Sample records for unrelated protein antigens

  1. HLA-C Antigen mismatches are associated with worse outcomes in unrelated donor peripheral blood stem cell transplantation

    PubMed Central

    Woolfrey, Ann; Klein, John P.; Haagenson, Michael; Spellman, Stephen; Petersdorf, Effie; Oudshoorn, Machteld; Gajewski, James; Hale, Gregory A.; Horan, John; Battiwalla, Minoo; Marino, Susana R.; Setterholm, Michelle; Ringden, Olle; Hurley, Carolyn; Flomenberg, Neal; Anasetti, Claudio; Fernandez-Vina, Marcelo; Lee, Stephanie J.

    2010-01-01

    Purpose The association between human leukocyte antigen (HLA) matching and outcome in unrelated donor, peripheral blood stem cell (PBSC) transplantation has not been established. Patients and Methods 1933 unrelated donor-recipient pairs transplanted between 1999-2006 for AML, ALL, MDS or CML and who had high resolution HLA typing for HLA-A, B, C, DRB1, DQA1 and DQB1 were included in the analysis. Outcomes were compared between HLA-matched and HLA-mismatched pairs, adjusting for patient and transplant characteristics. Results Matching for HLA-A, -B, -C and DRB1 alleles [8/8 match] was associated with better survival at one year compared with 7/8 HLA-matched pairs (56% vs. 47%). Using 8/8 HLA-matched patients as the baseline (n=1243), HLA-C antigen mismatches (n=189) were statistically significantly associated with lower LFS (RR 1.36 [95% CI 1.13-1.64] p=0.0010), and increased risk for mortality (RR=1.41 [1.16-1.70], p=0.0005), treatment-related mortality (RR=1.61 [1.25-2.08], p=0.0002), and grades III-IV graft-versus-host disease (RR=1.98 [1.50-2.62], p<0.0001). HLA-B antigen or allele mismatching was associated with a higher risk for acute GVHD grades III-IV. No statistically significant differences in outcome were observed for HLA-C allele mismatch (n=61), nor for mismatches at HLA-A antigen/allele (n=136), HLA-DRB1 allele (n=39) or HLA-DQ antigen/allele (n=114) compared to 8/8 HLA-matched pairs. HLA mismatching was not associated with relapse or chronic GVHD. Conclusion HLA-C antigen mismatched unrelated PBSC donors are associated with worse outcomes compared with 8/8 HLA-matched donors. Limited power due to small sample sized prevents comment about other mismatches. PMID:20870028

  2. Delayed Hypersensitivity to Toxoplasma and Unrelated Antigens in Toxoplasma-Infected Mice: Induction and Elicitation of Delayed-Type Hypersensitivity by Antigen-Pulsed Macrophages

    PubMed Central

    Handman, Emanuela; Chester, Patrice M.; Remington, Jack S.

    1980-01-01

    Delayed-type hypersensitivity (DTH) to Toxoplasma and unrelated antigens in Toxoplasma-infected BALB/c mice was investigated by the radioisotopic uptake method of Vadas et al. (Int. Arch. Allergy Appl. Immunol. 49: 670-692, 1975). DTH became positive on day 30 of infection and remained positive during chronic infection. The expression of DTH in mice infected with the relatively avirulent C37 strain of the parasite paralleled the Toxoplasma antibody response as detected by the Sabin-Feldman dye test. Mice sensitized with Toxoplasma, keyhole limpet hemocyanin, or sheep erythrocytes during the acute or chronic phase of Toxoplasma infection showed a DTH reaction similar to that of uninfected sensitized controls. No parasite antigens could be detected by immunofluorescence techniques on the surface of Toxoplasma-infected cells. When killed organisms were added to the cell cultures, specks of fluorescence appeared on cells containing intracellular parasites as well as on cells without intracellular organisms. That the antigens may be present in or on macrophages in a form readily recognizable by T cells is suggested by experiments in which we demonstrated that injection of uninfected normal macrophages pulsed with Toxoplasma-soluble antigens into the ears of chronically infected mice elicited a DTH reaction comparable to that observed when 106 Formalin-fixed tachyzoites were used as the test antigen. When macrophages pulsed with Toxoplasma antigen were used in attempts to induce DTH in naive uninfected mice, the intensity of the reaction was similar to that observed in infected mice. PMID:7399673

  3. C-Reactive Protein and Cognition Are Unrelated to Leukoaraiosis

    PubMed Central

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

    2014-01-01

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

  4. Allogeneic hematopoietic stem cell transplant for hematological malignancies from mismatched 9/10 human leukocyte antigen unrelated donors: comparison with transplants from 10/10 unrelated donors and human leukocyte antigen identical siblings.

    PubMed

    Michallet, Mauricette; Sobh, Mohamad; Serrier, Caroline; Morisset, Stéphane; Labussière, Hélène; Ducastelle, Sophie; Barraco, Fiorenza; Gilis, Lila; Thomas, Xavier; Nicolini, Franck E

    2015-04-01

    We studied the outcome of 213 patients who received allo-HSCT for hematological malignancies, 121 (57%) from HLA identical siblings, 63 (29%) from 10/10 HLA identical unrelated donors and 29 (14%) from 9/10 HLA mismatched unrelated donors. Engraftment was lower in the 9/10 HLA group (90%) than in the 10/10 HLA group (95%) than in the sibling group (99%); 3 months CI of aGVHD ? 2 was 32% (23-41), 20% (15-26) and 27% (23-32) respectively; the one year CI of extensive cGVHD was 21% (13-30), 9% (5-13) and 17% (14-21) respectively. The median OS was 10 months (5-21), 18 months (11-NR) and 60 months (31-NR) respectively with 2-years probability of 19% (8-44), 43% (31-59) and 63% (54-74) respectively. TRM was significantly higher in the 9/10 HLA group with 1 year CI of 45% (35-55), compared to 33% (27-39) in the unrelated 10/10 HLA group and 12% (9-15) in the identical siblings group (p < 0.001). PMID:25029640

  5. INVITED REVIEW: TARGETING AND EXPRESSION OF ANTIGENIC PROTEINS IN TRANSGENIC

    E-print Network

    Korban, Schuyler S.

    INVITED REVIEW: TARGETING AND EXPRESSION OF ANTIGENIC PROTEINS IN TRANSGENIC PLANTS FOR PRODUCTION, the tools of genetic engineering have allowed development of transgenic plants that can express various of accumulation of these proteins in transgenic plants by developing constructs whereby antigenic protein

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

    PubMed

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

    2015-06-01

    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

  7. Tresyl-Based Conjugation of Protein Antigen to Lipid Nanoparticles Increases Antigen Immunogencity

    PubMed Central

    Jain, Anekant; Yan, Weili; Miller, Keith R.; O'Carra, Ronan; Woodward, Jerold G.; Mumper, Russell J.

    2010-01-01

    The present studies were aimed at investigating the engineering of NPs with protein-conjugated-surfactant at their surface. In order to increase the immunogenicity of a protein antigen, Brij 78 was functionalized by tresyl chloride and then further reacted with the primary amine of the model proteins ovalbumin (OVA) or horseradish peroxide (HRP). The reaction yielded Brij 78-OVA and Brij 78-HRP conjugates which were then used directly to form NP-OVA or NP-HRP using a one-step warm oil-in-water microemulsion precursor method with emulsifying wax as the oil phase, and Brij 78 and the Brij 78-OVA or Brij 78-HRP conjugate as surfactants. Similarly, Brij 700 was conjugated to HIV p24 antigen to yield Brij 700-p24 conjugate. The utility of these NPs for enhancing the immune responses to protein-based vaccines was evaluated in vivo using ovalbumin (OVA) as model protein and p24 as a relevant HIV antigen. In separate in vivo studies, female BALB/c mice were immunized by subcutaneous (s.c.) injection with NP-OVA and NP-p24 formulations along with several control formulations. These results suggested that with multiple antigens, covalent attachment of the antigen to the NP significantly enhanced antigen-specific immune responses. This facile covalent conjugation and incorporation method may be utilized to further incorporate other protein antigens, even multiple antigens, into an enhanced vaccine delivery system. PMID:20837122

  8. Tresyl-based conjugation of protein antigen to lipid nanoparticles increases antigen immunogenicity.

    PubMed

    Jain, Anekant; Yan, Weili; Miller, Keith R; O'Carra, Ronan; Woodward, Jerold G; Mumper, Russell J

    2010-11-30

    The present studies were aimed at investigating the engineering of NPs with protein-conjugated-surfactant at their surface. In order to increase the immunogenicity of a protein antigen, Brij 78 was functionalized by tresyl chloride and then further reacted with the primary amine of the model proteins ovalbumin (OVA) or horseradish peroxide (HRP). The reaction yielded Brij 78-OVA and Brij 78-HRP conjugates which were then used directly to form NP-OVA or NP-HRP using a one-step warm oil-in-water microemulsion precursor method with emulsifying wax as the oil phase, and Brij 78 and the Brij 78-OVA or Brij 78-HRP conjugate as surfactants. Similarly, Brij 700 was conjugated to HIV p24 antigen to yield Brij 700-p24 conjugate. The utility of these NPs for enhancing the immune responses to protein-based vaccines was evaluated in vivo using ovalbumin (OVA) as model protein and p24 as a relevant HIV antigen. In separate in vivo studies, female BALB/c mice were immunized by subcutaneous (s.c.) injection with NP-OVA and NP-p24 formulations along with several control formulations. These results suggested that with multiple antigens, covalent attachment of the antigen to the NP significantly enhanced antigen-specific immune responses. This facile covalent conjugation and incorporation method may be utilized to further incorporate other protein antigens, even multiple antigens, into an enhanced vaccine delivery system. PMID:20837122

  9. Original article Permeability of milk protein antigens across the intestinal

    E-print Network

    Paris-Sud XI, Université de

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

  10. The ceramide transporter and the Goodpasture antigen binding protein: one protein--one function?

    PubMed

    Mencarelli, Chiara; Losen, Mario; Hammels, Caroline; De Vry, Jochen; Hesselink, Matthijs K C; Steinbusch, Harry W M; De Baets, Marc H; Martínez-Martínez, Pilar

    2010-06-01

    The Goodpasture antigen-binding protein (GPBP) and its splice variant the ceramide transporter (CERT) are multifunctional proteins that have been found to play important roles in brain development and biology. However, the function of GPBP and CERT is controversial because of their involvement in two apparently unrelated research fields: GPBP was initially isolated as a protein associated with collagen IV in patients with the autoimmune disease Goodpasture syndrome. Subsequently, a splice variant lacking a serine-rich domain of 26 amino acids (GPBPDelta26) was found to mediate the cytosolic transport of ceramide and was therefore (re)named CERT. The two splice forms likely carry out different functions in specific sub-cellular localizations. Selective GPBP knockdown induces extensive apoptosis and tissue loss in the brain of zebrafish. GPBP/GPBPDelta26 knock-out mice die as a result of structural and functional defects in endoplasmic reticulum and mitochondria. Because both mitochondria and ceramide play an important role in many biological events that regulate neuronal differentiation, cellular senescence, proliferation and cell death, we propose that GPBP and CERT are pivotal in neurodegenerative processes. In this review, we discuss the current state of knowledge on GPBP and CERT, including the molecular and biochemical characterization of GPBP in the field of autoimmunity as well as the fundamental research on CERT in ceramide transport, biosynthesis, localization, metabolism and cell homeostasis. PMID:20236389

  11. Prediction of protein antigenic determinants from amino acid sequences

    SciTech Connect

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

    1981-06-01

    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.

  12. The Role of Heat Shock Proteins in Antigen Cross Presentation

    PubMed Central

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

    2012-01-01

    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

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

    PubMed Central

    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

    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

  14. Unique gene organization: alternative splicing in Drosophila produces two structurally unrelated proteins

    Microsoft Academic Search

    Randy C. Mottus; Ian P. Whitehead; Michael O'Grady; Richard E. Sobel; Rod H. L. Burr; George B. Spiegelman; Thomas A. Grigliatti

    1997-01-01

    The Ub80 gene in eukaryotes produces a ubiquitin fusion protein in which ubiquitin is fused in frame to a tail protein (Redman and Rechsteiner, 1988; Finley et al., 1989; Barrio et al., 1994). The tail protein is incorporated into the ribosome, and ubiquitin is thought to act as a chaperone. The DUb80 gene of Drosophila melanogaster was cloned by Barrio

  15. Extracellular Release of Antigenic Proteins by Helicobacter pylori

    PubMed Central

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

    1998-01-01

    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

  16. Identification of Antigenic Proteins of the Nosocomial Pathogen Klebsiella pneumoniae

    PubMed Central

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

    2014-01-01

    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

  17. Protein antigens of Streptococcus mutans: purification and properties of a double antigen and its protease-resistant component.

    PubMed Central

    Russell, M W; Bergmeier, L A; Zanders, E D; Lehner, T

    1980-01-01

    A surface protein antigen of Streptococcus mutans having two sets of antigenic determinants (antigens I and II) was purified by column chromatography from culture supernatants of S. mutans serotype c. The protease-resistant component, antigen II, was purified from pronase-digested antigen I/II. The antigens were analyzed chemically and immunologically, and their physicochemical properties were investigated. Antigen I/II consisted of more than 80% protein, and its peptide chain molecular weight was estimated to be 185,000. Antigen II consisted of approximately 60% protein, with a peptide chain molecular weight of 48,000. Antisera to antigens I/II and II were raised in rabbits and used to investigate the presence of the antigens in cells of other streptococci. This indicated that not only serotype c but also serotypes e and f possessed antigen I and II determinants, whereas serotypes a, d, and g possessed a determinant related to antigen I but not one related to antigen II. Images Fig. 3 Fig. 5 Fig. 6 Fig. 7 PMID:6995311

  18. Antigenic protein fractions of Metagonimus yokogawai reacting with patient sera.

    PubMed

    Lee, S C; Chung, Y B; Kong, Y; Kang, S Y; Cho, S Y

    1993-03-01

    Antibody test is sometimes necessary for the diagnosis of acute human metagonimiasis because eggs may not be detected in stool. The antibody test (ELISA) was evaluated for its significance by reacting human sera from clinically diagnosed metagonimiasis, fascioliasis, clonorchiasis and paragonimiasis with 4 crude extracts of Metagonimus yokogawai (metacercariae), adults of Fasciola hepatica, Clonorchis sinensis and Paragonimus westermani. By ELISA, 10 of 11 metagonimiasis sera showed the highest absorbance (abs.) to the homologous antigen. Cross reactions to M. yokogawai antigen occurred most frequently in clonorchiasis sera. The antigenic protein fractions in M. yokogawai metacercarial extract were observed by SDS-PAGE/immunoblot using patients and control sera together with experimental cat sera. Out of 14 protein bands in the extract, 11 bands were reacting. Cross reacting bands to other trematodiasis sera were frequently observed. Of the reacting bands, 66 and 22 kDa proteins were recognized as specific for metagonimiasis. PMID:8512898

  19. Immunological Properties of Hepatitis B Core Antigen Fusion Proteins

    NASA Astrophysics Data System (ADS)

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

    1990-04-01

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

  20. Comparative Antigenicity and Immunogenicity of Hepadnavirus Core Proteins

    Microsoft Academic Search

    Jean-Noel Billaud; Darrell Peterson; Florian Schodel; Antony Chen; Matti Sallberg; Fermin Garduno; Phillip Goldstein; Wendy McDowell; Janice Hughes; Joyce Jones; David Milich

    2005-01-01

    The hepatitis B virus core protein (HBcAg) is a uniquely immunogenic particulate antigen and as such has been used as a vaccine carrier platform. The use of other hepadnavirus core proteins as vaccine carriers has not been explored. To determine whether the rodent hepadnavirus core proteins derived from the woodchuck (WHcAg), ground squirrel (GScAg), and arctic squirrel (AScAg) viruses possess

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

    E-print Network

    Teytelman, Leonid

    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 ...

  2. Development of a novel, quantitative protein microarray platform for the multiplexed serological analysis of autoantibodies to cancer-testis antigens.

    PubMed

    Beeton-Kempen, Natasha; Duarte, Jessica; Shoko, Aubrey; Serufuri, Jean-Michel; John, Thomas; Cebon, Jonathan; Blackburn, Jonathan

    2014-10-15

    The cancer-testis antigens are a group of unrelated proteins aberrantly expressed in various cancers in adult somatic tissues. This aberrant expression can trigger spontaneous immune responses, a phenomenon exploited for the development of disease markers and therapeutic vaccines. However, expression levels often vary amongst patients presenting the same cancer type, and these antigens are therefore unlikely to be individually viable as diagnostic or prognostic markers. Nevertheless, patterns of antigen expression may provide correlates of specific cancer types and disease progression. Herein, we describe the development of a novel, readily customizable cancer-testis antigen microarray platform together with robust bioinformatics tools, with which to quantify anti-cancer testis antigen autoantibody profiles in patient sera. By exploiting the high affinity between autoantibodies and tumor antigens, we achieved linearity of response and an autoantibody quantitation limit in the pg/mL range-equating to a million-fold serum dilution. By using oriented attachment of folded, recombinant antigens and a polyethylene glycol microarray surface coating, we attained minimal non-specific antibody binding. Unlike other proteomics methods, which typically use lower affinity interactions between monoclonal antibodies and tumor antigens for detection, the high sensitivity and specificity realized using our autoantibody-based approach may facilitate the development of better cancer biomarkers, as well as potentially enabling pre-symptomatic diagnosis. We illustrated the usage of our platform by monitoring the response of a melanoma patient cohort to an experimental therapeutic NY-ESO-1-based cancer vaccine; inter alia, we found evidence of determinant spreading in individual patients, as well as differential CT antigen expression and epitope usage. PMID:24604332

  3. Cell surface protein antigen from Wolinella recta ATCC 33238T.

    PubMed Central

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

    1989-01-01

    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

  4. Survival after transplantation of unrelated donor umbilical cord blood is comparable to that of human leukocyte antigen-matched unrelated donor bone marrow: results of a matched-pair analysis

    Microsoft Academic Search

    Juliet N. Barker; Stella M. Davies; Todd DeFor; Norma K. C. Ramsay; Daniel J. Weisdorf; John E. Wagner

    2001-01-01

    Umbilical cord blood (UCB) is being in- creasingly used for hematopoietic stem cell transplantation and has been associ- ated with a reduced incidence of severe graft-versus-host disease (GVHD). To fur- ther investigate the relative merits of unre- lated donor UCB versus bone marrow (BM), a matched-pair analysis comparing the outcomes of recipients of 0 to 3 human leukocyte antigen (HLA)-mis-

  5. Analysis of Proteins Induced by the Salmonella typhimurium Phage P221, a Hybrid between Serologically and Morphologically Unrelated Phages P22 and Fels 1

    Microsoft Academic Search

    MARY L. DROFFNER; NOBUTO YAMAMOTO

    1982-01-01

    SUMMARY The Salmonella phage P221 is a hybrid between the morphologically and serologically unrelated Salmonella phages P22 and Fels 1. P221 carries about 30% of P22 DNA containing a number of the early genes. Head and tail proteins are morphologically and serologically indistinguishable from those of Fels 1. The early proteins synthesized in Salmonella typhimurium strain Q 1 after P221

  6. Antigenic proteins of Helicobacter pylori of potential diagnostic value.

    PubMed

    Khalilpour, Akbar; Santhanam, Amutha; Wei, Lee Chun; Saadatnia, Geita; Velusamy, Nagarajan; Osman, Sabariah; Mohamad, Ahmad Munir; Noordin, Rahmah

    2013-01-01

    Helicobacter pylori antigen was prepared from an isolate from a patient with a duodenal ulcer. Serum samples were obtained from culture-positive H. pylori infected patients with duodenal ulcers, gastric ulcers and gastritis (n=30). As controls, three kinds of sera without detectable H. pylori IgG antibodies were used: 30 from healthy individuals without history of gastric disorders, 30 from patients who were seen in the endoscopy clinic but were H. pylori culture negative and 30 from people with other diseases. OFF-GEL electrophoresis, SDS-PAGE and Western blots of individual serum samples were used to identify protein bands with good sensitivity and specificity when probed with the above sera and HRP-conjugated anti-human IgG. Four H. pylori protein bands showed good (? 70%) sensitivity and high specificity (98-100%) towards anti-Helicobacter IgG antibody in culture- positive patients sera and control sera, respectively. The identities of the antigenic proteins were elucidated by mass spectrometry. The relative molecular weights and the identities of the proteins, based on MALDI TOF/ TOF, were as follows: CagI (25 kDa), urease G accessory protein (25 kDa), UreB (63 kDa) and proline/pyrroline- 5-carboxylate dehydrogenase (118 KDa). These identified proteins, singly and/or in combinations, may be useful for diagnosis of H. pylori infection in patients. PMID:23679248

  7. Antigenic differences among Campylobacter fetus S-layer proteins.

    PubMed Central

    Dubreuil, J D; Kostrzynska, M; Austin, J W; Trust, T J

    1990-01-01

    Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of S-layer proteins extracted from Campylobacter fetus strains by using acid glycine buffer showed that the predominant S-layer proteins of different strains had subunit molecular weights in the range of 90,000 to 140,000. Electron microscopy revealed oblique S-layer lattices with a spacing of approximately 5.6 nm (gamma = 75 degrees) on wild-type strains VC1, VC119, VC202, and VC203. Three variants of C. fetus VC119 producing a predominant S-layer subunit protein of different molecular weight (Mr) from that of the parent were also examined. Each variant produced an oblique lattice morphologically indistinguishable from that of the parent. Amino-terminal sequence analysis showed that the S-layer proteins of the VC119 parent and variants were identical up to residue 18 and that this sequence differed from but was related to the first 16 N-terminal residues shared by the S-layer proteins of the three other wild-type C. fetus isolates. Western immunoblot analysis with an antiserum prepared to the VC119 protein and an antiserum prepared to C. fetus 84-40 LP (Z. Pei, R. T. Ellison, R. V. Lewis, and M. J. Blaser, J. Biol. Chem. 263:6416-6420, 1988) showed that strains of C. fetus were capable of producing S-layer proteins with at least four different antigenic specificities. Immunoelectron microscopy with antiserum to the VC119 S-layer protein showed that C. fetus cultures contained cells with immunoreactive oblique S-layer lattices as well as cells with oblique S-layer lattices which did not bind antibody. This suggests that C. fetus S-layer proteins undergo antigenic variation. Thermal denaturation experiments indicated that the antigenicity conferred by the surface-exposed C. fetus S-layer epitopes was unusually resistant to heat, and the thermal stability appeared to be due to the highly organized lattice structure of the S. layer. Protease digestion of purified VC119 S-layer protein revealed a trypsin-, chymotrypsin-, and endoproteinase Glu-C-resistant domain with an apparent Mr of 110,000, which carried the majority of the epitopes of the S-layer protein, and a small enzyme-sensitive domain. The trypsin- and chymotrypsin-resistant polypeptides shared an overlapping sequence which differed from the N-terminal sequence of the intact S-layer protein. Images PMID:1697574

  8. Expression of the heat-modifiable major outer membrane protein of Haemophilus influenzae type b is unrelated to virulence.

    PubMed Central

    Hanson, M. S.; Cope, L. D.; Hansen, E. J.

    1989-01-01

    The heat-modifiable major outer membrane protein (P1) of Haemophilus influenzae type b (Hib) has been shown to be both exposed on the cell surface and capable of inducing the synthesis of antibodies protective against experimental Hib disease. Chemical mutagenesis of a recombinant plasmid containing the Hib gene encoding P1 resulted in inactivation of P1 expression by this plasmid. The mutated P1 gene was transformed into Hib to obtain an isogenic mutant lacking only the ability to synthesize this surface protein. In addition, the P1 gene was inserted into a plasmid shuttle vector and used to construct a recombinant Hib strain that overexpressed the P1 protein. Lack of P1 expression did not affect the ability of Hib to grow in vitro. Neither the absence nor the overproduction of P1 affected expression of capsular polysaccharide and lipooligosaccharide by Hib. The P1-negative mutant and the P1-overexpressing strain were both as susceptible to the bactericidal activity of pooled normal human serum as was the wild-type parent strain, while the P1-negative mutant was as resistant to the bactericidal activity of normal infant rat serum as was the wild-type parent strain. The P1-negative mutant was no less virulent than was the wild-type parent strain in an animal model system, such that both the numbers of animals infected by this mutant and the mean magnitudes of the resultant bacteremias were essentially identical to those obtained with challenge by the wild-type parent strain. Similarly, overexpression of P1 did not detectably affect the virulence of Hib. These data indicate that this protective protein antigen plays no detectable role in the expression of virulence by Hib, as assessed in an animal model system. Images PMID:2785959

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

    PubMed

    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

    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

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

    PubMed

    Pizza, Mariagrazia; Donnelly, John; Rappuoli, Rino

    2008-12-30

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

  11. Toxoplasma gondii excretory secretory antigenic proteins of diagnostic potential.

    PubMed

    Saadatnia, Geita; Mohamed, Zeehaida; Ghaffarifar, Fatemeh; Osman, Emelia; Moghadam, Zohreh Kazemi; Noordin, Rahmah

    2012-01-01

    Infection with Toxoplasma gondii is widespread and important in humans, especially pregnant women and immunosuppressed patients. A panel of tests is usually required for diagnosis toxoplasmosis. Excretory secretory antigen (ESA) is highly immunogenic, and thus it is a good candidate for investigation into new infection markers. ESA was prepared from tachyzoites of RH strain of T. gondii by mice intraperitoneal infection. Sera were obtained from several categories of individuals who differed in their status of anti-Toxoplasma IgM, IgG and IgG avidity antibodies. The ESA was subjected to SDS-PAGE, two-dimensional gel electrophoresis and Western blot analysis. Antigenic bands of approximate molecular weights of 12, 20 and 30 kDa, when probed with anti-human IgM-HRP and IgA-HRP, showed good potential as infection markers. The highest sensitivity of the bands was 98.7% with combination of IgM and IgA blots with sera of patients with anti-Toxoplasma IgM+ IgG+. The specificities were 84% and 70% with sera from other infections and healthy controls in IgM blots and IgA blots respectively. By mass spectrometry, the 12 kDa protein was identified as thioredoxin. The two top proteins identified for 20 kDa molecule were microneme protein 10 and dense granule protein 7; whereas that for 30 kDa were phosphoglycerate mutase 1 and phosphoglycerate mutase. PMID:22151308

  12. Antigenic structure of Coxiella burnetii. A comparison of lipopolysaccharide and protein antigens as vaccines against Q fever.

    PubMed

    Williams, J C; Hoover, T A; Waag, D M; Banerjee-Bhatnagar, N; Bolt, C R; Scott, G H

    1990-01-01

    The antigenic structure of Coxiella burnetii is being investigated by identifying both external and internal cellular epitopes of the morphologic cell types. Both the phase I lipopolysaccharide (LPSI) and several surface proteins are candidates for the development of subunit multivalent vaccines. The protective efficacy of purified LPSI was demonstrated in A/J mice. The purified LPSI preparations contained residual peptides detected by amino acid analysis. Therefore, the protection afforded by LPSI may be, in part, due to the presence of peptides. The purification of proteins free of LPSI must be accomplished before the protective efficacy of proteins or peptides can be established. We have identified three proteins that are both antigenic and immunogenic, as indicated by either enzyme immunoassay, radioimmunoprecipitation, immunoblot assay, or lymphocyte transformation. A 62-kDa protein antigen encoded by the htpB gene of C. burnetii was analyzed for immunogenicity. The purified protein antigen was immunogenic, as it elicited specific antibodies and performed as recall antigen in lymphocyte stimulation assays. The antigen was not detected on the surface of phase I cells but was highly represented on the surface of phase II cells. Therefore, the protein may not be a good candidate for vaccine development. The diagnostic utility of the 62-kDa protein antigen lies in the fact that convalescent and chronic Q fever sera from human patients reacted with the antigen, whereas acute sera did not. Although the 62-kDa protein is a "common antigen," specific peptide-based diagnostic reagents may be useful in the detection of Q fever disease progression. A major surface protein (P1) of roughly 29.5 kDa was purified from the phase I Nine Mile (clone 7) strain. No LPSI was detected in the P1 preparation by three different LPSI monoclonal antibodies. Monoclonal antibodies prepared against P1 were effective in localizing the protein on the cell surface, in the cell wall, and associated with the peptidoglycan of large cells of C. burnetii. Small, pressure-resistant cells did not contain P1. Mice immunized with two 25-micrograms injections of LPSI produced antibodies against LPSI and phase I whole cells. No antibody was detected against phase II whole cells. Immunization with P1 induced antibody against the LPSI fraction and phase I and phase II whole cells. P1 was more effective than LPSI in reducing the number of infectious C. burnetii in the spleens of challenged mice. The gene encoding another protein (P2) recognized by P1 monoclonal antibodies was cloned and sequenced.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2378463

  13. Subdominant Outer Membrane Antigens in Anaplasma marginale: Conservation, Antigenicity, and Protective Capacity Using Recombinant Protein

    PubMed Central

    Ducken, Deirdre R.; Brown, Wendy C.; Alperin, Debra C.; Brayton, Kelly A.; Reif, Kathryn E.; Turse, Joshua E.; Palmer, Guy H.; Noh, Susan M.

    2015-01-01

    Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP) extracts or a defined surface protein complex reproducibly induce protective immunity. However, there are several knowledge gaps limiting progress in vaccine development. First, are these OMPs conserved among the diversity of A. marginale strains circulating in endemic regions? Second, are the most highly conserved outer membrane proteins in the immunogens recognized by immunized and protected animals? Lastly, can this subset of OMPs recognized by antibody from protected vaccinates and conserved among strains recapitulate the protection of outer membrane vaccines? To address the first goal, genes encoding OMPs AM202, AM368, AM854, AM936, AM1041, and AM1096, major subdominant components of the outer membrane, were cloned and sequenced from geographically diverse strains and isolates. AM202, AM936, AM854, and AM1096 share 99.9 to 100% amino acid identity. AM1041 has 97.1 to 100% and AM368 has 98.3 to 99.9% amino acid identity. While all four of the most highly conserved OMPs were recognized by IgG from animals immunized with outer membranes, linked surface protein complexes, or unlinked surface protein complexes and shown to be protected from challenge, the highest titers and consistent recognition among vaccinates were to AM854 and AM936. Consequently, animals were immunized with recombinant AM854 and AM936 and challenged. Recombinant vaccinates and purified outer membrane vaccinates had similar IgG and IgG2 responses to both proteins. However, the recombinant vaccinates developed higher bacteremia after challenge as compared to adjuvant-only controls and outer membrane vaccinates. These results provide the first evidence that vaccination with specific antigens may exacerbate disease. Progressing from the protective capacity of outer membrane formulations to recombinant vaccines requires testing of additional antigens, optimization of the vaccine formulation and a better understanding of the protective immune response. PMID:26079491

  14. Subdominant Outer Membrane Antigens in Anaplasma marginale: Conservation, Antigenicity, and Protective Capacity Using Recombinant Protein.

    PubMed

    Ducken, Deirdre R; Brown, Wendy C; Alperin, Debra C; Brayton, Kelly A; Reif, Kathryn E; Turse, Joshua E; Palmer, Guy H; Noh, Susan M

    2015-01-01

    Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP) extracts or a defined surface protein complex reproducibly induce protective immunity. However, there are several knowledge gaps limiting progress in vaccine development. First, are these OMPs conserved among the diversity of A. marginale strains circulating in endemic regions? Second, are the most highly conserved outer membrane proteins in the immunogens recognized by immunized and protected animals? Lastly, can this subset of OMPs recognized by antibody from protected vaccinates and conserved among strains recapitulate the protection of outer membrane vaccines? To address the first goal, genes encoding OMPs AM202, AM368, AM854, AM936, AM1041, and AM1096, major subdominant components of the outer membrane, were cloned and sequenced from geographically diverse strains and isolates. AM202, AM936, AM854, and AM1096 share 99.9 to 100% amino acid identity. AM1041 has 97.1 to 100% and AM368 has 98.3 to 99.9% amino acid identity. While all four of the most highly conserved OMPs were recognized by IgG from animals immunized with outer membranes, linked surface protein complexes, or unlinked surface protein complexes and shown to be protected from challenge, the highest titers and consistent recognition among vaccinates were to AM854 and AM936. Consequently, animals were immunized with recombinant AM854 and AM936 and challenged. Recombinant vaccinates and purified outer membrane vaccinates had similar IgG and IgG2 responses to both proteins. However, the recombinant vaccinates developed higher bacteremia after challenge as compared to adjuvant-only controls and outer membrane vaccinates. These results provide the first evidence that vaccination with specific antigens may exacerbate disease. Progressing from the protective capacity of outer membrane formulations to recombinant vaccines requires testing of additional antigens, optimization of the vaccine formulation and a better understanding of the protective immune response. PMID:26079491

  15. Antibodies to citrullinated protein antigens (ACPAs): clinical and pathophysiologic significance.

    PubMed

    Demoruelle, M Kristen; Deane, Kevin

    2011-10-01

    Antibodies to citrullinated protein antigens (ACPAs) are highly specific for rheumatoid arthritis (RA) and are useful in the diagnosis of RA as well as the prediction of the course and outcomes of disease. Multiple methodologies exist for measuring ACPAs, including the widely available tests for anticyclic citrullinated peptide antibodies and for antibodies to mutated/modified citrullinated vimentin. These methodologies overall have similar diagnostic accuracies for RA, although there is some variability. The discovery of ACPAs and the biology of citrullination have also led to important advances in the understanding of the pathophysiology and development of RA, especially regarding the relationship between potential genetic and environmental risk factors for RA. Going forward, research into autoimmunity to citrullinated proteins may help identify the specific etiology of RA and provide approaches for the prediction of future risk of disease, and ultimately prevention of RA. PMID:21713412

  16. Antibodies to Citrullinated Protein Antigens (ACPAs): Clinical and Pathophysiologic Significance

    PubMed Central

    Demoruelle, M. Kristen; Deane, Kevin

    2014-01-01

    Antibodies to citrullinated protein antigens (ACPAs) are highly specific for rheumatoid arthritis (RA) and are useful in the diagnosis of RA as well as the prediction of the course and outcomes of disease. Multiple methodologies exist for measuring ACPAs, including the widely available tests for anticyclic citrullinated peptide antibodies and for antibodies to mutated/modified citrullinated vimentin. These methodologies overall have similar diagnostic accuracies for RA, although there is some variability. The discovery of ACPAs and the biology of citrullination have also led to important advances in the understanding of the pathophysiology and development of RA, especially regarding the relationship between potential genetic and environmental risk factors for RA. Going forward, research into autoimmunity to citrullinated proteins may help identify the specific etiology of RA and provide approaches for the prediction of future risk of disease, and ultimately prevention of RA. PMID:21713412

  17. 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

    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

    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

  18. Recombinant complexes of antigen with stress proteins are potent CD8 T-cell-stimulating immunogens

    Microsoft Academic Search

    Andreas Wieland; Markus Denzel; Erika Schmidt; Stefan Kochanek; Florian Kreppel; Jörg Reimann; Reinhold Schirmbeck

    2008-01-01

    Heat shock proteins (Hsp) of the Hsp70\\/90 families facilitate cellular immune responses to antigenic peptides or proteins\\u000a bound to them and have therefore been used as vaccine vehicles. We developed an expression system in which chimeric proteins\\u000a with an Hsp-capturing, viral J domain fused to diverse antigen-encoding sequences form stable complexes with eukaryotic (Hsp70,\\u000a Hsp73) or bacterial (DnaK) stress proteins

  19. Towards preserving the immunogenicity of protein antigens carried by nanoparticles while avoiding the cold chain.

    PubMed

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

    2010-06-30

    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 degrees 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

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

    PubMed Central

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

    2010-01-01

    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

  1. 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

    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

    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

  2. One-antigen mismatched related versus HLA-matched unrelated donor hematopoietic stem cell transplantation in adults with acute leukemia: Center for International Blood and Marrow Transplant Research results in the era of molecular HLA typing.

    PubMed

    Valcárcel, David; Sierra, Jorge; Wang, Tao; Kan, Fangyu; Gupta, Vikas; Hale, Gregory A; Marks, David I; McCarthy, Philip L; Oudshoorn, Machteld; Petersdorf, Effie W; Ringdén, Olle; Setterholm, Michelle; Spellman, Stephen R; Waller, Edmund K; Gajewski, James L; Marino, Susana R; Senitzer, David; Lee, Stephanie J

    2011-05-01

    Approximately 13% of patients lacking an HLA-identical sibling have a one-antigen-mismatched related donor (MMRD). Historically, outcomes from the use of a one-antigen MMRD were considered equivalent to those from the use of a matched unrelated donor (UD). Recent improvements in UD stem cell transplantation (SCT) resulting from better molecular HLA matching justifies investigating whether UD should be preferred over MMRD in adult patients with acute leukemia. Here, we compared the outcomes of MMRD (n = 89) and HLA-A, -B, -C, and -DRB1 allele-matched UD (n = 700) SCT reported to the Center for International Blood and Marrow Transplant Research between 1995 and 2005. The patients underwent transplantation for acute myelogenous leukemia or acute lymphoblastic leukemia in first or second complete remission. Donor type was not associated with hematologic recovery. Univariate and multivariate comparisons of MMRD versus HLA-matched UD transplants showed no statistically significant differences in overall survival, disease-free survival, treatment-related mortality, relapse, or 100-day grade III-IV acute graft-versus-host disease (GVHD). MMRD SCT was associated with a lower rate of chronic GVHD at 1 year (35% vs 47%; P = .03), which was confirmed by multivariate analysis (relative risk, 0.58; 95% confidence interval, 0.39-0.85; P < .01). According to our data, HLA-matched UD and MMRD SCT are associated with comparable survival. Given that less chronic GVHD was observed in the MMRD transplantations, this option, when available, remains the first choice in patients with acute leukemia without an HLA-identical sibling in need of allogeneic SCT. PMID:20674756

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

    PubMed

    Okuda, Kenji; Shikanai, Toshiharu

    2012-06-01

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

  4. Heat shock protein bystander antigens for peptide immunotherapy in autoimmune disease.

    PubMed

    Zonneveld-Huijssoon, E; Albani, S; Prakken, B J; van Wijk, F

    2013-01-01

    Mucosal administration of an antigen eliciting bystander suppression at the site of inflammation results in effective antigen-specific immunotherapy for autoimmune diseases. Heat shock proteins are bystander antigens that are effective in peptide-specific immunotherapy in both experimental and human autoimmune disease. The efficacy of preventive peptide immunotherapy is increased by enhancing peptide-specific immune responses with proinflammatory agents. Combining peptide-specific immunotherapy with general suppression of inflammation may improve its therapeutic effect. PMID:23199319

  5. Heat shock protein bystander antigens for peptide immunotherapy in autoimmune disease

    PubMed Central

    Zonneveld-Huijssoon, E; Albani, S; Prakken, B J; van Wijk, F

    2013-01-01

    Mucosal administration of an antigen eliciting bystander suppression at the site of inflammation results in effective antigen-specific immunotherapy for autoimmune diseases. Heat shock proteins are bystander antigens that are effective in peptide-specific immunotherapy in both experimental and human autoimmune disease. The efficacy of preventive peptide immunotherapy is increased by enhancing peptide-specific immune responses with proinflammatory agents. Combining peptide-specific immunotherapy with general suppression of inflammation may improve its therapeutic effect. PMID:23199319

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

    PubMed Central

    2014-01-01

    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

  7. Major Structural Proteins of Type B, Type C, and Type D Oncoviruses Share Interspecies Antigenic Determinants

    Microsoft Academic Search

    Mariano Barbacid; Linda K. Long; Stuart A. Aaronson

    1980-01-01

    Interspecies antigenic determinants have been used as markers to study the evolution of oncoviruses. By radioimmunologic techniques, it has been possible to detect crossreactivities among the major structural proteins of prototype viruses representing each of the three major Oncovirinae genera. Two different subsets of interspecies antigenic determinants were demonstrated. One encompasses known type B and type D viruses; the other

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

    Argos, P; Fuller, S D

    1988-01-01

    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

  10. Microsomal triglyceride transfer protein lipidation and control of CD1d on antigen-presenting cells

    Microsoft Academic Search

    Stephanie K. Dougan; Azucena Salas; Paul Rava; Amma Agyemang; Arthur Kaser; Jamin Morrison; Archana Khurana; Mitchell Kronenberg; Caroline Johnson; Mark Exley; M. Mahmood Hussain; Richard S. Blumberg

    Microsomal triglyceride transfer protein (MTP), an endoplasmic reticulum (ER) chaperone that loads lipids onto apolipoprotein B, also regulates CD1d presentation of glycolipid antigens in the liver and intestine. We show MTP RNA and protein in antigen-presenting cells (APCs) by reverse transcription-polymerase chain reaction and by immunoblotting of mouse liver mononuclear cells and mouse and human B cell lines. Functional MTP,

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

    PubMed Central

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

    2010-01-01

    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

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

    PubMed Central

    2014-01-01

    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

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

    PubMed

    Fiorucci, Sébastien; Zacharias, Martin

    2014-01-01

    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

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

    PubMed Central

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

    2014-01-01

    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

  15. Antigenic Mapping of the Surface Proteins of Infectious Hepatitis B Virus Particles

    Microsoft Academic Search

    M. A. Petit; F. Capel; M. M. Riottot; C. Dauguet; J. Pillot

    1987-01-01

    SUMMARY To identify further the surface proteins of the native virus, hepatitis B virus (HBV) particles purified from HBe antigen (Ag)-positive human sera were used as immunogens to produce murine monoclonal antibody (MAb)-secreting hybridomas. The specific binding of antibodies to the HBV envelope (env) proteins was determined in indirect radioimmunoassay and by Western blot analysis. Six MAbs directed against major

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

    Microsoft Academic Search

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

    2001-01-01

    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.

  17. Autoantigenicity of Ro\\/SSA Antigen is Related to a Nucleocapsid Protein of Vesicular Stomatitis Virus

    Microsoft Academic Search

    R. Hal Scofield; John B. Harley

    1991-01-01

    The fine specificity of a reference human anti-Ro\\/SSAautoantibody-containing serum has been analyzed by using sequential overlapping octapeptides from the human 60-kDa Ro\\/SSA antigen. From preliminary data, the most antigenic octapeptide in the carboxyl-terminal 120 amino acid residues of Ro\\/SSA shares seven of eight amino acids with the nucleocapsid (N) protein from the Indiana serotype of vesicular stomatitis virus. A sequence

  18. In vitro human immune reactivity of fast protein liquid chromatography fractionated Paracoccidioides brasiliensis soluble antigens

    Microsoft Academic Search

    Susana N Diniz; Patrícia S Cisalpino; Matilde C Koury; Glaucia M. Q Andrade; Maria G. S Nogueira; Alfredo M Goes

    1999-01-01

    Soluble antigens of Paracoccidioides brasiliensis yeast cells (PbAg) were fractionated in a fast protein liquid chromatography (FPLC) system, using Q-Sepharose anion-exchange resin, in order to characterize antigenic fractions that could elicit cell reactivity and antibody recognition in human paracoccidioidomycosis (PCM). PbAg fractions were eluted by 20 mM Tris-HCl solution (pH 9.6) with an increasing gradient up to 1 M NaCl. The

  19. A 125 I-protein A-binding assay detecting antibodies to cell surface antigens

    Microsoft Academic Search

    R. Fäldt; J. Ankerst

    1983-01-01

    Summary  A 125I-protein A-binding assay detecting antibodies to cell surface antigens on human blood cells was developed and evaluated using sera from multitransfused nonleukemic patients sensitized against HLA antigens. The binding assay was found to be reproducible and more sensitive than conventional HLA testing. Seven patients with acute myelogenous leukemia and two patients with acute lymphoblastic leukemia successfully treated by chemotherapy

  20. Human Dermal Dendritic Cells Process and Present Soluble Protein Antigens

    Microsoft Academic Search

    Frank O. Nestle; Luis Filgueira; Brian J. Nickoloff; Günter Burg

    1998-01-01

    Recently, a novel type of dendritic antigen-presenting cell has been identified in the dermis of normal human and mouse skin. These dermal dendritic cells (DDC) occur in higher numbers than epidermal Langerhans cells, represent a distinct differentiation pathway of dendritic cells, and are as potent as Langerhans cells in the activation of superantigen specific T cells. As yet, nothing is

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

    PubMed

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

    2010-01-01

    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

  2. Strong Antibody Responses Induced by Protein Antigens Conjugated onto the Surface of Lecithin-Based Nanoparticles

    PubMed Central

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

    2009-01-01

    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 200 nm. 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

  3. A Mutant Library Approach to Identify Improved Meningococcal Factor H Binding Protein Vaccine Antigens

    PubMed Central

    Konar, Monica; Rossi, Raffaella; Walter, Helen; Pajon, Rolando; Beernink, Peter T.

    2015-01-01

    Factor H binding protein (FHbp) is a virulence factor used by meningococci to evade the host complement system. FHbp elicits bactericidal antibodies in humans and is part of two recently licensed vaccines. Using human complement Factor H (FH) transgenic mice, we previously showed that binding of FH decreased the protective antibody responses to FHbp vaccination. Therefore, in the present study we devised a library-based method to identify mutant FHbp antigens with very low binding of FH. Using an FHbp sequence variant in one of the two licensed vaccines, we displayed an error-prone PCR mutant FHbp library on the surface of Escherichia coli. We used fluorescence-activated cell sorting to isolate FHbp mutants with very low binding of human FH and preserved binding of control anti-FHbp monoclonal antibodies. We sequenced the gene encoding FHbp from selected clones and introduced the mutations into a soluble FHbp construct. Using this approach, we identified several new mutant FHbp vaccine antigens that had very low binding of FH as measured by ELISA and surface plasmon resonance. The new mutant FHbp antigens elicited protective antibody responses in human FH transgenic mice that were up to 20-fold higher than those elicited by the wild-type FHbp antigen. This approach offers the potential to discover mutant antigens that might not be predictable even with protein structural information and potentially can be applied to other microbial vaccine antigens that bind host proteins. PMID:26057742

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

    Microsoft Academic Search

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

    2005-01-01

    The cDNA library of Clonorchis sinensis was screened for genes encoding antigenic proteins by using sera from clonorchiasis patients. A gene of 888 bp encoding a 28-kDa protein (Cs28) was cloned and found to contain a high percentage of glycine (20%), tyrosine (11%), and lysine (11%). The amino acid sequence of Cs28 showed 60% homology with the vitelline B precursor protein

  5. Antigenic and genetic characterization of the fusion (F) protein of mumps virus strains

    Microsoft Academic Search

    T. Tecle; B. Johansson; Z. Yun; C. Örvell

    2000-01-01

    Summary.  ?Twelve strains of mumps virus, belonging to the A, C and D genotypes of the small hydrophobic (SH) protein gene, were investigated\\u000a by nucleotide sequencing of the fusion protein gene. The nucleotide sequences and deduced amino acid sequences were aligned\\u000a and compared with previously reported sequences of the gene. In addition an antigenic comparison between the F protein of\\u000a different

  6. An analysis of bison erythrocyte antigens and blood proteins 

    E-print Network

    Zamora, Linda Elia

    1983-01-01

    -chairmen of Advisory Committee: Or. J. Caldwell Or. J. Templeton Six hundred and sixty four blood samples were collected from eight herds of American b1son. Hemolyt1c tests were made using 140 cattle blood typing reagents specific for 55 antigenic Factors and 20.... . Hemolytic Testing of Erythrocytes. . . . . . . . Isaunization Preparation and Technique. . . Collection and Processing of Antisera. . . . Antisera Analysis. Absorptions and Elutions Preparation of Blood Typing Reagents. . . . . Heeoglobin Typing...

  7. Nodal proteins are target antigens in Guillain-Barré syndrome.

    PubMed

    Devaux, Jérôme J; Odaka, Masaaki; Yuki, Nobuhiro

    2012-03-01

    Neurofascin-186 (NF186), neuronal cell adhesion molecule (NrCAM), and gliomedin are adhesion molecules playing a central role in the formation of nodes of Ranvier. In Guillain-Barré syndrome (GBS), immune attack toward the nodes may participate in the disabilities. Autoantibodies to NF186 and gliomedin have been detected in a rat model of GBS. Here, we investigated the prevalence of antibodies against nodal adhesion molecules in patients with GBS or chronic inflammatory demyelinating polyneuropathy (CIDP). Sera from 100 GBS patients, 50 CIDP patients, 80 disease controls, and 50 healthy controls were tested for their ability to bind the nodes of Ranvier. To characterize the antigens, we performed cell binding assays against NF186, gliomedin, contactin, and NrCAM. We found that 43% of patients with GBS and 30% of patients with CIDP showed IgG fixation at nodes or paranodes. In eight patients with GBS or CIDP, we identified that IgG antibodies recognized the native extracellular domain of NF186, gliomedin, or contactin. Also, 29 patients showed IgM against nodal adhesion molecules. However, we did not detect IgM fixation at nodes or paranodes. Antibodies to gliomedin or NF186 were mostly detected in demyelinating and axonal GBS, respectively. The adsorption of the antibodies to their soluble antigens abolished IgG deposition at nodes and paranodes in nerves, indicating these were specific to NF186, gliomedin, and contactin. In conclusion, gliomedin, NF186, and contactin are novel target antigens in GBS. At nodes, additional epitopes are also the targets of IgG. These results suggest that antibody attack against nodal antigens participates in the etiology of GBS. PMID:22462667

  8. An analysis of bison erythrocyte antigens and blood proteins

    E-print Network

    Zamora, Linda Elia

    1983-01-01

    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... 79 Page 6. Frequency of positive hemolytic reactions in bison using cattle blood typing reagents. . . . 87 7. Frequency oF positive hemolyti c reactions in bison usi ng experimental bison blood reagents. 8. Inheritance of carbonic anhydrase...

  9. Negatively charged liposomes show potent adjuvant activity when simply admixed with protein antigens.

    PubMed

    Yanasarn, Nijaporn; Sloat, Brian R; Cui, Zhengrong

    2011-08-01

    Liposomes have been investigated extensively as a vaccine delivery system. Herein the adjuvant activities of liposomes with different net surface charges (neutral, positive, or negative) were evaluated when admixed with protein antigens, ovalbumin (OVA, pI = 4.7), Bacillus anthracis protective antigen protein (PA, pI = 5.6), or cationized OVA (cOVA). Mice immunized subcutaneously with OVA admixed with different liposomes generated different antibody responses. Interestingly, OVA admixed with net negatively charged liposomes prepared with DOPA was as immunogenic as OVA admixed with positively charged liposomes prepared with DOTAP. Immunization of mice with the anthrax PA protein admixed with the net negatively charged DOPA liposomes also induced a strong and functional anti-PA antibody response. When the cationized OVA was used as a model antigen, liposomes with net neutral, negative, or positive charges showed comparable adjuvant activities. Immunization of mice with the OVA admixed with DOPA liposomes also induced OVA-specific CD8(+) cytotoxic T lymphocyte responses and significantly delayed the growth of OVA-expressing B16-OVA tumors in mice. However, not all net negatively charged liposomes showed a strong adjuvant activity. The adjuvant activity of the negatively charged liposomes may be related to the liposome's ability (i) to upregulate the expression of molecules related to the activation and maturation of antigen-presenting cells and (ii) to slightly facilitate the uptake of the antigens by antigen-presenting cells. Simply admixing certain negatively charged liposomes with certain protein antigens of interest may represent a novel platform for vaccine development. PMID:21615153

  10. Negatively charged liposomes show potent adjuvant activity when simply admixed with protein antigens

    PubMed Central

    Yanasarn, Nijaporn; Sloat, Brian R.; Cui, Zhengrong

    2011-01-01

    Liposomes have been investigated extensively as a vaccine delivery system. Herein the adjuvant activities of liposomes with different net surface charges (neutral, positive, or negative) were evaluated when admixed with protein antigens, ovalbumin (OVA, pI = 4.7), Bacillus anthracis protective antigen protein (PA, pI = 5.6), or cationized OVA (cOVA). Mice immunized subcutaneously with OVA admixed with different liposomes generated different antibody responses. Interestingly, OVA admixed with net negatively charged liposomes prepared with DOPA was as immunogenic as OVA admixed with positively charged liposomes prepared with DOTAP. Immunization of mice with the anthrax PA protein admixed with the net negatively charged DOPA liposomes also induced a strong and functional anti-PA antibody response. When the cationized OVA was used as a model antigen, liposomes with net neutral, negative, or positive charges showed comparable adjuvant activities. Immunization of mice with the OVA admixed with DOPA liposomes also induced OVA-specific CD8+ cytotoxic T lymphocyte responses and significantly delayed the growth of OVA-expressing B16-OVA tumors in mice. However, not all net negatively charged liposomes showed a strong adjuvant activity. The adjuvant activity of the negatively charged liposomes may be related to the liposome’s ability (i) to up-regulate the expression of molecules related to the activation and maturation of antigen-presenting cells and (ii) to slightly facilitate the uptake of the antigens by antigen-presenting cells. Simply admixing certain negatively charged liposomes with certain protein antigens of interest may represent a novel platform for vaccine development. PMID:21615153

  11. Modified anthrax fusion proteins deliver HIV antigens through MHC Class I and II pathways.

    PubMed

    McEvers, K; Elrefaei, M; Norris, P; Deeks, S; Martin, J; Lu, Y; Cao, H

    2005-07-14

    T cell-based HIV vaccine candidates have focused on eliciting both CD4- and CD8-mediated responses. One challenge in vaccine development is the successful introduction and presentation of exogenous antigen to elicit an immune response. Modified bacterial toxins have been studied extensively as intracellular delivery agents because of their unique capability to translocate antigen across the cell membrane without affecting cell viability. Modified anthrax toxin lethal factor (LFn) fusion protein is able to effectively induce anti-HIV cytotoxic T lymphocytes in the absence of protective antigen (PA) and is being evaluated as a vaccine candidate. Here we describe, for the first time, the processing and presentation of LFn fusion proteins by the MHC Class II pathway. The ability of LFn--HIV to induce both CD8- and CD4-mediated responses may have relevance in current approaches to vaccine design. Furthermore, the translocation and presentation of antigens occurs in the absence of PA, which proposes a modified molecular mechanism of antigen presentation by the anthrax toxin model. Additionally, we found that LFn--HIV is specific and sensitive in detecting HIV-specific CD4(+) and CD8(+) T cell responses in T cell assays, further broadening the value of this antigen delivery system as a useful immunologic tool. PMID:15964481

  12. The role of protein kinases in antigen-activation of peripheral blood mononuclear cells of Schistosoma mansoni infected individuals.

    PubMed

    Almeida, C A; Goes, A M

    1997-01-01

    T cell recognition of antigens displayed on the surface of antigen presenting cell results in rapid activation of protein tyrosine kinases and kinase C. This process leads to second messengers, such as inositol phosphates and diacylgycerol, and phosphorylation of multiple proteins. The role of different protein kinases in the activation of peripheral blood mononuclear cells (PBMC) from Schistosoma mansoni infected individuals was evaluated using genistein and H-7, specific inhibitors of protein tyrosine kinase and kinase C, respectively. Our results showed that proliferation in response to soluble egg antigen or adult worm antigen preparation of S. mansoni was reduced when PBMC were cultured in presence of protein kinase inhibitors. Using these inhibitors on in vitro granuloma reaction, we also observed a marked reduction of granuloma index. Taken together, our results suggest that S. mansoni antigen activation of PBMC involves protein kinases activity. PMID:9566227

  13. A Unique Human Mycoplasma Protein that Generically Blocks Antigen-Antibody Union

    PubMed Central

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

    2014-01-01

    We report the discovery and crystal structure of a human mycoplasma protein, Protein M, which binds with high affinity to antibodies, predominantly through attachment to the variable region of the ? and ? light chains. Protein M broadly blocks antibody-antigen union and its mechanism of inhibition is of considerable interest because, as a diversity system, the binding mode of each antibody is different. Protein M thus appears to function by a mechanism that is independent of the sequences of members of the extensive antibody repertoire. By anchoring to conserved regions of the antibody light chains, Protein M is in a position to extend its large C-terminal domain over the antibody combining site and block entrance to macromolecular antigens. PMID:24503852

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

    Microsoft Academic Search

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

    2011-01-01

    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,

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

    PubMed Central

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

    1997-01-01

    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

  16. Antigen-specificity of oligoclonal abnormal protein bands in multiple myeloma after allogeneic stem cell transplantation.

    PubMed

    Rahlff, Janina; Trusch, Maria; Haag, Friedrich; Bacher, Ulrike; Horst, Andrea; Schlüter, Hartmut; Binder, Mascha

    2012-10-01

    Myeloma patients may develop oligoclonal immunoglobulins, so-called abnormal protein bands (APB), after stem cell transplantation. APB do not correspond to the patient's paraprotein and confer a good prognosis. We set out to investigate whether such APB represent a humoral anti-myeloma immune response by screening immunoglobulins of 15 myeloma patients after allogeneic stem cell transplantation and a control group of healthy donors for reactivity with myeloma protein extracts. While the immunoglobulins of healthy donors did not react with myeloma protein extracts, patient-derived immunoglobulins showed variable levels of interaction, depending on the presence of APB on immunofixation. Most commonly, we detected interactions with heat-shock proteins, followed by neutral alpha-glucosidase, alpha-enolase and vimentin, as well as proliferating cell nuclear antigen and MAGEA4. More than 80% of targets were upregulated in myeloma. Heat-shock protein 60 (HSP60) was subsequently evaluated as an exemplary antigen. We found that HSP60 was aberrantly displayed on the surface of primary myeloma cells. Indeed, patient-derived APB-containing immunoglobulins recognized surface HSP60 suggesting that this antigen becomes accessible to the immune system after aberrant membrane exposition. We conclude that immunoglobulin fractions with APB recognize recurrent myeloma antigens and that this humoral response may contribute to the more favorable prognosis in patients with APB. PMID:22350072

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

    PubMed

    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

    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

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

    PubMed

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

    2005-02-01

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

  19. Protection against Mycobacterium avium by DNA Vaccines Expressing Mycobacterial Antigens as Fusion Proteins with Green Fluorescent Protein

    PubMed Central

    Velaz-Faircloth, Maria; Cobb, Alison J.; Horstman, Amanda L.; Henry, Stanley C.; Frothingham, Richard

    1999-01-01

    Mycobacterium avium causes disseminated disease in humans with AIDS, paratuberculosis in ruminants, lymphadenopathy in swine, and tuberculosis in birds. We constructed DNA vaccines expressing mycobacterial antigens as fusion proteins with enhanced green fluorescent protein (EGFP). Plasmids p65K-EGFP, p85A-EGFP, and p85B-EGFP expressed the M. avium 65-kDa antigen, the Mycobacterium bovis BCG 85A antigen, and the M. avium 85B antigen, respectively, as EGFP fusion proteins. We visualized protein expression directly in cultured murine fibroblasts and intact muscle. p65K-EGFP expressed fusion protein in a diffuse cytoplasmic pattern, and p85A-EGFP and p85B-EGFP produced a speckled pattern. We vaccinated C57BL/6 mice with three doses of plasmid DNA and then challenged them intraperitoneally with M. avium. Negative controls received saline, and positive controls received one dose of BCG vaccine. Mice in all groups developed disseminated infection with a high burden of organisms. Compared to negative controls, mice vaccinated with p85A-EGFP had an eightfold reduction in spleen M. avium CFU at 4 weeks after infection and a fourfold reduction at 8 weeks, reductions similar to those generated by BCG vaccine. Mice vaccinated with p65K-EGFP had a fourfold CFU reduction at 4 weeks and no effect at 8 weeks. This is the first report of DNA vaccines expressing foreign antigens as fusion proteins with EGFP and the first report of successful DNA vaccination against M. avium. PMID:10417198

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

    PubMed

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

    1983-01-01

    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

  1. Immunoassay using /sup 125/I- or enzyme-labeled protein A and antigen-coated tubes

    SciTech Connect

    Gee, A.P.; Langone, J.J.

    1981-09-15

    Antigen-coated plastic tubes were used with /sup 125/I- or enzyme-labeled stapylococcal protein A in a general immunoassay method for antigens and haptens. Protein A reacts with immunoglobulin G(IgG) regardless of antibody specificity at sites distal to the antigen combining site and does not inhibit the immune reaction. It therefore serves as a general tracer and its use eliminates the need to purify and to label individual components for each assay. Macromolecular antigens were bound to polystyrene or polypropylene tubes by direct passive absorption. Haptens with free carboxyl groups were bound covalently to poly-L-lysine and these conjugates passively absorbed to the tube surface. Optimal assay conditions were established for the quantitative determination of immunoglobulins and the folate derivatives, methotrexate and 5-methyltetrahydrofolate, using /sup 125/I-labeled protein A or protein A labeled with alkaline phosphatase. The method has been used to estimate levels of IgG, IgA, Igm, and IgE in serum in volumes up to 1 ml.

  2. The Leukocyte Common Antigen-Related Protein Tyrosine Phosphatase Receptor Regulates Regenerative Neurite Outgrowth In Vivo

    Microsoft Academic Search

    Youmei Xie; Tracy T. Yeo; Cheng Zhang; Tao Yang; Michelle A. Tisi; Stephen M. Massa; Frank M. Longo

    2001-01-01

    Drosophila and leech models of nervous system development demonstrate that protein tyrosine phosphatase (PTP) receptors regulate developmental neurite outgrowth. Whether PTP recep- tors regulate neurite outgrowth in adult systems or in regener- ative states remains unknown. The leukocyte common antigen- related (LAR) receptor is known to be present in rodent dorsal root ganglion (DRG) neurons; therefore, the well established model

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

    Microsoft Academic Search

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

    1997-01-01

    Recent studies have identified several epitopes in the N-terminal portion of the nucleocapsid protein which are predominantly recognized by sera of patients infected with hepatitis C virus (HCV). The characterization of the sequences recognized by theses antibodies and the evaluation of their reactivities have been performed mainly with synthetic peptides. However, synthetic peptides are notoriously unreliable as antigens when the

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

    Microsoft Academic Search

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

    1980-01-01

    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

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

    Microsoft Academic Search

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

    2000-01-01

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

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

    E-print Network

    Lieberman, Judy

    , 2000 (received for review January 24, 2000) Bacillus anthrax lethal toxin can be engineered to deliverGenetically modified anthrax lethal toxin safely delivers whole HIV protein antigens compartment of mammalian cells. The engineered anthrax toxin vaccine appears unlikely to induce an antibody

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

    E-print Network

    Lieberman, Judy

    A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective by Elkan R. Blout, Harvard Medical School, Cambridge, MA, April 2, 2003 (received for review March 10, 2003) Anthrax protective antigen (PA) is a 735-aa polypeptide that facili- tates the exit of anthrax lethal

  8. Transgenic carrot expressing fusion protein comprising M. tuberculosis antigens induces immune response in mice.

    PubMed

    Permyakova, Natalia V; Zagorskaya, Alla A; Belavin, Pavel A; Uvarova, Elena A; Nosareva, Olesya V; Nesterov, Andrey E; Novikovskaya, Anna A; Zav'yalov, Evgeniy L; Moshkin, Mikhail P; Deineko, Elena V

    2015-01-01

    Tuberculosis remains one of the major infectious diseases, which continues to pose a major global health problem. Transgenic plants may serve as bioreactors to produce heterologous proteins including antibodies, antigens, and hormones. In the present study, a genetic construct has been designed that comprises the Mycobacterium tuberculosis genes cfp10, esat6 and dIFN gene, which encode deltaferon, a recombinant analog of the human ?-interferon designed for expression in plant tissues. This construct was transferred to the carrot (Daucus carota L.) genome by Agrobacterium-mediated transformation. This study demonstrates that the fusion protein CFP10-ESAT6-dIFN is synthesized in the transgenic carrot storage roots. The protein is able to induce both humoral and cell-mediated immune responses in laboratory animals (mice) when administered either orally or by injection. It should be emphasized that M. tuberculosis antigens contained in the fusion protein have no cytotoxic effect on peripheral blood mononuclear cells. PMID:25949997

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

    PubMed

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

    2014-10-01

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

  10. Determinants of antigenicity and specificity in immune response for protein sequences

    Microsoft Academic Search

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

    2011-01-01

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

  11. Delivery of exogenous protein antigens to major histocompatibility complex class I pathway in cytosol.

    PubMed

    Cao, Huyen; Agrawal, Deepak; Kushner, Nicholas; Touzjian, Neal; Essex, Max; Lu, Yichen

    2002-01-15

    A fragment of anthrax lethal factor possesses the interesting function of delivering recombinant protein antigens through the classical major histocompatibility complex (MHC) class I pathway. This region of the lethal factor lacks the domain associated with anthrax cytotoxicity and functions independently of its binary partner, protective antigen. Experiments that used inhibitors at different steps of the MHC class I pathway supported this hypothesis. Application of this discovery to current T cell assays allows for the measurement of cytotoxic T lymphocyte function without resorting to live vectors and provides a useful new tool to design and test T cell-dependent vaccines. PMID:11807699

  12. Echinococcus antigens

    PubMed Central

    Kagan, Irving G.; Agosin, Moises

    1968-01-01

    Much of the work on immunology of hydatidosis has so far been devoted to the development of suitable methods for serological diagnosis. The precise nature of hydatid antigens and their chemical characterization has still not been worked out, largely because of the complex life-history of the parasite and the difficulties of in vitro cultivation. The most widely used antigen for routine serological testing in hydatidosis caused by Echinococcus granulosus is fluid taken from the cyst. This fluid is, however, a complex mixture of substances and contains several protein and carbohydrate fractions as well as end-products of carbohydrate and protein metabolism. The cyst fluid from different sources is variable in its antigenic properties, and the fluid from sterile cysts is especially lacking in antigenic activity. Antigens from tissue extracts of hydatid cysts appear to have greater specificity. Cyst extracts of E. multilocularis, the cysts of which contain relatively little fluid, have also been used but are poor antigens, and contain measurable amounts of host protein. Antigens prepared from other cestodes and metabolic antigens are also reviewed. Biochemical analysis of Echinococcus antigens covering polysaccharides, proteins, lipids, and blood-group substances is considered, together with the characterization of antigens by electrophoresis, column chromatography and gel-diffusion methods. The problems associated with the standardization of antigens are discussed. Recent data on the character and reactivity of antigens employed in Echinococcus studies are summarized. PMID:4973343

  13. Interspecies antigenic determinants of the reverse transcriptases and p30 proteins of mammalian type C viruses.

    PubMed Central

    Sherr, C J; Fedele, L A; Benveniste, R E; Todaro, G J

    1975-01-01

    The major internal structural proteins (p30) of type C viruses isolated from several mammalian species were studied by radioimmunoprecipitation and competitive radioimmunoassays. Three antigenically distinguishable sets of interspecies determinants could be demonstrated by both methods. One set of determinants shared by viruses of rodent origin (mouse and rat) can be detected readily in feline leukemia viruses but not in other type C viral groups. The p30 proteins of murine viruses also contain a second discrete set of antigenic determinants related to those in infectious primate viruses and endogenous porcine viruses, but not detected in the feline leukemia virus group. The p30 proteins of endogenous viruses of baboons and domestic cats share yet a third set of cross-reactive determinants not detected in type C viruses isolated from other species of animals. Enzyme inhibition studies performed with antisera raised toward the reverse transcriptases of these same groups of type C viruses showed the same patterns of immunological cross-reactions as observed with p30 proteins. The antigenic cross-reactions between the homologous proteins of type C virus isolated from genetically distant animals may reflect transmission of type C viruses across species barriers. PMID:49443

  14. Extracellular Release of Antigenic Proteins by Helicobacter pylori

    Microsoft Academic Search

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

    1998-01-01

    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

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

    PubMed Central

    2014-01-01

    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

  16. CHARACTERIZATION OF THE CARBOHYDRATE COMPONENTS OF Taenia solium ONCOSPHERE PROTEINS AND THEIR ROLE IN THE ANTIGENICITY

    PubMed Central

    Arana, Yanina; Verastegui, Manuela; Tuero, Iskra; Grandjean, Louis; Garcia, Hector H.; Gilman, Robert H.

    2015-01-01

    This study examines the carbohydrate composition of Taenia solium whole oncosphere antigens (WOAs), in order to improve the understanding of the antigenicity of the T. solium. Better knowledge of oncosphere antigens is crucial to accurately diagnose previous exposure to T. solium eggs and thus predict the development of neurocysticercosis. A set of seven lectins conjugates with wide carbohydrate specificity were used on parasite fixations and somatic extracts. Lectin fluorescence revealed that D-mannose, D-glucose, D-galactose and N-acetyl-D-galactosamine residues were the most abundant constituents of carbohydrate chains on the surface of T. solium oncosphere. Lectin blotting showed that post-translational modification with N-glycosylation was abundant while little evidence of O-linked carbohydrates was observed. Chemical oxidation and enzymatic deglycosylation in situ were performed to investigate the immunoreactivity of the carbohydrate moieties. Linearizing or removing the carbohydrate moieties from the protein backbones did not diminish the immunoreactivity of these antigens, suggesting that a substantial part of the host immune response against T. solium oncosphere is directed against the peptide epitopes on the parasite antigens. Finally, using carbohydrate probes, we demonstrated for the first time that the presence of several lectins on the surface of the oncosphere was specific to carbohydrates found in intestinal mucus, suggesting a possible role in initial attachment of the parasite to host cells. PMID:23982308

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

    PubMed Central

    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

    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

  18. A novel antigen-carrier system: the Mycobacterium tuberculosis Acr protein carried by raw starch microparticles.

    PubMed

    Moreno-Mendieta, S A; Guillén, D; Espitia, C; Hernández-Pando, R; Sanchez, S; Rodríguez-Sanoja, R

    2014-10-20

    Microparticles have been used as promising carriers for in vivo vaccine delivery. However, the processes for immobilizing peptides or proteins on microparticles usually require the use of undesirable compounds and complex protocols. In this work, we propose a new immobilization and delivery system with raw starch microparticles and a starch binding domain (SBD) tag fusion protein. The heat shock protein alpha crystallin from Mycobacterium tuberculosis was used as model. The immunogenicity of the system was investigated in BALB/c mice inoculated with purified Acr-SBDtag protein (pAcr-SBDtag) and starch immobilized Acr-SBDtag protein (?Acr-SBDtag) by oral and intranasal routes. We demonstrated mucosal immunization with the ?Acr-SBDtag protein induced systemic antibodies that were predominantly immunoglobulin G2a (IgG2a). An analysis of the cytokines from spleen cells and lung homogenates revealed that loaded microparticles induced the secretion of interferon-? (INF-?), suggesting an adjuvant effect from the immobilization. The immune responses induced by immobilized protein were primarily affected by the route of administration. These results demonstrate that the system exhibits the necessary characteristics to improve antigen release and presentation to antigen presenting cells (APCs) in the mucosae. Because no extra adjuvants were used, we posit that the system may be suitable for delivery and presentation to the field of subunit vaccine development. PMID:25093695

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

    Microsoft Academic Search

    JOHN D. NEILL; STANISLAV V. SOSNOVTSEV; KIM Y. GREEN

    2000-01-01

    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

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

    PubMed

    Grote, M; Vik, H; Elsayed, S

    1988-11-01

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

  1. Serological Expression Cloning of Novel Immunoreactive Antigens of Babesia microti

    PubMed Central

    Lodes, Michael J.; Houghton, Raymond L.; Bruinsma, Elizabeth S.; Mohamath, Raodoh; Reynolds, Lisa D.; Benson, Darin R.; Krause, Peter J.; Reed, Steven G.; Persing, David H.

    2000-01-01

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

  2. Antigenic determinants of a plant proteoglycan, the Gladiolus style arabinogalactan-protein.

    PubMed Central

    Gleeson, P A; Clarke, A E

    1980-01-01

    Antiserum has been raised to the arabinogalactan-protein of Gladiolus style mucilage. This macromolecule has been characterized and has a structure consistent with a 1 leads to 3-linked beta-galactan backbone with side branches of 1 leads to 6-linked beta-galactosyl residues, some of which carry terminal alpha-L-arabinofuranoside residues [Gleeson & Clarke (1979) Biochem. J. 181, 607-621]. The specificity of the antiserum has been investigated by immunoprecipitation with [3H]arabinogalactan-protein. THe 3H label was introduced into the arabinogalactan-protein by oxidation of the terminal galactose residues with galactose oxidase, followed by reduction with NaB3H4. The antigenic specificity of the antiserum was shown to be directed towards the carbohydrate component of the arabinogalactan-protein. D-galactose and L-arabinose were the most effective hapten inhibitors of the antiserum; other monosaccharides, N-acetyl-D-galactono-1,4-lactone, D-glucose, D-mannose, L-rhamnose. L-fucose and D-xylose, were all poor inhibitors. The antiserum showed preference for beta-galactosides over alpha-galactosides. Of the haptens examined, the disaccharide 6-O-beta-D-galactopyranosyl-D-galactopyranose was the most potent inhibitor. The antigenic features of the arabinogalactan-protein were investigated by examining the interaction of the antiserum with chemically and enzymically modified arabinogalactan-protein. Also, the cross-reactivity of structurally related polysaccharides and glycoproteins with the specific antiserum was assessed by a haemagglutination assay using erythrocytes coupled with specific antiserum. The results indicate that the dominant antigenic determinants of the arabinogalactan-protein are probably the side branches of 1 leads to 6 -linked beta-galactose residues bearing the terminal alpha-L-arabinose residues. PMID:6165351

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

    Microsoft Academic Search

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

    2008-01-01

    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

  4. Antigenic Properties of HpaA and Omp18, Two Outer Membrane Proteins of Helicobacter pylori

    Microsoft Academic Search

    Petra Voland; Nadia Hafsi; Marco Zeitner; Stephanie Laforsch; Hermann Wagner; Christian Prinz

    2003-01-01

    Outer membrane proteins (OMPs) are incorporated into the outer plasma membrane of Helicobacter pylori and are important for, e.g., ion transport, adherence, structural and osmotic stability, and bacterial virulence but may also be antigenic due to their surface exposure. Previous proteome-based approaches with H. pylori lysates determined a strong serological reaction towards two H. pylori OMPs, HpaA (TIGR HP0797) and

  5. A multiplex ELISA-based protein array for screening diagnostic antigens and diagnosis of Flaviviridae infection.

    PubMed

    Wang, D; Zheng, Y; Kang, X; Zhang, X; Hao, H; Chen, W; Liu, L; Li, X; Li, L; Yuan, Q; Chen, F; Yang, Y; Jiang, Y; Jiang, H

    2015-07-01

    Assays with the ability to detect multiple antibodies in parallel have a wide range of potential applications in epidemiologic research. Here, a multiplex enzyme-linked immunosorbent assay-based protein array (ELISA-array) was developed to simultaneously detect five Flaviviridae infections. The platform was based on an indirect ELISA and 15 antigens were constructed for specific antibody detection against five Flaviviridae viruses (Japanese B, tick-borne encephalitis, West Nile, dengue, and yellow fever viruses) and four serotypes of dengue virus. The specificity was evaluated by calculating the signal value cross-reacting with serum immunized with other viruses, and the sensitivity of antigens was compared with conventional ELISAs using immunized rabbit polyclonal antisera. IgG and IgM calibration curves were constructed to evaluate the reproducibility of the platform. Finally, 24 dengue fever (DF) infection and 15 tick-borne encephalitis (TBE) infection clinical sera were used to compare the advantage of ELISA-array to ELISA. After initial screening, 9 out of 15 antigens were chosen for ELISA-array printing. By using different virus-immunized rabbit antiserum, 7 out of 9 antigens showed good specificity in the ELISA-array. Eight out of 9 antigens showed four-fold greater sensitivity in ELISA-array compared to that in conventional ELISAs. The coefficients of determination (r (2)) close to 1 showed high reproducibility, and clinical sera test showed that ELISA-array had higher specificity and sensitivity than traditional ELISA. ELISA-array was a good platform for antigen screening and this multiplexed assay might be a useful and convenient tool for multiple immunological detection of infectious viral antibodies. PMID:25796511

  6. Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli.

    PubMed

    Feldman, Mario F; Wacker, Michael; Hernandez, Marcela; Hitchen, Paul G; Marolda, Cristina L; Kowarik, Michael; Morris, Howard R; Dell, Anne; Valvano, Miguel A; Aebi, Markus

    2005-02-22

    Campylobacter jejuni has a general N-linked protein glycosylation system that can be functionally transferred to Escherichia coli. In this study, we engineered E. coli cells in a way that two different pathways, protein N-glycosylation and lipopolysaccharide (LPS) biosynthesis, converge at the step in which PglB, the key enzyme of the C. jejuni N-glycosylation system, transfers O polysaccharide from a lipid carrier (undecaprenyl pyrophosphate) to an acceptor protein. PglB was the only protein of the bacterial N-glycosylation machinery both necessary and sufficient for the transfer. The relaxed specificity of the PglB oligosaccharyltransferase toward the glycan structure was exploited to create novel N-glycan structures containing two distinct E. coli or Pseudomonas aeruginosa O antigens. PglB-mediated transfer of polysaccharides might be valuable for in vivo production of O polysaccharides-protein conjugates for use as antibacterial vaccines. PMID:15703289

  7. Antigenic and immunogenic properties of a chimera of two immunodominant African swine fever virus proteins.

    PubMed

    Barderas, M G; Rodríguez, F; Gómez-Puertas, P; Avilés, M; Beitia, F; Alonso, C; Escribano, J M

    2001-01-01

    A chimera of the two immunodominant African swine fever (ASF) virus proteins p54 and p30 was constructed by insertion of the gene CP204L into a Not I restriction site of E183L gene. The resulting chimeric protein p54/30, expressed by a recombinant baculovirus in insect cells and in Trichoplusia ni larvae, retained antigenic determinants present in both proteins and reacted in Western blot with a collection of sera from inapparent ASF virus carrier pigs. Remarkably, pigs immunized with the chimeric protein developed neutralizing antibodies and survived the challenge with a virulent African swine fever virus, presenting a reduction of about two logs in maximum viremia titers with respect to control pigs. In conclusion, this study revealed that the constructed chimeric protein may have utility as a serological diagnostic reagent and for further immunological studies that may provide new insights on mechanisms of protective immunity to ASFV. PMID:11699955

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

    SciTech Connect

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

    1981-07-01

    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.

  9. Antigenic and immunogenic properties of defined physical forms of tick-borne encephalitis virus structural proteins.

    PubMed Central

    Heinz, F X; Tuma, W; Kunz, C

    1981-01-01

    Polymeric, delipidated glycoprotein complexes of defined size and composition were prepared from tick-borne encephalitis virus by solubilization with Triton X-100 or cetyltrimethylammonium bromide, followed by centrifugation into detergent-free sucrose density gradients. The antigenic reactivities and immunogenicities of these complexes were compared with those of complete inactivated virus. These glycoprotein preparations induced hemagglutination-inhibiting and neutralizing antibodies which proved to be protective in passive mouse protection tests and monospecifically reacted only with the viral envelope and not with the internal core. In a competitive radioimmunoassay the glycoprotein complexes revealed about 10-fold higher antigenicity than whole virus when tested at equal protein concentrations. The important implications of these results with respect to antigen quantification in vaccines are discussed. As shown in the mouse challenge potency test, glycoprotein complexes prepared after Triton X-100 solubilization actively protected mice almost as well as did complete inactivated virus at the same protein concentration, whereas those prepared after cetyltrimethylammonium bromide solubilization had a somewhat lower protective activity per microgram of protein. Images PMID:7263062

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

    PubMed Central

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

    2008-01-01

    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

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

    PubMed Central

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

    2014-01-01

    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

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

    PubMed Central

    2014-01-01

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

  13. Functional Variation of the Antigen I/II Surface Protein in Streptococcus mutans and Streptococcus intermedius

    PubMed Central

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

    2002-01-01

    Although Streptococcus intermedius and Streptococcus mutans are regarded as members of the commensal microflora of the body, S. intermedius is often associated with deep-seated purulent infections, whereas S. mutans is frequently associated with dental caries. In this study, we investigated the roles of the S. mutans and S. intermedius antigen I/II proteins in adhesion and modulation of cell surface characteristics. By using isogenic mutants, we show that the antigen I/II in S. mutans, but not in S. intermedius, was involved in adhesion to a salivary film under flowing conditions, as well as in binding to rat collagen type I. Binding to human fibronectin was a common function associated with the S. mutans and S. intermedius antigen I/II. Adhesion of S. mutans or S. intermedius to human collagen types I or IV was negligible. Hydrophobicity, as measured by water contact angles, and zeta potentials were unaltered in the S. intermedius mutant. The S. mutans isogenic mutants, on the other hand, exhibited more positive zeta potentials at physiological pH values than did the wild type. The results indicate common and species-specific roles for the antigen I/II in mediating the attachment of S. mutans and S. intermedius to host components and in determining cell surface properties. PMID:11748190

  14. Hepatitis delta virus: protein composition of delta antigen and its hepatitis B virus-derived envelope.

    PubMed Central

    Bonino, F; Heermann, K H; Rizzetto, M; Gerlich, W H

    1986-01-01

    Hepatitis delta virus (HDV)-associated particles were purified from the serum of an experimentally infected chimpanzee by size chromatography and by density centrifugation. Hepatitis delta antigen (HDAg) was detected after mild detergent treatment at a column elution volume corresponding to 36-nm particles and banded at a density of 1.25 g/ml. The serum had an estimated titer of 10(9) to 10(10) HDV-associated particles and had only a 10-fold excess of hepatitis B surface antigen (HBsAg) not associated with HDAg. Therefore, HDV appears to be much more efficiently packed and secreted than is its helper virus, hepatitis B virus (HBV), which is usually accompanied by a 1,000-fold excess of HBsAg. The protein compositions of the HDAg-containing particles were analyzed by immunoblotting with HDAg-, HBsAg-, and hepatitis B core antigen-specific antisera and monoclonal antibodies to HBV surface gene products. The HBsAg envelope of HDAg contained approximately 95% P24/GP27s, 5% GP33/36s, and 1% P39/GP42s proteins. This protein composition was more similar to that of the 22-nm particles of HBsAg than to that of complete HBV. The significant amount of GP33/36s suggests that the HBsAg component of the HDV-associated particle carries the albumin receptor. Two proteins of 27 and 29 kilodaltons which specifically bound antibody to HDAg but not HBV-specific antibodies were detected in the interior of the 36-nm particle. Since these proteins were structural components of HDAg and were most likely coded for by HDV, they were designated P27d and P29d. Images PMID:3701932

  15. Engineering the chloroplast genome for hyperexpression of human therapeutic proteins and vaccine antigens.

    PubMed

    Kumar, Shashi; Daniell, Henry

    2004-01-01

    The chloroplast genome is ideal for engineering because it offers a number of attractive advantages, including high-level gene expression, the feasibility of expressing multiple genes or pathways in a single transformation event, and transgene containment due to lack of pollen transmission. The chloroplast-based expression system is suitable for hyperexpression of foreign proteins, oral delivery of vaccine antigens and therapeutic proteins, via both leaves and fruits. Through the refinement of expression vectors and use of chaperones, chloroplasts produce up to 47% of foreign protein in the total cellular protein in transgenic tissues. This chapter describes various techniques for creating chloroplast transgenic plants and their biochemical and molecular characterization. Suitable examples for application of chloroplast genetic engineering in human medicine are provided. PMID:15269437

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

    PubMed

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

    2012-01-01

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

  17. Microsomal triglyceride transfer protein lipidation and control of CD1d on antigen-presenting cells.

    PubMed

    Dougan, Stephanie K; Salas, Azucena; Rava, Paul; Agyemang, Amma; Kaser, Arthur; Morrison, Jamin; Khurana, Archana; Kronenberg, Mitchell; Johnson, Caroline; Exley, Mark; Hussain, M Mahmood; Blumberg, Richard S

    2005-08-15

    Microsomal triglyceride transfer protein (MTP), an endoplasmic reticulum (ER) chaperone that loads lipids onto apolipoprotein B, also regulates CD1d presentation of glycolipid antigens in the liver and intestine. We show MTP RNA and protein in antigen-presenting cells (APCs) by reverse transcription-polymerase chain reaction and by immunoblotting of mouse liver mononuclear cells and mouse and human B cell lines. Functional MTP, demonstrated by specific triglyceride transfer activity, is present in both mouse splenocytes and a CD1d-positive mouse NKT hybridoma. In a novel in vitro transfer assay, purified MTP directly transfers phospholipids, but not triglycerides, to recombinant CD1d. Chemical inhibition of MTP lipid transfer does not affect major histocompatibility complex class II presentation of ovalbumin, but considerably reduces CD1d-mediated presentation of alpha-galactosylceramide (alpha-galcer) and endogenous antigens in mouse splenic and bone marrow-derived dendritic cells (DCs), as well as in human APC lines and monocyte-derived DCs. Silencing MTP expression in the human monocyte line U937 affects CD1d function, as shown by diminished presentation of alpha-galcer. We propose that MTP acts upstream of the saposins and functions as an ER chaperone by loading endogenous lipids onto nascent CD1d. Furthermore, our studies suggest that a small molecule inhibitor could be used to modulate the activity of NKT cells. PMID:16087713

  18. Antigenic stability of pecan [Carya illinoinensis (Wangenh.) K. Koch] proteins: effects of thermal treatments and in vitro digestion.

    PubMed

    Venkatachalam, Mahesh; Teuber, Suzanne S; Peterson, W Rich; Roux, Kenneth H; Sathe, Shridhar K

    2006-02-22

    Rabbit polyclonal antibody-based inhibition ELISA as well as immunoblotting analyses of proteins extracted from variously processed pecans (cv. Desirable) indicate that pecan proteins are antigenically stable. Pecan antigens were more sensitive to moist heat than dry heat processing treatments. SDS-PAGE and immunoblotting analysis of the native and heat-denatured proteins that were previously subjected to in vitro simulated gastric fluid digestions indicate that stable antigenic peptides were produced. Both enzyme-to-substrate ratio and digestion time were influential in determining the stability of pecan polypeptides. The stable antigenic polypeptides may serve as useful markers in developing assays suitable for the detection of trace amounts of pecans in foods. PMID:16478273

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

    PubMed Central

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

    1990-01-01

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

  20. Influenza virus-like particles engineered by protein transfer with tumor-associated antigens induces protective antitumor immunity.

    PubMed

    Patel, Jaina M; Vartabedian, Vincent F; Kim, Min-Chul; He, Sara; Kang, Sang-Moo; Selvaraj, Periasamy

    2015-06-01

    Delivery of antigen in particulate form using either synthetic or natural particles induces stronger immunity than soluble forms of the antigen. Among naturally occurring particles, virus-like particles (VLPs) have been genetically engineered to express tumor-associated antigens (TAAs) and have shown to induce strong TAA-specific immune responses due to their nano-particulate size and ability to bind and activate antigen-presenting cells. In this report, we demonstrate that influenza VLPs can be modified by a protein transfer technology to express TAAs for induction of effective antitumor immune responses. We converted the breast cancer HER-2 antigen to a glycosylphosphatidylinositol (GPI)-anchored form and incorporated GPI-HER-2 onto VLPs by a rapid protein transfer process. Expression levels on VLPs depended on the GPI-HER-2 concentration added during protein transfer. Vaccination of mice with protein transferred GPI-HER-2-VLPs induced a strong Th1 and Th2-type anti-HER-2 antibody response and protected mice against a HER-2-expressing tumor challenge. The Soluble form of GPI-HER-2 induced only a weak Th2 response under similar conditions. These results suggest that influenza VLPs can be enriched with TAAs by protein transfer to develop effective VLP-based subunit vaccines against cancer without chemical or genetic modifications and thus preserve the immune stimulating properties of VLPs for easier production of antigen-specific therapeutic cancer vaccines. Biotechnol. Bioeng. 2015;112: 1102-1110. © 2015 Wiley Periodicals, Inc. PMID:25689082

  1. Genome analysis of Excretory/Secretory proteins in Taenia solium reveals their Abundance of Antigenic Regions (AAR)

    PubMed Central

    Gomez, Sandra; Adalid-Peralta, Laura; Palafox-Fonseca, Hector; Cantu-Robles, Vito Adrian; Soberón, Xavier; Sciutto, Edda; Fragoso, Gladis; Bobes, Raúl J.; Laclette, Juan P.; Yauner, Luis del Pozo; Ochoa-Leyva, Adrián

    2015-01-01

    Excretory/Secretory (ES) proteins play an important role in the host-parasite interactions. Experimental identification of ES proteins is time-consuming and expensive. Alternative bioinformatics approaches are cost-effective and can be used to prioritize the experimental analysis of therapeutic targets for parasitic diseases. Here we predicted and functionally annotated the ES proteins in T. solium genome using an integration of bioinformatics tools. Additionally, we developed a novel measurement to evaluate the potential antigenicity of T. solium secretome using sequence length and number of antigenic regions of ES proteins. This measurement was formalized as the Abundance of Antigenic Regions (AAR) value. AAR value for secretome showed a similar value to that obtained for a set of experimentally determined antigenic proteins and was different to the calculated value for the non-ES proteins of T. solium genome. Furthermore, we calculated the AAR values for known helminth secretomes and they were similar to that obtained for T. solium. The results reveal the utility of AAR value as a novel genomic measurement to evaluate the potential antigenicity of secretomes. This comprehensive analysis of T. solium secretome provides functional information for future experimental studies, including the identification of novel ES proteins of therapeutic, diagnosis and immunological interest. PMID:25989346

  2. Novel protein isoforms of carcinoembryonic antigen are secreted from pancreatic, gastric and colorectal cancer cells

    PubMed Central

    2013-01-01

    Background Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is an oncofetal cell surface glycoprotein. Because of its high expression in cancer cells and secretion into serum, CEA has been widely used as a serum tumor marker. Although other members of CEACAM family were investigated for splice variants/variants-derived protein isoforms, few studies about the variants of CEACAM5 have been reported. In this study, we demonstrated the existence of novel CEACAM5 splice variants and splice variant-derived protein isoforms in gastrointestinal cancer cell lines. Results We identified two novel CEACAM5 splice variants in gastrointestinal (pancreatic, gastric, and colorectal) cancer cell lines. One of the variants possessed an alternative minor splice site that allowed generation of GC-AG intron. Furthermore, CEA protein isoforms derived from the novel splice variants were expressed in cancer cell lines and those protein isoforms were secreted into the culture medium. Although CEA protein isoforms always co-existed with the full-length protein, the secretion patterns of these isoforms did not correlate with the expression patterns. Conclusions This is the first study to identify the expression of CEA isoforms derived from the novel splice variants processed on the unique splice site. In addition, we also revealed the secretion of those isoforms from gastrointestinal cancer cell lines. Our findings suggested that discrimination between the full-length and identified protein isoforms may improve the clinical utility of CEA as a tumor marker. PMID:24070190

  3. Chlamydial disease pathogenesis. The 57-kD chlamydial hypersensitivity antigen is a stress response protein.

    PubMed

    Morrison, R P; Belland, R J; Lyng, K; Caldwell, H D

    1989-10-01

    Chlamydia trachomatis infection of humans is commonly a localized inflammation that can result in infertility, blindness, and perhaps arthritis. The pathogenic process(es) that cause these sequelae are thought to be immunological. A 57-kD protein that is common among Chlamydia elicits ocular inflammation when introduced onto the conjunctivae of guinea pigs or nonhuman primates previously sensitized by chlamydial infection. This protein is thought to mediate the immunopathology that follows chlamydial infection. To more thoroughly characterize this chlamydial component, we cloned its gene from a C. psittaci strain and identified a particular recombinant that produced the 57-kD polypeptide. The recombinant gene product was immunoreactive with a monospecific anti-57-kD serum, and elicited an ocular inflammation similar to that produced by the 57-kD antigen isolated from chlamydiae. Sequencing identified two ORFs that encode polypeptides of 11.2 and 58.1 kD and are co-transcribed. These two polypeptides show homology with Escherichia coli groE and Coxiella burnetii htp heat-shock proteins. Striking homology (greater than 50%) was found between the 57-kD protein and the HtpB, GroEL, 65-k Mycobacterium tuberculosis and Hsp60 proteins. Thus, the 57-kD chlamydial protein, previously implicated as mediating a deleterious immunologic response to chlamydial infections, is a stress-induced protein similar to those that occur universally in both prokaryotic and eukaryotic organisms. PMID:2571668

  4. Chlamydial disease pathogenesis. The 57-kD chlamydial hypersensitivity antigen is a stress response protein

    PubMed Central

    1989-01-01

    Chlamydia trachomatis infection of humans is commonly a localized inflammation that can result in infertility, blindness, and perhaps arthritis. The pathogenic process(es) that cause these sequelae are thought to be immunological. A 57-kD protein that is common among Chlamydia elicits ocular inflammation when introduced onto the conjunctivae of guinea pigs or nonhuman primates previously sensitized by chlamydial infection. This protein is thought to mediate the immunopathology that follows chlamydial infection. To more thoroughly characterize this chlamydial component, we cloned its gene from a C. psittaci strain and identified a particular recombinant that produced the 57-kD polypeptide. The recombinant gene product was immunoreactive with a monospecific anti-57-kD serum, and elicited an ocular inflammation similar to that produced by the 57-kD antigen isolated from chlamydiae. Sequencing identified two ORFs that encode polypeptides of 11.2 and 58.1 kD and are co-transcribed. These two polypeptides show homology with Escherichia coli groE and Coxiella burnetii htp heat-shock proteins. Striking homology (greater than 50%) was found between the 57-kD protein and the HtpB, GroEL, 65-k Mycobacterium tuberculosis and Hsp60 proteins. Thus, the 57-kD chlamydial protein, previously implicated as mediating a deleterious immunologic response to chlamydial infections, is a stress-induced protein similar to those that occur universally in both prokaryotic and eukaryotic organisms. PMID:2571668

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

    PubMed Central

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

    2009-01-01

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

  6. The keratin-related Ouroboros proteins function as immune antigens mediating tail regression in Xenopus metamorphosis

    PubMed Central

    Mukaigasa, Katsuki; Hanasaki, Akira; Maéno, Mitsugu; Fujii, Hiroshi; Hayashida, Shin-ichiro; Itoh, Mari; Kobayashi, Makoto; Tochinai, Shin; Hatta, Masayuki; Iwabuchi, Kazuya; Taira, Masanori; Onoé, Kazunori; Izutsu, Yumi

    2009-01-01

    Tail resorption during amphibian metamorphosis has been thought to be controlled mainly by a cell-autonomous mechanism of programmed cell death triggered by thyroid hormone. However, we have proposed a role for the immune response in metamorphosis, based on the finding that syngeneic grafts of tadpole tail skin into adult Xenopus animals are rejected by T cells. To test this, we identified two tail antigen genes called ouro1 and ouro2 that encode keratin-related proteins. Recombinant Ouro1 and Ouro2 proteins generated proliferative responses in vitro in T cells isolated from naive adult Xenopus animals. These genes were expressed specifically in the tail skin at the climax of metamorphosis. Overexpression of ouro1 and ouro2 induced T-cell accumulation and precocious tail degeneration after full differentiation of adult-type T cells when overexpressed in the tail region. When the expression of ouro1 and ouro2 were knocked down, tail skin tissue remained even after metamorphosis was complete. Our findings indicate that Ouro proteins participate in the process of tail regression as immune antigens and highlight the possibility that the acquired immune system contributes not only to self-defense but also to remodeling processes in vertebrate morphogenesis. PMID:19826093

  7. Medium tumor antigen of polyomavirus transformation-defective mutant NG59 is associated with 73-kilodalton heat shock protein.

    PubMed Central

    Walter, G; Carbone, A; Welch, W J

    1987-01-01

    Affinity-purified medium T antigen encoded by NG59, a nontransforming mutant of polyomavirus, is specifically associated with a protein of 72,000 daltons (72K protein). Medium T antigens of wild-type polyomavirus and the transformation-competent mutant dl8 are not associated with the 72K protein. Instead, they form a complex with another protein of 61,000 daltons. Several lines of evidence suggest that the medium T antigen-associated 72K protein is equivalent to the abundant and constitutive 73K heat shock protein. First, on two-dimensional polyacrylamide gels the 72K protein migrated with the same pI (5.6) as did the 73K heat shock protein. Second, the 72K protein was immunoprecipitable with antibodies against heat shock proteins. Third, when digested with V8 protease, the 72K protein gave rise to the same pattern of fragments as did the 73K heat shock protein. Images PMID:3027370

  8. 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

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

    2005-01-01

    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

  9. Effect of Ethanol on Antigenicity of Hepatitis B Virus Envelope Proteins

    Microsoft Academic Search

    Kenji Ito; Takumi Kajiura; Kenji Abe

    SUMMARY: Hepatitis B e antigen-positive human serum was treated with 50-90% ethanol at room temperature for 1-60 min, then the antigenicity of S antigen (hepatitis B surface antigen, in a narrow sense) was determined by radioimmunoassay and the antigenicities of pre-S1 and pre-S2 antigens were measured by enzyme immunoassay. In addition, hepatitis B virus (HBV) DNA in the treated serum

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

    PubMed

    Narmadha, Ganapathy; Yenugu, Suresh

    2015-04-01

    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

  11. Regional Distribution in the Mouse of Proteins Homologous to Artery-Specific Antigenic Proteins (ASAPs)

    Microsoft Academic Search

    J. Roberto M. Borromeo; Ninan Koshy; Shichao Xia; Karen Hardy; M. David Tilson

    1999-01-01

    We have reported the sequences of four novel proteins derived from extracts of human aortic tissue and a cDNA library from human aortic adventitia. These proteins are immunoreactive with serum immunoglobulins from patients with abdominal aortic aneurysms (AAA), and they have homologies of amino acid sequence with microfibrillar proteins of connective tissue. We are reporting separately that two of these

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

    Microsoft Academic Search

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

    1993-01-01

    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

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

    PubMed Central

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

    2002-01-01

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

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

    PubMed Central

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

    1986-01-01

    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

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

    PubMed

    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

    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

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

    PubMed

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

    2012-10-01

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

  17. Phosphotidylinositol-3 kinase-mediated signals in mice immunized with the 57 kDa major antigenic outer-membrane protein of Shigella dysenteriae type 1

    Microsoft Academic Search

    A K Bagchi

    2005-01-01

    Antigen-specific T-cell signalling via T-cell antigen receptor stimulation was carried out in BALB\\/c mice immunized with the 57 kDa major antigenic component of Shigella dysenteriae 1 outer- membrane proteins. In presence of anti-CD3, the 57 kDa antigen was found to increase the level of IL-2 significantly instead of IL-4. IL-2 production in T cells was consistent with an increase in

  18. Characterization of protein and mannan polysaccharide antigens of yeasts, moulds, and actinomycetes.

    PubMed

    Reiss, E; Huppert, M; Cherniak, R

    1985-01-01

    Antigens in coccidioidin were compared with purified subfractions via tandem immunoelectrophoresis (IEP) and by a combination of advancing line and crossed IEP. Rocket IEP was suitable for titrating the reactions and showing the relationship between column fractions. These techniques required multicomponent antisera produced by hyperimmunization over many months and by the use of known standard migration pairs. The IEP variations were used to chart the development of antisera against coccidioidin factors, to monitor antigen purifications, and to test the immunochemical homogeneity of an isolated antigen. Mannose-based heteroglycans of Cryptococcus neoformans were recovered from the culture filtrate. After precipitation of the major viscous glucuronoxylomannan (GXM) with ethanol or cetyltrimethylammonium bromide, the supernate is reserved because it contains a galactoxylomannan (GalXM). After removal of glucuronic acid from the GXM, the resulting xylomannan of serotype A was amenable to 13C-nuclear magnetic resonance (NMR) spectrometry; it revealed nonreducing xylose, alpha-1,3-mannose, and alpha-1,2/1,3 disubstituted mannose, thus confirming by an independent means what was previously known. The characterization sequence of GalXM included: (1) gas-liquid chromatography (GLC) of neutral sugars as peracetylated aldononitriles; (2) methylation-fragmentation GLC mass spectrometry to determine the glycosidic linkages; and (3) 13C-NMR showing similarities to mannan of Saccharomyces cerevisiae. Affinity chromatography of the GalXM on concanavalin A separated the galactoxylo component from an adsorbed mannoprotein. Selection of monoclonal antibodies (MAbs) relies on presumptive enzyme immunoassays (EIAs) or radioimmunoassays for rapid screening of clones and for determination of isotypes; however, higher resolution confirmatory tests are needed to obtain MAbs of desired specificity. MAbs against Candida tropicalis mannan were labeled with horseradish peroxidase to use for detecting mannan in serum. MAbs against the partially purified "m" factor of histoplasmin were characterized by the enzyme-linked immunoelectro-transfer blot technique (EITB), revealing unsuspected complexity in the antigen. Secreted proteins of Nocardia asteroides were isoelectrically focused; three proteins, identified by EITB as promising to be specific for that actinomycete, were cut out of gels and used to immunize mice for production of MAbs. The fimbriae of Actinomyces viscosus and A. naeslundii that mediate lactose-reversible coagglutination with Streptococcus sanguis have been used to evoke MAbs.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:3916766

  19. Cell proliferation-associated nuclear antigen defined by antibody Ki-67: a new kind of cell cycle-maintaining proteins.

    PubMed

    Duchrow, M; Schlüter, C; Key, G; Kubbutat, M H; Wohlenberg, C; Flad, H D; Gerdes, J

    1995-01-01

    A decade of studies on the human nuclear antigen defined by monoclonal antibody Ki-67 (the "Ki-67 protein") has made it abundantly clear that this structure is strictly associated with human cell proliferation and that the expression of this protein can be used to assess the growth fraction of a given cell population. Until recently the Ki-67 protein was described as a nonhistone protein that is highly susceptible to protease treatment. We have isolated and sequenced cDNAs encoding for this antigen and found two isoforms of the full length cDNA of 11.5 and 12.5 kb, respectively, sequence and structure of which are thus far unique. The gene encoding the Ki-67 protein is organized in 15 exons and is localized on chromosome 10. The center of this gene is formed by an extraordinary 6845 bp exon containing 16 successively repeated homologous segments of 366 bp ("Ki-67 repeats"), each containing a highly conserved new motif of 66 bp ("Ki-67 motif"). The deduced peptide sequence of this central exon possess 10 ProGluSerThr (PEST) motifs which are associated with high turnover proteins such as other cell cycle-related proteins, oncogenes and transcription factors, etc. Like the latter proteins the Ki-67 antigen plays a pivotal role in maintaining cell proliferation because Ki-67 protein antisense oligonucleotides significantly inhibit 3H-thymidine incorporation in permanent human tumor cell lines in a dose-dependent manner. PMID:8744726

  20. Polarity protein Par3 controls B-cell receptor dynamics and antigen extraction at the immune synapse

    PubMed Central

    Reversat, Anne; Yuseff, Maria-Isabel; Lankar, Danielle; Malbec, Odile; Obino, Dorian; Maurin, Mathieu; Penmatcha, Naga Venkata Gayathri; Amoroso, Alejandro; Sengmanivong, Lucie; Gundersen, Gregg G.; Mellman, Ira; Darchen, François; Desnos, Claire; Pierobon, Paolo; Lennon-Duménil, Ana-Maria

    2015-01-01

    B-cell receptor (BCR) engagement with surface-tethered antigens leads to the formation of an immune synapse, which facilitates antigen uptake for presentation to T-lymphocytes. Antigen internalization and processing rely on the early dynein-dependent transport of BCR–antigen microclusters to the synapse center, as well as on the later polarization of the microtubule-organizing center (MTOC). MTOC repositioning allows the release of proteases and the delivery of MHC class II molecules at the synapse. Whether and how these events are coordinated have not been addressed. Here we show that the ancestral polarity protein Par3 promotes BCR–antigen microcluster gathering, as well as MTOC polarization and lysosome exocytosis, at the synapse by facilitating local dynein recruitment. Par3 is also required for antigen presentation to T-lymphocytes. Par3 therefore emerges as a key molecule in the coupling of the early and late events needed for efficient extraction and processing of immobilized antigen by B-cells. PMID:25631815

  1. Use of in vivo-induced antigen technology (IVIAT) for the identification of Streptococcus suis serotype 2 in vivo-induced bacterial protein antigens

    PubMed Central

    2009-01-01

    Background Streptococcus suis serotype 2 (SS2) is a zoonotic agent that causes death and disease in both humans and swine. A better understanding of SS2-host molecular interactions is crucial for understanding SS2 pathogenesis and immunology. Conventional genetic and biochemical approaches used to study SS2 virulence factors are unable to take into account the complex and dynamic environmental stimuli associated with the infection process. In this study, in vivo-induced antigen technology (IVIAT), an immunoscreening technique, was used to identify the immunogenic bacterial proteins that are induced or upregulated in vivo during SS2 infection. Results Convalescent-phase sera from pigs infected with SS2 were pooled, adsorbed against in vitro antigens, and used to screen SS2 genomic expression libraries. Upon analysis of the identified proteins, we were able to assign a putative function to 40 of the 48 proteins. These included proteins implicated in cell envelope structure, regulation, molecule synthesis, substance and energy metabolism, transport, translation, and those with unknown functions. The in vivo-induced changes in the expression of 10 of these 40 genes were measured using real-time reverse transcription (RT)-PCR, revealing that the expression of 6 of the 10 genes was upregulated in the in vivo condition. The strain distribution of these 10 genes was analyzed by PCR, and they were found in the most virulent SS2 strains. In addition, protein sequence alignments of the newly identified proteins demonstrate that three are putative virulence-associated proteins. Conclusion Collectively, our results suggest that these in vivo-induced or upregulated genes may contribute to SS2 disease development. We hypothesize that the identification of factors specifically induced or upregulated during SS2 infection will aid in our understanding of SS2 pathogenesis and may contribute to the control SS2 outbreaks. In addition, the proteins identified using IVIAT may be useful potential vaccine candidates or virulence markers. PMID:19765272

  2. A major T cell antigen of Mycobacterium leprae is a 10-kD heat-shock cognate protein

    PubMed Central

    1992-01-01

    Several mycobacterial antigens, identified by monoclonal antibodies and patient sera, have been found to be homologous to stress or heat-shock proteins (hsp) defined in Escherichia coli and yeast. A major antigen recognized by most Mycobacterium leprae-reactive human T cell lines and cell wall-reactive T cell clones is a 10-kD protein that has now been cloned and sequenced. The predicted amino acid sequence of this protein is 44% homologous to the hsp 10 (GroES) of E. coli. The purified native and recombinant 10-kD protein was found to be a stronger stimulator of peripheral blood T cell proliferation than other native and recombinant M. leprae proteins tested. The degree of reactivity paralleled the response to intact M. leprae throughout the spectrum of leprosy. Limiting-dilution analysis of peripheral blood lymphocytes from a patient contact and a tuberculoid patient indicated that approximately one third of M. leprae-reactive T cell precursors responded to the 10- kD antigen. T cell lines derived from lepromin skin tests were strongly responsive to the 10-kD protein. T cell clones reactive to both the purified native and recombinant 10-kD antigens recognized M. leprae- specific epitopes as well as epitopes crossreactive with the cognate antigen of M. tuberculosis. Further, the purified hsp 10 elicited strong delayed-type hypersensitivity reactions in guinea pigs sensitized to M. leprae. The strong T cell responses against the M. leprae 10-kD protein suggest a role for this heat-shock cognate protein in the protective/resistant responses to infection. PMID:1730920

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

    Microsoft Academic Search

    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

    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

  4. Loss ofO-antigen increases cell shape abnormalities in penicillin-binding protein mutants of Escherichia coli

    PubMed Central

    Ghosh, Anindya S.; Melquist, Amy L.; Young, Kevin D.

    2013-01-01

    Escherichia coli mutants lacking multiple penicillin-binding proteins (PBPs) produce aberrantly shaped cells. However, most of these experiments have been performed in E. coli K12 strains, which do not attach a complete O-antigen to their outer membrane lipopolysaccharide. We constructed mutants in different genetic backgrounds and found that the frequency of morphological deformities was higher in strains lacking the O-antigen. Also, complementing O-negative mutants with a heterologous O-antigen from Klebsiella returned a substantial fraction of misshapen cells to a normal morphology. Thus, the O-antigen contributes to cell shape in E. coli, perhaps by reducing the number of ectopic poles, which may be the proximal cause of shape abnormalities. PMID:16978365

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

    PubMed Central

    2013-01-01

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

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

    PubMed

    Pignatelli, Jaime; Alonso-Padilla, Julio; Rodríguez, Dolores

    2013-01-01

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

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

    PubMed Central

    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

    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

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

    PubMed

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

    2013-03-11

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

  9. Vaccine protection from CD4+ T-cell loss caused by simian immunodeficiency virus (SIV) mac251 is afforded by sequential immunization with three unrelated vaccine vectors encoding multiple SIV antigens.

    PubMed

    Koopman, Gerrit; Mortier, Daniella; Hofman, Sam; Niphuis, Henk; Fagrouch, Zahra; Norley, Steve; Sutter, Gerd; Liljeström, Peter; Heeney, Jonathan L

    2004-10-01

    Candidate human immunodeficiency virus (HIV) vaccine strategies that induce strong cellular immune responses protect rhesus macaques that are infected with recombinant simian/human immunodeficiency virus SHIV89.6p from acute CD4+ T-cell loss and delay progression to AIDS. However, similar strategies have not proven as efficacious in the simian immunodeficiency virus (SIV)mac model of AIDS, an infection that causes a slow, steady loss of CD4+ T-cell function and numbers in rhesus macaques similar to that caused by HIV-1, the principal cause of AIDS in humans. Efforts to increase vaccine efficacy by repeated boosting with the same vector are quickly limited by rising anti-vector immune responses. Here, the sequential use of three different vectors (DNA, Semliki Forest virus and modified vaccinia virus Ankara) encoding the same SIVmac structural and regulatory antigens was investigated and demonstrated to prevent or slow the loss of CD4+ T-cells after mucosal challenge with the highly pathogenic SIVmac251 strain. Of particular interest was an inverse association between the extent of T-helper 2 cytokine responses and steady-state virus load. Although limited in the number of animals, this study provides important proof of the efficacy of the triple-vector vaccine strategy against chronic, progressive CD4+ T-cell loss in the rigorous SIVmac/rhesus macaque model of AIDS. PMID:15448353

  10. Label-free quantitative mass spectrometry for analysis of protein antigens in a meningococcal group B outer membrane vesicle vaccine.

    PubMed

    Dick, Lawrence W; Mehl, John T; Loughney, John W; Mach, Anna; Rustandi, Richard R; Ha, Sha; Zhang, Lan; Przysiecki, Craig T; Dieter, Lance; Hoang, Van M

    2015-06-01

    The development of a multivalent outer membrane vesicle (OMV) vaccine where each strain contributes multiple key protein antigens presents numerous analytical challenges. One major difficulty is the ability to accurately and specifically quantitate each antigen, especially during early development and process optimization when immunoreagents are limited or unavailable. To overcome this problem, quantitative mass spectrometry methods can be used. In place of traditional mass assays such as enzyme-linked immunosorbent assays (ELISAs), quantitative LC-MS/MS using multiple reaction monitoring (MRM) can be used during early-phase process development to measure key protein components in complex vaccines in the absence of specific immunoreagents. Multiplexed, label-free quantitative mass spectrometry methods using protein extraction by either detergent or 2-phase solvent were developed to quantitate levels of several meningococcal serogroup B protein antigens in an OMV vaccine candidate. Precision was demonstrated to be less than 15% RSD for the 2-phase extraction and less than 10% RSD for the detergent extraction method. Accuracy was 70 to 130% for the method using a 2-phase extraction and 90-110% for detergent extraction. The viability of MS-based protein quantification as a vaccine characterization method was demonstrated and advantages over traditional quantitative methods were evaluated. Implementation of these MS-based quantification methods can help to decrease the development time for complex vaccines and can provide orthogonal confirmation of results from existing antigen quantification techniques. PMID:25997113

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

    PubMed Central

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

    2000-01-01

    Bacillus anthrax lethal toxin can be engineered to deliver foreign proteins to the cytosol for antigen presentation to CD8 T cells. Vaccination with modified toxins carrying 8–9 amino acid peptide epitopes induces protective immunity in mice. To evaluate whether large protein antigens can be used with this system, recombinant constructs encoding several HIV antigens up to 500 amino acids were produced. These candidate HIV vaccines are safe in animals and induce CD8 T cells in mice. Constructs encoding gag p24 and nef stimulate gag-specific CD4 proliferation and a secondary cytotoxic T lymphocyte response in HIV-infected donor peripheral blood mononuclear cells in vitro. These results lay the foundation for future clinical vaccine studies. PMID:10884430

  12. Role of the charged tail in localization of a surface protein antigen of Streptococcus mutans.

    PubMed Central

    Murakami, Y; Yamashita, Y; Nakano, Y; Ito, H O; Yu, H; Koga, T

    1997-01-01

    To make clear the role of the C terminus of a surface protein antigen (PAc) of Streptococcus mutans, stepwise truncations beginning at the C terminus of PAc were performed by utilizing site-directed mutagenesis. A remarkable increase in the amount of cell-free PAc was observed upon deletion of four or more amino acid residues at the C terminus. On the other hand, the amount of cell surface PAc gradually decreased when increasing numbers (four or more) of amino acid residues were deleted at the C terminus, and deletion of six amino acids involving both the total charged tail and Leu, an amino acid residue immediately upstream of the charged tail, resulted in a drastic reduction in the amount of cell surface PAc. These results indicate that the cytoplasmic charged tail and Leu residue are required for cell surface localization of PAc in S. mutans. PMID:9119499

  13. Antigenic diversity of Theileria major piroplasm surface protein gene in Jeju black cattle

    PubMed Central

    Ko, Myung-Soon; Lee, Kyoung-Kap; Hwang, Kyu-Kye; Kim, Byung-Sun; Choi, Gui-Cheol

    2008-01-01

    Piroplasms are tick-transmitted, intracellular, hemoprotozoan parasites that cause anorexia, fever, anemia, and icterus. Theileriosis is caused by Theileria sergenti and causes major economic losses in grazing cattle in Japan and Korea. In May 2003, we examined the antigenic diversity of the major piroplasm surface protein (MPSP) gene in 35 healthy Jeju black cattle that were born and raised at the National Institute of Subtropical Agriculture. On microscopic examination of Giemsa-stained blood smears, 9 of 35 cattle had intra-erythrocytic piroplasms. Hematological data were within normal range for all 35 cattle. Amplification of DNA from all blood samples using universal MPSP gene primers showed mixed infections with C, I, and B type Theileria spp. Type C was identified in 20 of 35 blood samples, and type B was identified in 17 samples. Allelic variation was seen in type B. PMID:18487936

  14. Hypothyroidism attenuates protein tyrosine nitration, oxidative stress and renal damage induced by ischemia and reperfusion: effect unrelated to antioxidant enzymes activities

    PubMed Central

    Tenorio-Velázquez, Verónica M; Barrera, Diana; Franco, Martha; Tapia, Edilia; Hernández-Pando, Rogelio; Medina-Campos, Omar Noel; Pedraza-Chaverri, José

    2005-01-01

    Background It has been established that hypothyroidism protects rats against renal ischemia and reperfusion (IR) oxidative damage. However, it is not clear if hypothyroidism is able to prevent protein tyrosine nitration, an index of nitrosative stress, induced by IR or if antioxidant enzymes have involved in this protective effect. In this work it was explored if hypothyroidism is able to prevent the increase in nitrosative and oxidative stress induced by IR. In addition the activity of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase was studied. Control and thyroidectomized (HTX) rats were studied 24 h of reperfusion after 60 min ischemia. Methods Male Wistar rats weighing 380 ± 22 g were subjected to surgical thyroidectomy. Rats were studied 15 days after surgery. Euthyroid sham-operated rats were used as controls (CT). Both groups of rats underwent a right kidney nephrectomy and suffered a 60 min left renal ischemia with 24 h of reperfusion. Rats were divided in four groups: CT, HTX, IR and HTX+IR. Rats were sacrificed and samples of plasma and kidney were obtained. Blood urea nitrogen (BUN) and creatinine were measured in blood plasma. Kidney damage was evaluated by histological analysis. Oxidative stress was measured by immunohistochemical localization of protein carbonyls and 4-hydroxy-2-nonenal modified proteins. The protein carbonyl content was measured using antibodies against dinitrophenol (DNP)-modified proteins. Nitrosative stress was measured by immunohistochemical analysis of 3-nitrotyrosine modified proteins. The activity of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase was measured by spectrophotometric methods. Multiple comparisons were performed with ANOVA followed by Bonferroni t test. Results The histological damage and the rise in plasma creatinine and BUN induced by IR were significantly lower in HTX+IR group. The increase in protein carbonyls and in 3-nitrotyrosine and 4-hydroxy-2-nonenal modified proteins was prevented in HTX+IR group. IR-induced decrease in renal antioxidant enzymes was essentially not prevented by HTX in HTX+IR group. Conclusion Hypothyroidism was able to prevent not only oxidative but also nitrosative stress induced by IR. In addition, the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase seem not to play a protective role in this experimental model. PMID:16274486

  15. Antigenic analysis monoclonal antibodies against different epitopes of ?B protein of Muscovy duck reovirus.

    PubMed

    Li, Yongfeng; Yin, Xiuchen; Chen, Xiaodan; Li, Xiaojun; Li, Jinzhe; Liu, Chunguo; Liu, Ming; Zhang, Yun

    2012-02-01

    ?B is one of the major structural proteins of Muscovy duck reovirus (DRV), which is able to induce protective immune response in target birds. Four anti-DRV ?B MAbs were identified belong to two distinct epitopes, designated A (1E5, 4E3, and 5D8) and B (2F7) (Liu et al., 2010). To understand antigenic determinants of the ?B protein, a set of 20 (P1-P20), partially overlapping and consecutive peptides spanning ?B were expressed and then screened by MAbs. With Western blot and enzyme-linked immunosorbent assay (ELISA), two minimal units of the linear epitopes, 19YIRAPACWD27 (epitope B) and 65TDGVCFPHHK74 (epitope A), were identified within N-terminal region of the ?B protein. The epitope B was highly conserved among DRV and avian reovirus (ARV) strains through sequence alignment analysis. Immunofluorescence assays (IFA) and ELISA, confirmed that epitope B is a broad group-specific epitope among DRV and ARV. Epitope A could only react with chicken embyonated fibroblast cells (CEF) infected with DRV, but not ARV. However, both peptides have good immunogenicity and could induce antibodies against DRV in BALB/c mice. This report documents the first identification of ?B epitopes in the precise locations. The two probes would be useful in the development of discriminating diagnostic kits for DRV and ARV infection. PMID:22197425

  16. Targeting proliferating cell nuclear antigen and its protein interactions induces apoptosis in multiple myeloma cells.

    PubMed

    Müller, Rebekka; Misund, Kristine; Holien, Toril; Bachke, Siri; Gilljam, Karin M; Våtsveen, Thea K; Rø, Torstein B; Bellacchio, Emanuele; Sundan, Anders; Otterlei, Marit

    2013-01-01

    Multiple myeloma is a hematological cancer that is considered incurable despite advances in treatment strategy during the last decade. Therapies targeting single pathways are unlikely to succeed due to the heterogeneous nature of the malignancy. Proliferating cell nuclear antigen (PCNA) is a multifunctional protein essential for DNA replication and repair that is often overexpressed in cancer cells. Many proteins involved in the cellular stress response interact with PCNA through the five amino acid sequence AlkB homologue 2 PCNA-interacting motif (APIM). Thus inhibiting PCNA's protein interactions may be a good strategy to target multiple pathways simultaneously. We initially found that overexpression of peptides containing the APIM sequence increases the sensitivity of cancer cells to contemporary therapeutics. Here we have designed a cell-penetrating APIM-containing peptide, ATX-101, that targets PCNA and show that it has anti-myeloma activity. We found that ATX-101 induced apoptosis in multiple myeloma cell lines and primary cancer cells, while bone marrow stromal cells and primary healthy lymphocytes were much less sensitive. ATX-101-induced apoptosis was caspase-dependent and cell cycle phase-independent. ATX-101 also increased multiple myeloma cells' sensitivity against melphalan, a DNA damaging agent commonly used for treatment of multiple myeloma. In a xenograft mouse model, ATX-101 was well tolerated and increased the anti-tumor activity of melphalan. Therefore, targeting PCNA by ATX-101 may be a novel strategy in multiple myeloma treatment. PMID:23936203

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

    PubMed Central

    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

    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

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

    PubMed Central

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

    1997-01-01

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

  19. An acid dissociation bridging ELISA for detection of antibodies directed against therapeutic proteins in the presence of antigen

    Microsoft Academic Search

    Aaron Patton; Michael C. Mullenix; Steven J. Swanson; Eugen Koren

    2005-01-01

    Bridging Enzyme-Linked Immunosorbent Assay (ELISA) is commonly used in detection of antibodies directed against therapeutic proteins. Advantages of the bridging ELISA include the capability to detect antibodies regardless of their isotype or the species of origin. However, detection of antibodies can be difficult, if not impossible, in the presence of high levels of the antigen in the sample matrix. This

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

    Microsoft Academic Search

    JUNKO KIMURA-KURODA; KOTARO YASUI

    1986-01-01

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

  1. Conformational fluctuations in anthrax protective antigen: a possible role of calcium in the folding pathway of the protein

    Microsoft Academic Search

    Pradeep K Gupta; Harish Chandra; Reetika Gaur; Raj K Kurupati; Shantanu Chowdhury; Vibha Tandon; Yogendra Singh; Kapil Maithal

    2003-01-01

    Protective antigen (PA) is the central receptor binding component of anthrax toxin, which translocates catalytic components of the toxin into the cytosol of mammalian cells. Ever since the crystal structure of PA was solved, there have been speculations regarding the possible role of calcium ions present in domain I of the protein. We have carried out a systematic study to

  2. Effects of cytosine-phosphate-guanosine oligodinucleotides (CpG-ODNs) on oral immunization with protein antigen or replicating parasite 

    E-print Network

    Ameiss, Keith Allen

    2005-08-29

    immunomodulation of the host??s response to live vaccines may be a viable means to control enteric infections in commercial poultry. In the present investigations we evaluated a means for delivering protein antigen in the drinking water and the use of CpG-ODNs, a...

  3. THE BABESIA BOVIS MEROZOITE SURFACE ANTIGEN 2 LOCUS CONTAINS FOUR TANDEMLY ARRANGED AND EXPRESSED GENES ENCODING IMMUNOLOGICALLY DISTINCT PROTEINS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    SciTech Connect

    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

    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.

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2014-08-01

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

  7. Human H-Y: A Male-Specific Histocompatibility Antigen Derived from the SMCY Protein

    Microsoft Academic Search

    Wei Wang; Leslie R. Meadows; Joke M. M. den Haan; Nicholas E. Sherman; Ye Chen; Els Blokland; Jeffrey Shabanowitz; Alexander I. Agulnik; Ronald C. Hendrickson; Colin E. Bishop; Donald F. Hunt; Els Goulmy; Victor H. Engelhard

    1995-01-01

    H-Y is a transplantation antigen that can lead to rejection of male organ and bone marrow grafts by female recipients, even if the donor and recipient match at the major histocompatibility locus of humans, the HLA (human leukocyte antigen) locus. However, the origin and function of H-Y antigens has eluded researchers for 40 years. One human H-Y antigen presented by

  8. Identification of Novel Antigenic Proteins in a Complex Anaplasma marginale Outer Membrane Immunogen by Mass Spectrometry and Genomic Mapping

    PubMed Central

    Lopez, Job E.; Siems, William F.; Palmer, Guy H.; Brayton, Kelly A.; McGuire, Travis C.; Norimine, Junzo; Brown, Wendy C.

    2005-01-01

    Immunization with purified Anaplasma marginale outer membranes induces complete protection against infection that is associated with CD4+ T-lymphocyte-mediated gamma interferon secretion and immunoglobulin G2 (IgG2) antibody titers. However, knowledge of the composition of the outer membrane immunogen is limited. Recent sequencing and annotation of the A. marginale genome predicts at least 62 outer membrane proteins (OMP), enabling a proteomic and genomic approach for identification of novel OMP by use of IgG serum antibody from outer membrane vaccinates. Outer membrane proteins were separated by two-dimensional electrophoresis, and proteins recognized by total IgG and IgG2 in immune sera of outer membrane-vaccinated cattle were detected by immunoblotting. Immunoreactive protein spots were excised and subjected to liquid chromatography-tandem mass spectrometry. A database search of the A. marginale genome identified 24 antigenic proteins that were predicted to be outer membrane, inner membrane, or membrane-associated proteins. These included the previously characterized surface-exposed outer membrane proteins MSP2, operon associated gene 2 (OpAG2), MSP3, and MSP5 as well as recently identified appendage-associated proteins. Among the 21 newly described antigenic proteins, 14 are annotated in the A. marginale genome and include type IV secretion system proteins, elongation factor Tu, and members of the MSP2 superfamily. The identification of these novel antigenic proteins markedly expands current understanding of the composition of the protective immunogen and provides new candidates for vaccine development. PMID:16299305

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

    PubMed

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

    2010-04-19

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

  10. Autophagy proteins stabilize pathogen-containing phagosomes for prolonged MHC II antigen processing

    PubMed Central

    Romao, Susana; Gasser, Nathalie; Becker, Andrea C.; Guhl, Bruno; Bajagic, Milica; Vanoaica, Danusia; Ziegler, Urs; Roesler, Joachim; Dengjel, Jörn; Reichenbach, Janine

    2013-01-01

    Antigen preservation for presentation is a hallmark of potent antigen-presenting cells. In this paper, we report that in human macrophages and dendritic cells, a subset of phagosomes gets coated with Atg8/LC3, a component of the molecular machinery of macroautophagy, and maintains phagocytosed antigens for prolonged presentation on major histocompatibility complex class II molecules. These Atg8/LC3-positive phagosomes are formed around the antigen with TLR2 agonists and require reactive oxygen species production by NOX2 for their generation. A deficiency in the NOX2-dependent formation of these antigen storage phagosomes could contribute to compromise antifungal immune control in chronic granulomatous disease patients. PMID:24322427

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

    PubMed Central

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

    2008-01-01

    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

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

    PubMed

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

    2014-11-01

    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

  13. The expression of the Goodpasture antigen-binding protein (ceramide transporter) in adult rat brain.

    PubMed

    Mencarelli, Chiara; Hammels, Caroline; Van Den Broeck, Joost; Losen, Mario; Steinbusch, Hellen; Revert, Francisco; Saus, Juan; Hopkins, David A; De Baets, Marc H; Steinbusch, Harry W; Martinez-Martinez, Pilar

    2009-10-01

    The Goodpasture antigen-binding protein (GPBP) plays a critical role in brain development. Knockdown of GPBP leads to loss of myelinated tracts in the central nervous system and to extensive apoptosis in the brain during early embryogenesis. GPBP was initially identified as a protein associated with the autoantigen in Goodpasture autoimmune syndrome, where it was shown to be a kinase that regulates type IV collagen organization. GPBP isoforms bind and transport ceramide from the endoplasmic reticulum to the Golgi apparatus and are therefore also known as ceramide transporters (CERT). Ceramide dysregulation is involved in autoimmunity and neurodegenerative disorders. In order to analyze the possible role of GPBP in neuroinflammation and neurodegeneration we studied the basal GPBP expression in normal rat brain. High levels of immunoreactivity were detected in neurons of the cerebral cortex, hippocampal formation, the basal ganglia, the olfactory bulb and nuclei of the thalamus, the hypothalamus and the septal area. Lower expression levels of GPBP were observed widely throughout the brain, suggesting that GPBP plays an important role in central nervous system neuron function. PMID:19555756

  14. Evaluation of multiple antigenic peptides based on the Chikungunya E2 protein for improved serological diagnosis of infection.

    PubMed

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

    2015-03-01

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

  15. Variability of genes encoding surface proteins used as vaccine antigens in meningococcal endemic and epidemic strain panels from Norway.

    PubMed

    Holst, Johan; Comanducci, Maurizio; Bambini, Stefania; Muzzi, Alessandro; Comandi, Sara; Oksnes, Jan; DeTora, Lisa; Pizza, Mariagrazia; Rappuoli, Rino; Caugant, Dominique A

    2014-05-13

    Surface-expressed protein antigens such as factor H-binding protein (fHbp), Neisserial adhesin A (NadA), Neisserial heparin-binding antigen (NHBA) and Porin protein A (PorA); all express sequence variability that can affect their function as protective immunogens when used in meningococcal serogroup B vaccines like the recently-approved 4CMenB (Bexsero(®)). We assessed the sequence variation of genes coding for these proteins and two additional proteins ("fusion partners" to fHbp and NHBA) in pathogenic isolates from a recent low incidence period (endemic situation; 2005-2006) in Norway. Findings among strains from this panel were contrasted to what was found among isolates from a historic outbreak (epidemic situation; 1985-1990). Multilocus sequence typing revealed 14 clonal complexes (cc) among the 66 endemic strains, while cc32 vastly predominated in the 38-strain epidemic panel. Serogroup B isolates accounted for 50/66 among endemic strains and 28/38 among epidemic strains. Potential strain-coverage ("sequence match") for the 4CMenB vaccine was identified among the majority (>70%) of the endemic serogroup B isolates and all of the epidemic serogroup B isolates evaluated. Further information about the degree of expression, surface availability and the true cross-reactivity for the vaccine antigens will be needed to fully characterize the clinical strain-coverage of 4CMenB in various geographic and epidemiological situations. PMID:24631075

  16. Immune recognition of swine vesicular disease virus structural proteins: novel antigenic regions that are not exposed in the capsid.

    PubMed

    Jiménez-Clavero, M A; Douglas, A; Lavery, T; Garcia-Ranea, J A; Ley, V

    2000-04-25

    Swine vesicular disease virus (SVDV) is an enterovirus of the Picornaviridae family that belongs to the coxsackievirus B group. A number of antigenic sites have been identified in SVDV by analysis of neutralizing monoclonal antibody-resistant mutants and shown to be exposed on the surface of the capsid. In this paper we have identified seven new immunodominant antigenic regions in SVDV capsid proteins by a peptide scanning method, using a panel of sera from infected pigs. When these antigenic regions were located in the capsid by using a computer-generated three-dimensional model of the virion, one was readily exposed on the surface of the virus and the remaining sites were located facing the inner side of the capsid shell, at subunit contacts, or in the interior of the subunit structure. PMID:10772981

  17. Antigenic characterization of expressed complete and different truncated recombinant nucleocapsid proteins of hantaan virus by monoclonal antibodies.

    PubMed

    Lü, Xin; Zhang, Fanglin; Li, Yucheng; Xue, Xiaoping; Yin, Wen; Xu, Zhikai

    2011-10-01

    The nucleocapsid protein (NP) of hantavirus has been shown to be highly immunogenic in laboratory animals and humans, and it induces an early and long-lasting humoral immune response during hantavirus infection. In the present study, entire and three partially truncated open reading frames (ORF) of NP genes of Hantaan virus (HTNV) strain 76-118, a prototype of hantavirus, were amplified and then cloned in prokaryotic expression vectors. The recombinant whole NP and truncated NPs were expressed in Escherichia coli. In addition, their antigenic and biological activities were evaluated in an enzyme-linked immunosorbent assay (ELISA) for detection of a series of monoclonal antibodies (MAbs). Results showed that the expressed complete HTNV NP had a similar function as an authentic viral NP. The antigen sites on NP of hantavirus were mainly located in the N-terminus, and the region at 1-37 amino acid might be one important antigenic domain. PMID:22008071

  18. Membrane topology of the L6 antigen and identification of the protein epitope recognized by the L6 monoclonal antibody.

    PubMed

    Marken, J S; Bajorath, J; Edwards, C P; Farr, A G; Schieven, G L; Hellström, I; Hellström, K E; Aruffo, A

    1994-03-11

    The murine monoclonal antibody (mAb) L6 recognizes an integral membrane glycoprotein that is highly expressed on lung, breast, colon, and ovarian carcinomas and is referred to as the L6 antigen. This antigen is an attractive target for therapeutic intervention due to its high level expression on malignant cells. We have previously reported the isolation of a cDNA encoding the human L6 antigen (H-L6). Here, we report the isolation of a cDNA clone encoding the murine L6 antigen (M-L6). This cDNA contains one long open reading frame, which encodes a 220-amino acid polypeptide that is 78% homologous to H-L6. This protein contains short NH2- and COOH-terminal hydrophilic domains and four hydrophobic regions, each long enough to span the plasma membrane. Each of these hydrophobic domains is separated by a hydrophilic domain, the longest of which contains one possible N-linked glycosylation site and is located between the third and fourth hydrophobic domains. We have previously demonstrated that the murine L6 mAb recognizes a protein epitope expressed on human tumor-derived cell lines. Now, using chimeric cDNA constructs encoding human-murine L6 antigen hybrids in conjunction with monoclonal antibody binding experiments, we show that the 42-residue hydrophilic domain of the L6 antigen, located between the third and fourth hydrophobic domains, is outside the cell and that residues in the NH2-terminal region of this domain are critical for the binding of the murine L6 mAb to H-L6. PMID:7510285

  19. Marrow transplantation from unrelated donors.

    PubMed

    Sierra, J; Anasetti, C

    1995-11-01

    The use of an HLA-compatible unrelated donor is an option for patients who require an allogeneic transplant but lack a family member match. Grafts from unrelated volunteer donors have provided long-term disease-free survival for a variable proportion of patients, depending on degree of HLA matching with the donor, patient's disease, disease stage, and age. The number of volunteers in marrow donor registries worldwide has increased to more than 2.5 million. The number of unrelated donor transplants facilitated by the US National Marrow Donor Program alone will exceed 900 this year. Progress in HLA-typing technology results in a more precise definition of donor and recipient matching and new assays have been developed with initial success to measure alloreactive T-cell precursors for selection of donors with less antihost reactivity. Prevention and treatment of graft failure, graft-versus-host disease, opportunistic infections, and Epstein-Barr virus-associated lymphoproliferative disease remain a challenge. PMID:9372034

  20. QUANTITATIVE STUDIES ON ANTIGENIC RECOGNITION

    PubMed Central

    Ramseier, Hansruedy

    1969-01-01

    Lymphoid cells from normal animals from two genetically alien but isogenic strains of mice or rats elaborated a factor into the medium during in vitro cultivation. Similarly, lymphoid cells from specifically presensitized animals mixed and cultivated with donor-type lymphoid cells, as well as normal or specifically immunized mouse spleen cells exposed to heterologous red blood cell antigens, formed this factor. Culture supernatants of genetically identical lymphoid cells lacked activity. This factor was measured by its ability to induce accumulations of polymorphonuclear cells following injection into the skin of normal hamster hosts. Gentle trypsinization of excised 24 hr cutaneous reactions permitted quantitative estimation of the number of polymorphonuclear cells accumulated and, therefore, of the activity of the factor. The factor present in culture fluids could be defined as a product of antigenic recognition (PAR). PAR was formed only when immunologically competent cells were exposed to cells carrying histocompatibility antigens of recognizable foreignness. This view was based chiefly on the observation that, whereas lymphoid cells from specifically tolerant mice failed to recognize the paralytogen, they did recognize antigens of an unrelated mouse strain. In addition, immunocompetence was of paramount importance, since cocultivation of immunologically incompetent, yet strongly incompatible, rat thymocytes did not result in the formation of PAR. Formation of PAR was observed in very simple media. A source of energy and a suitable culture vessel were found to be the only essential nonimmunological factors for PAR to be elaborated. The presence of inhibitors of glycolysis in the medium during cultivation of mixtures of immunocompetent cells of disparate origin severely inhibited the recognition process. With cell mixtures of the immunological one-way variety, treatment of aggressor cells with inhibitors of glycolysis or of protein synthesis interfered with antigenic recognition, whereas similar treatments of the target cells were without effect. The findings presented sustain the thesis that the elaboration of PAR is a very early event in the immune process. PMID:5352782

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

    PubMed Central

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

    1998-01-01

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

  2. Differential regulation of T cell antigen responsiveness by isoforms of the src-related tyrosine protein kinase p59fyn

    PubMed Central

    1992-01-01

    Recent observations suggest that the src-related tyrosine protein kinase p59fyn may be involved in antigen-induced T lymphocyte activation. As a result of alternative splicing, p59fyn exists as two isoforms that differ exclusively within a short sequence spanning the end of the Src Homology 2 (SH2) region and the beginning of the tyrosine protein kinase domain. While one p59fyn isoform (fynB) is highly expressed in brain, the alternative product (fynT) is principally found in T lymphocytes. To further understand the role of p59fyn in T cell activation and to test the hypothesis that p59fynT serves a tissue-specific function in T lymphocytes, we have examined the effects of expression of activated versions (tyrosine 528 to phenylalanine 528 mutants) of either form of p59fyn on the physiology of an antigen-specific mouse T cell hybridoma. Our results demonstrated that the two forms of fyn, expressed in equivalent amounts, efficiently enhanced antibody-induced T cell receptor (TCR)-mediated signals. In contrast, only p59fynT increased interleukin 2 production in response to antigen stimulation. This finding implies that the distinct p59fyn isoform expressed in T lymphocytes regulates the coupling of TCR stimulation by antigen/major histocompatibility complex to lymphokine production. PMID:1375262

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

    PubMed Central

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

    2014-01-01

    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

  4. Leishmania donovani: Identification of stimulatory soluble antigenic proteins using cured human and hamster lymphocytes for their prophylactic potential against visceral leishmaniasis

    Microsoft Academic Search

    Ravendra Garg; Shraddha K. Gupta; Parul Tripathi; K. Hajela; S. Sundar; S. Naik; Anuradha Dube

    2006-01-01

    Most of the studies for the identification of prophylactic antigens that elicit T cell responses were concentrated on membrane proteins of Leishmania donovani. This study was taken up to assess L. donovani soluble promastigote antigens for their ability to stimulate proliferation of peripheral blood mononuclear cells (PBMCs) from cured visceral leishmaniasis (VL) patients, endemic and non-endemic controls and lymphocytes\\/peritoneal macrophages

  5. Recombinant Exosporium Protein BclA of Bacillus anthracis Is Effective as a Booster for Mice Primed with Suboptimal Amounts of Protective Antigen

    Microsoft Academic Search

    Trupti N. Brahmbhatt; Stephen C. Darnell; Humberto M. Carvalho; Patrick Sanz; Tae J. Kang; Robert L. Bull; Susan B. Rasmussen; Alan S. Cross; Alison D. O'Brien

    2007-01-01

    Bacillus collagen-like protein of anthracis (BclA) is an immunodominant glycoprotein located on the exosporium of Bacillus anthracis. We hypothesized that antibodies to this spore surface antigen are largely responsible for the augmented immunity to anthrax that has been reported for animals vaccinated with inactivated spores and protective antigen (PA) compared to vaccination with PA alone. To test this theory, we

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

    SciTech Connect

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

    1991-10-01

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

  7. Antigenic and Immunogenic Characterization of Recombinant Baculovirus-Expressed Severe Acute Respiratory Syndrome Coronavirus Spike Protein: Implication for Vaccine Design

    Microsoft Academic Search

    Yuxian He; Jingjing Li; Susanne Heck; Sara Lustigman; Shibo Jiang

    2006-01-01

    The spike (S) glycoprotein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates the receptor interaction and immune recognition and is considered a major target for vaccine design. However, its antigenic and immunogenic properties remain to be elucidated. In this study, we immunized mice with full-length S protein (FL-S) or its extracellular domain (EC-S) expressed by recombinant baculoviruses in insect cells.

  8. Antigenic Importance of the Carboxy-Terminal Beta-Strand of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein

    Microsoft Academic Search

    SARAH WOOTTON; GABRIELLA KOLJESAR; LIUZHAN YANG; KYOUNG-JIN YOON; DONGWAN YOO

    2001-01-01

    Five domains of antigenic importance were previously mapped on the nucleocapsid protein (N) of the porcine reproductive and respiratory syndrome virus (PRRSV), and a domain comprised of the 11 C-terminal-most amino acids (residues 112 to 123) was shown to be essential for binding of N-specific conformation-dependent monoclonal antibodies (MAbs). In the present study, the importance of individual residues within this

  9. Pan-Serotype Diagnostic for Foot-and-Mouth Disease Using the Consensus Antigen of Nonstructural Protein 3B.

    PubMed

    Van Dreumel, Alyssa K; Michalski, Wojtek P; McNabb, Leanne M; Shiell, Brian J; Singanallur, Nagendrakumar B; Peck, Grantley R

    2015-06-01

    An amino acid consensus sequence for the seven serotypes of foot-and-mouth disease virus (FMDV) nonstructural protein 3B, including all three contiguous repeats, and its use in the development of a pan-serotype diagnostic test for all seven FMDV serotypes are described. The amino acid consensus sequence of the 3B protein was determined from a multiple-sequence alignment of 125 sequences of 3B. The consensus 3B (c3B) protein was expressed as a soluble recombinant fusion protein with maltose-binding protein (MBP) using a bacterial expression system and was affinity purified using amylose resin. The MBP-c3B protein was used as the antigen in the development of a competition enzyme-linked immunosorbent assay (cELISA) for detection of anti-3B antibodies in bovine sera. The comparative diagnostic sensitivity and specificity at 47% inhibition were estimated to be 87.22% and 93.15%, respectively. Reactivity of c3B with bovine sera representing the seven FMDV serotypes demonstrated the pan-serotype diagnostic capability of this bioreagent. The consensus antigen and competition ELISA are described here as candidates for a pan-serotype diagnostic test for FMDV infection. PMID:25788546

  10. A Fusion Protein between Streptavidin and the Endogenous TLR4 Ligand EDA Targets Biotinylated Antigens to Dendritic Cells and Induces T Cell Responses In Vivo

    PubMed Central

    Durantez, Maika; Lozano, Teresa; Rudilla, Francesc; Rehberger, Federico; Casares, Noelia; Villanueva, Lorea; Martinez, Marta; Gorraiz, Marta; Borrás-Cuesta, Francisco; Sarobe, Pablo; Prieto, Jesús; Lasarte, Juan José

    2013-01-01

    The development of tools for efficient targeting of antigens to antigen presenting cells is of great importance for vaccine development. We have previously shown that fusion proteins containing antigens fused to the extra domain A from fibronectin (EDA), an endogenous TLR4 ligand, which targets antigens to TLR4-expressing dendritic cells (DC), are highly immunogenic. To facilitate the procedure of joining EDA to any antigen of choice, we have prepared the fusion protein EDAvidin by linking EDA to the N terminus of streptavidin, allowing its conjugation with biotinylated antigens. We found that EDAvidin, as streptavidin, forms tetramers and binds biotin or biotinylated proteins with a Kd ~ 2.6 × 10?14?mol/L. EDAvidin favours the uptake of biotinylated green fluorescent protein by DC. Moreover, EDAvidin retains the proinflammatory properties of EDA, inducing NF-?? by TLR4-expressing cells, as well as the production of TNF-? by the human monocyte cell line THP1 and IL-12 by DC. More importantly, immunization of mice with EDAvidin conjugated with the biotinylated nonstructural NS3 protein from hepatitis C virus induces a strong anti-NS3 T cell immune response. These results open a new way to use the EDA-based delivery tool to target any antigen of choice to DC for vaccination against infectious diseases and cancer. PMID:24093105

  11. Development of a multi-epitope antigen of S protein-based ELISA for antibodies detection against infectious bronchitis virus.

    PubMed

    Ding, Meng-Die; Wang, Hong-Ning; Cao, Hai-Peng; Fan, Wen-Qiao; Ma, Bing-Cun; Xu, Peng-Wei; Zhang, An-Yun; Yang, Xin

    2015-08-01

    An indirect enzyme-linked immunosorbent assay (ELISA) method based on a novel multi-epitope antigen of S protein (SE) was developed for antibodies detection against infectious bronchitis virus (IBV). The multi-epitope antigen SE protein was designed by arranging three S gene fragments (166-247 aa, S1 gene; 501-515 aa, S1 gene; 8-30 aa, S2 gene) in tandem. It was identified to be approximately 32 kDa as a His-tagged fusion protein and can bind IBV positive serum by western blot analysis. The conditions of the SE-ELISA method were optimized. The optimal concentration of the coating antigen SE was 3.689 ?g/mL and the dilution of the primary antibodies was identified as 1:1000 using a checkerboard titration. The cut-off OD450 value was established at 0.332. The relative sensitivity and specificity between the SE-ELISA and IDEXX ELISA kit were 92.38 and 89.83%, respectively, with an accuracy of 91.46%. This assay is sensitive and specific for detection of antibodies against IBV. PMID:25832890

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

    Microsoft Academic Search

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

    1992-01-01

    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

  13. Transfer of Proteins from Gels to Diazobenzyloxymethyl-Paper and Detection with Antisera: A Method for Studying Antibody Specificity and Antigen Structure

    Microsoft Academic Search

    Jaime Renart; Jakob Reiser; George R. Stark

    1979-01-01

    We describe a rapid and very sensitive method for detecting proteins as antigens after their separation in polyacrylamide\\/agarose composite gels, with or without sodium dodecyl sulfate. The polyacrylamide matrix is crosslinked with a reagent that can be cleaved with periodate or alkali to facilitate transfer of the protein bands to diazobenzyloxymethyl-paper, where they are coupled covalently. Specific proteins are detected

  14. Self-assembly, antigenicity, and immunogenicity of the rabbit haemorrhagic disease virus (Czechoslovakian strain V-351) capsid protein expressed in baculovirus

    Microsoft Academic Search

    H. S. Nagesha; L. F. Wang; A. D. Hyatt; C. J. Morrissy; C. Lenghaus; H. A. Westbury

    1995-01-01

    Summary Rabbit haemorrhagic disease virus (RHDV) capsid protein was expressed in a baculovirus system. Analysis of the expressed product showed that the recombinant protein, which is 60 kDa in size, was antigenic as revealed by its reactions in ELISA and Western blot with the antibodies raised against RHDV. Direct electron microscopy of the cell culture supernatant and the purified protein

  15. Origin of an 84-kDa protein with ABH blood-typing antigen activity in human seminal plasma.

    PubMed

    Sato, I; Nakamura, A; Yamazaki, K; Sakata, N; Ito, K; Ito, E; Sagi, M

    1999-01-01

    In order to investigate the origin of an 84-kDa protein with ABH blood-typing antigen activity (p 84) and its concentration in human seminal plasma, a monoclonal antibody (mAb p 84) was produced. The protein was recognized in breast milk as well as in seminal plasma by an indirect, enzyme-linked immunosorbent assay (ELISA) using this mAb. mAb p 84 identified 84-kDa and 83-kDa forms of the protein in seminal plasma and breast milk, respectively, on immunoblotting. The mean concentration of p 84 in seminal plasma was 949 microg/ml (n = 54 subjects). There was no significant difference in the concentration of p 84 between individuals who secreted (Se) or did not secrete (non-se) the ABH antigen into their seminal plasma, nor were there any significant correlations between the concentration of p 84 and the total seminal protein concentration. An immunohistochemical study using mAb p 84 with light microscopic detection showed that p 84 was located in the cytoplasm of the inner layer of pseudostratified cuboidal epithelial cells of the seminal vesicles, but no immunoreactivity was found in the prostate. These data indicate that p 84 originates from a single tissue, the seminal vesicles, and suggest that p 84 is an ABH epitope-bearing protein that has not previously been identified but possesses some immunological properties similar to those of lactotransferrin. PMID:10452591

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

    PubMed Central

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

    2014-01-01

    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

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

    PubMed Central

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

    2013-01-01

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

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

    E-print Network

    De Oliveira, Satiro N; Wang, Jiexin; Ryan, Christine; Morrison, Sherie L; Kohn, Donald B; Hollis, Roger P

    2013-01-01

    Fc: Fragment crystallizable region of an antibody; Fab: Fragment antigen-binding region of an antibody; HSPC: Hematopoietic stem/stem cells. Antibodies against the extracellular domain of the CAR molecule (anti-Fab, Fc

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

    E-print Network

    De Oliveira, Satiro N; Wang, Jiexin; Ryan, Christine; Morrison, Sherie L; Kohn, Donald B; Hollis, Roger P

    2013-01-01

    Fc: Fragment crystallizable region of an antibody; Fab: Fragment antigen-binding region of an antibody; HSPC: Hematopoietic stem/stem cells. Antibodies against the extracellular domain of the CAR molecule (anti-Fab, Fc

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

    SciTech Connect

    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

    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.

  1. Analysis of antigenic cross-reactivity between subgroup C avian pneumovirus and human metapneumovirus by using recombinant fusion proteins.

    PubMed

    Luo, L; Sabara, M I; Li, Y

    2009-10-01

    Avian pneumovirus subgroup C (APV/C) has recently been reported to be more closely related to human metapneumovirus (hMPV) as determined by sequence analysis. To examine the antigenic relationship between APV/C and hMPV, the APV/C fusion (F) gene was cloned and expressed as an uncleaved glycoprotein in a baculovirus system. The reactivity of the APV/C F protein with antibodies against APV subgroups A, B, C, and hMPV was examined by Western blot analysis. The results showed that the expressed APV/C F protein was not only recognized by APV/C-specific antibodies but also by antibodies raised against hMPV. Previously expressed recombinant hMPV F protein also reacted with APV/C-specific antibodies, suggesting that there was significant antigenic cross-reactivity and a potential evolutionary relationship between hMPV and APV/C. Interestingly, the recombinant F proteins from APV/C and hMPV were not recognized by polyclonal antibodies specific to APV subgroups A and B. PMID:19548897

  2. Computational Identification of Antigenicity-Associated Sites in the Hemagglutinin Protein of A/H1N1 Seasonal Influenza Virus

    PubMed Central

    Liu, Xiaoning; Shen, Xiping; Gao, Wenlong; Li, Juansheng

    2015-01-01

    The antigenic variability of influenza viruses has always made influenza vaccine development challenging. The punctuated nature of antigenic drift of influenza virus suggests that a relatively small number of genetic changes or combinations of genetic changes may drive changes in antigenic phenotype. The present study aimed to identify antigenicity-associated sites in the hemagglutinin protein of A/H1N1 seasonal influenza virus using computational approaches. Random Forest Regression (RFR) and Support Vector Regression based on Recursive Feature Elimination (SVR-RFE) were applied to H1N1 seasonal influenza viruses and used to analyze the associations between amino acid changes in the HA1 polypeptide and antigenic variation based on hemagglutination-inhibition (HI) assay data. Twenty-three and twenty antigenicity-associated sites were identified by RFR and SVR-RFE, respectively, by considering the joint effects of amino acid residues on antigenic drift. Our proposed approaches were further validated with the H3N2 dataset. The prediction models developed in this study can quantitatively predict antigenic differences with high prediction accuracy based only on HA1 sequences. Application of the study results can increase understanding of H1N1 seasonal influenza virus antigenic evolution and accelerate the selection of vaccine strains. PMID:25978416

  3. Proteomic approach for identification and characterization of novel immunostimulatory proteins from soluble antigens of Leishmania donovani promastigotes.

    PubMed

    Gupta, Shraddha Kumari; Sisodia, Brijesh Singh; Sinha, Sudhir; Hajela, Krishnan; Naik, Sita; Shasany, Ajit Kumar; Dube, Anuradha

    2007-03-01

    Visceral leishmaniasis (VL) caused by Leishmania donovani is a major parasitic disease prevalent in endemic regions of Bihar in India. In the absence of good chemotherapeutic options, there is a need to develop an effective vaccine against VL which should be dependent on the generation of a T helper type 1 (Th1) immune response. We have shown that soluble proteins from promastigote of a new clinical isolate of L. donovani (2001) ranging from 68 to 97.4 kDa (F2 fraction), induce Th1 responses in the peripheral blood mononuclear cells of cured Leishmania patients and hamsters and also showed significant prophylactic potential. To understand the nature of F2 proteins, it was further characterized using 2-DE, MALDI-TOF and MALDI-TOF/TOF-MS. In all, 63 spots were cut from a CBB stained gel for analysis and data was retrieved for 52 spots. A total of 33 proteins were identified including six hypothetical/unknown proteins. Major immunostimulatory proteins were identified as elongation factor-2, p45, heat shock protein (HSP)70, HSP83, aldolase, enolase, triosephosphate isomerase, protein disulfideisomerase and calreticulin. This study substantiates the usefulness of proteomics in characterizing a complex protein fraction (F2) map of soluble L. donovani promastigote antigen identified as Th1 stimulatory for its potential as vaccine targets against VL. PMID:17295358

  4. Human and guinea pig immune responses to Legionella pneumophila protein antigens OmpS and Hsp60.

    PubMed Central

    Weeratna, R; Stamler, D A; Edelstein, P H; Ripley, M; Marrie, T; Hoskin, D; Hoffman, P S

    1994-01-01

    We studied the immune responses of guinea pigs and humans to two Legionella pneumophila antigens. Guinea pigs surviving a lethal intraperitoneal challenge dose of virulent L. pneumophila exhibited strong cutaneous delayed-type hypersensitivity (DTH) reactions to purified OmpS (28-kDa major outer membrane protein) and Hsp60 (heat shock protein or common antigen), while weak DTH reactions were noted for extracellular protease (major secretory protein [MSP] [ProA]) and no reaction was observed with an ovalbumin (OA) control. Lymphocyte proliferation responses (LPRs) were measured for peripheral blood and spleen lymphocytes from guinea pigs surviving sublethal and lethal challenge doses of L. pneumophila. Lymphocytes from uninfected animals showed no proliferation to Hsp60 or OmpS, while lymphocytes from sublethally and lethally challenged animals exhibited strong proliferative responses to Hsp60 and OmpS. Guinea pigs vaccinated with purified OmpS exhibited low antibody titers and strong DTH and LPRs to OmpS, whereas lymphocytes from animals vaccinated with Hsp60 exhibited weak DTH responses and high antibody titers to Hsp60. All guinea pigs immunized with OmpS survived experimental challenge with L. pneumophila (two of two in a pilot study and seven of seven in trial 2) versus zero of seven OA-immunized controls (P = 0.006 by Fisher's exact test). In three vaccine trials in which animals were vaccinated with Hsp60, only 1 guinea pig of 15 survived lethal challenge. Peripheral blood lymphocytes (PBLs) from humans with legionellosis showed stronger LPRs to OmpS than PBLs from humans with no history of legionellosis (P = 0.0002 by Mann-Whitney test). PBLs of humans surviving legionellosis exhibited a lower but highly significant proliferative response to Hsp60 (P < 0.0001 compared with controls by Mann-Whitney test). These studies indicate that OmpS and Hsp60 are important antigens associated with the development of protective cellular immunity. However, as determined in vaccine trial studies in the guinea pig model for legionellosis, the species-specific antigen OmpS proved much more effective than the genus-common Hsp60 antigen. PMID:7913699

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

    PubMed

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

    1989-10-15

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

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

    PubMed

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

    1998-01-01

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

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

    PubMed

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

    2014-10-15

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

  8. Chagas disease: a homology model for the three-dimensional structure of the Trypanosoma cruzi ribosomal P0 antigenic protein.

    PubMed

    Gomez Barroso, Juan Arturo; Aguilar, Carlos Fernando

    2014-09-01

    Ribosomal P proteins form a "stalk" complex in the large subunit of the ribosomes. In Trypanosoma cruzi, the etiological agent of Chagas disease, the complex is formed by five P protein members: TcP0, TcP1?, TcP1?, TcP2? and TcP2?. The TcP0 protein has 34 kDa, and TcP1 and TcP2 proteins have 10 kDa. The structure of T. cruzi P0 and the stalk complex TcP0-TcP1?-TcP1?-TcP2?-TcP2? have not been solved to date. In this work, we constructed a three-dimensional molecular model for TcP0 using homology modeling as implemented in the MODELLER 9v12 software. The model was constructed using different templates: the X-ray structures of the protein P0 from Pirococcus horikoshii, a segment from the Danio renio Ca(+2)/K(+) channel and the C-terminal peptide (C13) from T. cruzi ribosomal P2 protein; the Cryo-EM structure of Triticum aestivum P0 protein and the NMR structure of Homo sapiens P1 ribosomal protein. TcP0 has a 200-residue-long N-terminal, which is an ?/? globular stable domain, and a flexible C-terminal, 120-residue-long domain. The molecular surface electrostatic potential and hydrophobic surface were calculated. The surface properties are important for the C-terminal's antigenic properties. They are also responsible for P0-specific binding to RNA26S and the binding to the P1-P2 proteins. We explored and identified protein interactions that may be involved in conformational stability. The structure proposed in this work represents a first structural report for the TcP0 protein. PMID:24986473

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

    PubMed Central

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

    2014-01-01

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

  10. Development and evaluation of an immunochromatographic strip for rapid detection of capsid protein antigen p27 of avian leukosis virus.

    PubMed

    Qian, Kun; Liang, You-Zhi; Yin, Li-Ping; Shao, Hong-Xia; Ye, Jian-Qiang; Qin, Ai-Jian

    2015-09-01

    A rapid immunochromatographic strip for detecting capsid protein antigen p27 of avian leukosis virus was successfully developed based on two high-affinity monoclonal antibodies. The test strip could detect not only 600pg purified recombinant p27 protein but also quantified avian leukosis virus as low as 70 TCID50, which has comparative sensitivity to the commercial enzyme-linked immunosorbent assay (ELISA) kit. For the evaluation of this test strip, 1100 samples consisting of cloacal swabs, meconium collected from the earliest stool of one day old chicken and virus isolates were assessed both by the strip and by the commercial ELISA kit. The agreement between these two tests was 93.91%, 93.42% and 100%, respectively. The sensitivity and specificity of the strip were also calculated by using the ELISA kit as the standard. This immunochromatographic strip provides advantages of rapid and simple detection of capsid protein antigen p27 of avian leukosis virus, which could be applied as an on-site testing assay and used for control and eradication programs of avian leukosis disease. PMID:25977186

  11. Mass-tag enhanced immuno-laser desorption/ionization mass spectrometry for sensitive detection of intact protein antigens.

    PubMed

    Lorey, Martina; Adler, Belinda; Yan, Hong; Soliymani, Rabah; Ekström, Simon; Yli-Kauhaluoma, Jari; Laurell, Thomas; Baumann, Marc

    2015-05-19

    A new read-out method for antibody arrays using laser desorption/ionization-mass spectrometry (LDI-MS) is presented. Small, photocleavable reporter molecules with a defined mass called "mass-tags" are used for detection of immunocaptured proteins from human plasma. Using prostate specific antigen (PSA), a biomarker for prostate cancer, as a model antigen, a high sensitivity generic detection methodology based immunocapture with a primary antibody and with a biotin labeled secondary antibody coupled to mass-tagged avidin is demonstrated. As each secondary antibody can bind several avidin molecules, each having a large number of mass-tags, signal amplification can be achieved. The developed PSA sandwich mass-tag analysis method provided a limit of detection below 200 pg/mL (6 pM) for a 10 ?L plasma sample, well below the clinically relevant cutoff value of 3-4 ng/mL. This brings the limit of detection (LOD) for detection of intact antigens with matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) down to levels comparable to capture by anti-peptide antibodies selected reaction monitoring (SISCAPA SRM) and enzyme linked immunosorbent assay (ELISA), as 6 pM corresponds to a maximal amount of 60 amol PSA captured on-spot. We propose the potential use of LDI (laser desorption/ionization) with mass-tag read-out implemented in a sandwich assay format for low abundant and/or early disease biomarker detection. PMID:25867450

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

    PubMed Central

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

    2014-01-01

    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

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

    PubMed

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

    2013-01-01

    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

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

    PubMed Central

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

    2008-01-01

    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

  15. Goodpasture antigen-binding protein (GPBP) directs myofibril formation: identification of intracellular downstream effector 130-kDa GPBP-interacting protein (GIP130).

    PubMed

    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

    2011-10-01

    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

  16. Characterization of Tula virus antigenic determinants defined by monoclonal antibodies raised against baculovirus-expressed nucleocapsid protein.

    PubMed

    Lundkvist, A; Vapalahti, O; Plyusnin, A; Sjölander, K B; Niklasson, B; Vaheri, A

    1996-11-01

    Tula virus was recently discovered by RT-PCR in lung samples from European common voles (Microtus arvalis and M. rossiaemeridionalis). Since virus isolation attempts had been unsuccessful, no antigen was available for analysis or for use in immunoassays. To circumvent this, complete Tula virus nucleocapsid protein (bac-TUL-N) was expressed in recombinant baculovirus. Rodent antibody end-point titers to bac-TUL-N and to truncated N fragments indicated that the NH2-terminal region is the major antigenic target and revealed a high cross-reactivity to Puumala virus N. Immunizations with crude bac-TUL-N preparations evoked high antibody responses to native hantavirus N in Balb/c mice and six monoclonal antibodies (Mabs) were generated. Epitope mapping of the Mabs, based on a competitive assay, reactivities to truncated recombinant N fragments, and reactivity patterns to different hantavirus strains, identified five recognition sites on Tula virus N. One epitope, which was identified as specific for Tula virus, was located in a region of N which is highly variable among the hantaviruses (aa 226-293), and four epitopes were mapped to the NH2-terminal region of the protein (aa 1-61). One epitope was expressed only in Tula and Prospect Hill viruses, one epitope in Tula, Prospect Hill, Khabarovsk, and Sin Nombre viruses, while two epitopes were conserved in all examined hantaviruses carried by rodents within the subfamily Arvicolinae of the Muridae family. PMID:8896239

  17. Biological Expression of Antigenic Determinants of Murine Leukemia Virus Proteins gp69/71 and p30

    PubMed Central

    Ikeda, H.; Pincus, T.; Yoshiki, T.; Strand, M.; August, J. T.; Boyse, E. A.; Mellors, R. C.

    1974-01-01

    Antisera to purified structural proteins of Rauscher murine leukemia virus, the major envelope glycoprotein, gp69/71, and the major internal protein, p30, were studied by immunofluorescence of viable and fixed virus-infected cells and by virus neutralization. Group-specific and type-specific determinants of gp69/71 were demonstrated by immunofluorescence and virus neutralization tests, indicating that these determinants are located in the cytoplasm and probably on the cell surface as well as on virus envelope. Antisera against p30 showed anti-group and anti-interspecies activities by immunofluorescence with no virus-neutralizing activity. Both antigenic determinants of gp69/71 were sensitive to guanidine-hydrochloride and to a lesser degree to ether treatment, whereas the group-specific determinants of p30 were relatively stable to these treatments. Images PMID:4139291

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

    PubMed Central

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

    2005-01-01

    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 of vaccine breakthrough isolates would also encode a diverse set of proteins, the complete msa-2 locus was characterized from 12 Australian B. bovis strains and isolates, including two vaccine strains and eight vaccine breakthrough isolates, and compared to the loci in previously and newly characterized American strains. In contrast to American strains, the msa-2 loci of all Australian strains and isolates examined contain, in addition to msa-2c, only a solitary gene (designated msa-2a/b) closely related to American strain msa-2a and msa-2b. Nevertheless, the proteins encoded by these genes are quite diverse both between and within geographic regions and harbor evidence of genetic exchange among other VMSA family members, including msa-1. Moreover, all but one of the Australian breakthrough isolate MSA-2a/b proteins is markedly different from the vaccine strain from which immune escape occurred, consistent with their role in strain-specific protective immunity. The densest distribution of polymorphisms occurs in a hypervariable region (HVR) within the carboxy third of the molecule that is highly proline rich. Variation in length and content of the HVR is primarily attributable to differences in the order and number of degenerate nucleotide repeats encoding three motifs of unknown function. PMID:16239512

  19. Comparison of purified 12 kDa and recombinant 15 kDa Fasciola hepatica antigens related to a Schistosoma mansoni fatty acid binding protein.

    PubMed

    Hillyer, G V

    1995-01-01

    Vaccines in schistosomiasis using homologous antigens have been studied extensively in experimentally infected mammalian hosts. Vaccines using heterologous antigens have received comparatively less attention. This review summarizes recent work on a heterologous 12 kDa Fasciola hepatica antigenic polypeptide which cross reacts with Schistosoma mansoni. A cDNA has been cloned and sequenced, and the predicted amino acid sequence of the recombinant protein has been shown to have significant (44%) identity with a 14 kDa S. mansoni fatty acid binding protein. Thus in the parasitic trematodes fatty acid binding proteins may be potential vaccine candidates. The F. hepatica recombinant protein has been overexpressed and purified and denoted rFh15. Preliminary rFh15 migrates more slowly (i.e. may be slightly larger) than nFh12 on SDS-PAGE and has a predicted pI of 6.01 vs. observed pI of 5.45. Mice infected with F. hepatica develop antibodies to nFh12 by 2 weeks of infection vs. 6 weeks of infection to rFh15; on the other hand, mice with schistosomiasis mansoni develop antibodies to both nFh12 and rFh15 by 6 weeks of infection. Both the F. hepatica and S. mansoni cross-reactive antigens may be cross-protective antigens with the protection inducing capability against both species. PMID:8531666

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

    PubMed

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

    2014-02-28

    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

  1. The 75-kilodalton antigen of Bartonella bacilliformis is a structural homolog of the cell division protein FtsZ.

    PubMed

    Padmalayam, I; Anderson, B; Kron, M; Kelly, T; Baumstark, B

    1997-07-01

    A genomic library of Bartonella bacilliformis was constructed and screened with human anti-Bartonella serum from a patient with the chronic, verruga peruana phase of bartonellosis. An immunoreactive clone isolated from this library was found to code for a 591-amino-acid protein with a high degree of sequence similarity to the FtsZ family of proteins. The degree of amino acid identity between the B. bacilliformis protein (FtsZ[Bb]) and the other FtsZ proteins is especially pronounced over the N-terminal 321 amino acids (N-terminal domain) of the sequence, with values ranging from 45% identity for the homolog from Micrococcus luteus (FtsZ[Ml]) to 91% identity for the homolog from Rhizobium melliloti, (FtsZ[Rm1]). All of the functional domains required for FtsZ activity are conserved in FtsZ(Bb) and are located within the N-terminal domain of the protein. FtsZ(Bb) is approximately twice as large as most of the other FtsZ proteins previously reported, a property it shares with FtsZ(Rm1). Like the Rhizobium homolog, FtsZ(Bb) has a C-terminal region of approximately 256 amino acids that is absent in the other FtsZ proteins. Evidence is presented that implicates this region in the protein's antigenicity and suggests that, unlike most other FtsZ homologs, FtsZ(Bb) is at least partly exposed at the cell surface. PCR analysis revealed that an ftsZ gene similar in size to the B. bacilliformis gene is present in Bartonella henselae, a bacterium that is closely related to B. bacilliformis. PMID:9226264

  2. Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen interacts with bromodomain protein Brd4 on host mitotic chromosomes.

    PubMed

    You, Jianxin; Srinivasan, Viswanathan; Denis, Gerald V; Harrington, William J; Ballestas, Mary E; Kaye, Kenneth M; Howley, Peter M

    2006-09-01

    The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is required for viral episome maintenance in host cells during latent infection. Two regions of the protein have been implicated in tethering LANA/viral episomes to the host mitotic chromosomes, and LANA chromosome-binding sites are subjects of high interest. Because previous studies had identified bromodomain protein Brd4 as the mitotic chromosome anchor for the bovine papillomavirus E2 protein, which tethers the viral episomes to host mitotic chromosomes (J. You, J. L. Croyle, A. Nishimura, K. Ozato, and P. M. Howley, Cell 117:349-360, 2004, and J. You, M. R. Schweiger, and P. M. Howley, J. Virol. 79:14956-14961, 2005), we examined whether KSHV LANA interacts with Brd4. We found that LANA binds Brd4 in vivo and in vitro and that the binding is mediated by a direct protein-protein interaction between the ET (extraterminal) domain of Brd4 and a carboxyl-terminal region of LANA previously implicated in chromosome binding. Brd4 associates with mitotic chromosomes throughout mitosis and demonstrates a strong colocalization with LANA and the KSHV episomes on host mitotic chromosomes. Although another bromodomain protein, RING3/Brd2, binds to LANA in a similar fashion in vitro, it is largely excluded from the mitotic chromosomes in KSHV-uninfected cells and is partially recruited to the chromosomes in KSHV-infected cells. These data identify Brd4 as an interacting protein for the carboxyl terminus of LANA on mitotic chromosomes and suggest distinct functional roles for the two bromodomain proteins RING3/Brd2 and Brd4 in LANA binding. Additionally, because Brd4 has recently been shown to have a role in transcription, we examined whether Brd4 can regulate the CDK2 promoter, which can be transactivated by LANA. PMID:16940503

  3. Epstein-Barr virus latent antigen 3C can mediate the degradation of the retinoblastoma protein through an SCF cellular ubiquitin ligase

    Microsoft Academic Search

    Jason S. Knight; Nikhil Sharma; Erle S. Robertson

    2005-01-01

    Epstein-Barr virus (EBV) stimulates the proliferation of latently infected B cells and promotes lymphoid malignancies in humans. To address the role of EBV latency protein Epstein-Barr nuclear antigen 3C (EBNA3C) in regulation of the retinoblastoma protein (Rb), we transfected EBNA3C into 293, BJAB, and SAOS-2 cells. In this context, a dominant effect of EBNA3C is to decrease Rb protein levels.

  4. Epstein-Barr Virus Latent Antigen 3C Can Mediate the Degradation of the Retinoblastoma Protein through an SCF Cellular Ubiquitin Ligase

    Microsoft Academic Search

    Jason S. Knight; Nikhil Sharma; Erle S. Robertson; Elliot D. Kieff

    2005-01-01

    Epstein-Barr virus (EBV) stimulates the proliferation of latently infected B cells and promotes lymphoid malignancies in humans. To address the role of EBV latency protein Epstein-Barr nuclear antigen 3C (EBNA3C) in regulation of the retinoblastoma protein (Rb), we transfected EBNA3C into 293, BJAB, and SAOS-2 cells. In this context, a dominant effect of EBNA3C is to decrease Rb protein levels.

  5. Fasciola hepatica: Vaccination of rabbits with native and recombinant antigens related to fatty acid binding proteins

    Microsoft Academic Search

    Antonio Muro; Vicente Ramajo; Julio López; Fernando Simón; George V. Hillyer

    1997-01-01

    The current study was designed to compare the immunogenic and immunoprophylactic properties of native (nFh 12) and recombinant (rFh 15) antigens from Fasciola hepatica in rabbits infected with the fluke. Levels of specific anti-nFh 12 and anti-rFh 15 antibodies were significantly higher in the rabbits vaccinated twice compared with non-vaccinated infection controls. A reduction of 40% in worm burdens was

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

    SciTech Connect

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

    2012-05-08

    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.

  7. 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...

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

    Microsoft Academic Search

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

    1999-01-01

    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

  9. Validation of a structural comparison of the antigenic characteristics of Usutu virus and West Nile virus envelope proteins.

    PubMed

    Nikolay, Birgit; Fall, Gamou; Boye, Cheikh Saad Bouh; Sall, Amadou Alpha; Skern, Tim

    2014-08-30

    Cross-reactions observed in serological assays between Usutu virus (USUV), the USUV outlier subtype strain CAR_1969 and West Nile virus (WNV) suggest that they share antigenic features amongst their structural outer proteins especially envelope (E) proteins. To investigate the molecular background of this observation, we compared the E protein sequences of seven USUV strains, USUV subtype strain CAR_1969 and WNV strain 2471, focusing on the binding site defined by the WNV neutralizing antibody E16. USUV SouthAfrica_1959 differs from WNV 2741 in three of four residues critical for E16 antibody binding and five of the 12 additionally involved residues. In contrast, USUV subtype CAR_1969 differs from WNV 2741 in two critical residues and five additional residues. Furthermore, USUV subtype CAR_1969 differs from other USUV strains in two critical residues. E16 antibody binding has previously been shown to be highly specific for WNV; thus, the observed variation in amino acid residues suggests that the region corresponding to the WNV E16 epitope is probably not responsible for the observed cross-reactions between WNV and USUV. Seroneutralisation assays confirmed these findings for WNV and USUV, however, showed occurring cross-reactivity between WNV and USUV subtype CAR_1969 at high antibody titers. The sequence diversity in this region might also explain some of the observed different antigenic characteristics of USUV strains and USUV subtype CAR_1969. A therapeutic effect of E16 antibody has been described in WNV infected mice; therefore, a USUV specific antibody generated against the region corresponding to the WNV E16 binding site might represent an approach for treating USUV infections. PMID:24874193

  10. Several Carcinoembryonic Antigens (CD66) Serve as Receptors for Gonococcal Opacity Proteins

    Microsoft Academic Search

    Tie Chen; Fritz Grunert; Andrew Medina-Marino; Emil C. Gotschlich

    2010-01-01

    Summary Neisseria gonorrhoeae (GC) is a human pathogen that adheres to and invades genital surfaces. Al- though pili are required for the initial adherence, the interaction of GC with epithelial cells is also promoted by a family of outer membrane proteins, the opacity (Opa) proteins such as OpaA protein from strain MS11. Studies have demonstrated that the interaction of the

  11. Structural analysis of the synthetic Duffy Binding Protein (DBP) antigen DEKnull relevant for Plasmodium vivax malaria vaccine design.

    PubMed

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

    2015-03-01

    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

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

    PubMed

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

    2013-04-01

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

  13. Temporally defined neocortical translation and polysome assembly are determined by the RNA-binding protein Hu antigen R

    PubMed Central

    Kraushar, Matthew L.; Thompson, Kevin; Wijeratne, H. R. Sagara; Viljetic, Barbara; Sakers, Kristina; Marson, Justin W.; Kontoyiannis, Dimitris L.; Buyske, Steven; Hart, Ronald P.; Rasin, Mladen-Roko

    2014-01-01

    Precise spatiotemporal control of mRNA translation machinery is essential to the development of highly complex systems like the neocortex. However, spatiotemporal regulation of translation machinery in the developing neocortex remains poorly understood. Here, we show that an RNA-binding protein, Hu antigen R (HuR), regulates both neocorticogenesis and specificity of neocortical translation machinery in a developmental stage-dependent manner in mice. Neocortical absence of HuR alters the phosphorylation states of initiation and elongation factors in the core translation machinery. In addition, HuR regulates the temporally specific positioning of functionally related mRNAs into the active translation sites, the polysomes. HuR also determines the specificity of neocortical polysomes by defining their combinatorial composition of ribosomal proteins and initiation and elongation factors. For some HuR-dependent proteins, the association with polysomes likewise depends on the eukaryotic initiation factor 2 alpha kinase 4, which associates with HuR in prenatal developing neocortices. Finally, we found that deletion of HuR before embryonic day 10 disrupts both neocortical lamination and formation of the main neocortical commissure, the corpus callosum. Our study identifies a crucial role for HuR in neocortical development as a translational gatekeeper for functionally related mRNA subgroups and polysomal protein specificity. PMID:25157170

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

    PubMed Central

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

    2015-01-01

    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

  15. Antigenic cross-reactivity among avian pneumoviruses of subgroups A, B, and C at the matrix but not nucleocapsid proteins.

    PubMed

    Lwamba, Humphrey C M; Halvorson, David A; Nagaraja, Kakambi V; Turpin, Elizabeth A; Swayne, David; Seal, Bruce S; Njenga, M Kariuki

    2002-01-01

    Earlier findings from our laboratory based on analysis of nucleotide and predicted amino acid sequence identities of 15 avian pneumoviruses (APVs) isolated from the United States (subgroup C) demonstrated that the viruses were phylogenetically separated from the European subgroup A and subgroup B viruses. Here, we investigated whether viruses from the three subgroups were cross-reactive by testing field sera positive for each of the APV subgroups in an enzyme-linked immunosorbent assay (ELISA) test with recombinant matrix (M) and nucleoprotein (N) proteins generated from a Minnesota APV isolate (APV/MN2A). Sera from turkeys infected with APV subgroup A, B, or C reacted with recombinant M protein derived from APV/MN2A. In contrast, recombinant N protein from APV/MN2A virus was reactive with sera from subtypes A and C viruses but not from subtype B virus. The results illustrate that viruses from the three APV subtypes share antigenic homology, and the M protein-based ELISA is adequate for monitoring APV outbreaks but not for distinguishing between different subtypes. PMID:12243541

  16. Coadministration of the fungal immunomodulatory protein FIP-Fve and a tumour-associated antigen enhanced antitumour immunity

    PubMed Central

    Ding, Ying; Seow, See Voon; Huang, Chiung Hui; Liew, Lee Mei; Lim, Yaw Chyn; Kuo, I Chun; Chua, Kaw Yan

    2009-01-01

    Fve is a fungal protein isolated from the golden needle mushroom Flammulina velutipes and has previously been reported to trigger immunological responses in both mouse and human lymphocytes. In this study, we evaluated the potential application of Fve as an adjuvant for tumour immunotherapy and examined the underlying mechanism(s). When the human papillomavirus (HPV)-16 E7 oncoprotein was used as a model antigen, mice coimmunized with HPV-16 E7 and Fve showed enhanced production of HPV-16 E7-specific antibodies as well as expansion of HPV-16 E7-specific interferon (IFN)-?-producing CD4+ and CD8+ T cells as compared with mice immunized with HPV-16 E7 alone. Tumour protection assays showed that 60% of mice coimmunized with HPV-16 E7 plus Fve, as compared with 20% of those immunized only with HPV-16 E7, remained tumour-free for up to 167 days after challenge with the tumour cells. Tumour therapeutic assays showed that HPV-16 E7 plus Fve treatment significantly prolonged the survival of tumour-bearing mice as compared with those treated only with HPV-16 E7. In vivo cell depletion and adoptive T-cell transfer assays showed that CD4+ and CD8+ T cells and IFN-? played critical roles in conferring the antitumour effects. Interestingly, Fve could stimulate the maturation of splenic dendritic cells in vivo and induce antigen-specific CD8+ T-cell immune responses. In summary, Fve has potent adjuvant properties that enhance T helper type 1 antigen-specific humoral and cellular immune responses which confer strong antitumour effects. The use of Fve as an adjuvant could be an attractive alternative to the current vaccination strategy for cancer immunotherapy. PMID:19740349

  17. Antigen-specific systemic and reproductive tract antibodies in foxes immunized with Salmonella typhimurium expressing bacterial and sperm proteins.

    PubMed

    de Jersey, J; Bird, P H; Verma, N K; Bradley, M P

    1999-01-01

    Attenuated Salmonella typhimurium strains are potential 'safe' delivery vectors of an oral immunocontraceptive vaccine for the European red fox (Vulpes vulpes). In the present study, model bacterial (Escherichia coli heat-labile enterotoxin B subunit, LTB) and fox sperm (fSP10) antigens were expressed in S. typhimurium SL3261 (delta aroA) under the control of the trc promoter. Adult female foxes were given three oral immunizations with SL3261 containing either LTB (SL3261/pLTB), fSP10 (SL3261/pFSP10) or a control plasmid (pKK233-2 or pTrc99A). All foxes raised serum (IgG) and vaginal (IgG and IgA) antibodies against S. typhimurium lipopolysaccharide (LPS). Each fox that received SL3261/pLTB raised high titre LTB-specific serum and vaginal IgG antibodies. However, only one of four foxes immunized with SL3261/pFSP10 raised an anti-fSP10 immune response, in the form of low titre serum and vaginal IgG antibodies. No vaginal IgA antibodies were raised against either LTB or fSP10 in these experiments. The immune responses against recombinant LTB and fSP10 resulted chiefly from the initial dose of antigen in the inocula and were minimally influenced by continued in vivo antigen expression. This study demonstrates for the first time in the female red fox that oral Salmonella can elicit specific systemic and reproductive tract antibodies against heterologous, recombinant proteins. PMID:10898286

  18. A ram epididymal secretory protein shares common anti-genic determinants with rat epididymal proteins and human

    E-print Network

    Boyer, Edmond

    in the prealbumin region on non-denaturing gels. The protein, synthesized by the epididymis under testosterone and is still present after ejaculation. This protein could thus be involved in sperm-egg interactionA ram epididymal secretory protein shares common anti- genic determinants with rat epididymal

  19. The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins.

    PubMed

    Schlüter, C; Duchrow, M; Wohlenberg, C; Becker, M H; Key, G; Flad, H D; Gerdes, J

    1993-11-01

    The antigen defined by mAb Ki-67 is a human nuclear protein the expression of which is strictly associated with cell proliferation and which is widely used in routine pathology as a "proliferation marker" to measure the growth fraction of cells in human tumors. Ki-67 detects a double band with apparent molecular weights of 395 and 345 kD in immunoblots of proteins from proliferating cells. We cloned and sequenced the full length cDNA, identified two differentially spliced isoforms of mRNA with open reading frames of 9,768 and 8,688 bp encoding for this cell proliferation-associated protein with calculated molecular weights of 358,761 D and 319,508 D, respectively. New mAbs against a bacterially expressed part and a synthetic polypeptide deduced from the isolated cDNA react with the native Ki-67 antigen, thus providing a circle of evidence that we have cloned the authentic Ki-67 antigen cDNA. The central part of the Ki-67 antigen cDNA contains a large 6,845-bp exon with 16 tandemly repeated 366-bp elements, the "Ki-67 repeats", each including a highly conserved new motif of 66 bp, the "Ki-67 motif", which encodes for the epitope detected by Ki-67. Computer analysis of the nucleic acid and the deduced amino acid sequence of the Ki-67 antigen confirmed that the cDNA encodes for a nuclear and short-lived protein without any significant homology to known sequences. Ki-67 antigen-specific antisense oligonucleotides inhibit the proliferation of IM-9 cell line cells, indicating that the Ki-67 antigen may be an absolute requirement for maintaining cell proliferation. We conclude that the Ki-67 antigen defines a new category of cell cycle-associated nuclear nonhistone proteins. PMID:8227122

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

    PubMed Central

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

    2014-01-01

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

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

    SciTech Connect

    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

    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.

  2. Citrullinated vimentin as an important antigen in immune complexes from synovial fluid of rheumatoid arthritis patients with antibodies against citrullinated proteins

    PubMed Central

    2010-01-01

    Introduction Rheumatoid arthritis (RA) is an inflammatory disease, which results in destruction of the joint. The presence of immune complexes (IC) in serum and synovial fluid of RA patients might contribute to this articular damage through different mechanisms, such as complement activation. Therefore, identification of the antigens from these IC is important to gain more insight into the pathogenesis of RA. Since RA patients have antibodies against citrullinated proteins (ACPA) in their serum and synovial fluid (SF) and since elevated levels of citrullinated proteins are detected in the joints of RA patients, citrullinated antigens are possibly present in IC from RA patients. Methods IC from serum of healthy persons, serum of RA patients and IC from synovial fluid of RA patients and Spondyloarthropathy (SpA) patients were isolated by immunoprecipitation. Identification of the antigens was performed by SDS-PAGE, mass spectrometry and immunodetection. The presence of citrullinated proteins was evaluated by anti-modified citrulline (AMC) staining. Results Circulating IC in the serum of RA patients and healthy controls contain fibrinogen? and fibronectin, both in a non-citrullinated form. Additionally, in IC isolated from RA SF, fibrinogen? and vimentin were identified as well. More importantly, vimentin and a minor portion of fibrinogen? were found to be citrullinated in the isolated complexes. Moreover these citrullinated antigens were only found in ACPA+ patients. No citrullinated antigens were found in IC from SF of SpA patients. Conclusions Citrullinated fibrinogen? and citrullinated vimentin were found in IC from SF of ACPA+ RA patients, while no citrullinated antigens were found in IC from SF of ACPA- RA patients or SpA patients or in IC from serum of RA patients or healthy volunteers. The identification of citrullinated vimentin as a prominent citrullinated antigen in IC from SF of ACPA+ RA patients strengthens the hypothesis that citrullinated vimentin plays an important role in the pathogenesis of RA. PMID:20609218

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

    PubMed

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

    2000-07-01

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

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

    PubMed Central

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

    2000-01-01

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

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

    PubMed Central

    1984-01-01

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

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

    PubMed Central

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

    2011-01-01

    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

  7. The CAP superfamily: cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins--roles in reproduction, cancer, and immune defense.

    PubMed

    Gibbs, Gerard M; Roelants, Kim; O'Bryan, Moira K

    2008-12-01

    The cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins (CAP) superfamily members are found in a remarkable range of organisms spanning each of the animal kingdoms. Within humans and mice, there are 31 and 33 individual family members, respectively, and although many are poorly characterized, the majority show a notable expression bias to the reproductive tract and immune tissues or are deregulated in cancers. CAP superfamily proteins are most often secreted and have an extracellular endocrine or paracrine function and are involved in processes including the regulation of extracellular matrix and branching morphogenesis, potentially as either proteases or protease inhibitors; in ion channel regulation in fertility; as tumor suppressor or prooncogenic genes in tissues including the prostate; and in cell-cell adhesion during fertilization. This review describes mammalian CAP superfamily gene expression profiles, phylogenetic relationships, protein structural properties, and biological functions, and it draws into focus their potential role in health and disease. The nine subfamilies of the mammalian CAP superfamily include: the human glioma pathogenesis-related 1 (GLIPR1), Golgi associated pathogenesis related-1 (GAPR1) proteins, peptidase inhibitor 15 (PI15), peptidase inhibitor 16 (PI16), cysteine-rich secretory proteins (CRISPs), CRISP LCCL domain containing 1 (CRISPLD1), CRISP LCCL domain containing 2 (CRISPLD2), mannose receptor like and the R3H domain containing like proteins. We conclude that overall protein structural conservation within the CAP superfamily results in fundamentally similar functions for the CAP domain in all members, yet the diversity outside of this core region dramatically alters target specificity and, therefore, the biological consequences. PMID:18824526

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

    PubMed Central

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

    1993-01-01

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

  9. A new lipid transfer protein homolog identified as an IgE-binding antigen from Japanese cedar pollen.

    PubMed

    Ibrahim, Ahmed Ragaa Nour; Kawamoto, Seiji; Nishimura, Minori; Pak, Syunka; Aki, Tsunehiro; Diaz-Perales, Araceli; Salcedo, Gabriel; Asturias, Juan A; Hayashi, Takaharu; Ono, Kazuhisa

    2010-01-01

    Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal rhinitis and conjunctivitis in Japan, and an understanding of its full allergen repertoire is prerequisite for the development of future molecular diagnostics and immunotherapeutic strategies. Here we report the identification of a new C. japonica pollen IgE-binding antigen (CJP-8) homologous to lipid transfer proteins (LTPs), a class of plant cross-reactive allergens found in foods, latex, and pollen grains. The cjp-8 cDNA encodes a 165-amino acid polypeptide possessing the conserved eight cysteines characteristic of plant LTP family members. Escherichia coli-expressed recombinant CJP-8 (r-CJP-8) reacted with IgE antibody from Japanese cedar pollinosis patients at a 37.5% frequency (6/16). PMID:20208354

  10. Expression of transforming region of Moloney murine sarcoma virus in Escherichia coli as a fusion protein with small tumor antigen of polyoma virus.

    PubMed Central

    Donoghue, D J; Hunter, T

    1982-01-01

    Bacterial expression of the transforming region of Moloney murine sarcoma virus, designated mos, was obtained as a fusion protein with a portion of the small tumor antigen of polyoma virus. This was accomplished by fusing the entire mos open reading frame, encoding a 41,000-dalton protein, with a plasmid that expresses a beta-galactosidase-polyoma fusion protein under lac operon control. The resulting plasmid directed synthesis of the predicted polyoma antigen-sarcoma virus fusion protein of 59,000 daltons. This protein was immunoprecipitated by an anti-polyoma tumor antigen antiserum that recognized polyoma determinants at the NH2 terminus of the hybrid protein. This protein was also immunoprecipitated by an antiserum directed against a synthetic peptide containing the 12 COOH-terminal amino acids encoded by the mos open reading frame. This work confirms the existence of a long open reading frame in the mos gene and resolves a discrepancy between different nucleotide sequences for its COOH-terminal coding region. Images PMID:6278495

  11. Identification of three antigen epitopes on the nucleocapsid protein of the genotype C of bovine parainfluenza virus type 3.

    PubMed

    Ren, Jian-Le; Zhu, Yuan-Mao; Zhou, Yue-Hui; Lv, Chuang; Yan, Hao; Ma, Lei; Shi, Hong-Fei; Xue, Fei

    2015-07-01

    Bovine parainfluenza virus type 3 (BPIV3) is an important respiratory tract pathogen for both young and adult cattle. So far, three genotypes A, B and C of BPIV3 have been described on the basis of genetic and phylogenetic analysis. But fine mapping of epitopes of BPIV3 is scant and the antigenic variations among the three genotypes of BPIV3 have not been reported. Nucleocapsid protein (NP) is the most abundant protein in the virion and highly conserved in BPIV3, which is crucial for the induction of protective immunity in host. To identify antigenic determinants of BPIV3 NP, a panel of monoclonal antibodies (mAbs) was tested against a series of overlapping recombinant NP fragments expressed in Escherichia coli. Firstly, six monoclonal antibodies (mAbs) against NP of the genotype C of BPIV3 (BPIV3c) were generated by using the purified BPIV3c strain SD0835 as immunogen and the recombinant NP of SD0835 as screening antigen. Then three antigen epitopes were identified with the six mAbs. One epitope (91)GNNADVKYVIYM(102) was recognized by mAb 5E5. The mAbs 7G5, 7G8, 7G9, and 7H5 were reactive with another epitope (407)FYKPTGG(413). The third epitope (428)ESRGDQDQ(435) was reactive with mAb 6F8. Further analysis showed that the epitope (91-102 amino acids [aa]) was the most conserved and reactive with mAb 5E5 for all three genotypes of BPIV3 and HPIV3. The epitope (407-413 aa) was relatively conserved and reactive with mAbs 7G5, 7G8, 7G9, and 7H5 for BPIV3a, BPIV3c and HPIV3, but not reactive with BPIV3b. The epitope (428-435 aa) was less conserved and was reactive only with mAb 6F8 for BPIV3a and BPIV3c. These results suggested that there were evident antigenic variations among the three genotypes of BPIV3 and HPIV3. The mAb 6F8 could be used to detect BPIV3a and BPIV3c. The mAbs 7G5, 7G8, 7G9, and 7H5 might be used for differentiate BPIV3a, BPIV3c and HPIV3 from BPIV3b. The mAb 5E5 might be used for detecting all three types of BPIV3 and HPIV3. The results in this study would have potential applications in the development of suitable diagnostic techniques for BPIV3, which was prevalent in China. PMID:25960335

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

    PubMed Central

    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

    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

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

    PubMed Central

    Haughney, Shannon L.; Petersen, Latrisha K.; Schoofs, Amy D.; Ramer-Tait, Amanda E.; King, Janice; Briles, David; Wannemuehler, Michael J.; Narasimhan, Balaji

    2013-01-01

    Pneumococcal surface protein A (PspA) is a choline-binding protein which is a virulence factor found on the surface of all Streptococcus pneumoniae strains. Vaccination with PspA has been shown to be protective against a lethal challenge with S. pneumoniae, making it a promising immunogen for use in vaccines. Herein, the design of a PspA-based subunit vaccine using polyanhydride nanoparticles as a delivery platform is described. Nanoparticles based on sebacic acid (SA), 1,6-bis-(p-carboxyphenoxy)hexane (CPH) and 1,8-bis-(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG), specifically 50:50 CPTEG:CPH and 20:80 CPH:SA, were used to encapsulate and release PspA. The protein released from the nanoparticle formulations retained its primary and secondary structure as well as its antigenicity. The released PspA was also biologically functional based on its ability to bind to apolactoferrin and prevent its bactericidal activity towards Escherichia coli. When the PspA nanoparticle formulations were administered subcutaneously to mice, the animals elicited a high titer and high avidity anti-PspA antibody response. Together, these studies provide a framework for the rational design of a vaccine against S. pneumoniae based on polyanhydride nanoparticles. PMID:23774257

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

    PubMed Central

    Otsuka, Taketo; Kirkham, Charmaine; Johnson, Antoinette; Jones, Megan M.

    2014-01-01

    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

  15. 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...

  16. Antigen related to mammalian type-C RNA viral p30 proteins is located in renal glomeruli in human systemic lupus erythematosus.

    PubMed Central

    Mellors, R C; Mellors, J W

    1976-01-01

    An antigen related to mammalian type-C RNA viral p30 proteins was shown by the use of anti-p30 sera and the indirect immunofluorescence method to be present in the kidneys and spleen in a fulminant fatal case of human systemic lupus erythematosus and to be located in the glomeruli, the site of active lupus diffuse glomerulonephritis. Images PMID:174099

  17. Multidrug Resistance-associated Protein 3 Is a Tumor Rejection Antigen Recognized by HLA-A2402-restricted Cytotoxic T Lymphocytes1

    Microsoft Academic Search

    Akira Yamada; Kouichiro Kawano; Makoto Koga; Tomoko Matsumoto; Kyogo Itoh

    The identification of tumor rejection antigens recognized by CTLs and its application in peptide-based specific immunotherapy against melano- mas have been extensively investigated in the past decade. However, only a small number of studies regarding these issues in other epithelial cancers have been reported. In this study, we show that a multidrug resistance- associated protein 3 (MRP3) is a tumor

  18. Expression of the bovine viral diarrhoea virus Osloss p80 protein: its use as ELISA antigen for cattle serum antibody detection

    Microsoft Academic Search

    Nathalie Vanderheijden; L. De Moerlooze; D. Vandenbergh; G. Chappuis; A. Renard; C. Lecomte

    1993-01-01

    The putative gene encoding the cytopathic bovine viral diarrhoea virus (BVDV) Osloss strain p80 protein was amplified by PCR and inserted into a T7 promoter- based vector for expression in Escherichia coli. Bacterial expression led to cytoplasmic insoluble inclusion bodies which were denatured by urea treatment and renatured by dialysis. Rabbit antisera were raised against this p80 recombinant antigen and

  19. Trypanosoma cruzi Recombinant Complement Regulatory Protein: a Novel Antigen for Use in an Enzyme-Linked Immunosorbent Assay for Diagnosis of Chagas' Disease

    Microsoft Academic Search

    Wendell S. F. Meira; Lucia M. C. Galvao; Eliane D. Gontijo; George L. L. Machado-Coelho; Karen A. Norris; Egler Chiari; Escola de Farmacia

    2002-01-01

    Currently, diagnosis of Chagas' disease is based on serological methods, but due to the high occurrence of inconclusive results, more reliable methods are needed. The use of recombinant antigens for serodiagnosis of Chagas' disease is recommended in order to increase the sensitivity and specificity of the serological tests. The Trypanosoma cruzi complement regulatory protein (CRP) is a surface glycoprotein present

  20. BABESIA BOVIS MEROZOITE SURFACE ANTIGEN 1 AND RHOPTRY-ASSOCIATED PROTEIN 1 ARE EXPRESSED IN SPOROZOITES, AND SPECIFIC ANTIBODIES INHIBIT SPOROZOITE ATTACHMENT TO ERYTHROCYTES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sporozoites of Babesia bovis were examined for the expression of merozoite surface antigen 1 (MSA-1) and rhoptry-associated protein 1 (RAP-1), two molecules postulated to be involved in the invasion of host erythrocytes. Both MSA-1 and RAP-1 were transcribed and expressed in infectious sporozoites....

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

    PubMed Central

    Sánchez-Jiménez, Carmen; Izquierdo, José M.

    2013-01-01

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

  2. The translocon protein sec61 mediates antigen transport from endosomes in the cytosol for cross-presentation to CD8(+) T cells.

    PubMed

    Zehner, Matthias; Marschall, Andrea L; Bos, Erik; Schloetel, Jan-Gero; Kreer, Christoph; Fehrenschild, Dagmar; Limmer, Andreas; Ossendorp, Ferry; Lang, Thorsten; Koster, Abraham J; Dübel, Stefan; Burgdorf, Sven

    2015-05-19

    The molecular mechanisms regulating antigen translocation into the cytosol for cross-presentation are under controversial debate, mainly because direct data is lacking. Here, we have provided direct evidence that the activity of the endoplasmic reticulum (ER) translocon protein Sec61 is essential for endosome-to-cytosol translocation. We generated a Sec61-specific intrabody, a crucial tool that trapped Sec61 in the ER and prevented its recruitment into endosomes without influencing Sec61 activity and antigen presentation in the ER. Expression of this ER intrabody inhibited antigen translocation and cross-presentation, demonstrating that endosomal Sec61 indeed mediates antigen transport across endosomal membranes. Moreover, we showed that the recruitment of Sec61 toward endosomes, and hence antigen translocation and cross-presentation, is dependent on dendritic cell activation by Toll-like receptor (TLR) ligands. These data shed light on a long-lasting question regarding antigen cross-presentation and point out a role of the ER-associated degradation machinery in compartments distinct from the ER. PMID:25979419

  3. Proteome analysis of Plasmodium falciparum extracellular secretory antigens at asexual blood stages reveals a cohort of proteins with possible roles in immune modulation and signaling.

    PubMed

    Singh, Meha; Mukherjee, Paushali; Narayanasamy, Krishnamoorthy; Arora, Reena; Sen, Som Dutta; Gupta, Shashank; Natarajan, Krishnamurthy; Malhotra, Pawan

    2009-09-01

    The highly co-evolved relationship of parasites and their hosts appears to include modulation of host immune signals, although the molecular mechanisms involved in the host-parasite interplay remain poorly understood. Characterization of these key genes and their cognate proteins related to the host-parasite interplay should lead to a better understanding of this intriguing biological phenomenon. The malaria agent Plasmodium falciparum is predicted to export a cohort of several hundred proteins to remodel the host erythrocyte. However, proteins actively exported by the asexual intracellular parasite beyond the host red blood cell membrane (before merozoite egress) have been poorly investigated so far. Here we used two complementary methodologies, two-dimensional gel electrophoresis/MS and LC-MS/MS, to examine the extracellular secreted antigens at asexual blood stages of P. falciparum. We identified 27 novel antigens exported by P. falciparum in the culture medium of which some showed clustering with highly polymorphic genes on chromosomes, suggesting that they may encode putative antigenic determinants of the parasite. Immunolocalization of four novel secreted proteins confirmed their export beyond the infected red blood cell membrane. Of these, preliminary functional characterization of two novel (Sel1 repeat-containing) parasite proteins, PfSEL1 and PfSEL2 revealed that they down-regulate expression of cell surface Notch signaling molecules in host cells. Also a novel protein kinase (PfEK) and a novel protein phosphatase (PfEP) were found to, respectively, phosphorylate/dephosphorylate parasite-specific proteins in the extracellular culture supernatant. Our study thus sheds new light on malaria parasite extracellular secreted antigens of which some may be essential for parasite development and could constitute promising new drug targets. PMID:19494339

  4. Study of IgE Antigenic Relationships in Hypersensitivity to Hydrolyzed Wheat Proteins and Wheat-Dependent Exercise-Induced Anaphylaxis

    Microsoft Academic Search

    Jacques Snégaroff; Isabelle Bouchez-Mahiout; Catherine Pecquet; Gérard Branlard; Michel Laurière

    2006-01-01

    Background: Wheat is involved in different forms of respiratory, food and contact allergy. The IgE of patients generally reacts with various flour proteins. It is not known if antigenic relationships could explain some of these reactions and if proteins could be involved in different pathologies. Methods: Two sera were selected as representative of patients with either wheat-dependent exercise-induced anaphylaxis (WDEIA)

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

    Microsoft Academic Search

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

    2005-01-01

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

  6. Comparative studies on thermoresistance of protein G-binding region and antigen determinant region of immunoglobulin G in acidic colostral whey

    Microsoft Academic Search

    Jinsong Cao; Xiaoqin Wang; Huina Zheng

    2007-01-01

    The kinetics of the denaturation of antigen determinant (AGD) region and protein G-binding (PGB) region of bovine immunoglobulin G (IgG) in acidic colostral whey were studied by single radial Immunodiffusion (SRID) and protein G affinity chromatography (PrGAC) during the same courses of heat treatment. The kinetic and thermodynamic parameters for heat-induced denaturation of AGD and PGB regions of IgG were

  7. Proteome Analysis of Plasmodium falciparum Extracellular Secretory Antigens at Asexual Blood Stages Reveals a Cohort of Proteins with Possible Roles in Immune Modulation and Signaling*

    PubMed Central

    Singh, Meha; Mukherjee, Paushali; Narayanasamy, Krishnamoorthy; Arora, Reena; Sen, Som Dutta; Gupta, Shashank; Natarajan, Krishnamurthy; Malhotra, Pawan

    2009-01-01

    The highly co-evolved relationship of parasites and their hosts appears to include modulation of host immune signals, although the molecular mechanisms involved in the host-parasite interplay remain poorly understood. Characterization of these key genes and their cognate proteins related to the host-parasite interplay should lead to a better understanding of this intriguing biological phenomenon. The malaria agent Plasmodium falciparum is predicted to export a cohort of several hundred proteins to remodel the host erythrocyte. However, proteins actively exported by the asexual intracellular parasite beyond the host red blood cell membrane (before merozoite egress) have been poorly investigated so far. Here we used two complementary methodologies, two-dimensional gel electrophoresis/MS and LC-MS/MS, to examine the extracellular secreted antigens at asexual blood stages of P. falciparum. We identified 27 novel antigens exported by P. falciparum in the culture medium of which some showed clustering with highly polymorphic genes on chromosomes, suggesting that they may encode putative antigenic determinants of the parasite. Immunolocalization of four novel secreted proteins confirmed their export beyond the infected red blood cell membrane. Of these, preliminary functional characterization of two novel (Sel1 repeat-containing) parasite proteins, PfSEL1 and PfSEL2 revealed that they down-regulate expression of cell surface Notch signaling molecules in host cells. Also a novel protein kinase (PfEK) and a novel protein phosphatase (PfEP) were found to, respectively, phosphorylate/dephosphorylate parasite-specific proteins in the extracellular culture supernatant. Our study thus sheds new light on malaria parasite extracellular secreted antigens of which some may be essential for parasite development and could constitute promising new drug targets. PMID:19494339

  8. Unrelated umbilical cord blood transplant for adult acute lymphoblastic leukemia in first and second complete remission: a comparison with allografts from adult unrelated donors.

    PubMed

    Marks, David I; Woo, Kwang Ahn; Zhong, Xiaobo; Appelbaum, Frederick R; Bachanova, Veronika; Barker, Juliet N; Brunstein, Claudio G; Gibson, John; Kebriaei, Partow; Lazarus, Hillard M; Olsson, Richard; Perales, Miguel-Angel; Pidala, Joseph; Savani, Bipin; Rocha, Vanderson; Eapen, Mary

    2014-02-01

    Allogeneic hematopoietic cell transplantation has an established role in the treatment of adults with acute lymphoblastic leukemia whose survival when recipients of grafts from adult unrelated donors approaches that of recipients of grafts from sibling donors. Our aim was to determine the role of mismatched unrelated cord blood grafts in transplantation for 802 adults with acute lymphoblastic leukemia in first or second complete remission. Using Cox regression we compared outcomes after 116 mismatched single or double cord blood transplants, 546 peripheral blood progenitor cell transplants and 140 bone marrow transplants. The characteristics of the recipients and their diseases were similar except cord blood recipients were younger, more likely to be non-Caucasians and more likely to have a low white blood cell count at diagnosis. There were differences in donor-recipient human leukocyte antigen-match depending on the source of the graft. Most adult donor transplants were matched at the allele-level considering human leukocyte antigens-A, -B, -C and -DRB1. In contrast, most cord blood transplants were mismatched and considered antigen-level matching; 57% were mismatched at two loci and 29% at one locus whereas only 29% of adult donor transplants were mismatched at one locus and none at two loci. There were no differences in the 3-year probabilities of survival between recipients of cord blood (44%), matched adult donor (44%) and mismatched adult donor (43%) transplants. Cord blood transplants engrafted slower and were associated with less grade 2-4 acute but similar chronic graft-versus-host disease, relapse, and transplant-related mortality. The survival of cord blood graft recipients was similar to that of recipients of matched or mismatched unrelated adult donor grafts and so cord blood should be considered a valid alternative source of stem cells for adults with acute lymphoblastic leukemia in the absence of a matched unrelated adult donor. PMID:24056817

  9. Unrelated umbilical cord blood transplant for adult acute lymphoblastic leukemia in first and second complete remission: a comparison with allografts from adult unrelated donors

    PubMed Central

    Marks, David I.; Woo, Kwang Ahn; Zhong, Xiaobo; Appelbaum, Frederick R.; Bachanova, Veronika; Barker, Juliet N.; Brunstein, Claudio G.; Gibson, John; Kebriaei, Partow; Lazarus, Hillard M.; Olsson, Richard; Perales, Miguel-Angel; Pidala, Joseph; Savani, Bipin; Rocha, Vanderson; Eapen, Mary

    2014-01-01

    Allogeneic hematopoietic cell transplantation has an established role in the treatment of adults with acute lymphoblastic leukemia whose survival when recipients of grafts from adult unrelated donors approaches that of recipients of grafts from sibling donors. Our aim was to determine the role of mismatched unrelated cord blood grafts in transplantation for 802 adults with acute lymphoblastic leukemia in first or second complete remission. Using Cox regression we compared outcomes after 116 mismatched single or double cord blood transplants, 546 peripheral blood progenitor cell transplants and 140 bone marrow transplants. The characteristics of the recipients and their diseases were similar except cord blood recipients were younger, more likely to be non-Caucasians and more likely to have a low white blood cell count at diagnosis. There were differences in donor-recipient human leukocyte antigen-match depending on the source of the graft. Most adult donor transplants were matched at the allele-level considering human leukocyte antigens-A, -B, -C and –DRB1. In contrast, most cord blood transplants were mismatched and considered antigen-level matching; 57% were mismatched at two loci and 29% at one locus whereas only 29% of adult donor transplants were mismatched at one locus and none at two loci. There were no differences in the 3-year probabilities of survival between recipients of cord blood (44%), matched adult donor (44%) and mismatched adult donor (43%) transplants. Cord blood transplants engrafted slower and were associated with less grade 2–4 acute but similar chronic graft-versus-host disease, relapse, and transplant-related mortality. The survival of cord blood graft recipients was similar to that of recipients of matched or mismatched unrelated adult donor grafts and so cord blood should be considered a valid alternative source of stem cells for adults with acute lymphoblastic leukemia in the absence of a matched unrelated adult donor. PMID:24056817

  10. Delayed (footpad) hypersensitivity and Arthus reactivity using protein-rich antigens and LPS in mice immunized with live attenuated aroA Salmonella vaccines.

    PubMed

    Mastroeni, P; Villarreal-Ramos, B; Demarco de Hormaeche, R; Hormaeche, C E

    1993-05-01

    Footpad reactions to protein-rich salmonella extracts and lipopolysaccharide (LPS) were studied in BALB/c mice 2 and 8 months after immunization with the Salmonella typhimurium SL3261 aroA live vaccine. T-cell depletion in vivo and adoptive serum transfer showed that protein-rich antigens induced T-cell dependent delayed hypersensitivity reactions, whereas LPS only elicited Arthus reactions. The footpad reactions to crude protein extracts were not always T-cell mediated, but depended on the nature and the dose of the antigen. Selective depletion of CD4+ T cells alone had a greater effect than depletion of CD8+ T cells alone, but neither was as marked as simultaneous depletion of both CD4+ and CD8+ T cells, which abolished the delayed type hypersensitivity (DTH) response. Crude protein-rich extracts subjected to alkaline hydrolysis (which removes some ester-linked fatty acids and causes disaggregation of LPS resulting in decreased toxicity while conserving O-specificity) still gave positive T-cell dependent reactions, but with reduced T-cell independent reactivity. Purified phenol-water LPS (2.5 micrograms) produced Arthus reactivity which could be confused with DTH. LPS induced positive reactions which still occurred in T-cell depleted mice and were transferable by immune serum. Arthus reactions did not occur when using alkali-treated LPS, which showed reduced complement fixation in vitro when using serum from immunized mice. The results indicate that footpad testing using salmonella antigens containing LPS elicit DTH but can also produce toxic reactions, some of which are T-cell independent and not necessarily a true measure of DTH. Arthus reactivity to LPS can be confused with DTH. Alkaline hydrolysis of the antigens can eliminate non-specific reactogenicity while retaining the ability of the (protein-rich) antigen to elicit a true T-cell dependent footpad response, which requires the participation of both CD4+ and CD8+ T cells. PMID:8366814

  11. Babesia gibsoni rhoptry-associated protein 1 and its potential use as a diagnostic antigen

    Microsoft Academic Search

    Jinlin Zhou; Honglin Jia; Yoshifumi Nishikawa; Kozo Fujisaki; Xuenan Xuan

    2007-01-01

    A cDNA encoding the rhoptry-associated protein 1 (RAP-1) homologue was obtained by immunoscreening an expression library prepared from Babesia gibsoni merozoite mRNA. The complete nucleotide sequence of the gene was 1740bp. Computer analysis suggested that the sequence contains an open reading frame of 1425bp encoding an expected protein with a molecular weight of 52kDa. Based on the sequence similarity, this

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

    PubMed

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

    2006-06-01

    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

  13. Babesia bovis Merozoite Surface Antigen 1 and Rhoptry-Associated Protein 1 Are Expressed in Sporozoites, and Specific Antibodies Inhibit Sporozoite Attachment to Erythrocytes

    PubMed Central

    Mosqueda, Juan; McElwain, Terry F.; Stiller, David; Palmer, Guy H.

    2002-01-01

    We examined Babesia bovis sporozoites for the expression of two molecules, merozoite surface antigen 1 (MSA-1) and rhoptry-associated protein 1 (RAP-1), that are postulated to be involved in the invasion of host erythrocytes. Both MSA-1 and RAP-1 were transcribed and expressed in infectious sporozoites. Importantly, monospecific MSA-1 and RAP-1 antisera each inhibited sporozoite invasion of erythrocytes in vitro. This is the first identification of antigens expressed in Babesia sp. sporozoites and establishes that, at least in part, sporozoites and merozoites share common targets of antibody mediated inhibition of erythrocyte invasion. PMID:11854249

  14. Efficient construction of a large nonimmune phage antibody library: The production of high-affinity human single-chain antibodies to protein antigens

    PubMed Central

    Sheets, Michael D.; Amersdorfer, Peter; Finnern, Ricarda; Sargent, Peter; Lindqvist, Ericka; Schier, Robert; Hemingsen, Grete; Wong, Cindy; Gerhart, John C.; Marks, James D.

    1998-01-01

    A large library of phage-displayed human single-chain Fv antibodies (scFv), containing 6.7 × 109 members, was generated by improving the steps of library construction. Fourteen different protein antigens were used to affinity select antibodies from this library. A panel of specific antibodies was isolated with each antigen, and each panel contained an average of 8.7 different scFv. Measurements of antibody–antigen interactions revealed several affinities below 1 nM, comparable to affinities observed during the secondary murine immune response. In particular, four different scFv recognizing the ErbB2 protein had affinities ranging from 220 pM to 4 nM. Antibodies derived from the library proved to be useful reagents for immunoassays. For example, antibodies generated to the Chlamydia trachomatis elementary bodies stained Chlamydia-infected cells, but not uninfected cells. These results demonstrate that phage antibody libraries are ideally suited for the rapid production of panels of high-affinity mAbs to a wide variety of protein antigens. Such libraries should prove especially useful for generating reagents to study the function of gene products identified by genome projects. PMID:9600934

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-01-01

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

  17. Processing and Cryopreservation of Placental\\/Umbilical Cord Blood for Unrelated Bone Marrow Reconstitution

    Microsoft Academic Search

    Pablo Rubinstein; Ludy Dobrila; Richard E. Rosenfield; John W. Adamson; Giovanni Migliaccio; Anna Rita Migliaccio; Patricia E. Taylor; Cladd E. Stevens

    1995-01-01

    Clinical evidence of hematopoietic restoration with placental\\/umbilical cord blood (PCB) grafts indicates that PCB can be a useful source of hematopoietic stem cells for routine bone marrow reconstitution. In the unrelated setting, human leukocyte antigen (HLA)-matched donors must be obtained for candidate patients and, hence, large panels of frozen HLA-typed PCB units must be established. The large volume of unprocessed

  18. Topical immunization onto mouse skin using a microemulsion incorporated with an anthrax protective antigen protein-encoding plasmid.

    PubMed

    Cui, Zhengrong; Sloat, Brian R

    2006-07-24

    The current anthrax vaccine in the U.S., the anthrax vaccine adsorbed, has several serious drawbacks, most notably the very lengthy and complicated dosing schedule. Thus, there is a critical need to develop an alternative anthrax vaccine with a simplified immunization schedule. To address this need, we evaluated the feasibility of topically priming or boosting onto the skin using an anthrax protective antigen (PA) protein-encoding DNA vaccine. To this end, we have shown that topical immunization of mice onto their skin with a perflubron-based microemulsion incorporated with a PA63-encoding plasmid, pGPA, led to significant PA-specific antibody responses, which have anthrax lethal toxin-neutralization activity. Moreover, topical boosting of mice primed with PA protein with the pGPA-incorporated, perflubron-based microemulsion significantly enhanced the anti-PA antibody responses induced. This topical anthrax DNA vaccine has the potential to be combined with a vaccine, such as the current AVA, to produce a simplified and more convenient dosing schedule. PMID:16730934

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

    PubMed Central

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

    1996-01-01

    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

  20. Synthesis of Phospholipid-Protein Conjugates as New Antigens for Autoimmune Antibodies.

    PubMed

    Maity, Arindam; Macaubas, Claudia; Mellins, Elizabeth; Astakhova, Kira

    2015-01-01

    Copper(I)-catalyzed azide-alkyne cycloaddition, or CuAAC click chemistry, is an efficient method for bioconjugation aiming at chemical and biological applications. Herein, we demonstrate how the CuAAC method can provide novel phospholipid-protein conjugates with a high potential for the diagnostics and therapy of autoimmune conditions. In doing this, we, for the first time, covalently bind via 1,2,3-triazole linker biologically complementary molecules, namely phosphoethanol amine with human ?2-glycoprotein I and prothrombin. The resulting phospholipid-protein conjugates show high binding affinity and specificity for the autoimmune antibodies against autoimmune complexes. Thus, the development of this work might become a milestone in further diagnostics and therapy of autoimmune diseases that involve the production of autoantibodies against the aforementioned phospholipids and proteins, such as antiphospholipid syndrome and systemic lupus erythematosus. PMID:26046322

  1. A conserved epitope on several human vitamin K-dependent proteins. Location of the antigenic site and influence of metal ions on antibody binding.

    PubMed

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

    1988-05-01

    A murine monoclonal antibody (designated H-11) produced by injecting mice with purified human protein C was found to bind several human vitamin K-dependent proteins. Using a solid-phase competitive radioimmunoassay with antibody immobilized onto microtiter plates, binding of 125I-labeled protein C to the antibody was inhibited by increasing amounts of protein C, prothrombin, and Factors X and VII over a concentration range of 1 X 10(-8) to 1 X 10(-6) M. Other vitamin K-dependent proteins including Factor IX and protein S did not inhibit or inhibited only at the highest concentration binding of radiolabeled protein C to the immobilized antibody. Chemical treatment of prothrombin with a variety of agents including denaturation by sodium dodecyl sulfate, reduction with mercaptoethanol followed by carboxymethylation with iodoacetic acid, citraconylation of lysine residues, removal of metal ion with EDTA, or heat decarboxylation did not destroy the antigenic site recognized by the antibody as measured by immunoblotting of prothrombin or prothrombin derivative immobilized onto nitrocellulose. Immunoblotting of purified vitamin K-dependent polypeptides with the monoclonal antibody following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose indicated that the antigenic site was found on the light chains of protein C and Factor X. Chymotrypsin digestion of prothrombin and isolation on QAE-Sephadex of the peptide representing amino-terminal residues 1-44 of prothrombin further localized the antigenic site recognized by the monoclonal antibody to the highly conserved gamma-carboxyglutamic acid-containing domain. The exact location of the antigenic determinant for antibody H-11 was established using synthetic peptides. Antibody H-11 bound specifically to synthetic peptides corresponding to residues 1-12 of Factor VII and 1-22 of protein C. Comparison of protein sequences of bovine and human vitamin K-dependent proteins suggests that the sequence Phe-Leu-Glu-Glu-Xaa-Arg/Lys is required for antibody binding. The glutamic acid residues in this peptide segment are the first 2 gamma-carboxyglutamic acid residues near the amino-terminal end in the native proteins. Increasing concentrations of Ca2+, Mg2+, or Mn2+ partially inhibited binding of 125I-protein C to the antibody in a solid-phase assay system with half-maximal binding observed at divalent metal ion concentrations of 2, 4, and 0.6 mM, respectively.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2452160

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  3. Antigenicity of Fusion Proteins from Sarcoma-associated Chromosomal Translocations1

    Microsoft Academic Search

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

    2001-01-01

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

  4. Herpes simplex virus 2 (HSV-2) infected cell proteins are among the most dominant antigens of a live-attenuated HSV-2 vaccine.

    PubMed

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

    2015-01-01

    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

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

    PubMed Central

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

    2015-01-01

    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

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

    PubMed Central

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

    2009-01-01

    CXC chemokines, which induce angiogenesis, 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 blood cells, endothelial cells undergoing neovascularization, and neuronal cells. The binding of chemokines by Duffy could modulate chemokine responses positively or negatively. Positive regulation could come through the presentation of chemokine to functional receptors, and negative regulation could come through Duffy competition with functional chemokine receptors for chemokine binding, thus serving as a decoy receptor. To determine whether Duffy has a role in angiogenesis and/or maintenance of homeostasis, we developed transgenic mice expressing mDuffy under the control of the preproendothelin promoter/enhancer (PPEP), which directs expression of the transgene to the endothelium. Two PPEP-mDuffy-transgenic founders were identified, and expression of the transgene in the endothelium was verified by Northern blot, RT-PCR, and immunostaining of tissues. The phenotype of the mice carrying the transgene appeared normal by all visual parameters. However, careful comparison of transgenic and nontransgenic mice revealed two phenotypic differences: mDuffy-transgenic mice exhibited a diminished angiogenic response to MIP-2 in the corneal micropocket assay, and mDuffy-transgenic mice exhibited enhanced hepatocellular toxicity and necrosis as compared with nontransgenic littermates in response to overdose of acetaminophen (APAP; 400 mg/kg body weight). Morover, APAP treatment was lethal in 50% of the mDuffy-transgenic mice 24 h post challenge, and 100% of the nontransgenic litter-mates survived this treatment at the 24 h time point. Our data suggest that enhanced expression of mDuffy on endothelial cells can lead to impaired angiogenic response to chemokines and impaired maintenance of homeostasis in response to toxic stresses. PMID:11781390

  7. Immunoglobulin A (IgA) and IgG serum antibodies to mycobacterial antigens in Crohn's disease patients and their relatives.

    PubMed Central

    Wayne, L G; Hollander, D; Anderson, B; Sramek, H A; Vadheim, C M; Rotter, J I

    1992-01-01

    Sera from patients with Crohn's disease, their relatives, their spouses, and unrelated healthy controls were assayed by enzyme-linked immunosorbent assay for immunoglobulin G (IgG) and IgA antibodies to Mycobacterium tuberculosis, M. avium, and M. gordonae. The patients had significantly higher IgA responses to mycobacterial antigens than did either their relatives or the controls. On the other hand, both the patients and their relatives had significantly higher IgG responses against these antigens than did the controls. The elevated IgA response was more pronounced against isopentanol-extracted whole bacterial cells than it was against soluble protein extracts, and it appeared to be directed against fixed surface antigens that lie under the loosely bound peptidoglycolipid or glycolipid antigens of mycobacteria. PMID:1500507

  8. 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

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

    2009-01-01

    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

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

    E-print Network

    Garlid, Keith

    is a thermogenic protein regulated by diet [1,2]. Several ¢ndings support this view. Thus, starvation in- creased-fat diet [8,9]; and UCP2 mRNA increased 5-fold in white fat of obese ob/ob and db/db mice [2]. Additional involvement has also been proposed for the e¡ect of thyroid hormone on resting metabolic rate [13,14]; however

  10. Antigenic Relationships Among Human Rotaviruses as Determined by Outer Capsid Protein VP4

    Microsoft Academic Search

    Mario Gorziglia; Gisela Larralde; Albert Z. Kapikian; Robert M. Chanock

    1990-01-01

    cDNA clones representing the VP4 gene of symptomatic human rotavirus strain KU (VP7 serotype 1) or DS-1 (VP7 serotype 2) or asymptomatic human rotavirus strain 1076 (VP7 serotype 2) were constructed and inserted into a baculovirus expression vector under the control of the polyhedrin promoter. The resulting recombinants expressed the appropriate authentic VP4 rotavirus outer capsid protein. Guinea pigs immunized

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

    PubMed

    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

    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

  12. Testis-expressed protein TSGA10 an auto-antigen in autoimmune polyendocrine syndrome type I.

    PubMed

    Reimand, Koit; Perheentupa, Jaakko; Link, Maire; Krohn, Kai; Peterson, Pärt; Uibo, Raivo

    2008-01-01

    Autoimmune polyendocrine syndrome type 1 (APS1) is a rare monogenic autosomal recessive disorder. Autoimmune gonadal failure is often one of its features. The aim of this study was to identify targets of immune reactions associated with male autoimmune hypogonadism in APS1. Human testis cDNA expression library immunoscreening with APS1 patients' sera identified the protein testis-specific protein 10 (TSGA10), which is a testis-expressed protein with a key role in spermatogenesis. The corresponding serum autoantibodies were detected by Radioimmunoprecipitation assay in 3 of 40 male (7.5%) and 2 of 26 female (7.7%) APS1 patients but in none of either 32 patients with Addison's disease or 116 healthy controls (p = 0.0055). However, the TSGA10 antibodies in APS1 patients showed no correlation with testicular or ovarian failure or with autoimmune hypogonadism markers. Nevertheless, their presence in a proportion of patients with APS1 highlights the role of TSGA10 as a target of immune reactions in APS1. PMID:18000009

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

    PubMed

    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

    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

  14. Salmonella Pathogenicity Island 2-Mediated Overexpression of Chimeric SspH2 Proteins for Simultaneous Induction of Antigen-Specific CD4 and CD8 T Cells

    PubMed Central

    Panthel, Klaus; Meinel, Katrin M.; Domènech, Victòria E. Sevil; Retzbach, Heike; Igwe, Emeka I.; Hardt, Wolf-Dietrich; Rüssmann, Holger

    2005-01-01

    Salmonella enterica serovar Typhimurium employs two different type III secretion systems (TTSS) encoded within Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) for targeting of effector proteins into the cytosol of eukaryotic cells during different stages of the infection cycle. The SPI1 TTSS translocates virulence factors across the plasma membrane when the bacterium initially contacts the host cell. In contrast, the SPI2 TTSS functions to translocate proteins across the membrane of the Salmonella-containing vacuole and promotes intracellular survival and replication. The aim of the present study was to directly compare the potentials of SPI1 and SPI2 type III effector proteins to act as carrier molecules for a heterologous antigen. The p60 protein of Listeria monocytogenes was used as a model antigen to construct chimeric SopE2 (SPI1), SifA (SPI2), and SspH2 (SPI2) proteins. SPI1- and SPI2-dependent up- and down-regulation of hybrid gene expression led to sequential translocation of p60 fusion proteins into the cytosol of Salmonella-infected macrophages. Mice orally immunized with recombinant Salmonella strains expressing these hybrid proteins revealed comparable numbers of p60-specific CD8 T cells. However, only overexpression of translocated SspH2/p60 from a medium-copy-number vector induced simultaneous antigen-specific CD4 and CD8 T-cell responses, suggesting that SspH2 is an attractive carrier molecule for foreign-protein delivery. PMID:15618170

  15. The C-terminal end of the capsid protein of Avian Nephritis Virus is antigenic and induces broadly cross-reactive antibodies.

    PubMed

    Hewson, K A; Wawegama, N K; O'Rourke, D; Putri, K; Chamings, A; Noormohammadi, A H; Ignjatovic, J

    2015-09-01

    Avian nephritis virus (ANV) has been isolated frequently from commercial broilers in many countries. The prevalence and economic impact of ANV however has been difficult to ascertain due to the lack of convenient serological techniques. In this study the full-length and fragments of the ANV capsid protein were expressed in Baculovirus and affinity purified recombinant proteins used for the detection of ANV antibodies in ELISA. The crystal structure of Human Astrovirus (HAstV) was used as a model to determine potential homologous C-terminal antigenic regions in ANV. The rp37 fragment from three ANV strains NSW_3, ANV-1 and ANV-2, and a shorter NSW_3 fragment (rp33) were compared for their ability to detect ANV antibodies in seven reference chicken sera. The ANV-1 rp37 antigen was the most strain specific whereas the NSW_3 rp37 and rp33 antigens detected antibodies in all heterologous sera, including ANV-1 serum. Irrespective of the strain used, the two NSW_3 protein fragments rp37 and rp33 were found to be superior as antigens for ELISA when compared to the full-length capsid protein rp75. An ELISA designed using the NSW_3 rp33 could reliably differentiate between uninfected and infected commercial broiler flocks, as demonstrated by statistically significant differences between the OD values. This study identified an ANV immunogenic region and successfully used recombinant protein expression of this region to detect cross-reactive ANV antibodies. The results of this study facilitate future studies into the epidemiology and importance of ANV infections in commercial poultry. PMID:25934419

  16. Comprehensive Analysis and Characterization of Linear Antigenic Domains on HN Protein from Genotype VII Newcastle Disease Virus Using Yeast Surface Display System

    PubMed Central

    Li, Tao; Wang, Gaoling; Shi, Bingtian; Liu, Peixin; Si, Wei; Wang, Bin; Jiang, Li; Zhou, Lunjiang; Xiu, Jinsheng; Liu, Henggui

    2015-01-01

    Circulation of genotype VII Newcastle disease virus (NDV) has posed a great threat for the poultry industry worldwide. Antibodies against Hemagglutinin-neuraminidase (HN), a membrane protein of NDV with critical roles in NDV infection, have been reported to provide chickens protection from NDV infection. In this study, we comprehensively analyzed the in vivo antibody responses against the linear antigenic domains of the HN protein from genotype VII NDV using a yeast surface display system. The results revealed four distinct regions of HN, P1 (1-52aa), P2 (53-192aa), P3 (193-302aa) and P4 (303-571aa), respectively, according to their antigenic potency. Analysis by FACS and ELISA assay indicated P2 to be the dominant linear antigenic domain, with the immunogenic potency to protect the majority of chickens from NDV challenge. In contrast, the P1, P3 and P4 domains showed weak antigenicity in vivo and could not protect chickens from NDV challenge. These results provide important insight into the characteristic of humoral immune responses elicited by HN of NDV in vivo. PMID:26121247

  17. Antigenicity and immunogenicity of a trimeric envelope protein from an Indian clade C HIV-1 isolate.

    PubMed

    Sneha Priya, Rangasamy; Veena, Menon; Kalisz, Irene; Whitney, Stephen; Priyanka, Dhopeshwarkar; LaBranche, Celia C; Sri Teja, Mullapudi; Montefiori, David C; Pal, Ranajit; Mahalingam, Sundarasamy; Kalyanaraman, Vaniambadi S

    2015-04-01

    Human immunodeficiency virus type 1 (HIV-1) isolates from India mainly belong to clade C and are quite distinct from clade C isolates from Africa in terms of their phylogenetic makeup, serotype, and sensitivity to known human broadly neutralizing monoclonal antibodies. Because many of these properties are associated with the envelope proteins of HIV-1, it is of interest to study the envelope proteins of Indian clade C isolates as part of the ongoing efforts to develop a vaccine against HIV-1. To this end, we purified trimeric uncleaved gp145 of a CCR5 tropic Indian clade C HIV-1 (93IN101) from the conditioned medium of 293 cells. The purified protein was shown to be properly folded with stable structure by circular dichroism. Conformational integrity was further demonstrated by its high affinity binding to soluble CD4, CD4 binding site antibodies such as b12 and VRC01, quaternary epitope-specific antibody PG9, and CD4-induced epitope-specific antibody 17b. Sera from rabbits immunized with gp145 elicited high titer antibodies to various domains of gp120 and neutralized a broad spectrum of clade B and clade C HIV-1 isolates. Similar to other clade B and clade C envelope immunogens, most of the Tier 1 neutralizing activity could be absorbed with the V3-specific peptide. Subsequent boosting of these rabbits with a clade B HIV-1 Bal gp145 resulted in an expanded breadth of neutralization of HIV-1 isolates. The present study strongly supports the inclusion of envelopes from Indian isolates in a future mixture of HIV-1 vaccines. PMID:25691567

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

    Microsoft Academic Search

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

    2006-01-01

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

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

    PubMed Central

    Lykakis, J. J.

    1968-01-01

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

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

    PubMed

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

    2009-02-01

    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 (Ca(2+)) influx occurs via highly selective Ca(2+) release-activated channels, and stromal interaction molecule 1 (STIM1) plays an important role in this pathway. We also demonstrate the temporal relation between Ca(2+)-dependent signaling events and formation of the immune synapse. Our data indicate that cytoplasmic Ca(2+) 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 Ca(2+) revealed the unanticipated finding that initial BCR-triggered, Ca(2+)-dependent tyrosine phosphorylation events involve predominantly Ca(2+) released from intracellular stores and that influx-derived Ca(2+) is not essential. This suggests a different role for this phase of Ca(2+) influx. PMID:19028960

  1. Bystander stimulation of activated CD4+ T cells of unrelated specificity following a booster vaccination with tetanus toxoid.

    PubMed

    Di Genova, Gianfranco; Savelyeva, Natalia; Suchacki, Amy; Thirdborough, Stephen M; Stevenson, Freda K

    2010-04-01

    Antigen-specific CD4(+) T cells are central to natural and vaccine-induced immunity. An ongoing antigen-specific T-cell response can, however, influence surrounding T cells with unrelated antigen specificities. We previously observed this bystander effect in healthy human subjects following recall vaccination with tetanus toxoid (TT). Since this interplay could be important for maintenance of memory, we have moved to a mouse model for further analysis. We investigated whether boosting memory CD4(+) T cells against TT in vivo would influence injected CD4(+) TCR transgenic T cells (OT-II) specific for an unrelated OVA peptide. If OT-II cells were pre-activated with OVA peptide in vitro, these cells showed a bystander proliferative response during the ongoing parallel TT-specific response. Bystander proliferation was dependent on boosting of the TT-specific memory response in the recipients, with no effect in naive mice. Bystander stimulation was also proportional to the strength of the TT-specific memory T-cell response. T cells activated in vitro displayed functional receptors for IL-2 and IL-7, suggesting these as potential mediators. This crosstalk between a stimulated CD4(+) memory T-cell response and CD4(+) T cells activated by an unrelated antigen could be important in human subjects continually buffeted by environmental antigens. PMID:20104490

  2. Partial purification and characterization of the major species-specific protein antigens of Rickettsia typhi and Rickettsia prowazekii identified by rocket immunoelectrophoresis.

    PubMed Central

    Dasch, G A; Samms, J R; Williams, J C

    1981-01-01

    Species-specific antigens from Rickettsia typhi and Rickettsia prowazekii were readily solubilized by French pressure cell extraction or sonication of Renografin density gradient-purified rickettsiae and were identified by rocket immunoelectrophoresis. As measured by quantitative rocket immunoelectrophoresis, the species-specific typhus rocket antigens (STRAs) appeared to be proteins; they were denatured by heating at 56 degrees C for 30 min but not by 50 degrees C treatment, and they were sensitive to pronase and trypsin but were not affected by periodate oxidation, glycosidases of various specificities, phospholipase A, or lipase. STRAs from both R. typhi and R. prowazekii were separated from common antigens by DE52 column chromatography of 100,000-X-g supernatant fractions of rickettsial extracts. The purified STRAs were characterized by crossed immunoelectrophoresis, by polyacrylamide gel electrophoresis on Davis and sodium dodecyl sulfate gels, and by an enzyme-linked immunosorbent assay. The two purified STRAs were proteins with similar native electrophoretic mobilities in agarose and polyacrylamide gels, and these proteins had similar polypeptide patterns on sodium dodecyl sulfate gels. Most of the STRA activity migrated as a single protein band on sodium dodecyl sulfate-polyacrylamide and Davis polyacrylamide gels, although minor protein bands with STRA activity were also detected. The major STRA proteins constituted 10 to 15% of the total cellular protein of R. typhi and R. prowazekii. According to sensitive enzyme-linked immunosorbent assay titrations, the STRA of R. prowazekii had substantial cross-reactivity with rabbit antiserum prepared against R. typhi, as shown also by rocket immunoelectrophoresis, whereas the STRA of R. typhi reacted only very weakly with antiserum prepared against R. prowazekii according to the enzyme-linked immunosorbent assay and not at all according to rocket immunoelectrophoresis. Images PMID:6783537

  3. The ESAT-6 protein of Mycobacterium tuberculosis interacts with beta-2-microglobulin (?2M) affecting antigen presentation function of macrophage.

    PubMed

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

    2014-10-01

    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

  4. Rap1 Is a Potent Activation Signal for Leukocyte Function-Associated Antigen 1 Distinct from Protein Kinase C and Phosphatidylinositol3OH Kinase

    Microsoft Academic Search

    KOKO KATAGIRI; MASAKAZU HATTORI; NAGAHIRO MINATO; SHIN-KICHI IRIE; KIYOSHI TAKATSU; TATSUO KINASHI

    2000-01-01

    To identify the intracellular signals which increase the adhesiveness of leukocyte function-associated antigen 1 (LFA-1), we established an assay system for activation-dependent adhesion through LFA-1\\/intercellular adhesion molecule 1 ICAM-1 using mouse lymphoid cells reconstituted with human LFA-1 and then introduced constitutively active forms of signaling molecules. We found that the phorbol myristate acetate (PMA)- responsive protein kinase C (PKC) isotypes

  5. Extracellular production of human hepatitis B virus preS2 antigen as hybrid proteins with Bacillus subtilis ?-amylases in high-salt-concentration media

    Microsoft Academic Search

    Kiyofumi Honda; Hideki Fujieda; Kou-ichiro Ogawa; Mitsunobu Imai; Hiroaki Yamamoto; Takayuki Ikeda; Kunio Yamane

    1993-01-01

    To produce PreS2 antigen of human hepatitis B virus extra-cellularly in Bacillus subtilis, it was fused with the COOH-termini of B. subtilis a-amylases of 522 (Amy+), 467 (Amy+) and 443 (Amy-) amino acids, Among them, a-amylase-A467, which has 467 amino acids, was a relatively stable carrier when the cells were cultured in Luria-Bertani (LB) medium. The a-amylase-A443-PreS2 hybrid protein (Amy-)

  6. LPS-binding protein and CD14-dependent attachment of hepatitis B surface antigen to monocytes is determined by the phospholipid moiety of the particles

    Microsoft Academic Search

    Peter Vanlandschoot; Freya Van Houtte; Annelies Roobrouck; Ali Farhoudi; Felix Stelter; Darell L. Peterson; Julian Gomez-Gutierrez; Francisco Gavilanes; Geert Leroux-Roels

    It was observed recently that recombinant yeast-derived hepatitis B surface antigen (rHBsAg) particles, which contain the S protein only, bind almost exclusively to monocytes. It is shown here that binding requires the presence of the LPS receptor CD14. Furthermore, evidence is presented that a domain on CD14 that is identical to or largely overlaps with the LPS-binding pocket is instrumental

  7. Evaluation of the recombinant Heat shock protein B (HspB) of Coxiella burnetii as a potential antigen for immunodiagnostic of Q fever in goats

    Microsoft Academic Search

    Isabelle Fernandes; Elodie Rousset; Philippe Dufour; Karim Sidi-Boumedine; Anny Cupo; Richard Thiéry; Véronique Duquesne

    2009-01-01

    Coxiella burnetii is an intracellular bacterium that causes a worldwide zoonosis, the Q fever. Currently, to diagnose the infection in ruminants, whole cell antigens-based ELISAs are used.In this study a heat shock protein, the HspB, was evaluated for its ability to be recognized by the goat immune system and its capacity to sign a stage of infection. The htpB gene

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

    Microsoft Academic Search

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

    1993-01-01

    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

  9. Expression and antigenic characterization of recombinant Mycoplasma agalactiae P48 major surface protein.

    PubMed

    Rosati, S; Robino, P; Fadda, M; Pozzi, S; Mannelli, A; Pittau, M

    2000-02-01

    The gene encoding the P48 major surface lipoprotein of M. agalactiae has been recently characterised. Since its product plays an important role in the immune response of infected animals, in this study we analysed a recombinant P48 expressed in E. coli. Multiple point mutations were introduced by site directed mutagenesis in order to convert four tryptophan TGA codons, which are a typical feature of the mycoplasma genetic code, into the standard TGG. The mutated p48 gene was subcloned into pGex-2T and expressed in fusion with glutathione-S transferase. Following purification steps, P48 was eluted from carrier protein by thrombin digestion and used in Western blot and indirect ELISA using well-characterised sheep sera. Results demonstrate that specific antibodies against P48 are detected 3 weeks after onset of clinical disease and the recombinant P48 is a diagnostically relevant marker of M. agalactiae infection. PMID:10703704

  10. Immunocontraceptive potential of major antigenic determinants of chicken riboflavin carrier protein in the female rat.

    PubMed

    Subramanian, S; Karande, A A; Adiga, P R

    2000-12-01

    Earlier investigations have demonstrated that antibodies generated against the N-terminal (10-17) and C-terminal (200-207) ends of chicken riboflavin carrier protein (RCP; 219 AA), but not towards the internal region (172-179), curtail pregnancy establishment in rodents and sub-human primates. In those studies, epitope peptides conjugated to diphtheria toxoid were used as immunogens. In the present study, linkage of these sequences to extraneous carriers was avoided to rule out the possibility of carrier-mediated suppression of hapten-specific antibody production in long-term immune response. The ability of these three free peptides to function as minivaccines was examined and the functional importance of these sequences in pregnancy establishment in rodents were evaluated. The results obtained reveal that the peptides YGC (residues 3-24) and HAC (residues 200-219) serve as immunocontraceptive vaccines. PMID:11137254

  11. Immune Recognition of Swine Vesicular Disease Virus Structural Proteins: Novel Antigenic Regions That Are Not Exposed in the Capsid

    Microsoft Academic Search

    M. A. Jiménez-Clavero; A. Douglas; T. Lavery; J. A. Garcia-Ranea; V. Ley

    2000-01-01

    Swine vesicular disease virus (SVDV) is an enterovirus of the Picornaviridae family that belongs to the coxsackievirus B group. A number of antigenic sites have been identified in SVDV by analysis of neutralizing monoclonal antibody-resistant mutants and shown to be exposed on the surface of the capsid. In this paper we have identified seven new immunodominant antigenic regions in SVDV

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

    Microsoft Academic Search

    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

    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

  13. A heat shock operon in Coxiella burnetti produces a major antigen homologous to a protein in both mycobacteria and Escherichia coli.

    PubMed Central

    Vodkin, M H; Williams, J C

    1988-01-01

    A gene library from the DNA of Coxiella burnetii has been constructed in the cosmid vector pHC79. A particular clone, pJB196, reacted strongly with Coxiella-specific antibodies elicited in a number of different species of animals. This clone produced two abundant C. burnetii-specific polypeptides, a 14-kilodalton nonimmunoreactive protein and a 62-kilodalton immunoreactive protein. Sequencing identified two open reading frames, encoding polypeptides of 10.5 and 58.3 kilodaltons. The only transcriptional control element observed on the 5' side of the initiation codon resembled a heat shock promoter. This heat shock promoter was functionally regulated in Escherichia coli, since both proteins were produced by growth conditions at 37 degrees C and neither protein was detected at 23 degrees C. There were four sequences from the literature that were highly homologous (greater than 50%) to the 62-kilodalton protein from C. burnetii. Three were from Mycobacterium species and represent the immunodominant antigen of this genus. The other was from E. coli, detected as a gene that complements or suppresses a temperature-sensitive RNase activity. Since the recombinant protein was immunogenic, it may serve as an efficacious vaccine against C. burnetii and other pathogenic microorganisms that express the conserved antigen. Images PMID:3343219

  14. Mycobacterium tuberculosis antigen 85B and ESAT-6 expressed as a recombinant fusion protein in Mycobacterium smegmatis elicits cell-mediated immune response in a murine vaccination model.

    PubMed

    Tsolaki, Anthony G; Nagy, Judit; Leiva, Sergio; Kishore, Uday; Rosenkrands, Ida; Robertson, Brian D

    2013-07-01

    In this study, we investigated the potential molecular and immunological differences of a recombinant fusion protein (Hybrid-1), comprising of the immunodominant antigens Ag85B and ESAT-6 from Mycobacterium tuberculosis, derived from two different expression systems, namely Mycobacterium smegmatis and Escherichia coli. The fusion protein was successfully expressed and purified from both bacterial hosts and analyzed for any host-dependent post-translational modifications that might affect the immunogenicity of the protein. We investigated the immunogenicity of Hybrid-1 expressed in the two host species in a murine vaccination model, together with a reference standard Hybrid-1 (expressed in E. coli) from the Statens Serum Institut. No evidence of any post-translation modification was found in the M. smegmatis-derived Hybrid-1 fusion protein, nor were there any significant differences in the T-cell responses obtained to the three antigens analyzed. In conclusion, the Hybrid-1 fusion protein was successfully expressed in a homologous expression system using M. smegmatis and this system is worth considering as a primary source for vaccination trials, as it provided protein of excellent yield, stability and free from lipopolysaccharide. PMID:23333882

  15. Effect of Chemical Modification and Fragmentation on Antigenic Determinants of Internal Protein p30 and Surface Glycoprotein gp70 of Type C Retroviruses

    PubMed Central

    Versteegen, Rosemary J.; Oroszlan, Stephen

    1980-01-01

    The effects of protein modification on the antigenic determinants of p30 and gp70 of type C retroviruses were investigated by using solid-phase competition radioimmunoassays. Proteins were modified by reduction with 2-mercaptoethanol and subsequent carboxymethylation of SH groups with iodoacetamide or by amidination of ? and ? amino groups with methylacetimidate. The type-specific determinants of gp70 were found to be conformational in nature, as they were destroyed by these chemical modifications. Group- and interspecies-specific determinants of gp70 antigens, however, appear to be sequential and do not involve residues susceptible to these chemical reagents. Conformation-dependent type-specific determinants of p30 were affected only by methylacetimidate. Group- and interspecies-specific determinants of p30 are similar to those of gp70 in that they also appear to be sequential antigenic sites. Therefore, the broadly reactive group- and interspecies-specific determinants of gp70 and p30 can be followed into small peptides. Accordingly, a cyanogen bromide cleavage fragment derived from the carboxyl-terminal one-third of Rauscher leukemia virus p30 was found to carry group-specific determinants but no detectable interspecies-specific determinants. In contrast, a peptide obtained by limited trypsin cleavage of p30, which was derived from the NH2-terminal region of the protein, contained at least one of the interspecies determinants shared with feline leukemia virus p27. Images PMID:6154154

  16. 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

    CHAUHAN, R; HANDA, R; DAS, T P; PATI, U

    2004-01-01

    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

  17. Local CD4 and CD8 T-Cell Reactivity to HSV-1 Antigens Documents Broad Viral Protein Expression and Immune Competence in Latently Infected Human Trigeminal Ganglia

    PubMed Central

    van Velzen, Monique; Jing, Lichen; Osterhaus, Albert D. M. E.; Sette, Alessandro

    2013-01-01

    Herpes simplex virus type 1 (HSV-1) infection results in lifelong chronic infection of trigeminal ganglion (TG) neurons, also referred to as neuronal HSV-1 latency, with periodic reactivation leading to recrudescent herpetic disease in some persons. HSV-1 proteins are expressed in a temporally coordinated fashion during lytic infection, but their expression pattern during latent infection is largely unknown. Selective retention of HSV-1 reactive T-cells in human TG suggests their role in controlling reactivation by recognizing locally expressed HSV-1 proteins. We characterized the HSV-1 proteins recognized by virus-specific CD4 and CD8 T-cells recovered from human HSV-1–infected TG. T-cell clusters, consisting of both CD4 and CD8 T-cells, surrounded neurons and expressed mRNAs and proteins consistent with in situ antigen recognition and antiviral function. HSV-1 proteome-wide scans revealed that intra-TG T-cell responses included both CD4 and CD8 T-cells directed to one to three HSV-1 proteins per person. HSV-1 protein ICP6 was targeted by CD8 T-cells in 4 of 8 HLA-discordant donors. In situ tetramer staining demonstrated HSV-1-specific CD8 T-cells juxtaposed to TG neurons. Intra-TG retention of virus-specific CD4 T-cells, validated to the HSV-1 peptide level, implies trafficking of viral proteins from neurons to HLA class II-expressing non-neuronal cells for antigen presentation. The diversity of viral proteins targeted by TG T-cells across all kinetic and functional classes of viral proteins suggests broad HSV-1 protein expression, and viral antigen processing and presentation, in latently infected human TG. Collectively, the human TG represents an immunocompetent environment for both CD4 and CD8 T-cell recognition of HSV-1 proteins expressed during latent infection. HSV-1 proteins recognized by TG-resident T-cells, particularly ICP6 and VP16, are potential HSV-1 vaccine candidates. PMID:23966859

  18. Anti-Group B Streptococcus Glycan-Conjugate Vaccines Using Pilus Protein GBS80 As Carrier and Antigen: Comparing Lysine and Tyrosine-directed Conjugation.

    PubMed

    Nilo, Alberto; Morelli, Laura; Passalacqua, Irene; Brogioni, Barbara; Allan, Martin; Carboni, Filippo; Pezzicoli, Alfredo; Zerbini, Francesca; Maione, Domenico; Fabbrini, Monica; Romano, Maria Rosaria; Hu, Qi-Ying; Margarit, Immaculada; Berti, Francesco; Adamo, Roberto

    2015-07-17

    Gram-positive Streptococcus agalactiae or group B Streptococcus (GBS) is a leading cause of invasive infections in pregnant women, newborns, and elderly people. Vaccination of pregnant women represents the best strategy for prevention of neonatal disease, and GBS polysaccharide-based conjugate vaccines are currently under clinical testing. The potential of GBS pilus proteins selected by genome-based reverse vaccinology as protective antigens for anti-streptococcal vaccines has also been demonstrated. Dressing pilus proteins with surface glycan antigens could be an attractive approach to extend vaccine coverage. We have recently developed an efficient method for tyrosine-directed ligation of large glycans to proteins via copper-free azide-alkyne [3 + 2] cycloaddition. This method enables targeting of predetermined sites of the protein, ensuring that protein epitopes are preserved prior to glycan coupling and a higher consistency in glycoconjugate batches. Herein, we compared conjugates of the GBS type II polysaccharide (PSII) and the GBS80 pilus protein obtained by classic lysine random conjugation and by the recently developed tyrosine-directed ligation. PSII conjugated to CRM197, a carrier protein used for vaccines in the market, was used as a control. We found that the constructs made from PSII and GBS80 were able to elicit murine antibodies recognizing individually the glycan and protein epitopes on the bacterial surface. The generated antibodies were efficacious in mediating opsonophagocytic killing of strains expressing exclusively PSII or GBS80 proteins. The two glycoconjugates were also effective in protecting newborn mice against GBS infection following vaccination of the dams. Altogether, these results demonstrated that polysaccharide-conjugated GBS80 pilus protein functions as a carrier comparably to CRM197, while maintaining its properties of protective protein antigen. Glycoconjugation and reverse vaccinology can, therefore, be combined to design vaccines with broad coverage. This approach opens a path to a new generation of vaccines. Tyrosine-ligation allows creation of more homogeneous vaccines, correlation of the immune response to defined connectivity points, and fine-tuning of the conjugation site in glycan-protein conjugates. PMID:25906283

  19. Epstein-Barr virus nuclear antigen 2 activates transcription of the terminal protein gene.

    PubMed Central

    Zimber-Strobl, U; Suentzenich, K O; Laux, G; Eick, D; Cordier, M; Calender, A; Billaud, M; Lenoir, G M; Bornkamm, G W

    1991-01-01

    Transcription of the terminal protein (TP) gene of Epstein-Barr virus (EBV) in Burkitt's lymphoma cells, in EBV-negative Burkitt's lymphoma cells converted with transformation-defective (P3HR1) and transformation-competent (B95-8, AG876) EBV strains, and in EBV-immortalized cell lines was studied. A TP1 cDNA probe spanning the boundary between exons 1 and 2 and discriminating between TP1 and TP2 transcripts was used for S1 analysis. TP RNA expression varied widely in Burkitt's lymphoma cells. TP-specific transcripts were not detectable or only hardly detectable in Burkitt's lymphoma cells with the group I phenotype (CD10+ CD77+ CD21- CD23- CD30- CDw70-) as well as in P3HR1 virus-converted Burkitt's lymphoma lines. TP expression was high in Burkitt's lymphoma lines with the group II and group III phenotypes (CD21+ CD23+ CD30+ CDw70+), in B95-8 and AG876 virus-converted lines, and in EBV-immortalized cells. Detection of TP1 RNA correlated with EBNA2 expression. TP1 transcription was shown to be dependent on EBNA2 expression by stable transfection of an EBNA2 expression vector into P3HR1 virus-converted BL41 cells. EBNA2 is activating the TP1 as well as the TP2 promoter, as shown by the analysis of TP promoter-chloramphenicol acetyltransferase constructs transiently transfected into EBNA2-positive and EBNA2-negative Burkitt's lymphoma cells. Images PMID:1845900

  20. Antibody production in vitamin A-depleted rats is impaired after immunization with bacterial polysaccharide or protein antigens.

    PubMed

    Pasatiempo, A M; Kinoshita, M; Taylor, C E; Ross, A C

    1990-05-01

    Vitamin A nutritional status has been implicated as important in maintaining the integrity of immune functions. We have determined the effect of vitamin A (retinol) depletion on the ability of young animals to produce antibodies after challenge with various bacterial antigens. Male Lewis rats raised on vitamin A-free or adequate diets were immunized either near 40 days of age, before signs of vitamin A deficiency were apparent, or near 47 days of age when symptoms of deficiency were beginning to be manifest. For rats immunized with polysaccharide antigens from Streptococcus pneumoniae or Neisseria meningitidis, antibody production did not exceed 0-19% of the response of control rats. Vitamin A depletion also severely compromised the response to two T cell-dependent antigens, tetanus toxoid and sheep red blood cells. In striking contrast, retinol-depleted rats immunized with lipopolysaccharides from Pseudomonas aeruginosa and Serratia marcesens produced an antibody response indistinguishable from retinol-sufficient animals. These lipopolysaccharides could elicit antibodies in rat pups, whereas the capsular polysaccharide antigens could not. This is consistent with the characteristics of type 1 and type 2 antigens, respectively. These studies indicate that retinol status is an important determinant of the humoral immune response to certain types of antigen and suggest that antibody production to capsular polysaccharides and T cell-dependent antigens is particularly dependent on adequate retinol status. PMID:2110538

  1. Genome Scale Identification of Treponema pallidum Antigens

    Microsoft Academic Search

    Matthew McKevitt; Mary Beth Brinkman; Melanie McLoughlin; Carla Perez; Jerrilyn K. Howell; George M. Weinstock; Steven J. Norris; Timothy Palzkill

    2005-01-01

    Antibody responses for 882 of the 1,039 proteins in the proteome of Treponema pallidum were examined. Sera collected from infected rabbits were used to systematically identify 106 antigenic proteins, including 22 previously identified antigens and 84 novel antigens. Additionally, sera collected from rabbits throughout the course of infection demonstrated a progression in the breadth and intensity of humoral immunoreactivity against

  2. Identification of Pigment Cell Antigens Defined by Vitiligo Antibodies

    Microsoft Academic Search

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

    1992-01-01

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

  3. Use of cloneable peptide-MBP fusion protein as a mimetic coating antigen in the standardized immunoassay for mycotoxin ochratoxin A.

    PubMed

    Xu, Yang; He, Zhenyun; He, Qinghua; Qiu, Yulou; Chen, Bo; Chen, Jing; Liu, Xing

    2014-09-01

    The quality of mycotoxin conjugates is essential to the development of reliability of immunoassays for mycotoxins. However, conventional mycotoxin conjugates are usually synthesized by chemical methods, which are harmful to the environment and yield unwanted cross-reactions. In this study, using ochratoxin A (OTA) as a model system, a selected OTA mimotope (phage-displayed peptide) that specifically binds to anti-OTA antibody was expressed as soluble and monovalent fusions to maltose binding protein (MBP). These prepared fusion proteins can serve as a mimetic coating antigen in both a quantitative chemiluminescent enzyme-linked immunoassay (CLEIA) and a qualitative dot immunoassay for OTA. One of the prepared mimetic coating antigen (L12-206-MBP)-based CLEIAs exhibited a half-inhibition concentration (IC50) of 0.82 ng/mL and a working range of 0.30-2.17 ng/mL, which resemble those of the conventional OTA-OVA conjugate-based immunoassay. The dot immunoassay developed with both the OTA-OVA conjugate and the mimetics showed identical visual cutoff values of 5 ng/mL. The mimetic coating antigen proposed here is an OTA-free product and can be prepared reproducibly as a homogeneous product and facilitates standardization of immunoassays for the mycotoxin OTA. PMID:25127400

  4. Tumor-associated antigen profiling in breast and ovarian cancer: mRNA, protein or T cell recognition?

    Microsoft Academic Search

    Simone Kayser; Iris Watermann; Christine Rentzsch; Toni Weinschenk; Diethelm Wallwiener; Brigitte Gückel

    2003-01-01

    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

  5. Bacterial Protein Secretion Is Required for Priming of CD8+ T Cells Specific for the Mycobacterium tuberculosis Antigen CFP10

    Microsoft Academic Search

    Joshua S. Woodworth; Sarah M. Fortune; Samuel M. Behar

    2008-01-01

    Mycobacterium tuberculosis infection elicits antigen-specific CD8 T cells that are required to control disease. It is unknown how the major histocompatibility complex class I (MHC-I) pathway samples mycobacterial antigens. CFP10 and ESAT6 are important virulence factors secreted by M. tuberculosis, and they are immu- nodominant targets of the human and murine T-cell response. Here, we test the hypothesis that CFP10

  6. Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: demonstration with region 145-151 (antigenic site 5) of myoglobin.

    PubMed

    Abaza, M S; Atassi, M Z

    1992-12-01

    Monoclonal antibodies of predetermined specificity were prepared by immunization with a free (i.e., without coupling to any protein carrier) synthetic peptide representing region 145-151 of sperm whale myoglobin (SpMb) and their cross-reactions with eight Mb variants were determined. Five Mbs--bottle-nose dolphin myoglobin (BdMb), pacific common dolphin myoglobin (PdMb), horse myoglobin (HsMb), dog myoglobin (DgMb), and badger myoglobin (BgMb)--have an identical sequence in that region. Nevertheless, these Mbs exhibited very different cross-reactivities. BdMb and PdMb exhibited cross-activities which were comparable to that of the reference antigen, SpMb; while the reactivity of HsMb was remarkedly decreased, DgMb and BgMb showed almost no cross-reactions with these mAbs. Since the region 145-151 has an identical sequence in all the five Mbs, it is concluded that the differences in their antigenic reactivities with anti-region 145-151 mAbs are due to the effects of amino acid substitutions outside the region 145-151. Another pair of myoglobins, echidna myoglobin (EdMb) and chicken myoglobin (ChMb), have the same sequence in that region, but reacted very differently with anti-region 145-151 mAbs. The reactivity and affinity of EdMb were substantially decreased while those of ChMb were almost completely absent, relative to SpMb. It is concluded, contrary to popular assumptions, that when an amino acid substitution influences the binding of a protein variant to a mAb, it is not necessary for that substitution to be an actual contact residue (i.e., a residue within the antigenic site where the mAb binds). Such effects, which are often very drastic, could be due to indirect influences of the substitution on the chemical and binding properties of the site residues. Furthermore, residues which had been postulated, on the basis of these assumptions, to constitute discontinuous antigenic sites in SpMb, were found [from the present studies and those recently reported with mAbs against the other four antigenic site of Mb (regions 15-22, 56-62, 94-100, and 113-120 of SpMb)] to merely be exerting indirect effects on the known five antigenic sites of Mb. The effects of substitutions, which can happen even in the absence of conformational changes, are determined by many factors, such as the chemical nature of the substitution, its environment, its distance from the site, and the nature of the site residue(s) being affected. PMID:1466763

  7. Intracellular localisation of the Ro 52kD auto-antigen in HeLa cells visualised with green fluorescent protein chimeras.

    PubMed

    Pourmand, N; Blange, I; Ringertz, N; Pettersson, I

    1998-01-01

    Autoantibodies to the Ro/SSA and La/SSB antigens are found in patients with Sjogren's syndrome and systemic lupus erythematosus. The Ro/SSA autoantigen consists of a 52 kD and a 60 kD protein, complexed with one of four small RNA molecules. The La protein can associate with the complex. The Ro/SSA autoantigens are present in all mammalian cells, but their intracellular location is subject of controversy and their function remains unclear. To study the intracellular sorting and targeting of Ro 52 kD we have constructed expression plasmids encoding fusion proteins between the full-length Ro 52 kD protein as well as Ro 52 kD fragments and the green fluorescent protein (GFP) from the jelly fish, Aequorea Victoria. The subcellular distribution of the GFP-Ro 52 kD fusion proteins was investigated in transient expression experiments using transfected HeLa cells. The GFP-full-length Ro 52 kD fusion protein was accumulated in the cytoplasm and excluded from the nucleus. When GFP was fused with the La protein, the fluorescence was located in the nucleus. Clones coding for Ro 52 kD fragments containing the hydrophilic central part of the Ro 52 kD protein gave the same intracellular location and type of cytoplasmic speckles as the full-length Ro 52 kD protein. In contrast, both amino terminal and carboxy terminal fragments were uniformly distributed throughout the cell just like the GFP protein itself. These observations indicated a cytoplasmic location of the Ro 52 kD protein and demonstrated the crucial role of the hydrophilic domain in restricting the Ro 52 kD protein to this intracellular compartment. PMID:9892504

  8. 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

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

    1977-01-01

    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

  9. The carboxy-terminal cysteine of the tetraspanin L6 antigen is required for its interaction with SITAC, a novel PDZ protein.

    PubMed

    Borrell-Pagès, M; Fernández-Larrea, J; Borroto, A; Rojo, F; Baselga, J; Arribas, J

    2000-12-01

    PDZ domains are protein modules that mediate protein-protein interactions. Here, we present the identification and characterization of a protein similar to the recently identified PDZ-containing protein TACIP18, which we have named SITAC (similar to TACIP18). SITAC is preferentially expressed in cells of the digestive tract, associated with intracellular membranes. Despite the high degree of sequence identity between the PDZ domains of TACIP18 and those of SITAC, none of the known ligands of the former shows interaction with the latter, as judged by two-hybrid analysis. SITAC interacts with peptides containing bulky hydrophobic amino acids two positions upstream of the C-terminal residue. Surprisingly, SITAC also shows interaction with peptides ending in C, a previously unacknowledged ability of PDZ domains. The sequence -Y-X-C-COOH, bound in vitro by SITAC, is present in the member of the tetraspanin superfamily, the L6 antigen. Coimmunoprecipitation experiments show that SITAC interacts with L6A, but not with an L6A C-terminal mutant, confirming the capacity of SITAC to interact with proteins ending in C. Confocal analysis shows that the interaction between L6A and SITAC is necessary for the precise colocalization of both molecules in the same subcellular compartment. In summary, the characterization of the protein SITAC has unveiled novel sequences recognized by PDZ domains, and it suggests that L6A is a natural ligand of this PDZ protein. PMID:11102519

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

    PubMed Central

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

    1991-01-01

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

  11. New PHA products using unrelated carbon sources

    PubMed Central

    Matias, Fernanda; de Andrade Rodrigues, Maria Filomena

    2011-01-01

    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

  12. Inter-comparison of unrelated fiber evidence.

    PubMed

    Houck, Max M

    2003-08-12

    The foreign textile fibers recovered from one item of evidence from each of 20 unrelated crimes in three categories (bank robbery, kidnapping, and homicide) were cross-compared. The items of evidence were scraped to remove the trace evidence and a sample of the collected fibers was examined using a standard scheme of analysis. The fibers were examined with light microscopy (including polarized light microscopy), fluorescence microscopy, and microspectrophotometry. The fibers were divided into natural and manufactured groups and then categorized by color and generic (polymer) class. Cross-comparing all 2083 fibers resulted in 2,168,403 comparisons, after removing duplicate (same fiber) comparisons. Colorless and denim fibers were excluded from this study. No two fibers were found to exhibit the same microscopic characteristics and analytical properties. Therefore, it is rare to find two unrelated items that have foreign fibers that are analytically indistinguishable. These results corroborate other population studies conducted in Europe and target fiber studies conducted both in the US and in Europe. PMID:12927416

  13. Immobilization antigen vaccine adjuvanted by parasitic heat shock protein 70C confers high protection in fish against cryptocaryonosis.

    PubMed

    Josepriya, T A; Chien, Kuo-Hsuan; Lin, Hsin-Yun; Huang, Han-Ning; Wu, Chang-Jer; Song, Yen-Ling

    2015-08-01

    The immobilization antigen (iAg) has been demonstrated as a protective immunogen against Cryptocaryon irritans infection. In this study, C-terminal domain of heat shock protein 70 cloned from C. irritans (Hsp70C) was tested for its immuno-stimulatory effects. The iAg and Hsp70C cDNAs were constructed independently in secretory forms and were encapsulated in chitosan nanoparticles. In the first immunization trial, grouper fingerlings orally intubated with iAg and iAg:Hsp70C presented 96% and 100% relative percent survival (RPS), respectively, after a lethal challenge. In the second trial, both iAg and iAg:Hsp70C groups showed 100% RPS and the skin trophont burden was significantly lowered. The iAg:Hsp70C still provides a significantly high protection of 51% RPS at 49 days post immunization, when an even more serious lethal infection occurs. RT-qPCR results showed that Hsp70C could up-regulate the expression of i) T cell markers: Cluster of Differentiation 8 alpha (CD8?) and CD4, ii) cytokine genes: Interferon gamma (IFN?), Tumor Necrosis Factor alpha (TNF?) and Interleukin 12 p40 (IL-12/P40), iii) antibody genes: Immunoglobulin M heavy chain (IgMH) and IgTH, and iv) major histocompatibility complex (MHC-I & MHC-II), in the spleen of iAg:Hsp70C group. Furthermore, significantly high levels of iAg-specific IgM was detected in skin mucus which efficiently immobilized live theronts in iAg- and iAg:Hsp70C-immunized fish at 5 weeks post immunization. Hsp70C significantly increased the number of nonspecific CD8(+) skin leucocytes which exerted cytotoxicity against theronts, although cytotoxic activity showed no difference among the various groups. Because of this complementary cooperation of cellular and humoral immune responses, Hsp70C enhances the efficacy of iAg vaccine and constrains C. irritans infection. In view of the severe loss caused by cryptocaryonosis, application of this parasitic vaccine in farmed and ornamental fish, is worthy to be considered. PMID:25957883

  14. Process development and analysis of liver-stage antigen 1, a preerythrocyte-stage protein-based vaccine for Plasmodium falciparum.

    PubMed

    Hillier, Collette J; Ware, Lisa A; Barbosa, Arnoldo; Angov, Evelina; Lyon, Jeffrey A; Heppner, D Gray; Lanar, David E

    2005-04-01

    Plasmodium falciparum liver-stage antigen 1 (LSA-1) is expressed solely in infected hepatocytes and is thought to have a role in liver schizogony and merozoite release. Specific humoral, cellular, and cytokine immune responses to LSA-1 are well documented, with epitopes identified that correlate with antibody production, proliferative T-cell responses, or cytokine induction. With the goal of developing a vaccine against this preerythrocyte-stage protein, we undertook the good manufacturing practices (GMP) manufacture of a recombinant LSA-1 construct, LSA-NRC, incorporating the N- and C-terminal regions of the protein and two of the centrally placed 17-amino-acid repeats. To improve the protein yield, a method of codon harmonization was employed to reengineer the gene sequence for expression in Escherichia coli. A 300-liter GMP fermentation produced 8 kg of bacterial cell paste, and a three-step column chromatographic method yielded 8 mg of purified antigen per g of paste. The final bulk protein was >98% pure, demonstrated long-term stability, and contained <0.005 endotoxin units per 50 microg of protein. To accomplish the initial stages of evaluation of this protein as a human-use vaccine against malaria, we immunized rabbits and mice with LSA-NRC in Montanide ISA 720. New Zealand White rabbits and A/J (H-2K) mice produced high-titer antibodies that recognized liver-stage parasites in infected cultured human hepatocytes. Gamma interferon-producing cells, which have been associated with LSA-1-mediated protection, were detected in splenocytes harvested from immunized mice. Finally, sera taken from people living in a region where malaria is holoendemic recognized LSA-NRC by Western blotting. PMID:15784552

  15. 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

    Aimin Jiang; Andrew Craxton; Tomohiro Kurosaki; Edward A. Clark

    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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

    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

    2014-01-01

    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

  18. Combining viral vectored and protein-in-adjuvant vaccines against the blood-stage malaria antigen AMA1: report on a phase 1a clinical trial.

    PubMed

    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

    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

  19. The gene coding for a major tumor rejection antigen of tumor P815 is identical to the normal gene of syngeneic DBA/2 mice

    PubMed Central

    1991-01-01

    We showed previously that mouse mastocytoma P815 expresses several distinct antigens that are recognized by cytolytic T lymphocytes (CTL) of syngeneic DBA/2 mice. Antigens P815A and P815B are usually lost jointly and are targets for immune rejection responses in vivo. We used a cosmid library and a CTL stimulation assay to obtain transfectants expressing tumor rejection antigen P815A. From these transfectants we retrieved gene P1A which transferred the expression of both P815A and B. This gene is unrelated to three previously isolated genes coding for tum-antigens. It encodes a putative protein of 224 amino acids which contains two highly acidic domains showing homology with similar regions of nuclear proteins. The P1A gene expressed by tumor P815 is completely identical to the gene present in normal DBA/2 cells. Expression of the gene was tested by Northern blots. Cells from liver, spleen, and a number of mast cell lines were negative, but mast cell line L138.8A produced a high level of P1A message and was lysed by CTL directed against antigens P815A and B. We conclude that major tumor rejection antigens of P815 are encoded by a gene showing little or no expression in most normal cells of adult mice. PMID:1903428

  20. Predicting Protein Function from Structure—The Roles of Short-chain Dehydrogenase/Reductase Enzymes in Bordetella O-antigen Biosynthesis

    PubMed Central

    King, Jerry D.; Harmer, Nicholas J.; Preston, Andrew; Palmer, Colin M.; Rejzek, Martin; Field, Robert A.; Blundell, Tom L.; Maskell, Duncan J.

    2007-01-01

    The pathogenic bacteria Bordetella parapertussis and Bordetella bronchiseptica express a lipopolysaccharide O antigen containing a polymer of 2,3-diacetamido-2,3-dideoxy-l-galacturonic acid. The O-antigen cluster contains three neighbouring genes that encode proteins belonging to the short-chain dehydrogenase/reductase (SDR) family, wbmF, wbmG and wbmH, and we aimed to elucidate their individual functions. Mutation and complementation implicate each gene in O-antigen expression but, as their putative sugar nucleotide substrates are not currently available, biochemical characterisation of WbmF, WbmG and WbmH is impractical at the present time. SDR family members catalyse a wide range of chemical reactions including oxidation, reduction and epimerisation. Because they typically share low sequence conservation, however, catalytic function cannot be predicted from sequence analysis alone. In this context, structural characterisation of the native proteins, co-crystals and small-molecule soaks enables differentiation of the functions of WbmF, WbmG and WbmH. These proteins exhibit typical SDR architecture and coordinate NAD. In the substrate-binding domain, all three enzymes bind uridyl nucleotides. WbmG contains a typical SDR catalytic TYK triad, which is required for oxidoreductase function, but the active site is devoid of additional acid–base functionality. Similarly, WbmH possesses a TYK triad, but an otherwise feature-poor active site. Consequently, 3,5-epimerase function can probably be ruled out for these enzymes. The WbmF active site contains conserved 3,5-epimerase features, namely, a positionally conserved cysteine (Cys133) and basic side chain (His90 or Asn213), but lacks the serine/threonine component of the SDR triad and therefore may not act as an oxidoreductase. The data suggest a pathway for synthesis of the O-antigen precursor UDP-2,3-diacetamido-2,3-dideoxy-l-galacturonic acid and illustrate the usefulness of structural data in predicting protein function. PMID:17950751

  1. Unrelated business income tax: an update.

    PubMed

    Fama, A J

    1984-02-01

    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

  2. Seroreactivity to a large panel of field-derived Plasmodium falciparum apical membrane antigen 1 and merozoite surface protein 1 variants reflects seasonal and lifetime acquired responses to malaria.

    PubMed

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

    2015-01-01

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

  3. Antigenic analysis of Toxoplasma gondii antigens by immunoelectrophoresis 

    E-print Network

    Mason, Edward Orys

    1970-01-01

    , particulate cell membrane fragment, and an aqueous lysate antigen. The soluble exudate was separated and seven antigens, unheated, heated to 56 C, and heated from 60 C to 100 C in 10 C increments were prepared. Each of these antigens were inoculated... that amount of time inactivated the toxic activity. Protein measure? ments of these toxins rems. ined static over the interva. l of heating. Immunoelectrophoresis of the crude, 56 C, 60 C and mouse antis era (the only antisera evoked by these antigen...

  4. A High Degree of HLA Disparity Arises From Limited Allelic Diversity: Analysis of 1775 Unrelated Bone Marrow Transplant Donor-Recipient Pairs

    Microsoft Academic Search

    Carolyn Katovich Hurley; Marcelo Fernandez-Vina; William H. Hildebrand; Harriet J. Noreen; Elizabeth Trachtenberg; Thomas M. Williams; Lee Ann Baxter-Lowe; Ann B. Begovich; Effie Petersdorf; Annamalai Selvakumar; Peter Stastny; Janet Hegland; Robert J. Hartzman; Michael Carston; Sharavi Gandham; Craig Kollman; Gene Nelson; Stephen Spellman; Michelle Setterholm

    2007-01-01

    The allelic diversity and associated human leukocyte antigen (HLA) disparity of 1775 bone marrow recipients and their unrelated donors, matched for six of six (1361\\/1775,77%), five of six (397\\/1775, 22%), or four of six (17\\/1775, 1%) HLA-A, -B, -DR antigens, were retrospectively evaluated. The comprehensive HLA analysis included the class I (A, B, C) and II (DRB1, DQA1, DQB1, DPA1,

  5. Pathways of Antigen Processing

    PubMed Central

    Blum, Janice S.; Wearsch, Pamela A.; Cresswell, Peter

    2014-01-01

    T cell recognition of antigen presenting cells depends on their expression of a spectrum of peptides bound to Major Histocompatibility Complex class I (MHC-I) and class II (MHC-II) molecules. Conversion of antigens from pathogens or transformed cells into MHC-I and MHC-II-bound peptides is critical for mounting protective T cell responses, and similar processing of self proteins is necessary to establish and maintain tolerance. Cells use a variety of mechanisms to acquire protein antigens, from translation in the cytosol to variations on the theme of endocytosis, and to degrade them once acquired. In this review we highlight the aspects of MHC-I and MHC-II biosynthesis and assembly that have evolved to intersect these pathways and sample the peptides that are produced. PMID:23298205

  6. Molecular Evolution and Genetic Analysis of the Major Capsid Protein VP1 of Duck Hepatitis A Viruses: Implications for Antigenic Stability

    PubMed Central

    Ma, Xiuli; Sheng, Zizhang; Huang, Bing; Qi, Lihong; Li, Yufeng; Yu, Kexiang; Liu, Cunxia; Qin, Zhuoming; Wang, Dan; Song, Minxun; Li, Feng

    2015-01-01

    The duck hepatitis A virus (DHAV), a member of the family Picornaviridae, is the major cause of outbreaks with high mortality rates in young ducklings. It has three distinctive serotypes and among them, serotypes 1 (DHAV-1) and 3 (DHAV-3) were recognized in China. To investigate evolutionary and antigenic properties of the major capsid protein VP1 of these two serotypes, a primary target of neutralizing antibodies, we determined the VP1 coding sequences of 19 DHAV-1 (spanning 2000-2012) and 11 DHAV-3 isolates (spanning 2008-2014) associated with disease outbreaks. By bioinformatics analysis of VP1 sequences of these isolates and other DHAV strains reported previously, we demonstrated that DHAV-1 viruses evolved into two genetic lineages, while DHAV-3 viruses exhibited three distinct lineages. The rate of nucleotide substitution for DHAV-1 VP1 genes was estimated to be 5.57 x 10-4 per site per year, which was about one-third times slower than that for DHAV-3 VP1 genes. The population dynamics analysis showed an upward trend for infection of DHAV-1 viruses over time with little change observed for DHAV-3 viruses. Antigenic study of representative DHAV-1 and DHAV-3 strains covering all observed major lineages revealed no detectable changes in viral neutralization properties within the serotype, despite the lack of cross-neutralization between serotypes 1 and 3 strains. Structural analysis identified VP1 mutations in DHAV-1 and DHAV-3 viruses that underpin the observed antigenic phenotypes. Results of our experiments described here shall give novel insights into evolution and antigenicity of duck picornaviruses. PMID:26173145

  7. A Small Antigenic Determinant of the Chikungunya Virus E2 Protein Is Sufficient to Induce Neutralizing Antibodies which Are Partially Protective in Mice

    PubMed Central

    Weber, Christopher; Büchner, Sarah M.; Schnierle, Barbara S.

    2015-01-01

    Background The mosquito-borne Chikungunya virus (CHIKV) causes high fever and severe joint pain in humans. It is expected to spread in the future to Europe and has recently reached the USA due to globalization, climate change and vector switch. Despite this, little is known about the virus life cycle and, so far, there is no specific treatment or vaccination against Chikungunya infections. We aimed here to identify small antigenic determinants of the CHIKV E2 protein able to induce neutralizing immune responses. Methodology/Principal Findings E2 enables attachment of the virus to target cells and a humoral immune response against E2 should protect from CHIKV infections. Seven recombinant proteins derived from E2 and consisting of linear and/or structural antigens were created, and were expressed in and purified from E. coli. BALB/c mice were vaccinated with these recombinant proteins and the mouse sera were screened for neutralizing antibodies. Whereas a linear N-terminally exposed peptide (L) and surface-exposed parts of the E2 domain A (sA) alone did not induce neutralizing antibodies, a construct containing domain B and a part of the ?-ribbon (called B+) was sufficient to induce neutralizing antibodies. Furthermore, domain sA fused to B+ (sAB+) induced the highest amount of neutralizing antibodies. Therefore, the construct sAB+ was used to generate a recombinant modified vaccinia virus Ankara (MVA), MVA-CHIKV-sAB+. Mice were vaccinated with MVA-CHIKV-sAB+ and/or the recombinant protein sAB+ and were subsequently challenged with wild-type CHIKV. Whereas four vaccinations with MVA-CHIKV-sAB+ were not sufficient to protect mice from a CHIKV infection, protein vaccination with sAB+ markedly reduced the viral titers of vaccinated mice. Conclusions/Significance The recombinant protein sAB+ contains important structural antigens for a neutralizing antibody response in mice and its formulation with appropriate adjuvants might lead to a future CHIKV vaccine. PMID:25905779

  8. A recombinant protein based on the Trypanosoma cruzi metacyclic trypomastigote 82-kilodalton antigen that induces and effective immune response to acute infection.

    PubMed Central

    Santori F, R; Paranhos-Bacalla, G S; Franco DA Silveira, J; Yamauchi, L M; Araya, J E; Yoshida, N

    1996-01-01

    To further investigate the immunological properties of the stage-specific 82-kDa glycoprotein (gp82) of Trypanosoma cruzi metacyclic trypomastigotes, previously shown to induce antigen-specific humoral and T-cell responses in mice, we performed a series of experiments with recombinant proteins containing sequences of gp82 fused to glutathione S-transferase. Of five fusion proteins tested, only J18b and J18b1, the carboxyproximal peptides containing amino acids 224 to 516 and 303 to 516, respectively, were recognized by monoclonal antibody 3F6 as well as by various anti-T. cruzi antisera and, when administered to mice, were capable of eliciting antibodies directed to the native gp82. The amino-terminal peptide and other carboxyterminal recombinant proteins lacking the central domain of gp82 (amino acids 224 to 356), which is exposed on the surface of live metacyclic forms, did not display any of these properties. Spleen cells derived from mice immunized with any of the five recombinant proteins proliferated in vitro in the presence of native gp82.J18b was the most stimulatory, whereas J18b3, the peptide containing amino acids 408 to 516, elicited the weakest response. When BALB/c mice immunized with J18b antigen plus A1(OH)3 as adjuvant were challenged 10 5 metacyclic trypomastigotes, 85% of them resisted acute infection, in comparison with control mice that received glutathione S-transferase plus adjuvant. Antibodies induced by J18b protein lacked agglutinating or complement-dependent lytic activity and failed to neutralize parasite infectivity. On the other hand, CD4+T cells from the spleens of J18b-immunized mice displayed an intense proliferative activity upon stimulation with 1.25 microgram of native gp82 per ml, which resulted in increased production of gamma interferon, a cytokine associated with resistance to T. cruzi infection. PMID:8606064

  9. Association of Streptococcus pneumoniae common protein antigen (CPA) antibodies and pneumococcal nasopharyngeal colonization in HIV-infected and HIV-uninfected African children.

    PubMed

    Ditse, Z; Adrian, P V; Kuwanda, L; Madhi, S A

    2013-09-13

    Due to the high cost and limited serotype coverage of pneumococcal conjugate vaccines (PCV), pneumococcal common protein antigens (CPAs) are being investigated as potential vaccine candidates. CPAs are likely to be immunogenic in infants and could confer serotype-independent protection. There are limited data on natural antibody kinetics against CPAs in African populations. We aimed to determine the prevalence of naturally acquired antibody titres to 15 CPAs and explore their association to concurrent pneumococcal nasopharyngeal colonization in children aged 4-7 years with and without underlying HIV-infection and/or previous PCV-vaccination. A 15-plex Luminex assay was established to measure serum IgG titres against "cell-wall associated or surface-exposed" proteins (PspA, PspC, LytB, IgA1-proteinase, SP0082, PdB and PcsB), "membrane-associated" proteins (PsaA, SP0609, SP0749, PpmA, SlrA, StkP and SP2194) as well as the hypothetical protein, SP2027. Archived serum samples from HIV-uninfected (n=212) and HIV-infected (n=74) children were analyzed. Concurrent pneumococcal nasopharyngeal colonization was determined with standard microbiological methods. HIV-uninfected children had significantly higher antibody titres against PspA, PspC, PdB, SP0082, LytB, IgA1 proteinase and PcsB compared to HIV-infected children. In contrast, antibody titres against membrane associated proteins (PsaA, SP2027, PpmA and SlrA) were significantly lower in HIV-uninfected compared to HIV-infected children. Higher antibody titres against PdB, and PcsB were associated with the absence of pneumococcal colonization. There was no association between anti-CPA titres and PCV vaccination. In conclusion PdB and PcsB antigens are potential vaccine-candidates which may protect against pneumococcal colonization and consequently pneumococcal disease. PMID:23845819

  10. A single amino acid substitution (R441A) in the receptor-binding domain of SARS coronavirus spike protein disrupts the antigenic structure and binding activity

    SciTech Connect

    He Yuxian [Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10021 (United States)]. E-mail: yhe@nybloodcenter.org; Li Jingjing [Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10021 (United States); Jiang Shibo [Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10021 (United States)

    2006-05-26

    The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) has two major functions: interacting with the receptor to mediate virus entry and inducing protective immunity. Coincidently, the receptor-binding domain (RBD, residues 318-510) of SAR-CoV S protein is a major antigenic site to induce neutralizing antibodies. Here, we used RBD-Fc, a fusion protein containing the RBD and human IgG1 Fc, as a model in the studies and found that a single amino acid substitution in the RBD (R441A) could abolish the immunogenicity of RBD to induce neutralizing antibodies in immunized mice and rabbits. With a panel of anti-RBD mAbs as probes, we observed that R441A substitution was able to disrupt the majority of neutralizing epitopes in the RBD, suggesting that this residue is critical for the antigenic structure responsible for inducing protective immune responses. We also demonstrated that the RBD-Fc bearing R441A mutation could not bind to soluble and cell-associated angiotensin-converting enzyme 2 (ACE2), the functional receptor for SARS-CoV and failed to block S protein-mediated pseudovirus entry, indicating that this point mutation also disrupted the receptor-binding motif (RBM) in the RBD. Taken together, these data provide direct evidence to show that a single amino acid residue at key position in the RBD can determine the major function of SARS-CoV S protein and imply for designing SARS vaccines and therapeutics.

  11. Immunodetection of Canine Parvovirus (CPV) in clinical samples by polyclonal antisera against CPV-VP2 protein expressed in Esherichia coli as an antigen.

    PubMed

    Park, Jae Sung; Choi, Bong Kum; Vijayachandran, Lakshmi Sumitra; Ayyappan, Vasudevan; Chong, Chom-Kyu; Lee, Ki-Sung; Kim, Sei Chang; Choi, Chang Won

    2007-12-01

    The entire virion protein 2 (VP2) gene of Canine Parvovirus (CPV) was amplified by polymerase chain reaction (PCR) and engineered to be expressed by a bacterial expression vector pET-28a, under the control of the IPTG-inducible T7lac promoter. SDS-PAGE gel revealed that VP2 expressed as a 67kDa, and found mainly in the pellet of the bacterial lysates, suggesting that cytoplasmic expression is not preferred. The recombinant protein VP2 fused with His-tag was purified from Esherichia coli using Ni-NTA resin under denaturing conditions. SDS-PAGE analysis also showed the high expression of several lower molecular weight (LMW) bands. Western blot analysis showed that polyclonal antisera produced by rabbit against E. coli-VP2 protein reacted specifically with the purified VP2 protein as well as two other LMW bands. Some of the resulting LMW products failed to keep their antigenic site in the N-terminal region of the VP2. The degradation of recombinant VP2 protein in E. coli could be due to the action of host proteases. The immunodetection ability of the polyclonal antisera was compared with that of a commercial monoclonal antibody to test numerous clinical specimens by immuno-dot blot assays. There were distinctive differences in the degree of immunodetection ability of polyclonal antisera and monoclonal antibody to react with CPV antigens. The reaction time of polyclonal antisera was much faster in visual color appearance than that of monoclonal antibody during NBT/BCIP staining. The result from diagnostic PCR assay confirmed the presence of CPV in 44 out of 46 specimens collected, consistent with polyclonal antisera-positive result. Therefore, the polyclonal antisera can be used for CPV detection in the faeces of diarrhoeic dogs, which was found to be more rapid, sensitive, broad but less specific than the monoclonal antibody. PMID:17825928

  12. Identification of a Specific Antigenic Region of the P82 Protein of Babesia equi and Its Potential Use in Serodiagnosis

    PubMed Central

    Hirata, Haruyuki; Xuan, Xuenan; Yokoyama, Naoaki; Nishikawa, Yoshifumi; Fujisaki, Kozo; Suzuki, Naoyoshi; Igarashi, Ikuo

    2003-01-01

    The efficacy of the Be82 gene product fused with glutathione S-transferase (GST/Be82) in an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Babesia equi infection was reported previously (H. Hirata et al., J. Clin. Microbiol. 40:1470-1474, 2002). However, the ELISA with the GST/Be82 antigen cross-reacted with Babesia caballi-infected horse sera, despite the high rate of detection of B. equi. These results suggested that GST/Be82 has an antigen in common with B. caballi or antigenicity similar to that of B. caballi. In the present study, we constructed a series of five clones with deletions in the Be82 gene product, each of which was fused with GST, and used them in ELISAs in order to overcome the cross-reactivity seen with B. caballi. One of the deletion clones, a clone with a deletion of the Be82 gene from positions 236 to 381 (Be82/236-381), specifically and sensitively detected B. equi-infected horse sera without cross-reactivity with B. caballi-infected horse sera. Assays with clones from which other gene products were deleted showed decreased sensitivities or remained nonspecific for the detection of B. equi-infected horse sera. These results suggest that the Be82/236-381 gene product is a novel antigen for the diagnosis of B. equi infection in horses. PMID:12574244

  13. Nuclear Export Signal-Interacting Protein Forms Complexes with Lamin A/C-Nups To Mediate the CRM1-Independent Nuclear Export of Large Hepatitis Delta Antigen

    PubMed Central

    Huang, Cheng; Jiang, Jia-Yin; Chang, Shin C.; Tsay, Yeou-Guang; Chen, Mei-Ru

    2013-01-01

    Nuclear export is an important process that not only regulates the functions of cellular factors but also facilitates the assembly of viral nucleoprotein complexes. Chromosome region maintenance 1 (CRM1) that mediates the transport of proteins bearing the classical leucine-rich nuclear export signal (NES) is the best-characterized nuclear export receptor. Recently, several CRM1-independent nuclear export pathways were also identified. The nuclear export of the large form of hepatitis delta antigen (HDAg-L), a nucleocapsid protein of hepatitis delta virus (HDV), which contains a CRM1-independent proline-rich NES, is mediated by the host NES-interacting protein (NESI). The mechanism of the NESI protein in mediating nuclear export is still unknown. In this study, NESI was characterized as a highly glycosylated membrane protein. It interacted and colocalized well in the nuclear envelope with lamin A/C and nucleoporins. Importantly, HDAg-L could be coimmunoprecipitated with lamin A/C and nucleoporins. In addition, binding of the cargo HDAg-L to the C terminus of NESI was detected for the wild-type protein but not for the nuclear export-defective HDAg-L carrying a P205A mutation [HDAg-L(P205A)]. Knockdown of lamin A/C effectively reduced the nuclear export of HDAg-L and the assembly of HDV. These data indicate that by forming complexes with lamin A/C and nucleoporins, NESI facilitates the CRM1-independent nuclear export of HDAg-L. PMID:23175358

  14. Nuclear export signal-interacting protein forms complexes with lamin A/C-Nups to mediate the CRM1-independent nuclear export of large hepatitis delta antigen.

    PubMed

    Huang, Cheng; Jiang, Jia-Yin; Chang, Shin C; Tsay, Yeou-Guang; Chen, Mei-Ru; Chang, Ming-Fu

    2013-02-01

    Nuclear export is an important process that not only regulates the functions of cellular factors but also facilitates the assembly of viral nucleoprotein complexes. Chromosome region maintenance 1 (CRM1) that mediates the transport of proteins bearing the classical leucine-rich nuclear export signal (NES) is the best-characterized nuclear export receptor. Recently, several CRM1-independent nuclear export pathways were also identified. The nuclear export of the large form of hepatitis delta antigen (HDAg-L), a nucleocapsid protein of hepatitis delta virus (HDV), which contains a CRM1-independent proline-rich NES, is mediated by the host NES-interacting protein (NESI). The mechanism of the NESI protein in mediating nuclear export is still unknown. In this study, NESI was characterized as a highly glycosylated membrane protein. It interacted and colocalized well in the nuclear envelope with lamin A/C and nucleoporins. Importantly, HDAg-L could be coimmunoprecipitated with lamin A/C and nucleoporins. In addition, binding of the cargo HDAg-L to the C terminus of NESI was detected for the wild-type protein but not for the nuclear export-defective HDAg-L carrying a P205A mutation [HDAg-L(P205A)]. Knockdown of lamin A/C effectively reduced the nuclear export of HDAg-L and the assembly of HDV. These data indicate that by forming complexes with lamin A/C and nucleoporins, NESI facilitates the CRM1-independent nuclear export of HDAg-L. PMID:23175358

  15. Solution Structure, Membrane Interactions, and Protein Binding Partners of the Tetraspanin Sm-TSP-2, a Vaccine Antigen from the Human Blood Fluke Schistosoma mansoni*

    PubMed Central

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

    2014-01-01

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

  16. Pre-transplant prognostic factors for patients with high-risk leukemia undergoing an unrelated cord blood transplantation

    Microsoft Academic Search

    A P Iori; R Cerretti; L De Felice; M Screnci; A Mengarelli; A Romano; M Caniglia; L Cerilli; G Gentile; M L Moleti; F Giona; F Agostini; I Pasqua; M P Perrone; M R Pinto; L Grapulin; A M Testi; P Martino; G De Rossi; F Mandelli; W Arcese

    2004-01-01

    From July 1995 to December 2001, 42 patients with leukemia aged 1–42 years underwent cord blood transplant (CBT) from unrelated, ?2 antigen HLA mismatched donors. In all, 26 patients were in ?2nd complete remission and 16 in more advanced phase. Conditioning regimens, graft-versus-host disease (GVHD) prophylaxis and supportive policy were uniform for all patients. The cumulative incidence of engraftment was

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

    PubMed Central

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

    1987-01-01

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

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

    Microsoft Academic Search

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

    2000-01-01

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

  19. Immunosuppression of in vivo and in vitro lymphocyte responses in swine induced by Trichinella spiralis or excretory-secretory antigens of the parasite.

    PubMed

    Gerencer, M; Marinculi?, A; Rapi?, D; Frankovi?, M; Valpoti?, I

    1992-10-01

    The in vivo and in vitro effects of Trichinella spiralis excretory-secretory (ES) antigens on porcine peripheral blood lymphocyte (PBL) responses induced with mitogens (phytohemagglutinin, PHA; concanavalin A, Con A; pokeweed mitogen, PWM) or unrelated antigen (Protein A) were studied to determine whether ES antigens depress lymphocyte responses in experimental swine trichinosis, and/or if this response was manifested after lymphocytes from infected pigs had been pretreated with ES antigens. Additionally, the range of inhibition of lymphocyte responses was tested in parasite-free pigs using different doses of ES antigens and compared with the responsiveness of control cultures from the same animals. The responses of lymphocytes from pigs inoculated with 4 x 10(3) muscle larvae (ML) were strongly depressed (P < 0.05) at post-inoculation days (PID) 7 (after stimulation with PHA), 14, 35 (Con A or PWM), and 49 (PWM). At PID 56 and 63 the lymphocytes from T. spiralis-infected pigs responded better (P < 0.05) to all three mitogens than those from non-infected controls. After 7 weeks post-inoculation, PBL which were pretreated with 10 or 250 micrograms ml-1 of ES antigens showed significantly weaker (P < 0.05, P < 0.001) responses to PWM or PHA, respectively, than those from non-infected animals. The responsiveness of lymphocytes from both groups of pigs to Protein A was not affected by the pretreatment with ES antigens in vitro. The responses of lymphocytes from the parasite-free pigs induced by PHA, PWM or Protein A were strongly depressed (P < 0.01) after in vitro pretreatment regardless of the dose of ES antigens (5, 10, 15, or 20 micrograms ml-1) applied. PMID:1466133

  20. Nonmyeloablative Unrelated Donor Hematopoietic Cell Transplantation for the Treatment of Patients with Poor-Risk, Relapsed or Refractory Multiple Myeloma

    PubMed Central

    Georges, George E.; Maris, Michael B.; Maloney, David G.; Sandmaier, Brenda M.; Sorror, Mohamed L.; Shizuru, Judith A.; Niederwieser, Dietger W.; Agura, Edward D.; Bruno, Benedetto; McSweeney, Peter A.; Pulsipher, Michael A.; Chauncey, Thomas R.; Mielcarek, Marco; Storer, Barry E.; Storb, Rainer

    2007-01-01

    The purpose of this study was to determine long-term outcome of unrelated donor nonmyeloablative hematopoietic cell transplantation in patients with poor-risk multiple myeloma. Twenty-four patients were enrolled. Seventeen patients (71%) had chemotherapy-refractory disease and 14 patients (58%) had disease relapse or progression after prior autologous transplantation. Thirteen patients underwent planned autologous transplantation followed 43–135 days later with unrelated transplantation, while 11 proceeded directly to unrelated transplantation. All 24 patients were treated with fludarabine (90 mg/m2) and 2 Gray total body irradiation and human leukocyte antigen (HLA)-matched unrelated peripheral blood stem cell transplantation. Postgrafting immunosuppression consisted of cyclosporine and mycophenolate mofetil. The median follow-up was 3 years after allografting. One patient experienced non-fatal graft rejection. The incidences of acute grades II, III and chronic graft-versus-host disease were 54%, 13% and 75%, respectively. The 3-year non-relapse mortality was 21%. Complete responses were observed in 10 patients (42%) and partial responses in 4 (17%). At 3 years, overall and progression-free survival rates were 61% and 33%, respectively. Patients receiving tandem autologous-unrelated transplantation had superior overall and progression-free survivals, 77% and 51%, compared to patients proceeding directly to unrelated donor transplantation, 44% and 11%, respectively (progression-free survival p-value, 0.03). In summary, for patients with poor-risk, relapsed or refractory multiple myeloma, cytoreductive autologous transplantation followed with nonmyeloablative conditioning and unrelated hematopoietic cell transplantation is effective treatment with low non-relapse mortality, high complete remission rates and prolonged disease-free survival. PMID:17287157

  1. Identification of two novel HSP90 proteins in Babesia orientalis: molecular characterization, and computational analyses of their structure, function, antigenicity and inhibitor interaction

    PubMed Central

    2014-01-01

    Background HSP90 protects the cells from heat stress and facilitates protein maturation and stability. The full genome sequences of piroplasms contain two putative HSP90 proteins, which are yet uncharacterized. To this end, the two putative HSP90 proteins of Babesia orientalis were identified and characterized by molecular and in silico methods. Methods The two putative proteins in B. orientalis genome showing homology with putative HSP90 of other piroplasms were cloned and sequenced. A computational analysis was carried out to predict the antigenic determinants, structure and function of these proteins. The interactions of two HSP90 isoforms with respective inhibitors were also examined through docking analysis. Results The length of BoHSP90-A gene (amplified from gDNA) was 2706 bp with one intron from position 997 to 1299 bp. This gene amplified from cDNA corresponded to full length CDS with an open reading frame (ORF) of 2403 bp encoding a 800 amino acid (AA) polypeptide with a predicted size of 91.02 kDa. The HSP90-B gene was intronless with an ORF of 2349 bp, and predicted polypeptide comprised of 797 AA with a size of 90.59 kDa. The AA sequences of these two proteins of B. orientalis were the most identical to those of B. bovis. The BoHSP90-A and BoHSP90-B were recognized as 90 kDa in the parasite lysate by the rabbit antisera raised against the recombinant BoHSP90 proteins. The anti-B. orientalis buffalo serum reacted with the rBoHSP90s expressed in E. coli, indicating that these proteins might be secreted by the parasite before entry into host cells. The overall structure and functional analyses showed several domains involved in ATPase activity, client protein binding and HSP90 dimerization. Likewise, several HSP90 inhibitors showed binding to ATP binding pockets of BoHSP90-A and BoHSP90-B, as observed through protein structure-ligand interaction analysis. Conclusions The two putative HSP90 proteins in B. orientalis were recognized as 90 kDa. The rBoHSP90-A and rBoHSP90-B were reacted with the B. orientalis infected buffalo serum. The computational structure and functional analyses revealed that these two proteins may have chaperonic activity. The protein structure-ligand interaction analyses indicated that these two proteins had many drug target sites. PMID:24970594

  2. Gene for proliferating-cell nuclear antigen (DNA polymerase delta auxiliary protein) is present in both mammalian and higher plant genomes.

    PubMed Central

    Suzuka, I; Daidoji, H; Matsuoka, M; Kadowaki, K; Takasaki, Y; Nakane, P K; Moriuchi, T

    1989-01-01

    Proliferating-cell nuclear antigen (PCNA; also called cyclin) was originally described in proliferating mammalian cells as a nuclear protein with an apparent Mr of 33,000-36,000 and recently was found to be a DNA polymerase delta auxiliary protein. To elucidate whether PCNA/cyclin is a universal protein necessary for proliferation of eukaryotes, a search was conducted for PCNA/cyclin homologues in higher plants. In Southern blot-hybridization analysis, a rat PCNA/cyclin cDNA probe hybridized with homologous sequences in genomic DNAs from rice, soybean, and tobacco. A PCNA/cyclin-related molecular clone (pCJ-1) was isolated from rice DNA and was partially sequenced. The pCJ-1 probe hybridized with a 1.2-kilobase transcript in RNA from rice root tips and shoots. Immunoblot analysis of the soluble extract of soybean root tips with monospecific anti-PCNA/cyclin identified an immunoreactive protein with an apparent Mr of 34,000. Immunohistochemical analysis revealed the presence of an immunoreactive PCNA/cyclin protein in the nuclei of cells in the meristem of soybean root tips. The highly homologous nature of the gene for PCNA/cyclin throughout the animal and plant kingdoms suggests that the product of the gene plays an essential role in DNA replication in eukaryotes. Images PMID:2566167

  3. Tandem Fusion of Hepatitis B Core Antigen Allows Assembly of Virus-Like Particles in Bacteria and Plants with Enhanced Capacity to Accommodate Foreign Proteins

    PubMed Central

    Peyret, Hadrien; Gehin, Annick; Thuenemann, Eva C.; Blond, Donatienne; El Turabi, Aadil; Beales, Lucy; Clarke, Dean; Gilbert, Robert J. C.; Fry, Elizabeth E.; Stuart, David I.; Holmes, Kris; Stonehouse, Nicola J.; Whelan, Mike; Rosenberg, William; Lomonossoff, George P.; Rowlands, David J.

    2015-01-01

    The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody. PMID:25830365

  4. The third subunit of protein phosphatase 2A (PP2A), a 55-kilodalton protein which is apparently substituted for by T antigens in complexes with the 36- and 63-kilodalton PP2A subunits, bears little resemblance to T antigens.

    PubMed Central

    Pallas, D C; Weller, W; Jaspers, S; Miller, T B; Lane, W S; Roberts, T M

    1992-01-01

    The small and middle T (tumor) antigens of polyomavirus have been shown previously to associate with the 36-kDa catalytic subunit and the 63-kDa regulatory subunit of protein phosphatase type 2A, apparently substituting for a normal third 55-kDa regulatory subunit (D.C. Pallas, L.K. Shahrik, B.L. Martin, S. Jaspers, T.B. Miller, D.L. Brautigan, and T.M. Roberts, Cell 60:167-176, 1990). To facilitate a comparison of the normal regulatory subunit and T antigens, we isolated a 2.14-kb cDNA clone encoding this 55-kDa subunit from a rat liver library. Using a probe from the coding region of this gene, we detected a major 2.4-kb mRNA transcript in liver and muscle RNAs. The 55-kDa protein phosphatase 2A subunit purified from rat skeletal muscle generates multiple species when analyzed on two-dimensional gels. Transcription and translation of the clone in vitro produced a full-length protein that comigrated precisely on two-dimensional gels with three of these species, indicating that the 55-kDa protein is apparently modified similarly in vivo and in reticulocyte lysates. Additional species in the purified preparation were not found in the translate, suggesting that there are probably two or more isoforms of this protein in rat muscle. Somewhat surprisingly, there was no clear homology with T-antigen amino acid sequences. Images PMID:1370560

  5. Virus-like particles from escherichia coli-derived untagged papaya ringspot virus capsid protein purified by immobilized metal affinity chromatography enhance the antibody response against a soluble antigen.

    PubMed

    Guerrero-Rodríguez, Jesús; Manuel-Cabrera, Carlos Alberto; Palomino-Hermosillo, Y Apatzingan; Delgado-Guzmán, Paola Guadalupe; Escoto-Delgadillo, Martha; Silva-Rosales, Laura; Herrera-Rodríguez, Sara Elisa; Sánchez-Hernández, Carla; Gutiérrez-Ortega, Abel

    2014-12-01

    There is a growing interest in using virus-like particles (VLPs) as scaffolds for the presentation of antigens of choice to the immune system. In this work, VLPs from papaya ringspot virus capsid protein expressed in Escherichia coli were evaluated as enhancers of antibody response against a soluble antigen. Interestingly, although the capsid protein lacks a histidine tag, its purification by immobilized metal affinity chromatography was achieved. The formation of VLPs was demonstrated by electron microscopy for the first time for this capsid protein. VLPs were enriched by polyethylene glycol precipitation. Additionally, these VLPs were chemically coupled to green fluorescent protein in order to evaluate them as antigen carriers; however, bioconjugate instability was observed. Nonetheless, the adjuvant effect of these VLPs on BALB/c mice was evaluated, using GFP as antigen, resulting in a significant increase in anti-GFP IgG response, particularly, IgG1 class, demonstrating that the VLPs enhance the immune response against the antigen chosen in this study. PMID:25119647

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

    PubMed Central

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

    2010-01-01

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

  7. Identification of a Meiosis-Specific Protein as a Member of the Class of Cancer\\/Testis Antigens

    Microsoft Academic Search

    Ozlem Tureci; Ugur Sahin; Carsten Zwick; Michael Koslowski; Gerhard Seitz; Michael Pfreundschuh

    1998-01-01

    Little is known about the function of human cancer\\/testis antigens (CTAs), such as MAGE, BAGE, GAGE, HOM-MEL-40, and NY-ESO-1, the expression of which is restricted to human malignancies and testis. When screening a cDNA expression library enriched for testis-specific representative long transcripts for reactivity with high-titered IgG antibodies from the serum of a patient with renal cell carcinoma, one repeatedly

  8. Identification of a Specific Antigenic Region of the P82 Protein of Babesia equi and Its Potential Use in Serodiagnosis

    Microsoft Academic Search

    Haruyuki Hirata; Xuenan Xuan; Naoaki Yokoyama; Yoshifumi Nishikawa; Kozo Fujisaki; Naoyoshi Suzuki; Ikuo Igarashi

    2003-01-01

    The efficacy of the Be82 gene product fused with glutathione S-transferase (GST\\/Be82) in an enzyme- linked immunosorbent assay (ELISA) for the diagnosis of Babesia equi infection was reported previously (H. Hirata et al., J. Clin. Microbiol. 40:1470-1474, 2002). However, the ELISA with the GST\\/Be82 antigen cross-reacted with Babesia caballi-infected horse sera, despite the high rate of detection of B. equi.

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

    Microsoft Academic Search

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

    2009-01-01

    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-medi- ated calcium ion (Ca2) influx occurs via highly selective Ca2 release-activated channels, and Ca2 regulatory molecule stromal interaction mol- ecule 1 plays an

  10. Evaluation of HLA matching in unrelated hematopoietic stem cell transplantation for nonmalignant disorders

    PubMed Central

    Wang, Tao; Haagenson, Michael; Spellman, Stephen R.; Dehn, Jason; Eapen, Mary; Frangoul, Haydar; Gupta, Vikas; Hale, Gregory A.; Hurley, Carolyn K.; Marino, Susana; Oudshoorn, Machteld; Reddy, Vijay; Shaw, Peter; Lee, Stephanie J.; Woolfrey, Ann

    2012-01-01

    The importance of human leukocyte antigen (HLA) matching in unrelated donor transplantation for nonmalignant diseases (NMD) has yet to be defined. We analyzed data from 663 unrelated marrow and peripheral blood stem cell transplants performed from 1995 to 2007 for treatment of NMD. Transplantation from a donor mismatched at the HLA-A, -B, -C, or -DRB1, but not -DQB1 or -DPB1, loci was associated with higher mortality in multivariate analyses (P = .002). The hazard ratio for mortality for single (7/8) and double mismatched (6/8) transplants was 1.29 (0.97-1.72; P = .079) and 1.82 (1.30-2.55; P = .0004), respectively, compared with 8/8 matched transplants. HLA mismatches were not associated with acute or chronic GVHD, but were strongly associated with graft failure. After adjustment for other factors, the odds ratio for graft failure for 7/8 and 6/8 (allele and/or antigen) matched pairs compared with 8/8 matched transplants was 2.81 (1.74-4.54; P < .0001) and 2.22 (1.26-3.97; P = .006), respectively. Patients with NMD should receive transplants from allele matched (8/8) donors if possible. Unlike the case with malignancies, HLA mismatching in NMD is associated with graft failure rather than GVHD. PMID:22829628

  11. Antigenic homogeneity of male Müllerian gland (MG) secretory proteins of a caecilian amphibian with secretory proteins of the mammalian prostate gland and seminal vesicles: evidence for role of the caecilian MG as a male accessory reproductive gland.

    PubMed

    Radha, Arumugam; Sree, Sreesha; Faisal, Kunnathodi; Kumar, G Pradeep; Oommen, Oommen V; Akbarsha, Mohammad A

    2014-10-01

    Whereas in all other vertebrates the Müllerian ducts of genetic males are aborted during development, under the influence of Müllerian-inhibiting substance, in the caecilian amphibians they are retained as a pair of functional glands. It has long been speculated that the Müllerian gland might be the male accessory reproductive gland but there has been no direct evidence to this effect. The present study was undertaken to determine whether the caecilian Müllerian gland secretory proteins would bear antigenic similarity to secretory proteins of the prostate gland and/or the seminal vesicles of a mammal. The secretory proteins of the Müllerian gland of Ichthyophis tricolor were evaluated for cross-reactivity with antisera raised against rat ventral prostate and seminal vesicle secretory proteins, adopting SDS-PAGE, two-dimensional electrophoresis and immunoblot techniques. Indeed there was a cross-reaction of five Müllerian gland secretory protein fractions with prostatic protein antiserum and of three with seminal vesicle protein antiserum. A potential homology exists because in mammals the middle group of the prostate primordia is derived from a diverticulum of the Müllerian duct. Thus this study, by providing evidence for expression of prostatic and seminal vesicle proteins in the Müllerian gland, substantiates the point that in caecilians the Müllerian glands are the male accessory reproductive glands. PMID:25160003

  12. Antigenic and immunogenic properties of recombinant hemagglutinin proteins from H1N1 A/Brisbane/59/07 and B/Florida/04/06 when produced in various protein expression systems

    PubMed Central

    Santiago, Felix W.; Emo, Kris Lambert; Fitzgerald, Theresa; Treanor, John J.

    2012-01-01

    Antibodies directed against the influenza hemagglutinin (HA) protein largely mediate virus neutralization and confer protection against infection. Consequently, many studies and assays of influenza vaccines are focused on HA-specific immune responses. Recombinant HA (rHA) proteins can be produced in a number of protein expression and cell culture systems. These range from baculovirus infection of insect cell cultures, to transient transfection of plants, to stably transfected human cell lines. Furthermore, the rHA proteins may contain genetic modifications, such as histidine tags or trimerization domains, intended to ease purification or enhance protein stability. However, no systematic study of these different forms of the HA protein have been conducted. It is not clear which, if any, of these different protein expression systems or structural modifications improve or diminish the biological behavior of the proteins as immunogens or antigens in immune assays. Therefore we set out to perform systematic evaluation of rHA produced in different proteins expression systems and with varied modifications. Five rHA proteins based on recent strains of seasonal influenza A and five based on influenza B HA were kindly provided by the Biodefense and Emerging Infections Reagent repository (BEIR). These proteins were evaluated in a combination of biochemical and structural assays, in vitro humoral and cellular immune assays, and in an animal vaccination model. Marked differences in the behavior of the individual proteins was evident suggesting that they are not equal when being used to detect an immune response. They were, nevertheless, similar at eliciting neutralizing antibody responses. PMID:22609035

  13. Leishmania donovani: identification of stimulatory soluble antigenic proteins using cured human and hamster lymphocytes for their prophylactic potential against visceral leishmaniasis.

    PubMed

    Garg, Ravendra; Gupta, Shraddha K; Tripathi, Parul; Hajela, K; Sundar, S; Naik, S; Dube, Anuradha

    2006-04-01

    Most of the studies for the identification of prophylactic antigens that elicit T cell responses were concentrated on membrane proteins of Leishmania donovani. This study was taken up to assess L. donovani soluble promastigote antigens for their ability to stimulate proliferation of peripheral blood mononuclear cells (PBMCs) from cured visceral leishmaniasis (VL) patients, endemic and non-endemic controls and lymphocytes/peritoneal macrophages of cured hamsters. The soluble protein was subjected to sequential precipitation with saturated ammonium sulphate (20%, 40%, 60% and 80%), of which largely 80% fractioned protein showed significant cellular responses in cured patients and hamsters. This fraction was further fractionated into five sub fractions by preparative SDS-PAGE and subjected to re-evaluation for their ability to induce cellular responses. Out of these, only F2 sub fraction belonging to the MW of 97.4-68 kDa stimulated remarkable lymphoproliferative and IFN-gamma responses in cured VL patients and in endemic controls. Similarly, significant lymphoproliferative responses and nitric oxide production were also noticed in cured Leishmania infected animals indicating an element of uniformity in responses between hamster and human. F2 sub fraction, when evaluated for its prophylactic efficacy with BCG against L. donovani challenge in hamster exhibited significant parasite inhibition in spleen (71.1%; p<0.001) and liver (68.2%; p<0.001) as compared to their unvaccinated counterpart. The vaccinated animals showed significant lymphoproliferative response and nitric oxide production but leishmania specific IgG level were suppressed. The results indicate the presence of immunostimulatory and protective molecules in F2 sub fraction which may further be exploited for the development of a vaccine against VL, hitherto an unrealized goal. PMID:16448729

  14. Oral immunization with recombinant Salmonella typhimurium expressing surface protein antigen A of Streptococcus sobrinus: persistence and induction of humoral responses in rats.

    PubMed Central

    Redman, T K; Harmon, C C; Michalek, S M

    1994-01-01

    Recombinant Salmonella typhimurium has been used as an oral vaccine for various microbial pathogens. Here we report immune responses in Fischer rats orally immunized with a recombinant S. typhimurium strain encoding surface protein antigen A (SpaA) of Streptococcus sobrinus. The attenuated S. typhimurium chi 4072 delta cya delta crp delta asd mutant used in this study contains the Asd+ plasmid pYA2905 expressing a fragment of the SpaA protein. Salmonella cells were cleared from spleens by 7 days and from Peyer's patches by 14 days in rats receiving a single oral immunization of 10(9) CFU of chi 4072. In animals receiving multiple (i.e., days 0 and 7 or days 0, 7, and 21) immunizations, Salmonella cells were cleared from the Peyer's patches by 25 days following the initial immunization. Antigen-specific systemic and mucosal antibody responses were greater in rats receiving multiple immunizations than in those receiving a single immunization. Serum anti-Salmonella activity was potentiated following boosting on day 21. Mucosal immunoglobulin A antibody responses were also greater in rats receiving multiple immunizations than in rats receiving a single immunization. Anti-Salmonella and anti-Streptococcus immunoglobulin A activity persisted longer in rats boosted on day 21 than in rats immunized on days 0 and 7. These data indicate that oral immunization of rats with the recombinant S. typhimurium chi 4072(pYA2905) vaccine induces systemic as well as mucosal antibody responses specific to the Salmonella cells and to the cloned SpaA protein. This is the first report of the use of an attenuated mutant of the murine pathogen S. typhimurium as an oral vaccine in rats. Images PMID:8039885

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

    Microsoft Academic Search

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

    2010-01-01

    Fasciola hepatica is responsible for human disease and economic livestock loss on a global scale. We report the first post-genomic investigation of cellular proteins expressed by embryonic F. hepatica via two-dimensional electrophoresis, image analysis and tandem mass spectrometry. Antioxidant proteins and protein chaperones are prominently expressed by embryonic F. hepatica. Molecular differences between the egg and other characterized F. hepatica

  16. Expression, purification, and characterization of protective MPT64 antigen protein and identification of its multimers isolated from nontoxic Mycobacterium tuberculosis H37Ra.

    PubMed

    Chu, Teng-Ping J; Yuann, Jeu-Ming P

    2011-05-01

    MPT64, a secreted protein of Mycobacterium tuberculosis (MTB), stimulates the immune reactions within cells and is a protective antigen that is lost by the bacilli Calmette-Guérin (BCG) vaccine during propagation. To minimize the toxicity caused by MTB, we used the MPT64 gene encoded by nontoxic H37Ra MTB to carry out genetic expansion via polymerase chain reaction and gene clone MPT64. The plasmid DNA encoded MPT64 was expressed at 20°C for 22 H, and a large quantity of MPT64 was obtained. In the absence of urea, MPT64 multimers with subunits being covalently connected via disulfide bonds were detected by Western blot showing strong protein-protein interactions, as evidenced by the formation of MPT64 tetramers. Finally, with urea of decreasing concentrations, we refolded MPT64 purified in the presence of urea and determined its secondary structures using circular dichroism. MPT64 was found to contain 2.2% ?-helix, 50.9% ?-sheet, 19.5% turn, and 27.4% random coil. The molecular weight of MPT64 was determined by a matrix-assisted laser desorption ionization-time of flight mass spectrometer and found to be 23,497 Da, very close to the theoretical molecular weight of MPT64. The results presented here provide a sound basis for future biochemical and biophysical studies of MPT64 or any other proteins encoded by nontoxic H37Ra MTB. PMID:21679242

  17. Sparse Seemingly Unrelated Regression Modelling: Applications in Econometrics and Finance

    E-print Network

    Wolpert, Robert L

    Sparse Seemingly Unrelated Regression Modelling: Applications in Econometrics and Finance By HAO) are frequently used in econometric, fi- nancial and sociological modelling (Zellner, 1962, 1971; Box & Tiao, 1973@stat.duke.edu Summary We present a sparse seemingly unrelated regression (SSUR) model to generate substan- tively

  18. Evaluation of a 14.5 kDa-Fasciola gigantica fatty acid binding protein as a diagnostic antigen for human fascioliasis.

    PubMed

    Allam, Gamal; Bauomy, Ibrahim R; Hemyeda, Zeinab M; Sakran, Thabet F

    2012-05-01

    The aim of the present study was to evaluate the efficiency of 14.5 kDa-Fasciola gigantica fatty acid binding protein (FABP) as a diagnostic antigen for human fascioliasis. 14.5 kDa FABP was isolated from the crude extract of adult F. gigantica worms by ion exchange chromatography followed by gel filtration chromatography and then analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing condition. Anti-FABP IgG polyclonal antibody (pAb) was generated in rabbits and purified by using sequential use of ammonium sulfate, caprylic acid, and then ion exchange chromatography. Conjugation of purified rabbit anti-FABP IgG with horse reddish peroxidase (HRP) was conducted and used in detecting the coproantigen in the stool and the circulating Fasciola antigen (CA) in the sera of Fasciola-infected patients using sandwich enzyme-linked immunosorbent assay (ELISA). The sensitivities of sandwich ELISA test were 96.43% and 94.74%, while the test specificities were 94.87% and 84.62% for the detection of coproantigen and CA, respectively. The parasitological diagnosis using the Kato-Katz technique revealed 64.29% sensitivity with 100% specificity. The diagnostic efficacy of sandwich ELISA was 95.52% for coproantigen and 87.93% for CA detection. In contrast, the diagnostic efficacy of Kato-Katz technique was 85.07%. It was concluded that 14.5 kDa FABP represented a valuable antigen for the immunodiagnosis of human fascioliasis using sandwich ELISA. PMID:22113304

  19. Major cytoplasmic membrane protein of Legionella pneumophila, a genus common antigen and member of the hsp 60 family of heat shock proteins, induces protective immunity in a guinea pig model of Legionnaires' disease.

    PubMed Central

    Blander, S J; Horwitz, M A

    1993-01-01

    We have examined the capacity of the major cytoplasmic membrane protein (MCMP) of Legionella pneumophila, a genus common antigen and member of the hsp 60 family of heat shock proteins, to induce protective immunity in a guinea pig model of Legionnaires' disease. We purified MCMP to homogeneity from L. pneumophila by buffer extraction, ion-exchange chromatography, and molecular sieve chromatography. Guinea pigs immunized with MCMP developed a strong cell-mediated immune response to the immunogen manifest by marked cutaneous delayed-type hypersensitivity. Guinea pigs immunized with MCMP and then challenged with a lethal aerosol dose of L. pneumophila exhibited a high level of protective immunity. Altogether, in four independent experiments, 55 of 64 (86%) animals immunized three times with 0.6-40 micrograms MCMP including 11 of 11 (100%) animals immunized three times with 40 micrograms MCMP survived aerosol challenge with L. pneumophila compared with 1 of 29 (3%) sham-immunized control animals (P < 0.0001, Cochran-Mantel-Haenszel X2 statistic for pooled data). To our knowledge, MCMP is the first member of the hsp 60 family of proteins shown to induce protective immunity to a microbial pathogen. MCMP has potential as a vaccine against Legionnaires' disease. Since MCMP is a genus common antigen, vaccination with a combination of MCMPs derived from different Legionella species has the potential of inducing protective immunity against all the major Legionella species causing human disease. Images PMID:8432872

  20. Exceptionally potent anti-tumor bystander activity of an scFv:sTRAIL fusion protein with specificity for EGP2 toward target antigen-negative tumor cells.

    PubMed

    Bremer, Edwin; Samplonius, Douwe; Kroesen, Bart-Jan; van Genne, Linda; de Leij, Lou; Helfrich, Wijnand

    2004-01-01

    Previously, we reported on the target cell-restricted fratricide apoptotic activity of scFvC54:sTRAIL, a fusion protein comprising human-soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) genetically linked to the antibody fragment scFvC54 specific for the cell surface target antigen EGP2. In the present study, we report that the selective binding of scFvC54:sTRAIL to EGP2-positive target cells conveys an exceptionally potent pro-apoptotic effect toward neighboring tumor cells that are devoid of EGP2 expression (bystander cells). The anti-tumor bystander activity of scFvC54:sTRAIL was detectable at target-to-bystander cell ratios as low as 1:100. Treatment in the presence of EGP2-blocking or TRAIL-neutralizing antibody strongly inhibited apoptosis in both target and bystander tumor cells. In the absence of target cells, bystander cell apoptosis induction was abrogated. The bystander apoptosis activity of scFvC54:sTRAIL did not require internalization, enzymatic conversion, diffusion, or communication (gap junctional intracellular communication) between target and bystander cells. Furthermore, scFvC54:sTRAIL showed no detectable signs of innocent bystander activity toward freshly isolated blood cells. Further development of this new principle is warranted for approaches where cancer cells can escape from antibody-based therapy due to partial loss of target antigen expression. PMID:15548373

  1. The 18-kDa cytoplasmic protein of Brucella species --an antigen useful for diagnosis--is a lumazine synthase.

    PubMed

    Goldbaum, F A; Velikovsky, C A; Baldi, P C; Mörtl, S; Bacher, A; Fossati, C A

    1999-09-01

    Previous studies have shown that the detection of antibodies to an 18-kDa cytoplasmic protein of Brucella spp. is useful for the diagnosis of human and animal brucellosis. This protein has now been expressed in recombinant form in Escherichia coli. The recombinant protein is soluble only under reducing conditions, but alkylation with iodoacetamide renders it soluble in non-reducing media. As shown by gel exclusion chromatography, this soluble form arranges in pentamers of 90 kDa. The reactivity of human and animal sera against the recombinant protein was similar to that found with the native protein present in brucella cytoplasmic fraction, suggesting that the recombinant protein is correctly folded. The protein has low but significant homology (30%) with lumazine synthases involved in bacterial riboflavin biosynthesis, which also arrange as pentamers. Biological tests on the crude extract of the recombinant bacteria and on the purified recombinant protein showed that the biological activity of the Brucella spp. 18-kDa protein is that of lumazine synthase. Preliminary crystallographic analysis showed that the Brucella spp. lumazine synthase arranges in icosahedric capsids similar to those formed by the lumazine synthases of other bacteria. The high immunogenicity of this protein, potentially useful for the design of acellular vaccines, could be explained by this polymeric arrangement. PMID:10482294

  2. Bioinformatic Analysis of Chlamydia trachomatis Polymorphic Membrane Proteins PmpE, PmpF, PmpG and PmpH as Potential Vaccine Antigens

    PubMed Central

    Nunes, Alexandra; Gomes, João P.; Karunakaran, Karuna P.; Brunham, Robert C.

    2015-01-01

    Chlamydia trachomatis is the most important infectious cause of infertility in women with important implications in public health and for which a vaccine is urgently needed. Recent immunoproteomic vaccine studies found that four polymorphic membrane proteins (PmpE, PmpF, PmpG and PmpH) are immunodominant, recognized by various MHC class II haplotypes and protective in mouse models. In the present study, we aimed to evaluate genetic and protein features of Pmps (focusing on the N-terminal 600 amino acids where MHC class II epitopes were mapped) in order to understand antigen variation that may emerge following vaccine induced immune selection. We used several bioinformatics platforms to study: i) Pmps’ phylogeny and genetic polymorphism; ii) the location and distribution of protein features (GGA(I, L)/FxxN motifs and cysteine residues) that may impact pathogen-host interactions and protein conformation; and iii) the existence of phase variation mechanisms that may impact Pmps’ expression. We used a well-characterized collection of 53 fully-sequenced strains that represent the C. trachomatis serovars associated with the three disease groups: ocular (N=8), epithelial-genital (N=25) and lymphogranuloma venereum (LGV) (N=20). We observed that PmpF and PmpE are highly polymorphic between LGV and epithelial-genital strains, and also within populations of the latter. We also found heterogeneous representation among strains for GGA(I, L)/FxxN motifs and cysteine residues, suggesting possible alterations in adhesion properties, tissue specificity and immunogenicity. PmpG and, to a lesser extent, PmpH revealed low polymorphism and high conservation of protein features among the genital strains (including the LGV group). Uniquely among the four Pmps, pmpG has regulatory sequences suggestive of phase variation. In aggregate, the results suggest that PmpG may be the lead vaccine candidate because of sequence conservation but may need to be paired with another protective antigen (like PmpH) in order to prevent immune selection of phase variants. PMID:26131720

  3. Release of mycobacterial antigens.

    PubMed

    Majlessi, Laleh; Prados-Rosales, Rafael; Casadevall, Arturo; Brosch, Roland

    2015-03-01

    Mycobacterium tuberculosis has evolved from a Mycobacterium canettii-like progenitor pool into one of the most successful and widespread human pathogens. The pathogenicity of M. tuberculosis is linked to its ability to secrete/export/release selected mycobacterial proteins, and it is also established that active release of mycobacterial antigens is a prerequisite for strong immune recognition. Recent research has enabled mycobacterial secretion systems and vesicle-based release of mycobacterial antigens to be elucidated, which together with host-related specificities constitute key variables that determine the outcome of infection. Here, we discuss recently discovered, novel aspects on the nature and the regulation of antigen release of the tuberculosis agent with particular emphasis on the biological characterization of mycobacteria-specific ESX/type VII secretion systems and their secreted proteins, belonging to the Esx, PE, and PPE categories. The importance of specific mycobacterial antigen release is probably best exemplified by the striking differences observed between the cellular events during infection with the ESX-1-deficient, attenuated Mycobacterium bovis BCG compared to the virulent M. tuberculosis, which are clearly important for design of more specific diagnostics and more efficient vaccines. PMID:25703550

  4. Size heterogeneity among antigenically related Giardia lamblia variant-specific surface proteins is due to differences in tandem repeat copy number.

    PubMed Central

    Mowatt, M R; Nguyen, B Y; Conrad, J T; Adam, R D; Nash, T E

    1994-01-01

    Giardia lamblia undergoes antigenic variation by modulating the expression of the different genes that comprise the trophozoite's variant-specific surface protein (VSP) repertoire. We studied an epitope that is conserved among VSPs expressed by cloned trophozoite lines derived from the independent G. lamblia isolates WB, G3M, Be-2, and CAT. The epitope recognized by monoclonal antibody 6E7 lies entirely within the region of tandemly repeated 65-amino-acid units that is characteristic of these size-variant VSPs. Northern (RNA) hybridization, cDNA cloning, and DNA sequence analysis indicate that size heterogeneity among these VSPs is due to differences in the number of repetitive units. Images PMID:7510666

  5. Goodpasture antigen-binding protein/ceramide transporter binds to human serum amyloid P-component and is present in brain amyloid plaques.

    PubMed

    Mencarelli, Chiara; Bode, Gerard H; Losen, Mario; Kulharia, Mahesh; Molenaar, Peter C; Veerhuis, Robert; Steinbusch, Harry W M; De Baets, Marc H; Nicolaes, Gerry A F; Martinez-Martinez, Pilar

    2012-04-27

    Serum amyloid P component (SAP) is a non-fibrillar glycoprotein belonging to the pentraxin family of the innate immune system. SAP is present in plasma, basement membranes, and amyloid deposits. This study demonstrates, for the first time, that the Goodpasture antigen-binding protein (GPBP) binds to human SAP. GPBP is a nonconventional Ser/Thr kinase for basement membrane type IV collagen. Also GPBP is found in plasma and in the extracellular matrix. In the present study, we demonstrate that GPBP specifically binds SAP in its physiological conformations, pentamers and decamers. The START domain in GPBP is important for this interaction. SAP and GPBP form complexes in blood and partly colocalize in amyloid plaques from Alzheimer disease patients. These data suggest the existence of complexes of SAP and GPBP under physiological and pathological conditions. These complexes are important for understanding basement membrane, blood physiology, and plaque formation in Alzheimer disease. PMID:22396542

  6. Identification of glutathione S-transferase omega 1 (GSTO1) protein as a novel tumor-associated antigen and its autoantibody in human esophageal squamous cell carcinoma.

    PubMed

    Li, Yang; Zhang, Qi; Peng, Bo; Shao, Qing; Qian, Wei; Zhang, Jian-Ying

    2014-11-01

    Esophageal squamous cell carcinoma (ESCC) is the main form of esophageal malignancy. The approach for early diagnosis of this malignancy is very limited. In the present study, we first evaluated glutathione S-transferase omega 1 (GSTO1), a protein related to metabolism, as a tumor-associated antigen in ESCC, and we also evaluated its autoantibody as a potential biomarker in early detection of ESCC. First, immunohistochemistry (IHC) analysis of GSTO1 protein expression in esophageal tissues showed that the percentage of positive staining of GSTO1 in ESCC tissues was 87.5% while there was no positive staining in adjacent tissues or normal tissues, indicating that overexpression of GSTO1 is closely related to ESCC. Then, enzyme-linked immunosorbent assay (ELISA) showed that the frequency of detectable autoantibody against GSTO1 in patients' sera totals 44.8%. In contrast, the frequency of detectable autoantibody was only 6.7% in normal human sera (p < 0.01). To further evaluate our ELISA results, western blotting and immunofluorescence assay were also performed. The results were consistent with the data from ELISA. In conclusion, the current study has demonstrated that GSTO1 protein is overexpressed in ESCC and can induce a detectable autoantibody response, which may serve as a potential biomarker in the early detection of ESCC. PMID:25085586

  7. Evaluation of the recombinant Heat shock protein B (HspB) of Coxiella burnetii as a potential antigen for immunodiagnostic of Q fever in goats.

    PubMed

    Fernandes, Isabelle; Rousset, Elodie; Dufour, Philippe; Sidi-Boumedine, Karim; Cupo, Anny; Thiéry, Richard; Duquesne, Véronique

    2009-03-01

    Coxiella burnetii is an intracellular bacterium that causes a worldwide zoonosis, the Q fever. Currently, to diagnose the infection in ruminants, whole cell antigens-based ELISAs are used. In this study a heat shock protein, the HspB, was evaluated for its ability to be recognized by the goat immune system and its capacity to sign a stage of infection. The htpB gene of C. burnetii was cloned and sequenced. A high identity (>90%) was observed among the htpB genes of four ruminant strains tested. A recombinant protein was expressed in a prokaryotic expression system. The rHspB protein was used to determine the IgG reactivity by ELISA. Sera from experimentally and naturally infected goats were tested. The rHspB is recognized early during the infection course, at 18 days post-infection. Moreover, 80-90% of the animals tested were positive at 39-60dpi. In addition, animals presenting a reactivation of the infection displayed a higher reactivity, statistically significant (p<0.05), than that of the animals in latent infection. These findings suggest that the rHspB could be a good candidate for the development of an ELISA test making possible the detection of recent C. burnetii infection in goats as well as reactivation in those with latent infection. PMID:18835111

  8. Reconstitution of B cell antigen receptor-induced signaling events in a nonlymphoid cell line by expressing the Syk protein-tyrosine kinase.

    PubMed

    Richards, J D; Gold, M R; Hourihane, S L; DeFranco, A L; Matsuuchi, L

    1996-03-15

    B cell antigen receptor (BCR) cross-linking activates both Src family and Syk tyrosine kinases, resulting in increased cellular protein-tyrosine phosphorylation and activation of several downstream signaling enzymes. To define the role of Syk in these events, we expressed the BCR in the AtT20 mouse pituitary cell line. These nonlymphoid cells endogenously expressed the Src family kinase Fyn but not Syk. Anti-IgM stimulation of these cells failed to induce most of the signaling events that occur in B cells. BCR-expressing AtT20 transfectants were generated that also expressed Syk. Syk expression reconstituted several signaling events upon anti-IgM stimulation, including Syk phosphorylation and association with the BCR, tyrosine phosphorylation of numerous proteins including Shc, and activation of mitogen-activated protein kinase. In contrast, Syk expression did not reconstitute anti-IgM-induced inositol phosphate production. A catalytically inactive Syk mutant could associate with the BCR and become tyrosine phosphorylated but could not reconstitute downstream signaling events. Expression of the Src family kinase Lck instead of Syk also did not reconstitute signaling. Thus, wild type Syk was required to reconstitute several BCR-induced signaling events but was not sufficient to couple the BCR to the phosphoinositide signaling pathway. PMID:8626447

  9. Antigenic structure of sperm whale myoglobin. I. Partition of specificities between antibodies reactive with peptides and native protein.

    PubMed

    Lando, G; Berzofsky, J A; Reichlin, M

    1982-07-01

    We have resolved anti-sperm whale Mb antibodies into two distinct populations: One population is reactive only with the native molecule, and the second is reaction with both Mb and peptide fragments. CNBr cleavage of sperm whale Mb yields three peptides: peptide I (1-55), peptide II (56-131), and peptide III (132-153). Immunoadsorbent columns made with these three peptides were used to fractionate antibody to sperm whale Mb. These columns cumulatively bind 60 to 70% of the total antibody present in the four sera studied (the amount of antibody bound by each peptide was proportional to its size). The remaining 30 to 40% of antibody could only bind specifically to an immunoadsorbent column of native Mb. This result indicates that not all antigenic determinants on the native Mb molecule are represented on peptide fragments. PMID:7086130

  10. Partial or total replacement of fish meal by soybean protein on growth, protein utilization, potential estrogenic or antigenic effects, cholesterolemia and flesh quality in rainbow trout, Oncorhynchus mykiss

    Microsoft Academic Search

    S. J. Kaushik; J. P. Cravedi; J. P. Lalles; J. Sumpter; B. Fauconneau; M. Laroche

    1995-01-01

    Groups of rainbow trout (initial body weight 83 ± 1 g) were fed diets (crude protein (CP) 46%; gross energy 21 kJ\\/g DM; crude fat 12%) containing graded levels of either a soyflour (CP 52% DM) or a soy protein concentrate (CP 72% DM) supplemented with L-methionine as partial or total replacement of fish meal protein. A growth trial was

  11. Tumor escape from immune recognition: lethal recurrent melanoma in a patient associated with downregulation of the peptide transporter protein TAP-1 and loss of expression of the immunodominant MART-1/Melan-A antigen.

    PubMed Central

    Maeurer, M J; Gollin, S M; Martin, D; Swaney, W; Bryant, J; Castelli, C; Robbins, P; Parmiani, G; Storkus, W J; Lotze, M T

    1996-01-01

    In the last few years, mutiple protein target antigens for immunorecognition by T cells have been identified on human melanoma. How melanoma lesions escape from functional antigen-specific immune recognition remains poorly understood. We have identified the concomitant loss of the immunodominant T cell-defined MART-1/Melan-A antigen and downregulation of the TAP-1 gene in a recurrent metastatic melanoma that was resected in 1993. This phenotype was not observed for an earlier autologous melanoma lesion resected in 1987. The "antigen loss" could be restored in the variant tumor cell line by simultaneously providing both the MART-1/Melan-A gene (by retroviral transfer) and the TAP-1 gene (by a bioballistic approach) resulting in tumor cell sensitivity to MART-1/Melan-A-specific cytotoxic T lymphocytes. This suggests that tumor escape from immune surveillance may have occurred in vivo as a sequential result of (a) antigen loss, and (b) downregulation of the peptide-transporter protein TAP-1 expression by this patient's tumor over a 6-yr period from 1987 to 1993. These results suggest that the characterization of the T cell response to melanoma in individual patients and definition of the immunologically relevant genetic defects in tumors may be required to select the most effective therapeutic strategies for a given patient. PMID:8833913

  12. A unique secreted adenovirus E3 protein binds to the leukocyte common antigen CD45 and modulates leukocyte functions.

    PubMed

    Windheim, Mark; Southcombe, Jennifer H; Kremmer, Elisabeth; Chaplin, Lucy; Urlaub, Doris; Falk, Christine S; Claus, Maren; Mihm, Janine; Braithwaite, Myles; Dennehy, Kevin; Renz, Harald; Sester, Martina; Watzl, Carsten; Burgert, Hans-Gerhard

    2013-12-10

    The E3 transcription unit of human adenoviruses (Ads) encodes immunomodulatory proteins. Interestingly, the size and composition of the E3 region differs considerably among Ad species, suggesting that distinct sets of immunomodulatory E3 proteins may influence their interaction with the human host and the disease pattern. However, to date, only common immune evasion functions of species C E3 proteins have been described. Here we report on the immunomodulatory activity of a species D-specific E3 protein, E3/49K. Unlike all other E3 proteins that act on infected cells, E3/49K seems to target uninfected cells. Initially synthesized as an 80- to 100-kDa type I transmembrane protein, E3/49K is subsequently cleaved, with the large ectodomain (sec49K) secreted. We found that purified sec49K exhibits specific binding to lymphoid cell lines and all primary leukocytes, but not to fibroblasts or epithelial cells. Consistent with this binding profile and the molecular mass, the sec49K receptor was identified as the cell surface protein tyrosine phosphatase CD45. Antibody-blocking studies suggested that sec49K binds to the membrane proximal domains present in all CD45 isoforms. Functional studies showed that sec49K can suppress the activation and cytotoxicity of natural killer cells as well as the activation, signaling, and cytokine production of T cells. Thus, we have discovered an adenovirus protein that is actively secreted and describe immunomodulatory activities of an E3 protein uniquely expressed by a single Ad species. PMID:24218549

  13. A unique secreted adenovirus E3 protein binds to the leukocyte common antigen CD45 and modulates leukocyte functions

    PubMed Central

    Windheim, Mark; Southcombe, Jennifer H.; Kremmer, Elisabeth; Chaplin, Lucy; Urlaub, Doris; Falk, Christine S.; Claus, Maren; Mihm, Janine; Braithwaite, Myles; Dennehy, Kevin; Renz, Harald; Sester, Martina; Watzl, Carsten; Burgert, Hans-Gerhard

    2013-01-01

    The E3 transcription unit of human adenoviruses (Ads) encodes immunomodulatory proteins. Interestingly, the size and composition of the E3 region differs considerably among Ad species, suggesting that distinct sets of immunomodulatory E3 proteins may influence their interaction with the human host and the disease pattern. However, to date, only common immune evasion functions of species C E3 proteins have been described. Here we report on the immunomodulatory activity of a species D-specific E3 protein, E3/49K. Unlike all other E3 proteins that act on infected cells, E3/49K seems to target uninfected cells. Initially synthesized as an 80- to 100-kDa type I transmembrane protein, E3/49K is subsequently cleaved, with the large ectodomain (sec49K) secreted. We found that purified sec49K exhibits specific binding to lymphoid cell lines and all primary leukocytes, but not to fibroblasts or epithelial cells. Consistent with this binding profile and the molecular mass, the sec49K receptor was identified as the cell surface protein tyrosine phosphatase CD45. Antibody-blocking studies suggested that sec49K binds to the membrane proximal domains present in all CD45 isoforms. Functional studies showed that sec49K can suppress the activation and cytotoxicity of natural killer cells as well as the activation, signaling, and cytokine production of T cells. Thus, we have discovered an adenovirus protein that is actively secreted and describe immunomodulatory activities of an E3 protein uniquely expressed by a single Ad species. PMID:24218549

  14. Proteolysis, proteasomes and antigen presentation

    NASA Technical Reports Server (NTRS)

    Goldberg, A. L.; Rock, K. L.

    1992-01-01

    Proteins presented to the immune system must first be cleaved to small peptides by intracellular proteinases. Proteasomes are proteolytic complexes that degrade cytosolic and nuclear proteins. These particles have been implicated in ATP-ubiquitin-dependent proteolysis and in the processing of intracellular antigens for cytolytic immune responses.

  15. Mycobacterium tuberculosis Rv0679c protein sequences involved in host-cell infection: Potential TB vaccine candidate antigen

    Microsoft Academic Search

    Diana P Cifuentes; Marisol Ocampo; Hernando Curtidor; Magnolia Vanegas; Martha Forero; Manuel E Patarroyo; Manuel A Patarroyo

    2010-01-01

    BACKGROUND: To date, the function of many hypothetical membrane proteins of Mycobacterium tuberculosis is still unknown and their involvement in pathogen-host interactions has not been yet clearly defined. In this study, the biological activity of peptides derived from the hypothetical membrane protein Rv0679c of M. tuberculosis and their involvement in pathogen-host interactions was assessed. Transcription of the Rv0679c gene was

  16. Role of the Antigenic Loop of the Hepatitis B Virus Envelope Proteins in Infectivity of Hepatitis Delta Virus

    Microsoft Academic Search

    Georges Abou Jaoude ´; Camille Sureau

    2005-01-01

    The infectious particles of hepatitis B virus (HBV) and hepatitis delta virus (HDV) are coated with the large, middle, and small envelope proteins encoded by HBV. While it is clear that the N-terminal pre-S1 domain of the large protein, which is exposed at the virion surface, is implicated in binding to a cellular receptor at viral entry, the role in

  17. Epstein-Barr virus latent antigen 3C can mediate the degradation of the retinoblastoma protein through an SCF cellular ubiquitin ligase.

    PubMed

    Knight, Jason S; Sharma, Nikhil; Robertson, Erle S

    2005-12-20

    Epstein-Barr virus (EBV) stimulates the proliferation of latently infected B cells and promotes lymphoid malignancies in humans. To address the role of EBV latency protein Epstein-Barr nuclear antigen 3C (EBNA3C) in regulation of the retinoblastoma protein (Rb), we transfected EBNA3C into 293, BJAB, and SAOS-2 cells. In this context, a dominant effect of EBNA3C is to decrease Rb protein levels. EBNA3C also rescues an Rb-induced flat cell phenotype and targets Rb for proteasome- and ubiquitin-dependent degradation. Further, EBNA3C forms a stable complex with Rb in cells when the proteasome machinery is inhibited and interacts with Rb in vitro, mapping to a conserved domain at the terminus of EBNA3C. Deletion analysis of EBNA3C identified a motif within amino acids 140-149 important for both the binding and regulation of Rb. This motif is of particular interest, because it has also been linked to regulation of the Skp1/Cul1/F-box complex, SCF(Skp2). Indeed, inhibition of Skp2 function with a dominant-negative molecule reduces the ability of EBNA3C to degrade Rb. Skp2 has no detectable effect on Rb levels in the absence of EBNA3C, suggesting that SCF(Skp2) is specifically usurped by EBNA3C for the enhancement of Rb degradation. That EBNA3C has exploited this association suggests that other human malignancies might use a similar strategy to regulate the Rb protein. PMID:16352731

  18. Sensitivity of immune response quality to influenza helix 190 antigen structure displayed on a modular virus-like particle.

    PubMed

    Anggraeni, Melisa R; Connors, Natalie K; Wu, Yang; Chuan, Yap P; Lua, Linda H L; Middelberg, Anton P J

    2013-09-13

    Biomolecular engineering enables synthesis of improved proteins through synergistic fusion of modules from unrelated biomolecules. Modularization of peptide antigen from an unrelated pathogen for presentation on a modular virus-like particle (VLP) represents a new and promising approach to synthesize safe and efficacious vaccines. Addressing a key knowledge gap in modular VLP engineering, this study investigates the underlying fundamentals affecting the ability of induced antibodies to recognize the native pathogen. Specifically, this quality of immune response is correlated to the peptide antigen module structure. We modularized a helical peptide antigen element, helix 190 (H190) from the influenza hemagglutinin (HA) receptor binding region, for presentation on murine polyomavirus VLP, using two strategies aimed to promote H190 helicity on the VLP. In the first strategy, H190 was flanked by GCN4 structure-promoting elements within the antigen module; in the second, dual H190 copies were arrayed as tandem repeats in the module. Molecular dynamics simulation predicted that tandem repeat arraying would minimize secondary structural deviation of modularized H190 from its native conformation. In vivo testing supported this finding, showing that although both modularization strategies conferred high H190-specific immunogenicity, tandem repeat arraying of H190 led to a strikingly higher immune response quality, as measured by ability to generate antibodies recognizing a recombinant HA domain and split influenza virion. These findings provide new insights into the rational engineering of VLP vaccines, and could ultimately enable safe and efficacious vaccine design as an alternative to conventional approaches necessitating pathogen cultivation. PMID:23845811

  19. Small interference RNA-mediated knockdown of sperm associated antigen 9 having structural homology with c-Jun N-terminal kinase-interacting protein

    SciTech Connect

    Rana, Ritu [Genes and Proteins Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, J.N.U. Campus, New Delhi-110 067 (India); Jagadish, Nirmala [Genes and Proteins Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, J.N.U. Campus, New Delhi-110 067 (India); Garg, Manoj [Genes and Proteins Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, J.N.U. Campus, New Delhi-110 067 (India); Mishra, Deepshikha [Genes and Proteins Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, J.N.U. Campus, New Delhi-110 067 (India); Dahiya, Neetu [Genes and Proteins Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, J.N.U. Campus, New Delhi-110 067 (India); Chaurasiya, Dipak [Genes and Proteins Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, J.N.U. Campus, New Delhi-110 067 (India); Suri, Anil [Genes and Proteins Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, J.N.U. Campus, New Delhi-110 067 (India)]. E-mail: anil@nii.res.in

    2006-02-03

    Recently, we reported a novel testis-specific sperm associated antigen 9 (SPAG9) protein, a new member of the JNK-interacting protein family, having a functional role in sperm-egg fusion [N. Jagadish, R. Rana, R. Selvi, D. Mishra, M. Garg, S. Yadav, J.C. Herr, K. Okumura, A. Hasegawa, K. Koyama, A. Suri, Biochem. J. 389 (2005) 73-82]. NCBI Blast searches revealed SPAG9 nucleotide sequence similarities with ESTs of various cancerous tissues. In the present study, we compared the efficiency of two independent SPAG9 specific small interfering RNA (siRNA) constructs, BS/U6/spag9 and BS/U6/spag9-I, to ablate the SPAG9 expression in mammalian cells. A positive correlation between the ratio of target gene versus siRNA and the suppression of SPAG9 expression was observed. Further, the cotransfection of BS/U6/spag9 with pcDNA-SPAG9 and pFlag-CMV2-JNK-3 resulted in specific suppression of SPAG9 without affecting JNK-3 expression. The present investigation will eventually extend the application of SPAG9 siRNA in in vivo targeting experiments that aim to define the SPAG9 functional genomics in tumor and reproductive biology.

  20. Epitope mapping and evaluation of specificity of T-helper sites in four major antigenic peptides of chicken riboflavin carrier protein in outbred rats.

    PubMed

    Subramanian, Sarada; Andal, S; Karande, Anjali A; Radhakantha Adiga, P

    2003-11-01

    This paper reviews our studies on synthetic peptides spanning the major antigenic determinants of the chicken riboflavin carrier protein (RCP; 219 AA). These determinants are composed of residues 4-24 (YGC), 64-83 (CED), 130-147 (GEN), and 200-219 (HAC) and function as minivaccines in terms of eliciting anti-peptide antibodies which recognize the native protein and are particularly promising contraceptive vaccine candidates. We have used 15-residue synthetic peptides to define short sequences involved in interaction with antibody and with T-cells. We have mapped the boundaries of T-cell epitopes of these peptides in outbred rats by immunizing the animals with each peptide and assaying the popliteal lymph node cell proliferation against a series of overlapping synthetic 15-mers covering the entire length of the individual peptides. The peptides YGC, GEN, and HAC harboured a single T-cell epitope each whereas the peptide CED exhibited bimodal response possessing two epitopes, one at N-terminus and the other at the C-terminus. These studies provide insight into the way in which an immunogen is viewed by the immune system. In addition, preferential T-cell helper function for B cells recognizing unique determinants on the same molecule was demonstrated. This information helps in exploiting synthetic peptides in the construction of designer immunogens which have potential as candidate vaccines. PMID:14575688

  1. The Epstein-Barr virus nuclear antigen 2 interacts with an EBNA2 responsive cis-element of the terminal protein 1 gene promoter.

    PubMed Central

    Zimber-Strobl, U; Kremmer, E; Grässer, F; Marschall, G; Laux, G; Bornkamm, G W

    1993-01-01

    The Epstein-Barr virus protein EBNA2 acts as a transcriptional activator of cellular and viral genes and plays a crucial role in the immortalization of human primary B-cells by EBV. We have shown previously that EBNA2 transactivates the promoters of the latent membrane antigens LMP, TP1 and TP2. The promoter of the TP1 gene was chosen as a model system to study the molecular mechanism of EBNA2 mediated transactivation. To identify an EBNA2 dependent cis-acting element, various TP1 promoter-reporter gene constructs were transfected in the absence and presence of an EBNA2 expression vector into the established B-cell line BL41-P3HR1. We were able to delineate an 81 bp EBNA2 responsive region between -258 and -177 relative to the TP1 RNA start site. The element worked in either orientation and could mediate EBNA2 dependent transactivation on a heterologous promoter. Electrophoretic mobility shift assays revealed three specific protein-DNA complexes formed with sequences of the EBNA2 responsive element. Two of these were not cell type specific, but the third was detected only in EBNA2 positive cell extracts. Gel-shift analysis in the presence of EBNA2 specific monoclonal antibodies revealed that EBNA2 is a component of the third complex. Thus, these experiments demonstrate that EBNA2 interacts with an EBNA2 responsive cis-element of the TP1 promoter. Images PMID:8381349

  2. Dual role of the tyrosine activation motif of the Ig-alpha protein during signal transduction via the B cell antigen receptor.

    PubMed Central

    Flaswinkel, H; Reth, M

    1994-01-01

    The B cell antigen receptor (BCR) is a multimeric protein complex consisting of the ligand binding immunoglobulin molecule and the Ig-alpha/beta heterodimer that mediates intracellular signalling by coupling the receptor to protein tyrosine kinases (PTKs). Transfection of the Ig-alpha deficient myeloma cell line J558L microns with expression vectors coding for mutated Ig-alpha allowed us to test the function of the tyrosines in the cytoplasmic region of Ig-alpha in the context of the BCR. Furthermore we expressed Ig-alpha mutations as chimeric CD8-Ig-alpha molecules on K46 B lymphoma cells and tested their signalling capacity in terms of PTK activation and release of calcium. We show here that the conserved tyrosine residues in the cytoplasmic portion of Ig-alpha have a dual role. First, they are required for efficient activation of PTKs during signal induction and second, one of them is subject to phosphorylation by activated src-related PTKs. Phosphorylation on tyrosine in the cytoplasmic portion of Ig-alpha is discussed as a possible mechanism to couple the BCR to SH2 domain-carrying molecules. Images PMID:8306975

  3. Antigen presentation subverted: Structure of the human cytomegalovirus protein US2 bound to the class I molecule HLA-A2.

    PubMed

    Gewurz, B E; Gaudet, R; Tortorella, D; Wang, E W; Ploegh, H L; Wiley, D C

    2001-06-01

    Many persistent viruses have evolved the ability to subvert MHC class I antigen presentation. Indeed, human cytomegalovirus (HCMV) encodes at least four proteins that down-regulate cell-surface expression of class I. The HCMV unique short (US)2 glycoprotein binds newly synthesized class I molecules within the endoplasmic reticulum (ER) and subsequently targets them for proteasomal degradation. We report the crystal structure of US2 bound to the HLA-A2/Tax peptide complex. US2 associates with HLA-A2 at the junction of the peptide-binding region and the alpha3 domain, a novel binding surface on class I that allows US2 to bind independently of peptide sequence. Mutation of class I heavy chains confirms the importance of this binding site in vivo. Available data on class I-ER chaperone interactions indicate that chaperones would not impede US2 binding. Unexpectedly, the US2 ER-luminal domain forms an Ig-like fold. A US2 structure-based sequence alignment reveals that seven HCMV proteins, at least three of which function in immune evasion, share the same fold as US2. The structure allows design of further experiments to determine how US2 targets class I molecules for degradation. PMID:11391001

  4. Oral immunization with recombinant Salmonella typhimurium expressing surface protein antigen A of Streptococcus sobrinus: dose response and induction of protective humoral responses in rats.

    PubMed Central

    Redman, T K; Harmon, C C; Lallone, R L; Michalek, S M

    1995-01-01

    An attenuated, recombinant Salmonella typhimurium mutant, chi 4072(pYA2905), expressing the surface protein antigen A (SpaA) of Streptococcus sobrinus was investigated for its effectiveness in inducing protective immune responses against S. sobrinus-induced dental caries in an experimental caries model. Fischer rats were orally immunized with either 10(8) or 10(9) CFU of S. typhimurium chi 4072(pYA2905). Persistence of salmonellae in Peyer's patches and spleens and the induction of immune responses were determined. Maximum numbers of salmonellae were recovered from Peyer's patches of rats within the first week of immunization, with higher numbers recovered from rats given 10(9) CFU than from those given 10(8) CFU. Serum anti-Salmonella and anti-SpaA responses increased more rapidly in rats given 10(9) CFU than in rats given 10(8) CFU. The salivary antibody response to SpaA increased with time, but the response varied in the two groups. In a separate study, rats were orally immunized with the recombinant Salmonella mutant and then challenged with cariogenic S. sobrinus 6715. The levels of serum and salivary antibody and caries activity were assessed at the termination of the experiment. Higher levels of salivary immunoglobulin A antibody to SpaA and Salmonella carrier were detected in rats given 10(9) CFU than in those given 10(8) CFU, and these responses were higher than those in nonimmunized controls. Mandibular molars from immunized rats had lower numbers of recoverable streptococci and less extensive carious lesions than those from nonimmunized, control rats. These data indicate that oral immunization with an attenuated recombinant S. typhimurium expressing SpaA of S. sobrinus induces the production of antigen-specific mucosal antibody and confers protection against dental caries. PMID:7729915

  5. In vitro binding of anthrax protective antigen on bacteriophage T4 capsid surface through Hoc-capsid interactions: A strategy for efficient display of large full-length proteins

    SciTech Connect

    Shivachandra, Sathish B. [Department of Biology, 103 McCort Ward Hall, Catholic University of America, 620 Michigan Ave., NE, Washington, DC 20064 (United States); Rao, Mangala [Division of Retrovirology, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910 (United States); Janosi, Laszlo [Division of Retrovirology, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910 (United States); Sathaliyawala, Taheri [Department of Biology, 103 McCort Ward Hall, Catholic University of America, 620 Michigan Ave., NE, Washington, DC 20064 (United States); Matyas, Gary R. [Division of Retrovirology, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910 (United States); Alving, Carl R. [Division of Retrovirology, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910 (United States); Leppla, Stephen H. [Bacterial Toxins and Therapeutics Section, National Institute of Allergy and Infectious Diseases, NIH, 30 Convent Dr., Bethesda, MD 20892 (United States); Rao, Venigalla B. [Department of Biology, 103 McCort Ward Hall, Catholic University of America, 620 Michigan Ave., NE, Washington, DC 20064 (United States)]. E-mail: rao@cua.edu

    2006-02-05

    An in vitro binding system is described to display large full-length proteins on bacteriophage T4 capsid surface at high density. The phage T4 icosahedral capsid features 155 copies of a nonessential highly antigenic outer capsid protein, Hoc, at the center of each major capsid protein hexon. Gene fusions were engineered to express the 83-kDa protective antigen (PA) from Bacillus anthracis fused to the N-terminus of Hoc and the 130-kDa PA-Hoc protein was expressed in Escherichia coli and purified. The purified PA-Hoc was assembled in vitro on hoc {sup -} phage particles. Binding was specific, stable, and of high affinity. This defined in vitro system allowed manipulation of the copy number of displayed PA and imposed no significant limitation on the size of the displayed antigen. In contrast to in vivo display systems, the in vitro approach allows all the capsid binding sites to be occupied by the 130-kDa PA-Hoc fusion protein. The PA-T4 particles were immunogenic in mice in the absence of an adjuvant, eliciting strong PA-specific antibodies and anthrax lethal toxin neutralizing antibodies. The in vitro display on phage T4 offers a novel platform for potential construction of customized vaccines against anthrax and other infectious diseases.

  6. Chemical Composition of Select Pecan [Carya illinoinensis (Wangenh.) K. Koch]Varieties and Antigenic Stability of Pecan Proteins

    Microsoft Academic Search

    Mahesh Venkatachalam

    2004-01-01

    Chemical composition of 25 pecan varieties revealed considerable differences in moisture (2.1-6.4%), protein (6.0-11.3%), lipid (65.9-78.0%), total soluble sugars (3.3-5.3%), ash (1.2-1.8%) and tannins (0.7-2.7%) when analyzed on an edible portion basis. Pecan varieties had similar protein polypeptide profiles as revealed by SDS-PAGE, IEF, and pecan pAb based Western blotting analysis.\\u000aBoth pH and ionic strength were important for pecan

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

    Microsoft Academic Search

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

    1993-01-01

    Summary The amino acid sequence at the F2\\/F1 cleavage site of the F0 fusion protein of 17 strains of Newcastle disease virus (NDV) was deduced from sequencing a 32 nucleotide area of the genome by reverse transcription and polymerase chain reaction (PCR) techniques. With the addition of sequences at the same area previously published for 9 other viruses comparisons were

  8. Nonpermissive HLA-DPB1 mismatch increases mortality after myeloablative unrelated allogeneic hematopoietic cell transplantation.

    PubMed

    Pidala, Joseph; Lee, Stephanie J; Ahn, Kwang Woo; Spellman, Stephen; Wang, Hai-Lin; Aljurf, Mahmoud; Askar, Medhat; Dehn, Jason; Fernandez Viña, Marcelo; Gratwohl, Alois; Gupta, Vikas; Hanna, Rabi; Horowitz, Mary M; Hurley, Carolyn K; Inamoto, Yoshihiro; Kassim, Adetola A; Nishihori, Taiga; Mueller, Carlheinz; Oudshoorn, Machteld; Petersdorf, Effie W; Prasad, Vinod; Robinson, James; Saber, Wael; Schultz, Kirk R; Shaw, Bronwen; Storek, Jan; Wood, William A; Woolfrey, Ann E; Anasetti, Claudio

    2014-10-16

    We examined current outcomes of unrelated donor allogeneic hematopoietic cell transplantation (HCT) to determine the clinical implications of donor-recipient HLA matching. Adult and pediatric patients who had first undergone myeloablative-unrelated bone marrow or peripheral blood HCT for acute myelogenous leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, and myelodysplastic syndrome between 1999 and 2011 were included. All had high-resolution typing for HLA-A, -B, -C, and -DRB1. Of the total (n = 8003), cases were 8/8 (n = 5449), 7/8 (n = 2071), or 6/8 (n = 483) matched. HLA mismatch (6-7/8) conferred significantly increased risk for grades II to IV and III to IV acute graft vs host disease (GVHD), chronic GVHD, transplant-related mortality (TRM), and overall mortality compared with HLA-matched cases (8/8). Type (allele/antigen) and locus (HLA-A, -B, -C, and -DRB1) of mismatch were not associated with overall mortality. Among 8/8 matched cases, HLA-DPB1 and -DQB1 mismatch resulted in increased acute GVHD, and HLA-DPB1 mismatch had decreased relapse. Nonpermissive HLA-DPB1 allele mismatch was associated with higher TRM compared with permissive HLA-DPB1 mismatch or HLA-DPB1 match and increased overall mortality compared with permissive HLA-DPB1 mismatch in 8/8 (and 10/10) matched cases. Full matching at HLA-A, -B, -C, and -DRB1 is required for optimal unrelated donor HCT survival, and avoidance of nonpermissive HLA-DPB1 mismatches in otherwise HLA-matched pairs is indicated. PMID:25161269

  9. Unrelated donor umbilical cord blood transplantation for the treatment of hematologic malignancies

    PubMed Central

    Sauter, Craig; Barker, Juliet N.

    2013-01-01

    Purpose of review This review summarizes the state of the art of unrelated donor (URD) umbilical cord blood transplantation (UCBT) for the treatment of hematologic malignancies and discusses the current issues associated with the use of this hematopoietic stem cell (HSC) source. Recent findings In contrast to the very high transplant-related mortality associated with the early experience of UCBT, recent series have been associated with comparable survival to that of human leucocyte antigen-matched URD transplantation in children with similarly promising results in adults with the use of double-unit grafts. In addition, utilization of reduced-intensity conditioning regimens has been successful extending access to patients unsuitable for myeloablation. Consequently, the use of umbilical cord blood as a HSC source and the global inventory of units in public banks is rapidly increasing although challenges associated with engraftment, unit quality, and infectious complications remain and will be discussed in this review. Summary URD umbilical cord blood is an alternative HSC source offering a unique set of advantages and disadvantages as compared with the transplantation of HSC from unrelated volunteers. Improved transplant outcomes are now making UCBT a rival to URD transplantation for the treatment of hematologic malignancies. PMID:18832927

  10. Evaluation of the immune response induced by a nasal anthrax vaccine based on the protective antigen protein in anaesthetized and non-anaesthetized mice.

    PubMed

    Sloat, Brian R; Cui, Zhengrong

    2006-04-01

    To better protect against inhalational anthrax infection, a nasal anthrax vaccine based on the protective antigen (PA) protein of Bacillus anthracis could be an attractive alternative to the current Anthrax-Vaccine-Adsorbed (AVA), which was licensed for cutaneous anthrax prevention. Previously, we have demonstrated that an anti-PA immune response comparable with that in mice subcutaneously immunized with PA protein adjuvanted with aluminium hydroxide was induced in both the systemic compartment and the mucosal secretions of the nose and lung of anaesthetized mice when they were nasally immunized with PA protein incorporated into previously reported LPD (Liposome-Protamine-DNA) particles. In this study, we evaluated the anti-PA immune response induced by the nasal PA/LPD particles in non-anaesthetized mice and compared it with that in anaesthetized mice. Our data showed that the anti-PA antibody response and the anthrax lethal toxin-neutralization activity induced by the nasal PA/LPD in non-anaesthetized mice was relatively weaker than that in anaesthetized mice. However, the splenocytes isolated from the nasally immunized mice, anaesthetized and non-anaesthetized, proliferated comparably after in-vitro re-stimulation. By evaluating the uptake of fluorescence-labelled LPD particles by phagocytes in the nasal and broncho-alveolar lavages of mice after the nasal administration, we concluded that the relatively weaker anti-PA immune response in the non-anaesthetized mice might be partially attributed to the reduced retention of the PA/LPD particles in the nasal cavity of the non-anaesthetized mice. Data collected in this study are expected to be useful for future anthrax nasal vaccine studies when mice are used as a model. PMID:16597361

  11. Characterization of an antigenic site that contains a dominant, type-specific neutralization determinant on the envelope protein domain III (ED3) of dengue 2 virus

    SciTech Connect

    Gromowski, Gregory D. [Department of Pathology, Sealy Center for Vaccine Development, Center for Biodefense and Emerging Infectious Diseases, and Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX 77555-0609 (United States); Barrett, Alan D.T. [Department of Pathology, Sealy Center for Vaccine Development, Center for Biodefense and Emerging Infectious Diseases, and Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX 77555-0609 (United States)], E-mail: abarrett@utmb.edu

    2007-09-30

    The surface of the mature dengue virus (DENV) particle consists of 90 envelope (E) protein dimers that mediate both receptor binding and fusion. The E protein ectodomain can be divided into three structural domains designated ED1, ED2, and ED3, of which ED3 contains the critical and dominant virus-specific neutralization sites. In this study the ED3 epitopes recognized by seven, murine, IgG1 DENV-2 type-specific, monoclonal antibodies (MAbs) were determined using site-directed mutagenesis of a recombinant DENV-2 ED3 (rED3) protein. A total of 41 single amino acid substitutions were introduced into the rED3 at 30 different surface accessible residues. The affinity of each MAb with the mutant rED3s was assessed by indirect ELISA and the results indicate that all seven MAbs recognize overlapping epitopes with residues K305 and P384 critical for binding. These residues are conserved among DENV-2 strains and cluster together on the upper lateral face of ED3. A linear relationship was observed between relative occupancy of ED3 on the virion by MAb and neutralization of the majority of virus infectivity ({approx} 90%) for all seven MAbs. Depending on the MAb, it is predicted that between 10% and 50% relative occupancy of ED3 on the virion is necessary for virus neutralization and for all seven MAbs occupancy levels approaching saturation were required for 100% neutralization of virus infectivity. Overall, the conserved antigenic site recognized by all seven MAbs is likely to be a dominant DENV-2 type-specific, neutralization determinant.

  12. Intracellular trafficking of bio-nanocapsule-liposome complex: Identification of fusogenic activity in the pre-S1 region of hepatitis B virus surface antigen L protein.

    PubMed

    Somiya, Masaharu; Sasaki, Yasuo; Matsuzaki, Takashi; Liu, Qiushi; Iijima, Masumi; Yoshimoto, Nobuo; Niimi, Tomoaki; Maturana, Andrés Daniel; Kuroda, Shun'ichi

    2015-08-28

    Bio-nanocapsules (BNCs) are a hollow nanoparticle consisting of about 100-nm liposome (LP) embedding about 110 molecules of hepatitis B virus (HBV) surface antigen (HBsAg) L protein as a transmembrane protein. Owing to the human hepatocyte-recognizing domains on the N-terminal region (pre-S1 region), BNCs have recently been shown to attach and enter into human hepatic cells using the early infection mechanism of HBV. Since BNCs could form a complex with an LP containing various drugs and genes, BNC-LP complexes have been used as a human hepatic cell-specific drug and gene-delivery system in vitro and in vivo. However, the role of BNCs in cell entry and intracellular trafficking of payloads in BNC-LP complexes has not been fully elucidated. In this study, we demonstrate that low pH-dependent fusogenic activity resides in the N-terminal part of pre-S1 region (NPLGFFPDHQLDPAFG), of which the first FF residues are essential for the activity, and which facilitates membrane fusion between LPs in vitro. Moreover, BNC-LP complexes can bind human hepatic cells specifically, enter into the cells via clathrin-mediated endocytosis, and release their payloads mostly into the cytoplasm. Taken together, the BNC portion of BNC-LP complexes can induce membrane fusion between LPs and endosomal membranes under low pH conditions, and thereby facilitate the endosomal escape of payloads. Furthermore, the fusogenic domain of the pre-S1 region of HBsAg L protein may play a pivotal role in the intracellular trafficking of not only BNC-LP complexes but also of HBV. PMID:26074149

  13. A FRET-Based High Throughput Screening Assay to Identify Inhibitors of Anthrax Protective Antigen Binding to Capillary Morphogenesis Gene 2 Protein

    PubMed Central

    Rogers, Michael S.; Cryan, Lorna M.; Habeshian, Kaiane A.; Bazinet, Lauren; Caldwell, Thomas P.; Ackroyd, P. Christine; Christensen, Kenneth A.

    2012-01-01

    Anti-angiogenic therapies are effective for the treatment of cancer, a variety of ocular diseases, and have potential benefits in cardiovascular disease, arthritis, and psoriasis. We have previously shown that anthrax protective antigen (PA), a non-pathogenic component of anthrax toxin, is an inhibitor of angiogenesis, apparently as a result of interaction with the cell surface receptors capillary morphogenesis gene 2 (CMG2) protein and tumor endothelial marker 8 (TEM8). Hence, molecules that bind the anthrax toxin receptors may be effective to slow or halt pathological vascular growth. Here we describe development and testing of an effective homogeneous steady-state fluorescence resonance energy transfer (FRET) high throughput screening assay designed to identify molecules that inhibit binding of PA to CMG2. Molecules identified in the screen can serve as potential lead compounds for the development of anti-angiogenic and anti-anthrax therapies. The assay to screen for inhibitors of this protein–protein interaction is sensitive and robust, with observed Z' values as high as 0.92. Preliminary screens conducted with a library of known bioactive compounds identified tannic acid and cisplatin as inhibitors of the PA-CMG2 interaction. We have confirmed that tannic acid both binds CMG2 and has anti-endothelial properties. In contrast, cisplatin appears to inhibit PA-CMG2 interaction by binding both PA and CMG2, and observed cisplatin anti-angiogenic effects are not mediated by interaction with CMG2. This work represents the first reported high throughput screening assay targeting CMG2 to identify possible inhibitors of both angiogenesis and anthrax intoxication. PMID:22768167

  14. A FRET-based high throughput screening assay to identify inhibitors of anthrax protective antigen binding to capillary morphogenesis gene 2 protein.

    PubMed

    Rogers, Michael S; Cryan, Lorna M; Habeshian, Kaiane A; Bazinet, Lauren; Caldwell, Thomas P; Ackroyd, P Christine; Christensen, Kenneth A

    2012-01-01

    Anti-angiogenic therapies are effective for the treatment of cancer, a variety of ocular diseases, and have potential benefits in cardiovascular disease, arthritis, and psoriasis. We have previously shown that anthrax protective antigen (PA), a non-pathogenic component of anthrax toxin, is an inhibitor of angiogenesis, apparently as a result of interaction with the cell surface receptors capillary morphogenesis gene 2 (CMG2) protein and tumor endothelial marker 8 (TEM8). Hence, molecules that bind the anthrax toxin receptors may be effective to slow or halt pathological vascular growth. Here we describe development and testing of an effective homogeneous steady-state fluorescence resonance energy transfer (FRET) high throughput screening assay designed to identify molecules that inhibit binding of PA to CMG2. Molecules identified in the screen can serve as potential lead compounds for the development of anti-angiogenic and anti-anthrax therapies. The assay to screen for inhibitors of this protein-protein interaction is sensitive and robust, with observed Z' values as high as 0.92. Preliminary screens conducted with a library of known bioactive compounds identified tannic acid and cisplatin as inhibitors of the PA-CMG2 interaction. We have confirmed that tannic acid both binds CMG2 and has anti-endothelial properties. In contrast, cisplatin appears to inhibit PA-CMG2 interaction by binding both PA and CMG2, and observed cisplatin anti-angiogenic effects are not mediated by interaction with CMG2. This work represents the first reported high throughput screening assay targeting CMG2 to identify possible inhibitors of both angiogenesis and anthrax intoxication. PMID:22768167

  15. Serotype-Converting Bacteriophage SfII Encodes an Acyltransferase Protein That Mediates 6-O-Acetylation of GlcNAc in Shigella flexneri O-Antigens, Conferring on the Host a Novel O-Antigen Epitope

    PubMed Central

    Knirel, Yuriy A.; Wang, Jianping; Luo, Xia; Senchenkova, Sofya N.; Lan, Ruiting; Shashkov, Alexander S.

    2014-01-01

    Shigella flexneri O-antigen is an important and highly variable cell component presented on the outer leaflet of the outer membrane. Most Shigella flexneri bacteria share an O-antigen backbone composed of ?2)-?-l-RhapIII-(1?2)-?-l-RhapII-(1?3)-?-l-RhapI-(1?3)-?-d-GlcpNAc-(1? repeats, which can be modified by adding various chemical groups to different sugars, giving rise to diverse O-antigen structures and, correspondingly, to various serotypes. The known modifications include glucosylation on various sugar residues, O-acetylation on RhaI or/and RhaIII, and phosphorylation with phosphoethanolamine on RhaII or/and RhaIII. Recently, a new O-antigen modification, namely, O-acetylation at position 6 of N-acetylglucosamine (GlcNAc), has been identified in S. flexneri serotypes 2a, 3a, Y, and Yv. In this study, the genetic basis of the 6-O-acetylation of GlcNAc in S. flexneri was elucidated. An O-acyltransferase gene designated oacD was found to be responsible for this modification. The oacD gene is carried on serotype-converting bacteriophage SfII, which is integrated into the host chromosome by lysogeny to form a prophage responsible for the evolvement of serotype 2 of S. flexneri. The OacD-mediated 6-O-acetylation also occurs in some other S. flexneri serotypes that carry a cryptic SfII prophage with a dysfunctional gtr locus for type II glucosylation. The 6-O-acetylation on GlcNAc confers to the host a novel O-antigen epitope, provisionally named O-factor 10. These findings enhance our understanding of the mechanisms of the O-antigen variation and enable further studies to understand the contribution of the O-acetylation to the antigenicity and pathogenicity of S. flexneri. PMID:25112477

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

    PubMed Central

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

    2013-01-01

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

  17. The T-cell antigen CD5 acts as a receptor and substrate for the protein-tyrosine kinase p56lck.

    PubMed Central

    Raab, M; Yamamoto, M; Rudd, C E

    1994-01-01

    CD5 is a T-cell-specific antigen which binds to the B-cell antigen CD72 and acts as a coreceptor in the stimulation of T-cell growth. CD5 associates with the T-cell receptor zeta chain (TcR zeta)/CD3 complex and is rapidly phosphosphorylated on tyrosine residues as a result of TcR zeta/CD3 ligation. However, despite this, the mechanism by which CD5 generates intracellular signals is unclear. In this study, we demonstrate that CD5 is coupled to the protein-tyrosine kinase p56lck and can act as a substrate for p56lck. Coexpression of CD5 with p56lck in the baculovirus expression system resulted in the phosphorylation of CD5 on tyrosine residues. Further, anti-CD5 and anti-p56lck coprecipitated each other in a variety of detergents, including Nonidet P-40 and Triton X-100. Anti-CD5 also precipitated the kinase from various T cells irrespective of the expression of TcR zeta/CD3 or CD4. No binding between p59fyn(T) and CD5 was detected in T cells. The binding of p56lck to CD5 induced a 10- to 15-fold increase in p56lck catalytic activity, as measured by in vitro kinase analysis. In vivo labelling with 32P(i) also showed a four- to fivefold increase in Y-394 occupancy in p56lck when associated with CD5. The use of glutathione S-transferase-Lck fusion proteins in precipitation analysis showed that the SH2 domain of p56lck could recognize CD5 as expressed in the baculovirus expression system. CD5 interaction with p56lck represents a novel variant of a receptor-kinase complex in which receptor can also serve as substrate. The CD5-p56lck interaction is likely to play roles in T-cell signalling and T-B collaboration. Images PMID:7513045

  18. Genetic and antigenic characterisation of the haemagglutinin protein of measles virus strains recently circulating in the UK.

    PubMed

    Jin, L; Knowles, W A; Rota, P A; Bellini, W J; Brown, D W

    1998-05-01

    The complete nucleotide sequence of the H protein gene of seven measles virus (MV) strains, representing three MV genotypes circulating in the UK in recent years, was determined. Compared to the MV vaccine strain Moraten (Mor-v), the divergence of the coded H gene (aal-600) of the seven UK strains was between 1.8% and 2.8%. Representative isolates from each of the genotypes were tested by radio-immunoprecipitation using a panel of H protein-specific MAbs. Different patterns of MAb reactivity were shown between the three genotypes and between the wild-type strains and the vaccine strain. Plaque reduction neutralising antibody titres against strains UK350/94 (genotype I) and UK226/94 (genotype III) were measured in sera from 11 vaccinees. Vaccine derived antibody neutralised both strains and the GMTs were not significantly lower against the wild-type strains than against strain Mor-v. PMID:9712517

  19. A Novel B Lymphocyte-associated Adaptor Protein, Bam32, Regulates Antigen Receptor Signaling Downstream of Phosphatidylinositol 3Kinase

    Microsoft Academic Search

    Aaron J. Marshall; Hiroaki Niiro; Cara G. Lerner; Theodore J. Yun; Sushma Thomas; Christine M. Disteche; Edward A. Clark

    We have identified and characterized a novel src homology 2 (SH2) and pleckstrin homology (PH) domain-containing adaptor protein, designated Bam32 (for B cell adaptor molecule of 32 kD). cDNAs encoding the human and mouse Bam32 coding sequences were isolated and the human bam32 gene was mapped to chromosome 4q25-q27. Bam32 is expressed by B lympho- cytes, but not T lymphocytes

  20. The Shc Family Protein Adaptor, Rai, Negatively Regulates T Cell Antigen Receptor Signaling by Inhibiting ZAP70 Recruitment and Activation

    Microsoft Academic Search

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

    2011-01-01

    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

  1. Importance of M-Protein C Terminus as Substrate Antigen for Serodetection of Equine Arteritis Virus Infection

    Microsoft Academic Search

    Célia Jeronimo; Denis Archambault

    2002-01-01

    Equine arteritis virus (EAV), an enveloped positive-stranded RNA virus, is the prototype of the arterivirus group. In a previous paper (A. Kheyar, S. Martin, G. St.-Laurent, P. J. Timoney, W. H. McCollum, and D. Archambault, Clin. Diagn. Lab. Immunol. 4:648-652, 1997), we have shown that the unglycosylated membrane (M) protein, which is composed of 162 amino acids (aa), is a

  2. An immunological study of corrinoid proteins from bacteria revealed homologous antigenic determinants of a soluble corrinoid-dependent methyltransferase and corrinoid-containing membrane proteins from Methanobacterium species

    Microsoft Academic Search

    Erhard Stupperich; Andreas Juza; Christoph Eckerskorn; Lillian Edelmann

    1990-01-01

    The hypothesis of common epitopes in corrinoid-dependent enzymes was tested by a monospecific polyclonal antiserum against the 33 kDa corrinoid-containing membrane protein from Methanobacterium thermoautotrophicum Marburg. Cross-reaction was detected with the 33 kDa and the 31 kDa subunits of the corrinoid-containing enriched 5-methyl-H4MPT: 5-hydroxybenzimidazolyl cobamide methyltransferase from the cytoplasmic fraction and a 33 kDa protein from the membrane fraction of

  3. Tumor-Associated Antigens in Breast Cancer

    PubMed Central

    Criscitiello, Carmen

    2012-01-01

    The targets for the immune system are antigens present on cancer cells; however, many are not cancer-specific and may also be found on normal tissues. These antigens are often products of mutated cellular genes, aberrantly expressed normal genes, or genes encoding viral proteins. Vaccines constitute an active and specific immunotherapy designed to stimulate the intrinsic antitumor immune response by presenting tumor-associated antigens expressed on normal tissues that are overexpressed on tumor cells. PMID:23904827

  4. Myelin proteolipid protein (PLP) as a marker antigen of central nervous system contaminations for routine food control.

    PubMed

    Villmann, Carmen; Sandmeier, Barbara; Seeber, Silke; Hannappel, Ewald; Pischetsrieder, Monika; Becker, Cord-Michael

    2007-08-22

    Spreading transmissible spongiform encephalopathies (TSE) have been widely attributed to transmission by ingestion of mammalian central nervous system (CNS) tissue. Reliable exclusion of this epidemiological important route of transmission relies on an effective surveillance of food contamination. Here, myelin proteolipid protein (PLP) is identified as a specific and largely heat-resistant marker for detection of food contaminations by CNS tissue. PLP is a component of oligodendritic glial sheaths of neuronal processes that is specifically expressed in the CNS. A highly selective polyclonal antibody was developed directed against an epitope present in the full-length PLP protein, but absent from the developmentally regulated splice variant DM-20. In combination with a hydrophobic extraction of PLP from tissue samples, the antibody reliably detected PLP from spinal cord, cerebellum, and cortex of different mammalian species. Consistent with earlier reports on PLP expression, no cross-reactivity was observed with peripheral nerve or extraneural tissue, except for a very faint signal obtained with heart. When applied to an artificial CNS contamination present in sausages, the antibody reliably detected a low concentration (1%) of the contaminant. Application of heat, as used during conventional sausage manufacturing, led to a predominant alteration of arginine residues in the PLP protein and a partial loss of immunoreactivity. In contrast, a stretch of hydrophilic amino acids(112-122) proved to be heat-resistant, preserving the immunogenicity of this PLP epitope during heating. Taken together, the excellent CNS specificity of PLP immunodetection and the presence of a heat-resistant epitope have permitted the development of a highly sensitive immunoassay for CNS contamination in routine food control. PMID:17629299

  5. Monoclonal antibody-based enzyme-linked immunosorbent assays (ELISA) for the measurement of vitamin K-dependent protein S: the effect of antibody immunoreactivity on plasma protein S antigen determinations.

    PubMed

    D'Angelo, S V; Tombesi, S; Marcovina, S; Albertini, A; Della Valle, P; D'Angelo, A

    1992-06-01

    Two monoclonal antibodies (Mabs) specifically directed to human protein S (PS) - named 5E9E9 and 3B10.25 - were produced and their properties compared to those of 2 previously characterized anti-PS-Mabs (HPS-2 and S10). 3B10.25, similar to S10, was directed to the calcium-free conformation of PS and had virtually identical affinity for free and C4b-binding protein (C4b-BP)-bound PS; 5E9E9 similar to HPS-2, had no calcium-dependency and was selectively directed to free PS. All Mabs were equally reactive to freshly purified and thrombin-cleaved PS. To evaluate the influence of C4b-BP bound PS on PS antigen determinations, ELISA systems employing the four Mabs individually as capture antibody (Ab) and peroxidase-conjugated polyclonal anti-PS IgG as detecting Ab were developed and compared to immunoelectrophoresis (EIA) and to an ELISA employing polyclonal anti-PS IgG as capture and detecting Ab, in the determination of PS in purified systems and in plasma. With all the ELISAs there was parallelism of dilution curves obtained with normal plasma and purified PS; however, supplementation of plasma with purified C4b-BP resulted in loss of parallelism when employing the Mabs directed to free PS as capture Ab. Influence of high C4b-BP on PS antigen determinations was confirmed in a series of plasma samples from patients with C4b-BP levels ranging from 70% to over 200%. Compared to the values obtained with the S10- or 3B10.25 - based ELISAs - which were similar despite a 10-fold difference in sample dilution - plasma PS was underestimated by the ELISAs employing 5E9E9 or HPS-2 while it was overestimated by EIA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1387263

  6. Antigenic and Molecular Properties of Type 3 Poliovirus Responsible for an Outbreak of Poliomyelitis in a Vaccinated Population

    Microsoft Academic Search

    D. I. Magrath; D. M. A. Evans; M. Ferguson; G. C. Schild; P. D. Minor; F. Horaud; R. Crainic; M. Stenvik; T. Hovi

    1986-01-01

    SUMMARY Virus isolated from an outbreak of poliomyelitis in Finland has been examined serologically and at the molecular level. The causative agent was an antigenically unusual strain of type 3 poliovirus, which was unrelated to the strains used to manufacture either live or killed poliovaccines. It is likely that the antigenic properties of the virus played a part in establishing

  7. The atypical GATA protein TRPS1 represses androgen-induced prostate-specific antigen expression in LNCaP prostate cancer cells.

    PubMed

    van den Bemd, Gert-Jan C M; Jhamai, Mila; Brinkmann, Albert O; Chang, Glenn T G

    2003-12-19

    Prostate-specific antigen (PSA) is considered as an important marker for prostate cancer. Regulation of PSA gene expression is mediated by androgens bound to androgen receptors via androgen response elements (AREs) in its promoter and far upstream enhancer regions. In addition, GATA proteins contribute to PSA gene transcription by interacting with GATA motifs present in the PSA enhancer sequence. The TRPS1 gene contains a single GATA zinc finger domain and not only binds to forward consensus GATA motifs but also to an inverse GATA motif overlapping the ARE III in the far upstream enhancer of the PSA gene. Overexpression of TRPS1 in androgen-dependent human LNCaP prostate cancer cells inhibited the transcription of a transiently transfected PSA enhancer/promoter-driven luciferase reporter construct. Furthermore, overexpression of TRPS1 reduced the androgen-induced endogenous PSA levels secreted in culture medium of LNCaP cells. Our results suggest a role of TRPS1 in androgen regulation of PSA gene expression. PMID:14680804

  8. Induction of the nuclear I{kappa}B protein I{kappa}B-{zeta} upon stimulation of B cell antigen receptor

    SciTech Connect

    Hijioka, Kuniaki [Department of Molecular and Cellular Biochemistry, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan); Matsuo, Susumu [Department of Molecular and Cellular Biochemistry, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan); Eto-Kimura, Akiko [Department of Molecular and Cellular Biochemistry, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan); Takeshige, Koichiro [Department of Molecular and Cellular Biochemistry, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan); Muta, Tatsushi [Department of Molecular and Cellular Biochemistry, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan) and Laboratory of Cell Recognition and Response, Graduate School of Life Sciences, Tohoku University, Sendai 980-8578 (Japan)]. E-mail: tmuta@biology.tohoku.ac.jp

    2007-05-04

    The nuclear I{kappa}B protein I{kappa}B-{zeta} is barely detectable in resting cells and is induced in macrophages and fibroblasts following stimulation of innate immunity via Toll-like receptors. The induced I{kappa}B-{zeta} associates with nuclear factor (NF)-{kappa}B in the nucleus and plays crucial roles in its transcriptional regulation. Here, we examined the induction of I{kappa}B-{zeta} in B lymphocytes, one of the major players in adaptive immunity. Upon crosslinking of the surface immunoglobulin complex, I{kappa}B-{zeta} mRNA was robustly induced in murine B-lymphoma cell line A20 cells. While the crosslinking activated NF-{kappa}B and induced its target gene, I{kappa}B-{alpha}, co-crosslinking of Fc{gamma} receptor IIB to the surface immunoglobulin complex inhibited NF-{kappa}B activation and the induction of I{kappa}B-{zeta} and I{kappa}B-{alpha}, suggesting critical roles for NF-{kappa}B in the induction. These results indicate that I{kappa}B-{zeta} is also induced by stimulation of B cell antigen receptor, suggesting that I{kappa}B-{zeta} is involved in the regulation of adaptive immune responses.

  9. Kinetics of Dengue Non-Structural Protein 1 Antigen and IgM and IgA Antibodies in Capillary Blood Samples from Confirmed Dengue Patients

    PubMed Central

    Matheus, Séverine; Pham, Thai Binh; Labeau, Bhetty; Huong, Vu Thi Que; Lacoste, Vincent; Deparis, Xavier; Marechal, Vincent

    2014-01-01

    Large-scale epidemiological surveillance of dengue in the field and dengue patient management require simple methods for sample collection, storage, and transportation as well as effective diagnostic tools. We evaluated the kinetics of three biological markers of dengue infection—non-structural protein 1 (NS1) antigen, immunoglobulin M (IgM), and IgA—in sequential capillary blood samples collected from fingertips of confirmed dengue patients. The overall sensitivities and specificities of the tests were 96% and 100%, respectively, for NS1, 58.1% and 100%, respectively, for IgM, and 33% and 100%, respectively, for IgA. During the acute phase of the disease, NS1 was the best marker of dengue infection, with a sensitivity of 98.7%, whereas from day 5, all three markers exhibited relevant levels of sensitivity. This first descriptive study of the kinetics of biological markers of dengue in capillary blood samples confirms the usefulness of this biological compartment for dengue diagnosis and argues for its exploitation in community-level and remote settings. PMID:24470561

  10. Protein Antigens Increase the Protective Efficacy of a Capsule-Based Vaccine against Staphylococcus aureus in a Rat Model of Osteomyelitis

    PubMed Central

    Lattar, Santiago M.; Noto Llana, Mariángeles; Denoël, Philippe; Germain, Sophie; Buzzola, Fernanda R.; Lee, Jean C.

    2014-01-01

    Staphylococcus aureus is an invasive bacterial pathogen, and antibiotic resistance has impeded adequate control of infections caused by this microbe. Moreover, efforts to prevent human infections with single-component S. aureus vaccines have failed. In this study, we evaluated the protective efficacy in rats of vaccines containing both S. aureus capsular polysaccharides (CPs) and proteins. The serotypes 5 CP (CP5) and 8 CP (CP8) were conjugated to tetanus toxoid and administered to rats alone or together with domain A of clumping factor A (ClfA) or genetically detoxified alpha-toxin (dHla). The vaccines were delivered according to a preventive or a therapeutic regimen, and their protective efficacy was evaluated in a rat model of osteomyelitis. Addition of dHla (but not ClfA) to the CP5 or CP8 vaccine induced reductions in bacterial load and bone morphological changes compared with immunization with either conjugate vaccine alone. Both the prophylactic and therapeutic regimens were protective. Immunization with dHla together with a pneumococcal conjugate vaccine used as a control did not reduce staphylococcal osteomyelitis. The emergence of unencapsulated or small-colony variants during infection was negligible and similar for all of the vaccine groups. In conclusion, addition of dHla to a CP5 or CP8 conjugate vaccine enhanced its efficacy against S. aureus osteomyelitis, indicating that the inclusion of multiple antigens will likely enhance the efficacy of vaccines against both chronic and acute forms of staphylococcal disease. PMID:24126523

  11. Protein antigens increase the protective efficacy of a capsule-based vaccine against Staphylococcus aureus in a rat model of osteomyelitis.

    PubMed

    Lattar, Santiago M; Noto Llana, Mariángeles; Denoël, Philippe; Germain, Sophie; Buzzola, Fernanda R; Lee, Jean C; Sordelli, Daniel O

    2014-01-01

    Staphylococcus aureus is an invasive bacterial pathogen, and antibiotic resistance has impeded adequate control of infections caused by this microbe. Moreover, efforts to prevent human infections with single-component S. aureus vaccines have failed. In this study, we evaluated the protective efficacy in rats of vaccines containing both S. aureus capsular polysaccharides (CPs) and proteins. The serotypes 5 CP (CP5) and 8 CP (CP8) were conjugated to tetanus toxoid and administered to rats alone or together with domain A of clumping factor A (ClfA) or genetically detoxified alpha-toxin (dHla). The vaccines were delivered according to a preventive or a therapeutic regimen, and their protective efficacy was evaluated in a rat model of osteomyelitis. Addition of dHla (but not ClfA) to the CP5 or CP8 vaccine induced reductions in bacterial load and bone morphological changes compared with immunization with either conjugate vaccine alone. Both the prophylactic and therapeutic regimens were protective. Immunization with dHla together with a pneumococcal conjugate vaccine used as a control did not reduce staphylococcal osteomyelitis. The emergence of unencapsulated or small-colony variants during infection was negligible and similar for all of the vaccine groups. In conclusion, addition of dHla to a CP5 or CP8 conjugate vaccine enhanced its efficacy against S. aureus osteomyelitis, indicating that the inclusion of multiple antigens will likely enhance the efficacy of vaccines against both chronic and acute forms of staphylococcal disease. PMID:24126523

  12. Block Copolymer Arrangement and Composition Effects on Protein Conformation Using AFM-Based Antigen-Antibody Adhesion

    PubMed Central

    Palacio, Manuel L. B.; Schricker, Scott R.; Bhushan, Bharat

    2013-01-01

    The conformational changes of fibronectin deposited on various block copolymers where one block is composed of poly(methyl methacrylate) (PMMA) and the other block is either poly(acrylic acid) (PAA) or poly(2-hydroxyethyl methacrylate) (PHEMA) were investigated using a functionalized atomic force microscope (AFM) tip. The tip was modified with an antibody sensitive to the exposure of the arginine-glycine-aspartic acid (RGD) groups in fibronectin. By studying the adhesive interactions between the antibody and the proteins adsorbed on the block copolymer surface and phase imaging, it was found that the triblock copolymers PAA-b-PMMA-b-PAA and PMMA-b-PHEMA-b-PMMA, which both have large domain sizes, are conducive to the exposure of the fibronectin RGD groups on the surface. Based on these results, it is concluded that the surface chemistry as well as the nanomorphology dictated by the block copolymer arrangement could both tune protein conformation and orientation and optimize cell adhesion to the biomaterial surface. PMID:22278846

  13. Recognition of YKL-39, a human cartilage related protein, as a target antigen in patients with rheumatoid arthritis

    PubMed Central

    Sekine, T; Masuko-Hongo, K; Matsui, T; Asahara, H; Takigawa, M; Nishioka, K; Kato, T

    2001-01-01

    OBJECTIVE—To investigate whether autoimmunity to YKL-39, a recently cloned cartilage protein, occurs in patients with rheumatoid arthritis (RA).?METHODS—Autoantibody to YKL-39 was assayed by enzyme linked immunosorbent assay (ELISA) and western blotting in serum samples from patients with RA, systemic lupus erythematosus (SLE), and healthy donors, using recombinant YKL-39 protein. This reactivity was compared with that against a YKL-39 homologue, YKL-40 (human cartilage gp-39/chondrex), which has been reported to be an autoantigen in RA.?RESULTS—Autoantibody to YKL-39 was detected in seven of 87 patients with RA (8%), but not in serum samples from patients with SLE or healthy donors. YKL-40 reactivity was found in only one of 87 RA serum samples (1%), with no cross reactivity to YKL-39.?CONCLUSION—The existence of anti-YKL-39 antibody in a subset of patients with RA is reported here for the first time. Further, it was shown that the immune response to YKL-39 was independent of that to YKL-40. Clarification of the antibody and T cell responses to autoantigens derived from chondrocyte, cartilage, or other joint components may lead to a better understanding of the pathophysiology of joint destruction in patients with RA.?? PMID:11114282

  14. Neisseria meningitidis factor H-binding protein fHbp: a key virulence factor and vaccine antigen.

    PubMed

    Seib, Kate L; Scarselli, Maria; Comanducci, Maurizio; Toneatto, Daniela; Masignani, Vega

    2015-06-01

    Neisseria meningitidis is a leading cause of meningitis and sepsis worldwide. The first broad-spectrum multicomponent vaccine against serogroup B meningococcus (MenB), 4CMenB (Bexsero(®)), was approved by the EMA in 2013, for prevention of MenB disease in all age groups, and by the US FDA in January 2015 for use in adolescents. A second protein-based MenB vaccine has also been approved in the USA for adolescents (rLP2086, Trumenba(®)). Both vaccines contain the lipoprotein factor H-binding protein (fHbp). Preclinical studies demonstrated that fHbp elicits a robust bactericidal antibody response that correlates with the amount of fHbp expressed on the bacterial surface. fHbp is able to selectively bind human factor H, the key regulator of the alternative complement pathway, and this has important implications both for meningococcal pathogenesis and for vaccine design. Here, we review the functional and structural properties of fHbp, the strategies that led to the design of the two fHbp-based vaccines and the data generated during clinical studies. PMID:25704037

  15. Identification and characterization of an Epstein-Barr virus nuclear antigen 2-responsive cis element in the bidirectional promoter region of latent membrane protein and terminal protein 2 genes.

    PubMed Central

    Laux, G; Dugrillon, F; Eckert, C; Adam, B; Zimber-Strobl, U; Bornkamm, G W

    1994-01-01

    Epstein-Barr virus (EBV) transforms resting B cells in vitro very efficiently. The nuclear viral protein EBV nuclear antigen 2 (EBNA2) is absolutely required for this process and also acts as a transcriptional activator of cellular and viral genes. As shown previously, EBNA2 transactivates the promoters of the viral latent membrane proteins. It interacts indirectly with an EBNA2-responsive cis element of the terminal protein 1 (TP1) promoter. To identify the sequences mediating EBNA2 transactivation of the bidirectional promoter region driving expression of the latent membrane proteins LMP and TP2 in opposite directions, we assayed the effects of EBNA2 on the activities of promoter deletion and site-directed mutants of TP2 and LMP promoter luciferase reporter gene constructs by cotransfections into EBNA2-negative Burkitt's lymphoma cells. We were able to delineate an 80-bp EBNA2-responsive region (EBNA2RE) between -232 and -152 relative to the LMP RNA start site which could also mediate EBNA2-dependent activation on a heterologous promoter. Sequences of 20 and 32 bp located at the 5' and 3' ends, respectively, of the EBNA2RE were both essential for EBNA2 responsiveness. Full transactivation of the LMP and TP2 promoters seemed to require 20 bp of 5' adjacent sequences in addition to the 80-bp element. Electrophoretic mobility shift assays revealed specific protein-DNA complexes formed at the EBNA2RE. Oligonucleotides from -181 to -152 and -166 to -132 relative to the LMP RNA start site visualized one B-cell and one B-cell-plus-HL60-specific retarded protein-DNA complex, respectively. Additionally, an oligonucleotide from -253 to -210 revealed two specific protein-DNA complexes with nuclear extracts from different B and non-B cells, suggesting also the binding of ubiquitously expressed proteins on the EBNA2RE. Thus, these experiments defined a 80-bp cis element sufficient for conferring EBNA2 inducibility and demonstrated specific interactions of cellular proteins at DNA sequences within the EBNA2RE, which are critical for transactivation by EBNA2. Images PMID:7933076

  16. The serodominant secreted effector protein of Salmonella, SseB, is a strong CD4 antigen containing an immunodominant epitope presented by diverse HLA class II alleles.

    PubMed

    Reynolds, Catherine J; Jones, Claire; Blohmke, Christoph J; Darton, Thomas C; Goudet, Amelie; Sergeant, Ruhena; Maillere, Bernard; Pollard, Andrew J; Altmann, Daniel M; Boyton, Rosemary J

    2014-11-01

    Detailed characterization of the protective T-cell response in salmonellosis is a pressing unmet need in light of the global burden of human Salmonella infections and the likely contribution of CD4 T cells to immunity against this intracellular infection. In previous studies screening patient sera against antigen arrays, SseB was noteworthy as a serodominant target of adaptive immunity, inducing significantly raised antibody responses in HIV-seronegative compared with seropositive patients. SseB is a secreted protein, part of the Espa superfamily, localized to the bacterial surface and forming part of the translocon of the type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2. We demonstrate here that SseB is also a target of CD4 T-cell immunity, generating a substantial response after experimental infection in human volunteers, with around 0.1% of the peripheral repertoire responding to it. HLA-DR/peptide binding studies indicate that this protein encompasses a number of peptides with ability to bind to several different HLA-DR alleles. Of these, peptide 11 (p11) was shown in priming of both HLA-DR1 and HLA-DR4 transgenic mice to contain an immunodominant CD4 epitope. Analysis of responses in human donors showed immunity focused on p11 and another epitope in peptide 2. The high frequency of SseB-reactive CD4 T cells and the broad applicability to diverse HLA genotypes coupled with previous observations of serodominance and protective vaccination in mouse challenge experiments, make SseB a plausible candidate for next-generation Salmonella vaccines. PMID:24891088

  17. Bacterial ClpB heat-shock protein, an antigen-mimetic of the anorexigenic peptide ?-MSH, at the origin of eating disorders

    PubMed Central

    Tennoune, N; Chan, P; Breton, J; Legrand, R; Chabane, Y N; Akkermann, K; Järv, A; Ouelaa, W; Takagi, K; Ghouzali, I; Francois, M; Lucas, N; Bole-Feysot, C; Pestel-Caron, M; do Rego, J-C; Vaudry, D; Harro, J; Dé, E; Déchelotte, P; Fetissov, S O

    2014-01-01

    The molecular mechanisms at the origin of eating disorders (EDs), including anorexia nervosa (AN), bulimia and binge-eating disorder (BED), are currently unknown. Previous data indicated that immunoglobulins (Igs) or autoantibodies (auto-Abs) reactive with ?-melanocyte-stimulating hormone (?-MSH) are involved in regulation of feeding and emotion; however, the origin of such auto-Abs is unknown. Here, using proteomics, we identified ClpB heat-shock disaggregation chaperone protein of commensal gut bacteria Escherichia coli as a conformational antigen mimetic of ?-MSH. We show that ClpB-immunized mice produce anti-ClpB IgG crossreactive with ?-MSH, influencing food intake, body weight, anxiety and melanocortin receptor 4 signaling. Furthermore, chronic intragastric delivery of E. coli in mice decreased food intake and stimulated formation of ClpB- and ?-MSH-reactive antibodies, while ClpB-deficient E. coli did not affect food intake or antibody levels. Finally, we show that plasma levels of anti-ClpB IgG crossreactive with ?-MSH are increased in patients with AN, bulimia and BED, and that the ED Inventory-2 scores in ED patients correlate with anti-ClpB IgG and IgM, which is similar to our previous findings for ?-MSH auto-Abs. In conclusion, this work shows that the bacterial ClpB protein, which is present in several commensal and pathogenic microorganisms, can be responsible for the production of auto-Abs crossreactive with ?-MSH, associated with altered feeding and emotion in humans with ED. Our data suggest that ClpB-expressing gut microorganisms might be involved in the etiology of EDs. PMID:25290265

  18. A Diacylglycerol-Protein Kinase C-RasGRP1 Pathway Directs Ras Activation upon Antigen Receptor Stimulation of T Cells

    PubMed Central

    Roose, Jeroen P.; Mollenauer, Marianne; Gupta, Vikas A.; Stone, James; Weiss, Arthur

    2005-01-01

    Ras GTPases are on/off switches regulating numerous cellular responses by signaling to various effector molecules. In T lymphocytes, Ras can be activated by two Ras exchange factors, SOS and RasGRP1, which are recruited through the adapters Grb2 and LAT and via the second-messenger diacylglycerol (DAG), respectively. Mitogen-activated protein (MAP) kinase phosphorylation patterns induced by active Ras can vary and contribute to distinct cellular responses. The different consequences of Ras activation by either guanine exchange factor are unknown. DAG also recruits and activates the kinase protein kinase C? (PKC?) turning on the Erk MAP kinase pathway, but the biochemical mechanism responsible is unclear. We generated T-cell clones deficient in phorbol myristate acetate (a surrogate for DAG)-induced Ras activation. Analysis of a RasGRP1-deficient Jurkat T-cell clone and RasGRP1 RNA interference in wild-type cells revealed that RasGRP1 is required for optimal, antigen receptor-triggered Ras-Erk activation. RasGRP1 relies on its DAG-binding domain to selectively activate Erk kinases. Activation of Erk correlates with the phosphorylation of threonine residue 184 in RasGRP1. This phosphorylation event requires the activities of novel PKC kinases. Conversely, active PKC? depends on RasGRP1 sufficiency to effectively trigger downstream events. Last, DAG-PKC-RasGRP1-driven Ras-Erk activation in T cells is a unique signaling event, not simply compensated for by SOS activity. PMID:15899849

  19. Cancer\\/testis antigens, gametogenesis and cancer

    Microsoft Academic Search

    Otavia L. Caballero; Achim Jungbluth; Yao-Tseng Chen; Lloyd J. Old; Andrew J. G. Simpson

    2005-01-01

    Cancer\\/testis (CT) antigens, of which more than 40 have now been identified, are encoded by genes that are normally expressed only in the human germ line, but are also expressed in various tumour types, including melanoma, and carcinomas of the bladder, lung and liver. These immunogenic proteins are being vigorously pursued as targets for therapeutic cancer vaccines. CT antigens are

  20. The putative capsid protein of the newly identified avian hepatitis E virus shares antigenic epitopes with that of swine and human hepatitis E viruses and chicken big liver and spleen disease virus

    Microsoft Academic Search

    G. Haqshenas; F. F. Huang; M. Fenaux; D. K. Guenette; F. W. Pierson; C. T. Larsen; H. L. Shivaprasad; T. E. Toth; X. J. Meng

    We recently identified a novel virus, designated avian hepatitis E virus (avian HEV), from chickens with hepatitis-splenomegaly (HS) syndrome in the USA. We showed that avian HEV is genetically related to swine and human HEVs. Here we report the antigenic cross-reactivity of the putative open reading frame 2 (ORF2) capsid protein of avian HEV with those of swine and human

  1. Differentiation of Foot-and-Mouth Disease Virus-Infected from Vaccinated Pigs by Enzyme-Linked Immunosorbent Assay Using Nonstructural Protein 3AB as the Antigen and Application to an Eradication Program

    Microsoft Academic Search

    Wen-Bin Chung; Karl Johan Sorensen; Pei-Chih Liao; Pin-Cheng Yang; Ming-Hwa Jong

    2002-01-01

    Baculovirus-expressed foot-and-mouth disease virus (FMDV) nonstructural protein 3AB was used as the antigen in an enzyme-linked immunosorbent assay. This assay allowed the differentiation of vaccinated from infected pigs. Serial studies were performed using sera collected from pigs in the field. Positive reactions were found in sera from fattening pigs and sows 16 weeks and 3.5 years postoutbreak, respectively. There was,

  2. Reduced Secretion of Virions and Hepatitis B Virus (HBV) Surface Antigen of a Naturally Occurring HBV Variant Correlates with the Accumulation of the Small S Envelope Protein in the Endoplasmic Reticulum and Golgi Apparatus

    Microsoft Academic Search

    Pong Kian Chua; Robert Yung-Liang Wang; Min-Hui Lin; Tetsuya Masuda; Fat-Moon Suk; Chiaho Shih

    2005-01-01

    We identified two novel naturally occurring mutations (W74L and L77R) in the small S envelope protein of hepatitis B virus (HBV). Mutation L77R alone resulted in >10-fold-reduced secretion of virions. In addition, the 2.8-fold reduction of the extracellular HBV surface antigen (HBsAg) of mutant L77R from transfected Huh7 cells appeared to be correlated with a 1.7-fold reduction of intracellular HBsAg,

  3. Molecular signatures distinguishing active from latent tuberculosis in peripheral blood mononuclear cells, after in vitro antigenic stimulation with purified protein derivative of tuberculin (PPD) or Candida: a preliminary report

    Microsoft Academic Search

    Joel N. H. Stern; Derin B. Keskin; Viviana Romero; Joaquin Zuniga; Liliana Encinales; Changlin Li; Carlos Awad; Edmond J. Yunis

    2009-01-01

    Purified protein derivative (PPD) or tuberculin skin testing is used to identify infected individuals with Mycobacterium tuberculosis (Mtb) and to assess cell-mediated immunity to Mtb. In the present study, we compared PBMC cultures in the presence of tuberculin\\u000a or Candida antigens using cytokine bead arrays and RNA microarrays. Measurements of different cytokines and chemokines in\\u000a supernatants of PMBC cultures in

  4. An oat coleoptile wall protein that induces wall extension in vitro and that is antigenically related to a similar protein from cucumber hypocotyls

    Microsoft Academic Search

    Zhen-Chang Li; Daniel M. Durachko; Daniel J. Cosgrove

    1993-01-01

    Plant cell walls expand considerably during cell enlargement, but the biochemical reactions leading to wall expansion are unknown. McQueen-Mason et al. (1992, Plant Cell 4, 1425) recently identified two proteins from cucumber (Cucumis sativus L.) that induced extension in walls isolated from dicotyledons, but were relatively ineffective on grass coleoptile walls. Here we report the identification and partial characterization of

  5. Control of tick infestations and pathogen prevalence in cattle and sheep farms vaccinated with the recombinant Subolesin-Major Surface Protein 1a chimeric antigen

    PubMed Central

    2014-01-01

    Background Despite the use of chemical acaricides, tick infestations continue to affect animal health and production worldwide. Tick vaccines have been proposed as a cost-effective and environmentally friendly alternative for tick control. Vaccination with the candidate tick protective antigen, Subolesin (SUB), has been shown experimentally to be effective in controlling vector infestations and pathogen infection. Furthermore, Escherichia coli membranes containing the chimeric antigen composed of SUB fused to Anaplasma marginale Major Surface Protein 1a (MSP1a) (SUB-MSP1a) were produced using a simple low-cost process and proved to be effective for the control of cattle tick, Rhipicephalus (Boophilus) microplus and R. annulatus infestations in pen trials. In this research, field trials were conducted to characterize the effect of vaccination with SUB-MSP1a on tick infestations and the prevalence of tick-borne pathogens in a randomized controlled prospective study. Methods Two cattle and two sheep farms with similar geographical locations and production characteristics were randomly assigned to control and vaccinated groups. Ticks were collected, counted, weighed and classified and the prevalence of tick-borne pathogens at the DNA and serological levels were followed for one year prior to and 9 months after vaccination. Results Both cattle and sheep developed antibodies against SUB in response to vaccination. The main effect of the vaccine in cattle was the 8-fold reduction in the percent of infested animals while vaccination in sheep reduced tick infestations by 63%. Female tick weight was 32-55% lower in ticks collected from both vaccinated cattle and sheep when compared to controls. The seroprevalence of Babesia bigemina was lower by 30% in vaccinated cattle, suggesting a possible role for the vaccine in decreasing the prevalence of this tick-borne pathogen. The effect of the vaccine in reducing the frequency of one A. marginale msp4 genotype probably reflected the reduction in the prevalence of a tick-transmitted strain as a result of the reduction in the percent of tick-infested cattle. Conclusions These data provide evidence of the dual effect of a SUB-based vaccine for controlling tick infestations and pathogen infection/transmission and provide additional support for the use of the SUB-MSP1a vaccine for tick control in cattle and sheep. PMID:24398155

  6. Unrelated donor hematopoietic stem cell transplantation for infantile enteropathy due to IL-10/IL-10 receptor defect.

    PubMed

    Gassas, Adam; Courtney, Sarah; Armstrong, Christine; Kapllani, Erilda; Muise, Aleixo M; Schechter, Tal

    2015-06-01

    Recent advances in genetic diagnosis have identified mutations in gene encoding interleukin-10 (IL-10) and IL-10 receptor (IL-10R) proteins as a cause for early-onset enterocolitis leading to hyperinflammatory immune response. Allogeneic HSCT offers a potential cure; however, it was only performed in a few infants and mainly from family-related donors. We report a case of a girl who presented very early in life with severe infantile enterocolitis. Gene sequencing confirmed IL-10R defect. Her older sister died at 13 months of age from severe undiagnosed enterocolitis. There was no family donor. An unrelated search identified a potential 10/10 high-resolution HLA-matched donor. There was some delay in donor activation because IL-10R defect was not on the standard list of indications for unrelated HSCT. Our patient received the unrelated HSCT at seven months of age, and she is currently nine months after transplant and doing very well. Because HSCT is the curative option of choice for this disorder, we encourage adding IL-10 and IL-10R protein defects to the list of HSCT indications for unrelated donor procurement. PMID:25761563

  7. Albright hereditary osteodystrophy and del(2)(q37.3) in four unrelated individuals

    SciTech Connect

    Phelan, M.C.; Rogers, R.C.; Clarkson, K.B. [Greenwood Genetic Center, Greenwood, SC (United States)] [and others

    1995-07-31

    Albright hereditary osteodystrophy (AHO) is a condition with characteristic physical findings (short stature, obesity, round face, brachydactyly) but variable biochemical changes (pseudohypoparathyroidism, pseudopseudohypoparathyroidism). Most patients with AHO have decreased activity of the guanine nucleotide-binding protein (G{sub s} protein) that stimulates adenylyl cyclase. The gene encoding the a subunit of the G{sub s} protein (GNAS1) has been mapped to the long arm of chromosome 20. We describe 4 unrelated individuals with apparent AHO, associated with small terminal deletions of chromosome 2. All 4 patients had normal serum calcium levels consistent with pseudopseudohypoparathyroidism. Del(2)(q37) is the first consistent karyotypic abnormality that has been documented in AHO. The finding of the same small terminal deletion in 4 unrelated individuals with a similar phenotype suggests that a gene locus in the 2q37 region is important in the pathogenesis of Albright syndrome. The association of Albright syndrome and the GNAS1 locus on chromosome 20 is well documented. The observation of a second potential disease locus on chromosome 2 may help explain the heterogeneity observed in this disorder. 44 refs., 7 figs.

  8. Development of antigen-specific ELISA for circulating autoantibodies to extracellular matrix protein 1 in lichen sclerosus

    PubMed Central

    Oyama, Noritaka; Chan, Ien; Neill, Sallie M.; South, Andrew P.; Wojnarowska, Fenella; Kawakami, Yoshio; D’Cruz, David; Mepani, Kirti; Hughes, Graham J.; Bhogal, Balbir S.; Kaneko, Fumio; Black, Martin M.; McGrath, John A.

    2004-01-01

    Lichen sclerosus is a common, acquired chronic inflammatory skin disease of unknown etiology, although circulating autoantibodies to the glycoprotein extracellular matrix protein 1 (ECM1) have been detected in most patients’ sera. We have examined the nature of ECM1 epitopes in lichen sclerosus sera, developed an ELISA system for serologic diagnosis, and assessed clinicopathological correlation between ELISA titer and disease. Epitope-mapping studies revealed that lichen sclerosus sera most frequently recognized the distal second tandem repeat domain and carboxyl-terminus of ECM1. We analyzed serum autoantibody reactivity against this immunodominant epitope in 413 individuals (95 subjects with lichen sclerosus, 161 normal control subjects, and 157 subjects with other autoimmune basement membrane or sclerosing diseases). The ELISA assay was highly sensitive; 76 of 95 lichen sclerosus patients (80.0%) exhibited IgG reactivity. It was also highly specific (93.7%) in discriminating between lichen sclerosus and other disease/control sera. Higher anti-ECM1 titers also correlated with more longstanding and refractory disease and cases complicated by squamous cell carcinoma. Furthermore, passive transfer of affinity-purified patient IgG reproduced some histologic and immunopathologic features of lichen sclerosus skin. This new ELISA is valuable for the accurate detection and quantification of anti-ECM1 autoantibodies. Moreover, the values may have clinical significance in patients with lichen sclerosus. PMID:15173881

  9. Screening of hepatocyte proteins binding with C?terminally truncated surface antigen middle protein of hepatitis B virus (MHBst167) by a yeast two?hybrid system.

    PubMed

    Li, Zhi Qun; Linghu, Enqiang; Jun, Wan; Cheng, Jun

    2014-09-01

    The function of middle hepatitis B surface protein C?terminally truncated at amino acid position 167 (MHBst167) is not currently clear. This study aimed to screen and identify the proteins that interact with MHBst167 in hepatocytes using a yeast two?hybrid system, and to explore the effects of MHBst167 in the development of hepatocellular carcinoma and precancerous diseases of the liver. The MHBst167 gene was amplified by polymerase chain reaction (PCR) and cloned into a pGEM?T vector. The target region was sequenced and the constructed bait plasmid, pGBKT7?MHBst167, was transformed into AH109 yeast cells. The transformed AH109 cells were then mated with Y187 yeast cells containing the fetal liver cDNA library plasmid using a yeast two?hybrid system. The false positives were eliminated and the true positive clones were selected by PCR and sequencing analysis. The pGBKT7?MHBst167 bait plasmid was successfully constructed and 66 clones grew in the selective synthetic defined media lacking leucine, tryptophan, histidine and adenine. Fifty?two clones were identified following X???Gal selection and segregation analysis. Seven proteins were found to be expressed that could interact with MHBst167 in hepatocytes by the yeast two?hybrid system. These results have provided novel insights into the biological functions of MHBst167. PMID:24968805

  10. Specific inhibition of GPI-anchored protein function by homing and self-association of specific GPI anchors.

    PubMed

    Nicholson, Thomas B; Stanners, Clifford P

    2006-11-20

    The functional specificity conferred by glycophosphatidylinositol (GPI) anchors on certain membrane proteins may arise from their occupancy of specific membrane microdomains. We show that membrane proteins with noninteractive external domains attached to the same carcinoembryonic antigen (CEA) GPI anchor, but not to unrelated neural cell adhesion molecule GPI anchors, colocalize on the cell surface, confirming that the GPI anchor mediates association with specific membrane domains and providing a mechanism for specific signaling. This directed targeting was exploited by coexpressing an external domain-defective protein with a functional protein, both with the CEA GPI anchor. The result was a complete loss of signaling capabilities (through integrin-ECM interaction) and cellular effect (differentiation blockage) of the active protein, which involved an alteration of the size of the microdomains occupied by the active protein. This work clarifies how the GPI anchor can determine protein function, while offering a novel method for its modulation. PMID:17101695

  11. Aptamer-targeted Antigen Delivery

    PubMed Central

    Wengerter, Brian C; Katakowski, Joseph A; Rosenberg, Jacob M; Park, Chae Gyu; Almo, Steven C; Palliser, Deborah; Levy, Matthew

    2014-01-01

    Effective therapeutic vaccines often require activation of T cell-mediated immunity. Robust T cell activation, including CD8 T cell responses, can be achieved using antibodies or antibody fragments to direct antigens of interest to professional antigen presenting cells. This approach represents an important advance in enhancing vaccine efficacy. Nucleic acid aptamers present a promising alternative to protein-based targeting approaches. We have selected aptamers that specifically bind the murine receptor, DEC205, a C-type lectin expressed predominantly on the surface of CD8?+ dendritic cells (DCs) that has been shown to be efficient at facilitating antigen crosspresentation and subsequent CD8+ T cell activation. Using a minimized aptamer conjugated to the model antigen ovalbumin (OVA), DEC205-targeted antigen crosspresentation was verified in vitro and in vivo by proliferation and cytokine production by primary murine CD8+ T cells expressing a T cell receptor specific for the major histocompatibility complex (MHC) I-restricted OVA257–264 peptide SIINFEKL. Compared with a nonspecific ribonucleic acid (RNA) of similar length, DEC205 aptamer-OVA-mediated antigen delivery stimulated strong proliferation and production of interferon (IFN)-? and interleukin (IL)-2. The immune responses elicited by aptamer-OVA conjugates were sufficient to inhibit the growth of established OVA-expressing B16 tumor cells. Our results demonstrate a new application of aptamer technology for the development of effective T cell-mediated vaccines. PMID:24682172

  12. Pediatric related and unrelated cord blood transplantation for malignant diseases

    Microsoft Academic Search

    V Rocha; N Kabbara; I Ionescu; A Ruggeri; D Purtill; E Gluckman

    2009-01-01

    The use of umbilical or placental donor cord blood transplantation (CBT) in children with malignant and non-malignant diseases has witnessed important progress, mainly because of better cord blood donor choice and patient selection translating into better patient outcome. Approximately 2000 children with malignant diseases (about 75 % with acute leukemias) have been transplanted with a related (n=199) or unrelated CBT

  13. Seemingly unrelated regression model with unequal size observations: computational aspects

    Microsoft Academic Search

    Paolo Foschi; Erricos John Kontoghiorghes

    2002-01-01

    The computational solution of the seemingly unrelated regression model with unequal size observations is considered. Two algorithms to solve the model when treated as a generalized linear least-squares problem are proposed. The algorithms have as a basic tool the generalized QR decomposition (GQRD) and efficiently exploit the block-sparse structure of the matrices. One of the algorithms reduces the computational burden

  14. Computational methods for modifying seemingly unrelated regressions models

    Microsoft Academic Search

    Erricos John Kontoghiorghes

    2004-01-01

    Computational efficient methods for updating seemingly unrelated regressions models with new observations are proposed. A recursive algorithm to solve a series of updating problems is developed. The algorithm is based on orthogonal transformations and has as main computational tool the updated generalized QR decomposition (UGQRD). Strategies to compute the orthogonal factorizations by exploiting the block-sparse structure of the matrices are

  15. Estimating seemingly unrelated regression models with vector autoregressive disturbances

    Microsoft Academic Search

    Paolo Foschi; Erricos J. Kontoghiorghes

    2003-01-01

    The numerical solution of seemingly unrelated regression (SUR) models with vector autoregressive disturbances is considered. Initially, an orthogonal transformation is applied to reduce the model to one with smaller dimensions. The transformed model is expressed as a reduced-size SUR model with stochastic constraints. The generalized QR decomposition is used as the main computational tool to solve this model. An iterative

  16. Mechanism Design for Fractional Scheduling on Unrelated Machines

    Microsoft Academic Search

    George Christodoulou; Elias Koutsoupias; Annamária Kovács

    2007-01-01

    Scheduling on unrelated machines is one of the most general and classical variants of the task scheduling problem. Fractional scheduling is the LP-relaxation of the problem, which is polynomially solvable in the offline setting, and is a useful tool to design deterministic and randomized approximation algorithms. The mechanism design version of the scheduling problem was introduced by Nisan and Ronen

  17. Characterization of the immune response to Leishmania infantum recombinant antigens

    Microsoft Academic Search

    Manuel Soto; Selma Jerônimo; Blaise Dondji; Olívia Bacellar; Viviane Luz; Glória Orge Orge; Carlos Alonso

    2003-01-01

    Leishmaniases have a high prevalence in tropical countries. In order to improve existing diagnostic systems based on total Leishmania proteins, and to identify antigen candidates for vaccine development, an intensive search for the identification of antigens was performed using molecular biology techniques. In this study, the immune response to three L. infantum recombinant antigens was evaluated. Upon stimulation with KMP11, mononuclear

  18. Natural Selection Promotes Antigenic Evolvability

    PubMed Central

    Graves, Christopher J.; Ros, Vera I. D.; Stevenson, Brian; Sniegowski, Paul D.; Brisson, Dustin

    2013-01-01

    The hypothesis that evolvability - the capacity to evolve by natural selection - is itself the object of natural selection is highly intriguing but remains controversial due in large part to a paucity of direct experimental evidence. The antigenic variation mechanisms of microbial pathogens provide an experimentally tractable system to test whether natural selection has favored mechanisms that increase evolvability. Many antigenic variation systems consist of paralogous unexpressed ‘cassettes’ that recombine into an expression site to rapidly alter the expressed protein. Importantly, the magnitude of antigenic change is a function of the genetic diversity among the unexpressed cassettes. Thus, evidence that selection favors among-cassette diversity is direct evidence that natural selection promotes antigenic evolvability. We used the Lyme disease bacterium, Borrelia burgdorferi, as a model to test the prediction that natural selection favors amino acid diversity among unexpressed vls cassettes and thereby promotes evolvability in a primary surface antigen, VlsE. The hypothesis that diversity among vls cassettes is favored by natural selection was supported in each B. burgdorferi strain analyzed using both classical (dN/dS ratios) and Bayesian population genetic analyses of genetic sequence data. This hypothesis was also supported by the conservation of highly mutable tandem-repeat structures across B. burgdorferi strains despite a near complete absence of sequence conservation. Diversification among vls cassettes due to natural selection and mutable repeat structures promotes long-term antigenic evolvability of VlsE. These findings provide a direct demonstration that molecular mechanisms that enhance evolvability of surface antigens are an evolutionary adaptation. The molecular evolutionary processes identified here can serve as a model for the evolution of antigenic evolvability in many pathogens which utilize similar strategies to establish chronic infections. PMID:24244173

  19. Protective Oral Vaccination against Infectious bursal disease virus Using the Major Viral Antigenic Protein VP2 Produced in Pichia pastoris

    PubMed Central

    Taghavian, Omid; Spiegel, Holger; Hauck, Rüdiger; Hafez, Hafez M.; Fischer, Rainer; Schillberg, Stefan

    2013-01-01

    Infectious bursal disease virus (IBDV) causes economically important immunosuppressive disease in young chickens. The self-assembling capsid protein (VP2) from IBDV strain IR01 was expressed in Pichia pastoris resulting in the formation of homomeric, 23-nm infectious bursal disease subviral particles (IBD-SVPs) with a yield of 76 mg/l before and 38 mg/l after purification. Anti-IBDV antibodies were detected in chickens injected with purified IBD-SVPs or fed with either purified IBD-SVPs or inactivated P. pastoris cells containing IBD-VP2 (cell-encapsulated). Challenge studies using the heterologous classical IBDV strain (MB3) showed that intramuscular vaccination with 20 µg purified IBD-SVPs conferred full protection, achieved complete virus clearance and prevented bursal damage and atrophy, compared with only 40% protection, 0–10% virus clearance accompanied by severe atrophy and substantial bursal damage in mock-vaccinated and challenge controls. The commercial IBDV vaccine also conferred full protection and achieved complete virus clearance, albeit with partial bursal atrophy. Oral administration of 500 µg purified IBD-SVPs with and without adjuvant conferred 100% protection but achieved only 60% virus clearance with adjuvant and none without it. Moderate bursal damage was observed in both cases but the inclusion of adjuvant resulted in bursal atrophy similar to that observed with live-attenuated vaccine and parenteral administration of 20 µg purified IBD-SVPs. The oral administration of 250 mg P. pastoris cells containing IBD-VP2 resulted in 100% protection with adjuvant and 60% without, accompanied by moderate bursal damage and atrophy in both groups, whereas 25 mg P. pastoris cells containing IBD-VP2 resulted in 90–100% protection with moderate bursal lesions and severe atrophy. Finally, the oral delivery of 50 µg purified IBD-SVPs achieved 40–60% protection with severe bursal lesions and atrophy. Both oral and parenteral administration of yeast-derived IBD-VP2 can therefore induce a specific and protective immune response against IBDV without affecting the growth rate of chickens. PMID:24376665

  20. THE LS-ANTIGEN OF VACCINIA

    PubMed Central

    Smadel, Joseph E.; Hoagland, Charles L.; Shedlovsky, Theodore

    1943-01-01

    Pure LS-antigen of vaccinia contains 15.8 per cent N and 50.6 per cent C. These analytical data together with the absence of lipids, phosphorus, nucleic acid, and glucosamine in preparations of the antigen confirm the protein nature of the substance. The action of proteolytic enzymes on LS offers further confirmation of the protein character of the antigen. Both the L- and S-activities of the antigen are destroyed by digestion with papain. The effects of crystalline chymotrypsin on LS-antigen are particularly interesting for, under proper conditions, this enzyme destroys the serological activity of the S-portion of the molecule without affecting the L-portion. This newly prepared degradation product of LS, called LS'', contains the same amount of N as the native substance but unlike LS, it forms needle-shaped crystalloids. PMID:19871272

  1. Plasmodium simium, a Plasmodium vivax-Related Malaria Parasite: Genetic Variability of Duffy Binding Protein II and the Duffy Antigen/Receptor for Chemokines.

    PubMed

    Camargos Costa, Daniela; Pereira de Assis, Gabriela Maíra; de Souza Silva, Flávia Alessandra; Araújo, Flávia Carolina; de Souza Junior, Júlio César; Braga Hirano, Zelinda Maria; Satiko Kano, Flora; Nóbrega de Sousa, Taís; Carvalho, Luzia Helena; Ferreira Alves de Brito, Cristiana

    2015-01-01

    Plasmodium simium is a parasite from New World monkeys that is most closely related to the human malaria parasite Plasmodium vivax; it also naturally infects humans. The blood-stage infection of P. vivax depends on Duffy binding protein II (PvDBPII) and its cognate receptor on erythrocytes, the Duffy antigen receptor for chemokines (hDARC), but there is no information on the P. simium erythrocytic invasion pathway. The genes encoding P. simium DBP (PsDBPII) and simian DARC (sDARC) were sequenced from Southern brown howler monkeys (Alouatta guariba clamitans) naturally infected with P. simium because P. simium may also depend on the DBPII/DARC interaction. The sequences of DBP binding domains from P. vivax and P. simium were highly similar. However, the genetic variability of PsDBPII was lower than that of PvDBPII. Phylogenetic analyses demonstrated that these genes were strictly related and clustered in the same clade of the evolutionary tree. DARC from A. clamitans was also sequenced and contained three new non-synonymous substitutions. None of these substitutions were located in the N-terminal domain of DARC, which interacts directly with DBPII. The interaction between sDARC and PvDBPII was evaluated using a cytoadherence assay of COS7 cells expressing PvDBPII on their surfaces. Inhibitory binding assays in vitro demonstrated that antibodies from monkey sera blocked the interaction between COS-7 cells expressing PvDBPII and hDARC-positive erythrocytes. Taken together, phylogenetic analyses reinforced the hypothesis that the host switch from humans to monkeys may have occurred very recently in evolution, which sheds light on the evolutionary history of new world plasmodia. Further invasion studies would confirm whether P. simium depends on DBP/DARC to trigger internalization into red blood cells. PMID:26107662

  2. A novel recombinant bivalent outer membrane protein of Vibrio vulnificus and Aeromonas hydrophila as a vaccine antigen of American eel (Anguilla rostrata).

    PubMed

    SongLin, Guo; PanPan, Lu; JianJun, Feng; JinPing, Zhao; Peng, Lin; LiHua, Duan

    2015-04-01

    The immogenicity of a novel vaccine antigen was evaluated after immunized American eels (Anguilla rostrata) with a recombinant bivalent expressed outer membrane protein (OMP) of Vibrio vulnificus and Aeromonas hydrophila. Three groups of eels were intraperitoneal (i.p) injected with phosphate-buffered saline (PBS group), formaline-killed-whole-cell (FKC) of A. hydrophila and V. vulnificus (FKC group) or the bivalent OMP (OMP group). On 14, 21, 28 and 42 days post-vaccination respectively, proliferation of the whole blood cells, titers of specific antibody and lysozyme activities of experimental eels were detected. On 28 day post-vaccination, eels from three groups were challenged by i.p injection of live A. hydrophila or V. vulnificus. The results showed that, compared with the PBS group, proliferation of whole blood cells in OMP group was significant enhanced on 28 days, and the serum titers of anti-A.hydrophila and anti-V. vulnificus antibody in eels of FKC and OMP group were significant increased on 14, 21 and 28d. Lysozyme Activities in serum, skin mucus, liver and kidney were significant changed between the three groups. Relative Percent Survival (RPS) after challenged A. hydrophila in KFC vs. PBS group and OMP vs. PBS group were 62.5% and 50% respectively, and the RPS challenged V. vulnificus in FKC and OMP vs. PBS group were 37.5% and 50% respectively. These results suggest that American eels immunized with the bivalent OMP would positively affect specific as well as non-specific immune parameters and protect against infection by the two pathogens in fresh water farming. PMID:25655329

  3. Value of detection of serum human epididymis secretory protein 4 and carbohydrate antigen 125 in diagnosis of early endometrial cancer of different pathological subtypes

    PubMed Central

    Liu, Xingui; Zhao, Fengting; Hu, Linli; Sun, Yingpu

    2015-01-01

    This study explored the value of detection of human epididymis secretory protein 4 (HE4) and carbohydrate antigen 125 (CA125) from serum in diagnosis of early endometrial cancer of different pathological subtypes and discussed the mechanism of HE4 and CA125 in diagnosis. In this study, enzyme-linked immunosorbent assay and chemiluminescent immunoassay were used to detect HE4 and CA125 from serum in endometrial cancer and control groups. Besides, the concentration of HE4 and CA125 was compared in these two groups, and the expression of CA125 and HE4 and clinicopathological characteristics in patients with endometrial cancer were also analyzed. Compared with the control group, the expression of HE4 was much higher in serum of patients with endometrial cancer, while there was no obvious change in the expression of CA125. The threshold detection value was acquired by receiver operating characteristic analysis method, that is, 141.5 pmol/L and 54.5 U/L, respectively. When comparing the concentration of HE4 in patients with endometrial cancer at the early stage (stage I) with healthy people, the difference therein had statistical significance, but there was no obvious difference in CA125. HE4 and CA125 in diagnosis of endometrial cancer in the stages I and II were found with no statistically significant difference. The difference of HE4 in the stages II and III had statistical significance while the difference of CA125 had no statistical significance. The specificity of both HE4 and CA125 was 95%, and the sensitivity of HE4 to uterine papillary serous carcinomas was higher than that to endometrioid adenocarcinoma. Thus, the serum HE4 is much better than CA125 in detecting the endometrial cancer at an early stage.

  4. Genome-wide association replicates the association of Duffy antigen receptor for chemokines (DARC) polymorphisms with serum monocyte chemoattractant protein-1 (MCP-1) levels in Hispanic children

    PubMed Central

    Voruganti, V. Saroja; Laston, Sandra; Haack, Karin; Mehta, Nitesh R.; Smith, C. Wayne; Cole, Shelley A.; Butte, Nancy F.; Comuzzie, Anthony G.

    2012-01-01

    Obesity is associated with a chronic low inflammatory state characterized by elevated levels of chemokines. Monocyte chemoattractant protein-1 (MCP-1) is a member of the cysteine-cysteine (CC) chemokine family and is increased in obesity. The purpose of this study was to identify loci regulating serum MCP-1 in obese Hispanic children from the Viva La Familia Study. A genome-wide association (GWA) analysis was performed in 815 children, ages 4-19 years, using genotypes assayed with the Illumina HumanOmni1-Quad v1.0 BeadChips. All analyses were performed in SOLAR using a linear regression-based test under an additive model of allelic effect, while accounting for the relatedness of family members via a kinship variance component. The strongest association for MCP-1 levels was found with a non-synonymous single nucleotide polymorphism (SNP), rs12075 resulting in an amino acid substitution (Asp42Gly) in the Duffy antigen receptor for chemokines (DARC) gene product (minor allele frequency = 43.6%, p = 1.3 × 10?21) on chromosome 1. Four other DARC SNPs were also significantly associated with MCP-1 levels(p < 10?16-10?6). The Asp42Gly variant was associated with higher levels of MCP-1 and accounted for approximately 10% of its variability. In addition, MCP-1 levels were significantly associated with SNPs in chemokine receptor 3 (CCR3) and caspase recruitment domain family, member 9 (CARD9). In summary, the association of the DARC Asp42Gly variant with MCP-1 levels replicates previous GWA results substantiating a potential role for DARC in the regulation of pro-inflammatory cytokines. PMID:23017229

  5. Pre-Treatment Levels of C-Reactive Protein and Squamous Cell Carcinoma Antigen for Predicting the Aggressiveness of Pharyngolaryngeal Carcinoma

    PubMed Central

    Chen, Hsuan-Ho; Wang, Hung-Ming; Fan, Kang-Hsing; Lin, Chien-Yu; Yen, Tzu-Chen; Liao, Chun-Ta; Chen, I-How; Kang, Chung-Jan; Huang, Shiang-Fu

    2013-01-01

    The levels of squamous cell carcinoma antigen (SCC-Ag) and C-reactive protein (CRP) can be used to predict tumor invasion, lymph node metastasis, staging and survival in patients with oral cavity cancer. The present study analyzed the relationship between pre-treatment levels of SCC-Ag and CRP in relation to clinicopathological factors in patients with pharyngolaryngeal cancer (PLC) and determined whether elevated levels of CRP and SCC-Ag were associated with tumor metabolic activity via [18F] fluorodeoxyglucose positron emission tomography (FDG-PET). We retrospectively recruited one hundred and six PLC patients between June 2008 and December 2011. All patients received computed tomography (CT)/magnetic resonance imaging (MRI) and FDG-PET staging analyses, and the serum levels of SCC-Ag and CRP in these patients were measured prior to treatment. A SCC-Ag level ?2.0 ng/ml and a CRP level ?5.0 mg/L were significantly associated with clinical stage (P<0.001), clinical tumor status (P<0.001), and clinical nodal status (P<0.001). The elevation of both SCC-Ag and CRP levels was correlated with the standardized uptake value (SUV) max of the tumor (?8.6 mg/L) and lymph nodes (?5.7 ng/ml) (P?=?0.019). The present study demonstrated that the presence of high levels of both pre-treatment SCC-Ag and CRP acts as a predictor of clinical stage, clinical tumor status, and clinical nodal status in patients with PLC. Moreover, elevated levels of SCC-Ag and CRP were associated with a high metabolic rate as well as the proliferative activity measured according to the SUVmax of the tumor and lymph nodes. Therefore, elevated levels of these two factors have the potential to serve as biomarkers for the prediction of tumor aggressiveness in cases of PLC. PMID:23383155

  6. Plasmodium simium, a Plasmodium vivax-Related Malaria Parasite: Genetic Variability of Duffy Binding Protein II and the Duffy Antigen/Receptor for Chemokines

    PubMed Central

    Camargos Costa, Daniela; Pereira de Assis, Gabriela Maíra; de Souza Silva, Flávia Alessandra; Araújo, Flávia Carolina; de Souza Junior, Júlio César; Braga Hirano, Zelinda Maria; Satiko Kano, Flora; Nóbrega de Sousa, Taís; Carvalho, Luzia Helena; Ferreira Alves de Brito, Cristiana

    2015-01-01

    Plasmodium simium is a parasite from New World monkeys that is most closely related to the human malaria parasite Plasmodium vivax; it also naturally infects humans. The blood-stage infection of P. vivax depends on Duffy binding protein II (PvDBPII) and its cognate receptor on erythrocytes, the Duffy antigen receptor for chemokines (hDARC), but there is no information on the P. simium erythrocytic invasion pathway. The genes encoding P. simium DBP (PsDBPII) and simian DARC (sDARC) were sequenced from Southern brown howler monkeys (Alouatta guariba clamitans) naturally infected with P. simium because P. simium may also depend on the DBPII/DARC interaction. The sequences of DBP binding domains from P. vivax and P. simium were highly similar. However, the genetic variability of PsDBPII was lower than that of PvDBPII. Phylogenetic analyses demonstrated that these genes were strictly related and clustered in the same clade of the evolutionary tree. DARC from A. clamitans was also sequenced and contained three new non-synonymous substitutions. None of these substitutions were located in the N-terminal domain of DARC, which interacts directly with DBPII. The interaction between sDARC and PvDBPII was evaluated using a cytoadherence assay of COS7 cells expressing PvDBPII on their surfaces. Inhibitory binding assays in vitro demonstrated that antibodies from monkey sera blocked the interaction between COS-7 cells expressing PvDBPII and hDARC-positive erythrocytes. Taken together, phylogenetic analyses reinforced the hypothesis that the host switch from humans to monkeys may have occurred very recently in evolution, which sheds light on the evolutionary history of new world plasmodia. Further invasion studies would confirm whether P. simium depends on DBP/DARC to trigger internalization into red blood cells. PMID:26107662

  7. Human biliverdin reductase suppresses Goodpasture antigen-binding protein (GPBP) kinase activity: the reductase regulates tumor necrosis factor-alpha-NF-kappaB-dependent GPBP expression.

    PubMed

    Miralem, Tihomir; Gibbs, Peter E M; Revert, Fernando; Saus, Juan; Maines, Mahin D

    2010-04-23

    The Ser/Thr/Tyr kinase activity of human biliverdin reductase (hBVR) and the expression of Goodpasture antigen-binding protein (GPBP), a nonconventional Ser/Thr kinase for the type IV collagen of basement membrane, are regulated by tumor necrosis factor (TNF-alpha). The pro-inflammatory cytokine stimulates kinase activity of hBVR and activates NF-kappaB, a transcriptional regulator of GPBP mRNA. Increased GPBP activity is associated with several autoimmune conditions, including Goodpasture syndrome. Here we show that in HEK293A cells hBVR binds to GPBP and down-regulates its TNF-alpha-stimulated kinase activity; this was not due to a decrease in GPBP expression. Findings with small interfering RNA to hBVR and to the p65 regulatory subunit of NF-kappaB show the hBVR role in the initial stimulation of GPBP expression by TNF-alpha-activated NF-kappaB; hBVR was not a factor in mediating GPBP mRNA stability. The interacting domain was mapped to the (281)CX(10)C motif in the C-terminal 24 residues of hBVR. A 7-residue peptide, KKRILHC(281), corresponding to the core of the consensus D(delta)-Box motif in the interacting domain, was as effective as the intact 296-residue hBVR polypeptide in inhibiting GPBP kinase activity. GPBP neither regulated hBVR expression nor TNF-alpha dependent NF-kappaB expression. Collectively, our data reveal that hBVR is a regulator of the TNF-alpha-GPBP-collagen type IV signaling cascade and uncover a novel biological interaction that may be of relevance in autoimmune pathogenesis. PMID:20177069

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

    PubMed Central

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

    2015-01-01

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

  9. Retroposition in a family of carcinoma-associated antigen genes.

    PubMed Central

    Linnenbach, A J; Seng, B A; Wu, S; Robbins, S; Scollon, M; Pyrc, J J; Druck, T; Huebner, K

    1993-01-01

    The gene encoding the carcinoma-associated antigen defined by the monoclonal antibody GA733 is a member of a family of at least two type I membrane proteins. This study describes the mechanism of evolution of the GA733-1 and GA733-2 genes. A full-length cDNA clone for GA733-1 was obtained by screening a human placental library with a genomic DNA probe. Comparative analysis of the cDNA sequence with the previously determined genomic sequence confirmed that GA733-1 is an intronless gene. The GA733-2 gene encoding the monoclonal antibody-defined antigen was molecularly cloned with a cDNA probe and partially sequenced. Comparison of GA733-2 gene sequences with the previously established cDNA sequence revealed that this gene consists of nine exons. The putative promoter regions of the GA733-1 and GA733-2 genes are unrelated. These findings suggest that the GA733-1 gene was formed by the retroposition of the GA733-2 gene via an mRNA intermediate. Prior to retroposition, the GA733-2 gene had been affected by exon shuffling. Analysis of GA733-2 exons revealed that many delineate structural motifs. The GA733-1 retroposon was localized either to chromosome region 1p32-1p31 or to 1p13-1q12, and the GA733-2 founder gene was localized to chromosome 4q. Images PMID:8382772

  10. Basophils as antigen presenting cells.

    PubMed

    Maddur, Mohan S; Kaveri, Srini V; Bayry, Jagadeesh

    2010-02-01

    Recent reports demonstrate that basophils act as antigen presenting cells to drive Th2 and IgE responses against protease and protein allergens and helminth parasites. Through MHC class II-dependent cognate interactions with CD4(+) T cells in the context of co-stimulatory molecules, and through secretion of IL-4, IL-13 and thymic stromal lymphopoietin, basophils drive antigen-specific Th2 responses. These results have uncovered previously unknown functions of basophils, and should aid in designing novel therapeutic strategies for asthma and allergic conditions. PMID:20060781

  11. Differential expression of S19 ribosomal protein, laminin-binding protein, and human lymphocyte antigen class I messenger RNAs associated with colon carcinoma progression and differentiation.

    PubMed

    Kondoh, N; Schweinfest, C W; Henderson, K W; Papas, T S

    1992-02-15

    Three complementary DNA encoding S19 ribosomal protein (S19), laminin-binding protein (LBP), and HLA class I (HLA-I) genes were isolated from a colon tumor-enriched subtraction library. To evaluate this mRNA expression, surgically removed colon tumors as well as matched normal tissue and human colon carcinoma cell lines showing various differentiation states, anchorage dependence, and proliferation states were examined by Northern blot analysis. The mRNA level of S19 mRNA (0.6 kilobase) was higher in primary colon carcinoma tissue than in matched normal colon tissue in 5 of 6 cases. In 2 of 4 cases, the expression of LBP mRNA (1.2 kilobases) was higher in carcinoma than in normal tissue. In 12 human colon cell lines, the level of LBP mRNA was higher in poorly differentiated cells. On the other hand, HLA-I mRNA (1.7 kilobases) was higher in well-differentiated cells. Although the S19 mRNA was expressed in both well- and poorly differentiated cells, a concomitant increase with tumor progression was observed in two pairs of cell lines derived from the same patients (SW480 and SW620; COLO201 and COLO205). Anchorage dependence of butyrate-treated HT29 colon carcinoma cells was correlated with lower levels of S19 and LBP mRNAs and higher levels of HLA-I mRNA expression compared with untreated cells. While the expression of S19 and LBP mRNAs was not changed due to cell growth states, HLA-I mRNA levels were found to be low in proliferating HT29 cells but highly induced in contact-inhibited cells. In summary, therefore, high expression of S19 and LBP combined with low expression of HLA-I were well correlated with colon carcinoma cells of higher malignant potential. PMID:1339304

  12. Monoclonal and polyclonal antibodies against Epstein-Barr virus nuclear antigen 5 (EBNA-5) detect multiple protein species in Burkitt's lymphoma and lymphoblastoid cell lines.

    PubMed Central

    Finke, J; Rowe, M; Kallin, B; Ernberg, I; Rosén, A; Dillner, J; Klein, G

    1987-01-01

    The Epstein-Barr virus nuclear antigen 5 (EBNA-5) is encoded by highly spliced mRNA from the major IR1 (BamHI-W) repeat region of the virus genome. A mouse monoclonal antibody, JF186, has been raised against a synthetic 18-amino-acid peptide deduced from the EBNA-5 message of B95-8 and Raji cells. The antibody showed characteristic coarse nuclear granules by indirect immunofluorescence and revealed multiple EBNA-5 species by immunoblotting and immunoprecipitation. The B95-8 line itself and all B95-8 virus-carrying cells, whether lymphoblastoid cell lines or in vitro-converted sublines of Epstein-Barr virus (EBV)-negative Burkitt's lymphoma (BL) lines, were EBNA-5 positive. Among 36 cell lines carrying different EBV strains, only 10 expressed the B95-8-Raji-prototype EBNA-5 recognized by JF186; this was probably due to genetic variation in the epitope recognized by JF186, as shown for P3HR-1. Human antibodies, affinity purified against EBNA-5-JF186 immunoprecipitates, detected EBNA-5 in the majority of EBV-positive BL lines and in all lymphoblastoid cell lines containing the BL-derived viruses. Thus, EBNA-5 can be expressed by all virus isolates examined, but is down-regulated, together with other latent gene products, in a minority of BL lines which have a particular cellular phenotype. EBNA-5 was detected as a ladder of protein species of 20 to 130 kilodaltons (kDa), with a regular spacing of 6 to 8 kDa, consistent with the coding capacity of the combined BamHI-W 66- and 132-base-pair exons, together with shifts of 2 to 4 kDa, consistent with the size of the separate 66- and 132-base-pair exons. Multiple EBNA-5 proteins can be expressed by the single cell as shown by cloning of newly infected cells. Images PMID:2824821

  13. Simian virus 40 large T antigen alters the phosphorylation state of the RB-related proteins p130 and p107.

    PubMed Central

    Stubdal, H; Zalvide, J; DeCaprio, J A

    1996-01-01

    p130 and p107 are nuclear phosphoproteins related to the retinoblastoma gene product (pRb). pRb, p107, and p130 each undergo cell cycle-dependent phosphorylation, form complexes with the E2F family of transcription factors, and associate with oncoproteins of DNA tumor viruses, including simian virus 40 (SV40) large T antigen (TAg) and adenovirus ElA protein. The results of recent studies with mouse embryo fibroblasts (MEFs) lacking the retinoblastoma gene (Rb-1) have suggested that p130 and p107 may be important targets for SV40 large TAg-mediated transformation (J.B. Christensen and M.J. Imperiale, J. Virol. 65:3945-3948, 1995; J. Zalvide and J.A. DeCaprio, Mol. Cell. Biol. 15:5800-5810, 1995). In this report, we demonstrate that the expression of TAg affects the phosphorylation state of p130 and p107. In cells expressing wild-type TAg, only un(der)phosphorylated p130 and p107 were detected. To determine the domains within TAg that contribute to this effect on the phosphorylation of p130, we performed transient expression assays. While transiently expressed p130 was apparently phosphorylated normally, only un(der)phosphorylated p130 was detected when p130 was coexpressed with TAg. Using this assay, we found that the first 147 amino acids of TAg were sufficient to alter the phosphorylation state of p130. Within this region, the LXCXE domain of TAg, required for binding to the retinoblastoma family of proteins, was necessary but not sufficient to affect p130 phosphorylation. Residues within the first 82 amino acids of TAg were also required. TAg with mutations in the N terminus retained the ability to efficiently associate with p130 but did not affect its phosphorylation state. This demonstrates that the effect of SV40 TAg on p130 is not simply the result of binding and suggests that TAg has a novel effect on p130 and p107 that differs from its effect on pRb. PMID:8627752

  14. Conformational Dependence and Conservation of an Immunodominant Epitope within the Babesia equi Erythrocyte-Stage Surface Protein Equi Merozoite Antigen 1

    PubMed Central

    Cunha, Cristina W.; Kappmeyer, Lowell S.; McGuire, Travis C.; Dellagostin, Odir A.; Knowles, Donald P.

    2002-01-01

    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 quantify the amino acid conservation of EMA-1, including the region containing the epitope bound by MAb 36/133.97. The alignment of the deduced amino acid sequence of full-length EMA-1 (Florida isolate) with 15 EMA-1 sequences from geographically distinct isolates showed 82.8 to 99.6% identities (median, 98.5%) and 90.5 to 99.6% similarities (median, 98.9%) between sequences. Full-length and truncated recombinant EMA-1 proteins were expressed and tested for their reactivities with MAb 36/133.97. Binding required the presence of amino acids on both N- and C-terminal regions of a truncated peptide (EMA-1.2) containing amino acids 1 to 98 of EMA-1. This result indicated that the epitope defined by MAb 36/133.97 is dependent on conformation. Sera from persistently infected horses inhibited the binding of MAb 36/133.97 to EMA-1.2 in a competitive ELISA, indicating that equine antibodies which inhibit binding of MAb 36/133.97 also recognize epitopes in the same region (the first 98 residues). Within this region, the deduced amino acid sequences had 85.7 to 100% identities (median, 99.0%), with similarities of 94.9 to 100% (median, 100%). Therefore, the region which binds to both MAb 36/133.97 and inhibiting equine antibodies has a median amino acid identity of 99.0% and a similarity of 100%. These data provide a molecular basis for the use of both EMA-1 and MAb 36/133.97 for the detection of antibodies against B. equi. PMID:12414764

  15. Purification and protective efficacy of monomeric and modified Yersinia pestis capsular F1-V antigen fusion proteins for vaccination against plague.

    PubMed

    Goodin, Jeremy L; Nellis, David F; Powell, Bradford S; Vyas, Vinay V; Enama, Jeffrey T; Wang, Lena C; Clark, Patrick K; Giardina, Steven L; Adamovicz, Jeffery J; Michiel, Dennis F

    2007-05-01

    The F1-V vaccine antigen, protective against Yersinia pestis, exhibits a strong tendency to multimerize that affects larger-scale manufacture and characterization. In this work, the sole F1-V cysteine was replaced with serine by site-directed mutagenesis for characterization of F1-V non-covalent multimer interactions and protective potency without participation by disulfide-linkages. F1-V and F1-V(C424S) proteins were overexpressed in Escherichia coli, recovered using mechanical lysis/pH-modulation and purified from urea-solubilized soft inclusion bodies, using successi