimum expression was observed at 72 h post-induction and the yield was 100 mg/L of culture. The expressed protein was precipitated with polyethylene glycol and analyzed by SDS-PAGE and Western blot using GTPV specific serum and GTPV-P32 protein specific monoclonal antibody. Further, the protein precipitated with acetone was evaluated as diagnostic antigen in indirect ELISA in order to replace the whole GTPV. The standardized P32 protein based indirect ELISA had relative specificity and sensitivity of 84.2% and 94.2-100%, respectively when compared with serum neutralization test and whole virus based indirect ELISA. This study showed a potential of the yeast expressed GTPV-P32 protein as safe antigen in ELISA for seroepidemiological study of the capripox infection in sheep and goats, in India as well as capripox enzootic countries. PMID:19733197

Bhanot, V; Balamurugan, V; Bhanuprakash, V; Venkatesan, G; Sen, A; Yadav, V; Yogisharadhya, R; Singh, R K

2009-12-01

290

Recombinant major outer membrane protein (MOMP) of Chlamydophila abortus, Chlamydophila pecorum, and Chlamydia suis as antigens to distinguish chlamydial species-specific antibodies in animal sera  

Microsoft Academic Search

Recombinant major outer membrane proteins (rMOMP) of Chlamydophila (Ch.) abortus, Ch. pecorum, and Chlamydia (C.) suis were used as antigens to distinguish chlamydial species-specific antibodies in (i) immune sera from six rabbits and three pigs raised against native purified elementary bodies, (ii) serum samples from 25 sows vaccinated with Ch. abortus, and (iii) 40 serum samples from four heifers experimentally

Ludwig E. Hoelzle; Katharina Hoelzle; Max M. Wittenbrink

2004-01-01

291

The effects of serial skin testing with purified protein derivative on the level and quality of antibodies to complex and defined antigens in Mycobacterium bovis-infected cattle  

Technology Transfer Automated Retrieval System (TEKTRAN)

Several serologic tests designed to detect antibodies to immunodominant Mycobacterium bovis antigens have recently emerged as ancillary tests for the detection of bovine tuberculosis in cattle, particularly when applied after injection of purified protein derivative (PPD) for skin test that signific...

292

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

PubMed Central

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

1997-01-01

293

An HLA-A2-restricte d Tyrosinase Antigen on Melanoma Cells Results from Posttranslational Modification and Suggests a Novel Pathway for Processing of Membrane Proteins  

Microsoft Academic Search

Summary T lymphocytes recognize antigens consisting ofpeptides presented by class I and II major histo- compatibility complex (MHC) molecules. The peptides identified so far have been predictable from the amino acid sequences of proteins. We have identified the natural peptide target of a CTL clone that recognizes the tyrosinase gene product on melanoma cells. The peptide results from posttranslational conversion

Jonathan C. A. Skipper; Ronald C. Hendrickson; Jeffrey Shabanowitz; Thomas Wolfel; Craig L. Slingluff; Thierry Boon; Donald F. Hunt; H. Engelhard

294

Major Trypanosoma cruzi antigenic determinant in Chagas' heart disease shares homology with the systemic lupus erythematosus ribosomal P protein epitope.  

PubMed Central

A Trypanosoma cruzi lambda gt11 cDNA clone, JL5, expressed a recombinant protein which was found to react predominantly with chronic Chagas' heart disease sera. The cloned 35-residue-long peptide was identified as the carboxyl-terminal portion of a T. cruzi ribosomal P protein. The JL5 13 carboxyl-terminal residues shared a high degree of homology with the systemic lupus erythematosus (SLE) ribosomal P protein epitope. Synthetic peptides comprising the 13 (R-13), 10 (R-10), and 7 (R-7) carboxyl-terminal residues of the JL5 protein were used to study, by enzyme-linked immunosorbent assay, the specificity of the Chagas' disease anti-JL5 and SLE anti-P antibodies. The R-13 peptide defined a linear antigenic determinant of the JL5 recombinant protein. As was proved for JL5, R-13 defined antibody specificities which were significantly increased in chronic Chagas' heart disease patients. Only SLE anti-P positive sera were found to react with JL5 and R-13. Fine epitope mapping showed that Chagas' disease anti-JL5 and SLE anti-P antibodies define similar epitopes within the R-13 peptide. The binding of the SLE sera to JL5 was completely blocked by the R-13 peptide, indicating that the shared specificity between anti-JL5 and anti-P autoantibodies was exclusively limited to the conserved linear epitope(s) within the R-13 peptide. The prevalence of high anti-R-13 antibody titers in Chagas' heart disease patients supports the hypothesis that postulates the existence of autoimmune disorders in Chagas' heart disease. PMID:1696282

Mesri, E A; Levitus, G; Hontebeyrie-Joskowicz, M; Dighiero, G; Van Regenmortel, M H; Levin, M J

1990-01-01

295

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

296

Isolation of T-Cell Antigens by Using a Recombinant Protein Library and Its Application to the Identification of Novel Vaccine Candidates against Schistosomiasis  

Microsoft Academic Search

We present here a novel approach to identify T-cell antigens from any infectious agent by use of a library of purified recombinant proteins. Essential features of this strategy include (i) a highly efficient cDNA cloning system which negatively selects against nonrecombinant transformants by making use of the bacterial EcoK restriction system, (ii) affinity staining of cDNA clones expressing recombinant proteins,

MATTHIAS EBERL; ADRIAN P. MOUNTFORD; DRAGANA JANKOVIC; EWALD BECK

1999-01-01

297

Using Human Sera to Identify a 52-kDa Exoantigen of Penicillium chrysogenum and Implications of Polyphasic Taxonomy of Anamorphic Ascomycetes in the Study of Antigenic Proteins  

Microsoft Academic Search

We are interested in isolating and identifying antigenic fungal proteins from species that grow on damp building materials.\\u000a The indoor clade of Penicillium chrysogenum, the so-called Fleming clade, is the most common species of Penicillium on moldy building materials. We have identified a 52-kDa marker protein for the indoor clade of P. chrysogenum not present in a taxonomically diverse selection

Aaron M. Wilson; Wen Luo; J. David Miller

2009-01-01

298

The Replication Protein A Binding Site in Simian Virus 40 (SV40) T Antigen and Its Role in the Initial Steps of SV40 DNA Replication  

Microsoft Academic Search

Physical interactions of simian virus 40 (SV40) large tumor (T) antigen with cellular DNA polymerase a- primase (Pol\\/Prim) and replication protein A (RPA) appear to be responsible for multiple functional inter- actions among these proteins that are required for initiation of viral DNA replication at the origin, as well as during lagging-strand synthesis. In this study, we mapped an RPA

KLAUS WEISSHART; POONAM TANEJA; ELLEN FANNING

1998-01-01

299

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

E-print Network

Many gram-negative bacterial pathogens employ type III secretion systems (TTSS) to transport effector proteins into eukaryotic host cell membranes and cytoplasms to subvert normal cellular functions. TTSSs contain a basal body which spans the inner...

Markham, Aaron Paul

2009-07-06

300

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

301

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

302

Effect of Pulsed Ultraviolet Light and High Hydrostatic Pressure on the Antigenicity of Almond Protein Extracts  

Microsoft Academic Search

The efficacy of pulsed ultraviolet light (PUV) and high hydrostatic pressure (HHP) on the IgE binding to the almond extracts\\u000a was studied using sodium dodecyl sulfate polyacrylamide-gel electrophoresis, Western blot, and enzyme-linked immunosorbent\\u000a assay (ELISA) probed with human plasma containing IgE antibodies to almond allergens and a polyclonal antibody against almond\\u000a major protein. Crude almond protein extracts were treated with

Yiqiao Li; Wade Yang; Si-Yin Chung; Haiqiang Chen; Mu Ye; Arthur A. Teixeira; Jesse F. Gregory; Bruce A. Welt; Sandra Shriver

303

Scrapie and Creutzfeldt-Jakob disease prion proteins share physical properties and antigenic determinants.  

PubMed Central

Scrapie of sheep and goats as well as Creutzfeldt-Jakob disease (CJD) of humans are neurologic disorders caused by slow infectious pathogens. The novel molecular properties of the pathogen causing scrapie have prompted introduction of the term "prion" to denote a small proteinaceous infectious particle that resists inactivation by nucleic acid-modifying procedures. Antiserum to the major hamster scrapie prion protein (PrP 27-30) was found to cross-react with murine CJD proteins. The CJD proteins had molecular weights similar to those observed for scrapie prion proteins as determined by NaDodSO4 gel electrophoresis. In addition, the CJD proteins were resistant to digestion by proteinase K and appear to polymerize into rod-shaped particles. The purification procedure developed for scrapie prions was found to be useful in purifying the CJD agent. Purification of the two infectious pathogens by virtually identical procedures provided further evidence for similarities in their molecular structures. We conclude that the molecular and biologic properties of the CJD agent are sufficiently similar to those of the scrapie prion protein that CJD should be classified as a prion disease. Images PMID:2579394

Bendheim, P E; Bockman, J M; McKinley, M P; Kingsbury, D T; Prusiner, S B

1985-01-01

304

Effects of antibodies against cell surface protein antigen PAc-glucosyltransferase fusion proteins on glucan synthesis and cell adhesion of Streptococcus mutans.  

PubMed Central

Cell surface protein antigen (PAc) and glucosyltransferases (GTFs) produced by Streptococcus mutans are considered to be major colonization factors of the organism, and the inhibition of these two factors is predicted to provide protection against dental caries. In this study, we have constructed fusion protein PAcA-GB, a fusion of the saliva-binding alanine-rich region (PAcA) of PAc with the glucan binding (GB) domain of GTF-I, an enzyme catalyzing the synthesis of water-insoluble glucan from sucrose, and fusion protein PAcA-SB, a fusion of PAcA with the sucrose binding (SB) domain of GTF-I. The recombinant fusion proteins were purified from cell extracts of Escherichia coli harboring the fusion genes, and rabbit antibodies against these fusion proteins were prepared. Water-insoluble glucan synthesis by cell-associated and cell-free GTF preparations from S. mutans as well as total glucan synthesis by GTF-I was markedly inhibited by anti-PAcA-GB immunoglobulin G (IgG) antibodies but not by anti-PAcA-SB IgG antibodies. Significant inhibition of the sucrose-independent and sucrose-dependent adhesion of S. mutans to saliva-coated hydroxyapatite beads was observed when anti-PAcA-GB antibodies were added to the reaction mixture. Anti-PAcA-SB antibodies inhibited the adhesion of S. mutans to the beads in the absence of sucrose but not in the presence of sucrose. Immunization with the fusion protein PAcA-GB may be useful for controlling the colonization of teeth by S. mutans. PMID:9169766

Yu, H; Nakano, Y; Yamashita, Y; Oho, T; Koga, T

1997-01-01

305

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

PubMed

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

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

2006-06-01

306

Proliferating cell nuclear antigen (PCNA)-binding protein C1orf124 is a regulator of translesion synthesis.  

PubMed

DNA damage-induced proliferating cell nuclear antigen (PCNA) ubiquitination serves as the key event mediating post-replication repair. Post-replication repair involves either translesion synthesis (TLS) or damage avoidance via template switching. In this study, we have identified and characterized C1orf124 as a regulator of TLS. C1orf124 co-localizes and interacts with unmodified and mono-ubiquitinated PCNA at UV light-induced damage sites, which require the PIP box and UBZ domain of C1orf124. C1orf124 also binds to the AAA-ATPase valosin-containing protein via its SHP domain, and cellular resistance to UV radiation mediated by C1orf124 requires its interactions with valosin-containing protein and PCNA. Interestingly, C1orf124 binds to replicative DNA polymerase POLD3 and PDIP1 under normal conditions but preferentially associates with TLS polymerase ? (POLH) upon UV damage. Depletion of C1orf124 compromises PCNA monoubiquitination, RAD18 chromatin association, and RAD18 localization to UV damage sites. Thus, C1orf124 acts at multiple steps in TLS, stabilizes RAD18 and ubiquitinated PCNA at damage sites, and facilitates the switch from replicative to TLS polymerase to bypass DNA lesion. PMID:22902628

Ghosal, Gargi; Leung, Justin Wai-Chung; Nair, Binoj C; Fong, Ka-Wing; Chen, Junjie

2012-10-01

307

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

308

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

309

Azurin-Like Protein Blocks Invasion of Toxoplasma gondii through Potential Interactions with Parasite Surface Antigen SAG1?  

PubMed Central

Some pathogenic bacteria produce factors that have evolved a capacity to neutralize competing microbes. The cupredoxin family protein azurin, produced by Pseudomonas aeruginosa, exhibits a remarkable ability to impede invasion of a number of diverse intracellular pathogens, including the human AIDS virus human immunodeficiency virus type 1 and the protozoan parasite Plasmodium falciparum (which causes malaria). Here we report that azurin and an azurin-like protein (Laz) from gonococci/meningococci have activity against Toxoplasma, an apicomplexan parasite that causes opportunistic infection in immunocompromised individuals. We demonstrate that the mechanism of action for Laz involves interfering with the ability of Toxoplasma to adhere to host cells. Computer structural analysis reveals that azurin shares structural features with the predominant surface antigen SAG1, which is known to play an important role in parasite attachment. Interestingly, azurin also has structural similarities to a monoclonal antibody to SAG1. Surface plasmon resonance binding studies validate that SAG1 interacts strongly with Laz and, to lesser extent, azurin. Moreover, Toxoplasma mutants lacking SAG1 are not as susceptible to the growth-inhibitory effects of Laz. Collectively, our data show that Toxoplasma adhesion can be significantly impaired by Laz, and to some extent by azurin, via interactions with SAG1. These observations indicate that Laz can serve as an important tool in the study of host-pathogen interactions and is worthy of further study for development into potential therapeutic agents. PMID:18070964

Naguleswaran, Arunasalam; Fialho, Arsenio M.; Chaudhari, Anita; Hong, Chang Soo; Chakrabarty, Ananda M.; Sullivan, William J.

2008-01-01

310

The cellular 107K protein that binds to adenovirus E1A also associates with the large T antigens of SV40 and JC virus.  

PubMed

The association between the retinoblastoma protein (p105-RB) and either the large T antigen of SV40 or the E1A proteins of adenovirus is thought to be an important step in transformation by these viral oncogenes. E1A and large T antigen share a small region of amino acid homology that is necessary for high affinity binding with p105-RB. Mutations of this homology region were shown to reduce drastically the frequency of transformation mediated by the E1A or large T oncogenes. Previously, this small region in E1A was shown to be sufficient for interaction with a second cellular protein of 107,000 daltons (107K). Here we show that in human cells, the large T antigens of SV40 or JC virus also form complexes with 107K. Demonstration of complexes between 107K and the large T antigens of SV40 and JC virus suggests that these associations may represent another component of a common mechanism for transformation between adenoviruses and polyoma viruses. PMID:2546678

Dyson, N; Buchkovich, K; Whyte, P; Harlow, E

1989-07-28

311

Reverse line blot assay for direct identification of seven Streptococcus agalactiae major surface protein antigen genes.  

PubMed

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 (Calpha), Rib, Epsilon (Epsilon/Alp1/Alp5), Alp2, Alp3, and Alp4, and the immunoglobulin A binding protein, Bac (Cbeta). We used the assay to identify these genes in a collection of well-characterized GBS isolates and reference strains. The results showed that mPCR/RLB avoids the common problems of cross-reaction and nontypability associated with protein typing using antisera. It is as sensitive as, but more practical than, separate gene-specific PCRs and would be suitable for large molecular epidemiological studies of GBS. PMID:16426012

Zhao, Zuotao; Kong, Fanrong; Gilbert, Gwendolyn L

2006-01-01

312

Antigenic Proteins Involved in Occupational Rhinitis and Asthma Caused by Obeche Wood (Triplochiton Scleroxylon)  

PubMed Central

Background Obeche wood dust is a known cause of occupational asthma where an IgE-mediated mechanism has been demonstrated. Objective To characterize the allergenic profile of obeche wood dust and evaluate the reactivity of the proteins by in vitro, ex vivo and in vivo assays in carpenters with confirmed rhinitis and/or asthma Materials and methods An in-house obeche extract was obtained, and two IgE binding bands were purified (24 and 12 kDa) and sequenced by N-terminal identity. Specific IgE and IgG, basophil activation tests and skin prick tests (SPTs) were performed with whole extract and purified proteins. CCD binding was analyzed by ELISA inhibition studies. Results Sixty-two subjects participated: 12 with confirmed occupational asthma/rhinitis (ORA+), 40 asymptomatic exposed (ORA?), and 10 controls. Of the confirmed subjects, 83% had a positive SPT to obeche. There was a 100% recognition by ELISA in symptomatic subjects vs. 30% and 10% in asymptomatic exposed subjects and controls respectively (p<0.05). Two new proteins were purified, a 24 kDa protein identified as a putative thaumatin-like protein and a 12 kDa gamma-expansin. Both showed allergenic activity in vitro, with the putative thaumatin being the most active, with 92% recognition by ELISA and 100% by basophil activation test in ORA+ subjects. Cross-reactivity due to CCD was ruled out in 82% of cases. Conclusions Two proteins of obeche wood were identified and were recognized by a high percentage of symptomatic subjects and by a small proportion of asymptomatic exposed subjects. Further studies are required to evaluate cross reactivity with other plant allergens. PMID:23349765

Aranda, Ana; Campo, Paloma; Palacin, Arantxa; Doña, Inmaculada; Gomez-Casado, Cristina; Galindo, Luisa; Díaz-Perales, Araceli; Blanca, Miguel

2013-01-01

313

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

314

Expression, purification, and improved antigenic specificity of a truncated recombinant bp26 protein of Brucella melitensis M5-90: a potential antigen for differential serodiagnosis of brucellosis in sheep and goats.  

PubMed

Antibodies produced in animals vaccinated using live attenuated vaccines against Brucella spp. are indistinguishable using current conventional serological tests from those produced in infected animals. One potential approach is to develop marker vaccines in which specific genes have been deleted from parental vaccine strains that show good immunogenicity and vaccine efficacy. Corresponding methods of detection for antibodies raised by the marker vaccine should also be developed. A specific fragment of the bp26 gene of Brucella melitensis M5-90 was cloned into vector pQE32 to construct the recombinant plasmid (pQE32-r?bp26). It was used to transform Escherichia coli M15 (pREP4) host cells, which expressed the r?bp26 protein. Subsequently, the recombinant protein was purified by immobilized metal affinity chromatography and size-exclusion chromatography. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified r?bp26 protein was represented by only one band, with a molecular weight of 14 kDa, and it showed good antigenic specificity on western blot and enzyme-linked immunosorbent assay (ELISA). The purified r?bp26 protein was intended to be used as an antigen to develop a novel ELISA to differentiate animals vaccinated with bp26 mutants of Brucella spp. from those infected naturally and those vaccinated with the parental vaccine strains. PMID:21446957

Liu, Wen-xing; Hu, Sen; Qiao, Zu-jian; Chen, Wei-ye; Liu, Lin-tao; Wang, Fang-kun; Hua, Rong-hong; Bu, Zhi-gao; Li, Xiang-rui

2011-01-01

315

Specific nongluten proteins of wheat are novel target antigens in celiac disease humoral response  

Technology Transfer Automated Retrieval System (TEKTRAN)

Background: Celiac disease is an immune-mediated enteropathy that is generally understood to be triggered by the ingestion of gluten proteins of wheat and related cereals. The skin manifestation of the condition is known as dermatitis herpetiformis. Antibody response to native and deamidated seque...

316

Effects of glycosylation on antigenicity and immunogenicity of classical swine fever virus envelope proteins  

Technology Transfer Automated Retrieval System (TEKTRAN)

Classical swine fever virus (CSFV) harbors three envelope glycoproteins (E(rns), E1 and E2). Previous studies have demonstrated that removal of specific glycosylation sites within these proteins yielded attenuated and immunogenic CSFV mutants. Here we analyzed the effects of lack of glycosylation of...

317

Rapid Expression of Mycobacterium avium subsp. paratuberculosis Recombinant Proteins for Antigen Discovery  

Microsoft Academic Search

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease, a chronic granulo- matous enteritis of ruminants and other species. Detection of infection in animals is hampered by the lack of sensitive and specific diagnostic assays. We describe here an approach that utilizes translationally active PCR fragments for the rapid in vitro transcription and translation of recombinant proteins for

Lingling Li; Shirin Munir; John P. Bannantine; Srinand Sreevatsan; Sagarika Kanjilal; Vivek Kapur

2007-01-01

318

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

Technology Transfer Automated Retrieval System (TEKTRAN)

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease, a chronic enteric disease of ruminant animals. In the present study, blue native PAGE electrophoresis and 2D SDS-PAGE were used to separate MAP envelope protein complexes, followed by mass spectrometry (MS) ...

319

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

PubMed

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

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

2015-02-01

320

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

PubMed Central

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

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

2010-01-01

321

WI-1, a novel 120-kilodalton surface protein on Blastomyces dermatitidis yeast cells, is a target antigen of cell-mediated immunity in human blastomycosis.  

PubMed Central

A large body of experimental data has demonstrated the central role of T cells in acquired resistance to the dimorphic fungus Blastomyces dermatitidis. We examined the human T-cell response to WI-1, a 120-kDa B. dermatitidis yeast cell surface protein recently shown to be an immunodominant antigen of the B-cell response in infected humans. Peripheral blood lymphocytes from 10 blastomycosis patients studied proliferated in response to WI-1 (mean, 19,431 cpm) and to the standard, crude cell wall antigen, Blastomyces alkali- and water-soluble antigen (B-ASWS) (mean, 19,131 cpm); lymphocytes from 10 histoplasmosis patients and 10 normal control subjects did not respond to WI-1. WI-1 stimulation of patient lymphocytes and rechallenge with WI-1 or B-ASWS showed that the antigens share immunodominant epitopes. Of 100 WI-1-responsive T-cell clones derived from peripheral blood, 10 were studied in detail to assess the phenotype, function, and ligands recognized. The clones exhibit the CD3+ CD4+ phenotype of helper T cells; 2 of 10 clones (and 21% of antigen-stimulated peripheral blood lymphocytes) use the V beta 8 T-cell receptor gene element to respond to WI-1. All the clones proliferate in response to both WI-1 and B-ASWS but not other fungal antigens, and some mediate potent cytolytic effects on WI-1- and B-ASWS-labeled targets. WI-1 recognition requires antigen processing and presentation of epitopes in association with HLA-DR (to noncytolytic clones) and HLA-DP (to cytolytic clones). From these findings, we conclude that CD4+ T cells with regulatory and cytolytic properties are involved in the development of acquired resistance of B. dermatitidis, that the cells are directed against WI-1, and that the manner of display of WI-1 peptide epitopes in conjunction with major histocompatibility complex class II may influence the profile of the immune response. PMID:1383148

Klein, B S; Sondel, P M; Jones, J M

1992-01-01

322

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

323

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

PubMed Central

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

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

2014-01-01

324

Cloning and characterization of the acidic ribosomal protein P2 of Cryptosporidium parvum, a new 17-kilodalton antigen.  

PubMed

Cryptosporidium infection is commonly observed among children and immunocompromised individuals in developing countries, but large-scale outbreaks of disease among adults have not been reported. In contrast, outbreaks of cryptosporidiosis in the United States and Canada are increasingly common among patients of all ages. Thus, it seems likely that residents of regions where Cryptosporidium is highly endemic acquire some level of immunity, while residents of the developed world do not. A new immunodominant Cryptosporidium parvum antigen in the 15- to 17-kDa size range was identified as the Cryptosporidium parvum 60S acidic ribosomal protein P2 (CpP2). We developed a recombinant protein-based enzyme-linked immunosorbent assay for serologic population surveillance for antibodies that was 89% sensitive and 92% specific relative to the results of the large-format Western blot assay. The human IgG response is directed almost exclusively toward the highly conserved, carboxy-terminal 15 amino acids of the protein. Although IgG antibody cross-reactivity was documented with sera from patients with acute babesiosis, the development of an anti-CpP2 antibody response in our Peru study population correlated better with Cryptosporidium infection than with infection by any other parasitic protozoan. In Haiti, the prevalence of antibodies to CpP2 plateaus at 11 to 20 years of age. Because anti-CpP2 IgG antibodies were found only among residents of countries in the developing world where Cryptosporidium infection occurs early and often, we propose that this response may be a proxy for the intensity of infection and for acquired immunity. PMID:20410328

Priest, Jeffrey W; Kwon, James P; Montgomery, Joel M; Bern, Caryn; Moss, Delynn M; Freeman, Amanda R; Jones, Cara C; Arrowood, Michael J; Won, Kimberly Y; Lammie, Patrick J; Gilman, Robert H; Mead, Jan R

2010-06-01

325

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

PubMed Central

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

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

2015-01-01

326

Wiskott-Aldrich syndrome protein controls antigen-presenting cell-driven CD4+ T-cell motility by regulating adhesion to intercellular adhesion molecule-1.  

PubMed

T-cell scanning for antigen-presenting cells (APC) is a finely tuned process. Whereas non-cognate APC trigger T-cell motility via chemokines and intercellular adhesion molecule-1 (ICAM-1), cognate APC deliver a stop signal resulting from antigen recognition. We tested in vitro the contribution of the actin cytoskeleton regulator Wiskott-Aldrich syndrome protein (WASP) to the scanning activity of primary human CD4(+)  T cells. WASP knock-down resulted in increased T-cell motility upon encounter with non-cognate dendritic cells or B cells and reduced capacity to stop following antigen recognition. The high motility of WASP-deficient T cells was accompanied by a diminished ability to round up and to stabilize pauses. WASP-deficient T cells migrated in a normal proportion towards CXCL12, CCL19 and CCL21, but displayed an increased adhesion and elongation on ICAM-1. The elongated morphology of WASP-deficient T cells was related to a reduced confinement of high-affinity lymphocyte function-associated antigen 1 to the mid-cell zone. Our data therefore indicate that WASP controls CD4(+) T-cell motility upon APC encounter by regulating lymphocyte function-associated antigen 1 spatial distribution. PMID:22804504

Lafouresse, Fanny; Cotta-de-Almeida, Vinicius; Malet-Engra, Gema; Galy, Anne; Valitutti, Salvatore; Dupré, Loïc

2012-10-01

327

Wiskott–Aldrich syndrome protein controls antigen-presenting cell-driven CD4+ T-cell motility by regulating adhesion to intercellular adhesion molecule-1  

PubMed Central

T-cell scanning for antigen-presenting cells (APC) is a finely tuned process. Whereas non-cognate APC trigger T-cell motility via chemokines and intercellular adhesion molecule-1 (ICAM-1), cognate APC deliver a stop signal resulting from antigen recognition. We tested in vitro the contribution of the actin cytoskeleton regulator Wiskott–Aldrich syndrome protein (WASP) to the scanning activity of primary human CD4+ T cells. WASP knock-down resulted in increased T-cell motility upon encounter with non-cognate dendritic cells or B cells and reduced capacity to stop following antigen recognition. The high motility of WASP-deficient T cells was accompanied by a diminished ability to round up and to stabilize pauses. WASP-deficient T cells migrated in a normal proportion towards CXCL12, CCL19 and CCL21, but displayed an increased adhesion and elongation on ICAM-1. The elongated morphology of WASP-deficient T cells was related to a reduced confinement of high-affinity lymphocyte function-associated antigen 1 to the mid-cell zone. Our data therefore indicate that WASP controls CD4+ T-cell motility upon APC encounter by regulating lymphocyte function-associated antigen 1 spatial distribution. PMID:22804504

Lafouresse, Fanny; Cotta-de-Almeida, Vinicius; Malet-Engra, Gema; Galy, Anne; Valitutti, Salvatore; Dupré, Loïc

2012-01-01

328

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

PubMed

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

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

2009-01-01

329

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

PubMed Central

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

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

2013-01-01

330

Tyrosinase family proteins are antigens specific to Vogt-Koyanagi-Harada disease.  

PubMed

Vogt-Koyanagi-Harada (VKH) disease (and sympathetic ophthalmia) is an ocular inflammatory disease that is considered to be a cell-mediated autoimmune disease against melanocytes. The purpose of this study was to determine the Ags specific to VKH disease and to develop an animal model of VKH disease. We found that exposure of lymphocytes from patients with VKH disease to peptides (30-mer) derived from the tyrosinase family proteins led to significant proliferation of the lymphocytes. Immunization of these peptides into pigmented rats induced ocular and extraocular changes that highly resembled human VKH disease, and we suggest that an experimental VKH disease was induced in these rats. We conclude that VKH disease is an autoimmune disease against the tyrosinase family proteins. PMID:11120868

Yamaki, K; Gocho, K; Hayakawa, K; Kondo, I; Sakuragi, S

2000-12-15

331

Identification of clone-9 antigenic protein of Theileria uilenbergi and evaluation of its application for serodiagnosis  

Microsoft Academic Search

The pathogenic protozoan parasite Theileria uilenbergi is one of the causative agents of theileriosis in small ruminants in China. The infection results in great economical losses\\u000a in the northwest part of China. Efforts are underway to establish an enzyme-linked immunosorbent assay (ELISA) based on a\\u000a T. uilenbergi immunodominant recombinantly expressed protein using different approaches in order to perform epidemiological studies

Jassim Abdo; Zhijie Liu; Hong Yin; Birgit Kullmann; Jabbar S. Ahmed; Ulrike Seitzer

2010-01-01

332

Structural and antigenic analysis of the yellow head virus nucleocapsid protein p20  

Microsoft Academic Search

Yellow head virus (YHV) is an invertebrate nidovirus that is highly pathogenic for marine shrimp. Nucleotide sequence analysis indicated that the YHV ORF2 gene encodes a basic protein (pI=9.9) of 146 amino acids with a predicted molecular weight of 16,325.5Da. The deduced amino acid sequence indicated a predominance of basic (15.1%), acidic (9.6%) and hydrophilic polar (34.3%) residues and a

Nusra Sittidilokratna; Natthida Phetchampai; Vichai Boonsaeng; Peter J. Walker

2006-01-01

333

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

Microsoft Academic Search

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

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

1984-01-01

334

Probing the equatorial groove of the hookworm protein and vaccine candidate antigen, Na-ASP-2.  

PubMed

Hookworm activation-associated secreted proteins can be structurally classified into at least three different groups. The hallmark feature of Group 1 activation-associated secreted proteins is a prominent equatorial groove, which is inferred to form a ligand binding site. Furthermore, a conserved tandem histidine motif is located in the centre of the groove and believed to provide or support a yet to be determined catalytic activity. Here, we report three-dimensional crystal structures of Na-ASP-2, an L3-secreted activation-associated secreted protein from the human hookworm Necator americanus, which demonstrate transition metal binding ability of the conserved tandem histidine motif. We further identified moderate phosphohydrolase activity of recombinant Na-ASP-2, which relates to the tandem histidine motif. By panning a random 12-mer peptide phage library, we identified a peptide with high similarity to the human calcium-activated potassium channel SK3, and confirm binding of the synthetic peptide to recombinant Na-ASP-2 by differential scanning fluorimetry. Potential binding modes of the peptide to Na-ASP-2 were studied by molecular dynamics simulations which clearly identify a preferred topology of the Na-ASP-2:SK3 peptide complex. PMID:24631931

Mason, Lyndel; Tribolet, Leon; Simon, Anne; von Gnielinski, Natascha; Nienaber, Lisa; Taylor, Paul; Willis, Charlene; Jones, Malcolm K; Sternberg, Paul W; Gasser, Robin B; Loukas, Alex; Hofmann, Andreas

2014-05-01

335

Antigenic and immunogenic properties of baculovirus-expressed infectious bursal disease viral proteins.  

PubMed

Genomic segment A of the variant Md infectious bursal disease virus (IBDV) was amplified by reverse transcriptase/polymerase chain reaction, cloned, and expressed in the baculovirus expression system. Three different baculovirus recombinants expressing the genes encoding VP2, VP2/VP4, and the complete polyprotein (VP2/VP4/VP3) were prepared. The three antigens were used in separate enzyme-linked immunosorbent assays, and each detected antibodies against the variant IBDV strains Md, Del-A, Del-E, and GLS and the classic IBDV strain D78 throughout a 14-wk period following vaccination. Eight-week-old specific-pathogen-free (SPF) chickens were inoculated subcutaneously using the VP2/VP4 or VP2/VP4/VP3 baculovirus recombinant or wild-type baculovirus-infected insect cell lysates. Virus-neutralizing antibodies were detected in the VP2/VP4 and VP2/VP4/VP3 baculovirus-inoculated birds at 13 days postinoculation (PI) and at 43 days PI when titrated against Md IBDV. No virus-neutralizing antibody titer was observed in the negative controls or wild-type baculovirus-inoculated birds. One-week-old SPF chickens were inoculated with the VP2, VP2/VP4/VP3 baculovirus recombinants or wild-type baculovirus-infected insect cell lysates. The birds were boosted 2 wk later and challenged at 4 wk of age with 0.5 ml/bird classic STC IBDV (10(2) median embryo infective dose [EID50]/ml) or variant Md IBDV (10(5) EID50/ml) via the oral/nasal route. The VP2 and VP2/VP4/VP3 baculovirus-inoculated birds were partially protected against challenge with the classic STC IBDV. The birds were protected against clinical disease and death but not bursal damage, whereas the wild-type baculovirus-inoculated birds exhibited clinical signs of disease, 13% mortality, and bursal damage. When challenged with the variant Md IBDV, neither the VP2, VP2/VP4/VP3 baculovirus recombinants nor the wild-type baculovirus elicited protection against bursal damage and atrophy. PMID:9533084

Dybing, J K; Jackwood, D J

1998-01-01

336

Affinity binding of antibodies to supermacroporous cryogel adsorbents with immobilized protein A for removal of anthrax toxin protective antigen.  

PubMed

Polymeric cryogels are efficient carriers for the immobilization of biomolecules because of their unique macroporous structure, permeability, mechanical stability and different surface chemical functionalities. The aim of the study was to demonstrate the potential use of macroporous monolithic cryogels for biotoxin removal using anthrax toxin protective antigen (PA), the central cell-binding component of the anthrax exotoxins, and covalent immobilization of monoclonal antibodies. The affinity ligand (protein A) was chemically coupled to the reactive hydroxyl and epoxy-derivatized monolithic cryogels and the binding efficiencies of protein A, monoclonal antibodies to the cryogel column were determined. Our results show differences in the binding capacity of protein A as well as monoclonal antibodies to the cryogel adsorbents caused by ligand concentrations, physical properties and morphology of surface matrices. The cytotoxicity potential of the cryogels was determined by an in vitro viability assay using V79 lung fibroblast as a model cell and the results reveal that the cryogels are non-cytotoxic. Finally, the adsorptive capacities of PA from phosphate buffered saline (PBS) were evaluated towards a non-glycosylated, plant-derived human monoclonal antibody (PANG) and a glycosylated human monoclonal antibody (Valortim(®)), both of which were covalently attached via protein A immobilization. Optimal binding capacities of 108 and 117 mg/g of antibody to the adsorbent were observed for PANG attached poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] and Valortim(®) attached poly(AAm-AGE) cryogels, respectively, This indicated that glycosylation status of Valortim(®) antibody could significantly increase (8%) its binding capacity relative to the PANG antibody on poly(AAm-AGE)-protien-A column (p < 0.05). The amounts of PA which remained in the solution after passing PA spiked PBS through PANG or Valortim bound poly(AAm-AGE) cryogel were significantly (p < 0.05) decreased relative to the amount of PA remained in the solution after passing through unmodified as well as protein A modified poly(AAm-AGE) cryogel columns, indicates efficient PA removal from spiked PBS over 60 min of circulation. The high adsorption capacity towards anthrax toxin PA of the cryogel adsorbents indicated potential application of these materials for treatment of Bacillus anthracis infection. PMID:25736504

Ingavle, Ganesh C; Baillie, Les W J; Zheng, Yishan; Lis, Elzbieta K; Savina, Irina N; Howell, Carol A; Mikhalovsky, Sergey V; Sandeman, Susan R

2015-05-01

337

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

SciTech Connect

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

Tilley, M.; Upton, S.J. (Kansas State Univ., Manhattan (USA))

1990-03-01

338

A protein fragment of streptococcal cell surface antigen I/II which prevents adhesion of Streptococcus mutans.  

PubMed Central

Attachment of Streptococcus mutans to the tooth surface involves a cell surface protein with an M(r) of 185,000, termed streptococcal antigen (SA) I/II. Four overlapping fragments of the gene encoding SA I/II were amplified by polymerase chain reaction, cloned, and expressed in Escherichia coli. The recombinant polypeptides were assayed for adhesion-binding activity to salivary receptors and for recognition by a panel of monoclonal antibodies (MAbs) raised against SA I/II. Two of the MAbs which are known to prevent colonization of S. mutans in vivo bound the recombinant polypeptide comprising residues 816 to 1161. In vitro adhesion of S. mutans to saliva-coated hydroxyapatite beads was also inhibited specifically by a polypeptide (residues 816 to 1213) encompassing the same region. The evidence from the MAbs preventing colonization of S. mutans and the adherence inhibition assay suggests that an adhesion-binding activity resides within the portion of SA I/II comprising residues 816 to 1213, which is highly conserved among oral streptococcal species. Images PMID:7691754

Munro, G H; Evans, P; Todryk, S; Buckett, P; Kelly, C G; Lehner, T

1993-01-01

339

Efficient generation of monoclonal antibodies against major structural proteins of rabies virus with suckling mouse brain antigen.  

PubMed

The rabies virus is a neurotropic virus that causes fatal disease in humans and animals. However, not all commercial antibodies against rabies virus (RABV) structural proteins are generally available, and production of high-quality monoclonal antibodies (MAbs) requires high purification of virus particles and special facilities and is time-consuming. By using RABV-infected suckling mouse brain as antigens in this study, 11 hybridoma cells secreting MAbs against RABV were obtained, which showed strong reactivity with RABV-infected Vero cells in immunofluorescence assay. Among the 11 MAbs, three MAbs (1B11, 1C8, and 8H12) showed a neutralizing effect to RABV, while MAb 4B7 recognized the recombinant nucleoprotein (N) of RABV expressed in Vero cells; seven MAbs (1H3, 3H7, 4E7, 4G3, 5A10, 6C9, and 7B3) reacted specifically with phosphoprotein (P) of RABV. The MAbs developed in this study will be useful in establishing a diagnostic test and study on the interactions between RABV and its host. PMID:24746150

Zhang, Jinyang; Ruan, Xizhen; Zan, Jie; Zheng, Xiaojuan; Yan, Yan; Liao, Min; Zhou, Jiyong

2014-04-01

340

Duffy antigen receptor for chemokines (Darc) polymorphism regulates circulating concentrations of monocyte chemoattractant protein-1 and other inflammatory mediators  

PubMed Central

To identify the genetic basis of circulating concentrations of monocyte chemoattractant protein-1 (MCP-1), we conducted genome-wide association analyses for MCP-1 in 3 independent cohorts (n = 9598). The strongest association was for serum MCP-1 with a nonsynonymous polymorphism, rs12075 (Asp42Gly) in DARC, the gene for Duffy antigen receptor for chemokines, a known vascular reservoir of proinflammatory cytokines (minor allele frequency, 45.6%; P < 1.0 * 10?323). This association was supported by family-based genetic linkage at a locus encompassing the DARC gene (genome-wide P = 8.0 * 10?13). Asp42Gly accounted for approximately 20% of the variability in serum MCP-1 concentrations and also was associated with serum concentrations of interleukin-8 and RANTES. While exploring a lack of association between this polymorphism and EDTA plasma MCP-1 concentrations (P = .82), we determined that both clotting and exogenous heparan sulfate (unfractionated heparin) released substantial amounts of MCP-1 from Darc. Quantitative immunoflow cytometry failed to identify meaningful Asp42Gly-associated differences in Darc expression, suggesting that a functional change is responsible for the differential cytokine binding. We conclude that Asp42Gly is a major regulator of erythrocyte Darc-mediated cytokine binding and thereby the circulating concentrations of several proinflammatory cytokines. We have also identified for the first time 2 mechanisms for the release of reservoir chemokines with possible clinical implications. PMID:20040767

Schnabel, Renate B.; Baumert, Jens; Barbalic, Maja; Dupuis, Josée; Ellinor, Patrick T.; Durda, Peter; Dehghan, Abbas; Bis, Joshua C.; Illig, Thomas; Morrison, Alanna C.; Jenny, Nancy S.; Keaney, John F.; Gieger, Christian; Tilley, Cathy; Yamamoto, Jennifer F.; Khuseyinova, Natalie; Heiss, Gerardo; Doyle, Margaret; Blankenberg, Stefan; Herder, Christian; Walston, Jeremy D.; Zhu, Yanyan; Vasan, Ramachandran S.; Klopp, Norman; Boerwinkle, Eric; Larson, Martin G.; Psaty, Bruce M.; Peters, Annette; Ballantyne, Christie M.; Witteman, Jacqueline C. M.

2010-01-01

341

Role of initial protein phosphorylation events and localized release-activated calcium influx in B cell antigen receptor signaling  

PubMed Central

An increase in intracellular calcium concentration is one of the major initial steps in B cell activation following antigen receptor (BCR) ligation. We show herein that in C57BL/6 murine B lymphocytes and in model cell lines, BCR-mediated calcium ion (Ca2+) influx occurs via highly selective Ca2+ release-activated channels, and stromal interaction molecule 1 (STIM1) plays an important role in this pathway. We also demonstrate the temporal relation between Ca2+-dependent signaling events and formation of the immune synapse. Our data indicate that cytoplasmic Ca2+ levels in areas adjacent to the immune synapse differ from those in the rest of the cytoplasm. Finally, a comparison of phosphorylation patterns of BCR-triggered signaling proteins in the presence or absence of Ca2+ revealed the unanticipated finding that initial BCR-triggered, Ca2+-dependent tyrosine phosphorylation events involve predominantly Ca2+ released from intracellular stores and that influx-derived Ca2+ is not essential. This suggests a different role for this phase of Ca2+ influx. PMID:19028960

Lyubchenko, Taras; Nielsen, J. Paul; Miller, Sara M.; Liubchenko, Ganna A.; Holers, V. Michael

2009-01-01

342

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

PubMed

Autophagy is an intracellular catabolic process that is required to maintain cellular homeostasis. Pathogen-elicited host cell autophagy may favour containment of infection or may help in bacterial survival. Pathogens have developed the ability to modulate host autophagy. The secreted antigen HP0175, a peptidyl prolyl cis,trans isomerase of Helicobacter pylori, has moonlighting functions with reference to host cells. Here we show that it executes autophagy in gastric epithelial cells. Autophagy is dependent on the unfolded protein response (UPR) that activates the expression of PKR-like ER?kinase (PERK). This is accompanied by phosphorylation of eukaryotic initiation factor 2? (eIF-2?) and transcriptional activation of ATF4 and CHOP. Knockdown of UPR-related genes inhibits the conversion of LC3I to LC3II, a marker of autophagy. The autophagy-inducing ability of H.?pylori is compromised when cells are infected with an isogenic hp0175 mutant. Autophagy precedes apoptosis. Silencing of BECLIN1 augments cleavage of caspase 3 as well as apoptosis. Increased apoptosis of gastric epithelial cells is known to be linked to H.?pylori-mediated gastric inflammation and carcinogenesis. To the best of our knowledge, this study provides the first demonstration of how HP0175 endowed with moonlighting functions links UPR-dependent autophagy and apoptosis during H.?pylori infection. PMID:25439545

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

2015-05-01

343

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

PubMed Central

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

2014-01-01

344

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

345

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

346

Soluble Human Leukocyte Antigen-G5 Activates Extracellular Signal-Regulated Protein Kinase Signaling and Stimulates Trophoblast Invasion  

PubMed Central

Soluble human leukocyte antigen-G (HLA-G) is a non-classical class Ib HLA molecule that is secreted from blastocysts. Soluble HLA-G modulates the immune tolerance of the mother and can be used as a prognostic factor for the clinical pregnancy rate. However, the underlying mechanism of how soluble HLA-G5 affects pregnancy remains largely unknown. We hypothesized that soluble HLA-G5 promotes successful implantation and pregnancy by modulating trophoblast invasion through receptor binding and activation of extracellular signal-regulated protein kinase (ERK) signaling pathway. Recombinant HLA-G5 protein over-expressed in E. coli BL21 was purified to near homogeneity. We studied the expression of HLA-G5 and its receptors, the leukocyte immunoglobulin-like receptor subfamily B1 (LILRB1) and killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), in primary trophoblasts and trophoblastic (JAr and JEG-3) cell lines by florescence-labeled HLA-G5. HLA-G5 was detected in the primary trophoblasts and JEG-3 cells. The LILRB1 and KIR2DL4 receptors were expressed in both primary trophoblasts and trophoblastic cell lines. HLA-G5 stimulated cell invasion (p<0.05) and increased urokinase (uPA) and matrix metalloproteinases (MMPs) transcripts and their activity (p<0.05) in trophoblastic cells. HLA-G5 activated the ERK signaling pathway and induced ERK1/2 phosphorylation in the trophoblastic cell lines. Addition of ERK inhibitors (U0126 and PD98059) nullified the stimulatory effect of HLA-G5 on trophoblastic cell invasion. Taken together, HLA-G5 induced trophoblast invasion by binding to KIR2DL4 and LILRB1, by increasing uPA and MMPs expressions and by activating the ERK signaling pathway. PMID:24098421

So, Kam-Hei; Gao, Jing; Yeung, William S. B.; Yao, YuanQing; Lee, Kai-Fai

2013-01-01

347

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

PubMed

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

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

2015-02-01

348

Soluble human leukocyte antigen-g5 activates extracellular signal-regulated protein kinase signaling and stimulates trophoblast invasion.  

PubMed

Soluble human leukocyte antigen-G (HLA-G) is a non-classical class Ib HLA molecule that is secreted from blastocysts. Soluble HLA-G modulates the immune tolerance of the mother and can be used as a prognostic factor for the clinical pregnancy rate. However, the underlying mechanism of how soluble HLA-G5 affects pregnancy remains largely unknown. We hypothesized that soluble HLA-G5 promotes successful implantation and pregnancy by modulating trophoblast invasion through receptor binding and activation of extracellular signal-regulated protein kinase (ERK) signaling pathway. Recombinant HLA-G5 protein over-expressed in E. coli BL21 was purified to near homogeneity. We studied the expression of HLA-G5 and its receptors, the leukocyte immunoglobulin-like receptor subfamily B1 (LILRB1) and killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), in primary trophoblasts and trophoblastic (JAr and JEG-3) cell lines by florescence-labeled HLA-G5. HLA-G5 was detected in the primary trophoblasts and JEG-3 cells. The LILRB1 and KIR2DL4 receptors were expressed in both primary trophoblasts and trophoblastic cell lines. HLA-G5 stimulated cell invasion (p<0.05) and increased urokinase (uPA) and matrix metalloproteinases (MMPs) transcripts and their activity (p<0.05) in trophoblastic cells. HLA-G5 activated the ERK signaling pathway and induced ERK1/2 phosphorylation in the trophoblastic cell lines. Addition of ERK inhibitors (U0126 and PD98059) nullified the stimulatory effect of HLA-G5 on trophoblastic cell invasion. Taken together, HLA-G5 induced trophoblast invasion by binding to KIR2DL4 and LILRB1, by increasing uPA and MMPs expressions and by activating the ERK signaling pathway. PMID:24098421

Guo, YiFan; Lee, Cheuk-Lun; So, Kam-Hei; Gao, Jing; Yeung, William S B; Yao, YuanQing; Lee, Kai-Fai

2013-01-01

349

p56lck protein-tyrosine kinase is cytoskeletal and does not bind to polyomavirus middle T antigen.  

PubMed Central

p56lck and p60c-src are closely related protein-tyrosine kinases that are activated by similar oncogenic mutations. We have used fibroblast cell lines that express p56lck from introduced DNA molecules to compare the subcellular localizations of p60c-src and p56lck and their abilities to bind polyomavirus middle T antigen (mT). p56lck is associated with the detergent-insoluble matrix, as defined by extraction with solutions containing nonionic detergents, whereas p60c-src is soluble under these conditions. p56lck is also associated with detergent-insoluble structures in a lymphoid cell line, LSTRA. p60c-src binds to mT, but p56lck does not bind detectably. In terms of both solubility and mT interactions, the nononcogenic p56lck more closely resembles oncogenically activated p60c-src mutants than it resembles p60c-src. Because published results show that an intact carboxy terminus is required for p60c-src to bind mT and be soluble, we tested whether the different localization and mT binding properties of p56lck and p60c-src were dictated by their different carboxy termini. A protein consisting largely of p60c-src sequences but carrying a p56lck carboxy terminus was soluble and bound to mT. We suggest that both the solubility and mT-binding properties of p60c-src not only require sequences common to the carboxy termini of p60c-src and p56lck, but also require sequences unique to the body of p60c-src. Images PMID:3184274

Louie, R R; King, C S; MacAuley, A; Marth, J D; Perlmutter, R M; Eckhart, W; Cooper, J A

1988-01-01

350

Initiation of autoimmunity to the p53 tumor suppressor protein by complexes of p53 and SV40 large T antigen.  

PubMed

Antinuclear antibodies (ANAs) reactive with a limited spectrum of nuclear antigens are characteristic of systemic lupus erythematosus (SLE) and other collagen vascular diseases, and are also associated with certain viral infections. The factors that initiate ANA production and determine ANA specificity are not well understood. In this study, high titer ANAs specific for the p53 tumor suppressor protein were induced in mice immunized with purified complexes of murine p53 and the Simian virus 40 large T antigen (SVT), but not in mice immunized with either protein separately. The autoantibodies to p53 in these mice were primarily of the IgG1 isotype, were not cross-reactive with SVT, and were produced at titers up to 1:25,000, without the appearance of other autoantibodies. The high levels of autoantibodies to p53 in mice immunized with p53/SVT complexes were transient, but low levels of the autoantibodies persisted. The latter may have been maintained by self antigen, since the anti-p53, but not the SVT, response in these mice could be boosted by immunizing with murine p53. Thus, once autoimmunity to p53 was established by immunizing with p53/SVT complexes, it could be maintained without a requirement for SVT. These data may be explained in at least two ways. First, altered antigen processing resulting from the formation of p53/SVT complexes might activate autoreactive T helper cells specific for cryptic epitopes of murine p53, driving anti-p53 autoantibody production. Alternatively, SVT-responsive T cells may provide intermolecular-intrastructural help to B cells specific for murine p53. In a second stage, these activated B cells might themselves process self p53, generating p53-responsive autoreactive T cells. The induction of autoantibodies during the course of an immune response directed against this naturally occurring complex of self and nonself antigens may be relevant to the generation of specific autoantibodies in viral infections, and may also have implications for understanding the pathogenesis of ANAs in SLE. In particular, our results imply that autoimmunity can be initiated by a "hit and run" mechanism in which the binding of a viral antigen to a self protein triggers an immune response that subsequently can be perpetuated by self antigen. PMID:8145041

Dong, X; Hamilton, K J; Satoh, M; Wang, J; Reeves, W H

1994-04-01

351

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

Microsoft Academic Search

The ideal immunological target for cancer vaccine development would meet the criteria of tumor specificity, immunogenicity and vital dependency of the tumor on the functional activities of the antigenic target so as to avoid antigenic loss by mutation. Given that at face value the brother of regulator of imprinted sites (BORIS) transcription factor meets these criteria, we have developed a

M Mkrtichyan; A Ghochikyan; D Loukinov; H Davtyan; T E Ichim; D H Cribbs; V V Lobanenkov; M G Agadjanyan

2008-01-01

352

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

Microsoft Academic Search

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

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

1996-01-01

353

Identification of Tyrosinase-related Protein 2 as a Tumor Rejection Antigen for the B16 Melanoma  

Microsoft Academic Search

Summary Recently, major advances have been made in the identification of antigens from human mela- noma which are recognized by T cells. In spite of this, little is known about the optimal ways to use these antigens to treat patients with cancer. Progress in this area is likely to require accu- rate preclinical animal models, but the availability of such

Matthew B. Bloom; Donna Perry-Lalley; Paul F. Robbins; Yong Li; Mona El-Gamil; Steven A. Rosenberg; James C. Yang

2010-01-01

354

Regulation of the antigen-induced F-actin response in rat basophilic leukemia cells by protein kinase C  

Microsoft Academic Search

Multivalent antigen that is capable of bind- ing to and crosslinking the IgE receptors on rat baso- philic leukemia (RBL) cells, induces a rapid and sus- tained rise in the content of filamentous actin. This reorganization of the actin may be responsible for changes in cellular morphology during the degranula- tion process. The antigen-stimulated polymerization of actin can be blocked

John R. Apgar

1991-01-01

355

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

356

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

357

Conformational Dependence and Conservation of an Immunodominant Epitope within the Babesia equi Erythrocyte-Stage Surface Protein Equi Merozoite Antigen 1  

Microsoft Academic Search

Equi merozoite antigen 1 (EMA-1) is an immunodominant Babesia equi erythrocyte-stage surface protein. A competitive enzyme-linked immunosorbent assay (ELISA), based on inhibition of monoclonal antibody (MAb) 36\\/133.97 binding to recombinant EMA-1 by equine anti-B. equi antibodies, detects horses infected with strains present throughout the world. The objectives of this study were to define the epitope bound by MAb 36\\/133.97 and

Cristina W. Cunha; Lowell S. Kappmeyer; Travis C. McGuire; Odir A. Dellagostin; Donald P. Knowles

2002-01-01

358

Noncapsulated Klebsiella pneumoniae Bearing Mannose-Containing O Antigens Is Rapidly Eradicated from Mouse Lung and Triggers Cytokine Production by Macrophages following Opsonization with Surfactant Protein D  

Microsoft Academic Search

To better understand the relationship between the surface polysaccharides of pulmonary pathogens and components of the lung innate immune system, we employed selected serotypes of Klebsiella pneumoniae expressing distinct capsular polysaccharides and\\/or O antigen in a murine model of K. pneumoniae infection. In addition, we examined the effect of surfactant protein D (SP-D) on the cytokine response of human monocyte-derived

Elena Kostina; Itzhak Ofek; Erika Crouch; Rotem Friedman; Lea Sirota; Gil Klinger; Hany Sahly; Yona Keisari

2005-01-01

359

Molecular characterization of a new gene, CEAL1, encoding for a carcinoembryonic antigen-like protein with a highly conserved domain of eukaryotic translation initiation factors  

Microsoft Academic Search

Carcinoembryonic antigen (CEA) is a complex immunoreactive glycoprotein belonging to a large and heterogeneous group of cross-reacting proteins known as the CEA gene family, which contains 29 genes\\/pseudogenes. CEA is used as a valuable serum tumor marker for monitoring response to therapy in patients with various solid tumors. Through the positional cloning approach we have identified and characterized a CEA-like

Andreas Scorilas; Pui Ming Chiang; Dionyssios Katsaros; George M. Yousef; Eleftherios P. Diamandis

2003-01-01

360

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

PubMed Central

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

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

2010-01-01

361

Diversity of ?? T-cell antigens  

PubMed Central

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

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

2013-01-01

362

Diversity of ?? T-cell antigens.  

PubMed

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

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

2013-01-01

363

New PHA products using unrelated carbon sources  

PubMed Central

Polyhydroxyalkanoates (PHA) are natural polyesters stored by a wide range of bacteria as carbon source reserve. Due to its chemical characteristics and biodegradability PHA can be used in chemical, medical and pharmaceutical industry for many human purposes. Over the past years, few Burkholderia species have become known for production of PHA. Aside from that, these bacteria seem to be interesting for discovering new PHA compositions which is important to different industrial applications. In this paper, we introduce two new strains which belong either to Burkholderia cepacia complex (Bcc) or genomovar-type, Burkholderia cepacia SA3J and Burkholderia contaminans I29B, both PHA producers from unrelated carbon sources. The classification was based on 16S rDNA and recA partial sequence genes and cell wall fatty acids composition. These two strains were capable to produce different types of PHA monomers or precursors. Unrelated carbon sources were used for growth and PHA accumulation. The amount of carbon source evaluated, or mixtures of them, was increased with every new experiment until it reaches eighteen carbon sources. As first bioprospection experiments staining methods were used with colony fluorescent dye Nile Red and the cell fluorescent dye Nile Blue A. Gas chromatography analysis coupled to mass spectrometry was used to evaluate the PHA composition on each strain cultivated on different carbon sources. The synthesized polymers were composed by short chain length-PHA (scl-PHA), especially polyhydroxybutyrate, and medium chain length-PHA (mcl-PHA) depending on the carbon source used. PMID:24031764

Matias, Fernanda; de Andrade Rodrigues, Maria Filomena

2011-01-01

364

New PHA products using unrelated carbon sources.  

PubMed

Polyhydroxyalkanoates (PHA) are natural polyesters stored by a wide range of bacteria as carbon source reserve. Due to its chemical characteristics and biodegradability PHA can be used in chemical, medical and pharmaceutical industry for many human purposes. Over the past years, few Burkholderia species have become known for production of PHA. Aside from that, these bacteria seem to be interesting for discovering new PHA compositions which is important to different industrial applications. In this paper, we introduce two new strains which belong either to Burkholderia cepacia complex (Bcc) or genomovar-type, Burkholderia cepacia SA3J and Burkholderia contaminans I29B, both PHA producers from unrelated carbon sources. The classification was based on 16S rDNA and recA partial sequence genes and cell wall fatty acids composition. These two strains were capable to produce different types of PHA monomers or precursors. Unrelated carbon sources were used for growth and PHA accumulation. The amount of carbon source evaluated, or mixtures of them, was increased with every new experiment until it reaches eighteen carbon sources. As first bioprospection experiments staining methods were used with colony fluorescent dye Nile Red and the cell fluorescent dye Nile Blue A. Gas chromatography analysis coupled to mass spectrometry was used to evaluate the PHA composition on each strain cultivated on different carbon sources. The synthesized polymers were composed by short chain length-PHA (scl-PHA), especially polyhydroxybutyrate, and medium chain length-PHA (mcl-PHA) depending on the carbon source used. PMID:24031764

Matias, Fernanda; de Andrade Rodrigues, Maria Filomena

2011-10-01

365

Over-expression of TATA binding protein (TBP) and p53 and autoantibodies to these antigens are features of systemic sclerosis, systemic lupus erythematosus and overlap syndromes  

PubMed Central

The aim of this study was to determine the expression levels of p53 and TATA binding protein (TBP) and the presence of autoantibodies to these antigens in Asian Indian patients with systemic sclerosis (SSc), overlap syndromes (OS) and systemic lupus erythematosus (SLE). Fifty patients with SSc, 20 with OS, including mixed connective tissue diseases (MCTD), 20 with SLE, 10 disease controls (DC) and 25 controls (C) were studied. The over-expression of p53 and TBP antigen was determined quantitatively by sandwich enzyme-linked immunosorbent assay (ELISA), varies between four- and sevenfold higher in patients with SSc, OS and SLE, in comparison to DC and C. The expressed protein antigens were not present as free antigens but as immune-complexes. Autoantibodies to p53 were detected by ELISA in 78% subjects with SSc, 100% with OS and 80% with SLE. Autoantibodies to TBP were observed in 28% patients with SSc, 25% with OS and 15% with SLE. In comparison to healthy controls, the titre of antibodies to p53 was significantly higher in patients with SSc (P = 0·00001) than the patients with OS (P = 0·00279) and SLE (P = 0·00289), whereas the titre of antibodies to TBP was higher in patients with OS (P = 0·00185) than the SLE (P = 0·00673) and the SSc (P = 0·00986) patients. Autoantibodies to p53 and TBP were detected in all these patients and the levels of these two autoantibodies showed weak negative correlation with each other. We propose that the over-expression of these antigens might be due to hyperactive regulatory regions in the p53 and TBP gene. PMID:15147363

CHAUHAN, R; HANDA, R; DAS, T P; PATI, U

2004-01-01

366

The antigenicity of a Plasmodium vivax reticulocyte binding protein-1 (PvRBP1) recombinant fragment in humans and its immunogenicity and protection studies in Aotus monkeys.  

PubMed

Plasmodium vivax merozoites have been found to specifically invade immature red blood cells (reticulocytes) and this preference has been associated with two proteins named reticulocyte binding protein-1 and protein-2 (PvRBP1 and PvRBP2). Previous reticulocyte binding assays using 15-mer synthetic peptides spanning the entire PvRBP1 sequence have shown that 25 out of the 195 peptides synthesised (grouped into 4 different regions) displayed high affinity binding to reticulocytes. The PvRBP1 region III (amino acids 1998-2348), encompassing 9 of the previously described high-affinity reticulocyte binding peptides, was expressed as a recombinant protein in the present study. This protein has been shown to be antigenic in humans and it has also been able to induce good humoral and cellular immune responses in Aotus nancymaae monkeys. Despite its high immunogenicity, no protective efficacy was observed in the immunised animals. PMID:17240494

Rojas Caraballo, Jose; Delgado, Gabriela; Rodriguez, Raul; Patarroyo, Manuel A

2007-05-01

367

Unrelated business income tax: an update.  

PubMed

To meet spiraling costs, tax-exempt hospitals increasingly are operating businesses unrelated to direct patient care. Knowing which activities may be open to challenge by the Internal Revenue Service (IRS) is essential to avoid the unrelated business income (UBI) tax. Three criteria must be met for an activity to be taxable as UBI: It must constitute a trade or business; It must be regularly carried on; and It must be unrelated to the organization's exempt purpose. The Internal Revenue Code and IRS rulings clearly exclude the following areas from UBI taxation: Activities performed by unpaid volunteers (e.g., hospital auxiliaries' fund-raising dinners and bazaars and the operation of thrift stores); Operations conducted for the convenience of the organization's members, students, patients, or employees (e.g., gift shops, cafeterias, coffee shops, parking lots, lounges, vending machines, pharmaceutical sales to inpatients and emergency room outpatients, and research activities for students' benefit; The sale of merchandise that has been received by gift (e.g., flea markets, baked goods sales, book sales, and rummage sales); Investment income such as dividends, interest, annuities, royalties, certain rents, and capital gains from the sale of investment assets; Gifts or contributions made directly to the facility; and Bingo games that are conducted commercially. Areas which may be subject to UBI taxation, or in which there have been controversial or contradictory court rulings, include: Pharmaceutical sales to the public or private physicians' patients; and Laboratory services provided to private physicians for treating their patients. IRS private letter rulings, though not precedential, have excluded from UBI taxation the x-ray income from a hospital's branch facility and rental income from property leased for use as a clinic or medical office building that is substantially related to the hospital's exempt functions. Private letter rulings have subjected to UBI taxation the income for a professional standards review organization's private review activities and debt-financed income from property that is not substantially related to the organization's exempt purpose. PMID:10265213

Fama, A J

1984-02-01

368

Efficient priming of protein antigen-specific human CD4(+) T cells by monocyte-derived dendritic cells.  

PubMed

Dendritic cells (DCs) have the unique ability to initiate an immune response in vivo by capturing antigens (Ags) in peripheral tissues and migrating to secondary lymphoid organs, where they sensitize naive CD4(+) T cells. To mimic this process in vitro, previous studies have shown that DCs directly isolated from peripheral blood can be used to elicit primary responses to neoantigens (neoAgs). In other studies, when monocyte-derived DCs have been utilized to sensitize total CD4(+) T cells in vitro, only secondary proliferation to neoAgs could be elicited. In the present study, the relative abilities of CD40 ligation, protein kinase C activation, and culture in tumor necrosis factor alpha (TNF-alpha) to induce functional and phenotypic maturation of human DCs from monocyte precursors were compared. Optimal TNF-alpha-induced maturation of DCs required a prolonged 4-day culture. It was then found that loading immature DCs with the neoAgs keyhole limpet hemocyanin or human immunodeficiency virus-1 p24 gag prior to TNF-alpha-induced maturation, rather than after maturation, was crucial to sensitize CD4(+) T cells to new Ags. This primary proliferation to neoAgs was initiated from the CD4(+) CD45RA(+) naive T-cell population. Finally, it was found that monocyte-derived DCs acquired the ability to secrete interleukin-12 p70, after contact with Ag-specific T cells. The ability to prime and expand Ag-specific CD4(+) T cells ex vivo to neoAgs in serum-free conditions has potential application for cellular vaccination and adoptive immunotherapy. PMID:11071646

Schlienger, K; Craighead, N; Lee, K P; Levine, B L; June, C H

2000-11-15

369

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

PubMed Central

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

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

2011-01-01

370

Tumor-associated antigen profiling in breast and ovarian cancer: mRNA, protein or T cell recognition?  

Microsoft Academic Search

PurposeThe absence of tumor-associated antigens (TAA) which might elicit an immune response is one reason for the disappointing results of therapeutical vaccines in cancer patients. Moreover, impaired expression of MHC class-I and components involved in antigen processing, such as TAP-1, -2, LMP-2, -7, and MECL-1, may lead to tumor escape from immune recognition. Expression profiling of TAA is one approach

Simone Kayser; Iris Watermann; Christine Rentzsch; Toni Weinschenk; Diethelm Wallwiener; Brigitte Gückel

2003-01-01

371

Diagnostic Value of Enzyme-linked Immunospot Assay Using CFP10/ESAT6 Fusion Protein as Antigen in Spinal Tuberculosis.  

PubMed

Objective To establish a method of detecting spinal tuberculosis(TB)infection by enzyme-linked immunospot(ELlSPOT)assay and evaluate the value of CFP10/ESAT6 fusion protein for diagnosis of spinal TB.Methods Suspected spinal TB patients were prospectively recruited in two hospitals(First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine;Nanfang Hospital,Southern Medical University)from May 2012 to December 2013. Data on clinical characteristics of the patients and conventional laboratory results were collected. Compare and analyze the positive detection rate in spinal TB diagnosis by different methods including ELISPOT detection and conventional detection methods. Results 47 patients with spinal TB had available biopsy or surgical specimens for histopathological examination and 41 specimens had pathological features consistent with a diagnosis of TB infection. Among the spinal TB patients and non-TB disease patients,the overall sensitivity,specificity,positive predictive value,and negative predictive value of the ELISPOT assay in spinal TB diagnosis were 82.7%,87.2%,89.6%,and 79.1%,respectively;the 4 indexes of the PPD skin test were 61.5%,46.2%,60.4%,and 47.4%,respectively;those of the antibody detection were 55.8%,61.5%,65.9%,and 51.1%. The positive rate of ELISPOT was significantly higher than those of PPD skin test and antibody detection test(82.7% vs. 61.5%,?(2)=5.786,P=0.016;82.7% vs. 55.8%,?(2)=8.847,P=0.003),but not significantly different from the positive rate of pathological examination(82.7% vs. 87.2%,?(2)=0.396,P=0.529). Moderate agreement was found between pathological examination and the ELISPOT assay(87.2%,?=0.498,P=0.001). Conclusion With high sensitivity and specificity,the ELISPOT assay using CFP10/ESAT6 fusion protein as antigen is an effective technique for auxiliary diagnosis of spinal TB. PMID:25676269

Yuan, Kai; Liang, De; Wu, Xue-Qiong; Yao, Zhen-Song; Jin, Da-Xiang; Yang, Zhi-Dong; Zhang, Shun-Cong; Ding, Jin-Yong; Jiang, Xiao-Bing; Chen, Jian-Ting

2015-02-12

372

Induction of potent CD8? T cell responses through the delivery of subunit protein vaccines to skin antigen-presenting cells using densely packed microprojection arrays.  

PubMed

The generation of both antibody and CD8? T cell responses against pathogens is considered important for many advanced vaccines for diseases including tuberculosis, HIV and malaria. However, most current vaccines are delivered into muscle by the needle and syringe method and induce protection via humoral (antibody) immune responses. In this paper, we test the hypothesis that delivering a model subunit protein antigen (ovalbumin) to the skin's abundant immune cell population using a densely packed microprojection array (Nanopatch) enhances CD8? T cell responses. We found that the Nanopatch significantly enhanced the CD8? T cell responses when compared to intramuscular delivery of both antigen-only and adjuvanted cases (Quil-A and CpG; separately). To our knowledge, this is the first published study demonstrating significantly improved CD8? T cell responses achieved by delivering subunit vaccines to the skin's abundant immune cell population. Successfully replicating these findings in humans could significantly advance the reach of vaccines. PMID:22841796

Ng, Hwee-Ing; Fernando, Germain J P; Kendall, Mark A F

2012-09-28

373

Ehrlichia ruminantium Major Antigenic Protein Gene (map1) Variants Are Not Geographically Constrained and Show No Evidence of Having Evolved under Positive Selection Pressure  

PubMed Central

In a search for tools to distinguish antigenic variants of Ehrlichia ruminantium, we sequenced the major antigenic protein genes (map1 genes) of 21 different isolates and found that the sequence polymorphisms were too great to permit the design of probes which could be used as markers for immunogenicity. Phylogenetic comparison of the 21 deduced MAP1 sequences plus another 9 sequences which had been previously published did not reveal any geographic clustering among the isolates. Maximum likelihood analysis of codon and amino acid changes over the phylogeny provided no statistical evidence that the gene is under positive selection pressure, suggesting that it may not be important for the evasion of host immune responses. PMID:11682561

Allsopp, M. T. E. P.; Dorfling, C. M.; Maillard, J. C.; Bensaid, A.; Haydon, D. T.; van Heerden, H.; Allsopp, B. A.

2001-01-01

374

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

375

Cross-reactions between serum proteins and water soluble liver tissue antigens of the nine-banded armadillo (Dasypus novemcinctus Linn.) and man.  

PubMed Central

Cross-reactions between serum proteins and water soluble liver antigens of the nine-banded armadillo (Dasypus novemcinctus Linn.) and man were studied by crossed immunoelectrophoresis (CIE). Armadillo serum tested with rabbit antiserum against human serum proteins gave twelve components in CIE. Nine of these cross-reacting proteins were identified and showed partial identity with the corresponding human proteins. The electrophoretic mobility of alpha 2-macroglobulin and Gc-globulin differed in the two species. An ultrasonicate of normal armadillo liver gave twenty-eight anodic and eight cathodic components in CIE. By absorption experiments with armadillo serum, twenty of the former and seven of the latter were shown to be liver tissue components. A combination of CIE and crossed-line immunoelectrophoresis (CLIE) revealed the presence of twelve anodic and six cathodic liver tissue components cross-reacting with man. A cathodic armadillo liver antigen called (CALA-17) showed partial identity with that of man both in tandem and fused rocket immunoelectrophoresis. The implications of the findings are discussed in relation to the use of armadillo-grown M. leprae for skin testing and other purposes in man. Images FIG. 1 FIG. 3 FIG. 4 FIG. 5 PMID:93527

Negassi, K; Closs, O; Harboe, M

1979-01-01

376

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

PubMed Central

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

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

2013-01-01

377

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

PubMed Central

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

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

1977-01-01

378

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

SciTech Connect

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

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

1990-02-01

379

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

380

Langerhans cells and dermal dendritic cells capture protein antigens in the skin: Possible targets for vaccination through the skin  

PubMed Central

Dendritic cells capture and process antigen and present it to T lymphocytes in the lymphoid organs. Dendritic cells of the skin, including epidermal Langerhans cells, langerin+ and langerinnegative dermal dendritic cells are ideally positioned to take up pathogens that enter the body through the skin or vaccines that are administered into (intradermal) or onto (epicutaneous) the skin. The antigen uptake properties of skin dendritic cells have not thoroughly been studied yet. We therefore investigated the uptake of the fluorochrome-conjugated model antigen ovalbumin (OVA) by skin dendritic cells in the mouse. OVA was readily taken up by immature Langerhans cells both in situ and in cell suspensions. When offered to Langerhans cells in situ either by “bathing” skin explants in OVA-containing culture medium or by intradermal injection they retained the captured OVA for at least 2–3 days when migrating into the culture medium and, importantly, into the draining lymph nodes. Also langerin+ and – to a larger extent – langerinnegative skin dendritic cells took up and transported OVA to the lymph nodes. Interestingly, mature Langerhans cells were still capable of ingesting substantial amounts of OVA, indicating that predominantly receptor-mediated endocytosis is operative in these cells. Unlike macropinocytosis, this pathway of endocytosis is not shut down upon dendritic cell maturation. These observations indicate that in intradermal vaccination schemes, Langerhans cells from the epidermis are prominently involved. They were recently shown to possess the capacity to induce functional cytotoxic T lymphocytes. Furthermore, the potential to markedly enhance antigen uptake and processing by targeting antigen to c-type lectin receptors on Langerhans cells was also recently demonstrated. Our data provide a rationale and an incentive to explore in more detail antigen targeting to Langerhans cells with the aim of harnessing it for immunotherapy. PMID:20599290

Sparber, Florian; Tripp, Christoph H.; Hermann, Martin; Romani, Nikolaus; Stoitzner, Patrizia

2010-01-01

381

Using human sera to identify a 52-kDa exoantigen of Penicillium chrysogenum and implications of polyphasic taxonomy of anamorphic ascomycetes in the study of antigenic proteins.  

PubMed

We are interested in isolating and identifying antigenic fungal proteins from species that grow on damp building materials. The indoor clade of Penicillium chrysogenum, the so-called Fleming clade, is the most common species of Penicillium on moldy building materials. We have identified a 52-kDa marker protein for the indoor clade of P. chrysogenum not present in a taxonomically diverse selection of fungi. It is found in high concentrations in protein extracted from the fungus grown on paper-faced gypsum wallboard. During this process, we illuminated the variability in response to patient sera and of strains of the fungus collected over a wide geographic area. From a collection of sera from all over the USA, 25 of the 48 patients reacted to the 52-kDa protein from this prescreened collection of sera. Most strain/antibody combinations had proportionate ELISA response associated with the presence of the target. However, approximately 25% of the strain/patient serum combinations included people who responded to many common allergens from the Penicillia. All the P. chrysogenum strains tested produced the target protein. However, there was considerable variability in patient IgG response to 32-, 30-, and 18-kDa antigens and in their production by the various clade 4 strains. The target protein was not found in spores or culture extracts of a wide selection of relevant fungi. It appears that the previous studies have been conducted on strains of the fungus from the three clades not those associated with the built environment. PMID:19590977

Wilson, Aaron M; Luo, Wen; Miller, J David

2009-11-01

382

The 56-kilodalton major protein antigen of Rickettsia tsutsugamushi: molecular cloning and sequence analysis of the sta56 gene and precise identification of a strain-specific epitope.  

PubMed Central

Lasting immunity against Rickettsia tsutsugamushi, the causative agent of scrub typhus fever, has been demonstrated to be strain specific. Two protein antigens of 110 and 56 kilodaltons (kDa) have been shown to exhibit strain-specific epitopes. The 56-kDa scrub typhus antigen (Sta56) is an abundant outer membrane protein of R. tsutsugamushi and is an antigen often recognized by humans infected with this obligate intracellular bacterium. In this study the complete gene encoding Sta56 (strain Karp) was cloned into pBR322 on a 2.3-kilobase genomic HindIII DNA fragment and the complete 56-kDa polypeptide was expressed in Escherichia coli. DNA sequence analysis of the 2.3-kilobase HindIII fragment revealed an open reading frame large enough to encode a 56-kDa polypeptide. A putative signal sequence was identified at the deduced amino terminus of the Sta56 polypeptide, and pulse-chase analysis of maxicells labeled with [35S]methionine demonstrated that a higher-molecular-weight precursor matures into the 56-kDa polypeptide. Epitope scanning analysis with synthetic peptides derived from the deduced amino acid sequence identified an octapeptide (located from amino acid residues 117 to 124) that was reactive with a Karp strain-specific monoclonal antibody (K13F88A). Other epitopes recognized by different monoclonal antibodies, including another Karp strain-specific monoclone (K1E106), were localized to different regions of the protein based on their reactivities with lambda gt11 recombinants expressing various portions of the sta56 gene. Images PMID:1694818

Stover, C K; Marana, D P; Carter, J M; Roe, B A; Mardis, E; Oaks, E V

1990-01-01

383

Combining viral vectored and protein-in-adjuvant vaccines against the blood-stage malaria antigen AMA1: report on a phase 1a clinical trial.  

PubMed

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

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

2014-12-01

384

Different Protein Tyrosine Kinases Are Required for B Cell Antigen Receptor-mediated Activation of Extracellular Signal-Regulated kinase, c-Jun NH 2 -terminal Kinase 1, and p38 Mitogen-activated Protein Kinase  

Microsoft Academic Search

Summary B cell antigen receptor (BCR) cross-linking activates three distinct families of nonreceptor pro- tein tyrosine kinases (PTKs): src-family kinases, Syk, and Btk; these PTKs are responsible for initiating downstream events. BCR cross-linking in the chicken DT40 B cell line also activates three distinct mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase (ERK)2, c-jun NH 2 -terminal kinase (JNK)1, and p38

Aimin Jiang; Andrew Craxton; Tomohiro Kurosaki; Edward A. Clark

385

The regions of the retinoblastoma protein needed for binding to adenovirus E1A or SV40 large T antigen are common sites for mutations.  

PubMed

The protein product of the retinoblastoma (RB) gene is thought to function in a pathway that restricts cell proliferation. Recently, transforming proteins from three different classes of DNA tumor viruses have been shown to form complexes with the RB protein. Genetic studies suggest that these interactions with the RB protein are important steps in transformation by these viruses. In order to understand better the function of the RB-viral oncoprotein complexes, we have mapped the regions of the RB protein that are necessary for these associations. Two non-contiguous regions of RB were found to be essential for complex formation with adenovirus E1A or SV40 large T antigen. These two regions are found between amino acids 393 and 572 and 646 and 772. Interestingly, these binding sites on RB overlap with the positions of naturally occurring, inactivating mutations of the RB gene. These results strongly suggest that these viral oncoproteins are targeting a protein domain that is an important site in the normal function of the RB protein. PMID:2138977

Hu, Q J; Dyson, N; Harlow, E

1990-04-01

386

Generating CTLs against the subdominant EBV LMP antigens by transient expression of an A20 inhibitor with EBV LMP proteins in human DCs.  

PubMed

Epstein-Barr virus (EBV) infection leads to Hodgkin's disease (HD) in some immunocompetent hosts. The malignant Reed-Sternberg cells of HD only express a limited array of subdominant EBV antigens to evade pre-existing immune responses to EBV. The EBV-encoded latent membrane proteins (LMP1 and LMP2), which are expressed by HD and various EBV-associated malignancies, have been proposed as a potential target for cytotoxic T lymphocyte (CTL)-based therapy. However, the precursor frequency for LMP-specific CTL is generally low in healthy EBV-infected hosts, and immunotherapy based on these antigens is often compromised by the poor immunogenicity and the oncogenic potential. In the present study, we report that transiently expressing an inhibitor of A20, a key negative regulator of inflammatory signaling pathways, together with the LMP antigens (truncated LMP1 and full-length LMP2) greatly enhances maturation and cytokine production of human (h) monocyte-derived dendritic cells (DCs). As a consequence, LMP1/2-expressed, A20-silenced hDCs have an enhanced potency to prime LMP-specific T-cell response. When the in vitro primed T cells are adoptively transferred into tumor-xenografted, severe-combined immunodeficient mice, some of the xenografted tumors approach complete regression. Thus, the study may provide an available resource of LMP-specific T cells for T-cell immunotherapy. PMID:22052242

Hong, B; Peng, G; Berry, L; Gottschalk, S; Jung, J U; Chen, S-Y; Huang, X F

2012-08-01

387

Generating CTLs against the subdominant EBV LMP antigens by transit expression of an A20 inhibitor with EBV LMP proteins in human DCs  

PubMed Central

Epstein-Barr virus (EBV) infection leads to Hodgkin’s disease (HD) in some immunocompetent hosts. The malignant Reed-Sternberg cells of HD only express a limited array of subdominant EBV antigens to evade preexisting immune responses to EBV. The EBV-encoded latent membrane proteins (LMP1 and LMP2), which are express