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Charlotte L Ownby - One of the best experts on this subject based on the ideXlab platform.

  • comparison of the immunogenicity and Antigenic Composition of ten central american snake venoms
    Toxicon, 1993
    Co-Authors: Steffan G Anderson, Josem Gutierrez, Charlotte L Ownby
    Abstract:

    S. G. Anderson, J. M. Gutierrez and C. L. Ownby. Comparison of the immunogenicity and Antigenic Composition of ten Central American snake venoms. Toxicon31, 1051–1059, 1993.—The immunological reactivity of five crotaline antivenoms for the venoms of ten Costa Rican snakes was determined. Venoms from Bothrops asper, B. godmani, B. lateralis, B. nasutus, B. ophryomegas, B. schlegelii, B. nummifer, B. picadoi, Crotalus durissus durissus and Lachesis muta stenophrys were separated by SDS-PAGE, transferred to cellulose nitrate membrane and reacted against five different antivenoms. Antisera used in the immunoblotting were prepared in rabbits to the crotaline venoms from Crotalus viridis viridis (prairie rattlesnake), Crotalus durissus terrificus (South American rattlesnake), Crotalus atrox (western diamondback rattlesnake), and Bothrops atrox (fer de lance). SDS-PAGE analysis of the ten venoms indicated that all venoms had components in the high-medium mol. wt ( > 15,000) and low mol. wt (< 15,000) range, but they all had at least twice as many components in the high-medium mol. wt range. The venoms of B. nummifer and B. nasutus have the greatest number of bands (24) whereas B. asper has the lowest (17). There appeared to be no difference in immunogenicity between high-medium mol. wt components and low mol. wt components; however, with the venoms of B. nasutus, B. ophryomegas, and B. schlegelii, there were few reactions between antivenoms and low mol. wt components. Half of the ten venoms tested had the highest reactivity with antivenom against B. atrox venom. Two venoms reacted most with antivenom against C. adamanteus venom; one with antivenom to C. atrox venom; one with antivenom to C. v. viridis venom and one with antivenom to C. d. terrificus venom. Antivenom to B. atrox and C. adamanteus venoms were the most reactive whereas antivenom to C. d. terrificus was the least reactive of the five antivenoms tested. The results also demonstrated considerable cross-reactivity between venoms from snakes in the Crotalus and Bothrops genera.

  • comparison of the immunogenicity and Antigenic Composition of several venoms of snakes in the family crotalidae
    Toxicon, 1990
    Co-Authors: Charlotte L Ownby, Terry R Colberg
    Abstract:

    Crude venoms from the prairie rattlesnake (Crotalus viridis viridis), the western diamondback rattlesnake (Crotalus atrox), the eastern diamondback rattlesnake (Crotalus adamanteus) and the timber rattlesnake (Crotalus horridus horridus) were used to prepare monovalent antivenoms in rabbits. Each of these four monovalent antivenoms was reacted against six different venoms using the technique of immunoblotting (Western blot) to determine the relative immunogenicity of the four venoms and to compare the Antigenic Composition of six venoms. In addition to the four venoms listed above, venoms from the South American rattlesnake (Crotalus durissus terrificus) and the fer-de-lance (Bothrops atrox) were tested. SDS-PAGE showed that C. v. viridis venom contains the greatest number of components with 20, and the greatest number (7) less than 15,000 in mol. wt. C. durissus terrificus venom contains the least number of components, having 11. Immunoblotting experiments showed that the greatest reaction between venom and antivenom is not always obtained with the homologous system although the two greatest reactions obtained in this study were for two homologous reactions: that between monovalent anti-C. v. viridis venom and C. v. viridis venom, and that between monovalent anti-C. atrox venom and C. atrox venom. For antivenoms made to C. h. horridus and C. adamanteus venoms, the greatest reaction was obtained with C. atrox venom. There appeared to be no difference in immunogenicity between high-medium mol. wt ( > 15,000) components and low mol. wt ( < 15,000) components in all systems tested except for C. atrox venom where two low mol. wt components gave a stronger reaction with the antivenom than would have been predicted based on their relative content in the venom as indicated by SDS-PAGE. If the immunoblots are scanned with a densitometer, both the qualitative (number of bands) and the quantitative (density of bands) reactions between venom and antivenoms can be taken into consideration by using a Reactivity Index (number of bands × density of bands). By comparing Reactivity Indexes of the various reactions obtained, the most cross-reactive antivenom tested was the monovalent antivenom to C. v. viridis venom, followed by anti-C. adamanteus, anti-C. atrox and anti-C. h. horridus in order of decreasing reactivity. The Reactivity Index can also be used to estimate the reactivity of a single antivenom with different venoms. The major limitation of this approach is the difficulty in standardizing the detection procedure using silver enhanced Protein A gold.

Terry R Colberg - One of the best experts on this subject based on the ideXlab platform.

  • comparison of the immunogenicity and Antigenic Composition of several venoms of snakes in the family crotalidae
    Toxicon, 1990
    Co-Authors: Charlotte L Ownby, Terry R Colberg
    Abstract:

    Crude venoms from the prairie rattlesnake (Crotalus viridis viridis), the western diamondback rattlesnake (Crotalus atrox), the eastern diamondback rattlesnake (Crotalus adamanteus) and the timber rattlesnake (Crotalus horridus horridus) were used to prepare monovalent antivenoms in rabbits. Each of these four monovalent antivenoms was reacted against six different venoms using the technique of immunoblotting (Western blot) to determine the relative immunogenicity of the four venoms and to compare the Antigenic Composition of six venoms. In addition to the four venoms listed above, venoms from the South American rattlesnake (Crotalus durissus terrificus) and the fer-de-lance (Bothrops atrox) were tested. SDS-PAGE showed that C. v. viridis venom contains the greatest number of components with 20, and the greatest number (7) less than 15,000 in mol. wt. C. durissus terrificus venom contains the least number of components, having 11. Immunoblotting experiments showed that the greatest reaction between venom and antivenom is not always obtained with the homologous system although the two greatest reactions obtained in this study were for two homologous reactions: that between monovalent anti-C. v. viridis venom and C. v. viridis venom, and that between monovalent anti-C. atrox venom and C. atrox venom. For antivenoms made to C. h. horridus and C. adamanteus venoms, the greatest reaction was obtained with C. atrox venom. There appeared to be no difference in immunogenicity between high-medium mol. wt ( > 15,000) components and low mol. wt ( < 15,000) components in all systems tested except for C. atrox venom where two low mol. wt components gave a stronger reaction with the antivenom than would have been predicted based on their relative content in the venom as indicated by SDS-PAGE. If the immunoblots are scanned with a densitometer, both the qualitative (number of bands) and the quantitative (density of bands) reactions between venom and antivenoms can be taken into consideration by using a Reactivity Index (number of bands × density of bands). By comparing Reactivity Indexes of the various reactions obtained, the most cross-reactive antivenom tested was the monovalent antivenom to C. v. viridis venom, followed by anti-C. adamanteus, anti-C. atrox and anti-C. h. horridus in order of decreasing reactivity. The Reactivity Index can also be used to estimate the reactivity of a single antivenom with different venoms. The major limitation of this approach is the difficulty in standardizing the detection procedure using silver enhanced Protein A gold.

Michael Lovett - One of the best experts on this subject based on the ideXlab platform.

  • temporal analysis of the Antigenic Composition of borrelia burgdorferi during infection in rabbit skin
    Infection and Immunity, 2004
    Co-Authors: Timothy R Crother, Cheryl I Champion, Xiaoyang Wu, David R Blanco, James N Miller, Julian P Whitelegge, Rodrigo Aguilera, Michael Lovett
    Abstract:

    There have been recent advances in understanding the molecular adaptations of the Lyme disease spirochete, Borrelia burgdorferi, relevant to tick transmission and to survival in mice. The spirochetal surface proteins that are downregulated or upregulated in response to tick feeding, OspA/B and OspC, have been a major focus of such work (7, 35). Recent gene knockouts have addressed the roles of OspA/B and OspC in tick and mouse infections. OspA/B are essential for colonization of the tick midgut but are not necessary for infection of mice (44). Pal et al. found that without OspC, B. burgdorferi strain N40 was unable to invade the salivary glands (32). In contrast, Grimm et al. found that ospC knockouts of strain B31-A3 invaded the salivary glands; however, this knockout strain could not infect either immunocompetent or SCID mice upon direct injection or by tick bite (14). Both groups directly visualized spirochetal OspC expression in salivary glands by using confocal microscopy, and the meaning of their divergent findings is unclear at this time. Most information about changes in B. burgdorferi gene expression during infection of mice is based upon detection of specific transcripts, not upon direct visualization in tissue, presumably due to the small numbers of spirochetes present. Recent studies have given discordant results regarding detection of ospA/B and ospC transcripts. Liang et al. have shown by reverse transcription-PCR that ospC is downregulated after 2 weeks in immunocompetent mice, following the appearance of OspC antibodies (25). Spirochetes infecting SCID mice expressed ospC as judged by reverse transcription-PCR (25). A microarray analysis of 137 lipoprotein genes revealed that many additional genes in addition to ospC became downregulated in immunocompetent mice by several weeks after infection, but not in SCID mice (26). ospB transcripts, but not ospA transcripts, were also detected throughout this period. However, Hodzic et al. recently reported that ospC transcripts were readily detected in immunocompetent C3H mice throughout an 8-week course of infection and that ospA transcripts were also detected (19). Transcription of the Antigenic variation lipoprotein gene, vlsE, and of the decorin binding protein gene, dbpA, was not reduced in normal mice during 33 days of infection (26). We have taken a different approach to assess B. burgdorferi gene expression during infection of murine tissues. Using a method termed HATTREX (hydrophobic antigen tissue Triton extraction), the constellation of hydrophobic protein antigens present during infection of SCID mice was visualized by immunoblotting two-dimensional (2D) gels (6). Using the SCID mouse, we showed the presence of several outer membrane proteins, including very high levels of the Antigenic variation protein, VlsE. Smaller forms of VlsE detected in infected tissues were also very prominent in mouse ear and joint tissues. OspC was readily detected in SCID mouse tissues. In the rabbit model of Lyme disease, B. burgdorferi infection is naturally cleared, with resulting immunity to reinfection (11). In mice, the immune response does not result in clearance of the infection, and chronic infection is established. The rabbit model therefore presents an opportunity to examine the spirochete and the host immune response during the ontogeny of protective immunity. This study presents real-time PCR-based data that indicate that the numbers of host-adapted Borrelia (HAB) in rabbit skin become maximal by 2 weeks after infection but then fall logarithmically in the following week. We used HATTREX to identify hydrophobic proteins of HAB in rabbit skin expressed at intervals ranging from 5 to 21 days after infection.

  • Antigenic Composition of borrelia burgdorferi during infection of scid mice
    Infection and Immunity, 2003
    Co-Authors: Timothy R Crother, Cheryl I Champion, Xiaoyang Wu, David R Blanco, James N Miller, Michael Lovett
    Abstract:

    The general concept that during infection of mice the Borrelia burgdorferi surface protein Composition differs profoundly from that of tick-borne or in vitro-cultivated spirochetes is well established. Specific knowledge concerning the differences is limited because the small numbers of spirochetes present in tissue have not been amenable to direct Compositional analysis. In this report we describe novel means for studying the Antigenic Composition of host-adapted Borrelia (HAB). The detergent Triton X-114 was used to extract the detergent-phase HAB proteins from mouse ears, ankles, knees, and hearts. Immunoblot analysis revealed a profile distinct from that of in vitro-cultivated Borrelia (IVCB). OspA and OspB were not found in the tissues of SCID mice 17 days after infection. The amounts of Antigenic variation protein VlsE and the relative amounts of its transcripts were markedly increased in ear, ankle, and knee tissues but not in heart tissue. VlsE existed as isoforms having both different unit sizes and discrete lower molecular masses. The hydrophobic smaller forms of VlsE were also found in IVCB. The amounts of the surface protein (OspC) and the decorin binding protein (DbpA) were increased in ear, ankle, knee, and heart tissues, as were the relative amounts of their transcripts. Along with these findings regarding VlsE, OspC, and DbpA, two-dimensional immunoblot analysis with immune sera also revealed additional details of the Antigenic Composition of HAB extracted from ear, heart, and joint tissues. A variety of novel antigens, including antigens with molecular masses of 65 and 30 kDa, were found to be upregulated in mouse tissues. Extraction of hydrophobic B. burgdorferi antigens from tissue provides a powerful tool for determining the Antigenic Composition of HAB.

Bozena Dziadek - One of the best experts on this subject based on the ideXlab platform.

  • the impact of the Antigenic Composition of chimeric proteins on their immunoprotective activity against chronic toxoplasmosis in mice
    Vaccine, 2019
    Co-Authors: Justyna Gatkowska, Katarzyna Dzitko, Bartlomiej Ferra, Lucyna Holecgąsior, Malwina Kawka, Bozena Dziadek
    Abstract:

    Toxoplasmosis may pose a serious threat for individuals with weakened or undeveloped immune systems. However, to date, there is no specific immunoprophylaxis for humans. Thus, the aim of this study was to evaluate the immunogenicity of three trivalent—SAG2-GRA1-ROP1L (SGR), SAG1L-MIC1-MAG1 (SMM), and GRA1-GRA2-GRA6 (GGG)—and two tetravalent—SAG2-GRA1-ROP1-GRA2 (SGRG) and SAG1-MIC1-MAG1-GRA2 (SMMG)—chimeric T. gondii proteins, as well as their protective potential against chronic toxoplasmosis in laboratory mice. All three trivalent recombinant proteins possessed immunogenic properties, as defined by specific humoral and cellular responses in vaccinated mice characterized by the synthesis of specific IgG (IgG1/IgG2a) antibodies in vivo and the release of Th1/Th2 cytokines by stimulated splenocytes in vitro. Immunization with all three recombinant proteins provided partial protection against toxoplasmosis, although the protective capacity strongly depended on the individual Antigenic Composition of each preparation. The antigens providing the highest (86%) and lowest (45%) protection, SGR and SMM, respectively, were supplemented with GRA2 antigen fragment, to form the tetravalent chimeric proteins SGRG and SMMG. Further study revealed that the tetravalent preparations exhibited high immunogenic potential; however, the addition of another antigen to the recombinant protein structure had distinct effects on the protection generated, compared to that of the trivalent counterparts, depending on the antigen tested.

Timothy R Crother - One of the best experts on this subject based on the ideXlab platform.

  • temporal analysis of the Antigenic Composition of borrelia burgdorferi during infection in rabbit skin
    Infection and Immunity, 2004
    Co-Authors: Timothy R Crother, Cheryl I Champion, Xiaoyang Wu, David R Blanco, James N Miller, Julian P Whitelegge, Rodrigo Aguilera, Michael Lovett
    Abstract:

    There have been recent advances in understanding the molecular adaptations of the Lyme disease spirochete, Borrelia burgdorferi, relevant to tick transmission and to survival in mice. The spirochetal surface proteins that are downregulated or upregulated in response to tick feeding, OspA/B and OspC, have been a major focus of such work (7, 35). Recent gene knockouts have addressed the roles of OspA/B and OspC in tick and mouse infections. OspA/B are essential for colonization of the tick midgut but are not necessary for infection of mice (44). Pal et al. found that without OspC, B. burgdorferi strain N40 was unable to invade the salivary glands (32). In contrast, Grimm et al. found that ospC knockouts of strain B31-A3 invaded the salivary glands; however, this knockout strain could not infect either immunocompetent or SCID mice upon direct injection or by tick bite (14). Both groups directly visualized spirochetal OspC expression in salivary glands by using confocal microscopy, and the meaning of their divergent findings is unclear at this time. Most information about changes in B. burgdorferi gene expression during infection of mice is based upon detection of specific transcripts, not upon direct visualization in tissue, presumably due to the small numbers of spirochetes present. Recent studies have given discordant results regarding detection of ospA/B and ospC transcripts. Liang et al. have shown by reverse transcription-PCR that ospC is downregulated after 2 weeks in immunocompetent mice, following the appearance of OspC antibodies (25). Spirochetes infecting SCID mice expressed ospC as judged by reverse transcription-PCR (25). A microarray analysis of 137 lipoprotein genes revealed that many additional genes in addition to ospC became downregulated in immunocompetent mice by several weeks after infection, but not in SCID mice (26). ospB transcripts, but not ospA transcripts, were also detected throughout this period. However, Hodzic et al. recently reported that ospC transcripts were readily detected in immunocompetent C3H mice throughout an 8-week course of infection and that ospA transcripts were also detected (19). Transcription of the Antigenic variation lipoprotein gene, vlsE, and of the decorin binding protein gene, dbpA, was not reduced in normal mice during 33 days of infection (26). We have taken a different approach to assess B. burgdorferi gene expression during infection of murine tissues. Using a method termed HATTREX (hydrophobic antigen tissue Triton extraction), the constellation of hydrophobic protein antigens present during infection of SCID mice was visualized by immunoblotting two-dimensional (2D) gels (6). Using the SCID mouse, we showed the presence of several outer membrane proteins, including very high levels of the Antigenic variation protein, VlsE. Smaller forms of VlsE detected in infected tissues were also very prominent in mouse ear and joint tissues. OspC was readily detected in SCID mouse tissues. In the rabbit model of Lyme disease, B. burgdorferi infection is naturally cleared, with resulting immunity to reinfection (11). In mice, the immune response does not result in clearance of the infection, and chronic infection is established. The rabbit model therefore presents an opportunity to examine the spirochete and the host immune response during the ontogeny of protective immunity. This study presents real-time PCR-based data that indicate that the numbers of host-adapted Borrelia (HAB) in rabbit skin become maximal by 2 weeks after infection but then fall logarithmically in the following week. We used HATTREX to identify hydrophobic proteins of HAB in rabbit skin expressed at intervals ranging from 5 to 21 days after infection.

  • Antigenic Composition of borrelia burgdorferi during infection of scid mice
    Infection and Immunity, 2003
    Co-Authors: Timothy R Crother, Cheryl I Champion, Xiaoyang Wu, David R Blanco, James N Miller, Michael Lovett
    Abstract:

    The general concept that during infection of mice the Borrelia burgdorferi surface protein Composition differs profoundly from that of tick-borne or in vitro-cultivated spirochetes is well established. Specific knowledge concerning the differences is limited because the small numbers of spirochetes present in tissue have not been amenable to direct Compositional analysis. In this report we describe novel means for studying the Antigenic Composition of host-adapted Borrelia (HAB). The detergent Triton X-114 was used to extract the detergent-phase HAB proteins from mouse ears, ankles, knees, and hearts. Immunoblot analysis revealed a profile distinct from that of in vitro-cultivated Borrelia (IVCB). OspA and OspB were not found in the tissues of SCID mice 17 days after infection. The amounts of Antigenic variation protein VlsE and the relative amounts of its transcripts were markedly increased in ear, ankle, and knee tissues but not in heart tissue. VlsE existed as isoforms having both different unit sizes and discrete lower molecular masses. The hydrophobic smaller forms of VlsE were also found in IVCB. The amounts of the surface protein (OspC) and the decorin binding protein (DbpA) were increased in ear, ankle, knee, and heart tissues, as were the relative amounts of their transcripts. Along with these findings regarding VlsE, OspC, and DbpA, two-dimensional immunoblot analysis with immune sera also revealed additional details of the Antigenic Composition of HAB extracted from ear, heart, and joint tissues. A variety of novel antigens, including antigens with molecular masses of 65 and 30 kDa, were found to be upregulated in mouse tissues. Extraction of hydrophobic B. burgdorferi antigens from tissue provides a powerful tool for determining the Antigenic Composition of HAB.