Academic literature on the topic 'Neisseria Heparin Binding Antigen'
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Journal articles on the topic "Neisseria Heparin Binding Antigen"
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Lucidarme, Jay, Stefanie Gilchrist, Lynne S. Newbold, et al. "Genetic Distribution of Noncapsular Meningococcal Group B Vaccine Antigens in Neisseria lactamica." Clinical and Vaccine Immunology 20, no. 9 (2013): 1360–69. http://dx.doi.org/10.1128/cvi.00090-13.
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ABSTRACTThe poor immunogenicity of the meningococcal serogroup B (MenB) capsule has led to the development of vaccines targeting subcapsular antigens, in particular the immunodominant and diverse outer membrane porin, PorA. These vaccines are largely strain specific; however, they offer limited protection against the diverse MenB-associated diseases observed in many industrialized nations. To broaden the scope of its protection, the multicomponent vaccine (4CMenB) incorporates a PorA-containing outer membrane vesicle (OMV) alongside relatively conserved recombinant protein components, including factor H-binding protein (fHbp),Neisseriaadhesin A (NadA), and neisserial heparin-binding antigen (NHBA). The expression of PorA is unique to meningococci (Neisseria meningitidis); however, many subcapsular antigens are shared with nonpathogenic members of the genusNeisseriathat also inhabit the nasopharynx. These organisms may elicit cross-protective immunity against meningococci and/or occupy a niche that might otherwise accommodate pathogens. The potential for 4CMenB responses to impact such species (and vice versa) was investigated by determining the genetic distribution of the primary 4CMenB antigens among diverse members of the common childhood commensal,Neisseria lactamica. All the isolates possessednhbabut were devoid offhbpandnadA. Thenhbaalleles were mainly distinct from but closely related to those observed among a representative panel of invasive MenB isolates from the same broad geographic region. We made similar findings for the immunogenic typing antigen, FetA, which constitutes a major part of the 4CMenB OMV. Thus, 4CMenB vaccine responses may impact or be impacted by nasopharyngeal carriage of commensal neisseriae. This highlights an area for further research and surveillance should the vaccine be routinely implemented.
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Semchenko, Evgeny A., Tsitsi D. Mubaiwa, Christopher J. Day, and Kate L. Seib. "Role of the Gonococcal Neisserial Heparin Binding Antigen in Microcolony Formation, and Serum Resistance and Adherence to Epithelial Cells." Journal of Infectious Diseases 221, no. 10 (2019): 1612–22. http://dx.doi.org/10.1093/infdis/jiz628.
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Abstract The sexually transmitted infection gonorrhoea is on the rise worldwide and an increased understanding of the mechanisms of colonization and pathogenesis of Neisseria gonorrhoeae is required to aid development of new treatment and prevention strategies. In the current study, we investigate the neisserial heparin-binding antigen (NHBA) of N. gonorrhoeae and confirm its role in binding to several glycans, including heparin, and identify interactions of NHBA with both gonococcal and host cells. Furthermore, we report that a gonococcal nhba mutant displays decreased cell aggregation and microcolony formation, as well as reduced survival in human serum and reduced adherence to human cervical and urethral epithelial cells, relative to the wild-type strain. These data indicate that the gonococcal NHBA contributes to several aspects of the colonization and survival of N. gonorrhoeae and may be a target for new antimicrobial or vaccines.
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Semchenko, Evgeny A., Christopher J. Day, and Kate L. Seib. "The Neisseria gonorrhoeae Vaccine Candidate NHBA Elicits Antibodies That Are Bactericidal, Opsonophagocytic and That Reduce Gonococcal Adherence to Epithelial Cells." Vaccines 8, no. 2 (2020): 219. http://dx.doi.org/10.3390/vaccines8020219.
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Due to the continuing emergence of multidrug resistant strains of Neisseria gonorrhoeae there is an urgent need for the development of a gonococcal vaccine. We evaluated the gonococcal Neisseria heparin binding antigen (NHBA) as a potential vaccine candidate, in terms of its sequence conservation and expression in a range of N. gonorrhoeae strains, as well as its immunogenicity and the functional activity of antibodies raised to either the full length NHBA or a C-terminal fragment of NHBA (NHBA-c). The gene encoding NHBA is highly conserved and expressed in all N. gonorrhoeae strains investigated. Recombinant NHBA is immunogenic, and mice immunized with either NHBA or NHBA-c adjuvanted with either Freund’s or aluminium hydroxide (alum) generated a humoral immune response, with predominantly IgG1 antibodies. Antibodies generated by both NHBA and NHBA-c antigens promoted complement activation and mediated bacterial killing via both serum bactericidal activity and opsonophagocytic activity, with slightly higher titers seen for the NHBA-c antigen. Anti-NHBA was also able to block the functional activity of NHBA by reducing binding to heparin and adherence to cervical and urethral epithelial cells. These data suggest that the gonococcal NHBA is a promising vaccine antigen to include in a vaccine to control N. gonorrhoeae.
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Semchenko, Evgeny A., Aimee Tan, Ray Borrow, and Kate L. Seib. "The Serogroup B Meningococcal Vaccine Bexsero Elicits Antibodies to Neisseria gonorrhoeae." Clinical Infectious Diseases 69, no. 7 (2018): 1101–11. http://dx.doi.org/10.1093/cid/ciy1061.
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Abstract Background Neisseria gonorrhoeae and Neisseria meningitidis are closely-related bacteria that cause a significant global burden of disease. Control of gonorrhoea is becoming increasingly difficult, due to widespread antibiotic resistance. While vaccines are routinely used for N. meningitidis, no vaccine is available for N. gonorrhoeae. Recently, the outer membrane vesicle (OMV) meningococcal B vaccine, MeNZB, was reported to be associated with reduced rates of gonorrhoea following a mass vaccination campaign in New Zealand. To probe the basis for this protection, we assessed the cross-reactivity to N. gonorrhoeae of serum raised to the meningococcal vaccine Bexsero, which contains the MeNZB OMV component plus 3 recombinant antigens (Neisseria adhesin A, factor H binding protein [fHbp]-GNA2091, and Neisserial heparin binding antigen [NHBA]-GNA1030). Methods A bioinformatic analysis was performed to assess the similarity of MeNZB OMV and Bexsero antigens to gonococcal proteins. Rabbits were immunized with the OMV component or the 3 recombinant antigens of Bexsero, and Western blots and enzyme-linked immunosorbent assays were used to assess the generation of antibodies recognizing N. gonorrhoeae. Serum from humans immunized with Bexsero was investigated to assess the nature of the anti-gonococcal response. Results There is a high level of sequence identity between MeNZB OMV and Bexsero OMV antigens, and between the antigens and gonococcal proteins. NHBA is the only Bexsero recombinant antigen that is conserved and surfaced exposed in N. gonorrhoeae. Bexsero induces antibodies in humans that recognize gonococcal proteins. Conclusions The anti-gonococcal antibodies induced by MeNZB-like OMV proteins could explain the previously-seen decrease in gonorrhoea following MeNZB vaccination. The high level of human anti-gonococcal NHBA antibodies generated by Bexsero vaccination may provide additional cross-protection against gonorrhoea.
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Vacca, Irene, Elena Del Tordello, Gianmarco Gasperini, et al. "Neisserial Heparin Binding Antigen (NHBA) Contributes to the Adhesion of Neisseria meningitidis to Human Epithelial Cells." PLOS ONE 11, no. 10 (2016): e0162878. http://dx.doi.org/10.1371/journal.pone.0162878.
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Di Fede, Martina, Massimiliano Biagini, Elena Cartocci, et al. "Neisseria Heparin Binding Antigen is targeted by the human alternative pathway C3-convertase." PLOS ONE 13, no. 3 (2018): e0194662. http://dx.doi.org/10.1371/journal.pone.0194662.
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Soler-Garcia, Aleix, Mariona Fernández de Sevilla, Raquel Abad, et al. "Meningococcal Serogroup B Disease in Vaccinated Children." Journal of the Pediatric Infectious Diseases Society 9, no. 4 (2019): 454–59. http://dx.doi.org/10.1093/jpids/piz071.
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Abstract Background Neisseria meningitidis serogroup B (MenB) is the most frequent cause of invasive meningococcal disease (IMD) in Spain. The multicomponent vaccine against MenB (4CMenB) was approved in Spain in January 2014. Methods We present 4 cases of children who developed MenB-associated IMD despite previous vaccination with 4CMenB. Extensive immunologic diagnostic work-up was performed in order to rule out any immunodeficiency. Also, molecular characterization of the MenB strain was conducted to determine whether bacterial antigens matched vaccine antigens. Results Among the 4 patients (2 girls), 2 had previous risk factors for IMD (recurrent bacterial meningitis of unknown origin and treatment with eculizumab). All patients developed meningitis, but only 2 developed septic shock; they were all cured without sequelae. No other primary or secondary immunodeficiencies were detected. MenB sequence type 213 was identified in 3 cases. With the exception of neisserial heparin-binding antigen peptide 465 present in 1 isolate, the rest of the isolated strains harbored vaccine antigen variants that did not match antigen variants included in the vaccine. Conclusions We present 4 children who developed MenB-associated IMD despite previous vaccination with 4CMenB. In 2 cases, the antibodies induced by 4CMenB likely were not effective against the isolated strains. A high level of suspicion for IMD seems advisable regardless of the patient’s vaccination history.
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Ispasanie, Emma, Gerd Pluschke, Abraham Hodgson, Ali Sie, Calman MacLennan, and Oliver Koeberling. "Characterization of vaccine antigens of meningococcal serogroup W isolates from Ghana and Burkina Faso from 2003 to 2009." F1000Research 3 (November 3, 2014): 264. http://dx.doi.org/10.12688/f1000research.3881.1.
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Neisseria meningitidis is a major cause of bacterial meningitis and a considerable health problem in the 25 countries of the ‘African Meningitis Belt’ that extends from Senegal in West Africa to Ethiopia in the East. Approximately 80% of cases of meningococcal meningitis in Africa have been caused by strains belonging to capsular serogroup A. After the introduction of a serogroup A conjugate polysaccharide vaccine, MenAfriVac™, that began in December 2010, the incidence of meningitis due to serogroup A has markedly declined in this region. Currently, serogroup W of N. meningitidis accounts for the majority of cases. Vaccines based on sub-capsular antigens, such as Generalized Modules for Membrane Antigens (GMMA), are under investigation for use in Africa. To analyse the antigenic properties of a serogroup W wave of colonisation and disease, we investigated the molecular diversity of the protein vaccine antigens PorA, Neisserial Adhesin A (NadA), Neisserial heparin-binding antigen (NHBA) and factor H binding protein (fHbp) of 31 invasive and carriage serogroup W isolates collected as part of a longitudinal study from Ghana and Burkina Faso between 2003 and 2009. We found that the isolates all expressed fHbp variant 2 ID 22 or 23, differing from each other by only one amino acid, and a single PorA subtype of P1.5,2. Of the isolates, 49% had a functional nhbA gene and 100% had the nadA allele 3, which contained the insertion sequence IS1301 in five isolates. Of the W isolates tested, 41% had high fHbp expression when compared with a reference serogroup B strain, known to be a high expresser of fHbp variant 2. Our results indicate that in this collection of serogroup W isolates, there is limited antigenic diversification over time of vaccine candidate outer membrane proteins (OMP), thus making them promising candidates for inclusion in a protein-based vaccine against meningococcal meningitis for Africa.
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Garcia, Yara Ruiz, Woo-Yun Sohn, Mariagrazia Pizza, and Rafik Bekkat-Berkani. "02. Beyond B Antigen Coverage: The Potential of the 4CMenB Vaccine for Cross-protection Against Pathogenic Neisseria Infections." Open Forum Infectious Diseases 8, Supplement_1 (2021): S125. http://dx.doi.org/10.1093/ofid/ofab466.205.
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Abstract Background Two human pathogenic Neisseria species exist: N. meningitidis (Nm) and N. gonorrhoeae (Ng). Although causing disparate clinical syndromes, invasive meningococcal disease (IMD) and gonorrhea, they are genetically similar and share key protein antigens. The 4CMenB vaccine, licensed against meningococcal B disease, comprises 4 antigenic components (factor H binding protein (fHbp), variant 1.1, subfamily B; Neisseria heparin binding antigen (NHBA) peptide 2; Neisserial adhesin A (NadA) variant 3; and Porin A (PorA) P1.4), and potentially protects against non-B invasive meningococcal and gonococcal strains. In this review, we summarize the similarities between these antigens and those in Nm serogroups A, C, W, X and Y and Ng. Methods Published data in humans were analyzed to conduct a narrative literature review of the potential extent of meningococcal vaccine-induced protection against non-B meningococcal strains and Ng. Techniques applied to indirectly measure this effect are based on genotype-phenotype modelling, strain coverage, bactericidal killing and direct impact on disease reduction. Results Data were identified from countries in America, Europe, Africa and Oceania. The genes encoding for fHbp and NHBA are also present in strains belonging to the five non-B serogroups, while NadA is present in several strains of serogroups C, W and Y, and PorA P1.4 mainly in serogroup W. At the genome level, Ng and Nm share up to 90% homology. Most of the outer membrane vesicle antigens, like PilQ, Omp85 (BamA), NspA, MtrE, MetQ, LbpA, PorB, FetA, OpcA and NHBA, are highly conserved in Ng. In addition, a synergistic effect might enhance immunogenicity against non-B serogroups as shown against serogroup B. Conclusion 4CMenB components are present and conserved in several Ng and Nm strains. Recent results demonstrate that 4CMenB reduces MenW disease incidence in infants and might generate cross-protection against other non-B serogroups. In addition, 4CMenB has been proven to be effective in reducing gonococcal infections in adolescents. Research on future genomic and proteomic characterizations of IMD and gonorrhea strains will provide information on the molecular basis of the underlying broad strain coverage, while informing decisions regarding prevention and immunization programmes. Disclosures Yara Ruiz Garcia, MSc, PhD, GSK group of companies (Employee) Woo-Yun Sohn, MD, GSK group of companies (Employee, Shareholder) Mariagrazia Pizza, Biological Sciences, PhD, GSK group of companies (Employee, Shareholder) Rafik Bekkat-Berkani, M.D, GSK group of companies (Employee, Shareholder)
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Chen, Tie, Fritz Grunert, Andrew Medina-Marino, and Emil C. Gotschlich. "Several Carcinoembryonic Antigens (CD66) Serve as Receptors for Gonococcal Opacity Proteins." Journal of Experimental Medicine 185, no. 9 (1997): 1557–64. http://dx.doi.org/10.1084/jem.185.9.1557.
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Neisseria gonorrhoeae (GC) is a human pathogen that adheres to and invades genital surfaces. Although 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 OpaA GC with epithelial cells involves binding to heparan sulfate attached to syndecan receptors. However, other Opa proteins interact with CEA gene family member 1 (CGM1) or biliary glycoprotein (BGP), members of the CD66 antigen family. In this study, we demonstrate that, in addition, the 180-kD carcinoembryonic antigen (CEA) is a receptor for Opa proteins. This conclusion was based on the following observations. First, transfected HeLa cells expressing CEA (HeLaCEA) and the CEA-expressing colon cancer cell line (LS 174T) bound and subsequently engulfed the Opa+ bacteria. These interactions were inhibited by anti-CEA antibody, but could not be inhibited by addition of heparin. Furthermore, OpaI E. coli directly bound purified CEA. We also compared the adherence and invasion by Opa+ bacteria of CD66 transfected HeLa cells: HeLa-BGPa, HeLa-CGM6, HeLa-NCA, HeLa-CGM1a, HeLa-CEA, and HeLa-Neo serving as negative control. Using OpaI as the prototype, the relative ability of the transfected HeLa cell lines to support adherence was (CEA = BGPa >CGM1a >NCA >>CGM6 = Neo). The ability to mediate invasion of the transfectant cells was (CGM1a >CEA >BGPa >NCA >CGM6 = Neo). Among the Opa proteins tested, OpaC proved to be bifunctional, able to mediate adherence to both syndecan receptors and to CD66 antigens.
Dissertations / Theses on the topic "Neisseria Heparin Binding Antigen"
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DI, FEDE MARTINA. "Dissecting the role of Neisseria Heparin Binding Antigen cleavage during adaptation of Neisseria meningitidis to mucosal surface." Doctoral thesis, Università di Siena, 2017. http://hdl.handle.net/11365/1009815.
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Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein specific for Neisseria and is one of the three main protein antigens of the Bexsero vaccine. Meningococcal and human proteases, including lactoferrin and kallikrein, cleave NHBA protein upstream or downstream a conserved Arg-rich motif, respectively. The cleavage results in the release of the C-terminal portion of the protein. C-terminal fragment originating from the processing of meningococcal proteases, referred as C2 fragment, exerts a toxic effect on endothelial cells altering their permeability. In this work, we reported that recombinant C2 fragment has no influence on the integrity of human airway epithelial cell monolayers, consistently with previous findings showing that N. meningitidis traverses the epithelial barrier without disrupting the junctional structures. Unexpectedly, epithelial cells constantly secreted proteases responsible for a rapid processing of C2 fragment, generating a new fragment that does not contain the Arg-rich motif. This cleavage might inactivate the toxic effect of C2 fragment by eliminating its docking domain. Epithelial cell proteases processed also the NHBA full-length protein, and we demonstrated it on live bacteria. Moreover, looking for the epithelial cell protease responsible for this processing, we identified the C3-convertase of alternative complement pathway as a novel human protease able to cleave NHBA during meningococcal infection. Overall, our data provide new insights on the cleavage of NHBA protein during meningococcal infection. This cleavage occurs at different stages of the infection, and it likely has a different role depending on the environment the bacterium is interacting with.
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Spadafina, Tiziana. "Characterization of Neisseria meningitidis GNA2132 antigen, a heparin binding protein cleaved by meningococcal NalP protease." Doctoral thesis, Università degli studi di Padova, 2009. http://hdl.handle.net/11577/3427201.
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ABSTRACT
GNA2132 (Genome-derived Neisseria Antigen 2132), a protein discovered by Reverse Vaccinology, is a surface-exposed lipoprotein expressed by genetically diverse Neisseria meningitidis strains. The protein induces bactericidal antibodies against most strains of meningococccus and has been included in a multivalent recombinant vaccine against N. meningitidis serogroup B. Sequence analysis of GNA2132 revealed the presence of an Arginine-rich region highly conserved among different strains.
In this study we described that in meningococcus the protein is cleaved upstream from the Arginine-rich region and the C-terminal fragment is released in the culture supernatant.
By genetic approaches we demonstrated that the processing of GNA2132 is strain-dependent and correlates with the expression of NalP, a phase variable autotransporter with a serine-protease activity, which has been previously shown to cleave known Neisseria virulence factors.
Sequence analysis of GNA2132 protein revealed the presence of an Arginine-rich region highly conserved among N. meningitidis strains. Our functional studies revealed that this region is crucial for the function of GNA2132 since recombinant GNA2132 binds heparin through the Arginine-rich region motif.<br>RIASSUNTO
GNA2132 (Genome-derived Neisseria Antigen 2132), una proteina scoperta tramite la Reverse Vaccinology, è una lipoproteina esposta sulla membrana esterna di Neisseria meningitidis. La proteina è in grado di indurre anticorpi battericidi contro la maggior parte dei ceppi di meningococco e per questa ragione è stata inclusa da Novartis nel vaccino contro meningococco serogruppo B. In questo studio abbiamo descritto come in meningococco GNA2132 è tagliata appena prima della regione ricca in arginine ed un frammento corrispondente alla porzione C-terminale della proteina è rilasciato nel supernatante di coltura.
Tramite un approccio genetico abbiamo dimostrato che il taglio di GNA2132 è un processo ceppo-dipendente. Inoltre abbiamo dimostrato che il processamento di GNA2132 correla con l’espressione di NalP, un autotransporter ad attività serino-proteasica, la cui espressione subisce variazione di fase e che è in grado di modulare il processamento di altri fattori di virulenza di meningococco.
GNA2132 contiene una regione ricca in arginine che analisi di sequenza hanno dimostrato essere altamente conservata in moltissimi ceppi di meningococco. I nostri studi sulla funzione della GNA2132 e dei frammenti generati indicano che la GNA2132 è capace di legare l’eparina e mostrano come questa regione sia fondamentale nel legame: infatti il frammento privo della regione ricca in arginine e le proteine ricombinanti (priva della zona in arginine oppure mutata nelle arginine) non mostrano avere il fenotipo di legame all’eparina.
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Vacca, Irene <1985>. "Analysis of the immunological and functional features of the Neisserial Heparin Binding Antigen (NHBA)." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6490/1/VACCA_IRENE_TESI.pdf.
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Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein ubiquitously expressed by genetically diverse Neisseria meningitidis strains and is an antigen of the multicomponent protein-based 4CMenB vaccine, able to induce bactericidal antibodies in humans and to bind heparin-like molecules. The aim of this study is to characterize the immunological and functional properties of NHBA. To evaluate immunogenicity and the contribution of aminoacid sequence variability to vaccine coverage, we constructed recombinant isogenic strains that are susceptible to bactericidal killing only by anti-NHBA antibodies and engineered them to express equal levels of selected NHBA peptides. In these recombinant strains, we observed different titres associated with the different peptide variants. These recombinant strains were then further engineered to express NHBA chimeric proteins to investigate the regions important for immunogenicity. In natural strains, anti-NHBA antibodies were found to be cross-protective against strains expressing different peptides.
To investigate the functional properties of this antigen, the recombinant purified NHBA protein was tested in in vitro binding studies and was found to be able to bind epithelial cells. The binding was abolished when cells were treated specifically with heparinase III, suggesting that the interaction with the cells is mediated by heparan sulfate proteoglycans (HSPG). Mutation of the Arg-rich tract of NHBA abrogated the binding, confirming the importance of this region in mediating the binding to heparin-like molecules.
In a panel of N. meningitidis strains, the deletion of nhba resulted in a reduction of adhesion with respect to each isogenic wild type strain. Furthermore, the adhesion of the wild-type strain was prevented by using anti-NHBA polyclonal sera, demonstrating the specificity of the interaction. These results suggest that NHBA could be a novel meningococcal adhesin contributing to host-cell interaction. Moreover, we analysed NHBA NalP-mediated cleavage in different NHBA peptides and showed that not all NHBA peptides are cleaved.
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Vacca, Irene <1985>. "Analysis of the immunological and functional features of the Neisserial Heparin Binding Antigen (NHBA)." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6490/.
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Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein ubiquitously expressed by genetically diverse Neisseria meningitidis strains and is an antigen of the multicomponent protein-based 4CMenB vaccine, able to induce bactericidal antibodies in humans and to bind heparin-like molecules. The aim of this study is to characterize the immunological and functional properties of NHBA. To evaluate immunogenicity and the contribution of aminoacid sequence variability to vaccine coverage, we constructed recombinant isogenic strains that are susceptible to bactericidal killing only by anti-NHBA antibodies and engineered them to express equal levels of selected NHBA peptides. In these recombinant strains, we observed different titres associated with the different peptide variants. These recombinant strains were then further engineered to express NHBA chimeric proteins to investigate the regions important for immunogenicity. In natural strains, anti-NHBA antibodies were found to be cross-protective against strains expressing different peptides.
To investigate the functional properties of this antigen, the recombinant purified NHBA protein was tested in in vitro binding studies and was found to be able to bind epithelial cells. The binding was abolished when cells were treated specifically with heparinase III, suggesting that the interaction with the cells is mediated by heparan sulfate proteoglycans (HSPG). Mutation of the Arg-rich tract of NHBA abrogated the binding, confirming the importance of this region in mediating the binding to heparin-like molecules.
In a panel of N. meningitidis strains, the deletion of nhba resulted in a reduction of adhesion with respect to each isogenic wild type strain. Furthermore, the adhesion of the wild-type strain was prevented by using anti-NHBA polyclonal sera, demonstrating the specificity of the interaction. These results suggest that NHBA could be a novel meningococcal adhesin contributing to host-cell interaction. Moreover, we analysed NHBA NalP-mediated cleavage in different NHBA peptides and showed that not all NHBA peptides are cleaved.
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Principato, Silvia, Luca Bini, and Brunella Brunelli. "Investigation of the protective mechanisms mediated by Neisserial Heparin Binding Antigen (NHBA) induced antibodies." Doctoral thesis, Università di Siena, 2021. http://hdl.handle.net/11365/1125830.
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Abstract
Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein ubiquitously expressed by Neisseria meningitidis strains and is one of the three main protein components of the Bexsero vaccine. NHBA binds heparin and heparan sulfates through an arginine-rich region and is cleaved by meningococcal and human proteases. Its expression is upregulated at 32°C [1] and it is sparsely distributed on the bacterial surface. Additionally, recent evidence suggests that NHBA plays a key role in bacterial adherence through its arginine-rich region [2].
NHBA induces bactericidal antibodies in humans and confers protective immunity in the infant rat animal model. Anti-NHBA antibodies (polyclonal and monoclonal) from mice and humans are functional, being able to induce complement-mediated bacterial killing, in the presence of rabbit complement (rSBA). However bactericidal activity is not measurable when human serum is used as the source of complement (hSBA).
The aim of this investigation was to elucidate the functional properties that determine the mechanisms of protection mediated by anti-NHBA antibodies.
Negative regulators of the complement system, such as factor H and vitronectin, have been investigated. The effects of antigen density have been explored using a genetically engineered NHBA overexpressing strain to characterize a panel of anti-NHBA monoclonal antibodies isolated from Bexsero immunized adults [3]. Monoclonal antibodies with enhanced C1q recruitment ability have been used to investigate the effect of antigen density and distribution. Moreover, the infant rat challenge model has been used to evaluate in vivo anti-NHBA antibodies induced passive protection.
Nonspecific downregulation of complement-mediated killing due to human factor H was observed to cause an underestimation of the anti-NHBA antibodies ability to efficiently kill bacteria in hSBA, despite the strong passive protection observed in the in vivo infection model. An interaction of NHBA with the complement down-regulator vitronectin was also demonstrated. By using a genetically engineered NHBA overexpressing strain, the relevance of antigen density on the bactericidal activity of antibodies was highlighted. A reduced antigen density on the bacterial surface was shown to be overcome by using engineered monoclonal antibodies with enhanced C1q recruitment ability.
It was demonstrated that multiple interactions with complement regulators interfere with the in vitro measurement of the bactericidal activity mediated by anti-NHBA antibodies in the presence of human complement. Interestingly NHBA, as the Neisseria Opc and NhhA, was found to interact with the extracellular matrix component vitronectin, opening the way to future studies to elucidate implications of this interaction for bacterial colonization. Taken together our findings further support the important role played by NHBA in meningococcal pathogenesis and immunity.
References
[1] M. Lappann et al., Impact of moderate temperature changes on Neisseria meningitidis adhesive phenotypes and proteome. Infection and immunity, 18, 3484-3495, 2016.
[2] I. Vacca et al., Neisserial heparin binding antigen (NHBA) contributes to the adhesion of Neisseria meningitidis to human epithelial cells. PloS One, 11, e0162878, 2016.
[3] M. Giuliani et al., Human protective response induced by meningococcus B vaccine is mediated by the synergy of multiple bactericidal epitopes. Scientific Reports, 8, 3700, 2018.
Disclosures
This work was sponsored by GlaxoSmithKline Biologicals SA.
Silvia Principato is a student of the University of Siena (Life Science Department) and participates in a PhD program at GSK. At present, SP is an employee of the GSK group of companies.
Bexsero is a trademark of the GSK group of companies.
Human monoclonal Antibodies were obtained from adults in a Phase I clinical study conducted in Krakow, Poland and sponsored by Novartis Vaccine, now part of the GSK group of Companies, using two doses of multicomponent serogroup B meningococcal vaccines. The Clinical trial protocol was approved by the Bioethics Committee of the District Medical Doctors’ Chamber in Krakow and the study was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from each of the subjects.
All animal studies were ethically reviewed and carried out in accordance with European Directive 2010/63/EEC and the GSK policy on the Care, Welfare and Treatment of Animals.
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NDONI, ENEA. "Characterization of the immune response and cross protection activity elicited by the Neisserial Heparin Binding Antigen (NHBA), a component of the 4CMenB vaccine." Doctoral thesis, Università di Siena, 2017. http://hdl.handle.net/11365/1011542.
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Invasive disease caused by capsular group B Neisseria meningitidis (MenB) is life threating disease causing hundred thousands of deaths every year, still remaining an unmet medical need in many countries. Although disease can be observed at all age groups, infants and adolescents are the most at risk populations showing the highest incidence in case numbers. Since the MenB capsule was not-immunogenic the development of a MenB vaccine which makes the use of other antigens becomes necessary. 4CMenB is a multicomponent vaccine against serogroup B N. meningitidis composed by three major protein antigens, factor H-binding protein (fHbp), Neisserial Heparin-Binding Antigen (NHBA) and Neisserial adhesin A (NadA), combined with outer membrane vesicles (OMVs) from the New-Zealand epidemic strain (NZ98/254). Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein expressed by all N. meningitidis strains analyzed so far and is composed of two major domains, a highly variable amino-terminal (N-term) domain which anchors the protein on the bacterial outer membrane through the lipobox motif, and a highly conserved carboxyl-terminal (C-term) domain. These domains are separated by a short and quite conserved Arginine-rich (Arg-rich) motif which has been reported to be involved in different mechanisms that mediate meningococci adhesion, infection and survival within the host’s blood stream. NHBA is susceptible to cleavage by NalP, a bacterial protease which has its cleavage site upstream of the arginine region. Moreover human proteases such as human lactoferrin (hLf) and kallikrein are able to process NHBA downstream the the Arg-rich region. Both bacterial and human proteases-mediated cleavage releases the C-term of NHBA in the supernatant, while the N-term of the protein remains anchored on the bacterial surface. NalP cleavage did not impact SBA titers elicited by anti-NHBA antibodies but little is known about the impact that host’s proteases have on bactericidal titers. Based on sequence analysis it has been reported that NHBA has two major alleles, the so called “short” and “long” variants, which differentiate by the presence or absence of a 190 bp long fragment. Despite its sequence variability, NHBA is able to induce a robust and broad immune response against meningococcal strains expressing vaccine homologous and heterologous variants. Although anti-NHBA antibodies are able to induce bacterial killing when tested in serum bactericidal activity assay (SBA), the regions involved in eliciting cross protective immune response remain still unknown. Aims of this study were to use monoclonal antibodies (mAbs) raised against the NHBA vaccine variant peptide 2 (NHBAp2) to (i) map the NHBA regions involved in eliciting the functional response, (ii) test their ability to induce cross protection against strains expressing epidemiologically relevant homologous and heterologous NHBA variants, and (iii) investigate the molecular mechanism of NHBA-mediated bactericidal activity. To this end we used a panel of anti-NHBA mAbs selected to recognize different regions of the protein. Our results showed that only anti-N-term mAbs were able to induce killing of bacterial strains expressing the homologous NHBAp2 and closely related heterologous NHBA variants. Synergy between monoclonal antibodies targeting the N-term and the C-term of NHBA resulted in a significant increase of bactericidal titers but cross protection remained restricted to closely phylogenetic NHBA variants. Anti C-term mAbs were not able to induce SBA activity when tested individually, but surprisingly they became bactericidal when tested in combination. Moreover they were able to induce full cross protection against a panel of strains expressing phylogenetically distant heterologous NHBA variants.
Our results suggest that the partial release of the NHBA C-terminal portion upon NalP and serum proteases could explain why anti-C-term mAbs are not able to induce complement mediated bactericidal killing when tested individually. However, the simultaneous binding of C-term mapping mAbs on the same NHBA molecule can induce the formation of a very stable ternary complex that probably allows a more efficient C1q engagement and C3 deposition, thus leading to the observed co-operative bactericidal activity. These results suggest that synergy between anti-NHBA antibodies is at the basis of the mechanism of NHBA-induced bactericidal activity, which could explain the robust and cross-protective immune response elicited by anti-NHBA polyclonal antibodies following immunization. Collectively, the body of experimental data suggests that both domains of NHBA are required to elicit complement mediated bactericidal activity against strains expressing the vaccine homologous and heterologous NHBA variants.
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Spinsanti, Marco <1987>. "Investigating the regulation of the vaccine antigen Factor H binding protein in Neisseria meningitidis." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2016. http://amsdottorato.unibo.it/7659/1/Marco_Spinsanti_PhD_thesis.pdf.
Full textAbstract:
Neisseria meningitidis is a strictly human pathogen and is a major cause of septicemia and meningitis worldwide. Factor H binding protein (fHbp) is a surface-exposed lipoprotein that binds human factor H (hfH) allowing the bacterium to evade the host innate immunity response. Of note, fHbp is a key antigen in two vaccines against N. meningitidis serogroup B. Although the fHbp gene is present in most circulating meningococcal strains, its level of expression varies among isolates and may influence strain susceptibility to anti-fHbp antisera. The aim of the study was to understand the sequence determinants that control fHbp expression in globally circulating strains. We analyzed the upstream fHbp intergenic region (fIR) of a panel of 105 invasive strains and we identified nine fIR sequence types which represent 77% of the isolates. By mass spectrometry we obtained an absolute quantification of fHbp in the same panel of strains and found a correlation between the fIR type and fHbp amounts. By the generation of a series of isogenic recombinant strains, where fHbp expression was under the control of each of the nine fIR types, we were able to confirm that the fIR sequence determines a specific level of expression and investigate the major determinants involved. The quantity of fHbp on the surface of the bacteria correlated directly with the susceptibility to killing mediated by anti-fHbp antibodies in immune sera. The influence of fHbp to mediate the evasion from generic complement-mediated killing presumably through binding of hfH was assessed and survival in human non-immune serum was less correlated with protein amounts measured from an in vitro growth culture. Overall, we demonstrated that the expression level of this antigen can be inferred by the DNA sequence of the fHbp intergenic region. Therefore, our findings can contribute to understand and predict vaccine coverage mediated by fHbp.
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Spinsanti, Marco <1987>. "Investigating the regulation of the vaccine antigen Factor H binding protein in Neisseria meningitidis." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2016. http://amsdottorato.unibo.it/7659/.
Full textAbstract:
Neisseria meningitidis is a strictly human pathogen and is a major cause of septicemia and meningitis worldwide. Factor H binding protein (fHbp) is a surface-exposed lipoprotein that binds human factor H (hfH) allowing the bacterium to evade the host innate immunity response. Of note, fHbp is a key antigen in two vaccines against N. meningitidis serogroup B. Although the fHbp gene is present in most circulating meningococcal strains, its level of expression varies among isolates and may influence strain susceptibility to anti-fHbp antisera. The aim of the study was to understand the sequence determinants that control fHbp expression in globally circulating strains. We analyzed the upstream fHbp intergenic region (fIR) of a panel of 105 invasive strains and we identified nine fIR sequence types which represent 77% of the isolates. By mass spectrometry we obtained an absolute quantification of fHbp in the same panel of strains and found a correlation between the fIR type and fHbp amounts. By the generation of a series of isogenic recombinant strains, where fHbp expression was under the control of each of the nine fIR types, we were able to confirm that the fIR sequence determines a specific level of expression and investigate the major determinants involved. The quantity of fHbp on the surface of the bacteria correlated directly with the susceptibility to killing mediated by anti-fHbp antibodies in immune sera. The influence of fHbp to mediate the evasion from generic complement-mediated killing presumably through binding of hfH was assessed and survival in human non-immune serum was less correlated with protein amounts measured from an in vitro growth culture. Overall, we demonstrated that the expression level of this antigen can be inferred by the DNA sequence of the fHbp intergenic region. Therefore, our findings can contribute to understand and predict vaccine coverage mediated by fHbp.
Book chapters on the topic "Neisseria Heparin Binding Antigen"
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Locht, Camille, Dominique Raze, Carine Rouanet, Christophe Genisset, Jérôme Segers, and Françoise Mascart. "The Mycobacterial Heparin-Binding Hemagglutinin: a Virulence Factor and Antigen Useful for Diagnostics and Vaccine Development." In The Mycobacterial Cell Envelope. ASM Press, 2014. http://dx.doi.org/10.1128/9781555815783.ch19.
Full textConference papers on the topic "Neisseria Heparin Binding Antigen"
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Jørgensen, M. "HEPARIN INDEPENDENT PURIFICATION OF ANTITHROMBIN III (AT III) BY IMMUNO-AFFINITY CHROMATOGRAPHY RESULTING IN A FUNCTIONALLY INTACT MOLECULE." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643682.
Full textAbstract:
Previous methods for purification of AT III are based on its heparin-binding capacity. However, in congenital AT III deficiency abnormal inhibitor molecules with impaired binding of heparin and/or thrombin has been reported. The aim of the present study was to develop a purification method based on immuno-affinity chromatography, and thus independent of the heparin binding capacity.Rabbits were immunized with human AT III purified by a three-step procedure involving dextran sulphate precipitation, affinity chromatography on heparin-Sepharose and ion-exchange chromatography on DEAE-Sephadex A-50. Rabbit immunoglobulins against human AT III were isolated by affinity chromatography using purified human AT III coupled to CNBr-activated Sepharose 4B. Trace amounts of immunoglobulin against human albumin, IgG and IgM were removed by solid phase immunoadsorption. The highly purified immunoglobulins against human AT III were coupled to CNBr-activated Sepharose 4B. This anti-AT III-Sepharose was used for single-step purification of AT III from plasma. Elution was performed by Na-citrate buffer at pH 3.0 and the eluted fractions immediately neutralized. The recovery was more than 60%.The purified AT III appeared as a single protein band in SDS-poly-acrylamide gel electrophoresis with or without reduction. Affinity purified AT III and AT III purified by the three-step procedure were indistinguishable when analyzed by crossed immunoelectrophoresis in the absence and the presence of heparin isoelectrical focusing in polyacrylamid gel at a pH 4-6.5 gradient, and SDS-polyacrylamide gel electrophoresis. AT III antigen concentration was determined by electroimmunoassay and the reactive site concentration determined by titration with purified human thrombin using Phe-Pip-Arg-Nan (S-2238) as substrate. The ratio (active site conc.)/(antigen conc.) was identical in the two AT III preparations. It is concluded that this single-step immuno-affinity chromatography gives a high recovery from plasma of a highly purified functionally intact AT III molecule. The purification method is independent of the heparin binding capacity of AT III. This is of particular importance for the purification and characterization of abnormal AT III molecules with impaired heparin-binding site.
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Wiman, B., T. Carlsson, and J. Chmielewska. "EVIDENCE FOR A PLASMINOGEN ACTIVATOR INHIBITOR BINDING PROTEIN IN PLASMA." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642859.
Full textAbstract:
For several years it has been known that plasminogen activator inhibitor in plasma behaves as a high molecular weight compound on gelfiltration, in spite of that the molecular weight is only 50,000 in the presence of sodium dodecylsul-phate. The reason for this has so far been unknown. On gelfiltration of plasma, to which purified latent PAI from HT 1080 cells was added, the PAI antigen gel-filtered as a 50,000 Mr protein. However, if the latent form of PAI was reactivated by guanidinium chloride prior to the gel-filtra-tion experiment, an apparent molecular weight of about 250.000 for PAI antigen and activity was observed. If more than 10,000 U of PAI activity was added/mL of normal human plasma, excess PAI occurred at 50,000 Mr on gel-filtration. Human normalplasma was subjected to gel-filtration on sepha-cryl S-300 or Sepharose 6B and the fractions were checked for capacity to transform low Mr functional PAI to high Mr functional PAI. This capacity was only found in the 150 - 200,000 Mr region of the chromatogram. These data suggest that human plasma contains a protein that binds active forms of PAI. The complex of this protein and PAI could be dissociated by gelfiltration in the presence of 3 mol/L guanidinium chloride or 0.1 % (w/v) sodium dodecylsulphate. The physiological or pathophysiological role of the PAI-binding protein is not known. Work with purification of the protein is in progress. Considerable purification have so far been obtained by precipitation with polyethylenglycol 6000 (0-6%), gel-filtration on Sephacry1 S-300, followed by affinity chromatography on heparin-Sepharose.
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Huisveld, I. A., E. C. G. Greven, and B. N. Bouma. "PROTEIN C AND PROTEIN S LEVELS IN TALL GIRLS TREATED WITH ETHINYL -OESTRADIOL." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644288.
Full textAbstract:
Increased levels of several coagulation factors and reduced levels of anti thrombin III (ATIII) have been observed in adults on oestrogen medication and in girls who were treated for excessive tallness with high doses of ethinyloestradiol (E2). In females using oral contraceptives an additional effect on the protein C system has been reported. In our study we evaluated the effect of e-thinyloestradiol treatment (0.1-0.3 mg, administered for at least 6 months) in 14 tall girls (age 14 + 2 yr and height 179 + 4 cm). 13 girls of comparable age (13 + 2 yr) and height 175 + 7 cm) served as controls. Protein C, protein S, C4-binding protein as well as Factor II, plasminogen and ATIII antigen levels were determined by rocket immunoelectrophoresis. ATI11 heparin cofactor ami do-lytic activity was assessed using S2222. Protein S antigen not in complex with C4b-binding protein (PSfree), was assayed in the supernatant plasma after selective precipitation of the protein S-C4b-binding protein complex with PEG 8000. Complete separation of free protein S and PS-C4b-bp complex was verified with crossed immunoelectrophoresis. Results were expressed as % of a normal pool. In the group of girls who were being treated with ethinyl oestradiol (E2-users) significantly higher levels (P<0.001) were observed for protein C (126 ± 29 vs 88 ± 10 in the controls), Factor II (109 ± 18 vs 81 ± 12 in the controls) and plasminogen (131 ± 19 vs 85 ± 9 in the controls). Protein S (58 ± 10 vs 97 ± 8 in the controls) and PSfree (54 ± 11 vs 88 ± 11 in the controls) were sig nificantly reduced (P<0.001) in the E2-users. Only a slight reduction was observed in ATIII antigen levels (95 ± 16 vs 104 ± 9 in the controls) and in the ATIII heparin cofactor activity (105 ± 14 vs 104 ± 9 in the controls). No difference between E2-users and controls was observed in the C4b-binding protein levels (111 ± 25 vs 114 ± 33).Results indicate that treatment of tall girls with high doses of synthetic oestrogens is associated with in vitro signs of hypercoagulability quite comparable to those observed in adult females. However, in contrast to observations made in adults no thromboembolic complications have been reported in juvenile subjects on E2-medication implying additional (aqe-related) variables in the qe-nesis of thrombosis.
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Vigano D'Angelo, S., F. Gilardoni, M. P. Seveso, A. Marassi, G. Mari, and A. D'Angelo. "REDUCTION OF THE ANTICOAGULANT ACTIVITY OF PROTEIN C AND PROTEIN S DURING THE POSTOPERATIVE PERIOD." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644287.
Full textAbstract:
Protein S circulates in plasma as free protein S and in complex with C4b-binding protein, an inhibitor of complement activation. Only free protein S functions as the cofactor for the anticoagulant and profibrinolytic effects of activated protein C. Since isolated reductions of protein C and protein S result in increased thrombotic risk, only measurement of both proteins permits comprehensive evaluation of the antithrombotic potential of the protein C system. No information is available on protein C and protein S functional levels during the postoperative period, an established prothrombotic condition. The plasma changes of protein C, protein S and C4b-binding protein were followed in 40 patients with no malignancy undergoing abdominal surgery. No significant change of protein C and protein S activi ty was observed following minor operations. After major surgery, protein C anticoagulant activity dropped to 80% of preoperative levels during the first postoperative week (p<0.00l). Significant increase of both total protein S antigen (110%, p< 0.01) and C4b-binding protein (130%, p<0.001) were observed after major surgery resulting in reduction of free protein S antigen to 86% of pre operative values (p<0.001). Protein S anticoagulant activity matched the changes of free protein S antigen.Albeit transient and moderate, the observed reductions of both protein C and protein S may act synergistically to cause significant impairment of the antithrombotic potential during the postoperative period. The effect of heparin prophylaxis on protein C and protein S postoperative levels is currently under investigation.
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Lesèche, G., G. Tobelem, J. Caen, and B. Andreassian. "ADULT HUMAN SAPHENOUS VEIN ENDOTHELIAL CELLS : ASSESMENT OF THEIR REPRODUCTIVE CAPACITY AND FUNCTIONAL INTEGRITY PRIOR TO IMPLANTATION ON A VASCULAR GRAFT." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643359.
Full textAbstract:
Establishment of an intact functioning endothelial monolayer on a graft, at or near the time of implantation might be one of the ultimate requirement to get a biocompatible intravascular prosthesis. Following this hypothesis, endothelial cells from human stripped varicose veins were harvested with collagenase and grown on a human fibronectin matrix. The cultured cells (either in primary culture or throughout their lifespan in culture, 1 to 10 passages) exhibited characteristic cobblestone morphology and consistently displayed immunofluorescent staining for factor VUI-related antigen. Functional integrity of this endothelial cells was assayed by their ability to produce prostacyclin. After 15 minutes stimulation with 1 U/ml thrombin, production of 6 Keto PGF1α determined by Elisa was 17 ± 1.2 ng/106 cells. Proliferation was investigated in defined medium supplemented with various concentrations of serum (5 up Jto 30 %), ECGS (25 up to 150 μg/ml) and heparin (10−8 to 10−5M). Optimal growth required both, 100 μg/ml ECGS and 10−5M heparin, under these conditions cells culture achieved cell densities at confluence of 1.2 105 cells per square centimeter with doubling times of one day. Using I-heparin. a binding was demonstrated with an apparent Kd of 0.35 × 10−6 M. After characterization according to morphological, proliferative and functional criteria., cells were freezed at −80°C and ultimately used to coat polytetrafluoro-ethylene grafts (PTFE) which are currently used for vascular reconstructive surgery . Protein-treated material did allow cell attachment and growth to a confluent monolayer as assayed by light and scanning electron microscopy.These data suggested that i) stripped varicose veins provide a readily available source of adult human endothelial cell, ii) these cells grow vigorously in long-term culture when both ECGS and heparin are added to the culture medium, iii) they can adhere and grow to a confluent monolayer on vascular graft prior to implantation, iv) lining graft materials in vitro may be useful in improving the performance of small caliber vascular grafts according to prostacyclin production and surface bound heparin of these cells.
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Sas, G. "DEFECTS IN SERINE PROTEASE INHIBITORS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643714.
Full textAbstract:
Several serine protease inhibitorsof plasma inhibit the activated coagulation enzymes but only antithrombin III(AT-III)and heparin cofactor II (HC-II) are implicated in the pathogenesisof the familial thrombosis. Since thefirst publication (1965) many thrombophilic families with reduced AT-III synthesis have been investigated. These studies have proved that the disorder is associated with a high risk forvenous thrombosis and the inheritanceis autosomal dominant. The AT-III activity in the plasma of the affected patients is about 50% of the normalvalue.In recent years the heterogeneity of the inherited AT-III deficiency has been verified. The various AT-III abnormalities are not mere interestingrarities but they provide naturally occurring models for the solution of theoretical problems such as the function of AT-III molecule, the physiological significance of heparin, etc. Furthermore, the clinical manifestationof the particular variants greatly differs from symptomless abnormality tosevere thrombotic cases.In the majority of cases, reduced functional activity is accompanied with a parallel decrease of antigen concentration of AT-III. This is the characteristic feature of the quantitative or Type I ("classical")AT-III deficiency. By means of crossed immunoelectrophoresis, electro-focussing and recombinant DNA techniques the heterogeneity of this group has been established. In one subgroup (Type la) AT-III molecules are normal as regards their biochemical characteristics. In Type lb, subnormal AT-IIIquantity is accompanied with decreased heparin affinity. Differentiation of these subgroups has practical consequences: therapeutic concentrations of heparin apparently does not decrease AT-III level in the plasma of patients with Type lb AT-III deficiency.The other main form is the qualitative deficiency of AT-III (Type II) which is characterised by reduced functional activity at normal antigen concentration. In general, two populations of AT-III molecules can be detected in the blood of these patients: a normal and an abnormal one. Up till now at least 24 different abnormalities were found and designatedwith toponymes. These disorders can be classified with relatively simple laboratory methods such as functional anti-IIaXaFirstDepartment of Medicine, Postgraduate Medical University, Budapest, Hungary.arin cofactor activity, crossed immunoelectrophoresiswith and without heparin, heparin-affinity chromatography. Type na is characterised by profound structural changes of the molecule,variably: reflected in reduced inactivation of F Ha and F Xa, abnormal heparin-AT-III reaction and aberrant immunochemical structure Seven different abnormalities fall into this group (Budapest I, Tokyo, MalmÖ, Chicago, Milano, Trento and Northwick Park). The last three abnormalities are very similar.In Type lib an isolated defect of protease inactivation can be detected and an isolated disturbance of the active centre ofthe molecule is assumed.Until now 6 apparently different variants belonging to this group have been described. (Aalborg, Vicenza, Denver, Hvidovre, Charleville, Milano 2.) Type lie abnormality is characterised by an isolated defect of the heparin-AT-III reaction. In these cases a disturbance of the heparin binding site(s) is assumed. Eleven families with this type of abnormality have been recorded (Ann Arbor, Basel, Paris 1 and 2, Toyama* Tours, Padova I and 2, Algers, Fontainebleu and Budapest 2). This subgroup is heterogeneous in respect ofheparin affinity: in the majority of cases the abnormal AT-III molecules have no heparin affinity at all while in rare cases (such as Basel, Budapest 2) they have reduced affinity.TheType lie AT-III deficiency has several distinctive features compared with the other subtypesJClinically, the thromboembolic complications are rare: in 4 families thrombosis has notoccurred at all. Only one member in each of 4 other families had thrombosis. In 3 families homozygous patients suffered severe thrombosis in young age and/or in unusual localisations (intraarterial, intracardiac, etc.) butthe other heterozygous members were free of thrombotic symptoms. No increased intravascular coagulation could be detected in Type lie heterozygous cases incontrast to the "classical" AT-III deficiency.These observations suggest a different mechanism and clinical manifestation of the deficiency of progressiveserine protease inactivation and of heparin cofactor activity. In case of progressive inactivation, reduction of 50% of the activity predisposes mainly to venous thrombosis as a consequence of the hypercoagulability of theblood. The isolated reduction of heparin cofactor activity seems to bringabout thrombosis in any part of the vascular system, but only if this reduction is as severe as that of the coagulant factors in case of coagulopathies.In accordance with this finding, rare cases of HCII deficiency give rise to thrombosis in both the arteries and the veins. Heparin cofactor activities may play an important role in the antithrombotic mechanism along theendothelial surface of the whole vascular system.
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Boyer-Neumann, C., M. Wolf, J. Amiral, A. M. Guyager, D. Meyer, and M. J. Larrieu. "FAMILIAL TYPE I PROTEIN S DEFICIENCY ASSOCIATED WITH SEVERE VENOUS THROMBOSIS. A STUDY OF FIVE CASES." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642943.
Full textAbstract:
Protein S deficiency has been demonstrated in 5 members from the same family with a history of severe recurrent venous thrombosis over three generations. The propositus, a 16 year old female, had a first spontaneous thrombotic episode at age 15. Phlebography revealed a total obstruction of her left ilio-femoral vein with an extension to the vena cava. She was treated with heparin followed by oral anticoagulant therapy. The four other affected members (mother, aunts and uncle of the propositus) had also presented recurrent thrombosis with onset at a young age. The grandfather, not tested, had died from massive pulmonary embolism at age 54. The immunological assay of protein S was performed in plasma by Laurell, using a monospecific antiserum to human protein S, or by an ELISA, using a kit from Diagnostica Stago (Asserachrom Protein S). In order to separate free protein S, the functionally active form, from protein S complexed with C4-binding protein, plasma was adsorbed with 3.75 % polyethyleneglycol (PEG 6000). Following PEG precipitation, the levels of free protein S antigen remaining in the supernatant were quantitated by the usual immunological methods. In addition, two-dimensional immunoelectrophoresis (DDIE) also provided information on the distribution of both forms. In plasma protein S levels were decreased (40 to 55 % of the normal range) in two untreated patients and lower levels (17 to 20 96) were observed in the three others, including the propositus, who were under dicoumarol therapy. In PEG treated-plasma, protein S was undetectable (less than 5 %) in all patients, indicating a lack of free protein S. This was confirmed by DDIE : whereas protein S migrated as two distinct peaks, corresponding to free and complexed protein S in normal plasma, only a single peak of complexed protein S was observed in all affected patients. These results clearly demonstrate a total lack of free protein S which appears to be responsible for the thromboembolic disorder in this family as there was no deficiency of the other plasma inhibitors (antithrombin III, heparin cofactor II and protein C). According to the classification recently proposed by Comp et al., this family belongs to type I protein S deficiency, with an autosomal dominant mode of inheritance.
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Sadler, J. Evan. "THE MOLECULAR BIOLOGY OF VON WILLEBRAND FACTOR." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643930.
Full textAbstract:
Human von Willebrand factor (vWF) is a plasma glycoprotein that is synthesized by endothelial cells and megakaryocytes, and perhaps by syncytiotrophoblast of placenta. The biosynthesis of vWF is very complex, involving proteolytic processing, glycosyla-tion, disulfide bond formation, and sulfation. Mature vWF consists of a single subunit of ∼ 250,000 daltons that is assembled into multimer ranging from dimers to species of over 10 million daltons. vWF performs its essential hemostatic function through several binding interactions, forming a bridge between specific receptors on the platelet surface and components of damaged vascular subendothelial connective tissue. Inherited deficiency of vWF, or von Willebrand disease (vWD), is the most common genetically transmitted bleeding disorder worldwide. The last two years has been a time of very rapid progress in understanding the molecular biology of vWF. Four research groups have independently isolated and sequenced the 9 kilobase full-length vWF cDNA. The predicted protein sequence has provided a foundation for understanding the biosynthetic processing of vWF, and has clarified the relationship between vWF and a 75-100 kilodalton plasma protein of unknown function, von Willebrand antigen II (vWAgll)/ vWAgll is co-distributed with vWF in endothelial cells and platelets, and is deficient in patients with vWD. The cDNA sequence of vWF shows that vWAgll is a rather large pro-peptide for vWF, explaining the biochemical and genetic association between the two proteins. vWF has a complex evolutionary history marked by many separate gene segment duplications. The primary structure of the protein contains four distinct types of repeated domains present in two to four copies each. Repeated domains account for over 90 percent of the protein sequence. This sequence provides a framework for ordering the functional domains that have been defined by protein chemistry methods. A tryptic peptide from the amino-terminus of vWF that overlaps domain D3 binds to factor VIII and also appears to bind to heparin. Peptides that include domain A1 bind to collagens, to heparin, and to platelet glycoprotein Ib. A second collagen binding site appears to lie within domain A3. The vWF cDNA has been expressed in heterologous cells to produce small amounts of functionally and structurally normal vWF, indicating that endothelial cells are not unique in their ability to process and assemble vWF multimers. Site-directed mutagenesis has been used to show that deletion of the propeptide of vWF prevents the formation of multimers. Cloned cDNA probes have been employed to isolate vWF genomic DNA from cosmid and λ-phage libraries, and the size of the vWF gene appears to be ∼ 150 kilobases. The vWF locus has been localized to human chromosome 12p12—pter. Several intragenic RFLPs have been characterized. With them, vWF has been placed on the human genetic linkage map as the most telomeric marker currently available for the short arm of chromosome 12. A second apparently homologous locus has been identified on chromosome 22, but the relationship of this locus to the authentic vWF gene is not yet known. The mechanism of vWD has been studied by Southern blotting of genomic DNA with cDNA probes in a few patients. Three unrelated pedigrees have been shown to have total deletions of the vWF gene as the cause of severe vWD (type III). This form of gene deletion appears to predispose to the development of inhibitory alloantibodies to vWF during therapy with cryoprecipitate. During the next several years recombinant DNA methods will continue to contribute our understanding of the evolution, biosynthesis, and structure-function relationships of vWF, as well as the mechanism of additional variants of vWD at the level of gene structure.
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Sala, N., and J. Fontcuberta. "STUDY ON THE EFFECT OF UNFRACTIONATED (UFH) AND LOW MOLECULAR WEIGHT (LMWH) HEPARINS ON THE DETERMINATION OF PROTEIN C ACTIVITY." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644315.
Full textAbstract:
In an attempt to see whether the presence of different heparins affected the determination of protein C activity (APC),this parameter was measured before and during treatment in 23 deep vein thrombosis patients that had been randomly treated with 3 different LMWH (Choay CY-216 and CY-222 and Kabi-2165) and UFH,for 10 days, Very low levels of APC (amidolytic assay that uses thrombin-thrombomodulin to activate the barium citrate eluted PC) were found in those patients receiving UFH and having an APTT more than 3 times that of control, as well as in those patients receiving LMWH CY-216 and having an APTT of only 8 to 10 seconds higher than that of control plasma, In patients receiving CY-222 and Kabi-2165, no significant differences were observed between APC levels before and during treatment, PC antigen (ELISA assay) was normal in all cases, In order to see if these low APC levels were due to interference of heparin with the assay and at which doses, control plasma pool was supplemented "in vitro" with 0 to 2.5 IU/ml (0 to 0,00252) of UFH and with 0 to 3 anti-Xa U/ml of LMWH CY-216, APTT, PCAg, APC and presence of ATI 11 in the barium citrate eluates (immunodiffusion), were determined in all plasma samples before and after treatment with protamine sulphate (PS) at 0,0032, The results showed that UFH, when not neutralized with PS, resulted in low APC values only at doses higher than 0,8 IU/ml, corresponding to an APTT of more than 3 times that of control plasma, LMWH CY-216 at doses above 1 anti-XaU/ml, corresponding to an APTT of only 10 seconds higher than that of control, also produced a gradual decrease in APC values, ATI 11 was clearly visualized in the barium citrate eluates of all those plasma samples having a low APC value, The addition of PS to all samples containing UFH resulted in a complete normalization of APC values, with almost normal AFTT values and disappearance of ATI 11 from the barium citrate eluates, On the contrary, addition of PS to plasma containing CY-216 resulted in low APC values and presence of ATI 11 in the eluates of those samples containing more than 4 antiXaU/ml, whose APTT still was about 10 seconds above that of control.It is concluded that at therapeutic doses not only UFH but also LMWH CY-216 interfere with the APC assay, probably through binding of hepar in-ATI 11 complexes to barium citrate and neutralization of the thrombin used to activate the barium citrate eluted PC, LMWH CY-222 and Kabi-2165, although increasing the APTT similarly to CY-216, do not seem to interfere with the APC assay, Protamine sulphate, at 0,0032 in plasma, completely abolishes the effect of UFH on APC assay but not that of LMWH CY-216, More studies are being performed to see if higher doses of PS can be used to neutralize the effect of this LMWH.
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Fujikawa, K., T. Funakoshi, R. L. Heimark, and J. F. Tait. "HUMAN PLACENTAL ANTICOAGULANT PROTEIN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642949.
Full textAbstract:
Endothelium is important to maintain blood fluidity preventing coagulation. Glycosaminoglycan in the endothelial cell plasma membrane has been thought to prevent activation of blood coagulation. Heparin-like compound, which is a potent anticoagulant activity, has been localized on the surface of the cultured endothelial cells. Anticoagulant action associated with thrombomodulin, which is present in endothelial cells, is another mechanism to provide hemostatic nature of endothelial cells.We wondered whether any other intracellular protein(s) is involved in coagulation. We looked for such a protein(s) in cultured bovine aortic endothelial cells. We soon found an anticoagulant activity in the soluble fraction of endothelial cells and it was partially purified. This activity was adsorbed to DEAE-Sepharose and eluted from a gel filtration column in a molecular weight range of 30,000-40,000. However, limited amounts of the cells made it difficult to purify this activity. We then chose human placenta as a substitute source of this protein and have continued the purification of this anticoagulant activity.In this communication, we describe the isolation and characterization of a placental anticoagulant protein, called "PAP", which is silmilar or possible same as the endothelial anticoaguant protein. PAP was purified from the soluble fraction of human placenta by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose, Sephadex G-75, and mono S (Pharmacia). Approximately 20 mg of the protein was purified from one placenta. The purified protein gave a single band by SDS polyacrylamide gel electrophoresis with a molecular weight of 36,500. This protein inhibited both kaolin- and thromboplastin-induced partial thromboplastin times of normal human plasma. It also inhibited the clotting time of platelet-rich plasma induced by factor Xa, but did not affect the thrombin activity of fibrinogen-fibrin conversion. The purified protein completely inhibited the prothrombin activation by reconstituted prothrombinase. The protein neither inhibited the amidolytic activity of factor Xa nor bound factor Xa. This protein specifically bound to phospholipid vesicles (20% phosphatidylserine and 80% phosphatidylcholine) in the presence of calcium ions. These results indicate that PAP inhibits coagulation through the binding to phospholipid vesicles. The study on the amino acid sequence of PAP is in progress in our laboratory. Surprisingly, the sequence analysis of the cyanogen bromide fragments revealed that PAP is a new member of the lipocortin or calpactin family. The sequences of several cyanogen bromide fragments of PAP aligns with the sequences of lipocortin I and II with over 50% identity.Since PAP interacts directly with phospholipid rather than factor Xa, other activation steps in the coagulation cascade, in which phospholipid is involved, are pro^|bly affected by PAP. These reactions are the activation of factor X by a complex of factor IXa-factor VIIIa-phospholipid-Ca++ and the activations of factor X and factor IX by a tissue factor-factor VIIa-Ca++ complex.Reutelingsperger et. al,, have reported the isolation of a novel inhibitor from arteries of human umbilical cord. This protein inhibited the prothrombin activation by prothrombinase. The authors proposed that the inhibition mechanism of this inhibitor was a competition with factor Xa for binding to phospholipid. This protein is very similar to PAP as to the mode of inhibition. The molecular weight of this inhibitor is 32,000, which is slightly smaller than PAP. With the limited chemical characterization of this protein, presently it is difficult to identify this inhibitor with PAP.At the present time, the physiological role and origin of PAP is not known. PAP may originate from the endothelium of placenta, because we have detected a PAP-like anticoagulant activity in bovine aortic endothelial cells. This activity and PAP were quite alike in the purification up to the gel filtration step. If PAP antibody recognizes the antigen in the endothelial cells, it is interesting to see whether PAP localizes on the surface or inside the cells. Nevertheless, if PAP is present in the endothelial cells, it may play an important role to maintain the hemostatic nature of endothelium. PAP may bind phospholipid components at injured sites, before coagulation factors come in contact with lipid components and initiate thrombolytic events.