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Sample records for immunity protein im7

  1. A structural comparison of the colicin immunity proteins Im7 and Im9 gives new insights into the molecular determinants of immunity-protein specificity.

    PubMed Central

    Dennis, C A; Videler, H; Pauptit, R A; Wallis, R; James, R; Moore, G R; Kleanthous, C

    1998-01-01

    We report the first detailed comparison of two immunity proteins which, in conjunction with recent protein engineering data, begins to explain how these structurally similar proteins are able to bind and inhibit the endonuclease domain of colicin E9 (E9 DNase) with affinities that differ by 12 orders of magnitude. In the present work, we have determined the X-ray structure of the Escherichia coli colicin E7 immunity protein Im7 to 2.0 A resolution by molecular replacement, using as a trial model the recently determined NMR solution structure of Im9. Whereas the two proteins adopt similar four-helix structures, subtle structural differences, in particular involving a conserved tyrosine residue critical for E9 DNase binding, and the identity of key residues in the specificity helix, lie at the heart of their markedly different ability to bind the E9 DNase. Two other crystal structures were reported recently for Im7; in one, Im7 was a monomer and was very similar to the structure reported here, whereas in the other it was a dimer to which functional significance was assigned. Since this previous work suggested that Im7 could exist either as a monomer or a dimer, we used analytical ultracentrifugation to investigate this question further. Under a variety of solution conditions, we found that Im7 only ever exists in solution as a monomer, even up to protein concentrations of 15 mg/ml, casting doubt on the functional significance of the crystallographically observed dimer. This work provides a structural framework with which we can understand immunity-protein specificity, and in addition we believe it to be the first successfully refined crystal structure solved by molecular replacement using an NMR trial model with less than 100% sequence identity. PMID:9639578

  2. Determination of an ensemble of structures representing the intermediate state of the bacterial immunity protein Im7

    PubMed Central

    Gsponer, Joerg; Hopearuoho, Harri; Whittaker, Sara B.-M.; Spence, Graham R.; Moore, Geoffrey R.; Paci, Emanuele; Radford, Sheena E.; Vendruscolo, Michele

    2006-01-01

    We present a detailed structural characterization of the intermediate state populated during the folding and unfolding of the bacterial immunity protein Im7. We achieve this result by incorporating a variety of experimental data available for this species in molecular dynamics simulations. First, we define the structure of the exchange-competent intermediate state of Im7 by using equilibrium hydrogen-exchange protection factors. Second, we use this ensemble to predict Φ-values and compare the results with the experimentally determined Φ-values of the kinetic refolding intermediate. Third, we predict chemical-shift measurements and compare them with the measured chemical shifts of a mutational variant of Im7 for which the kinetic folding intermediate is the most stable state populated at equilibrium. Remarkably, we found that the properties of the latter two species are predicted with high accuracy from the exchange-competent intermediate that we determined, suggesting that these three states are characterized by a similar architecture in which helices I, II, and IV are aligned in a native-like, but reorganized, manner. Furthermore, the structural ensemble that we obtained enabled us to rationalize the results of tryptophan fluorescence experiments in the WT protein and a series of mutational variants. The results show that the integration of diverse sets of experimental data at relatively low structural resolution is a powerful approach that can provide insights into the structural organization of this conformationally heterogeneous three-helix intermediate with unprecedented detail and highlight the importance of both native and non-native interactions in stabilizing its structure. PMID:16371468

  3. Determination of an ensemble of structures representing the intermediate state of the bacterial immunity protein Im7.

    PubMed

    Gsponer, Joerg; Hopearuoho, Harri; Whittaker, Sara B-M; Spence, Graham R; Moore, Geoffrey R; Paci, Emanuele; Radford, Sheena E; Vendruscolo, Michele

    2006-01-01

    We present a detailed structural characterization of the intermediate state populated during the folding and unfolding of the bacterial immunity protein Im7. We achieve this result by incorporating a variety of experimental data available for this species in molecular dynamics simulations. First, we define the structure of the exchange-competent intermediate state of Im7 by using equilibrium hydrogen-exchange protection factors. Second, we use this ensemble to predict Phi-values and compare the results with the experimentally determined Phi-values of the kinetic refolding intermediate. Third, we predict chemical-shift measurements and compare them with the measured chemical shifts of a mutational variant of Im7 for which the kinetic folding intermediate is the most stable state populated at equilibrium. Remarkably, we found that the properties of the latter two species are predicted with high accuracy from the exchange-competent intermediate that we determined, suggesting that these three states are characterized by a similar architecture in which helices I, II, and IV are aligned in a native-like, but reorganized, manner. Furthermore, the structural ensemble that we obtained enabled us to rationalize the results of tryptophan fluorescence experiments in the WT protein and a series of mutational variants. The results show that the integration of diverse sets of experimental data at relatively low structural resolution is a powerful approach that can provide insights into the structural organization of this conformationally heterogeneous three-helix intermediate with unprecedented detail and highlight the importance of both native and non-native interactions in stabilizing its structure.

  4. Ultraviolet resonance Raman studies reveal the environment of tryptophan and tyrosine residues in the native and partially folded states of the E colicin-binding immunity protein Im7.

    PubMed

    Rodriguez-Mendieta, Iñigo R; Spence, Graham R; Gell, Christopher; Radford, Sheena E; Smith, D Alastair

    2005-03-01

    Understanding the nature of partially folded proteins is a challenging task that is best accomplished when several techniques are applied in combination. Here we present ultraviolet resonance Raman (UVRR) spectroscopy studies of the E colicin-binding immunity proteins, Im7* and Im9*, together with a series of variants of Im7* that are designed to trap a partially folded state at equilibrium. We show that the environments of the tryptophan and tyrosine residues in native wild-type Im7* and Im9* are indistinguishable, in contrast with models for their structures based on X-ray and NMR methods. In addition, we show that there is a general increase in the hydrophobicity in the environment of Trp75 in all of the variants compared with wild-type Im7*. These data suggest that a significant rearrangement of the tryptophan pocket occurs in the variants, which, together with an overall decrease in solvent accessibility of Trp75 as judged by time-resolved fluorescence lifetime measurements and fluorescence quenching experiments, rationalize the unusual fluorescence properties of the variants reported previously. The data highlight the power of UVRR in analyzing the structural properties of different conformational states of the same protein and reveal new information about the structural rearrangements occurring during Im7* folding, not possible using other spectroscopic methods alone. Finally, we describe a previously unreported dependence of the tryptophan Fermi doublet on excitation wavelength in the ultraviolet region revealed by these protein spectra. We corroborated this observation using tryptophan-containing model compounds and conclude that the conventional interpretation of this UVRR feature at these wavelengths is unreliable.

  5. Single-Molecule Studies of the Im7 Folding Landscape

    PubMed Central

    Pugh, Sara D.; Gell, Christopher; Smith, D. Alastair; Radford, Sheena E.; Brockwell, David J.

    2010-01-01

    Under appropriate conditions, the four-helical Im7 (immunity protein 7) folds from an ensemble of unfolded conformers to a highly compact native state via an on-pathway intermediate. Here, we investigate the unfolded, intermediate, and native states populated during folding using diffusion single-pair fluorescence resonance energy transfer by measuring the efficiency of energy transfer (or proximity or P ratio) between pairs of fluorophores introduced into the side chains of cysteine residues placed in the center of helices 1 and 4, 1 and 3, or 2 and 4. We show that while the native states of each variant give rise to a single narrow distribution with high P values, the distributions of the intermediates trapped at equilibrium (denoted Ieqm) are fitted by two Gaussian distributions. Modulation of the folding conditions from those that stabilize the intermediate to those that destabilize the intermediate enabled the distribution of lower P value to be assigned to the population of the unfolded ensemble in equilibrium with the intermediate state. The reduced stability of the Ieqm variants allowed analysis of the effect of denaturant concentration on the compaction and breadth of the unfolded state ensemble to be quantified from 0 to 6 M urea. Significant compaction is observed as the concentration of urea is decreased in both the presence and absence of sodium sulfate, as previously reported for a variety of proteins. In the presence of Na2SO4 in 0 M urea, the P value of the unfolded state ensemble approaches that of the native state. Concurrent with compaction, the ensemble displays increased peak width of P values, possibly reflecting a reduction in the rate of conformational exchange among iso-energetic unfolded, but compact conformations. The results provide new insights into the initial stages of folding of Im7 and suggest that the unfolded state is highly conformationally constrained at the outset of folding. PMID:20211187

  6. Single-molecule studies of the Im7 folding landscape.

    PubMed

    Pugh, Sara D; Gell, Christopher; Smith, D Alastair; Radford, Sheena E; Brockwell, David J

    2010-04-23

    Under appropriate conditions, the four-helical Im7 (immunity protein 7) folds from an ensemble of unfolded conformers to a highly compact native state via an on-pathway intermediate. Here, we investigate the unfolded, intermediate, and native states populated during folding using diffusion single-pair fluorescence resonance energy transfer by measuring the efficiency of energy transfer (or proximity or P ratio) between pairs of fluorophores introduced into the side chains of cysteine residues placed in the center of helices 1 and 4, 1 and 3, or 2 and 4. We show that while the native states of each variant give rise to a single narrow distribution with high P values, the distributions of the intermediates trapped at equilibrium (denoted I(eqm)) are fitted by two Gaussian distributions. Modulation of the folding conditions from those that stabilize the intermediate to those that destabilize the intermediate enabled the distribution of lower P value to be assigned to the population of the unfolded ensemble in equilibrium with the intermediate state. The reduced stability of the I(eqm) variants allowed analysis of the effect of denaturant concentration on the compaction and breadth of the unfolded state ensemble to be quantified from 0 to 6 M urea. Significant compaction is observed as the concentration of urea is decreased in both the presence and absence of sodium sulfate, as previously reported for a variety of proteins. In the presence of Na(2)SO(4) in 0 M urea, the P value of the unfolded state ensemble approaches that of the native state. Concurrent with compaction, the ensemble displays increased peak width of P values, possibly reflecting a reduction in the rate of conformational exchange among iso-energetic unfolded, but compact conformations. The results provide new insights into the initial stages of folding of Im7 and suggest that the unfolded state is highly conformationally constrained at the outset of folding.

  7. Specificity in protein-protein interactions: the structural basis for dual recognition in endonuclease colicin-immunity protein complexes.

    PubMed

    Kühlmann, U C; Pommer, A J; Moore, G R; James, R; Kleanthous, C

    2000-09-01

    Bacteria producing endonuclease colicins are protected against their cytotoxic activity by virtue of a small immunity protein that binds with high affinity and specificity to inactivate the endonuclease. DNase binding by the immunity protein occurs through a "dual recognition" mechanism in which conserved residues from helix III act as the binding-site anchor, while variable residues from helix II define specificity. We now report the 1.7 A crystal structure of the 24.5 kDa complex formed between the endonuclease domain of colicin E9 and its cognate immunity protein Im9, which provides a molecular rationale for this mechanism. Conserved residues of Im9 form a binding-energy hotspot through a combination of backbone hydrogen bonds to the endonuclease, many via buried solvent molecules, and hydrophobic interactions at the core of the interface, while the specificity-determining residues interact with corresponding specificity side-chains on the enzyme. Comparison between the present structure and that reported recently for the colicin E7 endonuclease domain in complex with Im7 highlights how specificity is achieved by very different interactions in the two complexes, predominantly hydrophobic in nature in the E9-Im9 complex but charged in the E7-Im7 complex. A key feature of both complexes is the contact between a conserved tyrosine residue from the immunity proteins (Im9 Tyr54) with a specificity residue on the endonuclease directing it toward the specificity sites of the immunity protein. Remarkably, this tyrosine residue and its neighbour (Im9 Tyr55) are the pivots of a 19 degrees rigid-body rotation that relates the positions of Im7 and Im9 in the two complexes. This rotation does not affect conserved immunity protein interactions with the endonuclease but results in different regions of the specificity helix being presented to the enzyme.

  8. Isothermal aging of IM7/8320 and IM7/5260

    NASA Technical Reports Server (NTRS)

    Martin, Roderick H.; Siochi, Emilie J.; Gates, Thomas S.

    1992-01-01

    Isothermal aging was conducted on two composite systems being considered as possible candidates for the next generation supersonic transport. The composite systems were IM7/5260, a carbon/thermoset, and IM7/8320, a carbon/amorphous thermoplastic. The materials were isothermally aged for a total of 5000 hours at 125 C and 175 C. These temperatures are approximately equivalent to the upper skin temperatures of an aircraft flying at Mach 2.0 and Mach 2.4, respectively. The variations of the following properties were determined as a function of aging time: weight loss, moduli, glass transition temperature, microcracking, and modulus and strength of a +/- 45 laminate. The difficulties and accuracy of strain measurements are also discussed.

  9. Variola virus immune evasion proteins.

    PubMed

    Dunlop, Lance R; Oehlberg, Katherine A; Reid, Jeremy J; Avci, Dilek; Rosengard, Ariella M

    2003-09-01

    Variola virus, the causative agent of smallpox, encodes approximately 200 proteins. Over 80 of these proteins are located in the terminal regions of the genome, where proteins associated with host immune evasion are encoded. To date, only two variola proteins have been characterized. Both are located in the terminal regions and demonstrate immunoregulatory functions. One protein, the smallpox inhibitor of complement enzymes (SPICE), is homologous to a vaccinia virus virulence factor, the vaccinia virus complement-control protein (VCP), which has been found experimentally to be expressed early in the course of vaccinia infection. Both SPICE and VCP are similar in structure and function to the family of mammalian complement regulatory proteins, which function to prevent inadvertent injury to adjacent cells and tissues during complement activation. The second variola protein is the variola virus high-affinity secreted chemokine-binding protein type II (CKBP-II, CBP-II, vCCI), which binds CC-chemokine receptors. The vaccinia homologue of CKBP-II is secreted both early and late in infection. CKBP-II proteins are highly conserved among orthopoxviruses, sharing approximately 85% homology, but are absent in eukaryotes. This characteristic sets it apart from other known virulence factors in orthopoxviruses, which share sequence homology with known mammalian immune regulatory gene products. Future studies of additional variola proteins may help illuminate factors associated with its virulence, pathogenesis and strict human tropism. In addition, these studies may also assist in the development of targeted therapies for the treatment of both smallpox and human immune-related diseases.

  10. Circadian clock proteins and immunity.

    PubMed

    Curtis, Anne M; Bellet, Marina M; Sassone-Corsi, Paolo; O'Neill, Luke A J

    2014-02-20

    Immune parameters change with time of day and disruption of circadian rhythms has been linked to inflammatory pathologies. A circadian-clock-controlled immune system might allow an organism to anticipate daily changes in activity and feeding and the associated risk of infection or tissue damage to the host. Responses to bacteria have been shown to vary depending on time of infection, with mice being more at risk of sepsis when challenged ahead of their activity phase. Studies highlight the extent to which the molecular clock, most notably the core clock proteins BMAL1, CLOCK, and REV-ERBα, control fundamental aspects of the immune response. Examples include the BMAL1:CLOCK heterodimer regulating toll-like receptor 9 (TLR9) expression and repressing expression of the inflammatory monocyte chemokine ligand (CCL2) as well as REV-ERBα suppressing the induction of interleukin-6. Understanding the daily rhythm of the immune system could have implications for vaccinations and how we manage infectious and inflammatory diseases.

  11. Protein trafficking during plant innate immunity.

    PubMed

    Wang, Wen-Ming; Liu, Peng-Qiang; Xu, Yong-Ju; Xiao, Shunyuan

    2016-04-01

    Plants have evolved a sophisticated immune system to fight against pathogenic microbes. Upon detection of pathogen invasion by immune receptors, the immune system is turned on, resulting in production of antimicrobial molecules including pathogenesis-related (PR) proteins. Conceivably, an efficient immune response depends on the capacity of the plant cell's protein/membrane trafficking network to deploy the right defense-associated molecules in the right place at the right time. Recent research in this area shows that while the abundance of cell surface immune receptors is regulated by endocytosis, many intracellular immune receptors, when activated, are partitioned between the cytoplasm and the nucleus for induction of defense genes and activation of programmed cell death, respectively. Vesicle transport is an essential process for secretion of PR proteins to the apoplastic space and targeting of defense-related proteins to the plasma membrane or other endomembrane compartments. In this review, we discuss the various aspects of protein trafficking during plant immunity, with a focus on the immunity proteins on the move and the major components of the trafficking machineries engaged. PMID:26345282

  12. Visualizing chaperone-assisted protein folding.

    PubMed

    Horowitz, Scott; Salmon, Loïc; Koldewey, Philipp; Ahlstrom, Logan S; Martin, Raoul; Quan, Shu; Afonine, Pavel V; van den Bedem, Henry; Wang, Lili; Xu, Qingping; Trievel, Raymond C; Brooks, Charles L; Bardwell, James C A

    2016-07-01

    Challenges in determining the structures of heterogeneous and dynamic protein complexes have greatly hampered past efforts to obtain a mechanistic understanding of many important biological processes. One such process is chaperone-assisted protein folding. Obtaining structural ensembles of chaperone-substrate complexes would ultimately reveal how chaperones help proteins fold into their native state. To address this problem, we devised a new structural biology approach based on X-ray crystallography, termed residual electron and anomalous density (READ). READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the Escherichia coli chaperone Spy, and to capture a series of snapshots depicting the various folding states of Im7 bound to Spy. The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded to native-like states and reveals how a substrate can explore its folding landscape while being bound to a chaperone. PMID:27239796

  13. APOBEC3 Proteins in Viral Immunity.

    PubMed

    Stavrou, Spyridon; Ross, Susan R

    2015-11-15

    Apolipoprotein B editing complex 3 family members are cytidine deaminases that play important roles in intrinsic responses to infection by retroviruses and have been implicated in the control of other viruses, such as parvoviruses, herpesviruses, papillomaviruses, hepatitis B virus, and retrotransposons. Although their direct effect on modification of viral DNA has been clearly demonstrated, whether they play additional roles in innate and adaptive immunity to viruses is less clear. We review the data regarding the various steps in the innate and adaptive immune response to virus infection in which apolipoprotein B editing complex 3 proteins have been implicated. PMID:26546688

  14. The structure of pyogenecin immunity protein, a novel bacteriocin-like immunity protein from streptococcus pyogenes.

    SciTech Connect

    Chang, C.; Coggill, P.; Bateman, A.; Finn, R.; Cymborowski, M.; Otwinowski, Z.; Minor, W.; Volkart, L.; Joachimiak, A.; Wellcome Trust Sanger Inst.; Univ. of Virginia; UT Southwestern Medical Center

    2009-12-17

    Many Gram-positive lactic acid bacteria (LAB) produce anti-bacterial peptides and small proteins called bacteriocins, which enable them to compete against other bacteria in the environment. These peptides fall structurally into three different classes, I, II, III, with class IIa being pediocin-like single entities and class IIb being two-peptide bacteriocins. Self-protective cognate immunity proteins are usually co-transcribed with these toxins. Several examples of cognates for IIa have already been solved structurally. Streptococcus pyogenes, closely related to LAB, is one of the most common human pathogens, so knowledge of how it competes against other LAB species is likely to prove invaluable. We have solved the crystal structure of the gene-product of locus Spy-2152 from S. pyogenes, (PDB: 2fu2), and found it to comprise an anti-parallel four-helix bundle that is structurally similar to other bacteriocin immunity proteins. Sequence analyses indicate this protein to be a possible immunity protein protective against class IIa or IIb bacteriocins. However, given that S. pyogenes appears to lack any IIa pediocin-like proteins but does possess class IIb bacteriocins, we suggest this protein confers immunity to IIb-like peptides. Combined structural, genomic and proteomic analyses have allowed the identification and in silico characterization of a new putative immunity protein from S. pyogenes, possibly the first structure of an immunity protein protective against potential class IIb two-peptide bacteriocins. We have named the two pairs of putative bacteriocins found in S. pyogenes pyogenecin 1, 2, 3 and 4.

  15. Substrate protein folds while it is bound to the ATP-independent chaperone Spy.

    PubMed

    Stull, Frederick; Koldewey, Philipp; Humes, Julia R; Radford, Sheena E; Bardwell, James C A

    2016-01-01

    Chaperones assist in the folding of many proteins in the cell. Although the most well-studied chaperones use cycles of ATP binding and hydrolysis to assist in protein folding, a number of chaperones have been identified that promote folding in the absence of high-energy cofactors. Precisely how ATP-independent chaperones accomplish this feat is unclear. Here we characterized the kinetic mechanism of substrate folding by the small ATP-independent chaperone Spy from Escherichia coli. Spy rapidly associates with its substrate, immunity protein 7 (Im7), thereby eliminating Im7's potential for aggregation. Remarkably, Spy then allows Im7 to fully fold into its native state while it remains bound to the surface of the chaperone. These results establish a potentially widespread mechanism whereby ATP-independent chaperones assist in protein refolding. They also provide compelling evidence that substrate proteins can fold while being continuously bound to a chaperone. PMID:26619265

  16. Substrate protein folds while it is bound to the ATP-independent chaperone Spy.

    PubMed

    Stull, Frederick; Koldewey, Philipp; Humes, Julia R; Radford, Sheena E; Bardwell, James C A

    2016-01-01

    Chaperones assist in the folding of many proteins in the cell. Although the most well-studied chaperones use cycles of ATP binding and hydrolysis to assist in protein folding, a number of chaperones have been identified that promote folding in the absence of high-energy cofactors. Precisely how ATP-independent chaperones accomplish this feat is unclear. Here we characterized the kinetic mechanism of substrate folding by the small ATP-independent chaperone Spy from Escherichia coli. Spy rapidly associates with its substrate, immunity protein 7 (Im7), thereby eliminating Im7's potential for aggregation. Remarkably, Spy then allows Im7 to fully fold into its native state while it remains bound to the surface of the chaperone. These results establish a potentially widespread mechanism whereby ATP-independent chaperones assist in protein refolding. They also provide compelling evidence that substrate proteins can fold while being continuously bound to a chaperone.

  17. Functional Classification of Immune Regulatory Proteins

    SciTech Connect

    Rubinstein, Rotem; Ramagopal, Udupi A.; Nathenson, Stanley G.; Almo, Steven C.; Fiser, Andras

    2013-05-01

    Members of the immunoglobulin superfamily (IgSF) control innate and adaptive immunity and are prime targets for the treatment of autoimmune diseases, infectious diseases, and malignancies. We describe a computational method, termed the Brotherhood algorithm, which utilizes intermediate sequence information to classify proteins into functionally related families. This approach identifies functional relationships within the IgSF and predicts additional receptor-ligand interactions. As a specific example, we examine the nectin/nectin-like family of cell adhesion and signaling proteins and propose receptor-ligand interactions within this family. We were guided by the Brotherhood approach and present the high-resolution structural characterization of a homophilic interaction involving the class-I MHC-restricted T-cell-associated molecule, which we now classify as a nectin-like family member. The Brotherhood algorithm is likely to have a significant impact on structural immunology by identifying those proteins and complexes for which structural characterization will be particularly informative.

  18. Emerging Roles of Protein Deamidation in Innate Immune Signaling

    PubMed Central

    Zhao, Jun; Li, Junhua; Xu, Simin

    2016-01-01

    Protein deamidation has been considered a nonenzymatic process associated with protein functional decay or “aging.” Recent studies implicate protein deamidation in regulating signal transduction in fundamental biological processes, such as innate immune responses. Work investigating gammaherpesviruses and bacterial pathogens indicates that microbial pathogens deploy deamidases or enzyme-deficient homologues (pseudoenzymes) to induce deamidation of key signaling components and evade host immune responses. Here, we review studies on protein deamidation in innate immune signaling and present several imminent questions concerning the roles of protein deamidation in infection and immunity. PMID:26889032

  19. Stress-strain behaviour of IM7/977-2 and IM7/APC2 carbon fibre composites at low and high strain rates

    NASA Astrophysics Data System (ADS)

    Parry, D. J.; Al-Hazmi, F. S.

    2003-09-01

    The dynamic compressive behaviour of two IM7 carbon fibre composite systems has been studied at room temperature at strain rates from about 10^{-3} s^{-1} to 10^3 s^{-1}. A Hopkinson bar method was used for high rates and a conventional screw machine for low rates. One composite system (IM7/977-2) has a thermoset epoxy resin matrix toughened with thermoplastic material; the other system (IM7/APC2) has a thermoplastic PEEK matrix. Three types of fibre configuration were used for each system: (a) longitudinal unidirectional, 0° (LU), (b) transverse unidirectional, 90° (TU), and (c) transverse isotropic, (0°/90°/±45°)s (TI). At low rates, both systems showed similar stress-strain behaviour. For the LU types, the fracture stresses were about 790 MPa, while for the TU types the values were about 235 MPa. For the Tl specimens, the maximum available stress of 950 MPa was insufficient for fracture. At high rates, the fracture stress was about 25% greater than the quasistatic values for both LU types, and about 30% higher for both TU types. The major difference between the two systems was with the TI types: the energy density to fracture was about 65 MJ m^{-3} for the epoxy composite, while it was about 40 MJ m^{-3} for the PEEK composite.

  20. Pathogen mimicry of host protein-protein interfaces modulates immunity.

    PubMed

    Guven-Maiorov, Emine; Tsai, Chung-Jung; Nussinov, Ruth

    2016-10-01

    Signaling pathways shape and transmit the cell's reaction to its changing environment; however, pathogens can circumvent this response by manipulating host signaling. To subvert host defense, they beat it at its own game: they hijack host pathways by mimicking the binding surfaces of host-encoded proteins. For this, it is not necessary to achieve global protein homology; imitating merely the interaction surface is sufficient. Different protein folds often interact via similar protein-protein interface architectures. This similarity in binding surfaces permits the pathogenic protein to compete with a host target protein. Thus, rather than binding a host-encoded partner, the host protein hub binds the pathogenic surrogate. The outcome can be dire: rewiring or repurposing the host pathways, shifting the cell signaling landscape and consequently the immune response. They can also cause persistent infections as well as cancer by modulating key signaling pathways, such as those involving Ras. Mapping the rewired host-pathogen 'superorganism' interaction network - along with its structural details - is critical for in-depth understanding of pathogenic mechanisms and developing efficient therapeutics. Here, we overview the role of molecular mimicry in pathogen host evasion as well as types of molecular mimicry mechanisms that emerged during evolution.

  1. Out-Life Characteristics of IM7/977-3 Composites

    NASA Technical Reports Server (NTRS)

    Miller, Sandi G.; Sutter, James K.; Hou, Tan-Hung; Scheiman, Daniel A.; Martin, Richard E.; Maryanski, Michael; Schlea, Michelle; Gardner, John M.; Schiferl, Zack R.

    2010-01-01

    The capability to manufacture large structures leads to weight savings and reduced risk relative to joining smaller components. However, manufacture of increasingly large composite components is pushing the out-time limits of epoxy/ carbon fiber prepreg. IM7/977-3 is an autoclave processable prepreg material, commonly used in aerospace structures. The out-time limit is reported as 30 days by the manufacturer. The purpose of this work was to evaluate the material processability and composite properties of 977-3 resin and IM7/977-3 prepreg that had been aged at room temperature for up to 60 days. The effects of room temperature aging on the thermal and visco-elastic properties of the materials were investigated. Neat resin was evaluated by differential scanning calorimetry to characterize thermal properties and change in activation energy of cure. Neat resin was also evaluated by rheometry to characterize its processability in composite fabrication. IM7/977-3 prepreg was evaluated by dynamic mechanical analysis to characterize the curing behavior. Prepreg tack was also evaluated over 60 days. The overall test results suggested that IM7/977-3 was a robust material that offered quality laminates throughout this aging process when processed by autoclave.

  2. Differential protein network analysis of the immune cell lineage.

    PubMed

    Clancy, Trevor; Hovig, Eivind

    2014-01-01

    Recently, the Immunological Genome Project (ImmGen) completed the first phase of the goal to understand the molecular circuitry underlying the immune cell lineage in mice. That milestone resulted in the creation of the most comprehensive collection of gene expression profiles in the immune cell lineage in any model organism of human disease. There is now a requisite to examine this resource using bioinformatics integration with other molecular information, with the aim of gaining deeper insights into the underlying processes that characterize this immune cell lineage. We present here a bioinformatics approach to study differential protein interaction mechanisms across the entire immune cell lineage, achieved using affinity propagation applied to a protein interaction network similarity matrix. We demonstrate that the integration of protein interaction networks with the most comprehensive database of gene expression profiles of the immune cells can be used to generate hypotheses into the underlying mechanisms governing the differentiation and the differential functional activity across the immune cell lineage. This approach may not only serve as a hypothesis engine to derive understanding of differentiation and mechanisms across the immune cell lineage, but also help identify possible immune lineage specific and common lineage mechanism in the cells protein networks. PMID:25309909

  3. Inducible immune proteins in the dampwood termite Zootermopsis angusticollis

    NASA Astrophysics Data System (ADS)

    Rosengaus, Rebeca B.; Cornelisse, Tara; Guschanski, Katerina; Traniello, James F. A.

    2007-01-01

    Dampwood termites, Zootermopsis angusticollis (Isoptera: Termopsidae), mount an immune response to resist microbial infection. Here we report on results of a novel analysis that allowed us to electrophoretically assess changes in hemolymph proteins in the same individual before and after exposure to a pathogen. We demonstrate that contact with a sublethal concentration of the entomopathogenic fungus Metarhizium anisopliae (Deuteromycotina:Hypomycetes) induces the production of protective proteins in nymphs, pseudergates (false workers), and soldiers. Termites exposed to an immunizing dosage of fungal conidia consistently showed an enhancement of constitutive proteins (62-85 kDa) in the hemolymph as well as an induction of novel proteins (28-48 kDa) relative to preimmunization levels. No significant differences in protein banding patterns relative to baseline levels in control and naïve termites were observed. Incubating excised and eluted induced proteins produced by immunized pseudergates or immunized soldiers with conidia significantly reduced the germination of the fungus. The fungistatic effect of eluted proteins differed significantly among five colonies examined. Our results show that the upregulation of protective proteins in the hemolymph underscores the in vivo immune response we previously recorded in Z. angusticollis.

  4. [Cellular immunity in protein-caloric malnutrition].

    PubMed

    Khadraoui, S; Lopez, V; Hamza, B; Smith, N J

    1977-02-01

    In order to have a clearer idea on the relationship between infectious diseases and malnutrition, cellular immunity is studied in 3 to 18 months old malnourished and healthy infants. Nutritional status is evaluated by the food intake the anthropologic measurements and some biological parameters like transferrin. Cellular immunity is investigated by skin tests to tuberculin, monilia, P.H.A. and DNCB. Absolute number of peripheral lymphocytes and percentage of T cells are studied too. The results show that there is a cellular immunity deficiency: frequent non-response to antigens, weak sensitization to DNCB. Some patients have a low percentage of spontaneous rosettes. The transferrin titering is useful to appreciate the nutritional status and the iron therapy opportunity, the prognostic and the immunitary possibilities.

  5. Cell-mediated immune responses to COPV early proteins.

    PubMed

    Jain, Suchitra; Moore, Richard A; Anderson, Davina M; Gough, Gerald W; Stanley, Margaret A

    Cell-mediated immunity plays a key role in the regression of papillomavirus-induced warts and intra-epithelial lesions but the target antigens that induce this response are not clear. Canine oral papillomavirus (COPV) infection of the oral cavity in dogs is a well-characterized model of mucosal papillomavirus infection that permits analysis of the immune events during the infectious cycle. In this study we show that during the COPV infectious cycle, systemic T cell responses to peptides of several early proteins particularly the E2 protein, as assayed by delayed type hypersensitivity, lymphoproliferation and IFN-gamma ELISPOT, can be detected. The maximal response occurs in a narrow time window that coincides with maximal viral DNA replication and wart regression: thereafter, systemic T cell responses to early proteins decline quite rapidly. Vaccination using particle-mediated immunotherapeutic delivery (PMID) of codon-modified COPV E2 and E1 genes induces strong antigen-specific cell-mediated immune responses in the vaccinated animals. These data show that therapeutic immunization by PMID with codon-modified E2 is completely effective, that to E1 is partially protective, that this correlates with the intensity of antigen-specific cell-mediated immune responses and, further, they emphasize the importance of these responses and the route of immunization in the generation of protective immunity. PMID:16949120

  6. Nasal Immunization with Lactococcus lactis Expressing the Pneumococcal Protective Protein A Induces Protective Immunity in Mice▿

    PubMed Central

    Medina, Marcela; Villena, Julio; Vintiñi, Elisa; Hebert, Elvira María; Raya, Raúl; Alvarez, Susana

    2008-01-01

    Nisin-controlled gene expression was used to develop a recombinant strain of Lactococcus lactis that is able to express the pneumococcal protective protein A (PppA) on its surface. Immunodetection assays confirmed that after the induction with nisin, the PppA antigen was predictably and efficiently displayed on the cell surface of the recombinant strain, which was termed L. lactis PppA. The production of mucosal and systemically specific antibodies in adult and young mice was evaluated after mice were nasally immunized with L. lactis PppA. Immunoglobulin M (IgM), IgG, and IgA anti-PppA antibodies were detected in the serum and bronchoalveolar lavage fluid of adult and young mice, which showed that PppA expressed in L. lactis was able to induce a strong mucosal and systemic immune response. Challenge survival experiments demonstrated that immunization with L. lactis PppA was able to increase resistance to systemic and respiratory infection with different pneumococcal serotypes, and passive immunization assays of naïve young mice demonstrated a direct correlation between anti-PppA antibodies and protection. The results presented in this study demonstrate three major characteristics of the effectiveness of nasal immunization with PppA expressed as a protein anchored to the cell wall of L. lactis: it elicited cross-protective immunity against different pneumococcal serotypes, it afforded protection against both systemic and respiratory challenges, and it induced protective immunity in mice of different ages. PMID:18390997

  7. Characterization of the immune response of domestic fowl following immunization with proteins extracted from Dermanyssus gallinae.

    PubMed

    Harrington, David; Din, Hatem Mohi El; Guy, Jonathan; Robinson, Karen; Sparagano, Olivier

    2009-03-23

    Dermanyssus gallinae is the most significant ectoparasite of European poultry egg laying production systems due to high costs of control and associated production losses as well as adverse effects on bird welfare. In this study, soluble proteins were extracted from unfed D. gallinae (DGE) using a urea-based detergent and ultra-filtration, passed through a 0.22 microm filter and blended aseptically with adjuvant. One group of laying hens was immunized with DGE and adjuvant (Montanide ISA 50 V) whilst another group (Control) received physiological saline and adjuvant. All birds were immunized on two occasions, 21 days apart. Antibody response to immunization was determined by ELISA and western blotting using immunoglobulins (Igs) extracted from egg yolk. DGE immunization of hens resulted in a significant (P<0.05) IgY response compared to controls, although there was no significant difference in IgM response between treatments. A number of proteins were identified by western blotting using IgY antibodies from DGE immunized birds, most prominently at 40 and 230kDa. Analysis of proteins from approximately corresponding bands on SDS-PAGE confirmed the identity of tropomyosin, whilst other proteins showed high sequence homology with myosin and actin from other arachnid and insect species. Immunization of hens with DGE resulted in a 50.6% increase in mite mortality (P<0.001) 17h after feeding when tested by an in vitro mite feeding model. Data in this study demonstrate that somatic antigens from D. gallinae can be used to stimulate a protective immune response in laying hens. Further work is needed to identify other proteins of interest that could confer higher protection against D. gallinae, as well as optimization of the vaccination and in vitro testing protocol.

  8. Human immune system mice immunized with Plasmodium falciparum circumsporozoite protein induce protective human humoral immunity against malaria.

    PubMed

    Huang, Jing; Li, Xiangming; Coelho-dos-Reis, Jordana G A; Zhang, Min; Mitchell, Robert; Nogueira, Raquel Tayar; Tsao, Tiffany; Noe, Amy R; Ayala, Ramses; Sahi, Vincent; Gutierrez, Gabriel M; Nussenzweig, Victor; Wilson, James M; Nardin, Elizabeth H; Nussenzweig, Ruth S; Tsuji, Moriya

    2015-12-01

    In this study, we developed human immune system (HIS) mice that possess functional human CD4+ T cells and B cells, named HIS-CD4/B mice. HIS-CD4/B mice were generated by first introducing HLA class II genes, including DR1 and DR4, along with genes encoding various human cytokines and human B cell activation factor (BAFF) to NSG mice by adeno-associated virus serotype 9 (AAV9) vectors, followed by engrafting human hematopoietic stem cells (HSCs). HIS-CD4/B mice, in which the reconstitution of human CD4+ T and B cells resembles to that of humans, produced a significant level of human IgG against Plasmodium falciparum circumsporozoite (PfCS) protein upon immunization. CD4+ T cells in HIS-CD4/B mice, which possess central and effector memory phenotypes like those in humans, are functional, since PfCS protein-specific human CD4+ T cells secreting IFN-γ and IL-2 were detected in immunized HIS-CD4/B mice. Lastly, PfCS protein-immunized HIS-CD4/B mice were protected from in vivo challenge with transgenic P. berghei sporozoites expressing the PfCS protein. The immune sera collected from protected HIS-CD4/B mice reacted against transgenic P. berghei sporozoites expressing the PfCS protein and also inhibited the parasite invasion into hepatocytes in vitro. Taken together, these studies show that our HIS-CD4/B mice could mount protective human anti-malaria immunity, consisting of human IgG and human CD4+ T cell responses both specific for a human malaria antigen.

  9. Human immune system mice immunized with Plasmodium falciparum circumsporozoite protein induce protective human humoral immunity against malaria.

    PubMed

    Huang, Jing; Li, Xiangming; Coelho-dos-Reis, Jordana G A; Zhang, Min; Mitchell, Robert; Nogueira, Raquel Tayar; Tsao, Tiffany; Noe, Amy R; Ayala, Ramses; Sahi, Vincent; Gutierrez, Gabriel M; Nussenzweig, Victor; Wilson, James M; Nardin, Elizabeth H; Nussenzweig, Ruth S; Tsuji, Moriya

    2015-12-01

    In this study, we developed human immune system (HIS) mice that possess functional human CD4+ T cells and B cells, named HIS-CD4/B mice. HIS-CD4/B mice were generated by first introducing HLA class II genes, including DR1 and DR4, along with genes encoding various human cytokines and human B cell activation factor (BAFF) to NSG mice by adeno-associated virus serotype 9 (AAV9) vectors, followed by engrafting human hematopoietic stem cells (HSCs). HIS-CD4/B mice, in which the reconstitution of human CD4+ T and B cells resembles to that of humans, produced a significant level of human IgG against Plasmodium falciparum circumsporozoite (PfCS) protein upon immunization. CD4+ T cells in HIS-CD4/B mice, which possess central and effector memory phenotypes like those in humans, are functional, since PfCS protein-specific human CD4+ T cells secreting IFN-γ and IL-2 were detected in immunized HIS-CD4/B mice. Lastly, PfCS protein-immunized HIS-CD4/B mice were protected from in vivo challenge with transgenic P. berghei sporozoites expressing the PfCS protein. The immune sera collected from protected HIS-CD4/B mice reacted against transgenic P. berghei sporozoites expressing the PfCS protein and also inhibited the parasite invasion into hepatocytes in vitro. Taken together, these studies show that our HIS-CD4/B mice could mount protective human anti-malaria immunity, consisting of human IgG and human CD4+ T cell responses both specific for a human malaria antigen. PMID:26410104

  10. Heat shock proteins: stimulators of innate and acquired immunity.

    PubMed

    Colaco, Camilo A; Bailey, Christopher R; Walker, K Barry; Keeble, James

    2013-01-01

    Adjuvants were reintroduced into modern immunology as the dirty little secret of immunologists by Janeway and thus began the molecular definition of innate immunity. It is now clear that the binding of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) on antigen presenting cells (APCs) activates the innate immune response and provides the host with a rapid mechanism for detecting infection by pathogens and initiates adaptive immunity. Ironically, in addition to advancing the basic science of immunology, Janeway's revelation on induction of the adaptive system has also spurred an era of rational vaccine design that exploits PRRs. Thus, defined PAMPs that bind to known PRRs are being specifically coupled to antigens to improve their immunogenicity. However, while PAMPs efficiently activate the innate immune response, they do not mediate the capture of antigen that is required to elicit the specific responses of the acquired immune system. Heat shock proteins (HSPs) are molecular chaperones that are found complexed to client polypeptides and have been studied as potential cancer vaccines. In addition to binding PRRs and activating the innate immune response, HSPs have been shown to both induce the maturation of APCs and provide chaperoned polypeptides for specific triggering of the acquired immune response.

  11. Polyclonal Antibody Production for Membrane Proteins via Genetic Immunization

    PubMed Central

    Hansen, Debra T.; Robida, Mark D.; Craciunescu, Felicia M.; Loskutov, Andrey V.; Dörner, Katerina; Rodenberry, John-Charles; Wang, Xiao; Olson, Tien L.; Patel, Hetal; Fromme, Petra; Sykes, Kathryn F.

    2016-01-01

    Antibodies are essential for structural determinations and functional studies of membrane proteins, but antibody generation is limited by the availability of properly-folded and purified antigen. We describe the first application of genetic immunization to a structurally diverse set of membrane proteins to show that immunization of mice with DNA alone produced antibodies against 71% (n = 17) of the bacterial and viral targets. Antibody production correlated with prior reports of target immunogenicity in host organisms, underscoring the efficiency of this DNA-gold micronanoplex approach. To generate each antigen for antibody characterization, we also developed a simple in vitro membrane protein expression and capture method. Antibody specificity was demonstrated upon identifying, for the first time, membrane-directed heterologous expression of the native sequences of the FopA and FTT1525 virulence determinants from the select agent Francisella tularensis SCHU S4. These approaches will accelerate future structural and functional investigations of therapeutically-relevant membrane proteins. PMID:26908053

  12. Early innate immune response of immune proteins in juvenile channel catfish Ictalurus punctatus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Channel catfish (Ictalurus punctatus) are raised for aquaculture in the Southeast U.S. and are susceptible to bacterial and viral infections acquired from their pond environment. Innate immune proteins mannose-binding lectin (MBL) and lysozyme were studied during two consecutive years in channel cat...

  13. Hypervelocity Impact Testing of IM7/977-3 with Micro-Sized Particles

    NASA Technical Reports Server (NTRS)

    Smith, J. G.; Jegley, D. C.; Siochi, E. J.; Wells, B. K.

    2010-01-01

    Ground-based hypervelocity imapct testing was conducted on IM7/977-3 quasi-isotropic flat panels at normal incidence using micron-sized particles (i.e. less than or equal to 100 microns) of soda lime glass and olivine. Testing was performed at room temperature (RT) and 175 C with results from the 175 C test compared to those obtained at RT. Between 10 and 30 particles with velocities ranging from 5 to 13 km/s impacted each panel surface for each test temperature. Panels were ultrasonically scanned prior to and after impact testing to assess internal damage. Post-impact analysis included microscopic examination of the surface, determination of particle speed and location, and photomicroscopy for microcrack assessment. Internal damage was observed by ultrasonic inspection on panels impacted at 175 C, whereas damage for the RT impacted panels was confined to surface divets/craters as determined by microscopic analysis.

  14. Cure effects on microcracking in IM7 fiber/Matrimid 5292{reg_sign} BMI composites

    SciTech Connect

    Wilenski, M.S.; Shin, E.; Morgan, R.J.; Drzal, L.T.

    1995-12-31

    Initial results from a study of the microcracking behavior of the Matrimid 5292{reg_sign} BMI/IM7 carbon fiber system are presented. Studies were performed to determine the composite`s Stress Free Temperature (SFT) which is seen to control the presence and extent of microcracking. The SFT was determined using asymmetric laminates [0{sub 2}/90{sub 2}]. Varied post-cure cycles were utilized and it was found that until complete cure is obtained, the SFT is a function of the highest temperature experienced by the laminate, with little effect of previous thermal history. The reactions necessary to fully cure this system are not active at temperatures below 250 C, ruling out the possibility of obtaining a fully cured composite with a low SFT through extended post-cures at lower temperatures.

  15. Multifunctional antimicrobial proteins and peptides: natural activators of immune systems.

    PubMed

    Niyonsaba, François; Nagaoka, Isao; Ogawa, Hideoki; Okumura, Ko

    2009-01-01

    In addition to the physical barrier of the stratum corneum, cutaneous innate immunity also includes the release of various humoral mediators, such as cytokines and chemokines, recruitment and activation of phagocytes, and the production of antimicrobial proteins/peptides (AMPs). AMPs form an innate epithelial chemical shield, which provides a front-line component in innate immunity to inhibit microbial invasion; however, this might be an oversimplification of the diverse functions of these molecules. In fact, apart from exhibiting a broad spectrum of microbicidal properties, it is increasingly evident that AMPs display additional activities that are related to the stimulation and modulation of the cutaneous immune system. These diverse functions include chemoattraction and activation of immune and/or inflammatory cells, the production and release of cytokines and chemokines, acceleration of angiogenesis, promotion of wound healing, neutralization of harmful microbial products, and bridging of both innate and adaptive immunity. Thus, better understanding of the functions of AMPs in skin and identification of their signaling mechanisms may offer new strategies for the development of potential therapeutics for the treatment of infection- and/or inflammation-related skin diseases. Here, we briefly outline the structure, regulation of expression, and multifunctional roles of principal skin-derived AMPs.

  16. Human immune cell targeting of protein nanoparticles - caveospheres

    NASA Astrophysics Data System (ADS)

    Glass, Joshua J.; Yuen, Daniel; Rae, James; Johnston, Angus P. R.; Parton, Robert G.; Kent, Stephen J.; de Rose, Robert

    2016-04-01

    Nanotechnology has the power to transform vaccine and drug delivery through protection of payloads from both metabolism and off-target effects, while facilitating specific delivery of cargo to immune cells. However, evaluation of immune cell nanoparticle targeting is conventionally restricted to monocultured cell line models. We generated human caveolin-1 nanoparticles, termed caveospheres, which were efficiently functionalized with monoclonal antibodies. Using this platform, we investigated CD4+ T cell and CD20+ B cell targeting within physiological mixtures of primary human blood immune cells using flow cytometry, imaging flow cytometry and confocal microscopy. Antibody-functionalization enhanced caveosphere binding to targeted immune cells (6.6 to 43.9-fold) within mixed populations and in the presence of protein-containing fluids. Moreover, targeting caveospheres to CCR5 enabled caveosphere internalization by non-phagocytic CD4+ T cells--an important therapeutic target for HIV treatment. This efficient and flexible system of immune cell-targeted caveosphere nanoparticles holds promise for the development of advanced immunotherapeutics and vaccines.

  17. Melanosomal proteins as melanoma-specific immune targets.

    PubMed

    Sakai, C; Kawakami, Y; Law, L W; Furumura, M; Hearing, V J

    1997-04-01

    Pigmentation of our skin, hair and eyes is essential for photoprotection, embryological development, detoxification and protective/cosmetic coloration. A number of proteins important to the production of melanin within melanosomes have now been identified including enzymatic and structural proteins encoded at the murine albino, brown, pinkeyed-dilution, MART1, slaty and silver loci. Interestingly, many of those melanosomal proteins (including epitopes derived from tyrosinase, TRP1/gp75, silver/gp100 and MART1/melan-A) function in vivo as targets of humoral and cellular autoimmune responses directed specifically against normal or transformed melanocytes. These findings have provided new impetus to research on immune responses to melanoma and, perhaps more importantly, examining why they are insufficient to provide protection against tumour growth and what type of immune therapy can be designed to correct that. The melanosome must now be considered beyond its function in pigmentation, and assumes the role of a valuable source for specific immune targets for malignant melanoma. PMID:9167173

  18. Melanosomal proteins as melanoma-specific immune targets.

    PubMed

    Sakai, C; Kawakami, Y; Law, L W; Furumura, M; Hearing, V J

    1997-04-01

    Pigmentation of our skin, hair and eyes is essential for photoprotection, embryological development, detoxification and protective/cosmetic coloration. A number of proteins important to the production of melanin within melanosomes have now been identified including enzymatic and structural proteins encoded at the murine albino, brown, pinkeyed-dilution, MART1, slaty and silver loci. Interestingly, many of those melanosomal proteins (including epitopes derived from tyrosinase, TRP1/gp75, silver/gp100 and MART1/melan-A) function in vivo as targets of humoral and cellular autoimmune responses directed specifically against normal or transformed melanocytes. These findings have provided new impetus to research on immune responses to melanoma and, perhaps more importantly, examining why they are insufficient to provide protection against tumour growth and what type of immune therapy can be designed to correct that. The melanosome must now be considered beyond its function in pigmentation, and assumes the role of a valuable source for specific immune targets for malignant melanoma.

  19. Physical aging effects on the compressive linear viscoelastic creep of IM7/K3B composite

    NASA Technical Reports Server (NTRS)

    Veazie, David R.; Gates, Thomas S.

    1995-01-01

    An experimental study was undertaken to establish the viscoelastic behavior of 1M7/K3B composite in compression at elevated temperature. Creep compliance, strain recovery and the effects of physical aging on the time dependent response was measured for uniaxial loading at several isothermal conditions below the glass transition temperature (T(g)). The IM7/K3B composite is a graphite reinforced thermoplastic polyimide with a T(g) of approximately 240 C. In a composite, the two matrix dominated compliance terms associated with time dependent behavior occur in the transverse and shear directions. Linear viscoelasticity was used to characterize the creep/recovery behavior and superposition techniques were used to establish the physical aging related material constants. Creep strain was converted to compliance and measured as a function of test time and aging time. Results included creep compliance master curves, physical aging shift factors and shift rates. The description of the unique experimental techniques required for compressive testing is also given.

  20. Automated Fiber Placement of PEEK/IM7 Composites with Film Interleaf Layers

    NASA Technical Reports Server (NTRS)

    Hulcher, A. Bruce; Banks, William I., III; Pipes, R. Byron; Tiwari, Surendra N.; Cano, Roberto J.; Johnston, Norman J.; Clinton, R. G., Jr. (Technical Monitor)

    2001-01-01

    The incorporation of thin discrete layers of resin between plies (interleafing) has been shown to improve fatigue and impact properties of structural composite materials. Furthermore, interleafing could be used to increase the barrier properties of composites used as structural materials for cryogenic propellant storage. In this work, robotic heated-head tape placement of PEEK/IM7 composites containing a PEEK polymer film interleaf was investigated. These experiments were carried out at the NASA Langley Research Center automated fiber placement facility. Using the robotic equipment, an optimal fabrication process was developed for the composite without the interleaf. Preliminary interleaf processing trials indicated that a two-stage process was necessary; the film had to be tacked to the partially-placed laminate then fully melted in a separate operation. Screening experiments determined the relative influence of the various robotic process variables on the peel strength of the film-composite interface. Optimization studies were performed in which peel specimens were fabricated at various compaction loads and roller temperatures at each of three film melt processing rates. The resulting data were fitted with quadratic response surfaces. Additional specimens were fabricated at placement parameters predicted by the response surface models to yield high peel strength in an attempt to gage the accuracy of the predicted response and assess the repeatability of the process. The overall results indicate that quality PEEK/lM7 laminates having film interleaves can be successfully and repeatability fabricated by heated head automated fiber placement.

  1. High strain rate mechanical properties of IM7/8551-7 graphite epoxy composite

    SciTech Connect

    Powers, B.M.; Vinson, J.R.; Hall, I.W.

    1995-12-31

    Polymer matrix composites offer excellent mechanical properties such as high specific strength and stiffness which make them attractive for many naval, aerospace and automotive structural components. Although they are candidate materials for many applications where high strain rate loading is probable, little is known of the material responses to shock loading for most composite materials. Because mechanical properties vary significantly with strain rate, the use of static properties in the analysis and design of structures which undergo dynamic loadings can on one hand lead to a very conservative overweight design, or on the other hand can lead to designs which fail prematurely and unexpectedly. The use of dynamic material properties will ensure the design of composite structures which are weight efficient and structurally sound when they are subjected to dynamic loads. In this study, a Split Hopkinson Pressure Bar is used to obtain compressive mechanical properties of a unidirectional IM7/8551-7 graphite epoxy composite. For each of the three principal directions, the yield stress, yield strain, ultimate stress, ultimate strain, modulus of elasticity, elastic strain energy function and the total strain energy to failure are presented for strain rates varying from 49 sec{sup {minus}1} to 1430 sec{sup {minus}1}. The data from 72 tests are statistically analyzed, represented by equations, and discussed in some detail.

  2. Mechanical characterization of IM7/8551-7 carbon/epoxy under biaxial stress: (Final report)

    SciTech Connect

    Colvin, G.E. Jr.; Swanson, S.R.

    1987-11-13

    This is the final report on an investigation to evaluate the mechanical response of Hercules IM7/8551-7 carbon/epoxy, which is a high strength, high elongation fiber and a high toughness resin system used in a prepreg form. The material characterization involved testing both laminate and lamina forms under a wide range of biaxial stress states. Tubular specimens were employed that have been designed to eliminate undesirable end effects, permitting uniform stress states to be achieved. Quasi-isotropic (90/+-45/0)/sub ns/laminates and (90)/sub 16T/ lamina specimens were loaded under combinations of internal pressure, axial load, and torsion. Both stiffness and strength data were obtained under these multiaxial stress conditions. The measured laminate stiffnesses correlated well using classical laminated plate theory, and that laminate failure occurred in the two separate modes of matrix cracking and fiber failure. Like the previously examined carbon/epoxy systems, laminate failure could be predicted by using a fiber failure criterion to identify the critical plies and critical load levels. It was found that either maximum fiber stress or fiber direction strain could be used as a failure criterion on a ply level. 16 refs., 10 figs., 3 tabs.

  3. Ultrasonic measurements of stiffness in thermal-mechanically fatigued IM7/5260 composites

    SciTech Connect

    Seale, M.D.; Madaras, E.I. )

    1999-08-01

    In recent years, ultrasonic methods have been developed that can measure the mechanical stiffness of composites. The Lamb wave velocity is directly related to the material parameters, so an effective method exists to ascertain the stiffness of composites by measuring the velocity of these waves. In this study, a Lamb wave measurement system was used to measure the bending and out-of-plane stiffness coefficients of thermoset composite laminates undergoing thermal-mechanical loading. A series of 16 ply and 32 ply composite laminates were subjected to thermal-mechanical fatigue (TMF) in load frames equipped with special environmental chambers. The composite system studied was a graphite fiber-reinforced bismaleimide thermoset, IM7/5260. The samples were subjected to both high and low temperature profiles as well as high-strain and low-strain profiles. The bending and out-of-plane stiffnesses for composite samples that have undergone over 6,000 cycles of combined thermal and mechanical fatigue are reported. The Lamb wave generated elastic stiffness results have shown decreases of up to 64% at 4,706 cycles for samples subjected to TMF at high temperatures and less than a 10% decrease at over 6,000 cycles for samples subjected to TMF at low temperatures.

  4. Protein Kinase C δ: a Gatekeeper of Immune Homeostasis.

    PubMed

    Salzer, Elisabeth; Santos-Valente, Elisangela; Keller, Bärbel; Warnatz, Klaus; Boztug, Kaan

    2016-10-01

    Human autoimmune disorders present in various forms and are associated with a life-long burden of high morbidity and mortality. Many different circumstances lead to the loss of immune tolerance and often the origin is suspected to be multifactorial. Recently, patients with autosomal recessive mutations in PRKCD encoding protein kinase c delta (PKCδ) have been identified, representing a monogenic prototype for one of the most prominent forms of humoral systemic autoimmune diseases, systemic lupus erythematosus (SLE). PKCδ is a signaling kinase with multiple downstream target proteins and with functions in various signaling pathways. Interestingly, mouse models have indicated a special role of the ubiquitously expressed protein in the control of B-cell tolerance revealed by the severe autoimmunity in Prkcd (-/-) knockout mice as the major phenotype. As such, the study of PKCδ deficiency in humans has tremendous potential in enhancing our knowledge on the mechanisms of B-cell tolerance. PMID:27541826

  5. Protein Kinase C δ: a Gatekeeper of Immune Homeostasis.

    PubMed

    Salzer, Elisabeth; Santos-Valente, Elisangela; Keller, Bärbel; Warnatz, Klaus; Boztug, Kaan

    2016-10-01

    Human autoimmune disorders present in various forms and are associated with a life-long burden of high morbidity and mortality. Many different circumstances lead to the loss of immune tolerance and often the origin is suspected to be multifactorial. Recently, patients with autosomal recessive mutations in PRKCD encoding protein kinase c delta (PKCδ) have been identified, representing a monogenic prototype for one of the most prominent forms of humoral systemic autoimmune diseases, systemic lupus erythematosus (SLE). PKCδ is a signaling kinase with multiple downstream target proteins and with functions in various signaling pathways. Interestingly, mouse models have indicated a special role of the ubiquitously expressed protein in the control of B-cell tolerance revealed by the severe autoimmunity in Prkcd (-/-) knockout mice as the major phenotype. As such, the study of PKCδ deficiency in humans has tremendous potential in enhancing our knowledge on the mechanisms of B-cell tolerance.

  6. R proteins as fundamentals of plant innate immunity.

    PubMed

    Głowacki, Sylwester; Macioszek, Violetta K; Kononowicz, Andrzej K

    2011-03-01

    Plants are attacked by a wide spectrum of pathogens, being the targets of viruses, bacteria, fungi, protozoa, nematodes and insects. Over the course of their evolution, plants have developed numerous defense mechanisms including the chemical and physical barriers that are constitutive elements of plant cell responses locally and/or systemically. However, the modern approach in plant sciences focuses on the evolution and role of plant protein receptors corresponding to specific pathogen effectors. The recognition of an invader's molecules could be in most cases a prerequisite sine qua non for plant survival. Although the predicted three-dimensional structure of plant resistance proteins (R) is based on research on their animal homologs, advanced technologies in molecular biology and bioinformatics tools enable the investigation or prediction of interaction mechanisms for specific receptors with pathogen effectors. Most of the identified R proteins belong to the NBS-LRR family. The presence of other domains (including the TIR domain) apart from NBS and LRR is fundamental for the classification of R proteins into subclasses. Recently discovered additional domains (e.g. WRKY) of R proteins allowed the examination of their localization in plant cells and the role they play in signal transduction during the plant resistance response to biotic stress factors. This review focuses on the current state of knowledge about the NBS-LRR family of plant R proteins: their structure, function and evolution, and the role they play in plant innate immunity.

  7. Thermal Effects on the Compressive Behavior of IM7/PET15 Laminates

    NASA Technical Reports Server (NTRS)

    Walker, Sandra Polesky

    2003-01-01

    The effect of changing operating temperature on the compressive response of IM7/PETI5 composite laminates is investigated within this paper. The three temperatures evaluated for this study were 129 C, 21 C, and 177 C, a spectrum from cryogenic to an elevated operating temperature. Laminate compressive strength property testing was conducted using the Wyoming Combined Load Compression fixture to generate strength data at the three operating temperatures of interest for several lay-ups. A three-dimensional finite element analysis model of a [90/0]8s composite laminate subject to compressive loading is developed. The model is used to study the key attributes of the laminate that significantly influence the state of stress in the laminate. Both the resin rich layer located between lamina and the thermal residual stresses present in the laminate due to curing are included in the analysis model. For the laminate modeled, the effect of modeling temperature dependent material properties was determined to be insignificant for the operating temperatures studied. Simply using the material properties measured at the operating temperature of interest was sufficient for predicting stresses accurately in a linear analysis for the current problem. The three-dimensional analysis results revealed that the application of an applied compressive axial load in the 0-degree direction decreased the interlaminar stresses present in the laminate initially due to curing. Therefore, failure was concluded not be attributable to the interlaminar stresses in the composite laminate being studied when a compressive load is applied. The magnitude of the measured laminate compressive strength change with a change in temperature is concluded to be dominated by the change in the lamina compressive axial strength with a change in temperature.

  8. Immunization with extracellular proteins of Mycobacterium tuberculosis induces cell-mediated immune responses and substantial protective immunity in a guinea pig model of pulmonary tuberculosis.

    PubMed Central

    Pal, P G; Horwitz, M A

    1992-01-01

    We have studied the capacity of a selected fraction of Mycobacterium tuberculosis extracellular proteins (EP) released into broth culture by mid-logarithmic-growth-phase organisms to induce cell-mediated immune responses and protective immunity in a guinea pig model of pulmonary tuberculosis. Guinea pigs infected with M. tuberculosis by aerosol but not uninfected control guinea pigs exhibit strong cell-mediated immune responses to EP, manifest by dose-dependent cutaneous delayed-type hypersensitivity and splenic lymphocyte proliferation. Guinea pigs immunized subcutaneously with EP but not sham-immunized control guinea pigs also develop strong cell-mediated immune responses to EP, manifest by dose-dependent cutaneous delayed-type hypersensitivity and splenic lymphocyte proliferation. EP is nonlethal and nontoxic to guinea pigs upon subcutaneous immunization. Guinea pigs immunized with EP and then challenged with aerosolized M. tuberculosis exhibit protective immunity. In five independent experiments, EP-immunized guinea pigs were consistently protected against clinical illness, including weight loss. Compared with EP-immunized guinea pigs, sham-immunized control guinea pigs lost 12.9 +/- 2.0% (mean +/- SE) of their total weight. EP-immunized guinea pigs also had a 10-fold reduction in viable M. tuberculosis bacilli in their lungs and spleens (P = 0.004 and 0.001, respectively) compared with sham-immunized control animals. In the two experiments in which some guinea pigs died after aerosol challenge, EP-immunized animals were protected from death. Whereas all 12 (100%) EP-immunized guinea pigs survived challenge with aerosolized M. tuberculosis, only 6 of 12 (50%) sham-immunized control guinea pigs survived challenge (P = 0.007, Fisher exact test). This study demonstrates that actively growing M. tuberculosis cells release immunoprotective molecules extracellularly, that a subunit vaccine against tuberculosis is feasible, and that extracellular molecules of M

  9. Heat Shock Proteins: Conditional Mediators of Inflammation in Tumor Immunity

    PubMed Central

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

    2012-01-01

    Heat shock protein (HSP)-based anticancer vaccines have undergone successful preclinical testing and are now entering clinical trial. Questions still remain, however regarding the immunological properties of HSPs. It is now accepted that many of the HSPs participate in tumor immunity, at least in part by chaperoning tumor antigenic peptides, introducing them into antigen presenting cells such as dendritic cells (DC) that display the antigens on MHC class I molecules on the cell surface and stimulate cytotoxic lymphocytes (CTL). However, in order for activated CD8+ T cells to function as effective CTL and kill tumor cells, additional signals must be induced to obtain a sturdy CTL response. These include the expression of co-stimulatory molecules on the DC surface and inflammatory events that can induce immunogenic cytokine cascades. That such events occur is indicated by the ability of Hsp70 vaccines to induce antitumor immunity and overcome tolerance to tumor antigens such as mucin1. Secondary activation of CTL can be induced by inflammatory signaling through Toll-like receptors and/or by interaction of antigen-activated T helper cells with the APC. We will discuss the role of the inflammatory properties of HSPs in tumor immunity and the potential role of HSPs in activating T helper cells and DC licensing. PMID:22566956

  10. Study of Out-Time on the Processing and Properties of IM7/977-3 Composites

    NASA Technical Reports Server (NTRS)

    Miller, Sandi G.; Sutter, James K.; Scheiman, Daniel A.; Maryanski, Michael; Schlea, Michelle

    2010-01-01

    The capability to manufacture large structures leads to weight savings and reduced risk relative to joining smaller components. However, manufacture of increasingly large composite components is pushing the out-life limits of epoxy/ carbon fiber prepreg. IM7/977-3 is an autoclave processable prepreg material, commonly used in aerospace structures. The out-life limit is reported as 30 days by the manufacturer. The purpose of this work was to evaluate the material processability and composite properties of 977-3 resin and IM7/977-3 prepreg that had been aged at room temperature for up to 60 days. The neat resin was evaluated by differential scanning calorimetry, DSC, to characterize cure behavior of the aged material, as well as any change in activation energy. The rise in the modulus of the uncured prepreg was monitored throughout the 60 days by dynamic mechanical analysis, DMA. Composite panels made of the fresh and aged prepreg material were also characterized by DMA. The overall test results suggested that IM7/977-3 was a robust material that offered quality laminates throughout this aging process when processed by autoclave.

  11. ETRAP (efficient trapping and purification) of target protein polyclonal antibodies from GST-protein immune sera.

    PubMed

    Crimmins, Dan L; Brada, Nancy A; Lockwood, Christina M; Griest, Terry A; Waldemer, Rachel J; Cervinski, Mark A; Ohlendorf, Matthew F; McQuillan, Jay J; Ladenson, Jack H

    2010-12-01

    Recombinant GST (glutathione transferase) proteins are widely used as immunogens to generate polyclonal antibodies. Advantages of using GST proteins include: commercially available cloning vectors, vast literature for protein expression in Escherichia coli, the ease of protein purification, immunogen can be used as an ELISA standard and GST can be removed in some systems. However, there are disadvantages: GST oligomerization, inclusion body formation and target protein insolubility after GST removal. Perhaps the most detrimental is the significant generation of anti-GST antibodies by the host animal. A two-column procedure using a glutathione-GST column and a glutathione-(GST-protein) column can yield affinity-purified anti-(GST-protein) polyclonal antibody. Several passes over the first column are often required, though, to completely extract the anti-GST antibodies from the immune sera. We reasoned that knowledge of the target protein linear epitope(s) would allow construction of a peptide affinity resin for a single-pass 'one and done' purification termed ETRAP (efficient trapping and purification). In the present paper, we describe our efforts and present data on rabbits and sheep immunized with GST proteins having target protein molecular masses of ~8, 21 and 33 kDa. The titre and purity of the target antibodies using the ETRAP protocol were comparable to the more laborious multi-column purifications but with a considerable saving in time. PMID:21054278

  12. [RGS proteins (regulators of G protein signaling) and their roles in regulation of immune response].

    PubMed

    Lewandowicz, Anna M; Kowalski, Marek L; Pawliczak, Rafał

    2004-01-01

    RGS proteins (Regulators of G-protein Signaling) comprise a protein family responsible for regulating G proteins. By enhancing the GTPase activity of the a subunit, they speed up the reconstruction of the heterotrimeric structure of G protein, thus inhibiting its signal transduction. Sst2 protein in yeast Saccharomyces cervisiae, FlbA in fungus Aspergillus nidulans, and Egl-10 in the nematode Caenorhabditis elegans are the first native G regulators with GTPase activity (GAPs:--GTPase-activating proteins). The existence of over 30 RGS human proteins has been confirmed thus far, and they have been grouped and classified into six subfamilies. In immunocompetent cells, RGS proteins are entangled in a complicate net of different interrelating signal pathways. They are connected with B- and T-cell chemokine susceptibility, efficient T cell proliferation, and the regulation of B cell maturation. They also take an essential part in inflammation. High hopes are held for drugs, which handle would be RGS proteins and which would further provide the possibility of modifying the pharmacokinetics of drugs acting through G protein- coupled receptors. The aim of this review is to discuss the new RGS protein family and explain the potential involvement of RGS proteins in the modulation of the immune response PMID:15459549

  13. Immunity to heat shock proteins and arthritic disorders.

    PubMed Central

    van Eden, W

    1999-01-01

    Adjuvant arthritis (AA) is a frequently used model of experimental arthritis. Because of its histopathology, which is reminiscent of rheumatoid arthritis in humans, AA is used as a model for the development of novel anti-inflammatory drugs. Recently, it has become evident that AA is a typical T-cell-mediated autoimmune condition. Therefore, novel immunotherapies targeted to T cells can be developed in this model. Analysis of responding T cells in AA have now led to the definition of various antigens with potential relevance to arthritis, including human arthritic conditions. One such antigen defined in AA is the 60kD heat shock protein. Both T-cell vaccination approaches and active antigen immunizations and antigen toleration approaches have turned out to be effective in suppressing AA. PMID:10231009

  14. Glycation of extracellular matrix proteins impairs migration of immune cells.

    PubMed

    Haucke, Elisa; Navarrete-Santos, Alexander; Simm, Andreas; Silber, Rolf-Edgar; Hofmann, Britt

    2014-01-01

    The immune response during aging and diabetes is disturbed and may be due to the altered migration of immune cells in an aged tissue. Our study should prove the hypothesis that age and diabetes-related advanced glycation end products (AGEs) have an impact on the migration and adhesion of human T-cells. To achieve our purpose, we used in vitro AGE-modified proteins (soluble albumin and fibronectin [FN]), as well as human collagen obtained from bypass graft. A Boyden chamber was used to study cell migration. Migrated Jurkat T-cells were analyzed by flow cytometry and cell adhesion by crystal violet staining. Actin polymerization was determined by phalloidin-Alexa-fluor 488-labeled antibody and fluorescence microscopy. We found that significantly fewer cells (50%, p = 0.003) migrated through methylglyoxal modified FN. The attachment to FN in the presence of AGE-bovine serum albumin (BSA) was also reduced (p < 0.05). In ex vivo experiments, isolated collagen from human vein graft material negatively affected the migration of the cells depending on the grade of AGE modification of the collagen. Collagen with a low AGE level reduced the cell migration by 30%, and collagen with a high AGE level by 60%. Interaction of the cells with an AGE-modified matrix, but not with soluble AGEs like BSA-AGE per se, was responsible for a disturbed migration. The reduced migration was accompanied by an impaired actin polymerization. We conclude that AGEs-modified matrix protein inhibits cell migration and adhesion of Jurkat T-cells. PMID:24635174

  15. Glycation of extracellular matrix proteins impairs migration of immune cells.

    PubMed

    Haucke, Elisa; Navarrete-Santos, Alexander; Simm, Andreas; Silber, Rolf-Edgar; Hofmann, Britt

    2014-01-01

    The immune response during aging and diabetes is disturbed and may be due to the altered migration of immune cells in an aged tissue. Our study should prove the hypothesis that age and diabetes-related advanced glycation end products (AGEs) have an impact on the migration and adhesion of human T-cells. To achieve our purpose, we used in vitro AGE-modified proteins (soluble albumin and fibronectin [FN]), as well as human collagen obtained from bypass graft. A Boyden chamber was used to study cell migration. Migrated Jurkat T-cells were analyzed by flow cytometry and cell adhesion by crystal violet staining. Actin polymerization was determined by phalloidin-Alexa-fluor 488-labeled antibody and fluorescence microscopy. We found that significantly fewer cells (50%, p = 0.003) migrated through methylglyoxal modified FN. The attachment to FN in the presence of AGE-bovine serum albumin (BSA) was also reduced (p < 0.05). In ex vivo experiments, isolated collagen from human vein graft material negatively affected the migration of the cells depending on the grade of AGE modification of the collagen. Collagen with a low AGE level reduced the cell migration by 30%, and collagen with a high AGE level by 60%. Interaction of the cells with an AGE-modified matrix, but not with soluble AGEs like BSA-AGE per se, was responsible for a disturbed migration. The reduced migration was accompanied by an impaired actin polymerization. We conclude that AGEs-modified matrix protein inhibits cell migration and adhesion of Jurkat T-cells.

  16. Immunization with Brucella VirB proteins reduces organ colonization in mice through a Th1-type immune response and elicits a similar immune response in dogs.

    PubMed

    Pollak, Cora N; Wanke, María Magdalena; Estein, Silvia M; Delpino, M Victoria; Monachesi, Norma E; Comercio, Elida A; Fossati, Carlos A; Baldi, Pablo C

    2015-03-01

    VirB proteins from Brucella spp. constitute the type IV secretion system, a key virulence factor mediating the intracellular survival of these bacteria. Here, we assessed whether a Th1-type immune response against VirB proteins may protect mice from Brucella infection and whether this response can be induced in the dog, a natural host for Brucella. Splenocytes from mice immunized with VirB7 or VirB9 responded to their respective antigens with significant and specific production of gamma interferon (IFN-γ), whereas interleukin-4 (IL-4) was not detected. Thirty days after an intraperitoneal challenge with live Brucella abortus, the spleen load of bacteria was almost 1 log lower in mice immunized with VirB proteins than in unvaccinated animals. As colonization reduction seemed to correlate with a Th1-type immune response against VirB proteins, we decided to assess whether such a response could be elicited in the dog. Peripheral blood mononuclear cells (PBMCs) from dogs immunized with VirB proteins (three subcutaneous doses in QuilA adjuvant) produced significantly higher levels of IFN-γ than cells from control animals upon in vitro stimulation with VirB proteins. A skin test to assess specific delayed-type hypersensitivity was positive in 4 out of 5 dogs immunized with either VirB7 or VirB9. As both proteins are predicted to locate in the outer membrane of Brucella organisms, the ability of anti-VirB antibodies to mediate complement-dependent bacteriolysis of B. canis was assessed in vitro. Sera from dogs immunized with either VirB7 or VirB9, but not from those receiving phosphate-buffered saline (PBS), produced significant bacteriolysis. These results suggest that VirB-specific responses that reduce organ colonization by Brucella in mice can be also elicited in dogs. PMID:25540276

  17. Immunization with Brucella VirB proteins reduces organ colonization in mice through a Th1-type immune response and elicits a similar immune response in dogs.

    PubMed

    Pollak, Cora N; Wanke, María Magdalena; Estein, Silvia M; Delpino, M Victoria; Monachesi, Norma E; Comercio, Elida A; Fossati, Carlos A; Baldi, Pablo C

    2015-03-01

    VirB proteins from Brucella spp. constitute the type IV secretion system, a key virulence factor mediating the intracellular survival of these bacteria. Here, we assessed whether a Th1-type immune response against VirB proteins may protect mice from Brucella infection and whether this response can be induced in the dog, a natural host for Brucella. Splenocytes from mice immunized with VirB7 or VirB9 responded to their respective antigens with significant and specific production of gamma interferon (IFN-γ), whereas interleukin-4 (IL-4) was not detected. Thirty days after an intraperitoneal challenge with live Brucella abortus, the spleen load of bacteria was almost 1 log lower in mice immunized with VirB proteins than in unvaccinated animals. As colonization reduction seemed to correlate with a Th1-type immune response against VirB proteins, we decided to assess whether such a response could be elicited in the dog. Peripheral blood mononuclear cells (PBMCs) from dogs immunized with VirB proteins (three subcutaneous doses in QuilA adjuvant) produced significantly higher levels of IFN-γ than cells from control animals upon in vitro stimulation with VirB proteins. A skin test to assess specific delayed-type hypersensitivity was positive in 4 out of 5 dogs immunized with either VirB7 or VirB9. As both proteins are predicted to locate in the outer membrane of Brucella organisms, the ability of anti-VirB antibodies to mediate complement-dependent bacteriolysis of B. canis was assessed in vitro. Sera from dogs immunized with either VirB7 or VirB9, but not from those receiving phosphate-buffered saline (PBS), produced significant bacteriolysis. These results suggest that VirB-specific responses that reduce organ colonization by Brucella in mice can be also elicited in dogs.

  18. Immunization with Brucella VirB Proteins Reduces Organ Colonization in Mice through a Th1-Type Immune Response and Elicits a Similar Immune Response in Dogs

    PubMed Central

    Pollak, Cora N.; Wanke, María Magdalena; Estein, Silvia M.; Delpino, M. Victoria; Monachesi, Norma E.; Comercio, Elida A.; Fossati, Carlos A.

    2014-01-01

    VirB proteins from Brucella spp. constitute the type IV secretion system, a key virulence factor mediating the intracellular survival of these bacteria. Here, we assessed whether a Th1-type immune response against VirB proteins may protect mice from Brucella infection and whether this response can be induced in the dog, a natural host for Brucella. Splenocytes from mice immunized with VirB7 or VirB9 responded to their respective antigens with significant and specific production of gamma interferon (IFN-γ), whereas interleukin-4 (IL-4) was not detected. Thirty days after an intraperitoneal challenge with live Brucella abortus, the spleen load of bacteria was almost 1 log lower in mice immunized with VirB proteins than in unvaccinated animals. As colonization reduction seemed to correlate with a Th1-type immune response against VirB proteins, we decided to assess whether such a response could be elicited in the dog. Peripheral blood mononuclear cells (PBMCs) from dogs immunized with VirB proteins (three subcutaneous doses in QuilA adjuvant) produced significantly higher levels of IFN-γ than cells from control animals upon in vitro stimulation with VirB proteins. A skin test to assess specific delayed-type hypersensitivity was positive in 4 out of 5 dogs immunized with either VirB7 or VirB9. As both proteins are predicted to locate in the outer membrane of Brucella organisms, the ability of anti-VirB antibodies to mediate complement-dependent bacteriolysis of B. canis was assessed in vitro. Sera from dogs immunized with either VirB7 or VirB9, but not from those receiving phosphate-buffered saline (PBS), produced significant bacteriolysis. These results suggest that VirB-specific responses that reduce organ colonization by Brucella in mice can be also elicited in dogs. PMID:25540276

  19. Using viromes to predict novel immune proteins in non-model organisms.

    PubMed

    Quistad, Steven D; Lim, Yan Wei; Silva, Genivaldo Gueiros Z; Nelson, Craig E; Haas, Andreas F; Kelly, Linda Wegley; Edwards, Robert A; Rohwer, Forest L

    2016-08-31

    Immunity is mostly studied in a few model organisms, leaving the majority of immune systems on the planet unexplored. To characterize the immune systems of non-model organisms alternative approaches are required. Viruses manipulate host cell biology through the expression of proteins that modulate the immune response. We hypothesized that metagenomic sequencing of viral communities would be useful to identify both known and unknown host immune proteins. To test this hypothesis, a mock human virome was generated and compared to the human proteome using tBLASTn, resulting in 36 proteins known to be involved in immunity. This same pipeline was then applied to reef-building coral, a non-model organism that currently lacks traditional molecular tools like transgenic animals, gene-editing capabilities, and in vitro cell cultures. Viromes isolated from corals and compared with the predicted coral proteome resulted in 2503 coral proteins, including many proteins involved with pathogen sensing and apoptosis. There were also 159 coral proteins predicted to be involved with coral immunity but currently lacking any functional annotation. The pipeline described here provides a novel method to rapidly predict host immune components that can be applied to virtually any system with the potential to discover novel immune proteins. PMID:27581878

  20. Using viromes to predict novel immune proteins in non-model organisms

    PubMed Central

    Lim, Yan Wei; Silva, Genivaldo Gueiros Z.; Nelson, Craig E.; Haas, Andreas F.; Kelly, Linda Wegley; Edwards, Robert A.; Rohwer, Forest L.

    2016-01-01

    Immunity is mostly studied in a few model organisms, leaving the majority of immune systems on the planet unexplored. To characterize the immune systems of non-model organisms alternative approaches are required. Viruses manipulate host cell biology through the expression of proteins that modulate the immune response. We hypothesized that metagenomic sequencing of viral communities would be useful to identify both known and unknown host immune proteins. To test this hypothesis, a mock human virome was generated and compared to the human proteome using tBLASTn, resulting in 36 proteins known to be involved in immunity. This same pipeline was then applied to reef-building coral, a non-model organism that currently lacks traditional molecular tools like transgenic animals, gene-editing capabilities, and in vitro cell cultures. Viromes isolated from corals and compared with the predicted coral proteome resulted in 2503 coral proteins, including many proteins involved with pathogen sensing and apoptosis. There were also 159 coral proteins predicted to be involved with coral immunity but currently lacking any functional annotation. The pipeline described here provides a novel method to rapidly predict host immune components that can be applied to virtually any system with the potential to discover novel immune proteins. PMID:27581878

  1. Protective immunity against Naegleria fowleri infection on mice immunized with the rNfa1 protein using mucosal adjuvants.

    PubMed

    Lee, Jinyoung; Yoo, Jong-Kyun; Sohn, Hae-Jin; Kang, Hee-kyoung; Kim, Daesik; Shin, Ho-Joon; Kim, Jong-Hyun

    2015-04-01

    The free-living amoeba, Naegleria fowleri, causes a fatal disease called primary amoebic meningoencephalitis (PAM) in humans and experimental animals. Of the pathogenic mechanism of N. fowleri concerning host tissue invasion, the adherence of amoeba to hose cells is the most important. We previously cloned the nfa1 gene from N. fowleri. The protein displayed immunolocalization in the pseudopodia, especially the food-cups structure, and was related to the contact-dependent mechanism of the amoebic pathogenicity in N. fowleri infection. The cholera toxin B subunit (CTB) and Escherichia coli heat-labile enterotoxin B subunit (LTB) have been used as potent mucosal adjuvants via the parenteral route of immunization in most cases. In this study, to examine the effect of protective immunity of the Nfa1 protein for N. fowleri infection with enhancement by CTB or LTB adjuvants, intranasally immunized BALB/c mice were infected with N. fowleri trophozoites for the development of PAM. The mean time to death of mice immunized with the Nfa1 protein using LTB or CTB adjuvant was prolonged by 5 or 8 days in comparison with that of the control mice. In particular, the survival rate of mice immunized with Nfa1 plus CTB was 100% during the experimental period. The serum IgG levels were significantly increased in mice immunized with Nfa1 protein plus CTB or LTB adjuvants. These results suggest that the Nfa1 protein, with CTB or LTB adjuvants, induces strong protective immunity in mice with PAM due to N. fowleri infection. PMID:25604672

  2. Protective immunity against Naegleria fowleri infection on mice immunized with the rNfa1 protein using mucosal adjuvants.

    PubMed

    Lee, Jinyoung; Yoo, Jong-Kyun; Sohn, Hae-Jin; Kang, Hee-kyoung; Kim, Daesik; Shin, Ho-Joon; Kim, Jong-Hyun

    2015-04-01

    The free-living amoeba, Naegleria fowleri, causes a fatal disease called primary amoebic meningoencephalitis (PAM) in humans and experimental animals. Of the pathogenic mechanism of N. fowleri concerning host tissue invasion, the adherence of amoeba to hose cells is the most important. We previously cloned the nfa1 gene from N. fowleri. The protein displayed immunolocalization in the pseudopodia, especially the food-cups structure, and was related to the contact-dependent mechanism of the amoebic pathogenicity in N. fowleri infection. The cholera toxin B subunit (CTB) and Escherichia coli heat-labile enterotoxin B subunit (LTB) have been used as potent mucosal adjuvants via the parenteral route of immunization in most cases. In this study, to examine the effect of protective immunity of the Nfa1 protein for N. fowleri infection with enhancement by CTB or LTB adjuvants, intranasally immunized BALB/c mice were infected with N. fowleri trophozoites for the development of PAM. The mean time to death of mice immunized with the Nfa1 protein using LTB or CTB adjuvant was prolonged by 5 or 8 days in comparison with that of the control mice. In particular, the survival rate of mice immunized with Nfa1 plus CTB was 100% during the experimental period. The serum IgG levels were significantly increased in mice immunized with Nfa1 protein plus CTB or LTB adjuvants. These results suggest that the Nfa1 protein, with CTB or LTB adjuvants, induces strong protective immunity in mice with PAM due to N. fowleri infection.

  3. Processing and Properties of Fiber Reinforced Polymeric Matrix Composites. Part 2; Processing Robustness of IM7/PETI Polyimide Composites

    NASA Technical Reports Server (NTRS)

    Hou, Tan-Hung

    1996-01-01

    The processability of a phenylethynyl terminated imide (PETI) resin matrix composite was investigated. Unidirectional prepregs were made by coating an N-methylpyrrolidone solution of the amide acid oligomer onto unsized IM7. Two batches of prepregs were used: one was made by NASA in-house, and the other was from an industrial source. The composite processing robustness was investigated with respect to the effect of B-staging conditions, the prepreg shelf life, and the optimal processing window. Rheological measurements indicated that PETI's processability was only slightly affected over a wide range of B-staging temperatures (from 250 C to 300 C). The open hole compression (OHC) strength values were statistically indistinguishable among specimens consolidated using various B-staging conditions. Prepreg rheology and OHC strengths were also found not to be affected by prolonged (i.e., up to 60 days) ambient storage. An optimal processing window was established using response surface methodology. It was found that IM7/PETI composite is more sensitive to the consolidation temperature than to the consolidation pressure. A good consolidation was achievable at 371 C/100 Psi, which yielded an OHC strength of 62 Ksi at room temperature. However, processability declined dramatically at temperatures below 350 C.

  4. Determination of Interlaminar Toughness of IM7/977-2 Composites at Temperature Extremes and Different Thicknesses

    NASA Technical Reports Server (NTRS)

    Johnson, W. S.; Pavlick, M. M.; Oliver, M. S.

    2005-01-01

    Composite materials are being used in the aerospace industry as a means of reducing vehicle weight. In particular, polymer matrix composites (PMC) are good candidates due to their high strength-to-weight and high stiffness-to-weight ratios. Future reusable space launch vehicles and space exploration structures will need advanced light weight composites in order to minimize vehicle weight while demonstrating robustness and durability, guaranteeing high factors of safety. In particular, the implementation of composite cryogenic propellant fuel tanks (cryotanks) for future reusable launch vehicles (RLVs) could greatly reduce the vehicle's weight versus identically sized cryotanks constructed of metallic materials. One candidate composite material for future cryotank designs is IM7/977-2, which is a graphite/epoxy system. A successful candidate must demonstrate reasonable structural properties over a wide range of temperatures. Since the matrix material is normally the weak link in the composite, tests that emphasize matrix-dominated behavior need to be conducted. Therefore, the objective of this work is to determine the mode I interlaminar fracture toughness of "unidirectional" 8-ply and 16-ply IM7/977-2 through experimental testing. Tests were performed at -196 degrees Celsius (-320 degrees Fahrenheit), 22 degrees Celsius (72 degrees Fahrenheit), 93 degrees Celsius (200 degrees Fahrenheit) and 160 degrees C (320 degrees Fahrenheit). Low temperature testing was completed while the specimen was submerged in a liquid nitrogen bath. High temperature testing was completed in a temperature-controlled oven.

  5. Immune Response of Multiparous Hyper-Immunized Sows against Peptides from Non-Structural and Structural Proteins of PRRSV

    PubMed Central

    Rascón-Castelo, Edgar; Burgara-Estrella, Alexel; Reséndiz-Sandoval, Mónica; Hernández-Lugo, Andrés; Hernández, Jesús

    2015-01-01

    The purpose of this study was to evaluate the humoral and cellular responses of commercial multiparous and hyper-immunized sows against peptides from non-structural (nsp) and structural proteins of porcine reproductive and respiratory syndrome virus (PRRSV). We selected sows with different numbers of parities from a commercial farm. Management practices on this farm include the use of the MLV commercial vaccine four times per year, plus two vaccinations during the acclimation period. The humoral response was evaluated via the antibody recognition of peptides from nsp and structural proteins, and the cellular response was assessed by measuring the frequency of peptide and PRRSV-specific IFN-gamma-secreting cells (IFNγ-SC). Our results show that sows with six parities have more antibodies against peptides from structural proteins than against peptides from nsp. The analysis of the cellular response revealed that the number of immunizations did not affect the frequency of IFNγ-SC and that the response was stronger against peptides from structural proteins (M protein) than against nsp (nsp2). In summary, these results demonstrate that multiparous, hyper-immunized sows have a stronger immune humoral response to PRRSV structural peptides than nsp, but no differences in IFNγ-SC against the same peptides were observed. PMID:26633527

  6. Different protein of Echinococcus granulosus stimulates dendritic induced immune response.

    PubMed

    Wang, Yana; Wang, Qiang; Lv, Shiyu; Zhang, Shengxiang

    2015-06-01

    Cystic echinococcosis is a chronic infectious disease that results from a host/parasite interaction. Vaccination with ferritin derived from Echinococcus granulosus is a potential preventative treatment. To understand whether ferritin is capable of inducing a host immune response, we investigated the response of dendritic cells (DCs) to both recombinant ferritin protein and the hydatid fluid (HF) of E. granulosus. We evaluated the immunomodulatory potential of these antigens by performing, immunocytochemistry, electron microscopy and in vivo imaging of monocyte-derived murine DCs. During antigen stimulation of DCs, ferritin cause DCs maturation and induced higher levels of surface marker expression and activated T-cell proliferation and migration. On contrary, HF failed to induce surface marker expression and to stimulate T-cell proliferation. In response to HF, DCs produced interleukin-6 (IL-6), but no IL-12 and IL-10. DCs stimulated with ferritin produced high levels of cytokines. Overall, HF appears to induce host immunosuppression in order to ensure parasite survival via inhibits DC maturation and promotes Th2-dependent secretion of cytokines. Although ferritin also promoted DC maturation and cytokine release, it also activates CD4+T-cell proliferation, but regard of the mechanism of the Eg.ferritin induce host to eradicate E. granulosus were not clear.

  7. Isoform-specific targeting of ROCK proteins in immune cells

    PubMed Central

    Zanin-Zhorov, Alexandra; Flynn, Ryan; Waksal, Samuel D.; Blazar, Bruce R.

    2016-01-01

    ABSTRACT Rho-associated kinase 1 (ROCK1) and ROCK2 are activated by Rho GTPase and control cytoskeleton rearrangement through modulating the phosphorylation of their down-stream effector molecules. Although these 2 isoforms share more than 90% homology within their kinase domain the question of whether ROCK proteins function identically in different cell types is not clear. By using both pharmacological inhibition and genetic knockdown approaches recent studies suggest that the ROCK2 isoform plays an exclusive role in controlling of T-cell plasticity and macrophage polarization. Specifically, selective ROCK2 inhibition shifts the balance between pro-inflammatory and regulatory T-cell subsets via concurrent regulation of STAT3 and STAT5 phosphorylation, respectively. Furthermore, the administration of an orally available selective ROCK2 inhibitor effectively ameliorates clinical manifestations in experimental models of autoimmunity and chronic graft-vs.-host disease (cGVHD). Because ROCK2 inhibition results in the suppression of M2-type macrophages while favoring polarization of M1-type macrophages, ROCK2 inhibition can correct the macrophage imbalance seen during age-related macular degeneration (AMD). In summary, the exclusive role of ROCK2 in immune system modulation argues for the development and testing of isoform-specific ROCK2 inhibitors for the treatment of inflammatory disorders. PMID:27254302

  8. Intradermal powder immunization with protein-containing vaccines.

    PubMed

    Weissmueller, Nikolas T; Schiffter, Heiko A; Pollard, Andrew J

    2013-06-01

    The central importance for global public health policy of delivering life-saving vaccines for all children makes the development of efficacious and safe needle-free alternatives to hypodermic needles, preferably in a thermostable form, a matter of pressing urgency. This paper comprehensively reviews past in vivo studies on intradermal powder immunization with vaccine formulations that do not require refrigeration. Particular emphasis is given to the immune response in relation to antigen adjuvantation. While needle-free intradermal delivery of vaccines induces a predominantly Th2-type immune response, adjuvants powerfully enhance and modulate the magnitude and nature of the elicited immune response at various effector sites.

  9. Modulation of cellular immune response against hepatitis C virus nonstructural protein 3 by cationic liposome encapsulated DNA immunization.

    PubMed

    Jiao, Xuanmao; Wang, Richard Y-H; Feng, Zhiming; Alter, Harvey J; Shih, James Wai-Kuo

    2003-02-01

    A vaccine strategy directed to increase Th1 cellular immune responses, particularly to hepatitis C virus (HCV) nonstructural protein 3 (NS3), has considerable potential to overcome the infection with HCV. DNA vaccination can induce both humoral and cellular immune responses, but it became apparent that the cellular uptake of naked DNA injected into muscle was not very efficient, as much of the DNA is degraded by interstitial nucleases before it reaches the nucleus for transcription. In this paper, cationic liposomes composed of different cationic lipids, such as dimethyl-dioctadecylammonium bromide (DDAB), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), or 1,2-dioleoyl-sn-glycerol-3-ethylphosphocholine (DOEPC), were used to improve DNA immunization in mice, and their efficiencies were compared. It was found that cationic liposome-mediated DNA immunization induced stronger HCV NS3-specific immune responses than immunization with naked DNA alone. Cationic liposomes composed of DDAB and equimolar of a neutral lipid, egg yolk phosphatidylcholine (EPC), induced the strongest antigen-specific Th1 type immune responses among the cationic liposome investigated, whereas the liposomes composed of 2 cationic lipids, DDAB and DOEPC, induced an antigen-specific Th2 type immune response. All cationic liposomes used in this study triggered high-level, nonspecific IL-12 production in mice, a feature important for the development of maximum Th1 immune responses. In conclusion, the cationic liposome-mediated gene delivery is a viable HCV vaccine strategy that should be further tested in the chimpanzee model. PMID:12540796

  10. Immunization

    MedlinePlus

    ... a lot worse. Some are even life-threatening. Immunization shots, or vaccinations, are essential. They protect against things like measles, ... B, polio, tetanus, diphtheria, and pertussis (whooping cough). Immunizations are important for adults as well as children. ...

  11. Immunizations

    MedlinePlus

    ... How Can I Help a Friend Who Cuts? Immunizations KidsHealth > For Teens > Immunizations Print A A A ... That Shot? en español Las vacunas Why Are Vaccinations Important? Measles, mumps, and whooping cough may seem ...

  12. The Escherichia coli uropathogenic-specific-protein-associated immunity protein 3 (Imu3) has nucleic acid -binding activity

    PubMed Central

    2014-01-01

    Background The Escherichia coli uropathogenic-specific protein (Usp) is a bacteriocin-like genotoxin, active against mammalian cells and associated with E. coli strains that provoke pyelonephritis, prostatitis and bacteraemia. Usp is encoded by a small pathogenicity island with three downstream small open reading frames (Imu1-3) that are believed to provide immunity to the producer. To prevent host suicide, colicins, bacteriocins of E. coli, form tight complexes with their cognate immunity proteins. Colicin – immunity protein complexes are among the strongest protein complexes known. Here, the Usp associated immunity protein 3 (Imu3) was partially characterized to gain insight into its role and mechanism of activity. Results Isolation and partial characterisation of the Usp-associated immunity protein-3 (Imu3) revealed that, while Usp and Imu3 do not form a high affinity complex, Imu3 exhibits DNA and RNA binding activity. Imu3 was also shown to protect DNA against degradation by colicin E7. Conclusions Our data infer that nonspecific DNA binding of the Imu3 immunity protein, prevents suicide of E. coli producing the genotoxin Usp. PMID:24472116

  13. ApnI, a Transmembrane Protein Responsible for Subtilomycin Immunity, Unveils a Novel Model for Lantibiotic Immunity

    PubMed Central

    Deng, Yun; Li, Cong-Zhi; Zhu, Yi-Guang; Wang, Peng-Xia; Qi, Qing-Dong; Fu, Jing-Jing; Peng, Dong-Hai; Ruan, Li-Fang

    2014-01-01

    Subtilomycin was detected from the plant endophytic strain Bacillus subtilis BSn5 and was first reported from B. subtilis strain MMA7. In this study, a gene cluster that has been proposed to be related to subtilomycin biosynthesis was isolated from the BSn5 genome and was experimentally validated by gene inactivation and heterologous expression. Comparison of the subtilomycin gene cluster with other verified related lantibiotic gene clusters revealed a particular organization of the genes apnI and apnT downstream of apnAPBC, which may be involved in subtilomycin immunity. Through analysis of expression of the apnI and/or apnT genes in the subtilomycin-sensitive strain CU1065 and inactivation of apnI and apnT in the producer strain BSn5, we showed that the single gene apnI, encoding a putative transmembrane protein, was responsible for subtilomycin immunity. To our knowledge, evidence for lantibiotic immunity that is solely dependent on a transmembrane protein is quite rare. Further bioinformatic analysis revealed the abundant presence of ApnI-like proteins that may be responsible for lantibiotic immunity in Bacillus and Paenibacillus. We cloned the paeI gene, encoding one such ApnI-like protein, into CU1065 and showed that it confers resistance to paenibacillin. However, no cross-resistance was detected between ApnI and PaeI, even though subtilomycin and paenibacillin share similar structures, suggesting that the protection provided by ApnI/ApnI-like proteins involves a specific-sequence recognition mechanism. Peptide release/binding assays indicated that the recombinant B. subtilis expressing apnI interacted with subtilomycin. Thus, ApnI represents a novel model for lantibiotic immunity that appears to be common. PMID:25085495

  14. The effects of physical aging at elevated temperatures on the viscoelastic creep on IM7/K3B

    NASA Technical Reports Server (NTRS)

    Gates, Thomas S.; Feldman, Mark

    1994-01-01

    Physical aging at elevated temperature of the advanced composite IM7/K3B was investigated through the use of creep compliance tests. Testing consisted of short term isothermal, creep/recovery with the creep segments performed at constant load. The matrix dominated transverse tensile and in-plane shear behavior were measured at temperatures ranging from 200 to 230 C. Through the use of time based shifting procedures, the aging shift factors, shift rates and momentary master curve parameters were found at each temperature. These material parameters were used as input to a predictive methodology, which was based upon effective time theory and linear viscoelasticity combined with classical lamination theory. Long term creep compliance test data was compared to predictions to verify the method. The model was then used to predict the long term creep behavior for several general laminates.

  15. Experimental Verification of a Progressive Damage Model for IM7/5260 Laminates Subjected to Tension-Tension Fatigue

    NASA Technical Reports Server (NTRS)

    Coats, Timothy W.; Harris, Charles E.

    1995-01-01

    The durability and damage tolerance of laminated composites are critical design considerations for airframe composite structures. Therefore, the ability to model damage initiation and growth and predict the life of laminated composites is necessary to achieve structurally efficient and economical designs. The purpose of this research is to experimentally verify the application of a continuum damage model to predict progressive damage development in a toughened material system. Damage due to monotonic and tension-tension fatigue was documented for IM7/5260 graphite/bismaleimide laminates. Crack density and delamination surface area were used to calculate matrix cracking and delamination internal state variables to predict stiffness loss in unnotched laminates. A damage dependent finite element code predicted the stiffness loss for notched laminates with good agreement to experimental data. It was concluded that the continuum damage model can adequately predict matrix damage progression in notched and unnotched laminates as a function of loading history and laminate stacking sequence.

  16. Measurements of the compressive behavior of AS4/3501-6 and IM7/8551-7 carbon/epoxy

    SciTech Connect

    Swanson, S.R.; Colvin, G.E. Jr.; Haslam, C.L.

    1988-01-01

    The compressive strength of carbon/epoxy materials is of importance in design, and is typically somewhat lower than the strength in tension. The measurement of compressive strength has been subject to some uncertainty, as different experimental techniques can give somewhat different results. A new specimen for the determination of compressive strength has been developed, based on compression of a 38 mm (1.5 in) diameter tube. The tube has been designed to prevent overall structural buckling and also to minimize end effects. This and a larger tubular specimen have been utilized to determine the compressive strength of AS4/3501-6 and IM7/8551-7 carbon/epoxy prepregs. The results are compared with previous tests, indicating that the results are relatively independent of the test specimen. The comparison also supports a criterion for laminate failure prediction reported previously. 15 refs., 6 figs., 1 tab.

  17. Carotenoid and protein supplementation have differential effects on pheasant ornamentation and immunity.

    PubMed

    Smith, H G; Råberg, L; Ohlsson, T; Granbom, M; Hasselquist, D

    2007-01-01

    A currently popular hypothesis states that the expression of carotenoid-dependent sexual ornaments and immune function may be correlated because both traits are positively affected by carotenoids. However, such a correlation may arise for another reason: it is well known that immune function is dependent on nutritional condition. A recent study has suggested that the expression of ornaments may too depend on nutritional condition, as males in good nutritional condition are better at assimilating and/or modulating carotenoids. Thus, carotenoid-dependent ornaments and immune function may be correlated because both are dependent on nutritional condition. To elucidate if, and how, ornamentation and immune function are linked, pheasant diets were supplemented with carotenoid and/or protein in a fully factorial experiment. Carotenoid treatment affected wattle coloration and tail growth, but not cellular or humoral immunity. Immunity was unrelated to males' initial ornamentation including wattle colour. Males in better body condition, measured as residual mass, increased their wattle coloration more when carotenoid supplemented. Protein positively affected humoral but not cellular immunity, but had no effect on ornaments. Cellular, but not humoral, immunity increased with male body condition. Thus, there was no evidence that an immune-stimulatory effect of carotenoids resulted in wattle coloration honestly signalling immune function, but wattle coloration may still signal male body condition.

  18. Arabidopsis resistance protein SNC1 activates immune responses through association with a transcriptional corepressor

    PubMed Central

    Zhu, Zhaohai; Xu, Fang; Zhang, Yaxi; Cheng, Yu Ti; Wiermer, Marcel; Li, Xin; Zhang, Yuelin

    2010-01-01

    In both plants and animals, nucleotide-binding (NB) domain and leucine-rich repeat (LRR)-containing proteins (NLR) function as sensors of pathogen-derived molecules and trigger immune responses. Although NLR resistance (R) proteins were first reported as plant immune receptors more than 15 years ago, how these proteins activate downstream defense responses is still unclear. Here we report that the Toll-like/interleukin-1 receptor (TIR)-NB-LRR R protein, suppressor of npr1-1, constitutive 1 (SNC1) functions through its associated protein, Topless-related 1 (TPR1). Knocking out TPR1 and its close homologs compromises immunity mediated by SNC1 and several other TIR-NB-LRR–type R proteins, whereas overexpression of TPR1 constitutively activates SNC1-mediated immune responses. TPR1 functions as a transcriptional corepressor and associates with histone deacetylase 19 in vivo. Among the target genes of TPR1 are Defense no Death 1 (DND1) and Defense no Death 2 (DND2), two known negative regulators of immunity that are repressed during pathogen infection, suggesting that TPR1 activates R protein-mediated immune responses through repression of negative regulators. PMID:20647385

  19. Negative control of BAK1 by protein phosphatase 2A during plant innate immunity

    PubMed Central

    Segonzac, Cécile; Macho, Alberto P; Sanmartín, Maite; Ntoukakis, Vardis; Sánchez-Serrano, José Juan; Zipfel, Cyril

    2014-01-01

    Recognition of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern-recognition receptors (PRRs) activates plant innate immunity, mainly through activation of numerous protein kinases. Appropriate induction of immune responses must be tightly regulated, as many of the kinases involved have an intrinsic high activity and are also regulated by other external and endogenous stimuli. Previous evidences suggest that PAMP-triggered immunity (PTI) is under constant negative regulation by protein phosphatases but the underlying molecular mechanisms remain unknown. Here, we show that protein Ser/Thr phosphatase type 2A (PP2A) controls the activation of PRR complexes by modulating the phosphostatus of the co-receptor and positive regulator BAK1. A potential PP2A holoenzyme composed of the subunits A1, C4, and B’η/ζ inhibits immune responses triggered by several PAMPs and anti-bacterial immunity. PP2A constitutively associates with BAK1 in planta. Impairment in this PP2A-based regulation leads to increased steady-state BAK1 phosphorylation, which can poise enhanced immune responses. This work identifies PP2A as an important negative regulator of plant innate immunity that controls BAK1 activation in surface-localized immune receptor complexes. PMID:25085430

  20. Maltose-binding protein is a potential carrier for oral immunizations.

    PubMed

    Bellot, P; Tiels, P; Melkebeek, V; Devriendt, B; Goddeeris, B M; Cox, E

    2013-03-15

    Maltose binding protein (MBP) is often fused to a relevant protein to improve its yield and facilitate its purification, but MBP can also enhance the immunogenicity of the fused proteins. Recent data suggest that MBP may potentiate antigen-presenting functions in immunized animals by providing intrinsic maturation stimuli to dendritic cells through TLR4. The aim of this study was to examine if an MBP-specific immune response can be elicited by oral administration of MBP. Therefore, in a first experiment the MBP specific immune response was analyzed after oral immunization with MBP or MBP+CT to piglets and both the systemic and mucosal immune responses were examined Although no high systemic response was observed in the MBP-group, a local mucosal IgM MBP-specific response in the jejunal Peyer's patches was observed. In the second experiment MBPFedF was orally administered to piglets. A significant systemic response against MBP and a weak response against FedF were found after oral administration of MBPFedF+CT. Also the presence of MBP-specific IgA ASC in the lamina propria indicates that a local intestinal immune response against MBP was induced. Our data suggests that MBP can cross the epithelial barrier reaching the gut-associated lymphoid tissue after oral administration to pigs, which implicates that MBP could act as a carrier and delivery system for fused proteins to target the vaccine antigens to intestinal immune cells. PMID:23078905

  1. Modulation of host adaptive immunity by hRSV proteins

    PubMed Central

    Espinoza, Janyra A; Bohmwald, Karen; Céspedes, Pablo F; Riedel, Claudia A; Bueno, Susan M; Kalergis, Alexis M

    2014-01-01

    Globally, the human respiratory syncytial virus (hRSV) is the major cause of lower respiratory tract infections (LRTIs) in infants and children younger than 2 years old. Furthermore, the number of hospitalizations due to LRTIs has shown a sustained increase every year due to the lack of effective vaccines against hRSV. Thus, this virus remains as a major public health and economic burden worldwide. The lung pathology developed in hRSV-infected humans is characterized by an exacerbated inflammatory and Th2 immune response. In order to rationally design new vaccines and therapies against this virus, several studies have focused in elucidating the interactions between hRSV virulence factors and the host immune system. Here, we discuss the main features of hRSV biology, the processes involved in virus recognition by the immune system and the most relevant mechanisms used by this pathogen to avoid the antiviral host response. PMID:25513775

  2. Low cost delivery of proteins bioencapsulated in plant cells to human non-immune or immune modulatory cells.

    PubMed

    Xiao, Yuhong; Kwon, Kwang-Chul; Hoffman, Brad E; Kamesh, Aditya; Jones, Noah T; Herzog, Roland W; Daniell, Henry

    2016-02-01

    Targeted oral delivery of GFP fused with a GM1 receptor binding protein (CTB) or human cell penetrating peptide (PTD) or dendritic cell peptide (DCpep) was investigated. Presence of GFP(+) intact plant cells between villi of ileum confirm their protection in the digestive system from acids/enzymes. Efficient delivery of GFP to gut-epithelial cells by PTD or CTB and to M cells by all these fusion tags confirm uptake of GFP in the small intestine. PTD fusion delivered GFP more efficiently to most tissues or organs than the other two tags. GFP was efficiently delivered to the liver by all fusion tags, likely through the gut-liver axis. In confocal imaging studies of human cell lines using purified GFP fused with different tags, GFP signal of DCpep-GFP was only detected within dendritic cells. PTD-GFP was only detected within kidney or pancreatic cells but not in immune modulatory cells (macrophages, dendritic, T, B, or mast cells). In contrast, CTB-GFP was detected in all tested cell types, confirming ubiquitous presence of GM1 receptors. Such low-cost oral delivery of protein drugs to sera, immune system or non-immune cells should dramatically lower their cost by elimination of prohibitively expensive fermentation, protein purification cold storage/transportation and increase patient compliance. PMID:26706477

  3. Protein A suppresses immune responses during Staphylococcus aureus bloodstream infection in guinea pigs

    DOE PAGES

    Kim, Hwan Keun; Falugi, Fabiana; Thomer, Lena; Missiakas, Dominique M.; Schneewind, Olaf

    2015-01-06

    Staphylococcus aureus infection is not associated with the development of protective immunity, and disease relapses occur frequently. We hypothesize that protein A, a factor that binds immunoglobulin Fcγ and cross-links VH3 clan B cell receptors (IgM), is the staphylococcal determinant for host immune suppression. To test this, vertebrate IgM was examined for protein A cross-linking. High VH3 binding activity occurred with human and guinea immunoglobulin, whereas mouse and rabbit immunoglobulins displayed little and no binding, respectively. Establishing a guinea pig model of S. aureus bloodstream infection, we show that protein A functions as a virulence determinant and suppresses host Bmore » cell responses. Immunization with SpAKKAA, which cannot bind immunoglobulin, elicits neutralizing antibodies that enable guinea pigs to develop protective immunity.« less

  4. Therapeutic proteins and nanotechnology: immune response and stealth bioengineered constructs.

    PubMed

    Lopez-Marin, Luz M; Tamariz, Elisa; Acosta-Torres, Laura S; Castaño, Victor M

    2013-06-01

    With unique potentials for organ drug delivery and targeting, intravenous administration of drugs has represented a key tool in biomedicine. A major concern of this route is the rapid capture and destruction of foreign substances by circulating immune cells. Knowledge about the inter-relationships between drugs and blood cells is essential for a better control in drug stability and bioavailability. In this review, both classical pathways and novel insights into the immune mechanisms leading to drug clearance after systemic delivery are described. Drug surface chemistry and size have been identified as critical factors for the activation of host immune responses, and their modification has been extensively explored in order to evade immune surveillance. Common strategies to camouflage drug surfaces through polymer-grafting are presented, with special emphasis on Poly(Ethylene Glycol) (PEG) linkages, one of the most diverse strategies for modifying biomolecular surfaces. Finally, the use of "smart shields", such as PEG attachments shed at particular intracellular conditions, is briefly overviewed as an interesting approach for balancing circulation half lives VS bioavailability in polymer-grafted formulations.

  5. Comparative Proteomics Identifies Host Immune System Proteins Affected by Infection with Mycobacterium bovis.

    PubMed

    López, Vladimir; Villar, Margarita; Queirós, João; Vicente, Joaquín; Mateos-Hernández, Lourdes; Díez-Delgado, Iratxe; Contreras, Marinela; Alves, Paulo C; Alberdi, Pilar; Gortázar, Christian; de la Fuente, José

    2016-03-01

    Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly impact human and animal health worldwide. The mycobacterial life cycle is complex, and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Eurasian wild boar (Sus scrofa) are natural reservoir hosts for MTBC and a model for mycobacterial infection and tuberculosis (TB). In the wild boar TB model, mycobacterial infection affects the expression of innate and adaptive immune response genes in mandibular lymph nodes and oropharyngeal tonsils, and biomarkers have been proposed as correlates with resistance to natural infection. However, the mechanisms used by mycobacteria to manipulate host immune response are not fully characterized. Our hypothesis is that the immune system proteins under-represented in infected animals, when compared to uninfected controls, are used by mycobacteria to guarantee pathogen infection and transmission. To address this hypothesis, a comparative proteomics approach was used to compare host response between uninfected (TB-) and M. bovis-infected young (TB+) and adult animals with different infection status [TB lesions localized in the head (TB+) or affecting multiple organs (TB++)]. The results identified host immune system proteins that play an important role in host response to mycobacteria. Calcium binding protein A9, Heme peroxidase, Lactotransferrin, Cathelicidin and Peptidoglycan-recognition protein were under-represented in TB+ animals when compared to uninfected TB- controls, but protein levels were higher as infection progressed in TB++ animals when compared to TB- and/or TB+ adult wild boar. MHCI was the only protein over-represented in TB+ adult wild boar when compared to uninfected TB- controls. The results reported here suggest that M. bovis manipulates host immune response by reducing the production of immune system proteins. However, as infection progresses, wild boar immune response recovers to limit pathogen

  6. Comparative Proteomics Identifies Host Immune System Proteins Affected by Infection with Mycobacterium bovis

    PubMed Central

    López, Vladimir; Villar, Margarita; Queirós, João; Vicente, Joaquín; Mateos-Hernández, Lourdes; Díez-Delgado, Iratxe; Contreras, Marinela; Alves, Paulo C.; Alberdi, Pilar; Gortázar, Christian; de la Fuente, José

    2016-01-01

    Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly impact human and animal health worldwide. The mycobacterial life cycle is complex, and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Eurasian wild boar (Sus scrofa) are natural reservoir hosts for MTBC and a model for mycobacterial infection and tuberculosis (TB). In the wild boar TB model, mycobacterial infection affects the expression of innate and adaptive immune response genes in mandibular lymph nodes and oropharyngeal tonsils, and biomarkers have been proposed as correlates with resistance to natural infection. However, the mechanisms used by mycobacteria to manipulate host immune response are not fully characterized. Our hypothesis is that the immune system proteins under-represented in infected animals, when compared to uninfected controls, are used by mycobacteria to guarantee pathogen infection and transmission. To address this hypothesis, a comparative proteomics approach was used to compare host response between uninfected (TB-) and M. bovis-infected young (TB+) and adult animals with different infection status [TB lesions localized in the head (TB+) or affecting multiple organs (TB++)]. The results identified host immune system proteins that play an important role in host response to mycobacteria. Calcium binding protein A9, Heme peroxidase, Lactotransferrin, Cathelicidin and Peptidoglycan-recognition protein were under-represented in TB+ animals when compared to uninfected TB- controls, but protein levels were higher as infection progressed in TB++ animals when compared to TB- and/or TB+ adult wild boar. MHCI was the only protein over-represented in TB+ adult wild boar when compared to uninfected TB- controls. The results reported here suggest that M. bovis manipulates host immune response by reducing the production of immune system proteins. However, as infection progresses, wild boar immune response recovers to limit pathogen

  7. Comparative Proteomics Identifies Host Immune System Proteins Affected by Infection with Mycobacterium bovis.

    PubMed

    López, Vladimir; Villar, Margarita; Queirós, João; Vicente, Joaquín; Mateos-Hernández, Lourdes; Díez-Delgado, Iratxe; Contreras, Marinela; Alves, Paulo C; Alberdi, Pilar; Gortázar, Christian; de la Fuente, José

    2016-03-01

    Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly impact human and animal health worldwide. The mycobacterial life cycle is complex, and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Eurasian wild boar (Sus scrofa) are natural reservoir hosts for MTBC and a model for mycobacterial infection and tuberculosis (TB). In the wild boar TB model, mycobacterial infection affects the expression of innate and adaptive immune response genes in mandibular lymph nodes and oropharyngeal tonsils, and biomarkers have been proposed as correlates with resistance to natural infection. However, the mechanisms used by mycobacteria to manipulate host immune response are not fully characterized. Our hypothesis is that the immune system proteins under-represented in infected animals, when compared to uninfected controls, are used by mycobacteria to guarantee pathogen infection and transmission. To address this hypothesis, a comparative proteomics approach was used to compare host response between uninfected (TB-) and M. bovis-infected young (TB+) and adult animals with different infection status [TB lesions localized in the head (TB+) or affecting multiple organs (TB++)]. The results identified host immune system proteins that play an important role in host response to mycobacteria. Calcium binding protein A9, Heme peroxidase, Lactotransferrin, Cathelicidin and Peptidoglycan-recognition protein were under-represented in TB+ animals when compared to uninfected TB- controls, but protein levels were higher as infection progressed in TB++ animals when compared to TB- and/or TB+ adult wild boar. MHCI was the only protein over-represented in TB+ adult wild boar when compared to uninfected TB- controls. The results reported here suggest that M. bovis manipulates host immune response by reducing the production of immune system proteins. However, as infection progresses, wild boar immune response recovers to limit pathogen

  8. A core viral protein binds host nucleosomes to sequester immune danger signals.

    PubMed

    Avgousti, Daphne C; Herrmann, Christin; Kulej, Katarzyna; Pancholi, Neha J; Sekulic, Nikolina; Petrescu, Joana; Molden, Rosalynn C; Blumenthal, Daniel; Paris, Andrew J; Reyes, Emigdio D; Ostapchuk, Philomena; Hearing, Patrick; Seeholzer, Steven H; Worthen, G Scott; Black, Ben E; Garcia, Benjamin A; Weitzman, Matthew D

    2016-07-01

    Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses. Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important part in innate immune responses. Viral-encoded core basic proteins compact viral genomes, but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones. Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles, it is unknown whether protein VII affects cellular chromatin. Here we show that protein VII alters cellular chromatin, leading us to hypothesize that this has an impact on antiviral responses during adenovirus infection in human cells. We find that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified post-translational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter the protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in the chromatin of members of the high-mobility-group protein B family (HMGB1, HMGB2 and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses. We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together, our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signaling.

  9. A core viral protein binds host nucleosomes to sequester immune danger signals.

    PubMed

    Avgousti, Daphne C; Herrmann, Christin; Kulej, Katarzyna; Pancholi, Neha J; Sekulic, Nikolina; Petrescu, Joana; Molden, Rosalynn C; Blumenthal, Daniel; Paris, Andrew J; Reyes, Emigdio D; Ostapchuk, Philomena; Hearing, Patrick; Seeholzer, Steven H; Worthen, G Scott; Black, Ben E; Garcia, Benjamin A; Weitzman, Matthew D

    2016-07-01

    Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses. Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important part in innate immune responses. Viral-encoded core basic proteins compact viral genomes, but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones. Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles, it is unknown whether protein VII affects cellular chromatin. Here we show that protein VII alters cellular chromatin, leading us to hypothesize that this has an impact on antiviral responses during adenovirus infection in human cells. We find that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified post-translational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter the protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in the chromatin of members of the high-mobility-group protein B family (HMGB1, HMGB2 and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses. We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together, our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signaling. PMID:27362237

  10. Transdermal immunization with large proteins by means of ultradeformable drug carriers.

    PubMed

    Paul, A; Cevc, G; Bachhawat, B K

    1995-12-01

    By means of novel, ultradeformable and self-optimizing agent carriers called transfersomes, large molecules can be brought into the body through intact permeability barriers. This permits non-invasive immunization through normal skin and gives rise to a similar or even slightly higher antibody titer than subcutaneous injections of the same immunogen formulation. The former type of immunization also results in a higher IgA/IgG ratio in the blood than the repeated immunogen injections, as shown here for a soluble protein, human serum albumin, as well as for an integral membrane protein, gap junction protein, in mice.

  11. Translationally Controlled Tumor Protein, a Dual Functional Protein Involved in the Immune Response of the Silkworm, Bombyx mori

    PubMed Central

    Hua, Xiaoting; Song, Liang; Xia, Qingyou

    2013-01-01

    Insect gut immunity is the first line of defense against oral infection. Although a few immune-related molecules in insect intestine has been identified by genomics or proteomics approach with comparison to well-studied tissues, such as hemolymph or fat body, our knowledge about the molecular mechanism underlying the gut immunity which would involve a variety of unidentified molecules is still limited. To uncover additional molecules that might take part in pathogen recognition, signal transduction or immune regulation in insect intestine, a T7 phage display cDNA library of the silkworm midgut is constructed. By use of different ligands for biopanning, Translationally Controlled Tumor Protein (TCTP) has been selected. BmTCTP is produced in intestinal epithelial cells and released into the gut lumen. The protein level of BmTCTP increases at the early time points during oral microbial infection and declines afterwards. In vitro binding assay confirms its activity as a multi-ligand binding molecule and it can further function as an opsonin that promotes the phagocytosis of microorganisms. Moreover, it can induce the production of anti-microbial peptide via a signaling pathway in which ERK is required and a dynamic tyrosine phosphorylation of certain cytoplasmic membrane protein. Taken together, our results characterize BmTCTP as a dual-functional protein involved in both the cellular and the humoral immune response of the silkworm, Bombyx mori. PMID:23894441

  12. Processing and properties of fiber reinforced polymeric matrix composites: I. IM7/LARC(TM)-PETI-7 polyimide composites

    NASA Technical Reports Server (NTRS)

    Hou, Tan-Hung

    1995-01-01

    A phenylethynyl terminated imide oligomer formed from the reaction of benzophenone tetracarboxylic acid dianhydride, an 75:25 molar ratio of 4,4'-oxydianiline and meta-phenylenediamine and 4-phenylethynylphthalic anhydride as the endcapper at a theoretical number average molecular weight (Mn) of approximately 3,700 g/mol was evaluated as a composite resin matrix. A glass transition temperature (Tg) of 315 deg C was reached after 250 deg C/1 hr annealing of the matrix resin. Unidirectional prepreg was made by coating an N-methylpyrrolidinone solution of the amide acid oligomer onto unsized IM7 graphite fibers. The thermal and rheological properties and the solvent/volatile depletion rates of the amide acid/NMP system were determined. This information was used to successfully design a molding cycle for composite fabrication. Composites molded under 800 Psi at 371 C consistently yielded good consolidation as measured by C-scan and optical photomicrography. The composite's short beam shear strength (SBS), longitudinal and transverse flexural strengths and moduli were measured at various temperatures. These composites exhibited excellent room temperature (RT) longitudinal flexural strength and modulus and RT SBS strength retention at 177 C.

  13. Comparison of Intralaminar and Interlaminar Mode-I Fracture Toughness of Unidirectional IM7/8552 Graphite/Epoxy Composite

    NASA Technical Reports Server (NTRS)

    Czabaj, Michael W.; Ratcliffe, James

    2012-01-01

    The intralaminar and interlaminar mode-I fracture-toughness of a unidirectional IM7/8552 graphite/epoxy composite were measured using compact tension (CT) and double cantilever beam (DCB) test specimens, respectively. Two starter crack geometries were considered for both the CT and DCB specimen configurations. In the first case, starter cracks were produced by 12.5 micron thick, Teflon film inserts. In the second case, considerably sharper starter cracks were produced by fatigue precracking. For each specimen configuration, use of the Teflon film starter cracks resulted in initially unstable crack growth and artificially high initiation fracture-toughness values. Conversely, specimens with fatigue precracks exhibited stable growth onset and lower initiation fracture toughness. For CT and DCB specimens with fatigue precracks, the intralaminar and interlaminar initiation fracture toughnesses were approximately equal. However, during propagation, the CT specimens exhibited more extensive fiber bridging, and rapidly increasing R-curve behavior as compared to the DCB specimens. Observations of initiation and propagation of intralaminar and interlaminar fracture, and the measurements of fracture toughness, were supported by fractographic analysis using scanning electron microscopy.

  14. Effects of Aging-Time Reference on the Long Term Behavior of the IM7/K3B Composite

    NASA Technical Reports Server (NTRS)

    Veazie, David R.; Gates, Thomas S.

    1998-01-01

    An analytical study was undertaken to investigate the effects of the time-based shift reference on the long term behavior of the graphite reinforced thermoplastic polyimide composite IM7/K3B at elevated temperature. Creep compliance and the effects of physical aging on the time dependent response was measured for uniaxial loading at several isothermal conditions below the glass transition temperature (T(sub g). Two matrix dominated loading modes, shear and transverse, were investigated in tension and compression. The momentary sequenced creep/aging curves were collapsed through a horizontal (time) shift using the shortest, middle and longest aging time curve as the reference curve. Linear viscoelasticity was used to characterize the creep/recovery behavior and superposition techniques were used to establish the physical aging related material constants. The use of effective time expressions in a laminated plate model allowed for the prediction of long term creep compliance. The effect of using different reference curves with time/aging-time superposition was most sensitive to the physical aging shift rate at lower test temperatures. Depending on the loading mode, the reference curve used can result in a more accurate long term prediction, especially at lower test temperatures.

  15. Comparisons of Allergenic and Metazoan Parasite Proteins: Allergy the Price of Immunity.

    PubMed

    Tyagi, Nidhi; Farnell, Edward J; Fitzsimmons, Colin M; Ryan, Stephanie; Tukahebwa, Edridah; Maizels, Rick M; Dunne, David W; Thornton, Janet M; Furnham, Nicholas

    2015-10-01

    Allergic reactions can be considered as maladaptive IgE immune responses towards environmental antigens. Intriguingly, these mechanisms are observed to be very similar to those implicated in the acquisition of an important degree of immunity against metazoan parasites (helminths and arthropods) in mammalian hosts. Based on the hypothesis that IgE-mediated immune responses evolved in mammals to provide extra protection against metazoan parasites rather than to cause allergy, we predict that the environmental allergens will share key properties with the metazoan parasite antigens that are specifically targeted by IgE in infected human populations. We seek to test this prediction by examining if significant similarity exists between molecular features of allergens and helminth proteins that induce an IgE response in the human host. By employing various computational approaches, 2712 unique protein molecules that are known IgE antigens were searched against a dataset of proteins from helminths and parasitic arthropods, resulting in a comprehensive list of 2445 parasite proteins that show significant similarity through sequence and structure with allergenic proteins. Nearly half of these parasite proteins from 31 species fall within the 10 most abundant allergenic protein domain families (EF-hand, Tropomyosin, CAP, Profilin, Lipocalin, Trypsin-like serine protease, Cupin, BetV1, Expansin and Prolamin). We identified epitopic-like regions in 206 parasite proteins and present the first example of a plant protein (BetV1) that is the commonest allergen in pollen in a worm, and confirming it as the target of IgE in schistosomiasis infected humans. The identification of significant similarity, inclusive of the epitopic regions, between allergens and helminth proteins against which IgE is an observed marker of protective immunity explains the 'off-target' effects of the IgE-mediated immune system in allergy. All these findings can impact the discovery and design of molecules

  16. Comparisons of Allergenic and Metazoan Parasite Proteins: Allergy the Price of Immunity

    PubMed Central

    Tyagi, Nidhi; Farnell, Edward J; Fitzsimmons, Colin M; Ryan, Stephanie; Tukahebwa, Edridah; Maizels, Rick M; Dunne, David W; Thornton, Janet M; Furnham, Nicholas

    2015-01-01

    Allergic reactions can be considered as maladaptive IgE immune responses towards environmental antigens. Intriguingly, these mechanisms are observed to be very similar to those implicated in the acquisition of an important degree of immunity against metazoan parasites (helminths and arthropods) in mammalian hosts. Based on the hypothesis that IgE-mediated immune responses evolved in mammals to provide extra protection against metazoan parasites rather than to cause allergy, we predict that the environmental allergens will share key properties with the metazoan parasite antigens that are specifically targeted by IgE in infected human populations. We seek to test this prediction by examining if significant similarity exists between molecular features of allergens and helminth proteins that induce an IgE response in the human host. By employing various computational approaches, 2712 unique protein molecules that are known IgE antigens were searched against a dataset of proteins from helminths and parasitic arthropods, resulting in a comprehensive list of 2445 parasite proteins that show significant similarity through sequence and structure with allergenic proteins. Nearly half of these parasite proteins from 31 species fall within the 10 most abundant allergenic protein domain families (EF-hand, Tropomyosin, CAP, Profilin, Lipocalin, Trypsin-like serine protease, Cupin, BetV1, Expansin and Prolamin). We identified epitopic-like regions in 206 parasite proteins and present the first example of a plant protein (BetV1) that is the commonest allergen in pollen in a worm, and confirming it as the target of IgE in schistosomiasis infected humans. The identification of significant similarity, inclusive of the epitopic regions, between allergens and helminth proteins against which IgE is an observed marker of protective immunity explains the ‘off-target’ effects of the IgE-mediated immune system in allergy. All these findings can impact the discovery and design of

  17. A Probiotic Adjuvant Lactobacillus rhamnosus Enhances Specific Immune Responses after Ocular Mucosal Immunization with Chlamydial Polymorphic Membrane Protein C.

    PubMed

    Inic-Kanada, Aleksandra; Stojanovic, Marijana; Marinkovic, Emilija; Becker, Elisabeth; Stein, Elisabeth; Lukic, Ivana; Djokic, Radmila; Schuerer, Nadine; Hegemann, Johannes H; Barisani-Asenbauer, Talin

    2016-01-01

    Recent advances in the development of chlamydia vaccines, using live-attenuated or ultraviolet light-inactivated chlamydia, are paving the way for new possibilities to oppose the societal challenges posed by chlamydia-related diseases, such as blinding trachoma. An effective subunit vaccine would mitigate the risks associated with the use of a whole-cell vaccine. Our rationale for the design of an efficient subunit vaccine against Chlamydia trachomatis (Ct) is based on the membrane proteins involved in the initial Ct-host cell contact and on the route of immunization that mimics the natural infection process (i.e., via the ocular mucosa). The first aim of our study was to characterize the specific conjunctival and vaginal immune responses following eye drop immunization in BALB/c mice, using the N-terminal portion of the Ct serovar E polymorphic membrane protein C (N-PmpC) as the subunit vaccine antigen. Second, we aimed to examine the adjuvant properties of the probiotic Lactobacillus rhamnosus (LB) when formulated with N-PmpC. N-PmpC applied alone stimulated the production of N-PmpC- and Ct serovar B-specific antibodies in serum, tears and vaginal washes, whereas the combination with LB significantly enhanced these responses. The N-PmpC/LB combination initiated a T cell response characterized by an elevated percentage of CD25+ T cells and CD8+ effector T cells, enhanced CD4+ T-helper 1 skewing, and increased regulatory T cell responses. Together, these results show that eye drop vaccination with combined use of N-PmpC and a live probiotic LB stimulates specific cellular and humoral immune responses, not only locally in the conjunctiva but also in the vaginal mucosa, which could be a promising approach in Ct vaccine development. PMID:27636704

  18. A Probiotic Adjuvant Lactobacillus rhamnosus Enhances Specific Immune Responses after Ocular Mucosal Immunization with Chlamydial Polymorphic Membrane Protein C

    PubMed Central

    Marinkovic, Emilija; Becker, Elisabeth; Stein, Elisabeth; Lukic, Ivana; Djokic, Radmila; Schuerer, Nadine; Hegemann, Johannes H.; Barisani-Asenbauer, Talin

    2016-01-01

    Recent advances in the development of chlamydia vaccines, using live-attenuated or ultraviolet light-inactivated chlamydia, are paving the way for new possibilities to oppose the societal challenges posed by chlamydia-related diseases, such as blinding trachoma. An effective subunit vaccine would mitigate the risks associated with the use of a whole-cell vaccine. Our rationale for the design of an efficient subunit vaccine against Chlamydia trachomatis (Ct) is based on the membrane proteins involved in the initial Ct-host cell contact and on the route of immunization that mimics the natural infection process (i.e., via the ocular mucosa). The first aim of our study was to characterize the specific conjunctival and vaginal immune responses following eye drop immunization in BALB/c mice, using the N-terminal portion of the Ct serovar E polymorphic membrane protein C (N-PmpC) as the subunit vaccine antigen. Second, we aimed to examine the adjuvant properties of the probiotic Lactobacillus rhamnosus (LB) when formulated with N-PmpC. N-PmpC applied alone stimulated the production of N-PmpC- and Ct serovar B-specific antibodies in serum, tears and vaginal washes, whereas the combination with LB significantly enhanced these responses. The N-PmpC/LB combination initiated a T cell response characterized by an elevated percentage of CD25+ T cells and CD8+ effector T cells, enhanced CD4+ T-helper 1 skewing, and increased regulatory T cell responses. Together, these results show that eye drop vaccination with combined use of N-PmpC and a live probiotic LB stimulates specific cellular and humoral immune responses, not only locally in the conjunctiva but also in the vaginal mucosa, which could be a promising approach in Ct vaccine development. PMID:27636704

  19. Encapsulated cellular implants for recombinant protein delivery and therapeutic modulation of the immune system.

    PubMed

    Lathuilière, Aurélien; Mach, Nicolas; Schneider, Bernard L

    2015-05-08

    Ex vivo gene therapy using retrievable encapsulated cellular implants is an effective strategy for the local and/or chronic delivery of therapeutic proteins. In particular, it is considered an innovative approach to modulate the activity of the immune system. Two recently proposed therapeutic schemes using genetically engineered encapsulated cells are discussed here: the chronic administration of monoclonal antibodies for passive immunization against neurodegenerative diseases and the local delivery of a cytokine as an adjuvant for anti-cancer vaccines.

  20. Encapsulated Cellular Implants for Recombinant Protein Delivery and Therapeutic Modulation of the Immune System

    PubMed Central

    Lathuilière, Aurélien; Mach, Nicolas; Schneider, Bernard L.

    2015-01-01

    Ex vivo gene therapy using retrievable encapsulated cellular implants is an effective strategy for the local and/or chronic delivery of therapeutic proteins. In particular, it is considered an innovative approach to modulate the activity of the immune system. Two recently proposed therapeutic schemes using genetically engineered encapsulated cells are discussed here: the chronic administration of monoclonal antibodies for passive immunization against neurodegenerative diseases and the local delivery of a cytokine as an adjuvant for anti-cancer vaccines. PMID:26006227

  1. Recombinant lipidated dengue-3 envelope protein domain III stimulates broad immune responses in mice.

    PubMed

    Chiang, Chen-Yi; Liu, Shih-Jen; Hsieh, Chun-Hsiang; Chen, Mei-Yu; Tsai, Jy-Ping; Liu, Hsueh-Hung; Chen, I-Hua; Chong, Pele; Leng, Chih-Hsiang; Chen, Hsin-Wei

    2016-02-17

    The linkage of an immunogen with a toll-like receptor ligand has great potential to induce highly potent immune responses with the initial features of antigen-presenting cell activation. In the current study, we expressed recombinant dengue-3 envelope protein domain III (D3ED III) in lipidated form using an Escherichia coli-based system. The recombinant lipidated dengue-3 envelope protein domain III (LD3ED III) augments the expression levels of IL-12 family cytokines. LD3ED III-immunized mice enhance wide ranges of T cell responses as indicated by IFN-γ, IL-17, IL-21 production. Additionally, LD3ED III-immunized mice increase the frequencies of anti-D3ED III antibody producing cells. The boosted antibody titers cover various IgG isotypes, including IgG1, IgG2a, IgG2b, and IgG3. Importantly, LD3ED III-immunized mice induce neutralizing antibody capacity associated with a reduction of viremia levels after challenges. In contrast, mice that are immunized with D3ED III formulated with aluminum phosphate (D3ED III/Alum) only enhance Th2 responses and boost IgG1 antibody titers. Neither neutralizing antibody responses nor the inhibition of viremia levels after challenge is observed in mice that are immunized with D3ED III/Alum. These results suggest that LD3ED III can induce broad profiles of cellular and humoral immune responses.

  2. Immunization.

    ERIC Educational Resources Information Center

    Guerin, Nicole; And Others

    1986-01-01

    Contents of this double journal issue concern immunization and primary health care of children. The issue decribes vaccine storage and sterilization techniques, giving particular emphasis to the role of the cold chain, i.e., the maintenance of a specific temperature range to assure potency of vaccines as they are moved from a national storage…

  3. Possible association between phages, Hoc protein, and the immune system.

    PubMed

    Dabrowska, K; Switała-Jeleń, K; Opolski, A; Górski, A

    2006-02-01

    Mammals have become "an environment" for enterobacterial phage life cycles. Therefore it could be expected that bacteriophages adapt to them. This adaptation must comprise bacteriophage proteins. Gp Hoc seems to have significance neither for phage particle structure nor for phage antibacterial activity. It is evidently not necessary for the "typical" antibacterial actions of bacteriophages. But the rules of evolution make it improbable that gp Hoc really has no function, and non-essential genes of T4-type phages are probably important for phages' adaptation to their particular lifestyle. More interesting is the eukaryotic origin of gp Hoc: a resemblance to immunoglobulin-like proteins that reflects their evolutionary relation. Substantial differences in biological activity between T4 and a mutant that lacks gp Hoc were observed in a mammalian system. Hoc protein seems to be one of the molecules predicted to interact with mammalian organisms and/or modulate these interactions. PMID:16195787

  4. Arabidopsis heterotrimeric G proteins regulate immunity by directly coupling to the FLS2 receptor

    PubMed Central

    Liang, Xiangxiu; Ding, Pingtao; Lian, Kehui; Wang, Jinlong; Ma, Miaomiao; Li, Lin; Li, Lei; Li, Meng; Zhang, Xiaojuan; Chen, She; Zhang, Yuelin; Zhou, Jian-Min

    2016-01-01

    The Arabidopsis immune receptor FLS2 perceives bacterial flagellin epitope flg22 to activate defenses through the central cytoplasmic kinase BIK1. The heterotrimeric G proteins composed of the non-canonical Gα protein XLG2, the Gβ protein AGB1, and the Gγ proteins AGG1 and AGG2 are required for FLS2-mediated immune responses through an unknown mechanism. Here we show that in the pre-activation state, XLG2 directly interacts with FLS2 and BIK1, and it functions together with AGB1 and AGG1/2 to attenuate proteasome-mediated degradation of BIK1, allowing optimum immune activation. Following the activation by flg22, XLG2 dissociates from AGB1 and is phosphorylated by BIK1 in the N terminus. The phosphorylated XLG2 enhances the production of reactive oxygen species (ROS) likely by modulating the NADPH oxidase RbohD. The study demonstrates that the G proteins are directly coupled to the FLS2 receptor complex and regulate immune signaling through both pre-activation and post-activation mechanisms. DOI: http://dx.doi.org/10.7554/eLife.13568.001 PMID:27043937

  5. Modulation of proteinase-K resistant prion protein by prion peptide immunization.

    PubMed

    Souan, L; Tal, Y; Felling, Y; Cohen, I R; Taraboulos, A; Mor, F

    2001-08-01

    Prion diseases are caused by conformational alterations in the prion protein (PrP). The immune system has been assumed to be non-responsive to the self-prion protein, therefore, PrP autoimmunity has not been investigated. Here, we immunized various strains of mice with PrP peptides, some selected to fit the MHC class II-peptide binding motif. We found that specific PrP peptides elicited strong immune responses in NOD, C57BL/6 and A/J mice. To test the functional effect of this immunization, we examined the expression of proteinase-K-resistant PrP by a scrapie-infected tumor transplanted to immunized syngeneic A/J mice. PrP peptide vaccination did not affect the growth of the infected tumor transplant, but significantly reduced the level of protease-resistant PrP. Our results demonstrate that self-PrP peptides are immunogenic in mice and suggest that this immune response might affect PrP-scrapie levels in certain conditions.

  6. The Dependence of the Change in the Coefficient of Thermal Expansion of Graphite Fiber Reinforced Polyimide IM7-K3B on Microcracking due to Thermal Cycling

    NASA Technical Reports Server (NTRS)

    Stewart, Melissa C.

    1995-01-01

    Composite IM7-K3B was subjected to a simulated high speed aircraft thermal environment to determine the effects of microcracking on the change in CTE. IM7-K3B is a graphite fiber reinforced polyimide laminate, manufactured by Dupont. The lay-up for the material was (0.90((Sub 3)(Sub s))). The specimens were placed in a laser-interferometric dilatometer to obtain thermal expansion measurements and were then repeatedly cycled between -65 F and 350 F up to 1000 cycles. After cycling they were scanned for microcracks at a magnification of 400x. The material was expected not to crack and to have a near zero CTE. Some microcracking did occur in all specimens and extensive microcracking occurred in one specimen. Further testing is required to determine how closely the CTE and microcracking are related.

  7. Immune response in mice to ingested soya protein: antibody production, oral tolerance and maternal transfer.

    PubMed

    Christensen, Hanne R; Brix, Susanne; Frøkiaer, Hanne

    2004-05-01

    While allergic reactions to soya are increasingly investigated, the normal immune response to ingested soya is scarcely described. In the present study, we wanted to characterise the soya-specific immune response in healthy mice ingesting soya protein. Mice fed a soya-containing diet (F0) and mice of the first (F1) and second (F2) offspring generation bred on a soya protein-free diet were used either directly or were transferred between the soya-containing and soya protein-free diet during pregnancy or neonatal life. The mice were compared as to levels of naturally occurring specific antibodies analysed by ELISA, and to the presence of oral tolerance detected as a suppressed antibody and cell-proliferation response upon immunisation with soya protein. F0 mice generated soya-specific antibodies, while oral tolerance to the same soya proteins was also clearly induced. When F0 dams were transferred to soya protein-free feed before mating, the F1 and F2 offspring generations showed no significantly different response, indicating that soya-specific immune components were not maternally transmitted. However, the ingestion of dietary soya protein by F1 mice during late pregnancy and lactation caused a lasting antibody response in the offspring, but in this case in the absence of oral tolerance. This indicates that, under certain conditions, factors involved in spontaneous antibody production can be transmitted from mother to offspring. Understanding the immune response to soya protein ingested under healthy conditions is important in the assessment of adverse effects of soya protein and in the use of animal allergy models. The present results add to this understanding. PMID:15137924

  8. Heat Shock Proteins: A Review of the Molecular Chaperones for Plant Immunity

    PubMed Central

    Park, Chang-Jin; Seo, Young-Su

    2015-01-01

    As sessile organisms, plants are exposed to persistently changing stresses and have to be able to interpret and respond to them. The stresses, drought, salinity, chemicals, cold and hot temperatures, and various pathogen attacks have interconnected effects on plants, resulting in the disruption of protein homeostasis. Maintenance of proteins in their functional native conformations and preventing aggregation of non-native proteins are important for cell survival under stress. Heat shock proteins (HSPs) functioning as molecular chaperones are the key components responsible for protein folding, assembly, translocation, and degradation under stress conditions and in many normal cellular processes. Plants respond to pathogen invasion using two different innate immune responses mediated by pattern recognition receptors (PRRs) or resistance (R) proteins. HSPs play an indispensable role as molecular chaperones in the quality control of plasma membrane-resident PRRs and intracellular R proteins against potential invaders. Here, we specifically discuss the functional involvement of cytosolic and endoplasmic reticulum (ER) HSPs/chaperones in plant immunity to obtain an integrated understanding of the immune responses in plant cells. PMID:26676169

  9. Antibody study in canine distemper virus nucleocapsid protein gene-immunized mice.

    PubMed

    Yuan, B; Li, X Y; Zhu, T; Yuan, L; Hu, J P; Chen, J; Gao, W; Ren, W Z

    2015-01-01

    The gene for the nucleocapsid (N) protein of canine distemper virus was cloned into the pMD-18T vector, and positive recombinant plasmids were obtained by enzyme digestion and sequencing. After digestion by both EcoRI and KpnI, the plasmid was directionally cloned into the eukaryotic expression vector pcDNA; the positive clone pcDNA-N was screened by electrophoresis and then transfected into COS-7 cells. Immunofluorescence analysis results showed that the canine distemper virus N protein was expressed in the cytoplasm of transfected COS-7 cells. After emulsification in Freund's adjuvant, the recombinant plasmid pcDNA-N was injected into the abdominal cavity of 8-week-old BABL/c mice, with the pcDNA original vector used as a negative control. Mice were immunized 3 times every 2 weeks. The blood of immunized mice was drawn 2 weeks after completing the immunizations to measure titer levels. The antibody titer in the pcDNA-N test was 10(1.62 ± 0.164), while in the control group this value was 10(0.52 ± 0.56), indicating that specific humoral immunity was induced in canine distemper virus nucleocapsid protein-immunized mice. PMID:25966074

  10. Protein-poor diet reduces host-specific immune gene expression in Bombus terrestris.

    PubMed

    Brunner, Franziska S; Schmid-Hempel, Paul; Barribeau, Seth M

    2014-07-01

    Parasites infect hosts non-randomly as genotypes of hosts vary in susceptibility to the same genotypes of parasites, but this specificity may be modulated by environmental factors such as nutrition. Nutrition plays an important role for any physiological investment. As immune responses are costly, resource limitation should negatively affect immunity through trade-offs with other physiological requirements. Consequently, nutritional limitation should diminish immune capacity in general, but does it also dampen differences among hosts? We investigated the effect of short-term pollen deprivation on the immune responses of our model host Bombus terrestris when infected with the highly prevalent natural parasite Crithidia bombi. Bumblebees deprived of pollen, their protein source, show reduced immune responses to infection. They failed to upregulate a number of genes, including antimicrobial peptides, in response to infection. In particular, they also showed less specific immune expression patterns across individuals and colonies. These findings provide evidence for how immune responses on the individual-level vary with important elements of the environment and illustrate how nutrition can functionally alter not only general resistance, but also alter the pattern of specific host-parasite interactions. PMID:24850921

  11. Surfactant Protein-D Is Essential for Immunity to Helminth Infection.

    PubMed

    Thawer, Sumaiyya; Auret, Jennifer; Schnoeller, Corinna; Chetty, Alisha; Smith, Katherine; Darby, Matthew; Roberts, Luke; Mackay, Rosie-Marie; Whitwell, Harry J; Timms, John F; Madsen, Jens; Selkirk, Murray E; Brombacher, Frank; Clark, Howard William; Horsnell, William G C

    2016-02-01

    Pulmonary epithelial cell responses can enhance type 2 immunity and contribute to control of nematode infections. An important epithelial product is the collectin Surfactant Protein D (SP-D). We found that SP-D concentrations increased in the lung following Nippostrongylus brasiliensis infection; this increase was dependent on key components of the type 2 immune response. We carried out loss and gain of function studies of SP-D to establish if SP-D was required for optimal immunity to the parasite. N. brasiliensis infection of SP-D-/- mice resulted in profound impairment of host innate immunity and ability to resolve infection. Raising pulmonary SP-D levels prior to infection enhanced parasite expulsion and type 2 immune responses, including increased numbers of IL-13 producing type 2 innate lymphoid cells (ILC2), elevated expression of markers of alternative activation by alveolar macrophages (alvM) and increased production of the type 2 cytokines IL-4 and IL-13. Adoptive transfer of alvM from SP-D-treated parasite infected mice into naïve recipients enhanced immunity to N. brasiliensis. Protection was associated with selective binding by the SP-D carbohydrate recognition domain (CRD) to L4 parasites to enhance their killing by alvM. These findings are the first demonstration that the collectin SP-D is an essential component of host innate immunity to helminths. PMID:26900854

  12. Protein-poor diet reduces host-specific immune gene expression in Bombus terrestris.

    PubMed

    Brunner, Franziska S; Schmid-Hempel, Paul; Barribeau, Seth M

    2014-07-01

    Parasites infect hosts non-randomly as genotypes of hosts vary in susceptibility to the same genotypes of parasites, but this specificity may be modulated by environmental factors such as nutrition. Nutrition plays an important role for any physiological investment. As immune responses are costly, resource limitation should negatively affect immunity through trade-offs with other physiological requirements. Consequently, nutritional limitation should diminish immune capacity in general, but does it also dampen differences among hosts? We investigated the effect of short-term pollen deprivation on the immune responses of our model host Bombus terrestris when infected with the highly prevalent natural parasite Crithidia bombi. Bumblebees deprived of pollen, their protein source, show reduced immune responses to infection. They failed to upregulate a number of genes, including antimicrobial peptides, in response to infection. In particular, they also showed less specific immune expression patterns across individuals and colonies. These findings provide evidence for how immune responses on the individual-level vary with important elements of the environment and illustrate how nutrition can functionally alter not only general resistance, but also alter the pattern of specific host-parasite interactions.

  13. Transfer of Immunity from Mother to Offspring Is Mediated via Egg-Yolk Protein Vitellogenin.

    PubMed

    Salmela, Heli; Amdam, Gro V; Freitak, Dalial

    2015-07-01

    Insect immune systems can recognize specific pathogens and prime offspring immunity. High specificity of immune priming can be achieved when insect females transfer immune elicitors into developing oocytes. The molecular mechanism behind this transfer has been a mystery. Here, we establish that the egg-yolk protein vitellogenin is the carrier of immune elicitors. Using the honey bee, Apis mellifera, model system, we demonstrate with microscopy and western blotting that vitellogenin binds to bacteria, both Paenibacillus larvae--the gram-positive bacterium causing American foulbrood disease--and to Escherichia coli that represents gram-negative bacteria. Next, we verify that vitellogenin binds to pathogen-associated molecular patterns; lipopolysaccharide, peptidoglycan and zymosan, using surface plasmon resonance. We document that vitellogenin is required for transport of cell-wall pieces of E. coli into eggs by imaging tissue sections. These experiments identify vitellogenin, which is distributed widely in oviparous species, as the carrier of immune-priming signals. This work reveals a molecular explanation for trans-generational immunity in insects and a previously undescribed role for vitellogenin. PMID:26230630

  14. Transfer of Immunity from Mother to Offspring Is Mediated via Egg-Yolk Protein Vitellogenin

    PubMed Central

    Salmela, Heli; Amdam, Gro V.; Freitak, Dalial

    2015-01-01

    Insect immune systems can recognize specific pathogens and prime offspring immunity. High specificity of immune priming can be achieved when insect females transfer immune elicitors into developing oocytes. The molecular mechanism behind this transfer has been a mystery. Here, we establish that the egg-yolk protein vitellogenin is the carrier of immune elicitors. Using the honey bee, Apis mellifera, model system, we demonstrate with microscopy and western blotting that vitellogenin binds to bacteria, both Paenibacillus larvae – the gram-positive bacterium causing American foulbrood disease – and to Escherichia coli that represents gram-negative bacteria. Next, we verify that vitellogenin binds to pathogen-associated molecular patterns; lipopolysaccharide, peptidoglycan and zymosan, using surface plasmon resonance. We document that vitellogenin is required for transport of cell-wall pieces of E. coli into eggs by imaging tissue sections. These experiments identify vitellogenin, which is distributed widely in oviparous species, as the carrier of immune-priming signals. This work reveals a molecular explanation for trans-generational immunity in insects and a previously undescribed role for vitellogenin. PMID:26230630

  15. Surfactant Protein-D Is Essential for Immunity to Helminth Infection.

    PubMed

    Thawer, Sumaiyya; Auret, Jennifer; Schnoeller, Corinna; Chetty, Alisha; Smith, Katherine; Darby, Matthew; Roberts, Luke; Mackay, Rosie-Marie; Whitwell, Harry J; Timms, John F; Madsen, Jens; Selkirk, Murray E; Brombacher, Frank; Clark, Howard William; Horsnell, William G C

    2016-02-01

    Pulmonary epithelial cell responses can enhance type 2 immunity and contribute to control of nematode infections. An important epithelial product is the collectin Surfactant Protein D (SP-D). We found that SP-D concentrations increased in the lung following Nippostrongylus brasiliensis infection; this increase was dependent on key components of the type 2 immune response. We carried out loss and gain of function studies of SP-D to establish if SP-D was required for optimal immunity to the parasite. N. brasiliensis infection of SP-D-/- mice resulted in profound impairment of host innate immunity and ability to resolve infection. Raising pulmonary SP-D levels prior to infection enhanced parasite expulsion and type 2 immune responses, including increased numbers of IL-13 producing type 2 innate lymphoid cells (ILC2), elevated expression of markers of alternative activation by alveolar macrophages (alvM) and increased production of the type 2 cytokines IL-4 and IL-13. Adoptive transfer of alvM from SP-D-treated parasite infected mice into naïve recipients enhanced immunity to N. brasiliensis. Protection was associated with selective binding by the SP-D carbohydrate recognition domain (CRD) to L4 parasites to enhance their killing by alvM. These findings are the first demonstration that the collectin SP-D is an essential component of host innate immunity to helminths.

  16. Surfactant Protein-D Is Essential for Immunity to Helminth Infection

    PubMed Central

    Schnoeller, Corinna; Chetty, Alisha; Smith, Katherine; Darby, Matthew; Roberts, Luke; Mackay, Rosie-Marie; Whitwell, Harry J.; Timms, John F.; Madsen, Jens; Selkirk, Murray E.; Brombacher, Frank; Clark, Howard William; Horsnell, William G. C.

    2016-01-01

    Pulmonary epithelial cell responses can enhance type 2 immunity and contribute to control of nematode infections. An important epithelial product is the collectin Surfactant Protein D (SP-D). We found that SP-D concentrations increased in the lung following Nippostrongylus brasiliensis infection; this increase was dependent on key components of the type 2 immune response. We carried out loss and gain of function studies of SP-D to establish if SP-D was required for optimal immunity to the parasite. N. brasiliensis infection of SP-D-/- mice resulted in profound impairment of host innate immunity and ability to resolve infection. Raising pulmonary SP-D levels prior to infection enhanced parasite expulsion and type 2 immune responses, including increased numbers of IL-13 producing type 2 innate lymphoid cells (ILC2), elevated expression of markers of alternative activation by alveolar macrophages (alvM) and increased production of the type 2 cytokines IL-4 and IL-13. Adoptive transfer of alvM from SP-D-treated parasite infected mice into naïve recipients enhanced immunity to N. brasiliensis. Protection was associated with selective binding by the SP-D carbohydrate recognition domain (CRD) to L4 parasites to enhance their killing by alvM. These findings are the first demonstration that the collectin SP-D is an essential component of host innate immunity to helminths. PMID:26900854

  17. Protein-poor diet reduces host-specific immune gene expression in Bombus terrestris

    PubMed Central

    Brunner, Franziska S.; Schmid-Hempel, Paul; Barribeau, Seth M.

    2014-01-01

    Parasites infect hosts non-randomly as genotypes of hosts vary in susceptibility to the same genotypes of parasites, but this specificity may be modulated by environmental factors such as nutrition. Nutrition plays an important role for any physiological investment. As immune responses are costly, resource limitation should negatively affect immunity through trade-offs with other physiological requirements. Consequently, nutritional limitation should diminish immune capacity in general, but does it also dampen differences among hosts? We investigated the effect of short-term pollen deprivation on the immune responses of our model host Bombus terrestris when infected with the highly prevalent natural parasite Crithidia bombi. Bumblebees deprived of pollen, their protein source, show reduced immune responses to infection. They failed to upregulate a number of genes, including antimicrobial peptides, in response to infection. In particular, they also showed less specific immune expression patterns across individuals and colonies. These findings provide evidence for how immune responses on the individual-level vary with important elements of the environment and illustrate how nutrition can functionally alter not only general resistance, but also alter the pattern of specific host–parasite interactions. PMID:24850921

  18. Shigella Manipulates Host Immune Responses by Delivering Effector Proteins with Specific Roles

    PubMed Central

    Ashida, Hiroshi; Mimuro, Hitomi; Sasakawa, Chihiro

    2015-01-01

    The intestinal epithelium deploys multiple defense systems against microbial infection to sense bacterial components and danger alarms, as well as to induce intracellular signal transduction cascades that trigger both the innate and the adaptive immune systems, which are pivotal for bacterial elimination. However, many enteric bacterial pathogens, including Shigella, deliver a subset of virulence proteins (effectors) via the type III secretion system (T3SS) that enable bacterial evasion from host immune systems; consequently, these pathogens are able to efficiently colonize the intestinal epithelium. In this review, we present and select recently discovered examples of interactions between Shigella and host immune responses, with particular emphasis on strategies that bacteria use to manipulate inflammatory outputs of host-cell responses such as cell death, membrane trafficking, and innate and adaptive immune responses. PMID:25999954

  19. Capping protein integrates multiple MAMP signalling pathways to modulate actin dynamics during plant innate immunity.

    PubMed

    Li, Jiejie; Henty-Ridilla, Jessica L; Staiger, Benjamin H; Day, Brad; Staiger, Christopher J

    2015-01-01

    Plants and animals perceive diverse microbe-associated molecular patterns (MAMPs) via pattern recognition receptors and activate innate immune signalling. The actin cytoskeleton has been suggested as a target for innate immune signalling and a key transducer of cellular responses. However, the molecular mechanisms underlying actin remodelling and the precise functions of these rearrangements during innate immunity remain largely unknown. Here we demonstrate rapid actin remodelling in response to several distinct MAMP signalling pathways in plant epidermal cells. The regulation of actin dynamics is a convergence point for basal defence machinery, such as cell wall fortification and transcriptional reprogramming. Our quantitative analyses of actin dynamics and genetic studies reveal that MAMP-stimulated actin remodelling is due to the inhibition of capping protein (CP) by the signalling lipid, phosphatidic acid. In addition, CP promotes resistance against bacterial and fungal phytopathogens. These findings demonstrate that CP is a central target for the plant innate immune response. PMID:26018794

  20. Protein Defense Systems against the Lantibiotic Nisin: Function of the Immunity Protein NisI and the Resistance Protein NSR

    PubMed Central

    Khosa, Sakshi; Lagedroste, Marcel; Smits, Sander H. J.

    2016-01-01

    Lantibiotics are potential alternatives to antibiotics because of their broad-range killing spectrum. The producer strain is immune against its own synthesized lantibiotic via the expression of two proteins LanI and LanFEG. Recently, gene operons are found in mainly human pathogenic strains, which confer resistance against lantibiotics. Of all the lantibiotics discovered till date, nisin produced by some Lactococcus lactis strains is the most prominent member. Nisin has multiple mode of actions of which binding to the cell wall precursor lipid II and subsequent insertion into the bacterial membrane to form pores are the most effective. The nisin producing strains express the lipoprotein NisI to prevent a suicidal effect. NisI binds nisin, inducing a reversible cell clustering to prevent nisin from reaching the membrane. Importantly NisI does not modify nisin and releases it as soon as the concentration in the media drops below a certain level. The human pathogen Streptococcus agalactiae is naturally resistant against nisin by expressing a resistance protein called SaNSR, which is a nisin degrading enzyme. By cleaving off the last six amino acids of nisin, its effectiveness is 100-fold reduced. This cleavage reaction appears to be specific for nisin since SaNSR recognizes the C-terminal located lanthionine rings. Recently, the structures of both NisI and SaNSR were determined by NMR and X-ray crystallography, respectively. Furthermore, for both proteins the binding site for nisin was determined. Within this review, the structures of both proteins and their different defense mechanisms are described. PMID:27148193

  1. NY-ESO-1 protein glycosylated by yeast induces enhanced immune responses.

    PubMed

    Wadle, Andreas; Mischo, Axel; Strahl, Sabine; Nishikawa, Hiroyoshi; Held, Gerhard; Neumann, Frank; Wullner, Beate; Fischer, Eliane; Kleber, Sascha; Karbach, Julia; Jager, Elke; Shiku, Hiroshi; Odunsi, Kunle; Shrikant, Protul A; Knuth, Alexander; Cerundolo, Vincenzo; Renner, Christoph

    2010-11-01

    Vaccine strategies that target dendritic cells to elicit potent cellular immunity are the subject of intense research. Here we report that the genetically engineered yeast Saccharomyces cerevisiae, expressing the full-length tumour-associated antigen NY-ESO-1, is a versatile host for protein production. Exposing dendritic cells (DCs) to soluble NY-ESO-1 protein linked to the yeast a-agglutinin 2 protein (Aga2p) protein resulted in protein uptake, processing and MHC class I cross-presentation of NY-ESO-1-derived peptides. The process of antigen uptake and cross-presentation was dependent on the glycosylation pattern of NY-ESO-1-Aga2p protein and the presence of accessible mannose receptors. In addition, NY-ESO-1-Aga2p protein uptake by dendritic cells resulted in recognition by HLA-DP4 NY-ESO-1-specific CD4(+) T cells, indicating MHC class II presentation. Finally, vaccination of mice with yeast-derived NY-ESO-1-Aga2p protein led to an enhanced humoral and cellular immune response, when compared to the bacterially expressed NY-ESO-1 protein. Together, these data demonstrate that yeast-derived full-length NY-ESO-1-Aga2p protein is processed and presented efficiently by MHC class I and II complexes and warrants clinical trials to determine the potential value of S. cerevisiae as a host for cancer vaccine development.

  2. Comparative genomic study of arachnid immune systems indicates loss of beta-1,3-glucanase-related proteins and the immune deficiency pathway.

    PubMed

    Bechsgaard, J; Vanthournout, B; Funch, P; Vestbo, S; Gibbs, R A; Richards, S; Sanggaard, K W; Enghild, J J; Bilde, T

    2016-02-01

    Analyses of arthropod genomes have shown that the genes in the different innate humoral immune responses are conserved. These genes encode proteins that are involved in immune signalling pathways that recognize pathogens and activate immune responses. These immune responses include phagocytosis, encapsulation of the pathogen and production of effector molecules for pathogen elimination. So far, most studies have focused on insects leaving other major arthropod groups largely unexplored. Here, we annotate the immune-related genes of six arachnid genomes and present evidence for a conserved pattern of some immune genes, but also evolutionary changes in the arachnid immune system. Specifically, our results suggest that the family of recognition molecules of beta-1,3-glucanase-related proteins (βGRPs) and the genes from the immune deficiency (IMD) signalling pathway have been lost in a common ancestor of arachnids. These findings are consistent with previous work suggesting that the humoral immune effector proteins are constitutively produced in arachnids in contrast to insects, where these have to be induced. Further functional studies are needed to verify this. We further show that the full haemolymph clotting cascade found in the horseshoe crab is retrieved in most arachnid genomes. Tetranychus lacks at least one major component, although it is possible that this cascade could still function through recruitment of a different protein. The gel-forming protein in horseshoe crabs, coagulogen, was not recovered in any of the arachnid genomes; however, it is possible that the arachnid clot consists of a related protein, spätzle, that is present in all of the genomes. PMID:26528622

  3. Comparative genomic study of arachnid immune systems indicates loss of beta-1,3-glucanase-related proteins and the immune deficiency pathway.

    PubMed

    Bechsgaard, J; Vanthournout, B; Funch, P; Vestbo, S; Gibbs, R A; Richards, S; Sanggaard, K W; Enghild, J J; Bilde, T

    2016-02-01

    Analyses of arthropod genomes have shown that the genes in the different innate humoral immune responses are conserved. These genes encode proteins that are involved in immune signalling pathways that recognize pathogens and activate immune responses. These immune responses include phagocytosis, encapsulation of the pathogen and production of effector molecules for pathogen elimination. So far, most studies have focused on insects leaving other major arthropod groups largely unexplored. Here, we annotate the immune-related genes of six arachnid genomes and present evidence for a conserved pattern of some immune genes, but also evolutionary changes in the arachnid immune system. Specifically, our results suggest that the family of recognition molecules of beta-1,3-glucanase-related proteins (βGRPs) and the genes from the immune deficiency (IMD) signalling pathway have been lost in a common ancestor of arachnids. These findings are consistent with previous work suggesting that the humoral immune effector proteins are constitutively produced in arachnids in contrast to insects, where these have to be induced. Further functional studies are needed to verify this. We further show that the full haemolymph clotting cascade found in the horseshoe crab is retrieved in most arachnid genomes. Tetranychus lacks at least one major component, although it is possible that this cascade could still function through recruitment of a different protein. The gel-forming protein in horseshoe crabs, coagulogen, was not recovered in any of the arachnid genomes; however, it is possible that the arachnid clot consists of a related protein, spätzle, that is present in all of the genomes.

  4. Lipid Transfer Proteins As Components of the Plant Innate Immune System: Structure, Functions, and Applications

    PubMed Central

    Finkina, E. I.; Melnikova, D. N.; Bogdanov, I. V.; Ovchinnikova, T. V.

    2016-01-01

    Among a variety of molecular factors of the plant innate immune system, small proteins that transfer lipids and exhibit a broad spectrum of biological activities are of particular interest. These are lipid transfer proteins (LTPs). LTPs are interesting to researchers for three main features. The first feature is the ability of plant LTPs to bind and transfer lipids, whereby these proteins got their name and were combined into one class. The second feature is that LTPs are defense proteins that are components of plant innate immunity. The third feature is that LTPs constitute one of the most clinically important classes of plant allergens. In this review, we summarize the available data on the plant LTP structure, biological properties, diversity of functions, mechanisms of action, and practical applications, emphasizing their role in plant physiology and their significance in human life. PMID:27437139

  5. Mechanisms of Host-Pathogen Protein Complex Formation and Bacterial Immune Evasion of Streptococcus suis Protein Fhb.

    PubMed

    Li, Xueqin; Liu, Peng; Gan, Shuzhen; Zhang, Chunmao; Zheng, Yuling; Jiang, Yongqiang; Yuan, Yuan

    2016-08-12

    Streptococcus suis serotype 2 (S. suis 2)-induced sepsis and meningitis are often accompanied by bacteremia. The evasion of polymorphonuclear leukocyte-mediated phagocytic clearance is central to the establishment of bacteremia caused by S. suis 2 and is facilitated by the ability of factor H (FH)-binding protein (Fhb) to bind FH on the bacterial surface, thereby impeding alternative pathway complement activation and phagocytic clearance. Here, C3b/C3d was found to bind to Fhb, along with FH, forming a large immune complex. The formation of this immune complex was mediated by domain II of Fhb via electrostatic and hydrophobic interactions, which, to our knowledge, is a new type of interaction. Interestingly, Fhb was found to be associated with the cell envelope and also present in the culture supernatant, where secreted Fhb inhibited complement activation via interactions with domain II, thereby enhancing antiphagocytic clearance by polymorphonuclear leukocytes. Thus, Fhb is a multifunctional bacterial protein, which binds host complement component C3 as well as FH and interferes with innate immune recognition in a secret protein manner. S. suis 2 therefore appears to have developed a new strategy to combat host innate immunity and enhance survival in host blood. PMID:27342778

  6. The Solution Structure of the Lantibiotic Immunity Protein NisI and Its Interactions with Nisin.

    PubMed

    Hacker, Carolin; Christ, Nina A; Duchardt-Ferner, Elke; Korn, Sophie; Göbl, Christoph; Berninger, Lucija; Düsterhus, Stefanie; Hellmich, Ute A; Madl, Tobias; Kötter, Peter; Entian, Karl-Dieter; Wöhnert, Jens

    2015-11-27

    Many Gram-positive bacteria produce lantibiotics, genetically encoded and posttranslationally modified peptide antibiotics, which inhibit the growth of other Gram-positive bacteria. To protect themselves against their own lantibiotics these bacteria express a variety of immunity proteins including the LanI lipoproteins. The structural and mechanistic basis for LanI-mediated lantibiotic immunity is not yet understood. Lactococcus lactis produces the lantibiotic nisin, which is widely used as a food preservative. Its LanI protein NisI provides immunity against nisin but not against structurally very similar lantibiotics from other species such as subtilin from Bacillus subtilis. To understand the structural basis for LanI-mediated immunity and their specificity we investigated the structure of NisI. We found that NisI is a two-domain protein. Surprisingly, each of the two NisI domains has the same structure as the LanI protein from B. subtilis, SpaI, despite the lack of significant sequence homology. The two NisI domains and SpaI differ strongly in their surface properties and function. Additionally, SpaI-mediated lantibiotic immunity depends on the presence of a basic unstructured N-terminal region that tethers SpaI to the membrane. Such a region is absent from NisI. Instead, the N-terminal domain of NisI interacts with membranes but not with nisin. In contrast, the C-terminal domain specifically binds nisin and modulates the membrane affinity of the N-terminal domain. Thus, our results reveal an unexpected structural relationship between NisI and SpaI and shed light on the structural basis for LanI mediated lantibiotic immunity.

  7. The Solution Structure of the Lantibiotic Immunity Protein NisI and Its Interactions with Nisin.

    PubMed

    Hacker, Carolin; Christ, Nina A; Duchardt-Ferner, Elke; Korn, Sophie; Göbl, Christoph; Berninger, Lucija; Düsterhus, Stefanie; Hellmich, Ute A; Madl, Tobias; Kötter, Peter; Entian, Karl-Dieter; Wöhnert, Jens

    2015-11-27

    Many Gram-positive bacteria produce lantibiotics, genetically encoded and posttranslationally modified peptide antibiotics, which inhibit the growth of other Gram-positive bacteria. To protect themselves against their own lantibiotics these bacteria express a variety of immunity proteins including the LanI lipoproteins. The structural and mechanistic basis for LanI-mediated lantibiotic immunity is not yet understood. Lactococcus lactis produces the lantibiotic nisin, which is widely used as a food preservative. Its LanI protein NisI provides immunity against nisin but not against structurally very similar lantibiotics from other species such as subtilin from Bacillus subtilis. To understand the structural basis for LanI-mediated immunity and their specificity we investigated the structure of NisI. We found that NisI is a two-domain protein. Surprisingly, each of the two NisI domains has the same structure as the LanI protein from B. subtilis, SpaI, despite the lack of significant sequence homology. The two NisI domains and SpaI differ strongly in their surface properties and function. Additionally, SpaI-mediated lantibiotic immunity depends on the presence of a basic unstructured N-terminal region that tethers SpaI to the membrane. Such a region is absent from NisI. Instead, the N-terminal domain of NisI interacts with membranes but not with nisin. In contrast, the C-terminal domain specifically binds nisin and modulates the membrane affinity of the N-terminal domain. Thus, our results reveal an unexpected structural relationship between NisI and SpaI and shed light on the structural basis for LanI mediated lantibiotic immunity. PMID:26459561

  8. Opa proteins and CEACAMs: pathways of immune engagement for pathogenic Neisseria.

    PubMed

    Sadarangani, Manish; Pollard, Andrew J; Gray-Owen, Scott D

    2011-05-01

    Neisseria meningitidis and Neisseria gonorrhoeae are globally important pathogens, which in part owe their success to their ability to successfully evade human immune responses over long periods. The phase-variable opacity-associated (Opa) adhesin proteins are a major surface component of these organisms, and are responsible for bacterial adherence and entry into host cells and interactions with the immune system. Most immune interactions are mediated via binding to members of the carcinoembryonic antigen cell adhesion molecule (CEACAM) family. These Opa variants are able to bind to different receptors of the CEACAM family on epithelial cells, neutrophils, and T and B lymphocytes, influencing the innate and adaptive immune responses. Increased epithelial cell adhesion creates the potential for prolonged infection, invasion and dissemination. Furthermore, Opa proteins may inhibit T-lymphocyte activation and proliferation, B-cell antibody production, and innate inflammatory responses by infected epithelia, in addition to conferring increased resistance to antibody-dependent, complement-mediated killing. While vaccines containing Opa proteins could induce adhesion-blocking and bactericidal antibodies, the consequence of CEACAM binding by a candidate Opa-containing vaccine requires further investigation. This review summarizes current knowledge of the immunological consequences of the interaction between meningococcal and gonococcal Opa proteins and human CEACAMs, considering the implications for pathogenesis and vaccine development.

  9. Identifying Schistosoma japonicum excretory/secretory proteins and their interactions with host immune system.

    PubMed

    Liao, Qi; Yuan, Xiongying; Xiao, Hui; Liu, Changning; Lv, Zhiyue; Zhao, Yi; Wu, Zhongdao

    2011-01-01

    Schistosoma japonicum is a major infectious agent of schistosomiasis. It has been reported that large number of proteins excreted and secreted by S. japonicum during its life cycle are important for its infection and survival in definitive hosts. These proteins can be used as ideal candidates for vaccines or drug targets. In this work, we analyzed the protein sequences of S. japonicum and found that compared with other proteins in S. japonicum, excretory/secretory (ES) proteins are generally longer, more likely to be stable and enzyme, more likely to contain immune-related binding peptides and more likely to be involved in regulation and metabolism processes. Based on the sequence difference between ES and non-ES proteins, we trained a support vector machine (SVM) with much higher accuracy than existing approaches. Using this SVM, we identified 191 new ES proteins in S. japonicum, and further predicted 7 potential interactions between these ES proteins and human immune proteins. Our results are useful to understand the pathogenesis of schistosomiasis and can serve as a new resource for vaccine or drug targets discovery for anti-schistosome.

  10. Conserved Immune Recognition Hierarchy of Mycobacterial PE/PPE Proteins during Infection in Natural Hosts

    PubMed Central

    Vordermeier, H. Martin; Hewinson, R. Glyn; Wilkinson, Robert J.; Wilkinson, Katalin A.; Gideon, Hannah P.; Young, Douglas B.; Sampson, Samantha L.

    2012-01-01

    The Mycobacterium tuberculosis genome contains two large gene families encoding proteins of unknown function, characterized by conserved N-terminal proline and glutamate (PE and PPE) motifs. The presence of a large number of PE/PPE proteins with repetitive domains and evidence of strain variation has given rise to the suggestion that these proteins may play a role in immune evasion via antigenic variation, while emerging data suggests that some family members may play important roles in mycobacterial pathogenesis. In this study, we examined cellular immune responses to a panel of 36 PE/PPE proteins during human and bovine infection. We observed a distinct hierarchy of immune recognition, reflected both in the repertoire of PE/PPE peptide recognition in individual cows and humans and in the magnitude of IFN-γ responses elicited by stimulation of sensitized host cells. The pattern of immunodominance was strikingly similar between cattle that had been experimentally infected with Mycobacterium bovis and humans naturally infected with clinical isolates of M. tuberculosis. The same pattern was maintained as disease progressed throughout a four-month course of infection in cattle, and between humans with latent as well as active tuberculosis. Detailed analysis of PE/PPE responses at the peptide level suggests that antigenic cross-reactivity amongst related family members is a major determinant in the observed differences in immune hierarchy. Taken together, these results demonstrate that a subset of PE/PPE proteins are major targets of the cellular immune response to tuberculosis, and are recognized at multiple stages of infection and in different disease states. Thus this work identifies a number of novel antigens that could find application in vaccine development, and provides new insights into PE/PPE biology. PMID:22870206

  11. Peanut protein structure, polyphenol content and immune response to peanut proteins in vivo are modulated by laccase.

    PubMed

    Mihajlovic, L; Radosavljevic, J; Nordlund, E; Krstic, M; Bohn, T; Smit, J; Buchert, J; Cirkovic Velickovic, T

    2016-05-18

    Food texture can be improved by enzyme-mediated covalent cross-linking of different food components, such as proteins and carbohydrates. Cross-linking changes the biological and immunological properties of proteins and may change the sensitizing potential of food allergens. In this study we applied a microbial polyphenol oxidase, laccase, to cross-link peanut proteins. The size and morphology of the obtained cross-linked proteins were analyzed by electrophoresis and electron microscopy. Structural changes in proteins were analyzed by CD spectroscopy and by using specific antibodies to major peanut allergens. The bioavailability of peanut proteins was analyzed using a Caco-2 epithelial cell model. The in vivo sensitizing potential of laccase-treated peanut proteins was analyzed using a mouse model of food allergy. Finally, peanut polyphenols were analyzed by UHPLC-MS/MS, before and after the enzymatic reaction with laccase. Laccase treatment of peanut proteins yielded a covalently cross-linked material, with the modified tertiary structure of peanut proteins, improved bioavailability of Ara h 2 (by 70 fold, p < 0.05) and modulated allergic immune response in vivo. The modulation of the immune response was related to the increased production of IgG2a antibodies 11 fold (p < 0.05) and reduced IL-13 secretion in in vitro cultured splenocytes 7 fold (p < 0.05). Analysis of the peanut polyphenol content and profile by HPLC-MS/MS revealed that laccase treatment depleted the peanut extract of polyphenol compounds leaving mostly isorhamnetin derivatives and procyanidin dimer B-type in detectable amounts. Treatment of complex food extracts rich in polyphenols with laccase results in both protein cross-linking and modification of polyphenol compounds. These extensively cross-linked proteins have unchanged potency to induce allergic sensitization in vivo, but certain immunomodulatory changes were observed.

  12. Peanut protein structure, polyphenol content and immune response to peanut proteins in vivo are modulated by laccase.

    PubMed

    Mihajlovic, L; Radosavljevic, J; Nordlund, E; Krstic, M; Bohn, T; Smit, J; Buchert, J; Cirkovic Velickovic, T

    2016-05-18

    Food texture can be improved by enzyme-mediated covalent cross-linking of different food components, such as proteins and carbohydrates. Cross-linking changes the biological and immunological properties of proteins and may change the sensitizing potential of food allergens. In this study we applied a microbial polyphenol oxidase, laccase, to cross-link peanut proteins. The size and morphology of the obtained cross-linked proteins were analyzed by electrophoresis and electron microscopy. Structural changes in proteins were analyzed by CD spectroscopy and by using specific antibodies to major peanut allergens. The bioavailability of peanut proteins was analyzed using a Caco-2 epithelial cell model. The in vivo sensitizing potential of laccase-treated peanut proteins was analyzed using a mouse model of food allergy. Finally, peanut polyphenols were analyzed by UHPLC-MS/MS, before and after the enzymatic reaction with laccase. Laccase treatment of peanut proteins yielded a covalently cross-linked material, with the modified tertiary structure of peanut proteins, improved bioavailability of Ara h 2 (by 70 fold, p < 0.05) and modulated allergic immune response in vivo. The modulation of the immune response was related to the increased production of IgG2a antibodies 11 fold (p < 0.05) and reduced IL-13 secretion in in vitro cultured splenocytes 7 fold (p < 0.05). Analysis of the peanut polyphenol content and profile by HPLC-MS/MS revealed that laccase treatment depleted the peanut extract of polyphenol compounds leaving mostly isorhamnetin derivatives and procyanidin dimer B-type in detectable amounts. Treatment of complex food extracts rich in polyphenols with laccase results in both protein cross-linking and modification of polyphenol compounds. These extensively cross-linked proteins have unchanged potency to induce allergic sensitization in vivo, but certain immunomodulatory changes were observed. PMID:27138276

  13. Comparative biology of the pentraxin protein family: evolutionarily conserved component of innate immune system.

    PubMed

    Armstrong, Peter B

    2015-01-01

    The immune system is based on the actions of the collection of specialized immune defense cells and their secreted proteins and peptides that defend the host against infection by parasites. Parasites are organisms that live part or all of their lives in close physical association with the host and extract nutrients from the host and, by releasing toxins and virulence factors, cause disease with the potential for injury and premature death of that host. Parasites of the metazoa can be viruses, eubacteria, fungi, protozoans, and other metazoans. The immune system operates to kill or eliminate parasites and eliminate or detoxify their toxins and virulence factors. Although some of the elements of immune systems are specific to a particular phylum of metazoans, others show extensive evolutionary conservation, being present in several or all major phyla of the metazoa. The pentraxins display this latter character in their roles in immune defense. Pentraxins have been documented in vertebrates, nonvertebrate chordates, arthropods, and mollusks and may be present in other taxa of metazoans. Presumably the pentraxins appeared early in the evolution of metazoa, prior to their evolutionary divergence in the Precambrian epoch into many phyla present today, and have been preserved for the 542 million years since that explosive evolutionary radiation. The fidelity with which these phyla have preserved the pentraxins suggests that the functions of these proteins are important for survival of the members of these diverse taxa of animals.

  14. Comparative biology of the pentraxin protein family: evolutionarily conserved component of innate immune system.

    PubMed

    Armstrong, Peter B

    2015-01-01

    The immune system is based on the actions of the collection of specialized immune defense cells and their secreted proteins and peptides that defend the host against infection by parasites. Parasites are organisms that live part or all of their lives in close physical association with the host and extract nutrients from the host and, by releasing toxins and virulence factors, cause disease with the potential for injury and premature death of that host. Parasites of the metazoa can be viruses, eubacteria, fungi, protozoans, and other metazoans. The immune system operates to kill or eliminate parasites and eliminate or detoxify their toxins and virulence factors. Although some of the elements of immune systems are specific to a particular phylum of metazoans, others show extensive evolutionary conservation, being present in several or all major phyla of the metazoa. The pentraxins display this latter character in their roles in immune defense. Pentraxins have been documented in vertebrates, nonvertebrate chordates, arthropods, and mollusks and may be present in other taxa of metazoans. Presumably the pentraxins appeared early in the evolution of metazoa, prior to their evolutionary divergence in the Precambrian epoch into many phyla present today, and have been preserved for the 542 million years since that explosive evolutionary radiation. The fidelity with which these phyla have preserved the pentraxins suggests that the functions of these proteins are important for survival of the members of these diverse taxa of animals. PMID:25805121

  15. Systemic protein delivery by muscle-gene transfer is limited by a local immune response

    PubMed Central

    Wang, Lixin; Dobrzynski, Eric; Schlachterman, Alexander; Cao, Ou; Herzog, Roland W.

    2005-01-01

    Adeno-associated viral (AAV) vectors have been successfully used for therapeutic expression of systemic transgene products (such as factor IX or erythropoietin) following in vivo administration to skeletal muscle of animal models of inherited hematologic disorders. However, an immune response may be initiated if the transgene product represents a neoantigen. Here, we use ovalbumin (OVA) as a model antigen and demonstrate immune-mediated elimination of expression on muscle-directed AAV-2 gene transfer. Administration to immune competent mice resulted in transient systemic OVA expression. Within 10 days, OVA-specific T-helper cells had been activated in draining lymph nodes, an inflammatory immune response ensued, and OVA-expressing muscle fibers were destroyed by a cytotoxic CD8+ T-cell response. Use of a muscle-specific promoter did not prevent this immune response. Adoptively transferred CD4+ cells transgenic for a T-cell receptor specific to OVA peptide-major histocompatibility complex class II showed antigen-specific, vector dose-dependent proliferation confined to the draining lymph nodes of AAV-OVA–transduced muscle within 5 days after gene transfer and subsequently participated in lymphocytic infiltration of transduced muscle. This study documents that a local immune response limits sustained expression of a secreted protein in muscle gene transfer, a finding that may have consequences for design of clinical protocols. PMID:15713796

  16. Quantitative Proteomics Reveals the Roles of Peroxisome-associated Proteins in Antiviral Innate Immune Responses.

    PubMed

    Zhou, Mao-Tian; Qin, Yue; Li, Mi; Chen, Chen; Chen, Xi; Shu, Hong-Bing; Guo, Lin

    2015-09-01

    Compared with whole-cell proteomic analysis, subcellular proteomic analysis is advantageous not only for the increased coverage of low abundance proteins but also for generating organelle-specific data containing information regarding dynamic protein movement. In the present study, peroxisome-enriched fractions from Sendai virus (SeV)-infected or uninfected HepG2 cells were obtained and subjected to quantitative proteomics analysis. We identified 311 proteins that were significantly changed by SeV infection. Among these altered proteins, 25 are immune response-related proteins. Further bioinformatic analysis indicated that SeV infection inhibits cell cycle-related proteins and membrane attack complex-related proteins, all of which are beneficial for the survival and replication of SeV within host cells. Using Luciferase reporter assays on several innate immune-related reporters, we performed functional analysis on 11 candidate proteins. We identified LGALS3BP and CALU as potential negative regulators of the virus-induced activation of the type I interferons.

  17. Short Toxin-like Proteins Attack the Defense Line of Innate Immunity

    PubMed Central

    Tirosh, Yitshak; Ofer, Dan; Eliyahu, Tsiona; Linial, Michal

    2013-01-01

    ClanTox (classifier of animal toxins) was developed for identifying toxin-like candidates from complete proteomes. Searching mammalian proteomes for short toxin-like proteins (coined TOLIPs) revealed a number of overlooked secreted short proteins with an abundance of cysteines throughout their sequences. We applied bioinformatics and data-mining methods to infer the function of several top predicted candidates. We focused on cysteine-rich peptides that adopt the fold of the three-finger proteins (TFPs). We identified a cluster of duplicated genes that share a structural similarity with elapid neurotoxins, such as α-bungarotoxin. In the murine proteome, there are about 60 such proteins that belong to the Ly6/uPAR family. These proteins are secreted or anchored to the cell membrane. Ly6/uPAR proteins are associated with a rich repertoire of functions, including binding to receptors and adhesion. Ly6/uPAR proteins modulate cell signaling in the context of brain functions and cells of the innate immune system. We postulate that TOLIPs, as modulators of cell signaling, may be associated with pathologies and cellular imbalance. We show that proteins of the Ly6/uPAR family are associated with cancer diagnosis and malfunction of the immune system. PMID:23881252

  18. Role of SRC-like adaptor protein (SLAP) in immune and malignant cell signaling.

    PubMed

    Kazi, Julhash U; Kabir, Nuzhat N; Rönnstrand, Lars

    2015-07-01

    SRC-like adaptor protein (SLAP) is an adaptor protein structurally similar to the SRC family protein kinases. Like SRC, SLAP contains an SH3 domain followed by an SH2 domain but the kinase domain has been replaced by a unique C-terminal region. SLAP is expressed in a variety of cell types. Current studies suggest that it regulates signaling of various cell surface receptors including the B cell receptor, the T cell receptor, cytokine receptors and receptor tyrosine kinases which are important regulator of immune and cancer cell signaling. SLAP targets receptors, or its associated components, by recruiting the ubiquitin machinery and thereby destabilizing signaling. SLAP directs receptors to ubiquitination-mediated degradation and controls receptors turnover as well as signaling. Thus, SLAP appears to be an important component in regulating signal transduction required for immune and malignant cells.

  19. Partially Protective Immunity Induced by a 20 kDa Protein Secreted by Trichinella spiralis Stichocytes

    PubMed Central

    Wang, Lei; Gu, Yuan; Zhan, Bin; Zhu, Xinping

    2015-01-01

    Background Trichinella spiralis infection induces protective immunity against re-infection in animal models. Identification of the antigens eliciting acquired immunity during infection is important for vaccine development against Trichinella infection and immunodiagnosis. Methods and Findings The T. spiralis adult cDNA library was immunoscreened with sera from pigs experimentally infected with 20,000 infective T. spiralis larvae. Total 43 positive clones encoding for 28 proteins were identified; one of the immunodominant proteins was 20 kDa Ts-ES-1 secreted by Trichinella stichocytes and existing in the excretory/secretory (ES) products of T. spiralis adult and muscle larval worms. Ts-ES-1 contains 172 amino acids with a typical signal peptide in the first 20 amino acids. The expression of Ts-ES-1 was detected in both the adult and muscle larval stages at the mRNA and protein expression levels. Mice immunized with recombinant Ts-ES-1 (rTs-ES-1) formulated with ISA50v2 adjuvant exhibited a significant worm reduction in both the adult worm (27%) and muscle larvae burden (42.1%) after a challenge with T. spiralis compared to the adjuvant control group (p<0.01). The rTs-ES-1-induced protection was associated with a high level of specific anti-Ts-ES-1 IgG antibodies and a Th1/Th2 mixed immune response. Conclusion The newly identified rTs-ES-1 is an immunodominant protein secreted by Trichinella stichocytes during natural infection and enables to the induction of partial protective immunity in vaccinated mice against Trichinella infection. Therefore, rTs-ES-1 is a potential candidate for vaccine development against trichinellosis. PMID:26288365

  20. Dehydration decreases saliva antimicrobial proteins important for mucosal immunity.

    PubMed

    Fortes, Matthew B; Diment, Bethany C; Di Felice, Umberto; Walsh, Neil P

    2012-10-01

    The aim of the study was to investigate the effect of exercise-induced dehydration and subsequent overnight fluid restriction on saliva antimicrobial proteins important for host defence (secretory IgA (SIgA), α-amylase, and lysozyme). On two randomized occasions, 13 participants exercised in the heat, either without fluid intake to evoke progressive body mass losses (BML) of 1%, 2%, and 3% with subsequent overnight fluid restriction until 0800 h in the following morning (DEH) or with fluids to offset losses (CON). Participants in the DEH trial rehydrated from 0800 h until 1100 h on day 2. BML, plasma osmolality (Posm), and urine specific gravity (USG) were assessed as hydration indices. Unstimulated saliva samples were assessed for flow rate (SFR), SIgA, α-amylase, and lysozyme concentrations. Posm and USG increased during dehydration and remained elevated after overnight fluid restriction (BML = 3.5% ± 0.3%, Posm = 297 ± 6 mosmol·kg⁻¹, and USG = 1.026 ± 0.002; P < 0.001). Dehydration decreased SFR (67% at 3% BML, 70% at 0800 h; P < 0.01) and increased SIgA concentration, with no effect on SIgA secretion rate. SFR and SIgA responses remained unchanged in the CON trial. Dehydration did not affect α-amylase or lysozyme concentration but decreased secretion rates of α-amylase (44% at 3% BML, 78% at 0800 h; P < 0.01) and lysozyme (46% at 3% BML, 61% at 0800 h; P < 0.01), which were lower than in CON at these time points (P < 0.05). Rehydration returned all saliva variables to baseline. In conclusion, modest dehydration (~3% BML) decreased SFR, α-amylase, and lysozyme secretion rates. Whether the observed magnitude of decrease in saliva AMPs during dehydration compromises host defence remains to be shown.

  1. Structures of the Ultra-High-Affinity Protein-Protein Complexes of Pyocins S2 and AP41 and Their Cognate Immunity Proteins from Pseudomonas aeruginosa.

    PubMed

    Joshi, Amar; Grinter, Rhys; Josts, Inokentijs; Chen, Sabrina; Wojdyla, Justyna A; Lowe, Edward D; Kaminska, Renata; Sharp, Connor; McCaughey, Laura; Roszak, Aleksander W; Cogdell, Richard J; Byron, Olwyn; Walker, Daniel; Kleanthous, Colin

    2015-08-28

    How ultra-high-affinity protein-protein interactions retain high specificity is still poorly understood. The interaction between colicin DNase domains and their inhibitory immunity (Im) proteins is an ultra-high-affinity interaction that is essential for the neutralisation of endogenous DNase catalytic activity and for protection against exogenous DNase bacteriocins. The colicin DNase-Im interaction is a model system for the study of high-affinity protein-protein interactions. However, despite the fact that closely related colicin-like bacteriocins are widely produced by Gram-negative bacteria, this interaction has only been studied using colicins from Escherichia coli. In this work, we present the first crystal structures of two pyocin DNase-Im complexes from Pseudomonas aeruginosa, pyocin S2 DNase-ImS2 and pyocin AP41 DNase-ImAP41. These structures represent divergent DNase-Im subfamilies and are important in extending our understanding of protein-protein interactions for this important class of high-affinity protein complex. A key finding of this work is that mutations within the immunity protein binding energy hotspot, helix III, are tolerated by complementary substitutions at the DNase-Immunity protein binding interface. Im helix III is strictly conserved in colicins where an Asp forms polar interactions with the DNase backbone. ImAP41 contains an Asp-to-Gly substitution in helix III and our structures show the role of a co-evolved substitution where Pro in DNase loop 4 occupies the volume vacated and removes the unfulfilled hydrogen bond. We observe the co-evolved mutations in other DNase-Immunity pairs that appear to underpin the split of this family into two distinct groups.

  2. NAIPs: building an innate immune barrier against bacterial pathogens. NAIPs function as sensors that initiate innate immunity by detection of bacterial proteins in the host cell cytosol.

    PubMed

    Kofoed, Eric M; Vance, Russell E

    2012-07-01

    The innate immune system of mammals encodes several families of immune detector proteins that monitor the cytosol for signs of pathogen invasion. One important but poorly understood family of cytosolic immunosurveillance proteins is the NLR (nucleotide-binding domain, leucine-rich repeat containing) proteins. Recent work has demonstrated that one subfamily of NLRs, the NAIPs (NLR family, apoptosis inhibitory proteins), are activated by specific interaction with bacterial ligands, such as flagellin. NAIP activation leads to assembly of a large multiprotein complex called the inflammasome, which initiates innate immune responses by activation of the Caspase-1 protease. NAIPs therefore appear to detect pathogen molecules via a simple and direct receptor-ligand mechanism. Interestingly, other NLR family members appear to detect pathogens indirectly, perhaps by responding to host cell "stress" caused by the pathogen. Thus, the NLR family may have evolved surprisingly diverse mechanisms for detecting pathogens. PMID:22513803

  3. Induction of Mucosal and Systemic Immunity to a Recombinant Simian Immunodeficiency Viral Protein

    NASA Astrophysics Data System (ADS)

    Lehner, T.; Bergmeier, L. A.; Panagiotidi, C.; Tao, L.; Brookes, R.; Klavinskis, L. S.; Walker, P.; Walker, J.; Ward, R. G.; Hussain, L.; Gearing, A. J. H.; Adams, S. E.

    1992-11-01

    Heterosexual transmission through the cervico-vaginal mucosa is the principal route of human immunodeficiency virus (HIV) infection in Africa and is increasing in the United States and Europe. Vaginal immunization with simian immunodeficiency virus (SIV) had not yet been studied in nonhuman primates. Immune responses in macaques were investigated by stimulation of the genital and gut-associated lymphoid tissue with a recombinant, particulate SIV antigen. Vaginal, followed by oral, administration of the vaccine elicited three types of immunity: (i) gag protein p27-specific, secretory immunoglobulin A (IgA) and immunoglobulin G (IgG) in the vaginal fluid, (ii) specific CD4^+ T cell proliferation and helper function in B cell p27-specific IgA synthesis in the genital lymph nodes, and (iii) specific serum IgA and IgG, with CD4^+ T cell proliferative and helper functions in the circulating blood.

  4. Regulation of the Adaptive Immune Response by the IκB Family Protein Bcl-3

    PubMed Central

    Herrington, Felicity D.; Nibbs, Robert J. B.

    2016-01-01

    Bcl-3 is a member of the IκB family of proteins and an important regulator of Nuclear Factor (NF)-κB activity. The ability of Bcl-3 to bind and regulate specific NF-κB dimers has been studied in great depth, but its physiological roles in vivo are still not fully understood. It is, however, becoming clear that Bcl-3 is essential for the proper development, survival and activity of adaptive immune cells. Bcl-3 dysregulation can be observed in a number of autoimmune pathologies, and Bcl3-deficient animals are more susceptible to bacterial and parasitic infection. This review will describe our current understanding of the roles played by Bcl-3 in the development and regulation of the adaptive immune response, including lymphoid organogenesis, immune tolerance, lymphocyte function and dendritic cell biology. PMID:27023613

  5. Systemic immunization with papillomavirus L1 protein completely prevents the development of viral mucosal papillomas.

    PubMed

    Suzich, J A; Ghim, S J; Palmer-Hill, F J; White, W I; Tamura, J K; Bell, J A; Newsome, J A; Jenson, A B; Schlegel, R

    1995-12-01

    Infection of mucosal epithelium by papillomaviruses is responsible for the induction of genital and oral warts and plays a critical role in the development of human cervical and oropharyngeal cancer. We have employed a canine model to develop a systemic vaccine that completely protects against experimentally induced oral mucosal papillomas. The major capsid protein, L1, of canine oral papillomavirus (COPV) was expressed in Sf9 insect cells in native conformation. L1 protein, which self-assembled into virus-like particles, was purified on CsCl gradients and injected intradermally into the foot pad of beagles. Vaccinated animals developed circulating antibodies against COPV and became completely resistant to experimental challenge with COPV. Successful immunization was strictly dependent upon native L1 protein conformation and L1 type. Partial protection was achieved with as little as 0.125 ng of L1 protein, and adjuvants appeared useful for prolonging the host immune response. Serum immunoglobulins passively transferred from COPV L1-immunized beagles to naive beagles conferred protection from experimental infection with COPV. Our results indicate the feasibility of developing a human vaccine to prevent mucosal papillomas, which can progress to malignancy. PMID:8524802

  6. Systemic immunization with papillomavirus L1 protein completely prevents the development of viral mucosal papillomas.

    PubMed Central

    Suzich, J A; Ghim, S J; Palmer-Hill, F J; White, W I; Tamura, J K; Bell, J A; Newsome, J A; Jenson, A B; Schlegel, R

    1995-01-01

    Infection of mucosal epithelium by papillomaviruses is responsible for the induction of genital and oral warts and plays a critical role in the development of human cervical and oropharyngeal cancer. We have employed a canine model to develop a systemic vaccine that completely protects against experimentally induced oral mucosal papillomas. The major capsid protein, L1, of canine oral papillomavirus (COPV) was expressed in Sf9 insect cells in native conformation. L1 protein, which self-assembled into virus-like particles, was purified on CsCl gradients and injected intradermally into the foot pad of beagles. Vaccinated animals developed circulating antibodies against COPV and became completely resistant to experimental challenge with COPV. Successful immunization was strictly dependent upon native L1 protein conformation and L1 type. Partial protection was achieved with as little as 0.125 ng of L1 protein, and adjuvants appeared useful for prolonging the host immune response. Serum immunoglobulins passively transferred from COPV L1-immunized beagles to naive beagles conferred protection from experimental infection with COPV. Our results indicate the feasibility of developing a human vaccine to prevent mucosal papillomas, which can progress to malignancy. Images Fig. 1 PMID:8524802

  7. Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis

    PubMed Central

    Negm, Ola H.; Hamed, Mohamed R.; Dilnot, Elizabeth M.; Shone, Clifford C.; Marszalowska, Izabela; Lynch, Mark; Loscher, Christine E.; Edwards, Laura J.; Tighe, Patrick J.; Wilcox, Mark H.

    2015-01-01

    Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated <10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost. PMID:26178385

  8. Systemic Immunization with Papillomavirus L1 Protein Completely Prevents the Development of Viral Mucosal Papillomas

    NASA Astrophysics Data System (ADS)

    Suzich, Joann A.; Ghim, Shin-Je; Palmer-Hill, Frances J.; White, Wendy I.; Tamura, James K.; Bell, Judith A.; Newsome, Joseph A.; Bennett Jenson, A.; Schlegel, Richard

    1995-12-01

    Infection of mucosal epithelium by papillomaviruses is responsible for the induction of genital and oral warts and plays a critical role in the development of human cervical and oropharyngeal cancer. We have employed a canine model to develop a systemic vaccine that completely protects against experimentally induced oral mucosal papillomas. The major capsid protein, L1, of canine oral papillomavirus (COPV) was expressed in Sf9 insect cells in native conformation. L1 protein, which self-assembled into virus-like particles, was purified on CsCl gradients and injected intradermally into the foot pad of beagles. Vaccinated animals developed circulating antibodies against COPV and became completely resistant to experimental challenge with COPV. Successful immunization was strictly dependent upon native L1 protein conformation and L1 type. Partial protection was achieved with as little as 0.125 ng of L1 protein, and adjuvants appeared useful for prolonging the host immune response. Serum immunoglobulins passively transferred from COPV L1-immunized beagles to naive beagles conferred protection from experimental infection with COPV. Our results indicate the feasibility of developing a human vaccine to prevent mucosal papillomas, which can progress to malignancy.

  9. Humoral and cellular immune responses to matrix protein of measles virus in subacute sclerosing panencephalitis.

    PubMed Central

    Dhib-Jalbut, S; McFarland, H F; Mingioli, E S; Sever, J L; McFarlin, D E

    1988-01-01

    The immune response to matrix (M) protein of measles virus was examined in patients with subacute sclerosing panencephalitis (SSPE) and controls. Antibodies specific for M and nucleocapsid (NC) proteins in 11 serum and 8 cerebrospinal fluid (CSF) samples from patients with SSPE were quantitated by enzyme-linked immunosorbent assay by using affinity-purified measles virus proteins. Geometric mean anti-NC antibody titers were higher in the serum (6.58 +/- 0.98 [mean +/- standard deviation]) and CSF (4.38 +/- 0.74) of SSPE patients compared with controls. Anti-M antibodies were present in the serum and CSF of all SSPE samples tested but in titers lower than those of anti-NC antibodies. Geometric mean anti-M antibody titer was 3.35 +/- 0.53 in sera from patients with SSPE compared with 3.05 +/- 0.66 in sera from patients with other neurological diseases and 3.12 +/- 0.74 in sera from healthy individuals. Geometric mean anti-M antibody titer was 2.59 +/- 0.86 in the CSF of eight patients with SSPE compared with a mean less than 1.00 for patients with other neurological disease (controls). Intrathecal synthesis of anti-M or anti-NC antibodies was established in four patients with SSPE. The cellular immune responses to M, F, HA, and NC proteins were examined in four of the patients with SSPE by lymphoproliferation and were not significantly different from those in five healthy controls. The results demonstrate humoral and cellular immune responses to M protein in patients with SSPE and indicate that it is unlikely that a defect in the immune response to this virus component accounts for the disease process in the patients studied. Images PMID:3373575

  10. Repeat-enriched proteins are related to host cell invasion and immune evasion in parasitic protozoa.

    PubMed

    Mendes, T A O; Lobo, F P; Rodrigues, T S; Rodrigues-Luiz, G F; daRocha, W D; Fujiwara, R T; Teixeira, S M R; Bartholomeu, D C

    2013-04-01

    Proteins containing repetitive amino acid domains are widespread in all life forms. In parasitic organisms, proteins containing repeats play important roles such as cell adhesion and invasion and immune evasion. Therefore, extracellular and intracellular parasites are expected to be under different selective pressures regarding the repetitive content in their genomes. Here, we investigated whether there is a bias in the repetitive content found in the predicted proteomes of 6 exclusively extracellular and 17 obligate intracellular protozoan parasites, as well as 4 free-living protists. We also attempted to correlate the results with the distinct ecological niches they occupy and with distinct protein functions. We found that intracellular parasites have higher repetitive content in their proteomes than do extracellular parasites and free-living protists. In intracellular parasites, these repetitive proteins are located mainly at the parasite surface or are secreted and are enriched in amino acids known to be part of N- and O-glycosylation sites. Furthermore, in intracellular parasites, the developmental stages that are able to invade host cells express a higher proportion of proteins with perfect repeats relative to other life cycle stages, and these proteins have molecular functions associated with cell invasion. In contrast, in extracellular parasites, degenerate repetitive motifs are enriched in proteins that are likely to play roles in evading host immune response. Altogether, our results support the hypothesis that both the ability to invade host cells and to escape the host immune response may have shaped the expansion and maintenance of perfect and degenerate repeats in the genomes of intra- and extracellular parasites.

  11. Imaging murine NALT following intranasal immunization with flagellin-modified circumsporozoite protein malaria vaccines

    PubMed Central

    Nacer, Adéla; Carapau, Daniel; Mitchell, Robert; Meltzer, Abby; Shaw, Alan; Frevert, Ute; Nardin, Elizabeth H

    2013-01-01

    Intranasal (IN) immunization with a Plasmodium circumsporozoite (CS) protein conjugated to flagellin, a TLR5 agonist, was found to elicit antibody mediated protective immunity in our previous murine studies. To better understand IN elicited immune responses, we examined the nasopharynx-associated lymphoid tissue (NALT) in immunized mice and the interaction of flagellin-modified CS with murine dendritic cells (DC) in vitro. NALT of immunized mice contained a predominance of germinal center (GC) B cells and increased numbers of CD11c+ DC localized beneath the epithelium and within the GC T cell area. We detected microfold (M) cells distributed throughout the NALT epithelial cell layer and DC dendrites extending into the nasal cavity which could potentially function in luminal CS antigen uptake. Flagellin-modified CS taken up by DC in vitro was initially localized within intracellular vesicles followed by a cytosolic distribution. Vaccine modifications to enhance delivery to the NALT and specifically target NALT APC populations will advance development of an efficacious needle-free vaccine for the 40% of the world's population at risk of malaria. PMID:23820750

  12. Innovative immunization protocols using chimeric recombinant protein for the production of polyspecific loxoscelic antivenom in horses.

    PubMed

    Figueiredo, Luís F M; Dias-Lopes, Camila; Alvarenga, Larissa M; Mendes, Thais M; Machado-de-Ávila, Ricardo A; McCormack, Jessica; Minozzo, João C; Kalapothakis, Evanguedes; Chávez-Olórtegui, Carlos

    2014-08-01

    A chimeric protein (rCpLi) was constructed expressing three epitopes of rLiD1, a dermonecrotic toxin from the venom of Loxosceles intermedia spider. We have analyzed the neutralization potential of sera obtained by immunization of horses with rCpLi and rCpLi combined with initial doses of venoms and compared these with antivenom traditionally produced in horses using crude Loxosceles gaucho, Loxosceles laeta and L. intermedia venoms as antigens. We have demonstrated by ELISA that horses immunized with three initial doses of crude venom containing mixtures of L. intermedia, L. gaucho and L. laeta followed by nine doses of rCpLi generate antibodies with the same reactivity as those produced following immunization with traditional antivenom, towards the venoms of the three Loxosceles sp. species. Results from in vivo and in vitro neutralization assays showed that the new horse sera are able to neutralize the dermonecrotic activity of Loxosceles venoms, which are of medical importance in Brazil and some of these sera are capable of meeting the necessary potency requirements that could allow for their therapeutic use in humans. This immunization strategy combining both antigens used approximately 67% less crude Loxosceles venoms compared to traditional immunization protocol and can mean the development of Loxosceles antivenoms with the consequent reduction of devastation of arachnid fauna. PMID:24878371

  13. Protein kinase C in the immune system: from signalling to chromatin regulation.

    PubMed

    Lim, Pek Siew; Sutton, Christopher Ray; Rao, Sudha

    2015-12-01

    Protein kinase C (PKC) form a key family of enzymes involved in signalling pathways that specifically phosphorylates substrates at serine/threonine residues. Phosphorylation by PKC is important in regulating a variety of cellular events such as cell proliferation and the regulation of gene expression. In the immune system, PKCs are involved in regulating signal transduction pathways important for both innate and adaptive immunity, ultimately resulting in the expression of key immune genes. PKCs act as mediators during immune cell signalling through the immunological synapse. PKCs are traditionally known to be cytoplasmic signal transducers and are well embedded in the signalling pathways of cells to mediate the cells' response to a stimulus from the plasma membrane to the nucleus. PKCs are also found to transduce signals within the nucleus, a process that is distinct from the cytoplasmic signalling pathway. There is now growing evidence suggesting that PKC can directly regulate gene expression programmes through a non-traditional role as nuclear kinases. In this review, we will focus on the role of PKCs as key cytoplasmic signal transducers in immune cell signalling, as well as its role in nuclear signal transduction. We will also highlight recent evidence for its newly discovered regulatory role in the nucleus as a chromatin-associated kinase.

  14. The Stress Granule Protein G3BP1 Recruits Protein Kinase R To Promote Multiple Innate Immune Antiviral Responses

    PubMed Central

    Reineke, Lucas C.

    2014-01-01

    ABSTRACT Stress granules (SGs) are cytoplasmic storage sites containing translationally silenced mRNPs that can be released to resume translation after stress subsides. We previously showed that poliovirus 3C proteinase cleaves the SG-nucleating protein G3BP1, blocking the ability of cells to form SGs late in infection. Many other viruses also target G3BP1 and inhibit SG formation, but the reasons why these functions evolved are unclear. Previously, we also showed a link between G3BP1-induced SGs and protein kinase R (PKR)-mediated translational control, but the mechanism of PKR interplay with SG and the antiviral consequences are unknown. Here, we show that G3BP1 exhibits antiviral activity against several enteroviruses, whereas truncated G3BP1 that cannot form SGs does not. G3BP1-induced SGs are linked to activation of innate immune transcriptional responses through NF-κB and JNK. The G3BP1-induced SGs also recruit PKR and other antiviral proteins. We show that the PXXP domain within G3BP1 is essential for the recruitment of PKR to SGs, for eIF2α phosphorylation driven by PKR, and for nucleating SGs of normal composition. We also show that deletion of the PXXP domain in G3BP1 compromises its antiviral activity. These findings tie PKR activation to its recruitment to SGs by G3BP1 and indicate that G3BP1 promotes innate immune responses at both the transcriptional and translational levels and integrates cellular stress responses and innate immunity. IMPORTANCE Stress granules appear during virus infection, and their importance is not well understood. Previously, it was assumed that they were nonfunctional artifacts associated with cellular stress. PKR is a well-known antiviral protein; however, its regulation in cells is not well understood. Our work links cellular stress granules with activation of PKR and other innate immune pathways through the activity of G3BP1, a critical stress granule component. The ability of stress granules and G3BP1 to activate PKR and

  15. Inhibitory leukocyte immunoglobulin-like receptors: Immune checkpoint proteins and tumor sustaining factors.

    PubMed

    Kang, Xunlei; Kim, Jaehyup; Deng, Mi; John, Samuel; Chen, Heyu; Wu, Guojin; Phan, Hiep; Zhang, Cheng Cheng

    2016-01-01

    Inhibitory leukocyte immunoglobulin-like receptors (LILRBs 1-5) transduce signals via intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that recruit protein tyrosine phosphatase non-receptor type 6 (PTPN6 or SHP-1), protein tyrosine phosphatase non-receptor type 11 (PTPN11 or SHP-2), or Src homology 2 domain-containing inositol phosphatase (SHIP), leading to negative regulation of immune cell activation. Certain of these receptors also play regulatory roles in neuronal activity and osteoclast development. The activation of LILRBs on immune cells by their ligands may contribute to immune evasion by tumors. Recent studies found that several members of LILRB family are expressed by tumor cells, notably hematopoietic cancer cells, and may directly regulate cancer development and relapse as well as the activity of cancer stem cells. LILRBs thus have dual concordant roles in tumor biology - as immune checkpoint molecules and as tumor-sustaining factors. Importantly, the study of knockout mice indicated that LILRBs do not affect hematopoiesis and normal development. Therefore LILRBs may represent ideal targets for tumor treatment. This review aims to summarize current knowledge on expression patterns, ligands, signaling, and functions of LILRB family members in the context of cancer development. PMID:26636629

  16. The role of STIM and ORAI proteins in phagocytic immune cells

    PubMed Central

    Nunes, Paula

    2016-01-01

    Phagocytic cells, such as neutrophils, macrophages, and dendritic cells, migrate to sites of infection or damage and are integral to innate immunity through two main mechanisms. The first is to directly neutralize foreign agents and damaged or infected cells by secreting toxic substances or ingesting them through phagocytosis. The second is to alert the adaptive immune system through the secretion of cytokines and the presentation of the ingested materials as antigens, inducing T cell maturation into helper, cytotoxic, or regulatory phenotypes. While calcium signaling has been implicated in numerous phagocyte functions, including differentiation, maturation, migration, secretion, and phagocytosis, the molecular components that mediate these Ca2+ signals have been elusive. The discovery of the STIM and ORAI proteins has allowed researchers to begin clarifying the mechanisms and physiological impact of store-operated Ca2+ entry, the major pathway for generating calcium signals in innate immune cells. Here, we review evidence from cell lines and mouse models linking STIM and ORAI proteins to the control of specific innate immune functions of neutrophils, macrophages, and dendritic cells. PMID:26764049

  17. Evaluation of Th1/Th2-Related Immune Response against Recombinant Proteins of Brucella abortus Infection in Mice.

    PubMed

    Im, Young Bin; Park, Woo Bin; Jung, Myunghwan; Kim, Suk; Yoo, Han Sang

    2016-06-28

    Brucellosis is a zoonotic disease caused by Brucella, a genus of gram-negative bacteria. Cytokines have key roles in the activation of innate and acquired immunities. Despite several research attempts to reveal the immune responses, the mechanism of Brucella infection remains unclear. Therefore, immune responses were analyzed in mice immunized with nine recombinant proteins. Cytokine production profiles were analyzed in the RAW 264.7 cells and naive splenocytes after stimulation with three recombinant proteins, metal-dependent hydrolase (r0628), bacterioferritin (rBfr), and thiamine transporter substrate-binding protein (rTbpA). Immune responses were analyzed by ELISA and ELISpot assay after immunization with proteins in mice. The production levels of NO, TNF-α, and IL-6 were time-dependently increased after having been stimulated with proteins in the RAW 264.7 cells. In naive splenocytes, the production of IFN-γ and IL-2 was increased after stimulation with the proteins. It was concluded that two recombinant proteins, r0628 and rTbpA, showed strong immunogenicity that was induced with Th1-related cytokines IFN-γ, IL-2, and TNF-α more than Th2-related cytokines IL-6, IL-4, and IL-5 in vitro. Conversely, a humoral immune response was activated by increasing the number of antigen-secreting cells specifically. Furthermore, these could be candidate diagnosis antigens for better understanding of brucellosis.

  18. Oral and parenteral immunization of chickens (Gallus gallus) against West Nile virus with recombinant envelope protein

    USGS Publications Warehouse

    Fassbinder-Orth, C. A.; Hofmeister, E.K.; Weeks-Levy, C.; Karasov, W.H.

    2009-01-01

    West Nile virus (WNV) causes morbidity and mortality in humans, horses, and in more than 315 bird species in North America. Currently approved WNV vaccines are designed for parenteral administration and, as yet, no effective oral WNV vaccines have been developed. WNV envelope (E) protein is a highly antigenic protein that elicits the majority of virus-neutralizing antibodies during a WNV immune response. Leghorn chickens were given three vaccinations (each 2 wk apart) of E protein orally (20 ??g or 100 ??g/dose), of E protein intramuscularly (IM, 20 ??g/dose), or of adjuvant only (control group) followed by a WNV challenge. Viremias were measured post-WNV infection, and three new enzyme-linked immunosorbent assays were developed for quantifying IgM, IgY, and IgA-mediated immune response of birds following WNV infection. WNV viremia levels were significantly lower in the IM group than in both oral groups and the control group. Total WNV E protein-specific IgY production was significantly greater, and WNV nonstructural 1-specific IgY was significantly less, in the IM group compared to all other treatment groups. The results of this study indicate that IM vaccination of chickens with E protein is protective against WNV infection and results in a significantly different antibody production profile as compared to both orally vaccinated and nonvaccinated birds. ?? 2009 American Association of Avian Pathologists.

  19. Immunization against Rumen Methanogenesis by Vaccination with a New Recombinant Protein.

    PubMed

    Zhang, Litai; Huang, Xiaofeng; Xue, Bai; Peng, Quanhui; Wang, Zhisheng; Yan, Tianhai; Wang, Lizhi

    2015-01-01

    Vaccination through recombinant proteins against rumen methanogenesis provides a mitigation approach to reduce enteric methane (CH4) emissions in ruminants. The objective of present study was to evaluate the in vivo efficacy of a new vaccine candidate protein (EhaF) on methanogenesis and microbial population in the rumen of goats. We amplified the gene mru 1407 encoding protein EhaF using fresh rumen fluid samples of mature goats and successfully expressed recombinant protein (EhaF) in Escherichia coli Rosetta. This product was evaluated using 12 mature goats with half for control and other half injected with 400ug/goat the purified recombinant protein in day 1 and two subsequent booster immunizations in day 35 and 49. All measurements were undertaken from 63 to 68 days after the initial vaccination, with CH4 emissions determined using respiration calorimeter chambers. The results showed that the vaccination caused intensive immune responses in serum and saliva, although it had no significant effect on total enteric CH4 emissions and methanogen population in the rumen, when compared with the control goats. However, the vaccination altered the composition of rumen bacteria, especially the abundance of main phylum Firmicutes and genus Prevotella. The results indicate that protein EhaF might not be an effective vaccine to reduce enteric CH4 emissions but our vaccine have potential to influence the rumen ecosystem of goats. PMID:26445479

  20. An E4 Ligase Facilitates Polyubiquitination of Plant Immune Receptor Resistance Proteins in Arabidopsis[W

    PubMed Central

    Huang, Yan; Minaker, Sean; Roth, Charlotte; Huang, Shuai; Hieter, Philip; Lipka, Volker; Wiermer, Marcel; Li, Xin

    2014-01-01

    Proteins with nucleotide binding and leucine-rich repeat domains (NLRs) serve as immune receptors in animals and plants that recognize pathogens and activate downstream defense responses. As high accumulation of NLRs can result in unwarranted autoimmune responses, their cellular concentrations must be tightly regulated. However, the molecular mechanisms of this process are poorly detailed. The F-box protein Constitutive expressor of PR genes 1 (CPR1) was previously identified as a component of a Skp1, Cullin1, F-box protein E3 complex that targets NLRs, including Suppressor of NPR1, Constitutive 1 (SNC1) and Resistance to Pseudomonas syringae 2 (RPS2), for ubiquitination and further protein degradation. From a forward genetic screen, we identified Mutant, snc1-enhancing 3 (MUSE3), an E4 ubiquitin ligase involved in polyubiquitination of its protein targets. Knocking out MUSE3 in Arabidopsis thaliana results in increased levels of NLRs, including SNC1 and RPS2, whereas overexpressing MUSE3 together with CPR1 enhances polyubiquitination and protein degradation of these immune receptors. This report on the functional role of an E4 ligase in plants provides insight into the scarcely understood NLR degradation pathway. PMID:24449689

  1. Structural basis for concerted recruitment and activation of IRF-3 by innate immune adaptor proteins.

    PubMed

    Zhao, Baoyu; Shu, Chang; Gao, Xinsheng; Sankaran, Banumathi; Du, Fenglei; Shelton, Catherine L; Herr, Andrew B; Ji, Jun-Yuan; Li, Pingwei

    2016-06-14

    Type I IFNs are key cytokines mediating innate antiviral immunity. cGMP-AMP synthase, ritinoic acid-inducible protein 1 (RIG-I)-like receptors, and Toll-like receptors recognize microbial double-stranded (ds)DNA, dsRNA, and LPS to induce the expression of type I IFNs. These signaling pathways converge at the recruitment and activation of the transcription factor IRF-3 (IFN regulatory factor 3). The adaptor proteins STING (stimulator of IFN genes), MAVS (mitochondrial antiviral signaling), and TRIF (TIR domain-containing adaptor inducing IFN-β) mediate the recruitment of IRF-3 through a conserved pLxIS motif. Here we show that the pLxIS motif of phosphorylated STING, MAVS, and TRIF binds to IRF-3 in a similar manner, whereas residues upstream of the motif confer specificity. The structure of the IRF-3 phosphomimetic mutant S386/396E bound to the cAMP response element binding protein (CREB)-binding protein reveals that the pLxIS motif also mediates IRF-3 dimerization and activation. Moreover, rotavirus NSP1 (nonstructural protein 1) employs a pLxIS motif to target IRF-3 for degradation, but phosphorylation of NSP1 is not required for its activity. These results suggest a concerted mechanism for the recruitment and activation of IRF-3 that can be subverted by viral proteins to evade innate immune responses. PMID:27302953

  2. Immunization against Rumen Methanogenesis by Vaccination with a New Recombinant Protein

    PubMed Central

    Zhang, Litai; Huang, Xiaofeng; Xue, Bai; Peng, Quanhui; Wang, Zhisheng; Yan, Tianhai; Wang, Lizhi

    2015-01-01

    Vaccination through recombinant proteins against rumen methanogenesis provides a mitigation approach to reduce enteric methane (CH4) emissions in ruminants. The objective of present study was to evaluate the in vivo efficacy of a new vaccine candidate protein (EhaF) on methanogenesis and microbial population in the rumen of goats. We amplified the gene mru 1407 encoding protein EhaF using fresh rumen fluid samples of mature goats and successfully expressed recombinant protein (EhaF) in Escherichia coli Rosetta. This product was evaluated using 12 mature goats with half for control and other half injected with 400ug/goat the purified recombinant protein in day 1 and two subsequent booster immunizations in day 35 and 49. All measurements were undertaken from 63 to 68 days after the initial vaccination, with CH4 emissions determined using respiration calorimeter chambers. The results showed that the vaccination caused intensive immune responses in serum and saliva, although it had no significant effect on total enteric CH4 emissions and methanogen population in the rumen, when compared with the control goats. However, the vaccination altered the composition of rumen bacteria, especially the abundance of main phylum Firmicutes and genus Prevotella. The results indicate that protein EhaF might not be an effective vaccine to reduce enteric CH4 emissions but our vaccine have potential to influence the rumen ecosystem of goats. PMID:26445479

  3. Immunization against Rumen Methanogenesis by Vaccination with a New Recombinant Protein.

    PubMed

    Zhang, Litai; Huang, Xiaofeng; Xue, Bai; Peng, Quanhui; Wang, Zhisheng; Yan, Tianhai; Wang, Lizhi

    2015-01-01

    Vaccination through recombinant proteins against rumen methanogenesis provides a mitigation approach to reduce enteric methane (CH4) emissions in ruminants. The objective of present study was to evaluate the in vivo efficacy of a new vaccine candidate protein (EhaF) on methanogenesis and microbial population in the rumen of goats. We amplified the gene mru 1407 encoding protein EhaF using fresh rumen fluid samples of mature goats and successfully expressed recombinant protein (EhaF) in Escherichia coli Rosetta. This product was evaluated using 12 mature goats with half for control and other half injected with 400ug/goat the purified recombinant protein in day 1 and two subsequent booster immunizations in day 35 and 49. All measurements were undertaken from 63 to 68 days after the initial vaccination, with CH4 emissions determined using respiration calorimeter chambers. The results showed that the vaccination caused intensive immune responses in serum and saliva, although it had no significant effect on total enteric CH4 emissions and methanogen population in the rumen, when compared with the control goats. However, the vaccination altered the composition of rumen bacteria, especially the abundance of main phylum Firmicutes and genus Prevotella. The results indicate that protein EhaF might not be an effective vaccine to reduce enteric CH4 emissions but our vaccine have potential to influence the rumen ecosystem of goats.

  4. The Mitogen-Activated Protein Kinase (MAPK) Pathway: Role in Immune Evasion by Trypanosomatids.

    PubMed

    Soares-Silva, Mercedes; Diniz, Flavia F; Gomes, Gabriela N; Bahia, Diana

    2016-01-01

    Leishmania spp. and Trypanosoma cruzi are the causative agents of leishmaniasis and Chagas disease, respectively, two neglected tropical diseases that affect about 25 million people worldwide. These parasites belong to the family Trypanosomatidae, and are both obligate intracellular parasites that manipulate host signaling pathways and the innate immune system to establish infection. Mitogen-activated protein kinases (MAPKs) are serine and threonine protein kinases that are highly conserved in eukaryotes, and are involved in signal transduction pathways that modulate physiological and pathophysiological cell responses. This mini-review highlights existing knowledge concerning the mechanisms that Leishmania spp. and T. cruzi have evolved to target the host's MAPK signaling pathways and highjack the immune response, and, in this manner, promote parasite maintenance in the host. PMID:26941717

  5. The Mitogen-Activated Protein Kinase (MAPK) Pathway: Role in Immune Evasion by Trypanosomatids

    PubMed Central

    Soares-Silva, Mercedes; Diniz, Flavia F.; Gomes, Gabriela N.; Bahia, Diana

    2016-01-01

    Leishmania spp. and Trypanosoma cruzi are the causative agents of leishmaniasis and Chagas disease, respectively, two neglected tropical diseases that affect about 25 million people worldwide. These parasites belong to the family Trypanosomatidae, and are both obligate intracellular parasites that manipulate host signaling pathways and the innate immune system to establish infection. Mitogen-activated protein kinases (MAPKs) are serine and threonine protein kinases that are highly conserved in eukaryotes, and are involved in signal transduction pathways that modulate physiological and pathophysiological cell responses. This mini-review highlights existing knowledge concerning the mechanisms that Leishmania spp. and T. cruzi have evolved to target the host’s MAPK signaling pathways and highjack the immune response, and, in this manner, promote parasite maintenance in the host. PMID:26941717

  6. Characterization of IM7/8551-7 carbon/epoxy in compression and shear: Final report for the period October 1987 to September 1988

    SciTech Connect

    Swanson, S.R.; Colvin, G.E. Jr.

    1988-09-27

    This is a final report on a project entitled ''Characterization of IM7/8551-7 Carbon/Epoxy in Compression and Shear,'' for the period October 1987 to September 1988. The intent of this program is to determine the failure properties of carbon/epoxy laminates under multiaxial stress loadings, with particular emphasis on loadings involving compressive stresses. The work accomplished to date is summarized here, in the form of two published papers and a summary of other work. Appendix C contains a summary of the development of the multiaxial compression test fixture. 11 figs.

  7. E3 ubiquitin-ligases and their target proteins during the regulation of plant innate immunity.

    PubMed

    Duplan, Vincent; Rivas, Susana

    2014-01-01

    Reversible protein ubiquitination plays a crucial role during the regulation of plant immune signaling. E3 ubiquitin (Ub)-ligase enzymes, which are classified into different families depending on their structural and functional features, confer the specificity of substrate and are the best characterized components of the ubiquitination cascade. E3 Ub-ligases of different families have been shown to be involved in all steps of plant immune responses. Indeed, they have been involved in the first steps of pathogen perception, as they appear to modulate perception of pathogen-associated molecular patterns by pattern-recognition receptors at the plasma membrane and to regulate the accumulation of nucleotide-binding leucine-rich repeat-type intracellular immune receptors. In addition, E3 Ub-ligase proteins are also involved in the regulation of the signaling responses downstream of pathogen perception through targeting vesicle trafficking components or nuclear transcription factors, for instance. Finally, we also discuss the case of microbial effector proteins that are able to target host E3 Ub-ligases, or to act themselves as E3 Ub-ligases, in their attempt to subvert the host proteasome to promote disease. PMID:24592270

  8. Immune-Relevant and Antioxidant Activities of Vitellogenin and Yolk Proteins in Fish

    PubMed Central

    Sun, Chen; Zhang, Shicui

    2015-01-01

    Vitellogenin (Vtg), the major egg yolk precursor protein, is traditionally thought to provide protein- and lipid-rich nutrients for developing embryos and larvae. However, the roles of Vtg as well as its derived yolk proteins lipovitellin (Lv) and phosvitin (Pv) extend beyond nutritional functions. Accumulating data have demonstrated that Vtg, Lv and Pv participate in host innate immune defense with multifaceted functions. They can all act as multivalent pattern recognition receptors capable of identifying invading microbes. Vtg and Pv can also act as immune effectors capable of killing bacteria and virus. Moreover, Vtg and Lv are shown to possess phagocytosis-promoting activity as opsonins. In addition to these immune-relevant functions, Vtg and Pv are found to have antioxidant activity, which is able to protect the host from oxidant stress. These non-nutritional functions clearly deepen our understanding of the physiological roles of the molecules, and at the same time, provide a sound basis for potential application of the molecules in human health. PMID:26506386

  9. Merozoite surface proteins in red blood cell invasion, immunity and vaccines against malaria

    PubMed Central

    Beeson, James G.; Drew, Damien R.; Boyle, Michelle J.; Feng, Gaoqian; Fowkes, Freya J.I.; Richards, Jack S.

    2016-01-01

    Malaria accounts for an enormous burden of disease globally, with Plasmodium falciparum accounting for the majority of malaria, and P. vivax being a second important cause, especially in Asia, the Americas and the Pacific. During infection with Plasmodium spp., the merozoite form of the parasite invades red blood cells and replicates inside them. It is during the blood-stage of infection that malaria disease occurs and, therefore, understanding merozoite invasion, host immune responses to merozoite surface antigens, and targeting merozoite surface proteins and invasion ligands by novel vaccines and therapeutics have been important areas of research. Merozoite invasion involves multiple interactions and events, and substantial processing of merozoite surface proteins occurs before, during and after invasion. The merozoite surface is highly complex, presenting a multitude of antigens to the immune system. This complexity has proved challenging to our efforts to understand merozoite invasion and malaria immunity, and to developing merozoite antigens as malaria vaccines. In recent years, there has been major progress in this field, and several merozoite surface proteins show strong potential as malaria vaccines. Our current knowledge on this topic is reviewed, highlighting recent advances and research priorities. PMID:26833236

  10. Prokaryotic ancestry of eukaryotic protein networks mediating innate immunity and apoptosis.

    PubMed

    Dunin-Horkawicz, Stanislaw; Kopec, Klaus O; Lupas, Andrei N

    2014-04-01

    Protein domains characteristic of eukaryotic innate immunity and apoptosis have many prokaryotic counterparts of unknown function. By reconstructing interactomes computationally, we found that bacterial proteins containing these domains are part of a network that also includes other domains not hitherto associated with immunity. This network is connected to the network of prokaryotic signal transduction proteins, such as histidine kinases and chemoreceptors. The network varies considerably in domain composition and degree of paralogy, even between strains of the same species, and its repetitive domains are often amplified recently, with individual repeats sharing up to 100% sequence identity. Both phenomena are evidence of considerable evolutionary pressure and thus compatible with a role in the "arms race" between host and pathogen. In order to investigate the relationship of this network to its eukaryotic counterparts, we performed a cluster analysis of organisms based on a census of its constituent domains across all fully sequenced genomes. We obtained a large central cluster of mainly unicellular organisms, from which multicellular organisms radiate out in two main directions. One is taken by multicellular bacteria, primarily cyanobacteria and actinomycetes, and plants form an extension of this direction, connected via the basal, unicellular cyanobacteria. The second main direction is taken by animals and fungi, which form separate branches with a common root in the α-proteobacteria of the central cluster. This analysis supports the notion that the innate immunity networks of eukaryotes originated from their endosymbionts and that increases in the complexity of these networks accompanied the emergence of multicellularity.

  11. The safety of whey protein concentrate derived from the milk of cows immunized against Clostridium difficile.

    PubMed

    Young, Karen W H; Munro, Ian C; Taylor, Steve L; Veldkamp, Peter; van Dissel, Jaap T

    2007-04-01

    A whey protein concentrate prepared from the milk of cows that have been immunized against Clostridium difficile (C. difficile) and its toxins, toxin A and toxin B, is produced for use as a medical food for the dietary management of patients with C. difficile-associated diarrhea (CDAD) to prevent a relapse of the infection. The safety of anti-C. difficile whey protein concentrate (anti-CD WPC) is supported by analytical data comparing the composition of raw milk from immunized cows versus that from non-immunized cows, and the composition of anti-CD WPC versus that of regular whey protein concentrate. Additionally, a prospective clinical study was conducted in 77 patients with CDAD to demonstrate the safety of consuming anti-CD WPC to prevent relapse of the infection. This study, which included adverse event monitoring, physical examinations, and extensive hematological and biochemical assessments, showed that anti-CD WPC is safe to consume by patients with CDAD. The available analytical and clinical evidence demonstrate that anti-CD WPC is safe for use by individuals with CDAD, under the described conditions of use.

  12. Subacute sclerosing panencephalitis and multiple sclerosis: in vitro measles immunity and sensitization to myelin basic protein.

    PubMed Central

    Sheremata, W.; Sazant, A.; Watters, G.

    1978-01-01

    Three children with subacute sclerosing panencephalitis (SSPE), 12 patients with multiple sclerosis (MS) and 12 healthy persons were studied by the macrophage migration inhibition factor (MIF) assay with measles and rubella antigens and with myelin basic protein. For the SSPE patients the mean migration indexes +/- standard deviation were 44.1 +/- 10.9 for measles antigen, 38.7 +/- 12.3 for rubella antigen and 49.8 +/- 25.7 for myelin basic protein; for the MS patients the indexes were 103.0 +/- 10.6, 93.8 +/- 15.0 and 89.3 +/- 19.9; and for the healthy subjects the indexes were 68.8 +/- 22.6, 77.7 +/- 31.3 and 100.1 +/- 6.5. The results of this study showed increased cellular immunity to measles and rubella in SSPE patients as compared with healthy persons, and absence of immunity to measles in MS patients. Patients with MS showed hypersensitivity to myelin basic protein during clinical exacerbations that was not associated with changes in immunity to measles, whereas all SSPE patients showed a significant response regardless to stage of illness. PMID:343902

  13. Study on the immune response to recombinant Hsp70 protein from Megalobrama amblycephala.

    PubMed

    Chen, Nan; Wan, Xiao-Ling; Huang, Chun-Xiao; Wang, Wei-Min; Liu, Hong; Wang, Huan-Ling

    2014-11-01

    The expression of heat shock protein 70 (Hsp70) is induced in response to many factors including high temperature, infection, metal pollutants and toxic chemicals. In this study, Megalobrama amblycephala HSP70 promoter was cloned, and characteristic heat shock elements (HSEs) were identified in the promoter region. The recombinant M. amblycephala Hsp70 protein (rMaHsp70) was expressed and purified from Escherichia coli BL21 (DE3). To evaluate in vivo immune response of rMaHsp70, we administered intraperitoneal (IP) injection, and demonstrated that rMaHsp70 stimulated M. amblycephala immune activity by inducing the expression of HSP70, HIF-1α, HSC70, CXCR4b, TNF-α and IL-1β mRNAs in liver, headkidney, spleen and gill, as well as SOD, glutathione, lysozyme and interferon alpha proteins in serum and liver. The effect of rMaHsp70 as adjuvant against Aeromonas hydrophila was assessed by injecting a mixed vaccine of rMaHsp70 and A. hydrophila (A. hydrophila/Hsp70) into M. amblycephala, and the relative percent survival (RPS) in the A. hydrophila/Hsp70 group was 75% compared to 50% in the A. hydrophila/PBS group. Furthermore, rMaHsp70 also promoted the proliferation and suppressed apoptosis in M. amblycephala fin cells (MAF) in a dose-dependent manner. Taken together, these results suggest that rMaHsp70 can induce organic immune response and improve environmental tolerance. PMID:25113416

  14. The crystal structure of the immunity protein of colicin E7 suggests a possible colicin-interacting surface.

    PubMed Central

    Chak, K F; Safo, M K; Ku, W Y; Hsieh, S Y; Yuan, H S

    1996-01-01

    The immunity protein of colicin E7 (ImmE7) can bind specifically to the DNase-type colicin E7 and inhibit its bactericidal activity. Here we report the 1.8-angstrom crystal structure of the ImmE7 protein. This is the first x-ray structure determined in the superfamily of colicin immunity proteins. The ImmE7 protein consists of four antiparallel alpha-helices, folded in a topology similar to the architecture of a four-helix bundle structure. A region rich in acidic residues is identified. This negatively charged area has the greatest variability within the family of DNase-type immunity proteins; thus, it seems likely that this area is involved in specific binding to colicin. Based on structural, genetic, and kinetic data, we suggest that all the DNase-type immunity proteins, as well as colicins, share a "homologous-structural framework" and that specific interaction between a colicin and its cognate immunity protein relies upon how well these two proteins' charged residues match on the interaction surface, thus leading to specific immunity of the colicin. Images Fig. 1 Fig. 2 Fig. 5 PMID:8692833

  15. Identification of novel biomarkers in chronic immune thrombocytopenia (ITP) by microarray-based serum protein profiling.

    PubMed

    Bal, Gürkan; Futschik, Matthias E; Hartl, Daniela; Ringel, Frauke; Kamhieh-Milz, Julian; Sterzer, Viktor; Hoheisel, Jörg D; Alhamdani, Mohamed S S; Salama, Abdulgabar

    2016-02-01

    The pathological mechanisms underlying the development of immune thrombocytopenia (ITP) are unclear and its diagnosis remains a process of exclusion. Currently, there are no known specific biomarkers for ITP to support differential diagnosis and treatment decisions. Profiling of serum proteins may be valuable for identifying such biomarkers. Sera from 46 patients with primary chronic ITP and 34 healthy blood donors were analysed using a microarray of 755 antibodies. We identified 161 differentially expressed proteins. In addition to oncoproteins and tumour-suppressor proteins, including apoptosis regulator BCL2, breast cancer type 1 susceptibility protein (BRCA1), Fanconi anaemia complementation group C (FANCC) and vascular endothelial growth factor A (VEGFA), we detected six anti-nuclear autoantibodies in a subset of ITP patients: anti-PCNA, anti-SmD, anti-Ro/SSA60, anti-Ro/SSA52, anti-La/SSB and anti-RNPC antibodies. This finding may provide a rational explanation for the association of ITP with malignancies and other autoimmune diseases. While RUNX1mRNA expression in the peripheral blood mononuclear cells (PBMC) of patients was significantly downregulated, an accumulation of RUNX1 protein was observed in the platelets of ITP patients. This may indicate dysregulation of RUNX1 expression in PBMC and megakaryocytes and may lead to an imbalanced immune response and impaired thrombopoiesis. In conclusion, we provide novel insights into the pathogenic mechanisms of ITP that warrant further exploration.

  16. Recombinant Flagellin-Porcine Circovirus Type 2 Cap Fusion Protein Promotes Protective Immune Responses in Mice

    PubMed Central

    Zhang, Chunyan; Zhu, Shanshan; Wei, Li; Yan, Xu; Wang, Jing; Quan, Rong; She, Ruiping; Hu, Fengjiao; Liu, Jue

    2015-01-01

    The Cap protein of porcine circovirus type 2 (PCV2) that serves as a major host-protective immunogen was used to develop recombinant vaccines for control of PCV2-associated diseases. Growing research data have demonstrated the high effectiveness of flagellin as an adjuvant for humoral and cellular immune responses. Here, a recombinant protein was designed by fusing a modified version of bacterial flagellin to PCV2 Cap protein and expressed in a baculovirus system. When administered without adjuvant to BALB/c mice, the flagellin-Cap fusion protein elicited stronger PCV2-specific IgG antibody response, higher neutralizing antibody levels, milder histopathological changes and lower viremia, as well as higher secretion of cytokines such as TNF-α and IFN-γ that conferred better protection against virus challenge than those in the recombinant Cap alone-inoculated mice. These results suggest that the recombinant Cap protein when fused to flagellin could elicit better humoral and cellular immune responses against PCV2 infection in a mouse model, thereby acting as an attractive candidate vaccine for control of the PCV2-associated diseases. PMID:26070075

  17. Identification of novel biomarkers in chronic immune thrombocytopenia (ITP) by microarray-based serum protein profiling.

    PubMed

    Bal, Gürkan; Futschik, Matthias E; Hartl, Daniela; Ringel, Frauke; Kamhieh-Milz, Julian; Sterzer, Viktor; Hoheisel, Jörg D; Alhamdani, Mohamed S S; Salama, Abdulgabar

    2016-02-01

    The pathological mechanisms underlying the development of immune thrombocytopenia (ITP) are unclear and its diagnosis remains a process of exclusion. Currently, there are no known specific biomarkers for ITP to support differential diagnosis and treatment decisions. Profiling of serum proteins may be valuable for identifying such biomarkers. Sera from 46 patients with primary chronic ITP and 34 healthy blood donors were analysed using a microarray of 755 antibodies. We identified 161 differentially expressed proteins. In addition to oncoproteins and tumour-suppressor proteins, including apoptosis regulator BCL2, breast cancer type 1 susceptibility protein (BRCA1), Fanconi anaemia complementation group C (FANCC) and vascular endothelial growth factor A (VEGFA), we detected six anti-nuclear autoantibodies in a subset of ITP patients: anti-PCNA, anti-SmD, anti-Ro/SSA60, anti-Ro/SSA52, anti-La/SSB and anti-RNPC antibodies. This finding may provide a rational explanation for the association of ITP with malignancies and other autoimmune diseases. While RUNX1mRNA expression in the peripheral blood mononuclear cells (PBMC) of patients was significantly downregulated, an accumulation of RUNX1 protein was observed in the platelets of ITP patients. This may indicate dysregulation of RUNX1 expression in PBMC and megakaryocytes and may lead to an imbalanced immune response and impaired thrombopoiesis. In conclusion, we provide novel insights into the pathogenic mechanisms of ITP that warrant further exploration. PMID:26628061

  18. Transgenic Carrot Expressing Fusion Protein Comprising M. tuberculosis Antigens Induces Immune Response in Mice

    PubMed Central

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

    2015-01-01

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

  19. Gene Models, Expression Repertoire, and Immune Response of Plasmodium vivax Reticulocyte Binding Proteins.

    PubMed

    Hietanen, Jenni; Chim-Ong, Anongruk; Chiramanewong, Thanprakorn; Gruszczyk, Jakub; Roobsoong, Wanlapa; Tham, Wai-Hong; Sattabongkot, Jetsumon; Nguitragool, Wang

    2016-03-01

    Members of the Plasmodium vivax reticulocyte binding protein (PvRBP) family are believed to mediate specific invasion of reticulocytes by P. vivax. In this study, we performed molecular characterization of genes encoding members of this protein family. Through cDNA sequencing, we constructed full-length gene models and verified genes that are protein coding and those that are pseudogenes. We also used quantitative PCR to measure their in vivo transcript abundances in clinical P. vivax isolates. Like genes encoding related invasion ligands of P. falciparum, Pvrbp expression levels vary broadly across different parasite isolates. Through antibody measurements, we found that host immune pressure may be the driving force behind the distinctly high diversity of one of the family members, PvRBP2c. Mild yet significant negative correlation was found between parasitemia and the PvRBP2b antibody level, suggesting that antibodies to the protein may interfere with invasion. PMID:26712206

  20. A 35-kilodalton protein is a major target of the human immune response to Mycobacterium leprae.

    PubMed Central

    Triccas, J A; Roche, P W; Winter, N; Feng, C G; Butlin, C R; Britton, W J

    1996-01-01

    The control of leprosy will be facilitated by the identification of major Mycobacterium leprae-specific antigens which mirror the immune response to the organism across the leprosy spectrum. We have investigated the host response to a 35-kDa protein of M. leprae. Recombinant 35-kDa protein purified from Mycobacterium smegmatis resembled the native antigen in the formation of multimeric complexes and binding by monoclonal antibodies and sera from leprosy patients. These properties were not shared by two forms of 35-kDa protein purified from Escherichia coli. The M. smegmatis-derived 35-kDa protein stimulated a gamma interferon-secreting T-cell proliferative response in the majority of paucibacillary leprosy patients and healthy contacts of leprosy patients tested. Cellular responses to the protein in patients with multibacillary leprosy were weak or absent, consistent with hyporesponsiveness to M. leprae characteristic of this form of the disease. Almost all leprosy patients and contacts recognized the 35-kDa protein by either a T-cell proliferative or an immunoglobulin G antibody response, whereas few tuberculosis patients recognized the antigen. This specificity was confirmed in guinea pigs, with the 35-kDa protein eliciting strong delayed-type hypersensitivity in M. leprae-sensitized animals but not in those sensitized with Mycobacterium tuberculosis or Mycobacterium bovis BCG. Therefore, the M. leprae 35-kDa protein appears to be a major and relatively specific target of the human immune response to M. leprae and is a potential component of a diagnostic test to detect exposure to leprosy or a vaccine to combat the disease. PMID:8945562

  1. Proteomics of larval hemolymph in Bombyx mori reveals various nutrient-storage and immunity-related proteins.

    PubMed

    Zhang, Yan; Dong, Zhaoming; Wang, Dandan; Wu, Yong; Song, Qianru; Gu, Peiming; Zhao, Ping; Xia, Qingyou

    2014-04-01

    The silkworm, Bombyx mori, is an important economic insect for its production of silk. The larvae of many lepidopteran insects are major agricultural pests and often silkworm is explored as a model organism for other lepidopteran pest species. The hemolymph of caterpillars contains a lot of nutrient and immune components. In this study, we applied liquid chromatography-tandem mass spectrometry to gain a better understanding of the larval hemolymph proteomics in B. mori. We identified 752 proteins in hemolymph collected from day-4 fourth instar and day-7 fifth instar. Nearly half the identified proteins (49%) were predicted to function as binding proteins and 46% were predicted to have catalytic activities. Apolipophorins, storage proteins, and 30K proteins constituted the most abundant groups of nutrient-storage proteins. Of them, 30K proteins showed large differences between fourth instar larvae and fifth instar larvae. Besides nutrient-storage proteins, protease inhibitors are also expressed very highly in hemolymph. The analysis also revealed lots of immunity-related proteins, including recognition, signaling, effectors and other proteins, comprising multiple immunity pathways in hemolymph. Our data provide an exhaustive research of nutrient-storage proteins and immunity-related proteins in larval hemolymph, and will pave the way for future physiological and pathological studies of caterpillars. PMID:24402669

  2. Dissociation of immune determinants of outer membrane proteins of Chlamydia psittaci strain guinea pig inclusion conjunctivitis.

    PubMed

    Westbay, T D; Dascher, C C; Hsia, R C; Bavoil, P M; Zauderer, M

    1994-12-01

    Chlamydia trachomatis is an important human pathogen. Research to develop a Chlamydia vaccine has focused on the major outer membrane protein (MOMP). Determinants of this protein elicit serovar-specific neutralizing antibodies which are thought to play a critical role in protective immunity. MOMP-specific antibody responses are highly variable in the polymorphic population. Genetic factors which might influence the MOMP-specific immune response are consequently of particular interest. The C. psittaci strain guinea pig inclusion conjunctivitis (GPIC) is a natural pathogen of the guinea pig that causes both ocular and genital tract infections that closely resemble those caused by C. trachomatis in humans. As such, it provides an excellent model for disease. In this report, we explore the influence of major histocompatibility complex-linked genes on the MOMP-specific antibody response in mice immunized with either whole GPIC elementary bodies or recombinant GPIC MOMP. Our results indicate that the MOMP-specific antibody response is major histocompatibility complex linked such that mice of the H-2d haplotype are high responders while mice of the H-2k haplotype are low responders. We demonstrate that MOMP-specific B cells are present in H-2k strains which are, however, deficient in MOMP-specific helper T cells. Although immunization of low-MOMP-responder strains with whole chlamydial elementary bodies induces high levels of immunoglobulin G antibody specific for Omp2, the cysteine-rich outer membrane protein, MOMP-specific B cells are unable to receive help from Omp2-specific T cells. The failure of intermolecular help from Omp2-specific T cells and related observations raise important issues regarding the processing and presentation of chlamydial antigens and the design of optimal subunit vaccines.

  3. Serum immune-related proteins are differentially expressed during hibernation in the American black bear.

    PubMed

    Chow, Brian A; Donahue, Seth W; Vaughan, Michael R; McConkey, Brendan; Vijayan, Mathilakath M

    2013-01-01

    Hibernation is an adaptation to conserve energy in the face of extreme environmental conditions and low food availability that has risen in several animal phyla. This phenomenon is characterized by reduced metabolic rate (∼25% of the active basal metabolic rate in hibernating bears) and energy demand, while other physiological adjustments are far from clear. The profiling of the serum proteome of the American black bear (Ursus americanus) may reveal specific proteins that are differentially modulated by hibernation, and provide insight into the remarkable physiological adaptations that characterize ursid hibernation. In this study, we used differential gel electrophoresis (DIGE) analysis, liquid chromatography coupled to tandem mass spectrometry, and subsequent MASCOT analysis of the mass spectra to identify candidate proteins that are differentially expressed during hibernation in captive black bears. Seventy serum proteins were identified as changing by ±1.5 fold or more, out of which 34 proteins increased expression during hibernation. The majority of identified proteins are involved in immune system processes. These included α2-macroglobulin, complement components C1s and C4, immunoglobulin μ and J chains, clusterin, haptoglobin, C4b binding protein, kininogen 1, α2-HS-glycoprotein, and apoplipoproteins A-I and A-IV. Differential expression of a subset of these proteins identified by proteomic analysis was also confirmed by immunodetection. We propose that the observed serum protein changes contribute to the maintenance of the hibernation phenotype and health, including increased capacities for bone maintenance and wound healing during hibernation in bears.

  4. Serum Immune-Related Proteins are Differentially Expressed during Hibernation in the American Black Bear

    PubMed Central

    Chow, Brian A.; Donahue, Seth W.; Vaughan, Michael R.; McConkey, Brendan; Vijayan, Mathilakath M.

    2013-01-01

    Hibernation is an adaptation to conserve energy in the face of extreme environmental conditions and low food availability that has risen in several animal phyla. This phenomenon is characterized by reduced metabolic rate (∼25% of the active basal metabolic rate in hibernating bears) and energy demand, while other physiological adjustments are far from clear. The profiling of the serum proteome of the American black bear (Ursus americanus) may reveal specific proteins that are differentially modulated by hibernation, and provide insight into the remarkable physiological adaptations that characterize ursid hibernation. In this study, we used differential gel electrophoresis (DIGE) analysis, liquid chromatography coupled to tandem mass spectrometry, and subsequent MASCOT analysis of the mass spectra to identify candidate proteins that are differentially expressed during hibernation in captive black bears. Seventy serum proteins were identified as changing by ±1.5 fold or more, out of which 34 proteins increased expression during hibernation. The majority of identified proteins are involved in immune system processes. These included α2-macroglobulin, complement components C1s and C4, immunoglobulin μ and J chains, clusterin, haptoglobin, C4b binding protein, kininogen 1, α2-HS-glycoprotein, and apoplipoproteins A-I and A-IV. Differential expression of a subset of these proteins identified by proteomic analysis was also confirmed by immunodetection. We propose that the observed serum protein changes contribute to the maintenance of the hibernation phenotype and health, including increased capacities for bone maintenance and wound healing during hibernation in bears. PMID:23825529

  5. Serum immune-related proteins are differentially expressed during hibernation in the American black bear.

    PubMed

    Chow, Brian A; Donahue, Seth W; Vaughan, Michael R; McConkey, Brendan; Vijayan, Mathilakath M

    2013-01-01

    Hibernation is an adaptation to conserve energy in the face of extreme environmental conditions and low food availability that has risen in several animal phyla. This phenomenon is characterized by reduced metabolic rate (∼25% of the active basal metabolic rate in hibernating bears) and energy demand, while other physiological adjustments are far from clear. The profiling of the serum proteome of the American black bear (Ursus americanus) may reveal specific proteins that are differentially modulated by hibernation, and provide insight into the remarkable physiological adaptations that characterize ursid hibernation. In this study, we used differential gel electrophoresis (DIGE) analysis, liquid chromatography coupled to tandem mass spectrometry, and subsequent MASCOT analysis of the mass spectra to identify candidate proteins that are differentially expressed during hibernation in captive black bears. Seventy serum proteins were identified as changing by ±1.5 fold or more, out of which 34 proteins increased expression during hibernation. The majority of identified proteins are involved in immune system processes. These included α2-macroglobulin, complement components C1s and C4, immunoglobulin μ and J chains, clusterin, haptoglobin, C4b binding protein, kininogen 1, α2-HS-glycoprotein, and apoplipoproteins A-I and A-IV. Differential expression of a subset of these proteins identified by proteomic analysis was also confirmed by immunodetection. We propose that the observed serum protein changes contribute to the maintenance of the hibernation phenotype and health, including increased capacities for bone maintenance and wound healing during hibernation in bears. PMID:23825529

  6. Chondroitin sulphate proteoglycans: extracellular matrix proteins that regulate immunity of the central nervous system.

    PubMed

    Haylock-Jacobs, Sarah; Keough, Michael B; Lau, Lorraine; Yong, V Wee

    2011-10-01

    The extracellular matrix (ECM) is a complex network of scaffolding molecules that also plays an important role in cell signalling, migration and tissue structure. In the central nervous system (CNS), the ECM is integral to the efficient development/guidance and survival of neurons and axons. However, changes in distribution of the ECM in the CNS may significantly enhance pathology in CNS disease or following injury. One group of ECM proteins that is important for CNS homeostasis is the chondroitin sulphate proteoglycans (CSPGs). Up-regulation of these molecules has been demonstrated to be both desirable and detrimental following CNS injury. Taking cues from arthritis, where there is a strong anti-CSPG immune response, there is evidence that suggests that CSPGs may influence immunity during CNS pathological conditions. This review focuses on the role of CSPGs in CNS pathologies as well as in immunity, both from a viewpoint of how they may inhibit repair and exacerbate damage in the CNS, and how they are involved in activation and function of peripheral immune cells, particularly in multiple sclerosis. Lastly, we address how CSPGs may be manipulated to improve disease outcomes.

  7. The Arabidopsis Protein Phosphatase PP2C38 Negatively Regulates the Central Immune Kinase BIK1.

    PubMed

    Couto, Daniel; Niebergall, Roda; Liang, Xiangxiu; Bücherl, Christoph A; Sklenar, Jan; Macho, Alberto P; Ntoukakis, Vardis; Derbyshire, Paul; Altenbach, Denise; Maclean, Dan; Robatzek, Silke; Uhrig, Joachim; Menke, Frank; Zhou, Jian-Min; Zipfel, Cyril

    2016-08-01

    Plants recognize pathogen-associated molecular patterns (PAMPs) via cell surface-localized pattern recognition receptors (PRRs), leading to PRR-triggered immunity (PTI). The Arabidopsis cytoplasmic kinase BIK1 is a downstream substrate of several PRR complexes. How plant PTI is negatively regulated is not fully understood. Here, we identify the protein phosphatase PP2C38 as a negative regulator of BIK1 activity and BIK1-mediated immunity. PP2C38 dynamically associates with BIK1, as well as with the PRRs FLS2 and EFR, but not with the co-receptor BAK1. PP2C38 regulates PAMP-induced BIK1 phosphorylation and impairs the phosphorylation of the NADPH oxidase RBOHD by BIK1, leading to reduced oxidative burst and stomatal immunity. Upon PAMP perception, PP2C38 is phosphorylated on serine 77 and dissociates from the FLS2/EFR-BIK1 complexes, enabling full BIK1 activation. Together with our recent work on the control of BIK1 turnover, this study reveals another important regulatory mechanism of this central immune component. PMID:27494702

  8. The Arabidopsis Protein Phosphatase PP2C38 Negatively Regulates the Central Immune Kinase BIK1

    PubMed Central

    Liang, Xiangxiu; Bücherl, Christoph A.; Sklenar, Jan; Macho, Alberto P.; Ntoukakis, Vardis; Derbyshire, Paul; Altenbach, Denise; Robatzek, Silke; Uhrig, Joachim; Menke, Frank; Zhou, Jian-Min

    2016-01-01

    Plants recognize pathogen-associated molecular patterns (PAMPs) via cell surface-localized pattern recognition receptors (PRRs), leading to PRR-triggered immunity (PTI). The Arabidopsis cytoplasmic kinase BIK1 is a downstream substrate of several PRR complexes. How plant PTI is negatively regulated is not fully understood. Here, we identify the protein phosphatase PP2C38 as a negative regulator of BIK1 activity and BIK1-mediated immunity. PP2C38 dynamically associates with BIK1, as well as with the PRRs FLS2 and EFR, but not with the co-receptor BAK1. PP2C38 regulates PAMP-induced BIK1 phosphorylation and impairs the phosphorylation of the NADPH oxidase RBOHD by BIK1, leading to reduced oxidative burst and stomatal immunity. Upon PAMP perception, PP2C38 is phosphorylated on serine 77 and dissociates from the FLS2/EFR-BIK1 complexes, enabling full BIK1 activation. Together with our recent work on the control of BIK1 turnover, this study reveals another important regulatory mechanism of this central immune component. PMID:27494702

  9. Anticarrier immunity suppresses the antibody response to polysaccharide antigens after intranasal immunization with the polysaccharide-protein conjugate.

    PubMed Central

    Bergquist, C; Lagergård, T; Holmgren, J

    1997-01-01

    We have conjugated cholera toxin (CT) B subunit (CTB) to dextran and studied the effect in mice of previous immunization with CT and CTB on the response to dextran after intranasal immunizations with conjugate. Preexisting immunity to CTB was found to inhibit both the lung mucosal response and serum antibody response to dextran, but this effect could be overcome by using a higher dose of conjugate and delaying the conjugate immunization until the CTB antibody titers had declined. The role of anti-CTB antibodies on the mucosal surface was probably to prevent uptake of the conjugate through a mechanism of immune exclusion. Passively transferred serum antibodies against CTB, on the other hand, suppressed both the serum response and the local antibody response against CTB but did not affect the response to dextran after intranasal immunization with conjugate. PMID:9125533

  10. Co-expression of Interleukin-15 Enhances the Protective Immune Responses Induced by Immunization with a Murine Malaria MVA-Based Vaccine Encoding the Circumsporozoite Protein.

    PubMed

    Parra, Marcela; Liu, Xia; Derrick, Steven C; Yang, Amy; Molina-Cruz, Alvaro; Barillas-Mury, Carolina; Zheng, Hong; Thao Pham, Phuong; Sedegah, Martha; Belmonte, Arnel; Litilit, Dianne D; Waldmann, Thomas A; Kumar, Sanjai; Morris, Sheldon L; Perera, Liyanage P

    2015-01-01

    Malaria remains a major global public health problem with an estimated 200 million cases detected in 2012. Although the most advanced candidate malaria vaccine (RTS,S) has shown promise in clinical trials, its modest efficacy and durability have created uncertainty about the impact of RTS,S immunization (when used alone) on global malaria transmission. Here we describe the development and characterization of a novel modified vaccinia virus Ankara (MVA)-based malaria vaccine which co-expresses the Plasmodium yoelii circumsporozoite protein (CSP) and IL-15. Vaccination/challenge studies showed that C57BL/6 mice immunized with the MVA-CSP/IL15 vaccine were protected significantly better against a P. yoelii 17XNL sporozoite challenge than either mice immunized with an MVA vaccine expressing only CSP or naïve controls. Importantly, the levels of total anti-CSP IgG were elevated about 100-fold for the MVA-CSP/IL15 immunized group compared to mice immunized with the MVA-CSP construct that does not express IL-15. Among the IgG subtypes, the IL-15 expressing MVA-CSP vaccine induced levels of IgG1 (8 fold) and IgG2b (80 fold) higher than the MVA-CSP construct. The significantly enhanced humoral responses and protection detected after immunization with the MVA-CSP/IL15 vaccine suggest that this IL-15 expressing MVA construct could be considered in the development of future malaria immunization strategies.

  11. Trace levels of innate immune response modulating impurities (IIRMIs) synergize to break tolerance to therapeutic proteins.

    PubMed

    Verthelyi, Daniela; Wang, Vivian

    2010-12-22

    Therapeutic proteins such as monoclonal antibodies, replacement enzymes and toxins have significantly improved the therapeutic options for multiple diseases, including cancer and inflammatory diseases as well as enzyme deficiencies and inborn errors of metabolism. However, immune responses to these products are frequent and can seriously impact their safety and efficacy. Of the many factors that can impact protein immunogenicity, this study focuses on the role of innate immune response modulating impurities (IIRMIs) that could be present despite product purification and whether these impurities can synergize to facilitate an immunogenic response to therapeutic proteins. Using lipopolysaccharide (LPS) and CpG ODN as IIRMIs we showed that trace levels of these impurities synergized to induce IgM, IFNγ, TNFα and IL-6 expression. In vivo, trace levels of these impurities synergized to increase antigen-specific IgG antibodies to ovalbumin. Further, whereas mice treated with human erythropoietin showed a transient increase in hematocrit, those that received human erythropoietin containing low levels of IIRMIs had reduced response to erythropoietin after the 1(st) dose and developed long-lasting anemia following subsequent doses. This suggests that the presence of IIRMIs facilitated a breach in tolerance to the endogenous mouse erythropoietin. Overall, these studies indicate that the risk of enhancing immunogenicity should be considered when establishing acceptance limits of IIRMIs for therapeutic proteins.

  12. Serum protein changes in immune and nonimmune pigeons infected with various strains of Trichomonas gallinae

    USGS Publications Warehouse

    Kocan, R.M.; Herman, C.M.

    1970-01-01

    Serum protein changes were studied in immune and nonimmune pigeons infected with three different strains of Trichomonas gallinae. Strain I (nonvirulent) produced no change in the relative concentration of serum components. Strains II (oral canker) and III (Jones' Barn) produced decreases in albumin and alpha globulins, and increases in beta and gamma globulins between the 7th and 20th days post infection. Birds infected with strain II began to return to normal by the 20th day, while all those infected with strain III were dead between 10 and 14 days post infection. Two serum protein patterns resulted from infection of immune birds with the Jones' Barn strain. One showed no change in relative protein concentrations and no tissue invasion by the parasite while the other was similar to that seen in nonimmune birds infected with a strain producing oral canker. These also showed evidence of tissue invasion by the parasite. It was concluded that tissue invasion was necessary to evoke a quantitative change in serum protein concentrations.

  13. Oral immunization of mice with Lactococcus lactis expressing the rotavirus VP8* protein.

    PubMed

    Rodríguez-Díaz, Jesús; Montava, Rebeca; Viana, Rosa; Buesa, Javier; Pérez-Martínez, Gaspar; Monedero, Vicente

    2011-06-01

    The efficacy of recombinant Lactococcus lactis as a delivery vehicle for a rotavirus antigen was evaluated in a mouse model. The rotavirus VP8* protein was expressed intracellularly and extracellularly in L. lactis wild type and in an alr mutant deficient in alanine racemase activity, necessary for the synthesis of the cell-wall component D: -alanine. When the mucosal immune response was evaluated by measuring VP8*-specific IgA antibody in faeces, wild-type L. lactis triggered a low IgA synthesis only when the secreting strain was used. In contrast, VP8*-specific IgA was detected in faeces of both groups of mice orally given the alr mutant expressing extracellular VP8* and intracellular VP8*, which reached levels similar to that obtained with the wild type secreting strain. However, oral administration of the recombinant strains did not induce serum IgG or IgA responses. L. lactis cell-wall mutants may therefore provide certain advantages when low-antigenic proteins are expressed intracellularly. However, the low immune response obtained by using this antigen-bacterial host combination prompts to the use of new strains and vaccination protocols in order to develop acceptable rotavirus immunization levels.

  14. Genetic conjugation of components in two pneumococcal fusion protein vaccines enhances paediatric mucosal immune responses.

    PubMed

    Pope, Caroline; Oliver, Elizabeth H; Ma, Jiangtao; Langton Hewer, Claire; Mitchell, Tim J; Finn, Adam

    2015-03-30

    Streptococcus pneumoniae colonises the upper respiratory tract and can cause pneumonia, meningitis and otitis media. Existing pneumococcal conjugate vaccines are expensive to produce and only protect against 13 of the 90+ pneumococcal serotypes; hence there is an urgent need for the development of new vaccines. We have shown previously in mice that pneumolysin (Ply) and a non-toxic variant (Δ6Ply) enhance antibody responses when genetically fused to pneumococcal surface adhesin A (PsaA), a potentially valuable effect for future vaccines. We investigated this adjuvanticity in human paediatric mucosal primary immune cell cultures. Adenoidal mononuclear cells (AMNC) from children aged 0-15 years (n=46) were stimulated with conjugated, admixed or individual proteins, cell viability and CD4+ T-cell proliferative responses were assessed using flow cytometry and cytokine secretion was measured using multiplex technology. Proliferation of CD4+ T-cells in response to PsaAPly, was significantly higher than responses to individual or admixed proteins (p=0.002). In contrast, an enhanced response to PsaAΔ6Ply compared to individual or admixed proteins only occurred at higher concentrations (p<0.01). Evaluation of cytotoxicity suggested that responses occurred when Ply-induced cytolysis was inhibited, either by fusion or mutation, but importantly an additional toxicity independent immune enhancing effect was also apparent as a result of fusion. Responses were MHC class II dependent and had a Th1/Th17 profile. Genetic fusion of Δ6Ply to PsaA significantly modulates and enhances pro-inflammatory CD4+ T-cell responses without the cytolytic effects of some other pneumolysoids. Membrane binding activity of such proteins may confer valuable adjuvant properties as fusion may assist Δ6Ply to deliver PsaA to the APC surface effectively, contributing to the initiation of anti-pneumococcal CD4+ T-cell immunity.

  15. Immune response to synthetic peptides of dengue prM protein.

    PubMed

    Vázquez, Susana; Guzmán, María Guadalupe; Guillen, Gerardo; Chinea, Glay; Pérez, Ana Beatriz; Pupo, Maritza; Rodriguez, Rosmary; Reyes, Osvaldo; Garay, Hilda Elisa; Delgado, Iselys; García, Gissel; Alvarez, Mayling

    2002-03-15

    The immunological activities of five synthetic peptides of the prM protein of dengue-2 (DEN-2) virus containing B cell epitopes were evaluated in BALB/c mice. Two peptides elicited neutralizing antibodies against all four DEN serotypes. Virus-specific proliferative responses were demonstrated in mice immunized with four of the five peptides, demonstrating the presence of T cell epitopes. Mice immunized with three of the five peptides conjugated with bovine albumin showed statistically significant levels (P<0.05) of protection when challenged with DEN-2 virus. These results could constitute the basis for the establishment of the role of DEN virus pre and M antigens in the development of anti-flaviviral vaccines.

  16. Mapping the peptide and protein immune response in the larvae of the fleshfly Sarcophaga bullata.

    PubMed

    Ciencialová, Alice; Neubauerová, Tereza; Sanda, Miloslav; Sindelka, Radek; Cvacka, Josef; Voburka, Zdenek; Budesínský, Milos; Kasicka, Václav; Sázelová, Petra; Solínová, Veronika; Macková, Martina; Koutek, Bohumír; Jirácek, Jirí

    2008-06-01

    We chose the larvae of fleshfly Sarcophaga bullata to map the peptide and protein immune response. The hemolymph of the third-instar larvae of S. bullata was used for isolation. The larvae were injected with bacterial suspension to induce an antimicrobial response. The hemolymph was separated into crude fractions, which were subdivided by RP-HPLC, gel electrophoresis, and free-flow electrophoresis. In several fractions, we determined significant antimicrobial activities against the pathogenic bacteria Escherichia coli, Staphylococcus aureus, or Pseudomonas aeruginosa. Among antimicrobially active compounds we identified dipeptide beta-alanyl-L-tyrosine, protein transferrin, and two variants of peptide sapecin. We also partially characterized two novel antimicrobially active polypeptides; odorant-binding protein 99b, and a peptide which remains unidentified.

  17. Mucosal Immunization with Recombinant Fusion Protein DnaJ-ΔA146Ply Enhances Cross-Protective Immunity against Streptococcus pneumoniae Infection in Mice via Interleukin 17A

    PubMed Central

    Liu, Yusi; Wang, Hong; Zhang, Shuai; Zeng, Lingbin; Xu, Xiuyu; Wu, Kaifeng; Wang, Wei; Yin, Nanlin; Song, Zhixin

    2014-01-01

    Pneumolysin (Ply) and its variants are protective against pneumococcal infections in animal models, and as a Toll-like receptor 4 agonist, pneumolysin has been reported to be a mucosal adjuvant. DnaJ has been approved as a useful candidate vaccine protein; we therefore designed novel fusion proteins of DnaJ with a form of Ply that has a deletion of A146 (ΔA146Ply-DnaJ [the C terminus of ΔA146Ply connected with the N terminus of DnaJ] and DnaJ-ΔA146Ply [the C terminus of DnaJ connected with the N terminus of ΔA146Ply]) to test whether they are protective against focal and lethal pneumococcal infections and their potential protective mechanisms. The purified proteins were used to intranasally immunize the animals without additional adjuvant. Immunization with DnaJ-ΔA146Ply or DnaJ plus ΔA146Ply (Ply with a single deletion of A146) could significantly reduce S. pneumoniae colonization in the nasopharynx and lung relative with DnaJ alone. Additionally, we observed the best protection for DnaJ-ΔA146Ply-immunized mice after challenge with lethal doses of S. pneumoniae strains, which was comparable to that achieved by PPV23. Mice immunized with DnaJ-ΔA146Ply produced significantly higher levels of anti-DnaJ IgG in serum and secretory IgA (sIgA) in saliva than those immunized with DnaJ alone. The production of IL-17A was also striking in DnaJ-ΔA146Ply-immunized mice. IL-17A knockout (KO) mice did not benefit from DnaJ-ΔA146Ply immunization in colonization experiments, and sIgA production was impaired in IL-17A KO mice. Collectively, our results indicate a mucosal adjuvant potential for ΔA146Ply and that, without additional adjuvant, DnaJ-ΔA146Ply fusion protein exhibits extensive immune stimulation and is effective against pneumococcal challenges, properties which are partially attributed to the IL-17A-mediated immune responses. PMID:24491576

  18. Successful Interference with Cellular Immune Responses to Immunogenic Proteins Encoded by Recombinant Viral Vectors

    PubMed Central

    Sarukhan, Adelaida; Camugli, Sabine; Gjata, Bernard; von Boehmer, Harald; Danos, Olivier; Jooss, Karin

    2001-01-01

    Vectors derived from the adeno-associated virus (AAV) have been successfully used for the long-term expression of therapeutic genes in animal models and patients. One of the major advantages of these vectors is the absence of deleterious immune responses following gene transfer. However, AAV vectors, when used in vaccination studies, can result in efficient humoral and cellular responses against the transgene product. It is therefore important to understand the factors which influence the establishment of these immune responses in order to design safe and efficient procedures for AAV-based gene therapies. We have compared T-cell activation against a strongly immunogenic protein, the influenza virus hemagglutinin (HA), which is synthesized in skeletal muscle following gene transfer with an adenovirus (Ad) or an AAV vector. In both cases, cellular immune responses resulted in the elimination of transduced muscle fibers within 4 weeks. However, the kinetics of CD4+ T-cell activation were markedly delayed when AAV vectors were used. Upon recombinant Ad (rAd) gene transfer, T cells were activated both by direct transduction of dendritic cells and by cross-presentation of the transgene product, while upon rAAV gene transfer T cells were only activated by the latter mechanism. These results suggested that activation of the immune system by the transgene product following rAAV-mediated gene transfer might be easier to control than that following rAd-mediated gene transfer. Therefore, we tested protocols aimed at interfering with either antigen presentation by blocking the CD40/CD40L pathway or with the T-cell response by inducing transgene-specific tolerance. Long-term expression of the AAV-HA was achieved in both cases, whereas immune responses against Ad-HA could not be prevented. These data clearly underline the importance of understanding the mechanisms by which vector-encoded proteins are recognized by the immune system in order to specifically interfere with them and

  19. Immunization of Mice with Recombinant Brucella abortus Organic Hydroperoxide Resistance (Ohr) Protein Protects Against a Virulent Brucella abortus 544 Infection.

    PubMed

    Hop, Huynh Tan; Reyes, Alisha Wehdnesday Bernardo; Simborio, Hannah Leah Tadeja; Arayan, Lauren Togonon; Min, Won Gi; Lee, Hu Jang; Lee, Jin Ju; Chang, Hong Hee; Kim, Suk

    2016-01-01

    In this study, the Brucella abortus ohr gene coding for an organic hydroperoxide resistance protein (Ohr) was cloned into a maltose fusion protein expression system (pMAL), inserted into Escherichia coli, and purified, and its immunogenicity was evaluated by western blot analysis using Brucella-positive mouse sera. The purified recombinant Ohr (rOhr) was treated with adjuvant and injected intraperitoneally into BALB/c mice. A protective immune response analysis revealed that rOhr induced a significant increase in both the IgG1 and IgG2a titers, and IgG2a reached a higher level than IgG1 after the second and third immunizations. Additionally, immunization with rOhr induced high production of IFN-γ as well as proinflammatory cytokines such as TNF, MCP-1, IL-12p70, and IL-6, but a lesser amount of IL-10, suggesting that rOhr predominantly elicited a cell-mediated immune response. In addition, immunization with rOhr caused a significantly higher degree of protection against a virulent B. abortus infection compared with a positive control group consisting of mice immunized with maltose-binding protein. These findings showed that B. abortus rOhr was able to induce both humoral and cell-mediated immunity in mice, which suggested that this recombinant protein could be a potential vaccine candidate for animal brucellosis.

  20. Induction of immune responses by two recombinant proteins of brucella abortus, outer membrane proteins 2b porin and Cu/Zn superoxide dismutase, in mouse model.

    PubMed

    Sung, Kyung Yong; Jung, Myunghwan; Shin, Min-Kyoung; Park, Hyun-Eui; Lee, Jin Ju; Kim, Suk; Yoo, Han Sang

    2014-06-28

    The diagnosis of Brucella abortus is mainly based on serological methods using antibody against LPS, which has diagnostic problems. Therefore, to solve this problem, we evaluated two proteins of B. abortus, Cu/Zn superoxide dismutase (SodC) and outer membrane proteins 2b porin (Omp2b). The genes were cloned and expressed in a pMAL system, and the recombinant proteins, rOmp2b and rSodC, were purified as fusion forms with maltosebinding protein. The identity of the proteins was confirmed by SDS-PAGE and Western blot analysis with sera of mice infected with B. abortus. Production of cytokines and nitric oxide (NO) was investigated in RAW 264.7 cells and mouse splenocytes after stimulation with the proteins. Moreover, cellular and humoral immune responses were investigated in BALB/c mice after immunization with the proteins. TNF-α, IL-6, and NO were significantly inducible in RAW 264.7 cells. Splenocytes of naive mice produced IFN-γ and IL-4 significantly by stimulation. Moreover, number of IgG, IFN-γ, and IL-4 producing cells were increased in immunized mice with the two proteins. Production of IgG and IgM with rOmp2b was higher than those with rSodC in immunized mice. These results suggest that the two recombinant proteins of B. abortus may be potential LPS-free proteins for diagnosis.

  1. Immune recognition of Onchocerca volvulus proteins in the human host and animal models of onchocerciasis.

    PubMed

    Manchang, T K; Ajonina-Ekoti, I; Ndjonka, D; Eisenbarth, A; Achukwi, M D; Renz, A; Brattig, N W; Liebau, E; Breloer, M

    2015-05-01

    Onchocerca volvulus is a tissue-dwelling, vector-borne nematode parasite of humans and is the causative agent of onchocerciasis or river blindness. Natural infections of BALB/c mice with Litomosoides sigmodontis and of cattle with Onchocerca ochengi were used as models to study the immune responses to O. volvulus-derived recombinant proteins (OvALT-2, OvNLT-1, Ov103 and Ov7). The humoral immune response of O. volvulus-infected humans against OvALT-2, OvNLT-1 and Ov7 revealed pronounced immunoglobulin G (IgG) titres which were, however, significantly lower than against the lysate of O. volvulus adult female worms. Sera derived from patients displaying the hyperreactive form of onchocerciasis showed a uniform trend of higher IgG reactivity both to the single proteins and the O. volvulus lysate. Sera derived from L. sigmodontis-infected mice and from calves exposed to O. ochengi transmission in a hyperendemic area also contained IgM and IgG1 specific for O. volvulus-derived recombinant proteins. These results strongly suggest that L. sigmodontis-specific and O. ochengi-specific immunoglobulins elicited during natural infection of mice and cattle cross-reacted with O. volvulus-derived recombinant antigens. Monitoring O. ochengi-infected calves over a 26-month period, provided a comprehensive kinetic of the humoral response to infection that was strictly correlated with parasite load and occurrence of microfilariae. PMID:24721822

  2. A novel elicitor protein from Phytophthora parasitica induces plant basal immunity and systemic acquired resistance.

    PubMed

    Chang, Yi-Hsuan; Yan, Hao-Zhi; Liou, Ruey-Fen

    2015-02-01

    The interaction between Phytophthora pathogens and host plants involves the exchange of complex molecular signals from both sides. Recent studies of Phytophthora have led to the identification of various apoplastic elicitors known to trigger plant immunity. Here, we provide evidence that the protein encoded by OPEL of Phytophthora parasitica is a novel elicitor. Homologues of OPEL were identified only in oomycetes, but not in fungi and other organisms. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed that OPEL is expressed throughout the development of P. parasitica and is especially highly induced after plant infection. Infiltration of OPEL recombinant protein from Escherichia coli into leaves of Nicotiana tabacum (cv. Samsun NN) resulted in cell death, callose deposition, the production of reactive oxygen species and induced expression of pathogen-associated molecular pattern (PAMP)-triggered immunity markers and salicylic acid-responsive defence genes. Moreover, the infiltration conferred systemic resistance against a broad spectrum of pathogens, including Tobacco mosaic virus, the bacteria wilt pathogen Ralstonia solanacearum and P. parasitica. In addition to the signal peptide, OPEL contains three conserved domains: a thaumatin-like domain, a glycine-rich protein domain and a glycosyl hydrolase (GH) domain. Intriguingly, mutation of a putative laminarinase active site motif in the predicted GH domain abolished its elicitor activity, which suggests enzymatic activity of OPEL in triggering the defence response.

  3. Protein-energy malnutrition decreases immune response to Leishmania chagasi vaccine in BALB/c mice.

    PubMed

    Malafaia, G; Serafim, T D; Silva, M E; Pedrosa, M L; Rezende, S A

    2009-01-01

    Protein-energy malnutrition and visceral leishmaniasis are important problems of public health affecting millions of people worldwide. Vaccine efficacy depends on the ability of individuals to mount an appropriate immune response and may be inadequate in malnourished persons. In this study, we used a mouse model to verify the effect of combined protein, iron and zinc deficiency in the response to Leishmania chagasi antigen vaccine. BALB/c mice were fed with a low-protein (3% casein), iron- and zinc-deficient diet or control diet (14% casein and sufficient in zinc and iron). After malnutrition establishment, mice were vaccinated subcutaneously with L. chagasi Ag plus saponin. After vaccination, mice were nutritionally repleted and then all mice were challenged with L. chagasi promastigotes. Four weeks later, liver and spleen parasite load was evaluated. Our data show that vaccine caused a significant reduction in parasite load in spleen and liver from mice fed with control diet. However, splenic parasitism was increased in mice fed with deficient diet and this diet caused a reduction in splenocyte IFN-gamma production in response to the vaccine in repleted mice. These data suggest that malnutrition may alter immune response to L. chagasi vaccine in BALB/c model of infection, even after nutritional repletion.

  4. Minimal role for the circumsporozoite protein in the induction of sterile immunity by vaccination with live rodent malaria sporozoites.

    PubMed

    Mauduit, Marjorie; Tewari, Rita; Depinay, Nadya; Kayibanda, Michèle; Lallemand, Eliette; Chavatte, Jean-Marc; Snounou, Georges; Rénia, Laurent; Grüner, Anne Charlotte

    2010-05-01

    Immunization with live Plasmodium sporozoites under chloroquine prophylaxis (Spz plus CQ) induces sterile immunity against sporozoite challenge in rodents and, more importantly, in humans. Full protection is obtained with substantially fewer parasites than with the classic immunization with radiation-attenuated sporozoites. The sterile protection observed comprised a massive reduction in the hepatic parasite load and an additional effect at the blood stage level. Differences in the immune responses induced by the two protocols occur but are as yet little characterized. We have previously demonstrated that in mice immunized with irradiated sporozoites, immune responses against the circumsporozoite protein (CSP), the major component of the sporozoite's surface and the leading malaria vaccine candidate, were not essential for sterile protection. Here, we have employed transgenic Plasmodium berghei parasites in which the endogenous CSP was replaced by that of Plasmodium yoelii, another rodent malaria species, to assess the role of CSP in the sterile protection induced by the Spz-plus-CQ protocol. The data demonstrated that this role was minor because sterile immunity was obtained irrespective of the origin of CSP expressed by the parasites in this model of protection. The immunity was obtained through a single transient exposure of the host to the immunizing parasites (preerythrocytic and erythrocytic), a dose much smaller than that required for immunization with radiation-attenuated sporozoites.

  5. Conserved hypothetical protein Rv1977 in Mycobacterium tuberculosis strains contains sequence polymorphisms and might be involved in ongoing immune evasion.

    PubMed

    Jiang, Yi; Liu, Haican; Wang, Xuezhi; Li, Guilian; Qiu, Yan; Dou, Xiangfeng; Wan, Kanglin

    2015-01-01

    Host immune pressure and associated parasite immune evasion are key features of host-pathogen co-evolution. A previous study showed that human T cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved and thus it was deduced that M. tuberculosis lacks antigenic variation and immune evasion. Here, we selected 151 clinical Mycobacterium tuberculosis isolates from China, amplified gene encoding Rv1977 and compared the sequences. The results showed that Rv1977, a conserved hypothetical protein, is not conserved in M. tuberculosis strains and there are polymorphisms existed in the protein. Some mutations, especially one frameshift mutation, occurred in the antigen Rv1977, which is uncommon in M.tb strains and may lead to the protein function altering. Mutations and deletion in the gene all affect one of three T cell epitopes and the changed T cell epitope contained more than one variable position, which may suggest ongoing immune evasion.

  6. Protein A suppresses immune responses during Staphylococcus aureus bloodstream infection in guinea pigs

    SciTech Connect

    Kim, Hwan Keun; Falugi, Fabiana; Thomer, Lena; Missiakas, Dominique M.; Schneewind, Olaf

    2015-01-06

    Staphylococcus aureus infection is not associated with the development of protective immunity, and disease relapses occur frequently. We hypothesize that protein A, a factor that binds immunoglobulin Fcγ and cross-links VH3 clan B cell receptors (IgM), is the staphylococcal determinant for host immune suppression. To test this, vertebrate IgM was examined for protein A cross-linking. High VH3 binding activity occurred with human and guinea immunoglobulin, whereas mouse and rabbit immunoglobulins displayed little and no binding, respectively. Establishing a guinea pig model of S. aureus bloodstream infection, we show that protein A functions as a virulence determinant and suppresses host B cell responses. Immunization with SpAKKAA, which cannot bind immunoglobulin, elicits neutralizing antibodies that enable guinea pigs to develop protective immunity.

  7. Complex structure of type VI peptidoglycan muramidase effector and a cognate immunity protein

    SciTech Connect

    Wang, Tianyu; Ding, Jinjing; Zhang, Ying; Wang, Da-Cheng; Liu, Wei

    2013-10-01

    The structure of the Tse3–Tsi3 complex associated with the bacterial type VI secretion system of P. aeruginosa has been solved and refined at 1.9 Å resolution. The structural basis of the recognition of the muramidase effector and its inactivation by its cognate immunity protein is revealed. The type VI secretion system (T6SS) is a bacterial protein-export machine that is capable of delivering virulence effectors between Gram-negative bacteria. The T6SS of Pseudomonas aeruginosa transports two lytic enzymes, Tse1 and Tse3, to degrade cell-wall peptidoglycan in the periplasm of rival bacteria that are competing for niches via amidase and muramidase activities, respectively. Two cognate immunity proteins, Tsi1 and Tsi3, are produced by the bacterium to inactivate the two antibacterial effectors, thereby protecting its siblings from self-intoxication. Recently, Tse1–Tsi1 has been structurally characterized. Here, the structure of the Tse3–Tsi3 complex is reported at 1.9 Å resolution. The results reveal that Tse3 contains a C-terminal catalytic domain that adopts a soluble lytic transglycosylase (SLT) fold in which three calcium-binding sites were surprisingly observed close to the catalytic Glu residue. The electrostatic properties of the substrate-binding groove are also distinctive from those of known structures with a similar fold. All of these features imply that a unique catalytic mechanism is utilized by Tse3 in cleaving glycosidic bonds. Tsi3 comprises a single domain showing a β-sandwich architecture that is reminiscent of the immunoglobulin fold. Three loops of Tsi3 insert deeply into the groove of Tse3 and completely occlude its active site, which forms the structural basis of Tse3 inactivation. This work is the first crystallographic report describing the three-dimensional structure of the Tse3–Tsi3 effector–immunity pair.

  8. Unfolded protein response (UPR) signaling regulates arsenic trioxide-mediated macrophage innate immune function disruption

    SciTech Connect

    Srivastava, Ritesh K.; Li, Changzhao; Chaudhary, Sandeep C.; Ballestas, Mary E.; Elmets, Craig A.; Robbins, David J.; Matalon, Sadis; Deshane, Jessy S.; Afaq, Farrukh; Bickers, David R.; Athar, Mohammad

    2013-11-01

    Arsenic exposure is known to disrupt innate immune functions in humans and in experimental animals. In this study, we provide a mechanism by which arsenic trioxide (ATO) disrupts macrophage functions. ATO treatment of murine macrophage cells diminished internalization of FITC-labeled latex beads, impaired clearance of phagocytosed fluorescent bacteria and reduced secretion of pro-inflammatory cytokines. These impairments in macrophage functions are associated with ATO-induced unfolded protein response (UPR) signaling pathway characterized by the enhancement in proteins such as GRP78, p-PERK, p-eIF2α, ATF4 and CHOP. The expression of these proteins is altered both at transcriptional and translational levels. Pretreatment with chemical chaperon, 4-phenylbutyric acid (PBA) attenuated the ATO-induced activation in UPR signaling and afforded protection against ATO-induced disruption of macrophage functions. This treatment also reduced ATO-mediated reactive oxygen species (ROS) generation. Interestingly, treatment with antioxidant N-acetylcysteine (NAC) prior to ATO exposure, not only reduced ROS production and UPR signaling but also improved macrophage functions. These data demonstrate that UPR signaling and ROS generation are interdependent and are involved in the arsenic-induced pathobiology of macrophage. These data also provide a novel strategy to block the ATO-dependent impairment in innate immune responses. - Highlights: • Inorganic arsenic to humans and experimental animals disrupt innate immune responses. • The mechanism underlying arsenic impaired macrophage functions involves UPR signaling. • Chemical chaperon attenuates arsenic-mediated macrophage function impairment. • Antioxidant, NAC blocks impairment in arsenic-treated macrophage functions.

  9. Vaccination with Legionella pneumophila membranes induces cell-mediated and protective immunity in a guinea pig model of Legionnaires' disease. Protective immunity independent of the major secretory protein of Legionella pneumophila.

    PubMed Central

    Blander, S J; Horwitz, M A

    1991-01-01

    We have examined the capacity of Legionella pneumophila membranes to induce cell-mediated immune responses and protective immunity in a guinea pig model of Legionnaires' disease. Guinea pigs immunized by aerosol with L. pneumophila membranes developed strong cell-mediated immune responses to L. pneumophila membranes as demonstrated by cutaneous delayed-type hypersensitivity and in vitro splenic lymphocyte proliferation. Guinea pigs immunized by aerosol or by subcutaneous inoculation with L. pneumophila membranes developed strong protective immunity against lethal aerosol challenge with L. pneumophila. Overall, in six independent experiments, 39 of 49 (80%) guinea pigs immunized with L. pneumophila membranes survived challenge compared with 2 of 40 (5%) sham-immunized controls (P = 2 x 10(-13). In contrast, guinea pigs immunized by aerosol with formalin-killed L. pneumophila did not develop either a strong cell-mediated immune response to L. pneumophila antigens or protective immunity to lethal aerosol challenge. The capacity of L. pneumophila membranes to induce protective immunity was independent of the major secretory protein of L. pneumophila, which we previously demonstrated is an immunoprotective molecule. Purified L. pneumophila membranes did not contain detectable major secretory protein (MSP) on immunoblots; immunization of guinea pigs with L. pneumophila membranes did not induce anti-MSP antibody; and guinea pigs developed comparable protective immunity after immunization with membranes from either an L. pneumophila strain that secretes the major secretory protein or an isogenic mutant that does not. This study demonstrates that (a) immunization with L. pneumophila membranes but not formalin-killed L. pneumophila induces strong cell-mediated immune responses and protective immunity, (b) L. pneumophila membranes contain immunoprotective molecules distinct from the major secretory protein of L. pneumophila, and (c) L. pneumophila membranes have potential as

  10. Leptospiral Proteins Recognized during the Humoral Immune Response to Leptospirosis in Humans

    PubMed Central

    Guerreiro, Hygia; Croda, Júlio; Flannery, Brendan; Mazel, Mary; Matsunaga, James; Reis, Mitermayer Galvão; Levett, Paul N.; Ko, Albert I.; Haake, David A.

    2001-01-01

    Leptospirosis is an emerging zoonosis caused by pathogenic spirochetes belonging to the genus Leptospira. An understanding of leptospiral protein expression regulation is needed to develop new immunoprotective and serodiagnostic strategies. We used the humoral immune response during human leptospirosis as a reporter of protein antigens expressed during infection. Qualitative and quantitative immunoblot analysis was performed using sera from 105 patients from Brazil and Barbados. Sera from patients with other diseases and healthy individuals were evaluated as controls. Seven proteins, p76, p62, p48, p45, p41, p37, and p32, were identified as targets of the humoral response during natural infection. In both acute and convalescent phases of illness, antibodies to lipopolysaccharide were predominantly immunoglobulin M (IgM) while antibodies to proteins were exclusively IgG. Anti-p32 reactivity had the greatest sensitivity and specificity: positive reactions were observed in 37 and 84% of acute- and convalescent-phase sera, respectively, while only 5% of community control individuals demonstrated positive reactions. Six immunodominant antigens were expressed by all pathogenic leptospiral strains tested; only p37 was inconsistently expressed. Two-dimensional immunoblots identified four of the seven infection-associated antigens as being previously characterized proteins: LipL32 (the major outer membrane lipoprotein), LipL41 (a surface-exposed outer membrane lipoprotein), and heat shock proteins GroEL and DnaK. Fractionation studies demonstrated LipL32 and LipL41 reactivity in the outer membrane fraction and GroEL and DnaK in the cytoplasmic fraction, while p37 appeared to be a soluble periplasmic protein. Most of the other immunodominant proteins, including p48 and p45, were localized to the inner membrane. These findings indicate that leptospiral proteins recognized during natural infection are potentially useful for serodiagnosis and may serve as targets for vaccine

  11. Leptospiral proteins recognized during the humoral immune response to leptospirosis in humans.

    PubMed

    Guerreiro, H; Croda, J; Flannery, B; Mazel, M; Matsunaga, J; Galvão Reis, M; Levett, P N; Ko, A I; Haake, D A

    2001-08-01

    Leptospirosis is an emerging zoonosis caused by pathogenic spirochetes belonging to the genus Leptospira. An understanding of leptospiral protein expression regulation is needed to develop new immunoprotective and serodiagnostic strategies. We used the humoral immune response during human leptospirosis as a reporter of protein antigens expressed during infection. Qualitative and quantitative immunoblot analysis was performed using sera from 105 patients from Brazil and Barbados. Sera from patients with other diseases and healthy individuals were evaluated as controls. Seven proteins, p76, p62, p48, p45, p41, p37, and p32, were identified as targets of the humoral response during natural infection. In both acute and convalescent phases of illness, antibodies to lipopolysaccharide were predominantly immunoglobulin M (IgM) while antibodies to proteins were exclusively IgG. Anti-p32 reactivity had the greatest sensitivity and specificity: positive reactions were observed in 37 and 84% of acute- and convalescent-phase sera, respectively, while only 5% of community control individuals demonstrated positive reactions. Six immunodominant antigens were expressed by all pathogenic leptospiral strains tested; only p37 was inconsistently expressed. Two-dimensional immunoblots identified four of the seven infection-associated antigens as being previously characterized proteins: LipL32 (the major outer membrane lipoprotein), LipL41 (a surface-exposed outer membrane lipoprotein), and heat shock proteins GroEL and DnaK. Fractionation studies demonstrated LipL32 and LipL41 reactivity in the outer membrane fraction and GroEL and DnaK in the cytoplasmic fraction, while p37 appeared to be a soluble periplasmic protein. Most of the other immunodominant proteins, including p48 and p45, were localized to the inner membrane. These findings indicate that leptospiral proteins recognized during natural infection are potentially useful for serodiagnosis and may serve as targets for vaccine

  12. Immunization with Potato Plants Expressing VP60 Protein Protects against Rabbit Hemorrhagic Disease Virus

    PubMed Central

    Castañón, S.; Marín, M. S.; Martín-Alonso, J. M.; Boga, J. A.; Casais, R.; Humara, J. M.; Ordás, R. J.; Parra, F.

    1999-01-01

    The major structural protein VP60 of rabbit hemorrhagic disease virus (RHDV) has been produced in transgenic potato plants under the control of a cauliflower mosaic virus 35S promoter or a modified 35S promoter that included two copies of a strong transcriptional enhancer. Both types of promoters allowed the production of specific mRNAs and detectable levels of recombinant VP60, which were higher for the constructs carrying the modified 35S promoter. Rabbits immunized with leaf extracts from plants carrying this modified 35S promoter showed high anti-VP60 antibody titers and were fully protected against the hemorrhagic disease. PMID:10196345

  13. A cell wall protein-based vaccine candidate induce protective immune response against Sporothrix schenckii infection.

    PubMed

    Portuondo, Deivys Leandro; Batista-Duharte, Alexander; Ferreira, Lucas Souza; Martínez, Damiana Téllez; Polesi, Marisa Campos; Duarte, Roberta Aparecida; de Paula E Silva, Ana Carolina Alves; Marcos, Caroline Maria; Almeida, Ana Marisa Fusco de; Carlos, Iracilda Zeppone

    2016-02-01

    Sporotrichosis is a subcutaneous mycosis caused by several closely related thermo-dimorphic fungi of the Sporothrix schenckii species complex, affecting humans and other mammals. In the last few years, new strategies have been proposed for controlling sporotrichosis owning to concerns about its growing incidence in humans, cats, and dogs in Brazil, as well as the toxicity and limited efficacy of conventional antifungal drugs. In this study, we assessed the immunogenicity and protective properties of two aluminum hydroxide (AH)-adsorbed S. schenckii cell wall protein (ssCWP)-based vaccine formulations in a mouse model of systemic S. schenckii infection. Fractioning by SDS-PAGE revealed nine protein bands, two of which were functionally characterized: a 44kDa peptide hydrolase and a 47kDa enolase, which was predicted to be an adhesin. Sera from immunized mice recognized the 47kDa enolase and another unidentified 71kDa protein, whereas serum from S. schenckii-infected mice recognized both these proteins plus another unidentified 9.4kDa protein. Furthermore, opsonization with the anti-ssCWP sera led to markedly increased phagocytosis and was able to strongly inhibit the fungus' adhesion to fibroblasts. Immunization with the higher-dose AH-adjuvanted formulation led to increased ex vivo release of IL-12, IFN-γ, IL-4, and IL-17, whereas only IL-12 and IFN-γ were induced by the higher-dose non-adjuvanted formulation. Lastly, passive transference of the higher-dose AH-adjuvanted formulation's anti-ssCWP serum was able to afford in vivo protection in a subsequent challenge with S. schenckii, becoming a viable vaccine candidate for further testing.

  14. Effects of inadequate maternal dietary protein:carbohydrate ratios during pregnancy on offspring immunity in pigs

    PubMed Central

    2012-01-01

    Background Inadequate nutrition in utero may retard foetal growth and alter physiological development of offspring. This study investigated the effects of low and high protein diets fed to primiparous German Landrace sows throughout pregnancy on the immune function of their offspring at different ages. Sows were fed diets with adequate (AP, 12.1%; n = 13), low (LP, 6.5%; n = 15), or high (HP, 30%; n = 14) protein content, made isoenergetic by varying carbohydrate levels. Cortisol, total protein and immunoglobulin (IgG, IgM, IgA) concentrations were measured in the blood of sows over the course of pregnancy. Cortisol, total protein, immunoglobulins, lymphocyte proliferation, immune cell counts, and cytokines were assessed in the blood of offspring at baseline and under challenging conditions (weaning; lipopolysaccharide (LPS) administration). Results In sows, the LP diet increased cortisol (P < 0.05) and decreased protein levels (P < 0.01) at the end of pregnancy. Immunoglobulin concentrations were decreased in LP (IgA) and HP piglets (IgG, IgM and IgA) on the first day of life (P < 0.05), whereas the number of lymphocytes and mitogen-induced lymphocyte proliferation of the piglets were unaffected by the maternal diet. Mortality during the suckling period was higher in LP piglets compared with AP and HP offspring (P < 0.01). Furthermore, LP piglets showed an elevated cortisol response to weaning, and in HP piglets, the CD4+ cell percentage and the CD4+/CD8+ ratio increased after weaning (P < 0.05). The lipopolysaccharide-induced rise of IL-6 was higher in LP (P = 0.09) and HP (P < 0.01) compared with AP piglets, and LP piglets displayed higher IL-10 levels than AP piglets (P < 0.05). Conclusions Our results indicate that both low and high protein:carbohydrate ratios in the diet of pregnant sows can induce short-term as well as long-lasting effects on immune competence in piglets that may have serious consequences for host

  15. Extraordinary Diversity of Immune Response Proteins among Sea Urchins: Nickel-Isolated Sp185/333 Proteins Show Broad Variations in Size and Charge

    PubMed Central

    Sherman, Lauren S.; Schrankel, Catherine S.; Brown, Kristy J.; Smith, L. Courtney

    2015-01-01

    Effective protection against pathogens requires the host to produce a wide range of immune effector proteins. The Sp185/333 gene family, which is expressed by the California purple sea urchin Strongylocentrotus purpuratus in response to bacterial infection, encodes a highly diverse repertoire of anti-pathogen proteins. A subset of these proteins can be isolated by affinity to metal ions based on multiple histidines, resulting in one to four bands of unique molecular weight on standard Western blots, which vary depending on the individual sea urchin. Two dimensional gel electrophoresis (2DE) of nickel-isolated protein samples followed by Western blot was employed to detect nickel-isolated Sp185/333 (Ni-Sp185/333) proteins and to evaluate protein diversity in animals before and after immune challenge with marine bacteria. Ni-Sp185/333 proteins of the same molecular weight on standard Western blots appear as a broad complex of variants that differ in pI on 2DE Western blots. The Ni-Sp185/333 protein repertoire is variable among animals, and shows a variety of changes among individual sea urchins in response to immune challenges with both the same and different species of bacteria. The extraordinary diversity of the Ni-Sp185/333 proteins may provide significant anti-pathogen capabilities for sea urchins that survive solely on innate immunity. PMID:26406912

  16. SIGNR3-dependent immune regulation by Lactobacillus acidophilus surface layer protein A in colitis

    PubMed Central

    Lightfoot, Yaíma L; Selle, Kurt; Yang, Tao; Goh, Yong Jun; Sahay, Bikash; Zadeh, Mojgan; Owen, Jennifer L; Colliou, Natacha; Li, Eric; Johannssen, Timo; Lepenies, Bernd; Klaenhammer, Todd R; Mohamadzadeh, Mansour

    2015-01-01

    Intestinal immune regulatory signals govern gut homeostasis. Breakdown of such regulatory mechanisms may result in inflammatory bowel disease (IBD). Lactobacillus acidophilus contains unique surface layer proteins (Slps), including SlpA, SlpB, SlpX, and lipoteichoic acid (LTA), which interact with pattern recognition receptors to mobilize immune responses. Here, to elucidate the role of SlpA in protective immune regulation, the NCK2187 strain, which solely expresses SlpA, was generated. NCK2187 and its purified SlpA bind to the C-type lectin SIGNR3 to exert regulatory signals that result in mitigation of colitis, maintenance of healthy gastrointestinal microbiota, and protected gut mucosal barrier function. However, such protection was not observed in Signr3−/− mice, suggesting that the SlpA/SIGNR3 interaction plays a key regulatory role in colitis. Our work presents critical insights into SlpA/SIGNR3-induced responses that are integral to the potential development of novel biological therapies for autoinflammatory diseases, including IBD. PMID:25666591

  17. In Vivo Visualization of Tumor Antigen-containing Microparticles Generated in Fluorescent-protein-elicited Immunity

    PubMed Central

    Yang, Fei; Liu, Shun; Liu, Xiuli; Liu, Lei; Luo, Meijie; Qi, Shuhong; Xu, Guoqiang; Qiao, Sha; Lv, Xiaohua; Li, Xiangning; Fu, Ling; Luo, Qingming; Zhang, Zhihong

    2016-01-01

    In vivo optical spatio-temporal imaging of the tumor microenvironment is useful to explain how tumor immunotherapies work. However, the lack of fluorescent antigens with strong immunogenicity makes it difficult to study the dynamics of how tumors are eliminated by any given immune response. Here, we develop an effective fluorescent model antigen based on the tetrameric far-red fluorescent protein KatushkaS158A (tfRFP), which elicits both humoral and cellular immunity. We use this fluorescent antigen to visualize the dynamic behavior of immunocytes as they attack and selectively eliminate tfRFP-expressing tumors in vivo; swarms of immunocytes rush toward tumors with high motility, clusters of immunocytes form quickly, and numerous antigen-antibody complexes in the form of tfRFP+ microparticles are generated in the tumor areas and ingested by macrophages in the tumor microenvironment. Therefore, tfRFP, as both a model antigen and fluorescent reporter, is a useful tool to visualize specific immune responses in vivo. PMID:27375792

  18. The bacterial DNA repair protein Mfd confers resistance to the host nitrogen immune response.

    PubMed

    Guillemet, Elisabeth; Leréec, Alain; Tran, Seav-Ly; Royer, Corinne; Barbosa, Isabelle; Sansonetti, Philippe; Lereclus, Didier; Ramarao, Nalini

    2016-01-01

    Production of reactive nitrogen species (NO) is a key step in the immune response following infections. NO induces lesions to bacterial DNA, thus limiting bacterial growth within hosts. Using two pathogenic bacteria, Bacillus cereus and Shigella flexneri, we show that the DNA-repair protein Mfd (Mutation-Frequency-Decline) is required for bacterial resistance to the host-NO-response. In both species, a mutant deficient for mfd does not survive to NO, produced in vitro or by phagocytic cells. In vivo, the ∆mfd mutant is avirulent and unable to survive the NO-stress. Moreover, NO induces DNA-double-strand-breaks and point mutations in the Δmfd mutant. In overall, these observations demonstrate that NO damages bacterial DNA and that Mfd is required to maintain bacterial genomic integrity. This unexpected discovery reveals that Mfd, a typical housekeeping gene, turns out to be a true virulence factor allowing survival and growth of the pathogen in its host, due to its capacity to protect the bacterium against NO, a key molecule of the innate immune defense. As Mfd is widely conserved in the bacterial kingdom, these data highlight a mechanism that may be used by a large spectrum of bacteria to overcome the host immune response and especially the mutagenic properties of NO.

  19. Staphylococcus aureus infection induces protein A–mediated immune evasion in humans

    PubMed Central

    Pauli, Noel T.; Kim, Hwan Keun; Falugi, Fabiana; Huang, Min; Dulac, John; Henry Dunand, Carole; Zheng, Nai-Ying; Kaur, Kaval; Andrews, Sarah F.; Huang, Yunping; DeDent, Andrea; Frank, Karen M.; Charnot-Katsikas, Angella; Schneewind, Olaf

    2014-01-01

    Staphylococcus aureus bacterial infection commonly results in chronic or recurrent disease, suggesting that humoral memory responses are hampered. Understanding how S. aureus subverts the immune response is critical for the rescue of host natural humoral immunity and vaccine development. S. aureus expresses the virulence factor Protein A (SpA) on all clinical isolates, and SpA has been shown in mice to expand and ablate variable heavy 3 (VH3) idiotype B cells. The effects of SpA during natural infection, however, have not been addressed. Acutely activated B cells, or plasmablasts (PBs), were analyzed to dissect the ongoing immune response to infection through the production of monoclonal antibodies (mAbs). The B cells that were activated by infection had a highly limited response. When screened against multiple S. aureus antigens, only high-affinity binding to SpA was observed. Consistently, PBs underwent affinity maturation, but their B cell receptors demonstrated significant bias toward the VH3 idiotype. These data suggest that the superantigenic activity of SpA leads to immunodominance, limiting host responses to other S. aureus virulence factors that would be necessary for protection and memory formation. PMID:25348152

  20. Outer Surface Protein A Protects Lyme Disease Spirochetes from Acquired Host Immunity in the Tick Vector▿

    PubMed Central

    Battisti, James M.; Bono, James L.; Rosa, Patricia A.; Schrumpf, Merry E.; Schwan, Tom G.; Policastro, Paul F.

    2008-01-01

    The Lyme disease spirochete Borrelia burgdorferi alters the expression of outer surface protein (osp) genes as the bacterium cycles between ticks and mammals. OspA is produced as borreliae enter the tick vector and remains a major surface antigen during midgut colonization. To elucidate the role of OspA in the vector, we created an insertional deletion of ospA in strain B31-A3. The ospA mutant infects mice when it is injected intradermally and is acquired by larval ticks fed on these mice, where it persists through the molt to the nymph stage. Bacterial survival rates in artificially infected tick larvae fed on naïve mice were compared with those in the vector fed on immune mice. The ospA mutant proliferates in larvae if it is exposed to blood from naïve mice, but it declines in density after larval feeding if the blood is from immune mice. When uninfected larvae are fed on B-cell-deficient mice infected with the ospA mutant, larvae show borrelial densities and persistence that are significantly greater than those fed on infected, immunocompetent mice. We conclude that OspA serves a critical antibody-shielding role during vector blood meal uptake from immune hosts and is not required for persistence in the tick vector. PMID:18779341

  1. The bacterial DNA repair protein Mfd confers resistance to the host nitrogen immune response.

    PubMed

    Guillemet, Elisabeth; Leréec, Alain; Tran, Seav-Ly; Royer, Corinne; Barbosa, Isabelle; Sansonetti, Philippe; Lereclus, Didier; Ramarao, Nalini

    2016-01-01

    Production of reactive nitrogen species (NO) is a key step in the immune response following infections. NO induces lesions to bacterial DNA, thus limiting bacterial growth within hosts. Using two pathogenic bacteria, Bacillus cereus and Shigella flexneri, we show that the DNA-repair protein Mfd (Mutation-Frequency-Decline) is required for bacterial resistance to the host-NO-response. In both species, a mutant deficient for mfd does not survive to NO, produced in vitro or by phagocytic cells. In vivo, the ∆mfd mutant is avirulent and unable to survive the NO-stress. Moreover, NO induces DNA-double-strand-breaks and point mutations in the Δmfd mutant. In overall, these observations demonstrate that NO damages bacterial DNA and that Mfd is required to maintain bacterial genomic integrity. This unexpected discovery reveals that Mfd, a typical housekeeping gene, turns out to be a true virulence factor allowing survival and growth of the pathogen in its host, due to its capacity to protect the bacterium against NO, a key molecule of the innate immune defense. As Mfd is widely conserved in the bacterial kingdom, these data highlight a mechanism that may be used by a large spectrum of bacteria to overcome the host immune response and especially the mutagenic properties of NO. PMID:27435260

  2. The bacterial DNA repair protein Mfd confers resistance to the host nitrogen immune response

    PubMed Central

    Guillemet, Elisabeth; Leréec, Alain; Tran, Seav-Ly; Royer, Corinne; Barbosa, Isabelle; Sansonetti, Philippe; Lereclus, Didier; Ramarao, Nalini

    2016-01-01

    Production of reactive nitrogen species (NO) is a key step in the immune response following infections. NO induces lesions to bacterial DNA, thus limiting bacterial growth within hosts. Using two pathogenic bacteria, Bacillus cereus and Shigella flexneri, we show that the DNA-repair protein Mfd (Mutation-Frequency-Decline) is required for bacterial resistance to the host-NO-response. In both species, a mutant deficient for mfd does not survive to NO, produced in vitro or by phagocytic cells. In vivo, the ∆mfd mutant is avirulent and unable to survive the NO-stress. Moreover, NO induces DNA-double-strand-breaks and point mutations in the Δmfd mutant. In overall, these observations demonstrate that NO damages bacterial DNA and that Mfd is required to maintain bacterial genomic integrity. This unexpected discovery reveals that Mfd, a typical housekeeping gene, turns out to be a true virulence factor allowing survival and growth of the pathogen in its host, due to its capacity to protect the bacterium against NO, a key molecule of the innate immune defense. As Mfd is widely conserved in the bacterial kingdom, these data highlight a mechanism that may be used by a large spectrum of bacteria to overcome the host immune response and especially the mutagenic properties of NO. PMID:27435260

  3. Immune-checkpoint proteins VISTA and PD-1 nonredundantly regulate murine T-cell responses.

    PubMed

    Liu, Jun; Yuan, Ying; Chen, Wenna; Putra, Juan; Suriawinata, Arief A; Schenk, Austin D; Miller, Halli E; Guleria, Indira; Barth, Richard J; Huang, Yina H; Wang, Li

    2015-05-26

    V-domain immunoglobulin suppressor of T-cell activation (VISTA) is a negative immune-checkpoint protein that suppresses T-cell responses. To determine whether VISTA synergizes with another immune-checkpoint, programmed death 1 (PD-1), this study characterizes the immune responses in VISTA-deficient, PD-1-deficient (KO) mice and VISTA/PD-1 double KO mice. Chronic inflammation and spontaneous activation of T cells were observed in both single KO mice, demonstrating their nonredundancy. However, the VISTA/PD-1 double KO mice exhibited significantly higher levels of these phenotypes than the single KO mice. When bred onto the 2D2 T-cell receptor transgenic mice, which are predisposed to development of inflammatory autoimmune disease in the CNS, the level of disease penetrance was significantly enhanced in the double KO mice compared with in the single KO mice. Consistently, the magnitude of T-cell response toward foreign antigens was synergistically higher in the VISTA/PD-1 double KO mice. A combinatorial blockade using monoclonal antibodies specific for VISTA and PD-L1 achieved optimal tumor-clearing therapeutic efficacy. In conclusion, our study demonstrates the nonredundant role of VISTA that is distinct from the PD-1/PD-L1 pathway in controlling T-cell activation. These findings provide the rationale to concurrently target VISTA and PD-1 pathways for treating T-cell-regulated diseases such as cancer.

  4. Transient expression of protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus inhibits insect cellular immune responses

    NASA Astrophysics Data System (ADS)

    Ibrahim, Ahmed M. A.; Kim, Yonggyun

    2008-01-01

    Several immunosuppressive factors are associated with parasitism of an endoparasitoid wasp, Cotesia plutellae, on the diamondback moth, Plutella xylostella. C. plutellae bracovirus (CpBV) encodes a large number of putative protein tyrosine phosphatases (PTPs), which may play a role in inhibiting host cellular immunity. To address this inhibitory hypothesis of CpBV-PTPs, we performed transient expression of individual CpBV-PTPs in hemocytes of the beet armyworm, Spodoptera exigua, and analyzed their cellular immune responses. Two different forms of CpBV-PTPs were chosen and cloned into a eukaryotic expression vector under the control of the p10 promoter of baculovirus: one with the normal cysteine active site (CpBV-PTP1) and the other with a mutated active site (CpBV-PTP5). The hemocytes transfected with CpBV-PTP1 significantly increased in PTP activity compared to control hemocytes, but those with CpBV-PTP5 exhibited a significant decrease in the PTP activity. All transfected hemocytes exhibited a significant reduction in both cell spreading and encapsulation activities compared to control hemocytes. Co-transfection of CpBV-PTP1 together with its double-stranded RNA reduced the messenger RNA (mRNA) level of CpBV-PTP1 and resulted in recovery of both hemocyte behaviors. This is the first report demonstrating that the polydnaviral PTPs can manipulate PTP activity of the hemocytes to interrupt cellular immune responses.

  5. Protective Immunity and Reduced Renal Colonization Induced by Vaccines Containing Recombinant Leptospira interrogans Outer Membrane Proteins and Flagellin Adjuvant.

    PubMed

    Monaris, D; Sbrogio-Almeida, M E; Dib, C C; Canhamero, T A; Souza, G O; Vasconcellos, S A; Ferreira, L C S; Abreu, P A E

    2015-08-01

    Leptospirosis is a global zoonotic disease caused by different Leptospira species, such as Leptospira interrogans, that colonize the renal tubules of wild and domestic animals. Thus far, attempts to develop effective leptospirosis vaccines, both for humans and animals, have failed to induce immune responses capable of conferring protection and simultaneously preventing renal colonization. In this study, we evaluated the protective immunity induced by subunit vaccines containing seven different recombinant Leptospira interrogans outer membrane proteins, including the carboxy-terminal portion of the immunoglobulinlike protein A (LigA(C)) and six novel antigens, combined with aluminum hydroxide (alum) or Salmonella flagellin (FliC) as adjuvants. Hamsters vaccinated with the different formulations elicited high antigen-specific antibody titers. Immunization with LigA(C), either with alum or flagellin, conferred protective immunity but did not prevent renal colonization. Similarly, animals immunized with LigA(C) or LigA(C) coadministered with six leptospiral proteins with alum adjuvant conferred protection but did not reduce renal colonization. In contrast, immunizing animals with the pool of seven antigens in combination with flagellin conferred protection and significantly reduced renal colonization by the pathogen. The present study emphasizes the relevance of antigen composition and added adjuvant in the efficacy of antileptospirosis subunit vaccines and shows the complex relationship between immune responses and renal colonization by the pathogen. PMID:26108285

  6. Protective Immunity and Reduced Renal Colonization Induced by Vaccines Containing Recombinant Leptospira interrogans Outer Membrane Proteins and Flagellin Adjuvant

    PubMed Central

    Monaris, D.; Sbrogio-Almeida, M. E.; Dib, C. C.; Canhamero, T. A.; Souza, G. O.; Vasconcellos, S. A.; Ferreira, L. C. S.

    2015-01-01

    Leptospirosis is a global zoonotic disease caused by different Leptospira species, such as Leptospira interrogans, that colonize the renal tubules of wild and domestic animals. Thus far, attempts to develop effective leptospirosis vaccines, both for humans and animals, have failed to induce immune responses capable of conferring protection and simultaneously preventing renal colonization. In this study, we evaluated the protective immunity induced by subunit vaccines containing seven different recombinant Leptospira interrogans outer membrane proteins, including the carboxy-terminal portion of the immunoglobulinlike protein A (LigAC) and six novel antigens, combined with aluminum hydroxide (alum) or Salmonella flagellin (FliC) as adjuvants. Hamsters vaccinated with the different formulations elicited high antigen-specific antibody titers. Immunization with LigAC, either with alum or flagellin, conferred protective immunity but did not prevent renal colonization. Similarly, animals immunized with LigAC or LigAC coadministered with six leptospiral proteins with alum adjuvant conferred protection but did not reduce renal colonization. In contrast, immunizing animals with the pool of seven antigens in combination with flagellin conferred protection and significantly reduced renal colonization by the pathogen. The present study emphasizes the relevance of antigen composition and added adjuvant in the efficacy of antileptospirosis subunit vaccines and shows the complex relationship between immune responses and renal colonization by the pathogen. PMID:26108285

  7. Association of human immune response to Aedes aegypti salivary proteins with dengue disease severity.

    PubMed

    Machain-Williams, C; Mammen, M P; Zeidner, N S; Beaty, B J; Prenni, J E; Nisalak, A; Blair, C D

    2012-01-01

    Dengue viruses (DENV; family Flaviviridae, genus Flavivirus) are transmitted by Aedes aegypti mosquitoes and can cause dengue fever (DF), a relatively benign disease, or more severe dengue haemorrhagic fever (DHF). Arthropod saliva contains proteins delivered into the bite wound that can modulate the host haemostatic and immune responses to facilitate the intake of a blood meal. The potential effects on DENV infection of previous exposure to Ae. aegypti salivary proteins have not been investigated. We collected Ae. aegypti saliva, concentrated the proteins and fractionated them by nondenaturing polyacrylamide gel electrophoresis (PAGE). By the use of immunoblots, we analysed reactivity with the mosquito salivary proteins (MSP) of sera from 96 Thai children diagnosed with secondary DENV infections leading either to DF or DHF, or with no DENV infection, and found that different proportions of each patient group had serum antibodies reactive to specific Ae. aegypti salivary proteins. Our results suggest that prior exposure to MSP might play a role in the outcome of DENV infection in humans.

  8. Integration of decoy domains derived from protein targets of pathogen effectors into plant immune receptors is widespread.

    PubMed

    Kroj, Thomas; Chanclud, Emilie; Michel-Romiti, Corinne; Grand, Xavier; Morel, Jean-Benoit

    2016-04-01

    Plant immune receptors of the class of nucleotide-binding and leucine-rich repeat domain (NLR) proteins can contain additional domains besides canonical NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 (NB-ARC)) and leucine-rich repeat (LRR) domains. Recent research suggests that these additional domains act as integrated decoys recognizing effectors from pathogens. Proteins homologous to integrated decoys are suspected to be effector targets and involved in disease or resistance. Here, we scrutinized 31 entire plant genomes to identify putative integrated decoy domains in NLR proteins using the Interpro search. The involvement of the Zinc Finger-BED type (ZBED) protein containing a putative decoy domain, called BED, in rice (Oryza sativa) resistance was investigated by evaluating susceptibility to the blast fungus Magnaporthe oryzae in rice over-expression and knock-out mutants. This analysis showed that all plants tested had integrated various atypical protein domains into their NLR proteins (on average 3.5% of all NLR proteins). We also demonstrated that modifying the expression of the ZBED gene modified disease susceptibility. This study suggests that integration of decoy domains in NLR immune receptors is widespread and frequent in plants. The integrated decoy model is therefore a powerful concept to identify new proteins involved in disease resistance. Further in-depth examination of additional domains in NLR proteins promises to unravel many new proteins of the plant immune system. PMID:26848538

  9. Immune Protection of Retroviral Vectors Upon Molecular Painting with the Complement Regulatory Protein CD59.

    PubMed

    Heider, Susanne; Kleinberger, Sandra; Kochan, Feliks; Dangerfield, John A; Metzner, Christoph

    2016-07-01

    Glycosylphosphatidylinositol anchoring is a type of post-translational modification that allows proteins to be presented on the exterior side of the cell membrane. Purified glycosylphosphatidylinositol-anchored protein can spontaneously re-insert into lipid bilayer membranes in a process termed Molecular Painting. Here, we demonstrate the possibility of inserting purified, recombinant CD59 into virus particles produced from a murine retroviral producer cell line. CD59 is a regulator of the complement system that helps protect healthy cells from the lytic activity of the complement cascade. In this study, we could show that Molecular Painting confers protection from complement activity upon murine retroviral vector particles. Indeed, increased infectivity of CD59-modified virus particles was observed upon challenge with human serum, indicating that Molecular Painting is suitable for modulating the immune system in gene therapy or vaccination applications. PMID:27170144

  10. Vaccines displaying mycobacterial proteins on biopolyester beads stimulate cellular immunity and induce protection against tuberculosis.

    PubMed

    Parlane, Natalie A; Grage, Katrin; Mifune, Jun; Basaraba, Randall J; Wedlock, D Neil; Rehm, Bernd H A; Buddle, Bryce M

    2012-01-01

    New improved vaccines are needed for control of both bovine and human tuberculosis. Tuberculosis protein vaccines have advantages with regard to safety and ease of manufacture, but efficacy against tuberculosis has been difficult to achieve. Protective cellular immune responses can be preferentially induced when antigens are displayed on small particles. In this study, Escherichia coli and Lactococcus lactis were engineered to produce spherical polyhydroxybutyrate (PHB) inclusions which displayed a fusion protein of Mycobacterium tuberculosis, antigen 85A (Ag85A)-early secreted antigenic target 6-kDa protein (ESAT-6). L. lactis was chosen as a possible production host due its extensive use in the food industry and reduced risk of lipopolysaccharide contamination. Mice were vaccinated with PHB bead vaccines with or without displaying Ag85A-ESAT-6, recombinant Ag85A-ESAT-6, or M. bovis BCG. Separate groups of mice were used to measure immune responses and assess protection against an aerosol M. bovis challenge. Increased amounts of antigen-specific gamma interferon, interleukin-17A (IL-17A), IL-6, and tumor necrosis factor alpha were produced from splenocytes postvaccination, but no or minimal IL-4, IL-5, or IL-10 was produced, indicating Th1- and Th17-biased T cell responses. Decreased lung bacterial counts and less extensive foci of inflammation were observed in lungs of mice receiving BCG or PHB bead vaccines displaying Ag85A-ESAT-6 produced in either E. coli or L. lactis compared to those observed in the lungs of phosphate-buffered saline-treated control mice. No differences between those receiving wild-type PHB beads and those receiving recombinant Ag85A-ESAT-6 were observed. This versatile particulate vaccine delivery system incorporates a relatively simple production process using safe bacteria, and the results show that it is an effective delivery system for a tuberculosis protein vaccine. PMID:22072720

  11. PBL13 Is a Serine/Threonine Protein Kinase That Negatively Regulates Arabidopsis Immune Responses.

    PubMed

    Lin, Zuh-Jyh Daniel; Liebrand, Thomas W H; Yadeta, Koste A; Coaker, Gitta

    2015-12-01

    Receptor-like cytoplasmic kinases (RLCKs) are a subset of plant receptor-like kinases lacking both extracellular and transmembrane domains. Some of the 46 members in the Arabidopsis (Arabidopsis thaliana) RLCK subfamily VII have been linked to plant innate immunity; however, most remain uncharacterized. Thus, multiple subfamily VII members are expected to be involved in plant immune signaling. Here, we investigate the role of AvrPphB SUSCEPTIBLE1-LIKE13 (PBL13), a subfamily VII RLCK with unique domain architecture. Unlike other characterized RLCKs, PBL13 transfer DNA insertion lines exhibit enhanced disease resistance after inoculation with virulent Pseudomonas syringae. The pbl13-2 knockout also exhibits elevated basal-level expression of the PATHOGENESIS-RELATED GENE1 defense marker gene, enhanced reactive oxygen species (ROS) burst in response to perception of bacterial microbial patterns, and accelerated flagellin-induced activation of mitogen-activated protein kinases. Recombinant PBL13 is an active kinase, and its primary autophosphorylated sites map to a 15-amino acid repeat motif unique to PBL13. Complementation of pbl13-2 with PBL13-3xFLAG converts the enhanced resistance and elevated ROS phenotypes back to wild-type levels. In contrast, kinase-dead PBL13(K111A)-3xFLAG was unable to rescue pbl13-2 disease phenotypes. Consistent with the enhanced ROS burst in the pbl13-2 knockout, PBL13 is able to associate with the nicotinamide adenine dinucleotide phosphate, reduced oxidase RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN D (RBOHD) by split-luciferase complementation assay, and this association is disrupted by flagellin treatment. We conclude that the PBL13 kinase negatively regulates plant innate immunity to pathogenic bacteria and can associate with RBOHD before pathogen perception. These data are consistent with the hypothesis that PBL13 acts to prevent inappropriate activation of defense responses in the absence of pathogen challenge.

  12. Role of protein tyrosine phosphatases in regulating the immune system: implications for chronic intestinal inflammation.

    PubMed

    Spalinger, Marianne R; McCole, Declan F; Rogler, Gerhard; Scharl, Michael

    2015-03-01

    Current hypothesis suggests that genetic, immunological, and bacterial factors contribute essentially to the pathogenesis of inflammatory bowel disease. Variations within the gene loci encoding protein tyrosine phosphatases (PTPs) have been associated with the onset of inflammatory bowel disease. PTPs modulate the activity of their substrates by dephosphorylation of tyrosine residues and are critical for the regulation of fundamental cellular signaling processes. Evidence emerges that expression levels of PTPN2, PTPN11, and PTPN22 are altered in actively inflamed intestinal tissue. PTPN2 seems to be critical for protecting intestinal epithelial barrier function, regulating innate and adaptive immune responses and finally for maintaining intestinal homeostasis. These observations have been confirmed in PTPN2 knockout mice in vivo. Those animals are clearly more susceptible to intestinal and systemic inflammation and feature alterations in innate and adaptive immune responses. PTPN22 controls inflammatory signaling in lymphocytes and mononuclear cells resulting in aberrant cytokine secretion pattern and autophagosome formation. PTPN22 deficiency in vivo results in more severe colitis demonstrating the relevance of PTPN22 for intestinal homeostasis in vivo. Of note, loss of PTPN22 promotes mitogen-activated protein kinase-induced cytokine secretion but limits secretion of nuclear factor κB-associated cytokines and autophagy in mononuclear cells. Loss of PTPN11 is also associated with increased colitis severity in vivo. In summary, dysfunction of those PTPs results in aberrant and uncontrolled immune responses that result in chronic inflammatory conditions. This way, it becomes more and more evident that dysfunction of PTPs displays an important factor in the pathogenesis of chronic intestinal inflammation, in particular inflammatory bowel disease.

  13. Potent stimulation of the innate immune system by a Leishmania brasiliensis recombinant protein.

    PubMed

    Borges, M M; Campos-Neto, A; Sleath, P; Grabstein, K H; Morrissey, P J; Skeiky, Y A; Reed, S G

    2001-09-01

    The interaction of the innate immune system with the microbial world involves primarily two sets of molecules generally known as microbial pattern recognition receptors and microbial pattern recognition molecules, respectively. Examples of the former are the Toll receptors present particularly in macrophages and dendritic cells. Conversely, the microbial pattern recognition molecules are conserved protist homopolymers, such as bacterial lipopolysaccharides, lipoteichoic acids, peptidoglycans, glucans, mannans, unmethylated bacterial DNA, and double-strand viral RNA. However, for protists that lack most of these molecules, such as protozoans, the innate immune system must have evolved receptors that recognize other groups of microbial molecules. Here we present evidence that a highly purified protein encoded by a Leishmania brasiliensis gene may be one such molecule. This recombinant leishmanial molecule, a homologue of eukaryotic ribosomal elongation and initiation factor 4a (LeIF), strongly stimulates spleen cells from severe combined immunodeficient (SCID) mice to produce interleukin-12 (IL-12), IL-18, and high levels of gamma interferon. In addition, LeIF potentiates the cytotoxic activity of the NK cells of these animals. Because LeIF is a conserved molecule and because SCID mice lack T and B lymphocytes but have a normal innate immune system (normal reticuloendothelial system and NK cells), these results suggest that proteins may also be included as microbial pattern recognition molecules. The nature of the receptor involved in this innate recognition is unknown. However, it is possible to exclude the Toll receptor Tlr4 as a putative LeIF receptor because the gene encoding this receptor is defective in C3H/HeJ mice, the mouse strain used in the present studies.

  14. Stability of plant immune-receptor resistance proteins is controlled by SKP1-CULLIN1-F-box (SCF)-mediated protein degradation

    PubMed Central

    Cheng, Yu Ti; Li, Yingzhong; Huang, Shuai; Huang, Yan; Dong, Xinnian; Zhang, Yuelin; Li, Xin

    2011-01-01

    The nucleotide-binding domain and leucine-rich repeats containing proteins (NLRs) serve as immune receptors in both plants and animals. Overaccumulation of NLRs often leads to autoimmune responses, suggesting that the levels of these immune receptors must be tightly controlled. However, the mechanism by which NLR protein levels are regulated is unknown. Here we report that the F-box protein CPR1 controls the stability of plant NLR resistance proteins. Loss-of-function mutations in CPR1 lead to higher accumulation of the NLR proteins SNC1 and RPS2, as well as autoactivation of immune responses. The autoimmune responses in cpr1 mutant plants can be largely suppressed by knocking out SNC1. Furthermore, CPR1 interacts with SNC1 and RPS2 in vivo, and overexpressing CPR1 results in reduced accumulation of SNC1 and RPS2, as well as suppression of immunity mediated by these two NLR proteins. Our data suggest that SKP1-CULLIN1-F-box (SCF) complex-mediated stability control of plant NLR proteins plays an important role in regulating their protein levels and preventing autoimmunity. PMID:21873230

  15. Ternary WD40 Repeat-Containing Protein Complexes: Evolution, Composition and Roles in Plant Immunity

    PubMed Central

    Miller, Jimi C.; Chezem, William R.; Clay, Nicole K.

    2016-01-01

    Plants, like mammals, rely on their innate immune system to perceive and discriminate among the majority of their microbial pathogens. Unlike mammals, plants respond to this molecular dialog by unleashing a complex chemical arsenal of defense metabolites to resist or evade pathogen infection. In basal or non-host resistance, plants utilize signal transduction pathways to detect “non-self,” “damaged-self,” and “altered-self”- associated molecular patterns and translate these “danger” signals into largely inducible chemical defenses. The WD40 repeat (WDR)-containing proteins Gβ and TTG1 are constituents of two independent ternary protein complexes functioning at opposite ends of a plant immune signaling pathway. They are also encoded by single-copy genes that are ubiquitous in higher plants, implying the limited diversity and functional conservation of their respective complexes. In this review, we summarize what is currently known about the evolutionary history of these WDR-containing ternary complexes, their repertoire and combinatorial interactions, and their downstream effectors and pathways in plant defense. PMID:26779203

  16. Pediatric Sepsis – Part V: Extracellular Heat Shock Proteins: Alarmins for the Host Immune System

    PubMed Central

    Giuliano, John S; Lahni, Patrick M.; Wong, Hector R.; Wheeler, Derek S.

    2012-01-01

    Heat shock proteins (HSPs) are molecular chaperones that facilitate the proper folding and assembly of nascent polypeptides and assist in the refolding and stabilization of damaged polypeptides. Through these largely intracellular functions, the HSPs maintain homeostasis and assure cell survival. However, a growing body of literature suggests that HSPs have important effects in the extracellular environment as well. Extracellular HSPs are released from damaged or stressed cells and appear to act as local “danger signals” that activate stress response programs in surrounding cells. Importantly, extracellular HSPs have been shown to activate the host innate and adaptive immune response. With this in mind, extracellular HSPs are commonly included in a growing list of a family of proteins known as danger-associated molecular patterns (DAMPs) or alarmins, which trigger an immune response to tissue injury, such as may occur with trauma, ischemia-reperfusion injury, oxidative stress, etc. Extracellular HSPs, including Hsp72 (HSPA), Hsp27 (HSPB1), Hsp90 (HSPC), Hsp60 (HSPD), and Chaperonin/Hsp10 (HSPE) are especially attractrive candidates for DAMPs or alarmins which may be particularly relevant in the pathophysiology of the sepsis syndrome. PMID:24765217

  17. A bacterial cysteine protease effector protein interferes with photosynthesis to suppress plant innate immune responses.

    PubMed

    Rodríguez-Herva, José J; González-Melendi, Pablo; Cuartas-Lanza, Raquel; Antúnez-Lamas, María; Río-Alvarez, Isabel; Li, Ziduo; López-Torrejón, Gema; Díaz, Isabel; Del Pozo, Juan C; Chakravarthy, Suma; Collmer, Alan; Rodríguez-Palenzuela, Pablo; López-Solanilla, Emilia

    2012-05-01

    The bacterial pathogen Pseudomonas syringae pv tomato DC3000 suppresses plant innate immunity with effector proteins injected by a type III secretion system (T3SS). The cysteine protease effector HopN1, which reduces the ability of DC3000 to elicit programmed cell death in non-host tobacco, was found to also suppress the production of defence-associated reactive oxygen species (ROS) and callose when delivered by Pseudomonas fluorescens heterologously expressing a P. syringae T3SS. Purified His(6) -tagged HopN1 was used to identify tomato PsbQ, a member of the oxygen evolving complex of photosystem II (PSII), as an interacting protein. HopN1 localized to chloroplasts and both degraded PsbQ and inhibited PSII activity in chloroplast preparations, whereas a HopN1(D299A) non-catalytic mutant lost these abilities. Gene silencing of NtPsbQ in tobacco compromised ROS production and programmed cell death by DC3000. Our data reveal PsbQ as a contributor to plant immunity responses and a target for pathogen suppression.

  18. Immune System Dysfunction and Autoimmune Disease in Mice Lacking Emk (Par-1) Protein Kinase

    PubMed Central

    Hurov, Jonathan B.; Stappenbeck, Thaddeus S.; Zmasek, Christian M.; White, Lynn S.; Ranganath, Sheila H.; Russell, John H.; Chan, Andrew C.; Murphy, Kenneth M.; Piwnica-Worms, Helen

    2001-01-01

    Emk is a serine/threonine protein kinase implicated in regulating polarity, cell cycle progression, and microtubule dynamics. To delineate the role of Emk in development and adult tissues, mice lacking Emk were generated by targeted gene disruption. Emk−/− mice displayed growth retardation and immune cell dysfunction. Although B- and T-cell development were normal, CD4+T cells lacking Emk exhibited a marked upregulation of the memory marker CD44/pgp-1 and produced more gamma interferon and interleukin-4 on stimulation through the T-cell receptor in vitro. In addition, B-cell responses to T-cell-dependent and -independent antigen challenge were altered in vivo. As Emk−/− animals aged, they developed splenomegaly, lymphadenopathy, membranoproliferative glomerulonephritis, and lymphocytic infiltrates in the lungs, parotid glands and kidneys. Taken together, these results demonstrate that the Emk protein kinase is essential for maintaining immune system homeostasis and that loss of Emk may contribute to autoimmune disease in mammals. PMID:11287624

  19. Anti-apoptotic seminal vesicle protein IV inhibits cell-mediated immunity.

    PubMed

    Fuggetta, M P; Lanzilli, G; Cottarelli, A; Ravagnan, G; Cartenì, M; De Maria, S; Metafora, B M; Metafora, V; Metafora, S

    2008-07-01

    The in vitro effect of seminal vesicle protein IV (SV-IV) on the cytotoxic activity of human natural or acquired cellular immunity has been investigated by standard immunological procedures, a (51)Cr-release cytotoxicity assay, and labeled-ligand binding experiments. The data obtained demonstrate that: (1) fluoresceinated or [(125)I]-labeled SV-IV binds specifically to the surface of human purified non-adherent mononuclear cells (NA-MNC); (2) SV-IV suppresses the cytotoxicity of natural killer (NK) cells against K562 target cells, that of IL-2-stimulated NK (LAK) cells against DAUDI target cells, and that of VEL antigen-sensitized cytotoxic T lymphocytes (CTLs) against VEL target cells; (3) treatment of K562 target cells alone with SV-IV decreases their susceptibility to NK-induced lysis. These findings indicate that the protein SV-IV has a marked in vitro inhibitory effect on NK, LAK and CTL cytotoxicity, providing a better understanding of its immune regulatory functions.

  20. Evaluation of humoral, mucosal, and cellular immune responses following co-immunization of HIV-1 Gag and Env proteins expressed by Newcastle disease virus.

    PubMed

    Khattar, Sunil K; Palaniyandi, Senthilkumar; Samal, Sweety; LaBranche, Celia C; Montefiori, David C; Zhu, Xiaoping; Samal, Siba K

    2015-01-01

    The combination of multiple HIV antigens in a vaccine can broaden antiviral immune responses. In this study, we used NDV vaccine strain LaSota to generate rNDV (rLaSota/optGag) expressing human codon optimized p55 Gag protein of HIV-1. We examined the effect of co-immunization of rLaSota/optGag with rNDVs expressing different forms of Env protein gp160, gp120, gp140L [a version of gp140 that lacked cytoplasmic tail and contained complete membrane-proximal external region (MPER)] and gp140S (a version of gp140 that lacked cytoplasmic tail and distal half of MPER) on magnitude and breadth of humoral, mucosal and cellular immune responses in guinea pigs and mice. Our results showed that inclusion of rLaSota/optGag with rNDVs expressing different forms of Env HIV Gag did not affect the Env-specific humoral and mucosal immune responses in guinea pigs and that the potent immune responses generated against Env persisted for at least 13 weeks post immunization. The highest Env-specific humoral and mucosal immune responses were observed with gp140S+optGag group. The neutralizing antibody responses against HIV strains BaL.26 and MN.3 induced by gp140S+optGag and gp160+optGag were higher than those elicited by other groups. Inclusion of Gag with gp160, gp140S and gp140L enhanced the level of Env-specific IFN-γ-producing CD8(+) T cells in mice. Inclusion of Gag with gp160 and gp140L also resulted in increased Env-specific CD4(+) T cells. The level of Gag-specific CD8(+) and CD4(+) T cells was also enhanced in mice immunized with Gag along with gp140S and gp120. These results indicate lack of antigen interference in a vaccine containing rNDVs expressing Env and Gag proteins.

  1. Effects of immunization with the rNfa1 protein on experimental Naegleria fowleri-PAM mice.

    PubMed

    Lee, Y J; Kim, J H; Sohn, H J; Lee, J; Jung, S Y; Chwae, Y J; Kim, K; Park, S; Shin, H J

    2011-07-01

    Free-living Naegleria fowleri causes primary amoebic meningoencephalitis (PAM) in humans and animals. To examine the effect of immunization with Nfa1 protein on experimental murine PAM because of N. fowleri, BALB/c mice were intra-peritoneally or intra-nasally immunized with a recombinant Nfa1 protein. We analysed Nfa1-specific antibody and cytokine induction, and the mean survival time of infected mice. Mice immunized intra-peritoneally or intra-nasally with rNfa1 protein developed specific IgG, IgA and IgE antibodies; the IgG response was dominated by IgG1, followed by IgG2b, IgG2a and IgG3. High levels of the Th1 cytokine, IFN-γ, and the regulatory cytokine, IL-10, were also induced. The mean survival time of mice immunized intra-peritoneally with rNfa1 protein was prolonged compared with controls, (25.0 and 15.5 days, respectively). Similarly, the mean survival time of mice immunized intra-nasally with rNfa1 protein was 24.7 days, compared with 15.0 days for controls.

  2. Protein disulfide isomerases are antibody targets during immune-mediated tumor destruction

    PubMed Central

    Fonseca, Catia; Soiffer, Robert; Ho, Vincent; Vanneman, Matthew; Jinushi, Masahisa; Ritz, Jerome; Neuberg, Donna; Stone, Richard; DeAngelo, Dan

    2009-01-01

    The identification of cancer antigens that contribute to transformation and are linked with immune-mediated tumor destruction is an important goal for immunotherapy. Toward this end, we screened a murine renal cell carcinoma cDNA expression library with sera from mice vaccinated with irradiated tumor cells engineered to secrete granulocyte macrophage colony-stimulating factor (GM-CSF). Multiple nonmutated, overexpressed proteins that function in tumor cell migration, protein/nucleic acid homeostasis, metabolism, and stress responses were detected. Among these, the most frequently recognized clone was protein disulfide isomerase (PDI). High titer antibodies to human PDI were similarly induced in an acute myeloid leukemia patient who achieved a complete response after vac-cination with irradiated, autologous GM-CSF–secreting tumor cells in the setting of nonmyeloablative allogeneic bone marrow transplantation. Moreover, ERp5, a closely related disulfide isomerase involved in major histocompatibility complex (MHC) class I chain-related protein A (MICA) shedding, also evoked potent humoral reactions in diverse solid and hematologic malignancy patients who responded to GM-CSF–secreting tumor cell vaccines or antibody blockade of cytotoxic T lymphocyte–associated antigen 4 (CTLA-4). Together, these findings reveal the unexpected immunogenicity of PDIs and raise the possibility that these gene products might serve as targets for therapeutic monoclonal antibodies. PMID:19008459

  3. SPRYSEC Effectors: A Versatile Protein-Binding Platform to Disrupt Plant Innate Immunity

    PubMed Central

    Diaz-Granados, Amalia; Petrescu, Andrei-José; Goverse, Aska; Smant, Geert

    2016-01-01

    Persistent infections by sedentary plant-parasitic nematodes are a major threat to important food crops all over the world. These roundworms manipulate host plant cell morphology and physiology to establish sophisticated feeding structures. Key modifications to plant cells during their transition into feeding structures are largely attributed to the activity of effectors secreted by the nematodes. The SPRYSEC effectors were initially identified in the potato cyst nematodes Globodera rostochiensis and G. pallida, and are characterized by a single SPRY domain, a non-catalytic domain present in modular proteins with different functions. The SPRY domain is wide-spread among eukaryotes and thought to be involved in mediating protein–protein interactions. Thus far, the SPRY domain is only reported as a functional domain in effectors of plant-parasitic nematodes, but not of other plant pathogens. SPRYSEC effectors have been implicated in both suppression and activation of plant immunity, but other possible roles in nematode virulence remain undefined. Here, we review the latest reports on the structure, function, and sequence diversity of SPRYSEC effectors, which provide support for a model featuring these effectors as a versatile protein-binding platform for the nematodes to target a wide range of host proteins during parasitism. PMID:27812363

  4. An Immunoglobulin Binding Protein (Antigen 5) of the Stable Fly (Diptera: Muscidae) Salivary Gland Stimulates Bovine Immune Responses

    PubMed Central

    AMERI, M.; WANG, X.; WILKERSON, M. J.; KANOST, M. R.; BROCE, A. B.

    2008-01-01

    The stable fly, Stomoxys calcitrans, is an economically important pest of livestock. Prior studies demonstrated lymphocyte suppression by crude salivary gland extract (SGE) of the stable fly. A dominant 27 kDa protein identified in the SGE was reported to stimulate immunodominant antibody responses in exposed cattle. The purpose of this study was to determine if this protein, now identified as a homolog of insect proteins named antigen 5 (Ag5), was responsible for the lymphocyte suppression and if naïve calves can mount an immune response to it. Calves raised in the winter months were immunized with recombinant Ag5 (rAg5) expressed in Drosophila S2 cells or with “natural” Ag5 protein isolated by preparative gel electrophoresis of SGE. Control calves were immunized with adjuvant alone. Rising antibody concentrations to rAg5 were detected in two of three calves immunized with rAg5 and one of three calves immunized with natural Ag5. Recall lymphocyte responses to rAg5 were detected at 21 and 28 DPI in calves immunized with rAg5 but not in calves immunized with the natural Ag5 or those exposed to adjuvant alone. Mitogen-stimulated bovine lymphocyte responses were not suppressed by rAg5. Further investigation using immunoblotting revealed that rAg5 binds to the Fc and F(ab’)2 portions of bovine IgG, but not to an Fab fragment. These findings suggest that Ag5 of the stable fly salivary gland is not immunosuppressive, but has immunoglobulin binding properties and can invoke specific antibody and memory lymphocyte responses in immunized calves. PMID:18283948

  5. Effect of immunization against prostate- and testis-expressed (PATE) proteins on sperm function and fecundity in the rat.

    PubMed

    Rajesh, Angireddy; Yenugu, Suresh

    2015-08-01

    Evaluating the immunocontraceptive potential of sperm-bound proteins is an active area of investigation. In this study, we analyzed the role of prostate- and testes-expressed (PATE) and PATE-F proteins in sperm function. Capacitation was measured as a function of tyrosine phosphorylation of sperm membrane proteins. Ionophore-induced acrosome reaction was assessed by measuring the fluorescence intensity of calcium-bound Fluo 3-AM and sperm-bound PNA-FITC in a flow cytometer. Rat spermatozoa subjected to capacitation and acrosome reaction in vitro displayed changes in the PATE and PATE-F protein localization on their surface, indicating the role of these proteins in sperm function. Capacitation and ionophore-induced acrosome reaction in vitro were inhibited in spermatozoa pre-incubated with antiserum raised in rabbit against PATE or PATE-F. Male rats were immunized with PATE proteins to assess their role in sperm function and fecundity. Antibody titer in the serum, testicular, and epididymal fluid was measured by ELISA. The motility parameters were recorded using CASA. High antibody titer was observed in serum, epididymal, and testicular fluid in rats immunized with PATE or PATE-F protein. Immunization did not cause any structural damage and inflammation in the testis and epididymis. PATE and PATE-F antisera obtained from the immunized rats inhibited acrosome reaction. Motility parameters, capacitation, acrosome reaction, and fecundity were compromised in PATE-F-immunized rats, whereas the same were not affected in rats immunized with PATE. These results suggest that PATE-F might play an important role in sperm function and fecundity and can be explored further to determine its immunocontraceptive potential.

  6. Modified recombinant adenoviruses increase porcine circovirus 2 capsid protein expression and induce enhanced immune responses in mice.

    PubMed

    Li, D L; Huang, Y; Chang, L L; DU, Q; Chen, Y; Wang, T T; Luo, X M; Zhao, X M; Tong, D W

    2016-01-01

    Porcine circovirus type 2 (PCV2) is the primary viral pathogen of porcine circovirus associated disease (PCVAD) and vaccination is an important method to prevent and control the disease. The expression of PCV2 capsid protein (Cap) in adenovirus vector system has been investigated, but the poor immune responses limit its application. In this study, transcriptional enhancer element largest intron of the human cytomegalovirus (Intron A) and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) were applied to increase the immunogenicity of PCV2 Cap adenovirus-based vaccine. Western blot and indirect immunofluorescence assay (IFA) analysis showed that modified adenoviruses with Intron A and WPRE alone or both could significantly increase the expression of Cap compared to the unmodified adenoviruses. Furthermore, the humoral and cellular immune responses of the constructed recombinant adenoviruses were evaluated in mice. Indirect ELISA, virus neutralizing test and western blot showed that modified adenoviruses elicited higher humoral immune responses than unmodified adenovirus, and Intron A-WPRE-modified virus immunized group had better immune response than the others. Besides, the results of lymphocyte proliferation response and cytokines release assay showed that enhanced cellular immune responses were induced by modified adenoviruses. These results demonstrated that Intron A and WPRE significantly improved the expression of the Cap protein in adenovirus vector system and enhanced the immune responses in mice, making the adenovirus vector system more applicable against PCV2. PMID:27640437

  7. Maintenance of immune hyporesponsiveness to melanosomal proteins by DHICA-mediated antioxidation: Possible implications for autoimmune vitiligo.

    PubMed

    Liu, Xiao-Ming; Zhou, Qiong; Xu, Shi-Zheng; Wakamatsu, Kazumasa; Lei, Tie-Chi

    2011-05-01

    Melanocyte destruction in the skin of vitiligo patients has been considered to be a consequence of an autoimmune response against melanosomal proteins. However, little is known about the molecular mechanisms by which the immune system recognizes these sequestered intracellular self-proteins, which are confined in specialized organelles termed melanosomes, and is provoked into an autoimmune response to melanocytes. Here, we utilize a sucrose density-gradient ultracentrifugation protocol to enrich melanosomal components from dopachrome tautomerase (Dct)-mutant or wild-type melanocytes exposed to a pulse of hydrogen peroxide at a noncytotoxic concentration to evaluate their immunogenicity in mice challenged with the corresponding melanosomal proteins. The results demonstrate that enhanced humoral and cellular immune responses to a challenge with late-stage melanosomal proteins, especially with those derived from Dct-mutant melanocytes, are found in the immunized mice. To elucidate whether a reduced 5,6-dihydroxyindole-2-carboxylic acid (DHICA) content in melanin might cause a loss in antioxidative protection to the proteins, we incubated these melanosomal proteins in vitro with synthetic 5,6-dihydroindole (DHI)-melanin or DHI/DHICA (1:1)-melanin and then used them to immunize mice. T cell proliferation and IgG antibody responsiveness to the challenges were significantly induced by melanosomal proteins treated with DHI-melanin, but not by those treated with DHI/DHICA (1:1)-melanin. Moreover, we observed that melanosomal proteins derived from Dct-mutant melanocytes are subject to oxidative modifications that alter their antigenic configurations to attain an enhanced immunogenicity compared with those derived from wild-type melanocytes. From these results, we conclude that DHICA-mediated antioxidation plays a critical role in the maintenance of immune hyporesponsiveness to melanosomal proteins.

  8. Immune responses to a recombinant Rv0057-Rv1352 fusion protein of Mycobacterium tuberculosis.

    PubMed

    Yang, Yourong; Feng, Jindong; Zhang, Junxian; Zhao, Weiguo; Liu, Yu; Liang, Yan; Bai, Xuejuan; Wang, Lan; Wu, Xueqiong

    2015-01-01

    The identification and characterization of antigens of Mycobacterium tuberculosis help in understanding the mechanisms of protective immunity and in improving diagnostic methods for TB. Rv0057 and Rv1352 proteins are new T-cell antigens, found to play roles in TB infection. In this study, a recombinant fusion protein Rv0057-Rv1352 was made and analyzed for its immunological characteristics and potential utility. It showed good immunoreactivity with serum from TB patients by western blotting. The antibody levels against Rv0057-Rv1352 were significantly higher in sera from 69 TB patients than in sera from 60 patients with non-TB respiratory diseases (P<0.001). The sensitivities of a diagnostic ELISA test based on detecting Rv0057-Rv1352 antibody (60.3%) or 38 kDa-16 kDa antibody (58.8%) were comparable to commercial rapid test B (75.4%), and significantly higher (p<0.001) than bacteriological methods (15.9%), rapid test A (20.3%), or rapid test C (29.0%). The specificities of Rv0057-Rv1352 (93.3%) or 38 kDa-16 kDa antibody tests (93.3%) were equivalent to the rapid tests A (93.3%) and C (86.7%), and significantly higher than rapid test B (63.3%, p<0.001). When 38 kDa-16 kDa was used together with Rv0057-Rv1352, the test sensitivity reached 85.5%, and its specificity remained high (86.7%). The test was as sensitive with bacterium-positive TB patients as with bacterium-negative. In an ELISPOT assay for cellular immunity, Rv0057-Rv1352 stimulated T lymphocytes to produce fewer spots secreting IFN-γ than CFP10-ESAT6 fusion protein did (p>0.05). These results suggest that Rv0057-Rv1352 has potential for the serodiagnosis of active pulmonary TB. PMID:25696009

  9. Fine specificity of cellular immune responses in humans to human cytomegalovirus immediate-early 1 protein.

    PubMed Central

    Alp, N J; Allport, T D; Van Zanten, J; Rodgers, B; Sissons, J G; Borysiewicz, L K

    1991-01-01

    Cell-mediated immunity is important in maintaining the virus-host equilibrium in persistent human cytomegalovirus (HCMV) infection. The HCMV 72-kDa major immediate early 1 protein (IE1) is a target for CD8+ cytotoxic T cells in humans, as is the equivalent 89-kDa protein in mouse. Less is known about responses against this protein by CD4+ T cells, which may be important as direct effector cells or helper cells for antibody and CD8+ responses. Proliferative-T-cell responses to HCMV IE1 were studied in normal seropositive subjects. Peripheral blood mononuclear cells from 85% of seropositive subjects proliferated in response to HCMV from infected fibroblasts, and of these, 73% responded to recombinant baculovirus IE1. Responding cells were predominantly CD3+ CD4+. IE1 antigen preparations, including baculovirus recombinant protein, transfected rat cell nuclei, and synthetic peptides, induced IE1-specific T-cell lines which cross-reacted between the preparations. The fine specificity of these IE1-specific T-cell lines was studied by using overlapping synthetic peptides encompassing the entire sequence of the IE1 protein. The regions of the IE1 molecule recognized were identified and these varied between individuals, possibly reflecting differences in major histocompatibility complex (MHC) class II haplotype. In one subject, the peptide specificities of proliferative and MHC class I-restricted cytotoxic determinants on IE1 were spatially distinct. Thus, no single immunodominant T-cell determinant within HCMV IE1 was identified, suggesting that multiple peptides or a region of the 72-kDa IE1 protein would be required to induce specific T-cell responses in humans. PMID:1714519

  10. Recombinant Streptococcus equi Proteins Protect Mice in Challenge Experiments and Induce Immune Response in Horses

    PubMed Central

    Flock, Margareta; Jacobsson, Karin; Frykberg, Lars; Hirst, Timothy R.; Franklin, Anders; Guss, Bengt; Flock, Jan-Ingmar

    2004-01-01

    Horses that have undergone infection caused by Streptococcus equi subspecies equi (strangles) were found to have significantly increased serum antibody titers against three previously characterized proteins, FNZ (cell surface-bound fibronectin binding protein), SFS (secreted fibronectin binding protein), and EAG (α2-macroglobulin, albumin, and immunoglobulin G [IgG] binding protein) from S. equi. To assess the protective efficacy of vaccination with these three proteins, a mouse model of equine strangles was utilized. Parts of the three recombinant proteins were used to immunize mice, either subcutaneously or intranasally, prior to nasal challenge with S. equi subsp. equi. The adjuvant used was EtxB, a recombinant form of the B subunit of Escherichia coli heat-labile enterotoxin. It was shown that nasal colonization of S. equi subsp. equi and weight loss due to infection were significantly reduced after vaccination compared with a mock-vaccinated control group. This effect was more pronounced after intranasal vaccination than after subcutaneous vaccination; nearly complete eradication of nasal colonization was obtained after intranasal vaccination (P < 0.001). When the same antigens were administered both intranasally and subcutaneously to healthy horses, significant mucosal IgA and serum IgG antibody responses against FNZ and EAG were obtained. The antibody response was enhanced when EtxB was used as an adjuvant. No adverse effects of the antigens or EtxB were observed. Thus, FNZ and EAG in conjunction with EtxB are promising candidates for an efficacious and safe vaccine against strangles. PMID:15155624

  11. Francisella tularensis LVS surface and membrane proteins as targets of effective post-exposure immunization for tularemia.

    PubMed

    Chandler, Jeffrey C; Sutherland, Marjorie D; Harton, Marisa R; Molins, Claudia R; Anderson, Rebecca V; Heaslip, Darragh G; Bosio, Catharine M; Belisle, John T

    2015-02-01

    Francisella tularensis causes disease (tularemia) in a large number of mammals, including man. We previously demonstrated enhanced efficacy of conventional antibiotic therapy for tularemia by postexposure passive transfer of immune sera developed against a F. tularensis LVS membrane protein fraction (MPF). However, the protein composition of this immunogenic fraction was not defined. Proteomic approaches were applied to define the protein composition and identify the immunogens of MPF. MPF consisted of at least 299 proteins and 2-D Western blot analyses using sera from MPF-immunized and F. tularensis LVS-vaccinated mice coupled to liquid chromatography-tandem mass spectrometry identified 24 immunoreactive protein spots containing 45 proteins. A reverse vaccinology approach that applied labeling of F. tularensis LVS surface proteins and bioinformatics was used to reduce the complexity of potential target immunogens. Bioinformatics analyses of the immunoreactive proteins reduced the number of immunogen targets to 32. Direct surface labeling of F. tularensis LVS resulted in the identification of 31 surface proteins. However, only 13 of these were reactive with MPF and/or F. tularensis LVS immune sera. Collectively, this use of orthogonal proteomic approaches reduced the complexity of potential immunogens in MPF by 96% and allowed for prioritization of target immunogens for antibody-based immunotherapies against tularemia.

  12. Major Basic Protein from Eosinophils and Myeloperoxidase from Neutrophils Are Required for Protective Immunity to Strongyloides stercoralis in Mice ▿

    PubMed Central

    O'Connell, Amy E.; Hess, Jessica A.; Santiago, Gilberto A.; Nolan, Thomas J.; Lok, James B.; Lee, James J.; Abraham, David

    2011-01-01

    Eosinophils and neutrophils contribute to larval killing during the primary immune response, and neutrophils are effector cells in the secondary response to Strongyloides stercoralis in mice. The objective of this study was to determine the molecular mechanisms used by eosinophils and neutrophils to control infections with S. stercoralis. Using mice deficient in the eosinophil granule products major basic protein (MBP) and eosinophil peroxidase (EPO), it was determined that eosinophils kill the larvae through an MBP-dependent mechanism in the primary immune response if other effector cells are absent. Infecting PHIL mice, which are eosinophil deficient, with S. stercoralis resulted in development of primary and secondary immune responses that were similar to those of wild-type mice, suggesting that eosinophils are not an absolute requirement for larval killing or development of secondary immunity. Treating PHIL mice with a neutrophil-depleting antibody resulted in a significant impairment in larval killing. Naïve and immunized mice with neutrophils deficient in myeloperoxidase (MPO) infected with S. stercoralis had significantly decreased larval killing. It was concluded that there is redundancy in the primary immune response, with eosinophils killing the larvae through an MBP-dependent mechanism and neutrophils killing the worms through an MPO-dependent mechanism. Eosinophils are not required for the development or function of secondary immunity, but MPO from neutrophils is required for protective secondary immunity. PMID:21482685

  13. Simulated climate change causes immune suppression and protein damage in the crustacean Nephrops norvegicus.

    PubMed

    Hernroth, Bodil; Sköld, Helen Nilsson; Wiklander, Kerstin; Jutfelt, Fredrik; Baden, Susanne

    2012-11-01

    Rising atmospheric carbon dioxide concentration is causing global warming, which affects oceans by elevating water temperature and reducing pH. Crustaceans have been considered tolerant to ocean acidification because of their retained capacity to calcify during subnormal pH. However, we report here that significant immune suppression of the Norway lobster, Nephrops norvegicus, occurs after a 4-month exposure to ocean acidification (OA) at a level predicted for the year 2100 (hypercapnic seawater with a pH lowered by 0.4 units). Experiments carried out at different temperatures (5, 10, 12, 14, 16, and 18°C) demonstrated that the temperature within this range alone did not affect lobster immune responses. In the OA-treatment, hemocyte numbers were reduced by almost 50% and the phagocytic capacity of the remaining hemocytes was inhibited by 60%. The reduction in hemocyte numbers was not due to increased apoptosis in hematopoetic tissue. Cellular responses to stress were investigated through evaluating advanced glycation end products (AGE) and lipid oxidation in lobster hepatopancreata, and OA-treatment was shown to significantly increase AGEs', indicating stress-induced protein alterations. Furthermore, the extracellular pH of lobster hemolymph was reduced by approximately 0.2 units in the OA-treatment group, indicating either limited pH compensation or buffering capacity. The negative effects of OA-treatment on the nephropidae immune response and tissue homeostasis were more pronounced at higher temperatures (12-18°C versus 5°C), which may potentially affect disease severity and spread. Our results signify that ocean acidification may have adverse effects on the physiology of lobsters, which previously had been overlooked in studies of basic parameters such as lobster growth or calcification.

  14. Cellular immunity to the P6 outer membrane protein of nontypeable Haemophilus influenzae.

    PubMed Central

    Kodama, H; Faden, H

    1995-01-01

    Cellular immunity to nontypeable Haemophilus influenzae in a population of 10 healthy, immune adults was determined by measuring lymphocyte blast transformation and antibody secretion in response to the P6 outer membrane protein. P6 (200 microliters/ml) induced lymphocyte blast transformation that peaked on day 10 of incubation. The peak induction of antibody-secreting cells occurred on day 8 of incubation. In comparison with the response to tetanus toxoid stimulation, the peak lymphocyte blast transformation response to P6 was reduced (mean counts per minute +/- standard error of the mean [SEM], 3,457 +/- 503 versus 9,414 +/- 1,464; P = 0.0051) and delayed (mean days +/- SEM, 10.3 +/- 0.4 versus 8.4 +/- 0.5; P = 0.0169); however, P6 was a better stimulus of antibody secretion from lymphocytes, particularly antibody of the immunoglobulin M (IgM) class (mean peak numbers of antibody-secreting cells per 10(5) peripheral blood mononuclear cells +/- SEM: IgG, 85 +/- 29 versus 42 +/- 16 [P = 0.0469]; IgM, 81 +/- 20 versus 25 +/- 7 [P = 0.0125]; IgA, 24 +/- 8 versus 16 +/- 6 [P = 0.0526]). Thus, lymphocytes from immune individuals recognize P6 of nontypeable H. influenzae as an immunogen. These data provide a basis for future studies with otitis-prone children who fail to develop a normal antibody response to P6 antigen (N. Yamanaka and H. Faden, J. Pediatr. 122:212-218, 1993). PMID:7790058

  15. The alternative complement pathway control protein H binds to immune complexes and serves their detection

    SciTech Connect

    Nydegger, U.E.; Corvetta, A.; Spaeth, P.J.; Spycher, M.

    1983-01-01

    During solubilization of immune complexes C3b becomes fixed to the immunoglobulin part and serves as a receptor for the alternative complement pathway control protein H. The H-C3b immune complex interaction can be made detectable using 4% polyethyleneglycol to separate free from bound /sup 125/I-H. Tetanus toxoid (Te)/anti-Te complexes kept soluble with fresh serum and containing 125 IU of specific antibody bound 18% of /sup 125/I-H; when fresh serum was chelated with 10 mM EDTA, /sup 125/I-H binding was only 5%. On sucrose density gradients, the H-binding material sedimented in the range of 12 to 30 S. In 36 serum samples from rheumatoid arthritis (RA) patients and in 12 serum samples from patients with systemic lupus erythematosus (SLE), /sup 125/I-H binding was significantly elevated to 9.5 +/- 4.7% (mean +/- 1 SD) and 13.3 +/- 5.6%, respectively, while /sup 125/I-H binding by 36 normal human sera was 4 +/- 2%. RA samples (17/36, 47%) and SLE samples (9/12, 75%) had H-binding values increased by more than 2 SD above the normal mean. The serum samples were also assessed for conglutinin- and C1q-binding activities; a significant correlation between H and C1q binding was observed (P less than 0.001); there was no correlation between H and conglutinin binding. Although binding to immune complexes through its interaction with C3b, H clearly detects a population of complexes other than conglutinin, thus expanding the possibilities of further characterizing pathological complexes.

  16. Secretion of Rhoptry and Dense Granule Effector Proteins by Nonreplicating Toxoplasma gondii Uracil Auxotrophs Controls the Development of Antitumor Immunity

    PubMed Central

    Fox, Barbara A.; Sanders, Kiah L.; Rommereim, Leah M.; Bzik, David J.

    2016-01-01

    Nonreplicating type I uracil auxotrophic mutants of Toxoplasma gondii possess a potent ability to activate therapeutic immunity to established solid tumors by reversing immune suppression in the tumor microenvironment. Here we engineered targeted deletions of parasite secreted effector proteins using a genetically tractable Δku80 vaccine strain to show that the secretion of specific rhoptry (ROP) and dense granule (GRA) proteins by uracil auxotrophic mutants of T. gondii in conjunction with host cell invasion activates antitumor immunity through host responses involving CD8α+ dendritic cells, the IL-12/interferon-gamma (IFN-γ) TH1 axis, as well as CD4+ and CD8+ T cells. Deletion of parasitophorous vacuole membrane (PVM) associated proteins ROP5, ROP17, ROP18, ROP35 or ROP38, intravacuolar network associated dense granule proteins GRA2 or GRA12, and GRA24 which traffics past the PVM to the host cell nucleus severely abrogated the antitumor response. In contrast, deletion of other secreted effector molecules such as GRA15, GRA16, or ROP16 that manipulate host cell signaling and transcriptional pathways, or deletion of PVM associated ROP21 or GRA3 molecules did not affect the antitumor activity. Association of ROP18 with the PVM was found to be essential for the development of the antitumor responses. Surprisingly, the ROP18 kinase activity required for resistance to IFN-γ activated host innate immunity related GTPases and virulence was not essential for the antitumor response. These data show that PVM functions of parasite secreted effector molecules, including ROP18, manipulate host cell responses through ROP18 kinase virulence independent mechanisms to activate potent antitumor responses. Our results demonstrate that PVM associated rhoptry effector proteins secreted prior to host cell invasion and dense granule effector proteins localized to the intravacuolar network and host nucleus that are secreted after host cell invasion coordinately control the

  17. Folded state of the integral membrane colicin E1 immunity protein in solvents of mixed polarity.

    PubMed

    Taylor, R M; Zakharov, S D; Bernard Heymann, J; Girvin, M E; Cramer, W A

    2000-10-10

    The colicin E1 immunity protein (ImmE1), a 13.2-kDa hydrophobic integral membrane protein localized in the Escherichia coli cytoplasmic membrane, protects the cell from the lethal, channel-forming activity of the bacteriocin, colicin E1. Utilizing its solubility in organic solvents, ImmE1 was purified by 1-butanol extraction of isolated membranes, followed by gel filtration and ion-exchange chromatography in a chloroform/methanol/H(2)O (4:4:1) solvent system. Circular dichroism analysis indicated that the alpha-helical content of ImmE1 is approximately 80% in 1-butanol or 2,2,2-trifluoroethanol, consistent with a previous membrane-folding model with three extended hydrophobic transmembrane helical domains, H1-H3. Each of these extended hydrophobic domains contains a centrally located single Cys residue that could be used as a probe of protein structure. The presence of tertiary structure of purified ImmE1 in a solvent of mixed polarity, chloroform/methanol/H(2)O (4:4:1) was demonstrated by (i) the constraints on Tyr residues shown by the amplitude of near-UV circular dichroism spectra in the wavelength interval, 270-285 nm; (ii) the correlation between the near-UV Tyr CD spectrum of single and double Cys-to-X mutants of the Imm protein and their in vivo activity; (iii) the upfield shift of methyl groups in a 1D NMR spectrum, a 2D- HSQC NMR spectrum of ImmE1 in the mixed polarity solvent mixture, and a broadening and disappearance of the indole (1)H proton resonance from Trp94 in H3 by a spin label attached to Cys16 in the H2 hydrophobic domain; (iv) near-UV circular dichroism spectra with a prominent ellipticity band centered at 290 nm from a single Trp inserted into the extended hydrophobic domains. It was concluded that the colicin E1 immunity protein adopts a folded conformation in chloroform/methanol/H(2)O (4:4:1) that is stabilized by helix-helix interactions. Analysis of the probable membrane folding topology indicated that several Tyr residues in the bilayer

  18. Altered immune response in mice deficient for the G protein-coupled receptor GPR34.

    PubMed

    Liebscher, Ines; Müller, Uwe; Teupser, Daniel; Engemaier, Eva; Engel, Kathrin M Y; Ritscher, Lars; Thor, Doreen; Sangkuhl, Katrin; Ricken, Albert; Wurm, Antje; Piehler, Daniel; Schmutzler, Sandra; Fuhrmann, Herbert; Albert, Frank W; Reichenbach, Andreas; Thiery, Joachim; Schöneberg, Torsten; Schulz, Angela

    2011-01-21

    The X-chromosomal GPR34 gene encodes an orphan G(i) protein-coupled receptor that is highly conserved among vertebrates. To evaluate the physiological relevance of GPR34, we generated a GPR34-deficient mouse line. GPR34-deficient mice were vital, reproduced normally, and showed no gross abnormalities in anatomical, histological, laboratory chemistry, or behavioral investigations under standard housing. Because GPR34 is highly expressed in mononuclear cells of the immune system, mice were specifically tested for altered functions of these cell types. Following immunization with methylated BSA, the number of granulocytes and macrophages in spleens was significantly lower in GPR34-deficient mice as in wild-type mice. GPR34-deficient mice showed significantly increased paw swelling in the delayed type hypersensitivity test and higher pathogen burden in extrapulmonary tissues after pulmonary infection with Cryptococcus neoformans compared with wild-type mice. The findings in delayed type hypersensitivity and infection tests were accompanied by significantly different basal and stimulated TNF-α, GM-CSF, and IFN-γ levels in GPR34-deficient animals. Our data point toward a functional role of GPR34 in the cellular response to immunological challenges. PMID:21097509

  19. Altered Immune Response in Mice Deficient for the G Protein-coupled Receptor GPR34*

    PubMed Central

    Liebscher, Ines; Müller, Uwe; Teupser, Daniel; Engemaier, Eva; Engel, Kathrin M. Y.; Ritscher, Lars; Thor, Doreen; Sangkuhl, Katrin; Ricken, Albert; Wurm, Antje; Piehler, Daniel; Schmutzler, Sandra; Fuhrmann, Herbert; Albert, Frank W.; Reichenbach, Andreas; Thiery, Joachim; Schöneberg, Torsten; Schulz, Angela

    2011-01-01

    The X-chromosomal GPR34 gene encodes an orphan Gi protein-coupled receptor that is highly conserved among vertebrates. To evaluate the physiological relevance of GPR34, we generated a GPR34-deficient mouse line. GPR34-deficient mice were vital, reproduced normally, and showed no gross abnormalities in anatomical, histological, laboratory chemistry, or behavioral investigations under standard housing. Because GPR34 is highly expressed in mononuclear cells of the immune system, mice were specifically tested for altered functions of these cell types. Following immunization with methylated BSA, the number of granulocytes and macrophages in spleens was significantly lower in GPR34-deficient mice as in wild-type mice. GPR34-deficient mice showed significantly increased paw swelling in the delayed type hypersensitivity test and higher pathogen burden in extrapulmonary tissues after pulmonary infection with Cryptococcus neoformans compared with wild-type mice. The findings in delayed type hypersensitivity and infection tests were accompanied by significantly different basal and stimulated TNF-α, GM-CSF, and IFN-γ levels in GPR34-deficient animals. Our data point toward a functional role of GPR34 in the cellular response to immunological challenges. PMID:21097509

  20. Oral administration with attenuated Salmonella encoding a Trichinella cystatin-like protein elicited host immunity.

    PubMed

    Liu, X D; Wang, X L; Bai, X; Liu, X L; Wu, X P; Zhao, Y; Sun, S M; Yu, L; Su, X Z; Wang, Z Q; Wang, F; Liu, M Y

    2014-06-01

    Trichinellosis is a public health problem and is regarded as an emergent/re-emergent disease in various countries. The cDNA encoding a cystatin-like protein (Ts-cystatin) was identified by immunoscreening intestinal muscle larvae cDNA libraries with serum from pigs experimentally infected with 20,000 Trichinella spiralis muscle larvae. To study its impact on host immunity, we chose a eukaryotic expression system based on several comparisons of immunogenicity between the two Salmonella typhimurium administration schemes, which indicated that the eukaryotic expression system was superior. Humoral IgG and mucosal IgA were measured to determine the antibody response. To explore whether Th1 and Th2 responses were responsible for the induced protection, Th1- and Th2-specific cellular transcription factors and the cytokine profile were examined. Changes in the T lymphocyte and macrophage populations were detected by flow cytometry. Lastly, parasitological examination was examined. The results showed that Ts-cystatin induced a Th1/Th2-mixed type of immune response and decreased STAT6 transcription. The intestinal adult recovery increased by 10.9% in the Ts-cystatin group, the Ts-cystatin group fecundity rate was decreased by 91%. Furthermore, the number of muscle larvae did not change compared with the control group. In conclusion, our results suggest that Ts-cystatin plays an important role in Trichinella resistance to rapid expulsion by the host and is worth further study.

  1. Induction of immune tolerance to caseins and whey proteins by oral intubation in mouse allergy model.

    PubMed

    Shandilya, U K; Kapila, R; Singh, S; Dahiya, D; Kapila, S; Kansal, V K

    2014-06-01

    This study was designed to evaluate the effect of oral tolerance of caseins (CSN) and whey proteins (WP) in alleviating the allergic response to cow's milk proteins in Swiss albino mice raised on a milk protein-free diet. Oral tolerance was induced by feeding mice with 20 mg of CSN or WP once in a day for 4 days consecutively before immunization with respective protein by intraperitoneal (i.p.) injections (20 μg 200 per μl of PBS) using 2% of alum Al(OH)3 as adjuvant. Three weeks later, oral tolerance induction was analysed in humoral and cellular compartments of CSN- and WP-fed versus saline-fed control mice groups by measuring seric and intestinal antibody responses, mRNA abundance in splenic tissue and cytokine secretion patterns. The specific serum immunoglobulin-E (IgE) levels were significantly suppressed (p < 0.05), while sIgA was enhanced in these groups when compared with their respective saline-fed mice. Moreover, the mRNA levels of interferon-γ (IFN-γ) and interleukin-4 (IL-4) in both CSN- and WP-tolerized mice were found to be significantly decreased, while the abundance of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) was increased significantly, as compared to respective control groups. Finally, cytokine profiles indicated a reciprocal decrease in IL-4 and IFN-γ versus an increase in IL-10 secretions in supernatants of cultured splenocytes of tolerized mice. Taken together, these results clearly showed that oral administration of cows' milk caseins and whey proteins can induce significant hyposensitization in mice, with the participation of suppressor cytokines.

  2. Structural characterization of the pulmonary innate immune protein SPLUNC1 and identification of lipid ligands

    PubMed Central

    Ning, Fangkun; Wang, Chao; Berry, Karin Zemski; Kandasamy, Pitchaimani; Liu, Haolin; Murphy, Robert C.; Voelker, Dennis R.; Nho, Chu Won; Pan, Choel-Ho; Dai, Shaodong; Niu, Liwen; Chu, Hong-Wei; Zhang, Gongyi

    2014-01-01

    The short palate, lung and nasal epithelial clone 1 (SPLUNC1) protein is a member of the palate, lung, and nasal epithelium clone (PLUNC) family, also known as bactericidal/permeability-increasing (BPI) fold-containing protein, family A, member 1 (BPIFA1). SPLUNC1 is an abundant protein in human airways, but its function remains poorly understood. The lipid ligands of SPLUNC1 as well as other PLUNC family members are largely unknown, although some reports provide evidence that lipopolysaccharide (LPS) could be a lipid ligand. Unlike previous hypotheses, we found significant structural differences between SPLUNC1 and BPI. Recombinant SPLUNC1 produced in HEK 293 cells harbored several molecular species of sphingomyelin and phosphatidylcholine as its ligands. Significantly, in vitro lipid-binding studies failed to demonstrate interactions between SPLUNC1 and LPS, lipoteichoic acid, or polymyxin B. Instead, one of the major and most important pulmonary surfactant phospholipids, dipalmitoylphosphatidylcholine (DPPC), bound to SPLUNC1 with high affinity and specificity. We found that SPLUNC1 could be the first protein receptor for DPPC. These discoveries provide insight into the specific determinants governing the interaction between SPLUNC1 and lipids and also shed light on novel functions that SPLUNC1 and other PLUNC family members perform in host defense.—Ning, F., Wang, C., Berry, K. Z., Kandasamy, P., Liu, H., Murphy, R. C., Voelker, D. R., Nho, C. W., Pan, C.-H., Dai, S., Niu, L., Chu, H.-W., Zhang, G. Structural characterization of the pulmonary innate immune protein SPLUNC1 and identification of lipid ligands. PMID:25223608

  3. A multi-objective evolutionary algorithm for protein structure prediction with immune operators.

    PubMed

    Judy, M V; Ravichandran, K S; Murugesan, K

    2009-08-01

    Genetic algorithms (GA) are often well suited for optimisation problems involving several conflicting objectives. It is more suitable to model the protein structure prediction problem as a multi-objective optimisation problem since the potential energy functions used in the literature to evaluate the conformation of a protein are based on the calculations of two different interaction energies: local (bond atoms) and non-local (non-bond atoms) and experiments have shown that those types of interactions are in conflict, by using the potential energy function, Chemistry at Harvard Macromolecular Mechanics. In this paper, we have modified the immune inspired Pareto archived evolutionary strategy (I-PAES) algorithm and denoted it as MI-PAES. It can effectively exploit some prior knowledge about the hydrophobic interactions, which is one of the most important driving forces in protein folding to make vaccines. The proposed MI-PAES is comparable with other evolutionary algorithms proposed in literature, both in terms of best solution found and the computational time and often results in much better search ability than that of the canonical GA.

  4. A single vertebrate DNA virus protein disarms invertebrate immunity to RNA virus infection

    PubMed Central

    Gammon, Don B; Duraffour, Sophie; Rozelle, Daniel K; Hehnly, Heidi; Sharma, Rita; Sparks, Michael E; West, Cara C; Chen, Ying; Moresco, James J; Andrei, Graciela; Connor, John H; Conte, Darryl; Gundersen-Rindal, Dawn E; Marshall, William L; Yates, John R; Silverman, Neal; Mello, Craig C

    2014-01-01

    Virus-host interactions drive a remarkable diversity of immune responses and countermeasures. We found that two RNA viruses with broad host ranges, vesicular stomatitis virus (VSV) and Sindbis virus (SINV), are completely restricted in their replication after entry into Lepidopteran cells. This restriction is overcome when cells are co-infected with vaccinia virus (VACV), a vertebrate DNA virus. Using RNAi screening, we show that Lepidopteran RNAi, Nuclear Factor-κB, and ubiquitin-proteasome pathways restrict RNA virus infection. Surprisingly, a highly conserved, uncharacterized VACV protein, A51R, can partially overcome this virus restriction. We show that A51R is also critical for VACV replication in vertebrate cells and for pathogenesis in mice. Interestingly, A51R colocalizes with, and stabilizes, host microtubules and also associates with ubiquitin. We show that A51R promotes viral protein stability, possibly by preventing ubiquitin-dependent targeting of viral proteins for destruction. Importantly, our studies reveal exciting new opportunities to study virus-host interactions in experimentally-tractable Lepidopteran systems. DOI: http://dx.doi.org/10.7554/eLife.02910.001 PMID:24966209

  5. Group A Streptococcal M1 Protein Sequesters Cathelicidin to Evade Innate Immune Killing

    PubMed Central

    LaRock, Christopher N.; Döhrmann, Simon; Todd, Jordan; Corriden, Ross; Olson, Joshua; Johannssen, Timo; Lepenies, Bernd; Gallo, Richard L.; Ghosh, Partho; Nizet, Victor

    2015-01-01

    SUMMARY The antimicrobial peptide LL-37 is generated upon proteolytic cleavage of cathelicidin and limits invading pathogens by directly targeting microbial membranes as well as stimulating innate immune cell function. However, some microbes evade LL-37-mediated defense. Notably, Group A Streptococcus (GAS) strains belonging to the hypervirulent M1T1 serogroup are more resistant to human LL-37 than other GAS serogroups. We show that the GAS surface-associated M1 protein sequesters and neutralizes LL-37 antimicrobial activity through its N-terminal domain. M1 protein also binds the cathelicidin precursor hCAP-18, preventing its proteolytic maturation into antimicrobial forms. Exogenous M1 protein rescues M1-deficient GAS from killing by neutrophils and within neutrophil extracellular traps and neutralizes LL-37 chemotactic properties. M1 also binds murine cathelicidin, and its virulence contribution in a murine model of necrotizing skin infection is largely driven by its ability to neutralize this host defense peptide. Thus, cathelicidin resistance is essential for the pathogenesis of hyperinvasive M1T1 GAS. PMID:26468750

  6. Urea-induced unfolding of the immunity protein Im9 monitored by spFRET.

    PubMed

    Tezuka-Kawakami, Tomoko; Gell, Chris; Brockwell, David J; Radford, Sheena E; Smith, D Alastair

    2006-09-01

    We have studied the urea-induced unfolding of the E colicin immunity protein Im9 using diffusion single-pair fluorescence resonance energy transfer. Detailed examination of the proximity ratio of the native and denatured molecules over a wide range of urea concentrations suggests that the conformational properties of both species are denaturant-dependent. Whereas native molecules become gradually more expanded as urea concentration increases, denatured molecules show a dramatic dependence of the relationship between proximity ratio and denaturant concentration, consistent with substantial compaction of the denatured ensemble at low denaturant concentrations. Analysis of the widths of the proximity ratio distributions for each state suggests that whereas the native state ensemble is relatively narrow and homogeneous, the denatured state may possess heterogeneity in mildly denaturing conditions.

  7. Urea-Induced Unfolding of the Immunity Protein Im9 Monitored by spFRET

    PubMed Central

    Tezuka-Kawakami, Tomoko; Gell, Chris; Brockwell, David J.; Radford, Sheena E.; Smith, D. Alastair

    2006-01-01

    We have studied the urea-induced unfolding of the E colicin immunity protein Im9 using diffusion single-pair fluorescence resonance energy transfer. Detailed examination of the proximity ratio of the native and denatured molecules over a wide range of urea concentrations suggests that the conformational properties of both species are denaturant-dependent. Whereas native molecules become gradually more expanded as urea concentration increases, denatured molecules show a dramatic dependence of the relationship between proximity ratio and denaturant concentration, consistent with substantial compaction of the denatured ensemble at low denaturant concentrations. Analysis of the widths of the proximity ratio distributions for each state suggests that whereas the native state ensemble is relatively narrow and homogeneous, the denatured state may possess heterogeneity in mildly denaturing conditions. PMID:16798813

  8. Recombinant outer membrane protein C of Aeromonas hydrophila elicits mixed immune response and generates agglutinating antibodies.

    PubMed

    Yadav, Sunita Kumari; Meena, Jitendra Kumar; Sharma, Mahima; Dixit, Aparna

    2016-08-01

    Aeromonas hydrophila is a gram-negative fish pathogenic bacterium, also responsible for causing opportunistic pathological conditions in humans. It causes a number of diseases in fish due to which the fish industry incurs huge economic losses annually. Due to problems of antibiotic resistance, and the rapidity with which the infection spreads among fishes, vaccination remains the most effective strategy to combat this infection in fish populations. Among various virulence factors associated with bacterial virulence, outer membrane proteins have been widely evaluated for their vaccine potential owing to their surface exposure and related role in pathogenicity. In the present study, we have investigated the immunogenic potential of a non-specific porin, outer membrane protein C (OmpC) whose expression is regulated by the two-component regulatory system and plays a major role in the survival of A. hydrophila under different osmolaric conditions. The full-length gene (~1 kb) encoding OmpC of A. hydrophila was cloned, characterized and expressed in E. coli. High yield (~112 mg/L at shake flask level) of the recombinant OmpC (rOmpC) (~40 kDa) of A. hydrophila was obtained upon purification from inclusion bodies using Ni(2+)-NTA affinity chromatography. Immunization with purified rOmpC in murine model generated high endpoint (>1:40,000) titers. IgG isotyping, ELISA and ELISPOT assay indicated mixed immune response with a TH2 bias. Also, the anti-rOmpC antibodies were able to agglutinate A. hydrophila in vitro and exhibited specific cross-reactivity with different Aeromonas strains, which will facilitate easy detection of different Aeromonas isolates in infected samples. Taken together, these data clearly indicate that rOmpC could serve as an effective vaccine against different strains of Aeromonas, a highly heterogenous group of bacteria. PMID:27328672

  9. Comparison of acute and chronic protein-energy malnutrition on host antitumor immune mechanisms.

    PubMed

    Lieberman, M D; Reynolds, J; Redmond, H P; Leon, P; Shou, J; Daly, J M

    1991-01-01

    Protein-calorie malnutrition (PCM) is prevalent in cancer patients. However, the effect of PCM on anti-tumor immunity is unclear and critically important in an era of improving results with adoptive immunotherapy. This study examined the effect of short- and long-term PCM on tumor-specific and natural immune effector mechanisms in a murine neuroblastoma (C1300 NRB) model. A/J mice received an isocaloric 2.5% or 24% casein diet for 3 or 8 weeks before inoculation with tumor. Three weeks later lymphocytes from tumor-bearing mice were harvested for determination of cytotoxic T lymphocyte (CTL) generation and natural killer (NK) cell cytotoxicity. Both 3 and 8 weeks of PCM significantly reduced mean total body weight by 25% (p less than 0.001) and 41% (p less than 0.001), respectively, compared with regularly nourished mice. Short-term PCM did not inhibit CTL or NK cytotoxicity, whereas long-term PCM significantly diminished CTL generation (p less than 0.001) but preserved NK cytotoxic function. These results indicate that CTL development against autologous tumor, in contrast to basal NK function, is dependent on host nutritional status. Mean tumor growth, determined by tumor-weight to carcass-weight ratio, was unchanged for both short- and long-term protein-energy deprived groups compared with results in regularly nourished mice. These findings suggest that NK function is the predominant effector mechanism inhibiting C1300 NRB growth and that NK tumoricidal capacity is preserved during PCM.

  10. Immune-Signatures for Lung Cancer Diagnostics: Evaluation of Protein Microarray Data Normalization Strategies

    PubMed Central

    Brezina, Stefanie; Soldo, Regina; Kreuzhuber, Roman; Hofer, Philipp; Gsur, Andrea; Weinhaeusel, Andreas

    2015-01-01

    New minimal invasive diagnostic methods for early detection of lung cancer are urgently needed. It is known that the immune system responds to tumors with production of tumor-autoantibodies. Protein microarrays are a suitable highly multiplexed platform for identification of autoantibody signatures against tumor-associated antigens (TAA). These microarrays can be probed using 0.1 mg immunoglobulin G (IgG), purified from 10 µL of plasma. We used a microarray comprising recombinant proteins derived from 15,417 cDNA clones for the screening of 100 lung cancer samples, including 25 samples of each main histological entity of lung cancer, and 100 controls. Since this number of samples cannot be processed at once, the resulting data showed non-biological variances due to “batch effects”. Our aim was to evaluate quantile normalization, “distance-weighted discrimination” (DWD), and “ComBat” for their effectiveness in data pre-processing for elucidating diagnostic immune-signatures. “ComBat” data adjustment outperformed the other methods and allowed us to identify classifiers for all lung cancer cases versus controls and small-cell, squamous cell, large-cell, and adenocarcinoma of the lung with an accuracy of 85%, 94%, 96%, 92%, and 83% (sensitivity of 0.85, 0.92, 0.96, 0.88, 0.83; specificity of 0.85, 0.96, 0.96, 0.96, 0.83), respectively. These promising data would be the basis for further validation using targeted autoantibody tests.

  11. Complex structure of type VI peptidoglycan muramidase effector and a cognate immunity protein

    PubMed Central

    Wang, Tianyu; Ding, Jinjing; Zhang, Ying; Wang, Da-Cheng; Liu, Wei

    2013-01-01

    The type VI secretion system (T6SS) is a bacterial protein-export machine that is capable of delivering virulence effectors between Gram-negative bacteria. The T6SS of Pseudomonas aeruginosa transports two lytic enzymes, Tse1 and Tse3, to degrade cell-wall peptidoglycan in the periplasm of rival bacteria that are competing for niches via amidase and muramidase activities, respectively. Two cognate immunity proteins, Tsi1 and Tsi3, are produced by the bacterium to inactivate the two antibacterial effectors, thereby protecting its siblings from self-intoxication. Recently, Tse1–Tsi1 has been structurally characterized. Here, the structure of the Tse3–Tsi3 complex is reported at 1.9 Å resolution. The results reveal that Tse3 contains a C-terminal catalytic domain that adopts a soluble lytic transglycosylase (SLT) fold in which three calcium-binding sites were surprisingly observed close to the catalytic Glu residue. The electrostatic properties of the substrate-binding groove are also distinctive from those of known structures with a similar fold. All of these features imply that a unique catalytic mechanism is utilized by Tse3 in cleaving glycosidic bonds. Tsi3 comprises a single domain showing a β-sandwich architecture that is reminiscent of the immunoglobulin fold. Three loops of Tsi3 insert deeply into the groove of Tse3 and completely occlude its active site, which forms the structural basis of Tse3 inactivation. This work is the first crystallographic report describing the three-dimensional structure of the Tse3–Tsi3 effector–immunity pair. PMID:24100309

  12. Immunity to malaria elicited by hybrid hepatitis B virus core particles carrying circumsporozoite protein epitopes

    PubMed Central

    1994-01-01

    The hepatitis B virus (HBV) nucleocapsid antigen (HBcAg) was investigated as a carrier moiety for the immunodominant circumsporozoite (CS) protein repeat epitopes of Plasmodium falciparum and the rodent malaria agent P. berghei. For this purpose hybrid genes coding for [NANP]4 (C75CS2) or [DP4NPN]2 (C75CS1) as internal inserts in HBcAg (between amino acids 75 and 81) were constructed and expressed in recombinant Salmonella typhimurium. The resulting hybrid HBcAg-CS polypeptides purified from S. typhimurium were particulate and displayed CS and HBc antigenicity, however, the HBc antigenicity was reduced compared to native recombinant HBcAg. Immunization of several mouse strains with HBcAg-CS1 and HBcAg-CS2 particles resulted in high titer, P.berghei- or P.falciparum-specific anti-CS antibodies representing all murine immunoglobulin G isotypes. The possible influence of carrier-specific immunosuppression was examined, and preexisting immunity to HBcAg did not significantly affect the immunogenicity of the CS epitopes within HBcAg-CS1 particles. Similarly, the choice of adjuvant did not significantly alter the immunogenicity of HBcAg-CS hybrid particles. Immunization in complete or incomplete Freund's adjuvant or alum resulted in equivalent anti-HBc and anti-CS humoral responses. Examination of T cell recognition of HBcAg-CS particles revealed that HBcAg-specific T cells were universally primed and CS-specific T cells were primed if the insert contained a CS-specific T cell recognition site. This indicates that the internal site in HBcAg is permissive for the inclusion of heterologous pathogen-specific T as well as B cell epitopes. Most importantly, 90 and 100% of BALB/c mice immunized with HBcAg-CS1 particles were protected against a P. berghei challenge infection in two independent experiments. Therefore, hybrid HBcAg-CS particles may represent a useful approach for future malaria vaccine development. PMID:7520465

  13. The rice immune receptor XA21 recognizes a tyrosine-sulfated protein from a Gram-negative bacterium.

    PubMed

    Pruitt, Rory N; Schwessinger, Benjamin; Joe, Anna; Thomas, Nicholas; Liu, Furong; Albert, Markus; Robinson, Michelle R; Chan, Leanne Jade G; Luu, Dee Dee; Chen, Huamin; Bahar, Ofir; Daudi, Arsalan; De Vleesschauwer, David; Caddell, Daniel; Zhang, Weiguo; Zhao, Xiuxiang; Li, Xiang; Heazlewood, Joshua L; Ruan, Deling; Majumder, Dipali; Chern, Mawsheng; Kalbacher, Hubert; Midha, Samriti; Patil, Prabhu B; Sonti, Ramesh V; Petzold, Christopher J; Liu, Chang C; Brodbelt, Jennifer S; Felix, Georg; Ronald, Pamela C

    2015-07-01

    Surveillance of the extracellular environment by immune receptors is of central importance to eukaryotic survival. The rice receptor kinase XA21, which confers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo), is representative of a large class of cell surface immune receptors in plants and animals. We report the identification of a previously undescribed Xoo protein, called RaxX, which is required for activation of XA21-mediated immunity. Xoo strains that lack RaxX, or carry mutations in the single RaxX tyrosine residue (Y41), are able to evade XA21-mediated immunity. Y41 of RaxX is sulfated by the prokaryotic tyrosine sulfotransferase RaxST. Sulfated, but not nonsulfated, RaxX triggers hallmarks of the plant immune response in an XA21-dependent manner. A sulfated, 21-amino acid synthetic RaxX peptide (RaxX21-sY) is sufficient for this activity. Xoo field isolates that overcome XA21-mediated immunity encode an alternate raxX allele, suggesting that coevolutionary interactions between host and pathogen contribute to RaxX diversification. RaxX is highly conserved in many plant pathogenic Xanthomonas species. The new insights gained from the discovery and characterization of the sulfated protein, RaxX, can be applied to the development of resistant crop varieties and therapeutic reagents that have the potential to block microbial infection of both plants and animals.

  14. The rice immune receptor XA21 recognizes a tyrosine-sulfated protein from a Gram-negative bacterium

    PubMed Central

    Pruitt, Rory N.; Schwessinger, Benjamin; Joe, Anna; Thomas, Nicholas; Liu, Furong; Albert, Markus; Robinson, Michelle R.; Chan, Leanne Jade G.; Luu, Dee Dee; Chen, Huamin; Bahar, Ofir; Daudi, Arsalan; De Vleesschauwer, David; Caddell, Daniel; Zhang, Weiguo; Zhao, Xiuxiang; Li, Xiang; Heazlewood, Joshua L.; Ruan, Deling; Majumder, Dipali; Chern, Mawsheng; Kalbacher, Hubert; Midha, Samriti; Patil, Prabhu B.; Sonti, Ramesh V.; Petzold, Christopher J.; Liu, Chang C.; Brodbelt, Jennifer S.; Felix, Georg; Ronald, Pamela C.

    2015-01-01

    Surveillance of the extracellular environment by immune receptors is of central importance to eukaryotic survival. The rice receptor kinase XA21, which confers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo), is representative of a large class of cell surface immune receptors in plants and animals. We report the identification of a previously undescribed Xoo protein, called RaxX, which is required for activation of XA21-mediated immunity. Xoo strains that lack RaxX, or carry mutations in the single RaxX tyrosine residue (Y41), are able to evade XA21-mediated immunity. Y41 of RaxX is sulfated by the prokaryotic tyrosine sulfotransferase RaxST. Sulfated, but not nonsulfated, RaxX triggers hallmarks of the plant immune response in an XA21-dependent manner. A sulfated, 21–amino acid synthetic RaxX peptide (RaxX21-sY) is sufficient for this activity. Xoo field isolates that overcome XA21-mediated immunity encode an alternate raxX allele, suggesting that coevolutionary interactions between host and pathogen contribute to RaxX diversification. RaxX is highly conserved in many plant pathogenic Xanthomonas species. The new insights gained from the discovery and characterization of the sulfated protein, RaxX, can be applied to the development of resistant crop varieties and therapeutic reagents that have the potential to block microbial infection of both plants and animals. PMID:26601222

  15. Immunization with H1, HASPB1 and MML Leishmania proteins in a vaccine trial against experimental canine leishmaniasis

    PubMed Central

    Moreno, J.; Nieto, J.; Masina, S.; Cañavate, C.; Cruz, I.; Chicharro, C.; Carrillo, E.; Napp, S.; Reymond, C.; Kaye, P.M.; Smith, D.F.; Fasel, N.; Alvar, J.

    2007-01-01

    The protective capabilities of three Leishmania recombinant proteins – histone 1 (H1) and hydrophilic acylated surface protein B1 (HASPB1) immunized singly, or together as a protein cocktail vaccine with Montanide™, and the polyprotein MML immunized with MPL®-SE adjuvant – were assessed in beagle dogs. Clinical examination of the dogs was carried out periodically under blinded conditions and the condition of the dogs defined as asymptomatic or symptomatic. At the end of the trial, we were able to confirm that following infection with L. infantum promastigotes, five out of eight dogs immunized with H1 Montanide™, and four out of eight dogs immunized with either the combination of HASPB1 with Montanide™ or the combination of H1 + HASPB1 with Montanide™, remained free of clinical signs, compared with two out of seven dogs immunized with the polyprotein MML and adjuvant MPL®-SE, and two out of eight dogs in the control group. The results demonstrate that HASPB1 and H1 antigens in combination with Montanide™ were able to induce partial protection against canine leishmaniasis, even under extreme experimental challenge conditions. PMID:17576026

  16. Multi-species protein similarity clustering reveals novel expanded immune gene families in the eastern oyster Crassostrea virginica.

    PubMed

    McDowell, Ian C; Modak, Tejashree H; Lane, Chris E; Gomez-Chiarri, Marta

    2016-06-01

    Comparative genomics research in non-model species has highlighted how invertebrate hosts possess complex diversified repertoires of immune molecules. The levels of diversification in particular immune gene families appear to differ between invertebrate lineages and even between species within lineages, reflecting differences not only in evolutionary histories, but also in life histories, environmental niches, and pathogen exposures. The goal of this research was to identify immune-related gene families experiencing high levels of diversification in eastern oysters, Crassostrea virginica. Families containing 1) transcripts differentially expressed in eastern oysters in response to bacterial challenge and 2) a larger number of transcripts compared to other species included those coding for the C1q and C-type lectin domain containing proteins (C1qDC and CTLDC), GTPase of the immune-associated proteins (GIMAP), scavenger receptors (SR), fibrinogen-C domain containing proteins (also known as FREPs), dopamine beta-hydrolase (DBH), interferon-inducible 44 (IFI44), serine protease inhibitors, apextrin, and dermatopontin. Phylogenetic analysis of two of the families significantly expanded in bivalves, IFI44 and GIMAP, showed a patchy distribution within both protostomes and deuterostomes, suggesting multiple independent losses and lineage-specific expansions. Increased availability of genomic information for a broader range of non-model species broadly distributed through vertebrate and invertebrate phyla will likely lead to improved knowledge on mechanisms of immune-gene diversification. PMID:27033806

  17. Evaluation of the immune response to Anaplasma marginale MSP5 protein using a HSV-1 amplicon vector system or recombinant protein.

    PubMed

    Palacios, Carlos; Torioni de Echaide, Susana; Mattion, Nora

    2014-12-01

    Anaplasma marginale is an intraerythrocytic vector-borne infectious agent of cattle. Immunization with the current vaccine, based on parasitized erythrocytes with live Anaplasma centrale, shows some constraints and confers partial protection, suggesting the feasibility for the development of new generation of vaccines. The aim of the present study was to assess the effect of sequential immunization of BALB/c mice, with herpesvirus amplicon vector-based vaccines combined with protein-based vaccines, on the quality of the immune response against the major surface protein 5 of A. marginale. The highest antibody titers against MSP5 were elicited in mice that received two doses of adjuvanted recombinant protein (p < 0.0001). Mice treated with a heterologous prime-boost strategy generated sustained antibody titers at least up to 200 days, and a higher specific cellular response. The results presented here showed that sequential immunization with HSV-based vectors and purified antigen enhances the quality of the immune response against A. marginale. PMID:25458492

  18. Evaluation of the immune response to Anaplasma marginale MSP5 protein using a HSV-1 amplicon vector system or recombinant protein.

    PubMed

    Palacios, Carlos; Torioni de Echaide, Susana; Mattion, Nora

    2014-12-01

    Anaplasma marginale is an intraerythrocytic vector-borne infectious agent of cattle. Immunization with the current vaccine, based on parasitized erythrocytes with live Anaplasma centrale, shows some constraints and confers partial protection, suggesting the feasibility for the development of new generation of vaccines. The aim of the present study was to assess the effect of sequential immunization of BALB/c mice, with herpesvirus amplicon vector-based vaccines combined with protein-based vaccines, on the quality of the immune response against the major surface protein 5 of A. marginale. The highest antibody titers against MSP5 were elicited in mice that received two doses of adjuvanted recombinant protein (p < 0.0001). Mice treated with a heterologous prime-boost strategy generated sustained antibody titers at least up to 200 days, and a higher specific cellular response. The results presented here showed that sequential immunization with HSV-based vectors and purified antigen enhances the quality of the immune response against A. marginale.

  19. Effect of lysophosphatidic acid on the immune inflammatory response and the connexin 43 protein in myocardial infarction

    PubMed Central

    ZHANG, DUODUO; ZHANG, YAN; ZHAO, CHUNYAN; ZHANG, WENJIE; SHAO, GUOGUANG; ZHANG, HONG

    2016-01-01

    Lysophosphatidic acid (LPA) is an intermediate product of membrane phospholipid metabolism. Recently, LPA has gained attention for its involvement in the pathological processes of certain cardiovascular diseases. The aim of the present study was to clarify the association between the effect of LPA and the immune inflammatory response, and to investigate the effects of LPA on the protein expression levels of connexin 43 during myocardial infarction. Surface electrocardiograms of myocardial infarction rats and isolated rat heart tissue samples were obtained in order to determine the effect of LPA on the incidence of arrhythmia in rats that exhibited changes in immune status. The results demonstrated that the incidence of arrhythmia decreased when the rat immune systems were suppressed, and the incidence of arrhythmia increased when the rat immune systems were enhanced. The concentration levels of tumor necrosis factor (TNF)-α were determined by ELISA, and the results demonstrated that LPA induced T lymphocyte synthesis and TNF-α release. Using a patch-clamp technique, LPA was shown to increase the current amplitude of the voltage-dependent potassium channels (Kv) and calcium-activated potassium channels (KCa) in Jurkat T cells. The protein expression of connexin 43 (Cx43) was determined by immunohistochemical staining. The results indicated that LPA caused the degradation of Cx43 and decreased the expression of Cx43. This effect was associated with the immune status of the rats. There was a further decrease in Cx43 expression in the rats of the immune-enhanced group. To the best of our knowledge, these results provide the first evidence that LPA causes arrhythmia through the regulation of immune inflammatory cells and the decrease of Cx43 protein expression. The present study provided an experimental basis for the treatment of arrhythmia and may guide clinical care. PMID:27168781

  20. Food proteins from different allergen families sensitize Balb/c mice to family-specific immune responses.

    PubMed

    Wang, Jing; Sun, Na; Zhou, Cui; Zhou, Xin; Lu, Jing; Wang, Cuiyan; Che, Huilian

    2014-01-01

    The classification of food allergens based on the structure and function of proteins contributes to the study of the relationship between bioinformatics and potential allergenicity of allergens. Food allergens always share sequence similarity with the allergens in the same allergen families. For that reason, food proteins from different allergen families may induce different patterns of immune responses in animal models. Female Balb/c mice (3-4-weeks-old) were sensitized with food proteins once per week for 4 weeks, and then challenged 2 weeks later (on Day 42 of study). Blood was collected (to obtain serum levels of histamine and protein-specific IgG1 and IgE antibodies) and measures of vascular permeability were performed 20 min after the challenge. Five food proteins (11S globulin, OVA [ovalbumin], HAS [human serum albumin] and LRP [lysine-responsive storage protein] of different allergen families, and Cry 1Ab/Ac [crystal protein]) were used to assess patterns of immune responses for each allergen family and then bioinformatics and digestive stability in simulated gastric fluid were employed to assess the overall utility of the Balb/c. The assay results indicated that, in this model, histamine and protein-specific IgE antibody levels and vascular permeability could be used to identify allergenicity of 11S globulin, OVA, and PAP (potato acid phosphatase) only. However, the results of the protein-specific IgG1 measures could only distinguish allergic food proteins with negative control. Based on bioinformatic analyses, the five different food proteins clearly induced distinct patterns of immune responses in the Balb/c model.

  1. Genetic immunization: a new monoclonal antibody for the detection of BCL-6 protein in paraffin sections.

    PubMed

    García, José-Francisco; García, Juan-Fernando; Maestre, Lorena; Lucas, Elena; Sánchez-Verde, Lydia; Romero-Chala, Silvia; Piris, Miguel-Angel; Roncador, Giovanna

    2006-01-01

    Genetic immunization can be combined with hybridoma technology to generate high-affinity monoclonal antibodies (MAbs). A new anti-BCL-6 MAb (GI191E/A8) was produced by cloning full-length BCL-6 cDNA into a eukaryotic vector and delivering this into mouse epidermis using a helium gene gun. A comparative study was made of the specificity and the effects of formalin fixation on immunohistochemistry quality of GI191E/A8 and two other anti-BCL-6 MAbs. To evaluate its possible application to differential diagnosis of lymphomas, two tissue microarrays (89 diffuse large B-cell lymphomas and 24 B-cell chronic lymphocytic leukemia cases) were stained with GI191E/A8 and another anti-BCL-6 MAb produced by conventional means. Using GI191E/A8, the detection of BCL-6 protein was significantly increased, and its specificity was independent of formalin-fixation time. Using automatic quantified analysis, the correlation between the two anti-BCL-6 MAbs tested was identical in cases with overexpression or absence of BCL-6. In cases with intermediate BCL-6 protein expression, detection with GI191E/A8 was more sensitive. A significant association of higher BCL-6 expression and longer median overall survival times in diffuse large B-cell lymphomas was found. Using conventionally produced MAbs in the same patient group, the association was not significant.

  2. RNase 7, a novel innate immune defense antimicrobial protein of healthy human skin.

    PubMed

    Harder, Jurgen; Schroder, Jens-Michael

    2002-11-29

    We analyzed healthy human skin for the presence of endogenous antimicrobial proteins that might explain the unusually high resistance of human skin against infections. A novel 14.5-kDa antimicrobial ribonuclease, termed RNase 7, was isolated from skin-derived stratum corneum. RNase 7 exhibited potent ribonuclease activity and thus may contribute to the well known ribonuclease activity of human skin. RNase 7 revealed broad spectrum antimicrobial activity against many pathogenic microorganisms and remarkably potent activity (lethal dose of 90% < 30 nm) against a vancomycin-resistant Enterococcus faecium. Molecular cloning from skin-derived primary keratinocytes and purification of RNase 7 from supernatants of cultured primary keratinocytes indicate that keratinocytes represent the major cellular source in skin and that RNase 7 is secreted. RNase 7 mRNA expression was detected in various epithelial tissues including skin, respiratory tract, genitourinary tract, and at a low level, in the gut. In addition to a constitutive expression, RNase 7 mRNA was induced in cultured primary keratinocytes by interleukin-1beta, interferon-gamma, and bacterial challenge. This is the first report demonstrating RNases as a novel class of epithelial inducible antimicrobial proteins, which may play an important role in the innate immune defense system of human epithelia.

  3. Roles of Autophagy and Autophagy-Related Proteins in Antifungal Immunity

    PubMed Central

    Kanayama, Masashi; Shinohara, Mari L.

    2016-01-01

    Autophagy was initially characterized as a process to digest cellular components, including damaged cell organelles or unused proteins. However, later studies showed that autophagy plays an important role to protect hosts from microbial infections. Accumulating evidences showed the contribution of autophagy itself and autophagy-related proteins (ATGs) in the clearance of bacteria, virus, and parasites. A number of studies also revealed the molecular mechanisms by which autophagy is initiated and developed. Furthermore, it is now understood that some ATGs are shared between two distinct processes; autophagy and LC3-associated phagocytosis (LAP). Thus, our understanding on autophagy has been greatly enhanced in the last decade. By contrast, roles of autophagy and ATGs in fungal infections are still elusive relative to those in bacterial and viral infections. Based on limited numbers of reports, ATG-mediated host responses appear to significantly vary depending on invading fungal species. In this review, we discuss how autophagy and ATGs are involved in antifungal immune responses based on recent discoveries. PMID:26925060

  4. Immunity Elicited by an Experimental Vaccine Based on Recombinant Flagellin-Porcine Circovirus Type 2 Cap Fusion Protein in Piglets.

    PubMed

    Zhu, Shanshan; Zhang, Chunyan; Wang, Jing; Wei, Li; Quan, Rong; Yang, Jiayu; Yan, Xu; Li, Zixuan; She, Ruiping; Hu, Fengjiao; Liu, Jue

    2016-01-01

    In a recent study, we reported that a recombinant protein from fusion expression of flagellin to porcine circovirus type 2 (PCV2) Cap induced robust humoral and cell-mediated immunity that afforded full protection for PCV2 infection using BALB/c mice. Here, we further evaluated the immunogenicity and protection of the recombinant protein using specific pathogen free (SPF) pigs. Twenty-five 3-week-old piglets without passively acquired immunity were divided into 5 groups. All piglets except negative controls were challenged with a virulent PCV2 at 21 days after booster vaccination and necropsied at 21 days post-challenge. Vaccination of piglets with the recombinant protein without adjuvant induced strong humoral and cellular immune responses as observed by high levels of PCV2-specific IgG antibodies and neutralizing antibodies, as well as frequencies of PCV2-specific IFN-γ-secreting cells that conferred good protection against PCV2 challenge, with significant reduced PCV2 viremia, mild lesions, low PCV2 antigen-positive cells, as well as improved body weight gain, comparable to piglets vaccinated with a commercial PCV2 subunit vaccine. These results further demonstrated that the recombinant flagellin-Cap fusion protein is capable of inducing solid protective humoral and cellular immunity when administered to pigs, thereby becoming an effective PCV2 vaccine candidate for control of PCV2 infection. PMID:26848967

  5. Die another day: molecular mechanisms of effector-triggered immunity elicited by type III secreted effector proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial pathogens inject type III secreted effector (T3SE) proteins into their hosts where they display dual roles depending on the host genotype. T3SEs promote bacterial virulence in susceptible hosts, and elicit immunity in resistant hosts. T3SEs are typically recognized when they modify a host ...

  6. Immunity Elicited by an Experimental Vaccine Based on Recombinant Flagellin-Porcine Circovirus Type 2 Cap Fusion Protein in Piglets

    PubMed Central

    Wang, Jing; Wei, Li; Quan, Rong; Yang, Jiayu; Yan, Xu; Li, Zixuan; She, Ruiping; Hu, Fengjiao; Liu, Jue

    2016-01-01

    In a recent study, we reported that a recombinant protein from fusion expression of flagellin to porcine circovirus type 2 (PCV2) Cap induced robust humoral and cell-mediated immunity that afforded full protection for PCV2 infection using BALB/c mice. Here, we further evaluated the immunogenicity and protection of the recombinant protein using specific pathogen free (SPF) pigs. Twenty-five 3-week-old piglets without passively acquired immunity were divided into 5 groups. All piglets except negative controls were challenged with a virulent PCV2 at 21 days after booster vaccination and necropsied at 21 days post-challenge. Vaccination of piglets with the recombinant protein without adjuvant induced strong humoral and cellular immune responses as observed by high levels of PCV2-specific IgG antibodies and neutralizing antibodies, as well as frequencies of PCV2-specific IFN-γ-secreting cells that conferred good protection against PCV2 challenge, with significant reduced PCV2 viremia, mild lesions, low PCV2 antigen-positive cells, as well as improved body weight gain, comparable to piglets vaccinated with a commercial PCV2 subunit vaccine. These results further demonstrated that the recombinant flagellin-Cap fusion protein is capable of inducing solid protective humoral and cellular immunity when administered to pigs, thereby becoming an effective PCV2 vaccine candidate for control of PCV2 infection. PMID:26848967

  7. Screen of Non-annotated Small Secreted Proteins of Pseudomonas syringae Reveals a Virulence Factor That Inhibits Tomato Immune Proteases

    PubMed Central

    Shindo, Takayuki; Kaschani, Farnusch; Kovács, Judit; Tian, Fang; Kourelis, Jiorgos; Hong, Tram Ngoc; Colby, Tom; Shabab, Mohammed; Chawla, Rohini; Kumari, Selva; Ilyas, Muhammad; Hörger, Anja C.; Alfano, James R.; van der Hoorn, Renier A. L.

    2016-01-01

    Pseudomonas syringae pv. tomato DC3000 (PtoDC3000) is an extracellular model plant pathogen, yet its potential to produce secreted effectors that manipulate the apoplast has been under investigated. Here we identified 131 candidate small, secreted, non-annotated proteins from the PtoDC3000 genome, most of which are common to Pseudomonas species and potentially expressed during apoplastic colonization. We produced 43 of these proteins through a custom-made gateway-compatible expression system for extracellular bacterial proteins, and screened them for their ability to inhibit the secreted immune protease C14 of tomato using competitive activity-based protein profiling. This screen revealed C14-inhibiting protein-1 (Cip1), which contains motifs of the chagasin-like protease inhibitors. Cip1 mutants are less virulent on tomato, demonstrating the importance of this effector in apoplastic immunity. Cip1 also inhibits immune protease Pip1, which is known to suppress PtoDC3000 infection, but has a lower affinity for its close homolog Rcr3, explaining why this protein is not recognized in tomato plants carrying the Cf-2 resistance gene, which uses Rcr3 as a co-receptor to detect pathogen-derived protease inhibitors. Thus, this approach uncovered a protease inhibitor of P. syringae, indicating that also P. syringae secretes effectors that selectively target apoplastic host proteases of tomato, similar to tomato pathogenic fungi, oomycetes and nematodes. PMID:27603016

  8. Screen of Non-annotated Small Secreted Proteins of Pseudomonas syringae Reveals a Virulence Factor That Inhibits Tomato Immune Proteases.

    PubMed

    Shindo, Takayuki; Kaschani, Farnusch; Yang, Fan; Kovács, Judit; Tian, Fang; Kourelis, Jiorgos; Hong, Tram Ngoc; Colby, Tom; Shabab, Mohammed; Chawla, Rohini; Kumari, Selva; Ilyas, Muhammad; Hörger, Anja C; Alfano, James R; van der Hoorn, Renier A L

    2016-09-01

    Pseudomonas syringae pv. tomato DC3000 (PtoDC3000) is an extracellular model plant pathogen, yet its potential to produce secreted effectors that manipulate the apoplast has been under investigated. Here we identified 131 candidate small, secreted, non-annotated proteins from the PtoDC3000 genome, most of which are common to Pseudomonas species and potentially expressed during apoplastic colonization. We produced 43 of these proteins through a custom-made gateway-compatible expression system for extracellular bacterial proteins, and screened them for their ability to inhibit the secreted immune protease C14 of tomato using competitive activity-based protein profiling. This screen revealed C14-inhibiting protein-1 (Cip1), which contains motifs of the chagasin-like protease inhibitors. Cip1 mutants are less virulent on tomato, demonstrating the importance of this effector in apoplastic immunity. Cip1 also inhibits immune protease Pip1, which is known to suppress PtoDC3000 infection, but has a lower affinity for its close homolog Rcr3, explaining why this protein is not recognized in tomato plants carrying the Cf-2 resistance gene, which uses Rcr3 as a co-receptor to detect pathogen-derived protease inhibitors. Thus, this approach uncovered a protease inhibitor of P. syringae, indicating that also P. syringae secretes effectors that selectively target apoplastic host proteases of tomato, similar to tomato pathogenic fungi, oomycetes and nematodes. PMID:27603016

  9. Pyrosequence analysis of expressed sequence tags for Manduca sexta hemolymph proteins involved in immune responses.

    PubMed

    Zou, Zhen; Najar, Fares; Wang, Yang; Roe, Bruce; Jiang, Haobo

    2008-06-01

    The tobacco hornworm Manduca sexta is widely used as a model organism to investigate the biochemical basis of insect physiological processes but little transcriptome information is available. To get a broad view of the larval hemolymph proteins, particularly those related to immunity, we synthesized and sequenced cDNA fragments from a mixture of eight total RNA samples: fat body and hemocytes from larvae injected with killed bacteria, fat body, hemocytes, integument and trachea from naïve larvae, and fat body and hemocytes from wandering larvae. Using massively parallel pyrosequencing, we obtained 95,458 M. sexta expressed sequence tags (ESTs) at an average size of 185bp per read. A majority of the sequences (69,429 reads) could be assembled into 7231 contigs with an average size of 300bp, 1178 of which had significant similarity with Drosophila genes from various functional groups. Only approximately 8% (606) of the contigs matched known M. sexta cDNA sequences, representing 186 of the 375 unique NCBI entries. The remaining 6625 contigs represented newly discovered cDNA segments from this well studied biochemical model insect. A search of the 7231 contigs using Tribolium castaneum, Drosophila melanogaster, and Bombyx mori immunity-related sequences revealed 424 cDNA contigs with significant similarity (E-value <1 x 10(-5)). These included 218 previously unknown M. sexta sequences coding for putative defense molecules such as pattern recognition receptors, serine proteinases, serpins, Spätzle, Toll-like receptors, intracellular signaling molecules, and antimicrobial peptides. PMID:18510979

  10. Receptor-Interacting Protein Kinase-2 Inhibition by CYLD Impairs Antibacterial Immune Responses in Macrophages

    PubMed Central

    Wex, Katharina; Schmid, Ursula; Just, Sissy; Wang, Xu; Wurm, Rebecca; Naumann, Michael; Schlüter, Dirk; Nishanth, Gopala

    2016-01-01

    Upon infection with intracellular bacteria, nucleotide oligomerization domain protein 2 recognizes bacterial muramyl dipeptide and binds, subsequently, to receptor-interacting serine/threonine kinase 2 (RIPK2), which activates immune responses via the nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) and extracellular signal-regulated kinase (ERK) pathways. Activation of RIPK2 depends on its K63 ubiquitination by E3 ligases, whereas the deubiquitinating enzyme A20 counter regulates RIPK2 activity by cleaving K63-polyubiquitin chains from RIPK2. Here, we newly identify the deubiquitinating enzyme CYLD as a new inhibitor of RIPK2. We show that CYLD binds to and removes K63-polyubiquitin chains from RIPK2 in Listeria monocytogenes (Lm) infected murine bone marrow-derived macrophages. CYLD-mediated K63 deubiquitination of RIPK2 resulted in an impaired activation of both NF-κB and ERK1/2 pathways, reduced production of proinflammatory cytokines interleukin-6 (IL-6), IL-12, anti-listerial reactive oxygen species (ROS) and nitric oxide (NO), and, finally, impaired pathogen control. In turn, RIPK2 inhibition by siRNA prevented activation of NF-κB and ERK1/2 and completely abolished the protective effect of CYLD deficiency with respect to the production of IL-6, NO, ROS, and pathogen control. Noteworthy, CYLD also inhibited autophagy of Listeria in a RIPK2-ERK1/2-dependent manner. The protective function of CYLD deficiency was dependent on interferon gamma (IFN-γ) prestimulation of infected macrophages. Interestingly, the reduced NF-κB activation in CYLD-expressing macrophages limited the protective effect of IFN-γ by reducing NF-κB-dependent signal transducers and activators of transcription-1 (STAT1) activation. Taken together, our study identifies CYLD as an important inhibitor of RIPK2-dependent antibacterial immune responses in macrophages. PMID:26834734

  11. Vaccinia virus protein K7 is a virulence factor that alters the acute immune response to infection.

    PubMed

    Benfield, Camilla T O; Ren, Hongwei; Lucas, Stuart J; Bahsoun, Basma; Smith, Geoffrey L

    2013-07-01

    Vaccinia virus (VACV) encodes many proteins that antagonize the innate immune system including a family of intracellular proteins with a B-cell lymphoma (Bcl)-2-like structure. One of these Bcl-2 proteins called K7 binds Toll-like receptor-adaptor proteins and the DEAD-box RNA helicase DDX3 and thereby inhibits the activation of NF-κB and interferon regulatory factor 3. However, the contribution of K7 to virus virulence is not known. Here a VACV lacking the K7R gene (vΔK7) was constructed and compared with control viruses that included a plaque purified wt (vK7), a revertant with the K7R gene reinserted (vK7-rev) and a frame-shifted virus in which the translational initiation codon was mutated to prevent K7 protein expression (vK7-fs). Data presented show that loss of K7 does not affect virus replication in cell culture or in vivo; however, viruses lacking the K7 protein were less virulent than controls in murine intradermal (i.d.) and intranasal (i.n.) infection models and there was an altered acute immune response to infection. In the i.d. model, vΔK7 induced smaller lesions than controls, and after i.n. infection vΔK7 induced a reduced weight loss and signs of illness, and more rapid clearance of virus from infected tissue. Concomitantly, the intrapulmonary innate immune response to infection with vΔK7 showed increased infiltration of NK cells and CD8⁺ T-cells, enhanced MHC class II expression by macrophages, and enhanced cytolysis of target cells by NK cells and VACV-specific CD8⁺ T-cells. Thus protein K7 is a virulence factor that affects the acute immune response to infection.

  12. Human cytomegalovirus tegument protein pUL83 inhibits IFI16-mediated DNA sensing for immune evasion.

    PubMed

    Li, Tuo; Chen, Jin; Cristea, Ileana M

    2013-11-13

    Nuclear sensing of viral DNA has emerged as an essential step in innate immune responses against herpesviruses. Here, we provide mechanistic insight into host recognition of human cytomegalovirus (HCMV) and subsequent immune evasion by this prominent DNA virus. We establish that the interferon-inducible protein IFI16 acts as a nuclear DNA sensor following HCMV infection, binding viral DNA and triggering expression of antiviral cytokines via the STING-TBK1-IRF3 signaling pathway. The HCMV tegument protein pUL83 inhibits this response by interacting with the IFI16 pyrin domain, blocking its oligomerization upon DNA sensing and subsequent immune signals. pUL83 disrupts IFI16 by concerted action of its N- and C-terminal domains, in which an evolutionarily conserved N-terminal pyrin association domain (PAD) binds IFI16. Additionally, phosphorylation of the N-terminal domain modulates pUL83-mediated inhibition of pyrin aggregation. Collectively, our data elucidate the interplay between host DNA sensing and HCMV immune evasion, providing targets for restoring antiviral immunity.

  13. Elongation Factor Tu and Heat Shock Protein 70 Are Membrane-Associated Proteins from Mycoplasma ovipneumoniae Capable of Inducing Strong Immune Response in Mice.

    PubMed

    Jiang, Fei; He, Jinyan; Navarro-Alvarez, Nalu; Xu, Jian; Li, Xia; Li, Peng; Wu, Wenxue

    2016-01-01

    Chronic non-progressive pneumonia, a disease that has become a worldwide epidemic has caused considerable loss to sheep industry. Mycoplasma ovipneumoniae (M. ovipneumoniae) is the causative agent of interstitial pneumonia in sheep, goat and bighorn. We here have identified by immunogold and immunoblotting that elongation factor Tu (EF-Tu) and heat shock protein 70 (HSP 70) are membrane-associated proteins on M. ovipneumonaiea. We have evaluated the humoral and cellular immune responses in vivo by immunizing BALB/c mice with both purified recombinant proteins rEF-Tu and rHSP70. The sera of both rEF-Tu and rHSP70 treated BALB/c mice demonstrated increased levels of IgG, IFN-γ, TNF-α, IL-12(p70), IL-4, IL-5 and IL-6. In addition, ELISPOT assay showed significant increase in IFN-γ+ secreting lymphocytes in the rHSP70 group when compared to other groups. Collectively our study reveals that rHSP70 induces a significantly better cellular immune response in mice, and may act as a Th1 cytokine-like adjuvant in immune response induction. Finally, growth inhibition test (GIT) of M. ovipneumoniae strain Y98 showed that sera from rHSP70 or rEF-Tu-immunized mice inhibited in vitro growth of M. ovipneumoniae. Our data strongly suggest that EF-Tu and HSP70 of M. ovipneumoniae are membrane-associated proteins capable of inducing antibody production, and cytokine secretion. Therefore, these two proteins may be potential candidates for vaccine development against M. ovipneumoniae infection in sheep.

  14. Elongation Factor Tu and Heat Shock Protein 70 Are Membrane-Associated Proteins from Mycoplasma ovipneumoniae Capable of Inducing Strong Immune Response in Mice

    PubMed Central

    Jiang, Fei; He, Jinyan; Navarro-Alvarez, Nalu; Xu, Jian; Li, Xia; Li, Peng; Wu, Wenxue

    2016-01-01

    Chronic non-progressive pneumonia, a disease that has become a worldwide epidemic has caused considerable loss to sheep industry. Mycoplasma ovipneumoniae (M. ovipneumoniae) is the causative agent of interstitial pneumonia in sheep, goat and bighorn. We here have identified by immunogold and immunoblotting that elongation factor Tu (EF-Tu) and heat shock protein 70 (HSP 70) are membrane-associated proteins on M. ovipneumonaiea. We have evaluated the humoral and cellular immune responses in vivo by immunizing BALB/c mice with both purified recombinant proteins rEF-Tu and rHSP70. The sera of both rEF-Tu and rHSP70 treated BALB/c mice demonstrated increased levels of IgG, IFN-γ, TNF-α, IL-12(p70), IL-4, IL-5 and IL-6. In addition, ELISPOT assay showed significant increase in IFN-γ+ secreting lymphocytes in the rHSP70 group when compared to other groups. Collectively our study reveals that rHSP70 induces a significantly better cellular immune response in mice, and may act as a Th1 cytokine-like adjuvant in immune response induction. Finally, growth inhibition test (GIT) of M. ovipneumoniae strain Y98 showed that sera from rHSP70 or rEF-Tu-immunized mice inhibited in vitro growth of M. ovipneumoniae. Our data strongly suggest that EF-Tu and HSP70 of M. ovipneumoniae are membrane-associated proteins capable of inducing antibody production, and cytokine secretion. Therefore, these two proteins may be potential candidates for vaccine development against M. ovipneumoniae infection in sheep. PMID:27537186

  15. Elongation Factor Tu and Heat Shock Protein 70 Are Membrane-Associated Proteins from Mycoplasma ovipneumoniae Capable of Inducing Strong Immune Response in Mice.

    PubMed

    Jiang, Fei; He, Jinyan; Navarro-Alvarez, Nalu; Xu, Jian; Li, Xia; Li, Peng; Wu, Wenxue

    2016-01-01

    Chronic non-progressive pneumonia, a disease that has become a worldwide epidemic has caused considerable loss to sheep industry. Mycoplasma ovipneumoniae (M. ovipneumoniae) is the causative agent of interstitial pneumonia in sheep, goat and bighorn. We here have identified by immunogold and immunoblotting that elongation factor Tu (EF-Tu) and heat shock protein 70 (HSP 70) are membrane-associated proteins on M. ovipneumonaiea. We have evaluated the humoral and cellular immune responses in vivo by immunizing BALB/c mice with both purified recombinant proteins rEF-Tu and rHSP70. The sera of both rEF-Tu and rHSP70 treated BALB/c mice demonstrated increased levels of IgG, IFN-γ, TNF-α, IL-12(p70), IL-4, IL-5 and IL-6. In addition, ELISPOT assay showed significant increase in IFN-γ+ secreting lymphocytes in the rHSP70 group when compared to other groups. Collectively our study reveals that rHSP70 induces a significantly better cellular immune response in mice, and may act as a Th1 cytokine-like adjuvant in immune response induction. Finally, growth inhibition test (GIT) of M. ovipneumoniae strain Y98 showed that sera from rHSP70 or rEF-Tu-immunized mice inhibited in vitro growth of M. ovipneumoniae. Our data strongly suggest that EF-Tu and HSP70 of M. ovipneumoniae are membrane-associated proteins capable of inducing antibody production, and cytokine secretion. Therefore, these two proteins may be potential candidates for vaccine development against M. ovipneumoniae infection in sheep. PMID:27537186

  16. Structures of the Ultra-High-Affinity Protein–Protein Complexes of Pyocins S2 and AP41 and Their Cognate Immunity Proteins from Pseudomonas aeruginosa

    PubMed Central

    Joshi, Amar; Grinter, Rhys; Josts, Inokentijs; Chen, Sabrina; Wojdyla, Justyna A.; Lowe, Edward D.; Kaminska, Renata; Sharp, Connor; McCaughey, Laura; Roszak, Aleksander W.; Cogdell, Richard J.; Byron, Olwyn; Walker, Daniel; Kleanthous, Colin

    2015-01-01

    How ultra-high-affinity protein–protein interactions retain high specificity is still poorly understood. The interaction between colicin DNase domains and their inhibitory immunity (Im) proteins is an ultra-high-affinity interaction that is essential for the neutralisation of endogenous DNase catalytic activity and for protection against exogenous DNase bacteriocins. The colicin DNase–Im interaction is a model system for the study of high-affinity protein–protein interactions. However, despite the fact that closely related colicin-like bacteriocins are widely produced by Gram-negative bacteria, this interaction has only been studied using colicins from Escherichia coli. In this work, we present the first crystal structures of two pyocin DNase–Im complexes from Pseudomonas aeruginosa, pyocin S2 DNase–ImS2 and pyocin AP41 DNase–ImAP41. These structures represent divergent DNase–Im subfamilies and are important in extending our understanding of protein–protein interactions for this important class of high-affinity protein complex. A key finding of this work is that mutations within the immunity protein binding energy hotspot, helix III, are tolerated by complementary substitutions at the DNase–Immunity protein binding interface. Im helix III is strictly conserved in colicins where an Asp forms polar interactions with the DNase backbone. ImAP41 contains an Asp-to-Gly substitution in helix III and our structures show the role of a co-evolved substitution where Pro in DNase loop 4 occupies the volume vacated and removes the unfulfilled hydrogen bond. We observe the co-evolved mutations in other DNase–Immunity pairs that appear to underpin the split of this family into two distinct groups. PMID:26215615

  17. Evaluation of the immune response elicited by vaccination with viral vectors encoding FMDV capsid proteins and boosted with inactivated virus.

    PubMed

    Romanutti, Carina; D'Antuono, Alejandra; Palacios, Carlos; Quattrocchi, Valeria; Zamorano, Patricia; La Torre, Jose; Mattion, Nora

    2013-08-30

    The aim of the present study was to assess the effect of introducing a priming step with replication-defective viral vectors encoding the capsid proteins of FMDV, followed by a boost with killed virus vaccines, using a suitable BALB/c mice model. Additionally, the immune response to other combined vector immunization regimens was studied. For this purpose, we analyzed different prime-boost immunizations with recombinant adenovirus (Ad), herpesvirus amplicons (Hs) and/or killed virus (KV) vaccines. The highest antibody titers were found in the group that received two doses of adjuvanted KV (P<0.002). Antibody titers were higher in those groups receiving a mixed regimen of vectors, compared to immunization with either vector alone (P<0.0001). Priming with any of the viral vectors induced a shift of the cytokine balance toward a Th1 type immune response regardless of the delivery system used for boosting. The highest IgG1 titer was induced by two doses of adjuvanted KV (P=0.0002) and the highest IgG2a titer corresponded to the group primed with Ad and boosted with KV (P=0.01). Re-stimulation of all groups of mice with 0.5 μg of inactivated virus five months later resulted in a fast increase of antibody titers in all the groups tested. After virus stimulation, antibody titers in the groups that received KV alone or Ad prime-KV boost, were indistinguishable (P=0.800). Protection from challenge was similar (75%) in the groups of animals that received Ad prime-Hs boost or Ad prime-KV boost, or two doses of oil-adjuvanted KV. The data presented in this study suggest that sequential immunization with viral vectors-based vaccines combined with protein-based vaccines have the potential to enhance the quality of the immune response against FMDV. PMID:23683999

  18. Evaluation of the immune response elicited by vaccination with viral vectors encoding FMDV capsid proteins and boosted with inactivated virus.

    PubMed

    Romanutti, Carina; D'Antuono, Alejandra; Palacios, Carlos; Quattrocchi, Valeria; Zamorano, Patricia; La Torre, Jose; Mattion, Nora

    2013-08-30

    The aim of the present study was to assess the effect of introducing a priming step with replication-defective viral vectors encoding the capsid proteins of FMDV, followed by a boost with killed virus vaccines, using a suitable BALB/c mice model. Additionally, the immune response to other combined vector immunization regimens was studied. For this purpose, we analyzed different prime-boost immunizations with recombinant adenovirus (Ad), herpesvirus amplicons (Hs) and/or killed virus (KV) vaccines. The highest antibody titers were found in the group that received two doses of adjuvanted KV (P<0.002). Antibody titers were higher in those groups receiving a mixed regimen of vectors, compared to immunization with either vector alone (P<0.0001). Priming with any of the viral vectors induced a shift of the cytokine balance toward a Th1 type immune response regardless of the delivery system used for boosting. The highest IgG1 titer was induced by two doses of adjuvanted KV (P=0.0002) and the highest IgG2a titer corresponded to the group primed with Ad and boosted with KV (P=0.01). Re-stimulation of all groups of mice with 0.5 μg of inactivated virus five months later resulted in a fast increase of antibody titers in all the groups tested. After virus stimulation, antibody titers in the groups that received KV alone or Ad prime-KV boost, were indistinguishable (P=0.800). Protection from challenge was similar (75%) in the groups of animals that received Ad prime-Hs boost or Ad prime-KV boost, or two doses of oil-adjuvanted KV. The data presented in this study suggest that sequential immunization with viral vectors-based vaccines combined with protein-based vaccines have the potential to enhance the quality of the immune response against FMDV.

  19. Immune responses elicited against rotavirus middle layer protein VP6 inhibit viral replication in vitro and in vivo.

    PubMed

    Lappalainen, Suvi; Pastor, Ana Ruth; Tamminen, Kirsi; López-Guerrero, Vanessa; Esquivel-Guadarrama, Fernando; Palomares, Laura A; Vesikari, Timo; Blazevic, Vesna

    2014-01-01

    Rotavirus (RV) is a common cause of severe gastroenteritis (GE) in children worldwide. Live oral RV vaccines protect against severe RVGE, but the immune correlates of protection are not yet clearly defined. Inner capsid VP6 protein is a highly conserved, abundant, and immunogenic RV protein, and VP6-specific mucosal antibodies, especially IgA, have been implicated to protect against viral challenge in mice. In the present study systemic and mucosal IgG and IgA responses were induced by immunizing BALB/c mice intranasally with a combination of recombinant RV VP6 protein (subgroup II [SGII]) and norovirus (NoV) virus-like particles (VLPs) used in a candidate vaccine. Following immunization mice were challenged orally with murine RV strain EDIMwt (SG non-I-non-II, G3P10[16]). In order to determine neutralizing activity of fecal samples, sera, and vaginal washes (VW) against human Wa RV (SGII, G1P1A[8]) and rhesus RV (SGI, G3P5B[3]), the RV antigen production was measured with an ELISA-based antigen reduction neutralization assay. Only VWs of immunized mice inhibited replication of both RVs, indicating heterotypic protection of induced antibodies. IgA antibody depletion and blocking experiments using recombinant VP6 confirmed that neutralization was mediated by anti-VP6 IgA antibodies. Most importantly, after the RV challenge significant reduction in viral shedding was observed in feces of immunized mice. These results suggest a significant role for mucosal RV VP6-specific IgA for the inhibition of RV replication in vitro and in vivo. In addition, these results underline the importance of non-serotype-specific immunity induced by the conserved subgroup-specific RV antigen VP6 in clearance of RV infection. PMID:25424814

  20. Immune responses elicited against rotavirus middle layer protein VP6 inhibit viral replication in vitro and in vivo

    PubMed Central

    Lappalainen, Suvi; Pastor, Ana Ruth; Tamminen, Kirsi; López-Guerrero, Vanessa; Esquivel-Guadarrama, Fernando; Palomares, Laura A; Vesikari, Timo; Blazevic, Vesna

    2014-01-01

    Rotavirus (RV) is a common cause of severe gastroenteritis (GE) in children worldwide. Live oral RV vaccines protect against severe RVGE, but the immune correlates of protection are not yet clearly defined. Inner capsid VP6 protein is a highly conserved, abundant, and immunogenic RV protein, and VP6-specific mucosal antibodies, especially IgA, have been implicated to protect against viral challenge in mice. In the present study systemic and mucosal IgG and IgA responses were induced by immunizing BALB/c mice intranasally with a combination of recombinant RV VP6 protein (subgroup II [SGII]) and norovirus (NoV) virus-like particles (VLPs) used in a candidate vaccine. Following immunization mice were challenged orally with murine RV strain EDIMwt (SG non-I-non-II, G3P10[16]). In order to determine neutralizing activity of fecal samples, sera, and vaginal washes (VW) against human Wa RV (SGII, G1P1A[8]) and rhesus RV (SGI, G3P5B[3]), the RV antigen production was measured with an ELISA-based antigen reduction neutralization assay. Only VWs of immunized mice inhibited replication of both RVs, indicating heterotypic protection of induced antibodies. IgA antibody depletion and blocking experiments using recombinant VP6 confirmed that neutralization was mediated by anti-VP6 IgA antibodies. Most importantly, after the RV challenge significant reduction in viral shedding was observed in feces of immunized mice. These results suggest a significant role for mucosal RV VP6-specific IgA for the inhibition of RV replication in vitro and in vivo. In addition, these results underline the importance of non-serotype-specific immunity induced by the conserved subgroup-specific RV antigen VP6 in clearance of RV infection. PMID:25424814

  1. Immune evasion activities of accessory proteins Vpu, Nef and Vif are conserved in acute and chronic HIV-1 infection.

    PubMed

    Mlcochova, Petra; Apolonia, Luis; Kluge, Silvia F; Sridharan, Aishwarya; Kirchhoff, Frank; Malim, Michael H; Sauter, Daniel; Gupta, Ravindra K

    2015-08-01

    Heterosexual HIV-1 transmission has been identified as a genetic bottleneck and a single transmitted/founder (T/F) variant with reduced sensitivity to type I interferon initiates productive infection in most cases. We hypothesized that particularly active accessory protein(s) may confer T/F viruses with a selective advantage in establishing HIV infection. Thus, we tested vpu, vif and nef alleles from six T/F and six chronic (CC) viruses in assays for 9 immune evasion activities involving the counteraction of interferon-stimulated genes and modulation of ligands known to activate innate immune cells. All functions were highly conserved with no significant differences between T/F and CC viruses, suggesting that these accessory protein functions are important throughout the course of infection.

  2. Functions of innate and acquired immune system are reduced in domestic pigeons (Columba livia domestica) given a low protein diet

    PubMed Central

    Mabuchi, Yuko; Frankel, Theresa L.

    2016-01-01

    Racing pigeons are exposed to and act as carriers of diseases. Dietary protein requirement for their maintenance has not been determined experimentally despite their being domesticated for over 7000 years. A maintenance nitrogen (protein) requirement (MNR) for pigeons was determined in a balance study using diets containing 6, 10 and 14% crude protein (CP). Then, the effects of feeding the diets were investigated to determine whether they were adequate to sustain innate and acquired immune functions. Nitrogen intake from the 6% CP diet was sufficient to maintain nitrogen balance and body weight in pigeons. However, the immune functions of phagocytosis, oxidative burst and lymphocyte proliferation in pigeons fed this diet were reduced compared with those fed 10 and 14% CP diets. Pigeons given the 6 and 10% CP diets had lower antibody titres following inoculation against Newcastle disease (ND) than those on the 14% CP diet. A confounding factor found on autopsy was the presence of intestinal parasites in some of the pigeons given the 6 and 10% CP diets; however, none of the pigeons used to measure MNR or acquired immunity to ND were infested with parasites. In conclusion, neither the 6 nor 10% CP diets adequately sustained acquired immune function of pigeons. PMID:27069640

  3. Immune responses to Mycobacterial heat shock protein 70 accompany self-reactivity to human BiP in rheumatoid arthritis

    PubMed Central

    Shoda, Hirofumi; Hanata, Norio; Sumitomo, Shuji; Okamura, Tomohisa; Fujio, Keishi; Yamamoto, Kazuhiko

    2016-01-01

    Rheumatoid arthritis (RA) is an autoimmune disease, and a member of human heat shock protein (HSP) 70 protein family, Binding Immunoglobulin Protein (BiP), has been identified as an important autoantigen for T and B cells. We herein focused on Mycobacterial (Myc) HSPs and immune responses to MycHSPs in RA patients. Serum titers of antibodies against MycHSP70 were significantly elevated in RA patients and correlated with serum anti-BiP antibody titers. A MycHSP70-derived HLA-DR4 major epitope was identified using the proliferative capacity of RA PBMCs as an indicator. The major epitope, MycHSP70287–306, was located at the corresponding position in the major epitope for human BiP336–355, and a strong correlation was found between the proliferation of PBMCs in response to MycHSP70287–306 and BiP336–355. The immunization of HLA-DR4 transgenic mice with MycHSP70 induced the proliferation of T cells and development of anti-BiP antibodies. In contrast, the oral administration of MycHSP70287–306 resulted in the amelioration of collagen-induced arthritis, serum antibody responses, and T cell proliferation. In conclusion, immune responses to MycHSP70 were associated with adaptive immunity against BiP in RA, and could be an important mechanism underlying the development of autoimmunity. PMID:26927756

  4. Functions of innate and acquired immune system are reduced in domestic pigeons (Columba livia domestica) given a low protein diet.

    PubMed

    Mabuchi, Yuko; Frankel, Theresa L

    2016-03-01

    Racing pigeons are exposed to and act as carriers of diseases. Dietary protein requirement for their maintenance has not been determined experimentally despite their being domesticated for over 7000 years. A maintenance nitrogen (protein) requirement (MNR) for pigeons was determined in a balance study using diets containing 6, 10 and 14% crude protein (CP). Then, the effects of feeding the diets were investigated to determine whether they were adequate to sustain innate and acquired immune functions. Nitrogen intake from the 6% CP diet was sufficient to maintain nitrogen balance and body weight in pigeons. However, the immune functions of phagocytosis, oxidative burst and lymphocyte proliferation in pigeons fed this diet were reduced compared with those fed 10 and 14% CP diets. Pigeons given the 6 and 10% CP diets had lower antibody titres following inoculation against Newcastle disease (ND) than those on the 14% CP diet. A confounding factor found on autopsy was the presence of intestinal parasites in some of the pigeons given the 6 and 10% CP diets; however, none of the pigeons used to measure MNR or acquired immunity to ND were infested with parasites. In conclusion, neither the 6 nor 10% CP diets adequately sustained acquired immune function of pigeons.

  5. A small molecule inhibitor for ATPase activity of Hsp70 and Hsc70 enhances the immune response to protein antigens

    NASA Astrophysics Data System (ADS)

    Baek, Kyung-Hwa; Zhang, Haiying; Lee, Bo Ryeong; Kwon, Young-Guen; Ha, Sang-Jun; Shin, Injae

    2015-12-01

    The ATPase activities of Hsp70 and Hsc70 are known to be responsible for regulation of various biological processes. However, little is known about the roles of Hsp70 and Hsc70 in modulation of immune responses to antigens. In the present study, we investigated the effect of apoptozole (Az), a small molecule inhibitor of Hsp70 and Hsc70, on immune responses to protein antigens. The results show that mice administered with both protein antigen and Az produce more antibodies than those treated with antigen alone, showing that Az enhances immune responses to administered antigens. Treatment of mice with Az elicits production of antibodies with a high IgG2c/IgG1 ratio and stimulates the release of Th1 and Th2-type cytokines, suggesting that Az activates the Th1 and Th2 immune responses. The observations made in the present study suggest that inhibition of Hsp70 and Hsc70 activities could be a novel strategy designing small molecule-based adjuvants in protein vaccines.

  6. Functional analysis of membrane-bound complement regulatory protein on T-cell immune response in ginbuna crucian carp.

    PubMed

    Nur, Indriyani; Abdelkhalek, Nevien K; Motobe, Shiori; Nakamura, Ryota; Tsujikura, Masakazu; Somamoto, Tomonori; Nakao, Miki

    2016-02-01

    Complements have long been considered to be a pivotal component in innate immunity. Recent researches, however, highlight novel roles of complements in T-cell-mediated adaptive immunity. Membrane-bound complement regulatory protein CD46, a costimulatory protein for T cells, is a key molecule for T-cell immunomodulation. Teleost CD46-like molecule, termed Tecrem, has been newly identified in common carp and shown to function as a complement regulator. However, it remains unclear whether Tecrem is involved in T-cell immune response. We investigated Tecrem function related to T-cell responses in ginbuna crucian carp. Ginbuna Tecrem (gTecrem) proteins were detected by immunoprecipitation using anti-common carp Tecrem monoclonal antibody (mAb) and were ubiquitously expressed on blood cells including CD8α(+) and CD4(+) lymphocytes. gTecrem expression on leucocyte surface was enhanced after stimulation with the T-cell mitogen, phytohaemagglutinin (PHA). Coculture with the anti-Tecrem mAb significantly inhibited the proliferative activity of PHA-stimulated peripheral blood lymphocytes, suggesting that cross-linking of Tecrems on T-cells interferes with a signal transduction pathway for T-cell activation. These findings indicate that Tecrem may act as a T-cell moderator and imply that the complement system in teleost, as well as mammals, plays an important role for linking adaptive and innate immunity.

  7. Protective immunity in gibel carp, Carassius gibelio of the truncated proteins of cyprinid herpesvirus 2 expressed in Pichia pastoris.

    PubMed

    Zhou, Yong; Jiang, Nan; Ma, Jie; Fan, Yuding; Zhang, Linlin; Xu, Jin; Zeng, Lingbing

    2015-12-01

    Cyprinid herpesvirus 2 (CyHV-2) infection is a newly emerged infectious disease of farmed gibel carp (Carassius gibelio) in China and causes huge economic losses to the aquaculture industry. In this study, the three membrane proteins encoded by genes ORF25, ORF25C, and ORF25D of CyHV-2 were truncated and expressed in yeast, Pichia pastoris. Screening of the recombinant yeasts was done by detecting the truncated proteins using Western blot. Through immunogold labeling, it was shown that proteins binding the colloidal gold were presented on the surface of cells. In the experiment of inhibition of virus binding by the recombinant truncated proteins, the TCID50 of the tORF25 group (10(4.1)/ml) was lower than that of tORF25C (10(4.6)/ml) or tORF25D groups (10(5)/ml). These results suggested that the proteins may be involved in attachment of the virus to the cell surface. Healthy gibel carp were immunized with 20 μg of tORF25, tORF25C, and tORF25D proteins, and the control group received PBS. Interleukin 11 (IL-11) expression in the spleens of the immunized fish peaked at day 4 and the complement component C3 (C3) genes were significantly up-regulated at day 7 post-immunization. Specific antibodies were measured in the three immunized groups and the titer detected in the tORF25 group reached 327, that was significantly higher than the tORF25C (247) or tORF25D (228) groups. When the immunized fish were challenged with live CyHV-2 by intraperitoneal injection the relative percent survival (RPS) of the tORF25, tORF25C, and tORF25D immunized groups was 75%, 63%, and 54%, respectively. The feasibility of the P. pastoris yeast expression system for the production of the recombinant truncated proteins and their apparent bioactivity suggests that tORF25, tORF25C, and tORF25D are potential candidate vaccines against Cyprinid herpesvirus 2 infection in gibel carp. PMID:26564473

  8. Induction of autoantibodies against mouse soluble proteins after immunization with living cells presenting the autoantigen at the cell surface in fusion with a human type 2 transmembrane protein.

    PubMed

    Lemaire, Muriel M; Vanhaudenarde, Aurélie; Nizet, Yannick; Dumoutier, Laure; Renauld, Jean-Christophe

    2011-03-31

    Induction of autoantibodies towards immune regulatory proteins, such as cytokines or their receptors, is a powerful strategy for functional studies on the role of these factors in vivo. Here we describe a new procedure to elicit autoantibodies by taking advantage of tumor cells as a vaccine against peptides presented at their surface in fusion with the human CD134L transmembrane protein. P1.HTR, an immunogenic variant of the P815 mastocytoma cell line, was used to generate stably transfected cell clones with expression vectors encoding the human CD134L transmembrane protein fused with either mouse IL-22BP or IL-9. Following repeated injections of living tumor cells expressing the mIL-22BP construct, mice developed autoantibodies that bind to mIL-22BP and inhibit its interaction with IL-22 in vitro. Mice similarly immunized against mIL-9 produced high titers of autoantibodies that block the activity of this cytokine in the TS1 bioassay. This procedure also inhibits IL-9 activity in vivo as no increase of serum MMCP-1 mast cell protease concentration was observed following IL-9 administration to immunized mice. As an alternative to the injection of living tumor cells expressing the CD134L-antigen fusion protein, intramuscular electrotransfer of the corresponding DNA construct also induced autoantibodies. These results validate this method as a simple and convenient approach to knock down the in vivo activity of soluble regulatory proteins, including cytokines and their receptors.

  9. Performance, immunity, and physiological responses of broilers to dietary energy and protein sequential variations.

    PubMed

    Ale Saheb Fosoul, S S; Toghyani, M; Gheisari, A; Tabeidiyan, S A; Mohammadrezaei, M; Azarfar, A

    2016-09-01

    The current trial was designed to examine effects of sequential feeding of diets varying in energy and protein contents on performance, gut development, welfare indices, and immune responses of broiler chicks. A total of 240 one-day-old broiler chicks (Ross 308) were randomly allotted to 4 treatments with 5 replicates in a completely randomized design. Sequential feeding was evaluated in 48-h cycles during 8 to 28 d of age. Diets varied in energy (E+ = 3,210 kcal/kg and E- = 2,790 kcal/kg) and protein (P+ = 25.14% CP and P- = 16.76% CP) contents. The 4 feeding programs were: control (ME: 3,000 kcal/kg, CP: 20.95%); P+/P- (P+ followed by P- containing 3,000 kcal/kg energy); E-/E+ (E- followed by E+ containing 20.95% CP), and E-P+ / E+P- (E-P+ followed by E+P-). The experiment lasted 42 d and birds were fed by a standard finishing diet from d 28 to 42. Broilers fed on E-/E+ and E-P+/E+P- had lower daily feed intake than control ones during 8 to 28 d of age (P < 0.05), while daily weight gain and feed conversion ratio were not affected significantly. Footpad dermatitis was lower in birds sequentially fed E-/E+ and E-P+/E+P- at 35 d of age (P < 0.05). Sequential feeding of diets varying in either energy or protein or both increased duration of tonic immobility at d 39 of age (P < 0.05). Feeding with E-/E+ regime decreased jejunal crypt depth, while feeding P+/P- regime increased villus height and crypt depth in the duodenum and ileum (P < 0.05). However, no relationship was found between intestinal morphology and growth performance of broiler chickens. Antibody production against sheep red blood cells as well as Newcastle and influenza disease viruses was not affected by sequential feeding. In conclusion, digestive organs and intestinal morphology might be adjusted to energy and protein variations. Sequential feeding increased the fear level in chickens. PMID:26994199

  10. T-cell immune responses to Wilms tumor 1 protein in myelodysplasia responsive to immunosuppressive therapy.

    PubMed

    Sloand, Elaine M; Melenhorst, J Joseph; Tucker, Zachary C G; Pfannes, Loretta; Brenchley, Jason M; Yong, Agnes; Visconte, Valeria; Wu, Colin; Gostick, Emma; Scheinberg, Phillip; Olnes, Matthew J; Douek, Daniel C; Price, David A; Barrett, A John; Young, Neal S

    2011-03-01

    Clinical observations and laboratory evidence link bone marrow failure in myelodysplastic syndrome (MDS) to a T cell-mediated immune process that is responsive to immunosuppressive treatment (IST) in some patients. Previously, we showed that trisomy 8 MDS patients had clonally expanded CD8(+) T-cell populations that recognized aneuploid hematopoietic progenitor cells (HPC). Furthermore, microarray analyses showed that Wilms tumor 1 (WT1) gene was overexpressed by trisomy 8 hematopoietic progenitor (CD34(+)) cells compared with CD34(+) cells from healthy donors. Here, we show that WT1 mRNA expression is up-regulated in the bone marrow mononuclear cells of MDS patients with trisomy 8 relative to healthy controls and non-trisomy 8 MDS; WT1 protein levels were also significantly elevated. In addition, using a combination of physical and functional assays to detect the presence and reactivity of specific T cells, respectively, we demonstrate that IST-responsive MDS patients exhibit significant CD4(+) and CD8(+) T-cell responses directed against WT1. Finally, WT1-specific CD8(+) T cells were present within expanded T-cell receptor Vβ subfamilies and inhibited hematopoiesis when added to autologous patient bone marrow cells in culture. Thus, our results suggest that WT1 is one of the antigens that triggers T cell-mediated myelosuppression in MDS.

  11. Fos/AP-1 proteins in bone and the immune system.

    PubMed

    Wagner, Erwin F; Eferl, Robert

    2005-12-01

    The skeleton and the immune system share a variety of different cytokines and transcription factors, thereby mutually influencing each other. These interactions are not confined to the bone marrow cavity where bone cells and hematopoietic cells exist in proximity but also occur at locations that are target sites for inflammatory bone diseases. The newly established research area termed 'osteoimmunology' attempts to unravel these skeletal/immunological relationships. Studies towards a molecular understanding of inflammatory bone diseases from an immunological as well as a bone-centered perspective have been very successful and led to the identification of several signaling pathways that are causally involved in inflammatory bone loss. Induction of receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) signals by activated T cells and subsequent activation of the key transcription factors Fos/activator protein-1 (AP-1), NF-kappaB, and NF for activation of T cells c1 (NFATc1) are in the center of the signaling networks leading to osteoclast-mediated bone loss. Conversely, nature has employed the interferon system to antagonize excessive osteoclast differentiation, although this counteracting activity appears to be overruled under pathological conditions. Here, we focus on Fos/AP-1 functions in osteoimmunology, because this osteoclastogenic transcription factor plays a central role in inflammatory bone loss by regulating genes like NFATc1 as well as the interferon system. We also attempt to put potential therapeutic strategies for inflammatory bone diseases in perspective.

  12. Proline Isomerization of the Immune Receptor-Interacting Protein RIN4 by a Cyclophilin Inhibits Effector-Triggered Immunity in Arabidopsis

    PubMed Central

    Li, Meng; Ma, Xiqing; Chiang, Yi-Hsuan; Yadeta, Koste A.; Ding, Pengfei; Dong, Liansai; Zhao, Yan; Li, Xiuming; Yu, Yufei; Zhang, Ling; Shen, Qian-Hua; Xia, Bin; Coaker, Gitta; Liu, Dong; Zhou, Jian-Min

    2016-01-01

    SUMMARY In the absence of pathogen infection, plant effector-triggered immune (ETI) receptors are maintained in a preactivation state by intermolecular interactions with other host proteins. Pathogen effector-induced alterations activate the receptor. In Arabidopsis, the ETI receptor RPM1 is activated via bacterial effector AvrB-induced phosphorylation of the RPM1-interacting protein RIN4 at Threonine 166. We find that RIN4 also interacts with the prolyl-peptidyl isomerase (PPIase) ROC1, which is reduced upon RIN4 Thr166 phosphorylation. ROC1 suppresses RPM1 immunity in a PPIase-dependent manner. Consistent with this, RIN4 Pro149 undergoes cis/trans isomerization in the presence of ROC1. While the RIN4P149V mutation abolishes RPM1 resistance, the deletion of Pro149 leads to RPM1 activation in the absence of RIN4 phosphorylation. These results support a model in which RPM1 directly senses conformational changes in RIN4 surrounding Pro149 that is controlled by ROC1. RIN4 Thr166 phosphorylation indirectly regulates RPM1 resistance by modulating the ROC1-mediated RIN4 isomerization. PMID:25299333

  13. The C-type lectin-like domain containing proteins Clec-39 and Clec-49 are crucial for Caenorhabditis elegans immunity against Serratia marcescens infection.

    PubMed

    Miltsch, S M; Seeberger, P H; Lepenies, B

    2014-07-01

    Caenorhabditis elegans exhibits protective immunity against a variety of fungal and bacterial pathogens. Since C. elegans lacks an adaptive immune system, pathogen recognition is mediated entirely by innate immunity. To date, little is known about the involvement of pattern recognition receptors (PRRs) in pathogen sensing as part of the C. elegans immunity. C-type lectin-like domain (CTLD) containing proteins represent a superfamily of PRRs. A large number of genes encoding for CTLD proteins are present in the C. elegans genome, however the role of CTLD proteins in bacterial recognition and antibacterial immunity has not yet been determined. In this study, we investigated the function of selected C. elegans CTLD proteins during infection with the Gram-negative bacterium Serratia marcescens. Wild-type and CTLD gene-deficient C. elegans strains were compared in their susceptibility to S. marcescens infection. Interestingly, survival and egg laying were significantly reduced in strains deficient for clec-39 and clec-49 indicating a role for both CTLD proteins in C. elegans immune defense against bacteria as evidenced by using S. marcescens infection. Binding studies with recombinantly expressed Clec-39-Fc and Clec-49-Fc fusion proteins revealed that both CTLD proteins recognized live bacteria in a Ca(2+)-independent manner. This study provides insight into the role of CTLD proteins in C. elegans immunity and demonstrates their function during bacterial infection.

  14. Double-stranded RNA-activated protein kinase regulates early innate immune responses during respiratory syncytial virus infection.

    PubMed

    Minor, Radiah A Corn; Limmon, Gino V; Miller-DeGraff, Laura; Dixon, Darlene; Andrews, Danica M K; Kaufman, Randal J; Imani, Farhad

    2010-04-01

    Respiratory syncytial virus (RSV) is the most common cause of childhood viral bronchiolitis and lung injury. Inflammatory responses significantly contribute to lung pathologies during RSV infections and bronchiolitis but the exact mechanisms have not been completely defined. The double-stranded RNA-activated protein kinase (PKR) functions to inhibit viral replication and participates in several signaling pathways associated with innate inflammatory immune responses. Using a functionally defective PKR (PKR(-/-)) mouse model, we investigated the role of this kinase in early events of RSV-induced inflammation. Our data showed that bronchoalveolar lavage (BAL) fluid from infected PKR(-/-) mice had significantly lower levels of several innate inflammatory cytokines and chemokines. Histological examinations revealed that there was less lung injury in infected PKR(-/-) mice as compared to the wild type. A genome-wide analysis showed that several early antiviral and immune regulatory genes were affected by PKR activation. These data suggest that PKR is a signaling molecule for immune responses during RSV infections.

  15. The mitochondrial fission factor dynamin-related protein 1 modulates T-cell receptor signalling at the immune synapse

    PubMed Central

    Baixauli, Francesc; Martín-Cófreces, Noa B; Morlino, Giulia; Carrasco, Yolanda R; Calabia-Linares, Carmen; Veiga, Esteban; Serrador, Juan M; Sánchez-Madrid, Francisco

    2011-01-01

    During antigen-specific T-cell activation, mitochondria mobilize towards the vicinity of the immune synapse. We show here that the mitochondrial fission factor dynamin-related protein 1 (Drp1) docks at mitochondria, regulating their positioning and activity near the actin-rich ring of the peripheral supramolecular activation cluster (pSMAC) of the immune synapse. Mitochondrial redistribution in response to T-cell receptor engagement was abolished by Drp1 silencing, expression of the phosphomimetic mutant Drp1S637D and the Drp1-specific inhibitor mdivi-1. Moreover, Drp1 knockdown enhanced mitochondrial depolarization and T-cell receptor signal strength, but decreased myosin phosphorylation, ATP production and T-cell receptor assembly at the central supramolecular activation cluster (cSMAC). Our results indicate that Drp1-dependent mitochondrial positioning and activity controls T-cell activation by fuelling central supramolecular activation cluster assembly at the immune synapse. PMID:21326213

  16. Deficiency of prolactin-inducible protein leads to impaired Th1 immune response and susceptibility to Leishmania major in mice.

    PubMed

    Li, Jintao; Liu, Dong; Mou, Zhirong; Ihedioha, Olivia C; Blanchard, Anne; Jia, Ping; Myal, Yvonne; Uzonna, Jude E

    2015-04-01

    Although the strategic production of prolactin-inducible protein (PIP) at several ports of pathogen entry into the body suggests it might play a role in host defense, no study has directly implicated it in immunity against any infectious agent. Here, we show for the first time that PIP deficiency is associated with reduced numbers of CD4(+) T cells in peripheral lymphoid tissues and impaired CD4(+) Th1-cell differentiation in vitro. In vivo, CD4(+) T cells from OVA-immunized, PIP-deficient mice showed significantly impaired proliferation and IFN-γ production following in vitro restimulation. Furthermore, PIP-deficient mice were highly susceptible to Leishmani major infection and failed to control lesion progression and parasite proliferation. This susceptibility was associated with impaired NO production and leishmanicidal activity of PIP KO macrophages following IFN-γ and LPS stimulation. Collectively, our findings implicate PIP as an important regulator of CD4(+) Th1-cell-mediated immunity.

  17. T cell metabolism. The protein LEM promotes CD8⁺ T cell immunity through effects on mitochondrial respiration.

    PubMed

    Okoye, Isobel; Wang, Lihui; Pallmer, Katharina; Richter, Kirsten; Ichimura, Takahuru; Haas, Robert; Crouse, Josh; Choi, Onjee; Heathcote, Dean; Lovo, Elena; Mauro, Claudio; Abdi, Reza; Oxenius, Annette; Rutschmann, Sophie; Ashton-Rickardt, Philip G

    2015-05-29

    Protective CD8(+) T cell-mediated immunity requires a massive expansion in cell number and the development of long-lived memory cells. Using forward genetics in mice, we identified an orphan protein named lymphocyte expansion molecule (LEM) that promoted antigen-dependent CD8(+) T cell proliferation, effector function, and memory cell generation in response to infection with lymphocytic choriomeningitis virus. Generation of LEM-deficient mice confirmed these results. Through interaction with CR6 interacting factor (CRIF1), LEM controlled the levels of oxidative phosphorylation (OXPHOS) complexes and respiration, resulting in the production of pro-proliferative mitochondrial reactive oxygen species (mROS). LEM provides a link between immune activation and the expansion of protective CD8(+) T cells driven by OXPHOS and represents a pathway for the restoration of long-term protective immunity based on metabolically modified cytotoxic CD8(+) T cells.

  18. Protein malnutrition impairs the immune response and influences the severity of infection in a hamster model of chronic visceral leishmaniasis.

    PubMed

    Carrillo, Eugenia; Jimenez, Maria Angeles; Sanchez, Carmen; Cunha, Joana; Martins, Camila Marinelli; da Paixão Sevá, Anaiá; Moreno, Javier

    2014-01-01

    Leishmaniasis remains one of the world's most devastating neglected tropical diseases. It mainly affects developing countries, where it often co-exists with chronic malnutrition, one of the main risk factors for developing the disease. Few studies have been published, however, on the relationship between leishmaniasis progression and malnutrition. The present paper reports the influence of protein malnutrition on the immune response and visceral disease development in adult hamsters infected with Leishmania infantum fed either standard or low protein diets. The low protein diet induced severe malnutrition in these animals, and upon infection with L. infantum 33% had severe visceral leishmaniasis compared to only 8% of animals fed the standard diet. The infected, malnourished animals showed notable leukocyte depletion, mild specific antibody responses, impairment of lymphoproliferation, presence of parasites in blood (16.67% of the hamsters) and significant increase of the splenic parasite burden. Animals fed standard diet suffered agranulocytosis and monocytopenia, but showed stronger specific immune responses and had lower parasite loads than their malnourished counterparts. The present results show that protein malnutrition promotes visceral leishmaniasis and provide clues regarding the mechanisms underlying the impairment of the immune system.

  19. Protein Malnutrition Impairs the Immune Response and Influences the Severity of Infection in a Hamster Model of Chronic Visceral Leishmaniasis

    PubMed Central

    Carrillo, Eugenia; Jimenez, Mª Angeles; Sanchez, Carmen; Cunha, Joana; Martins, Camila Marinelli; da Paixão Sevá, Anaiá; Moreno, Javier

    2014-01-01

    Leishmaniasis remains one of the world's most devastating neglected tropical diseases. It mainly affects developing countries, where it often co-exists with chronic malnutrition, one of the main risk factors for developing the disease. Few studies have been published, however, on the relationship between leishmaniasis progression and malnutrition. The present paper reports the influence of protein malnutrition on the immune response and visceral disease development in adult hamsters infected with Leishmania infantum fed either standard or low protein diets. The low protein diet induced severe malnutrition in these animals, and upon infection with L. infantum 33% had severe visceral leishmaniasis compared to only 8% of animals fed the standard diet. The infected, malnourished animals showed notable leukocyte depletion, mild specific antibody responses, impairment of lymphoproliferation, presence of parasites in blood (16.67% of the hamsters) and significant increase of the splenic parasite burden. Animals fed standard diet suffered agranulocytosis and monocytopenia, but showed stronger specific immune responses and had lower parasite loads than their malnourished counterparts. The present results show that protein malnutrition promotes visceral leishmaniasis and provide clues regarding the mechanisms underlying the impairment of the immune system. PMID:24586759

  20. Protective immunity provided by HLA-A2 epitopes for fusion and hemagglutinin proteins of measles virus

    SciTech Connect

    Oh, Sang Kon . E-mail: sangkono@baylorhealth.edu; Stegman, Brian; Pendleton, C. David; Ota, Martin O.; Pan, C.-H.; Griffin, Diane E.; Burke, Donald S.; Berzofsky, Jay A. . E-mail: berzofsk@helix.nih.gov

    2006-09-01

    Natural infection and vaccination with a live-attenuated measles virus (MV) induce CD8{sup +} T-cell-mediated immune responses that may play a central role in controlling MV infection. In this study, we show that newly identified human HLA-A2 epitopes from MV hemagglutinin (H) and fusion (F) proteins induced protective immunity in HLA-A2 transgenic mice challenged with recombinant vaccinia viruses expressing F or H protein. HLA-A2 epitopes were predicted and synthesized. Five and four peptides from H and F, respectively, bound to HLA-A2 molecules in a T2-binding assay, and four from H and two from F could induce peptide-specific CD8{sup +} T cell responses in HLA-A2 transgenic mice. Further experiments proved that three peptides from H (H9-567, H10-250, and H10-516) and one from F protein (F9-57) were endogenously processed and presented on HLA-A2 molecules. All peptides tested in this study are common to 5 different strains of MV including Edmonston. In both A2K{sup b} and HHD-2 mice, the identified peptide epitopes induced protective immunity against recombinant vaccinia viruses expressing H or F. Because F and H proteins induce neutralizing antibodies, they are major components of new vaccine strategies, and therefore data from this study will contribute to the development of new vaccines against MV infection.

  1. Proteomic Identification of Proteins Suggestive of Immune-Mediated Response or Neuronal Degeneration in Serum of Achalasia Patients

    PubMed Central

    Im, Seon Kyo; Yeo, Mari

    2013-01-01

    Background/Aims The primary pathophysiologic abnormality in achalasia is known to be a loss of inhibitory myenteric ganglion cells, which may result from an immune-mediated response or neuronal degeneration. The aim of this study was to identify proteins suggestive of an immune-mediated response or neuronal degeneration in the serum of achalasia patients using a proteomic analysis. Methods Blood samples were collected from five symptomatic achalasia patients and five sex- and age-matched healthy controls. Serum proteomic analysis was conducted, and the protein spots were identified using matrix-assisted laser desorption ionization/time-of-flight and a proteomics analyzer. The serum level of C3 was measured by enzyme-linked immunosorbent assay in nine patients with achalasia and 18 sex- and age-matched healthy controls. Results Of the 658 matched protein spots, 28 spots were up-regulated over 2-fold in the serum from achalasia patients compared with that from controls. The up-regulated proteins included complement C4B5, complement C3, cyclin-dependent kinase 5, transthyretin, and alpha 2 macroglobulin. The serum levels of C3 in achalasia patients were significantly higher than those of controls. Conclusions The serum proteomic analysis of achalasia patients suggests an immune-mediated response or neuronal degeneration. Further validation studies in larger samples and the esophageal tissue of achalasia patients are required. PMID:23898380

  2. Immunization with Pneumococcal Surface Protein K of Nonencapsulated Streptococcus pneumoniae Provides Protection in a Mouse Model of Colonization

    PubMed Central

    Keller, Lance E.; Luo, Xiao; Thornton, Justin A.; Moon, Bo Youn; Robinson, D. Ashley

    2015-01-01

    Current vaccinations are effective against encapsulated strains of Streptococcus pneumoniae, but they do not protect against nonencapsulated Streptococcus pneumoniae (NESp), which is increasing in colonization and incidence of pneumococcal disease. Vaccination with pneumococcal proteins has been assessed for its ability to protect against pneumococcal disease, but several of these proteins are not expressed by NESp. Pneumococcal surface protein K (PspK), an NESp virulence factor, has not been assessed for immunogenic potential or host modulatory effects. Mammalian cytokine expression was determined in an in vivo mouse model and in an in vitro cell culture system. Systemic and mucosal mouse immunization studies were performed to determine the immunogenic potential of PspK. Murine serum and saliva were collected to quantitate specific antibody isotype responses and the ability of antibody and various proteins to inhibit epithelial cell adhesion. Host cytokine response was not reduced by PspK. NESp was able to colonize the mouse nasopharynx as effectively as encapsulated pneumococci. Systemic and mucosal immunization provided protection from colonization by PspK-positive (PspK+) NESp. Anti-PspK antibodies were recovered from immunized mice and significantly reduced the ability of NESp to adhere to human epithelial cells. A protein-based pneumococcal vaccine is needed to provide broad protection against encapsulated and nonencapsulated pneumococci in an era of increasing antibiotic resistance and vaccine escape mutants. We demonstrate that PspK may serve as an NESp target for next-generation pneumococcal vaccines. Immunization with PspK protected against pneumococcal colonization, which is requisite for pneumococcal disease. PMID:26311246

  3. Production and Release of Antimicrobial and Immune Defense Proteins by Mammary Epithelial Cells following Streptococcus uberis Infection of Sheep

    PubMed Central

    Pisanu, Salvatore; Marogna, Gavino; Cubeddu, Tiziana; Pagnozzi, Daniela; Cacciotto, Carla; Campesi, Franca; Schianchi, Giuseppe; Rocca, Stefano

    2013-01-01

    Investigating the innate immune response mediators released in milk has manifold implications, spanning from elucidation of the role played by mammary epithelial cells (MECs) in fighting microbial infections to the discovery of novel diagnostic markers for monitoring udder health in dairy animals. Here, we investigated the mammary gland response following a two-step experimental infection of lactating sheep with the mastitis-associated bacterium Streptococcus uberis. The establishment of infection was confirmed both clinically and by molecular methods, including PCR and fluorescent in situ hybridization of mammary tissues. Proteomic investigation of the milk fat globule (MFG), a complex vesicle released by lactating MECs, enabled detection of enrichment of several proteins involved in inflammation, chemotaxis of immune cells, and antimicrobial defense, including cathelicidins and calprotectin (S100A8/S100A9), in infected animals, suggesting the consistent involvement of MECs in the innate immune response to pathogens. The ability of MECs to produce and release antimicrobial and immune defense proteins was then demonstrated by immunohistochemistry and confocal immunomicroscopy of cathelicidin and the calprotectin subunit S100A9 on mammary tissues. The time course of their release in milk was also assessed by Western immunoblotting along the course of the experimental infection, revealing the rapid increase of these proteins in the MFG fraction in response to the presence of bacteria. Our results support an active role of MECs in the innate immune response of the mammary gland and provide new potential for the development of novel and more sensitive tools for monitoring mastitis in dairy animals. PMID:23774600

  4. Protection against lethal measles virus infection in mice by immune-stimulating complexes containing the hemagglutinin or fusion protein.

    PubMed Central

    Varsanyi, T M; Morein, B; Löve, A; Norrby, E

    1987-01-01

    The importance of each of the two surface glycoproteins of measles virus in active and passive immunization was examined in mice. Infected-cell lysates were depleted of either the hemagglutinin (H) or fusion (F) glycoprotein by using multiple cycles of immunoaffinity chromatography. The products were used to prepare immune-stimulating complexes (iscoms) containing either F or H glycoprotein. Such complexes are highly immunogenic, possibly as a result of effective presentation of viral proteins to the immune system [B. Morein, B. Sundquist, S. Höglund, K. Dalsgaard, and A. Osterhaus, Nature (London) 308:457-460, 1984]. Groups of 3-week-old BALB/c mice were inoculated with the iscom preparations. All animals developed hemolysis-inhibiting antibodies, whereas only sera of animals immunized with the iscoms containing the H glycoprotein had hemagglutination-inhibiting antibodies. Sera from animals immunized with the H or F preparation only precipitated the homologous glycoprotein in radioimmune precipitation assays. The immunized animals were challenged with a lethal dose of the hamster neurotropic variant of measles virus. Of the 7-week-old animals in the nonimmunized control group, 50% died within 10 days after challenge. No animals in the immunized groups showed symptoms of disease throughout the observation period of 3 months. Passive administration of anti-H monoclonal antibodies gave full protection against the 100% lethal acute infection with the hamster neurotropic variant of measles virus in newborn mice, whereas anti-F monoclonal antibodies failed to protect the animals. This study emphasizes that both H and F glycoproteins need to be considered in the development of measles virus subunit vaccines. Images PMID:2960833

  5. Targeting tumor-associated immune suppression with selective protein kinase A type I (PKAI) inhibitors may enhance cancer immunotherapy.

    PubMed

    Hussain, Muzammal; Shah, Zahir; Abbas, Nasir; Javeed, Aqeel; Mukhtar, Muhammad Mahmood; Zhang, Jiancun

    2016-01-01

    Despite the tremendous progress in last few years, the cancer immunotherapy has not yet improved disease-free because of the tumor-associated immune suppression being a major barrier. Novel trends to enhance cancer immunotherapy aims at harnessing the therapeutic manipulation of signaling pathways mediating the tumor-associated immune suppression, with the general aims of: (a) reversing the tumor immune suppression; (b) enhancing the innate and adaptive components of anti-tumor immunosurveillance, and (c) protecting immune cells from the suppressive effects of T regulatory cells (Tregs) and the tumor-derived immunoinhibitory mediators. A particular striking example in this context is the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A type I (PKAI) pathway. Oncogenic cAMP/PKAI signaling has long been implicated in the initiation and progression of several human cancers. Emerging data indicate that cAMP/PKAI signaling also contributes to tumor- and Tregs-derived suppression of innate and adaptive arms of anti-tumor immunosurveillance. Therapeutically, selective PKAI inhibitors have been developed which have shown promising anti-cancer activity in pre-clinical and clinical settings. Rp-8-Br-cAMPS is a selective PKAI antagonist that is widely used as a biochemical tool in signal transduction research. Collateral data indicate that Rp-8-Br-cAMPS has shown immune-rescuing potential in terms of enhancing the innate and adaptive anti-tumor immunity, as well as protecting adaptive T cells from the suppressive effects of Tregs. Therefore, this proposal specifically implicates that combining selective PKAI antagonists/inhibitors with cancer immunotherapy may have multifaceted benefits, such as rescuing the endogenous anti-tumor immunity, enhancing the efficacy of cancer immunotherapy, and direct anti-cancer effects.

  6. PGRP-LB is a maternally transmitted immune milk protein that influences symbiosis and parasitism in tsetse’s offspring

    PubMed Central

    Wang, Jingwen; Aksoy, Serap

    2012-01-01

    Beneficial microbe functions range from host dietary supplementation to development and maintenance of host immune system. In mammals, newborn progeny are quickly colonized with a symbiotic fauna that is provisioned in mother’s milk and that closely resembles that of the parent. Tsetse fly (Diptera: Glossinidae) also depends on the obligate symbiont Wigglesworthia for nutritional supplementation, optimal fecundity, and immune system development. Tsetse progeny develop one at a time in an intrauterine environment and receive nourishment and symbionts in mother’s milk. We show that the host Peptidoglycan Recognition Protein (PGRP-LB) is expressed only in adults and is a major component of the milk that nourishes the developing progeny. The amidase activity associated with PGRP-LB may scavenge the symbiotic peptidoglycan and prevent the induction of tsetse's Immune Deficiency pathway that otherwise can damage the symbionts. Reduction of PGRP-LB experimentally diminishes female fecundity and damages Wigglesworthia in the milk through induction of antimicrobial peptides, including Attacin. Larvae that receive less maternal PGRP-LB give rise to adults with fewer Wigglesworthia and hyperimmune responses. Such adults also suffer dysregulated immunity, as indicated by the presence of higher trypanosome densities in parasitized adults. We show that recPGRP-LB has antimicrobial and antitrypanosomal activities that may regulate symbiosis and impact immunity. Thus, PGRP-LB plays a pivotal role in tsetse’s fitness by protecting symbiosis against host-inflicted damage during development and by controlling parasite infections in adults that can otherwise reduce host fecundity. PMID:22689989

  7. Echinoderm immunity.

    PubMed

    Smith, L Courtney; Ghosh, Julie; Buckley, Katherine M; Clow, Lori A; Dheilly, Nolwenn M; Haug, Tor; Henson, John H; Li, Chun; Lun, Cheng Man; Majeske, Audrey J; Matranga, Valeria; Nair, Sham V; Rast, Jonathan P; Raftos, David A; Roth, Mattias; Sacchi, Sandro; Schrankel, Catherine S; Stensvåg, Klara

    2010-01-01

    A survey for immune genes in the genome for the purple sea urchin has shown that the immune system is complex and sophisticated. By inference, immune responses of all echinoderms maybe similar. The immune system is mediated by several types of coelomocytes that are also useful as sensors of environmental stresses. There are a number of large gene families in the purple sea urchin genome that function in immunity and of which at least one appears to employ novel approaches for sequence diversification. Echinoderms have a simpler complement system, a large set of lectin genes and a number of antimicrobial peptides. Profiling the immune genes expressed by coelomocytes and the proteins in the coelomic fluid provide detailed information about immune functions in the sea urchin. The importance of echinoderms in maintaining marine ecosystem stability and the disastrous effects of their removal due to disease will require future collaborations between ecologists and immunologists working towards understanding and preserving marine habitats. PMID:21528703

  8. A Small Cysteine-Rich Protein from the Asian Soybean Rust Fungus, Phakopsora pachyrhizi, Suppresses Plant Immunity

    PubMed Central

    Qi, Mingsheng; Link, Tobias I.; Müller, Manuel; Hirschburger, Daniela; Pudake, Ramesh N.; Pedley, Kerry F.; Braun, Edward; Voegele, Ralf T.; Baum, Thomas J.

    2016-01-01

    The Asian soybean rust fungus, Phakopsora pachyrhizi, is an obligate biotrophic pathogen causing severe soybean disease epidemics. Molecular mechanisms by which P. pachyrhizi and other rust fungi interact with their host plants are poorly understood. The genomes of all rust fungi encode many small, secreted cysteine-rich proteins (SSCRP). While these proteins are thought to function within the host, their roles are completely unknown. Here, we present the characterization of P. pachyrhizi effector candidate 23 (PpEC23), a SSCRP that we show to suppress plant immunity. Furthermore, we show that PpEC23 interacts with soybean transcription factor GmSPL12l and that soybean plants in which GmSPL12l is silenced have constitutively active immunity, thereby identifying GmSPL12l as a negative regulator of soybean defenses. Collectively, our data present evidence for a virulence function of a rust SSCRP and suggest that PpEC23 is able to suppress soybean immune responses and physically interact with soybean transcription factor GmSPL12l, a negative immune regulator. PMID:27676173

  9. Immune responses induced by recombinant Bacillus subtilis expressing the spike protein of transmissible gastroenteritis virus in pigs.

    PubMed

    Mou, Chunxiao; Zhu, Liqi; Xing, Xianping; Lin, Jian; Yang, Qian

    2016-07-01

    Transmissible gastroenteritis (TGE) causes severe diarrhea in suckling piglets, results in enormous economic loss in swine-producing areas of the world. To develop an effective, safe, and convenient vaccine for the prevention of TGE, we have constructed a recombinant Bacillus subtilis strain (B. subtilis CotGSG) displaying the transmissible gastroenteritis virus (TGEV) spike (S) protein and discussed its immune function to intestinal submucosal dendritic cells (DCs). Our results showed that the recombinant B. subtilis had the ability to recruit more DCs to sample B. subtilis CotGSG, migrate to MLNs, and induce immune responses. Immunized piglets with B. subtilis CotGSG could significantly elevate the specific SIgA titers in feces, IgG titers and neutralizing antibodies in serum. Collectively, our results suggested that recombinant B. subtilis CotGSG expressing the TGEV S protein could effectively induce immune responses via DCs, and provided a perspective on potential novel strategy and approach that may be applicable to the development of the next generation of TGEV vaccines. PMID:26988122

  10. In silico interaction of methyl isocyanate with immune protein responsible for Mycobacterium tuberculosis infection using molecular docking.

    PubMed

    Shrivastava, Rahul; Yasir, Mohammad; Tripathi, Manish; Singh, Pushpendra

    2016-01-01

    This article reports in silico analysis of methyl isocyanate (MIC) on different key immune proteins against Mycobacterium tuberculosis. The analysis shows that MIC is released in the Bhopal gas tragedy in 1984, which is highly toxic and extremely hazardous to human health. In this study, we have selected immune proteins to perform molecular docking with the help of Autodock 4.0. Results show that the CD40 ligand and alpha5beta1 integrin have higher inhibition compared to plasminogen activator urokinase, human glutathione synthetase, mitogen-activated protein kinase (P38 MAPK 14), surfactant protein-B, -D (SP-D), and pulmonary SP-D. MIC interacted with His-125, Try-146 residue of CD40 ligand and Ala-149, and Arg-152 residue of alpha5beta1 integrin and affects the proteins functioning by binding on their active sites. These inhibitory conformations were energetically and statistically favored and supported the evidence from wet laboratory experiments reported in the literature. We can conclude that MIC directly or indirectly affects these proteins, which shows that survivals of the disaster suffer from the diseases like tuberculosis infection and lung cancer.

  11. Protective immunity of a Pichia pastoris expressed recombinant iridovirus major capsid protein in the Chinese giant salamander, Andrias davidianus.

    PubMed

    Zhou, Yong; Fan, Yuding; LaPatra, Scott E; Ma, Jie; Xu, Jin; Meng, Yan; Jiang, Nan; Zeng, Lingbing

    2015-10-13

    The major capsid protein (MCP) is the main immunogenic protein of iridoviruses, that has been widely used as an immunogen in vaccination trials. In this study, the codon-optimized giant salamander iridovirus (GSIV) MCP gene (O-MCP) was synthesized and cloned into a pPICZα B vector for secretory expression in the methylotrophic yeast Pichia pastoris after methanol induction. The expression of the O-MCP protein was detected by the Bradford protein assay, SDS-PAGE, Western blotting and electron microscopy. The Bradford protein assay indicated that the concentration of the O-MCP expressed was about 40 μg/ml in culture supernatants. SDS-PAGE analysis revealed that the O-MCP had a molecular weight of about 66 kDa and reacted with a His-specific MAb that was confirmed by Western blotting. Electron microscopy observations revealed that the purified O-MCP could self-assemble into virus-like particles. Healthy giant salamanders were vaccinated by intramuscular injection with the O-MCP antigen at a dose of 20 μg/individual. The numbers of erythrocytes and leukocytes in the peripheral blood of immunized Chinese giant salamanders increased significantly at day 3 and reached a peak at day 5 post-immunization. Meanwhile, the differential leukocyte counts of monocytes and neutrophils increased significantly at day 5 post-immunization compared to that of the control group. The percentage of lymphocytes was 71.33 ± 3.57% at day 21 post-immunization. The neutralization assay showed that the serum neutralizing antibody titer reached 321 at day 21 post-immunization. The GSIV challenge test revealed that the relative percent survival of Chinese giant salamanders vaccinated with O-MCP was 78%. These results indicated that the O-MCP antigen expressed by the Pichia pastoris system elicited significant immune response in the Chinese giant salamander against GSIV and might represent a potential yeast-derived vaccine candidate that could be used for the control of disease caused by the

  12. Ultrastructural localization of highly variable 185/333 immune response proteins in the coelomocytes of the sea urchin, Heliocidaris erythrogramma.

    PubMed

    Dheilly, Nolwenn M; Birch, Debra; Nair, Sham V; Raftos, David A

    2011-11-01

    The 185/333 proteins of sea urchins represent a family of highly variable immune response molecules with unknown functions. In this study, we show that 185/333 proteins are expressed by three cell types: amoebocytes, colourless spherule cells and gut-associated amoebocytes. A sub-population of amoebocytes express 185/333 proteins on the membranes of vesicles emanating from the trans-Golgi and which later fuse with the plasma membranes of the cells. The previously uncharacterized gut-associated amoebocytes also show a high level of 185/333 protein expression on their internal vesicles and plasma membranes. Colourless spherule cells contain 185/333 proteins within large spherules (specialized intracellular vesicles). In the presence of bacteria and yeast, the ultrastucture of colourless spherule cells changes and 185/333 proteins disappear. In contrast, 185/333 proteins were not found in the phagosomes of coelomocytes. The 185/333-positive gut amoebocytes were often associated with anuclear bodies, which appeared to incorporate material of microbial origin that was surrounded by 185/333 proteins. The association between 185/333 proteins on gut amoebocytes and anuclear bodies suggests that these proteins may be involved in the phagocytosis of microbes in the gut epithelium.

  13. Salmonella typhimurium impedes innate immunity with a mast-cell-suppressing protein tyrosine phosphatase, SptP.

    PubMed

    Choi, Hae Woong; Brooking-Dixon, Rhea; Neupane, Subham; Lee, Chul-Jin; Miao, Edward A; Staats, Herman F; Abraham, Soman N

    2013-12-12

    The virulence of Salmonella is linked to its invasive capacity and suppression of adaptive immunity. This does not explain, however, the rapid dissemination of the pathogen after it breaches the gut. In our study, S. Typhimurium suppressed degranulation of local mast cells (MCs), resulting in limited neutrophil recruitment and restricting outflow of vascular contents into infection sites, thus facilitating bacterial spread. MC suppression was mediated by secreted effector protein (SptP), which shares structural homology with Yersinia YopH. SptP functioned by dephosphorylating the vesicle fusion protein N-ethylmalemide-sensitive factor and by blocking phosphorylation of Syk. Without SptP, orally challenged S. Typhimurium failed to suppress MC degranulation and exhibited limited colonization of the mesenteric lymph nodes. Administration of SptP to sites of E. coli infection markedly enhanced its virulence. Thus, SptP-mediated inactivation of local MCs is a powerful mechanism utilized by S. Typhimurium to impede early innate immunity.

  14. Binding of the Fap2 protein of Fusobacterium nucleatum to human inhibitory receptor TIGIT protects tumors from immune cell attack.

    PubMed

    Gur, Chamutal; Ibrahim, Yara; Isaacson, Batya; Yamin, Rachel; Abed, Jawad; Gamliel, Moriya; Enk, Jonatan; Bar-On, Yotam; Stanietsky-Kaynan, Noah; Coppenhagen-Glazer, Shunit; Shussman, Noam; Almogy, Gideon; Cuapio, Angelica; Hofer, Erhard; Mevorach, Dror; Tabib, Adi; Ortenberg, Rona; Markel, Gal; Miklić, Karmela; Jonjic, Stipan; Brennan, Caitlin A; Garrett, Wendy S; Bachrach, Gilad; Mandelboim, Ofer

    2015-02-17

    Bacteria, such as Fusobacterium nucleatum, are present in the tumor microenvironment. However, the immunological consequences of intra-tumoral bacteria remain unclear. Here, we have shown that natural killer (NK) cell killing of various tumors is inhibited in the presence of various F. nucleatum strains. Our data support that this F. nucleatum-mediated inhibition is mediated by human, but not by mouse TIGIT, an inhibitory receptor present on all human NK cells and on various T cells. Using a library of F. nucleatum mutants, we found that the Fap2 protein of F. nucleatum directly interacted with TIGIT, leading to the inhibition of NK cell cytotoxicity. We have further demonstrated that tumor-infiltrating lymphocytes expressed TIGIT and that T cell activities were also inhibited by F. nucleatum via Fap2. Our results identify a bacterium-dependent, tumor-immune evasion mechanism in which tumors exploit the Fap2 protein of F. nucleatum to inhibit immune cell activity via TIGIT.

  15. The μ Subunit of Arabidopsis Adaptor Protein-2 Is Involved in Effector-Triggered Immunity Mediated by Membrane-Localized Resistance Proteins.

    PubMed

    Hatsugai, Noriyuki; Hillmer, Rachel; Yamaoka, Shohei; Hara-Nishimura, Ikuko; Katagiri, Fumiaki

    2016-05-01

    Endocytosis has been suggested to be important in the cellular processes of plant immune responses. However, our understanding of its role during effector-triggered immunity (ETI) is still limited. We have previously shown that plant endocytosis, especially clathrin-coated vesicle formation at the plasma membrane, is mediated by the adaptor protein-2 (AP-2) complex and that loss of the μ subunit of AP-2 (AP2M) affects plant growth and floral organ development. Here, we report that AP2M is required for full-strength ETI mediated by the disease resistance (R) genes RPM1 and RPS2 in Arabidopsis. Reduced ETI was observed in an ap2m mutant plant, measured by growth of Pseudomonas syringae pv. tomato DC3000 strains carrying the corresponding effector genes avrRpm1 or avrRpt2 and by hypersensitive cell death response and defense gene expression triggered by these strains. In contrast, RPS4-mediated ETI and its associated immune responses were not affected by the ap2m mutation. While RPM1 and RPS2 are localized to the plasma membrane, RPS4 is localized to the cytoplasm and nucleus. Our results suggest that AP2M is involved in ETI mediated by plasma membrane-localized R proteins, possibly by mediating endocytosis of the immune receptor complex components from the plasma membrane.

  16. Immunization with the cysteine proteinase Ldccys1 gene from Leishmania (Leishmania) chagasi and the recombinant Ldccys1 protein elicits protective immune responses in a murine model of visceral leishmaniasis.

    PubMed

    Ferreira, Josie Haydée L; Gentil, Luciana Girotto; Dias, Suzana Souza; Fedeli, Carlos Eduardo C; Katz, Simone; Barbiéri, Clara Lúcia

    2008-01-30

    The gene Ldccys1 encoding a cysteine proteinase of 30 kDa from Leishmania (Leishmania) chagasi, as well as the recombinant cysteine proteinase rLdccys1, obtained by cloning and expression of the Ldccys1 gene in the pHIS vector, were used to evaluate their ability to induce immune protective responses in BALB/c mice against L. (L.) chagasi infection. Mice were immunized subcutaneously with rLdccys1 plus Bacille Calmette Guerin (BCG) or Propionibacterium acnes as adjuvants or intramuscularly with a plasmid carrying the Ldccys1 gene (Ldccys1/pcDNA3) and CpG ODN as the adjuvant, followed by a booster with rLdccys1 plus CpG ODN. Two weeks after immunization the animals were challenged with 1 x 10(7) amastigotes of L. (L.) chagasi. Both immunization protocols induced significant protection against L. (L.) chagasi infection as shown by a very low parasite load in the spleen of immunized mice compared to the non-immunized controls. However, DNA immunization was 10-fold more protective than immunization with the recombinant protein. Whereas rLdccys1 induced a significant secretion of IFN-gamma and nitric oxide (NO), animals immunized with the Ldccys1 gene increased the production of IgG2a antibodies, IFN-gamma and NO. These results indicated that protection triggered by the two immunization protocols was correlated to a predominant Th1 response.

  17. The First Structure of a Lantibiotic Immunity Protein, SpaI from Bacillus subtilis, Reveals a Novel Fold*

    PubMed Central

    Christ, Nina A.; Bochmann, Sophie; Gottstein, Daniel; Duchardt-Ferner, Elke; Hellmich, Ute A.; Düsterhus, Stefanie; Kötter, Peter; Güntert, Peter; Entian, Karl-Dieter; Wöhnert, Jens

    2012-01-01

    Lantibiotics are peptide-derived antibiotics that inhibit the growth of Gram-positive bacteria via interactions with lipid II and lipid II-dependent pore formation in the bacterial membrane. Due to their general mode of action the Gram-positive producer strains need to express immunity proteins (LanI proteins) for protection against their own lantibiotics. Little is known about the immunity mechanism protecting the producer strain against its own lantibiotic on the molecular level. So far, no structures have been reported for any LanI protein. We solved the structure of SpaI, a LanI protein from the subtilin producing strain Bacillus subtilis ATCC 6633. SpaI is a 16.8-kDa lipoprotein that is attached to the outside of the cytoplasmic membrane via a covalent diacylglycerol anchor. SpaI together with the ABC transporter SpaFEG protects the B. subtilis membrane from subtilin insertion. The solution-NMR structure of a 15-kDa biologically active C-terminal fragment reveals a novel fold. We also demonstrate that the first 20 N-terminal amino acids not present in this C-terminal fragment are unstructured in solution and are required for interactions with lipid membranes. Additionally, growth tests reveal that these 20 N-terminal residues are important for the immunity mediated by SpaI but most likely are not part of a possible subtilin binding site. Our findings are the first step on the way of understanding the immunity mechanism of B. subtilis in particular and of other lantibiotic producing strains in general. PMID:22904324

  18. Innate immunity of surfactant proteins A and D in urinary tract infection with uropathogenic Escherichia coli

    PubMed Central

    Hu, Fengqi; Ding, Guohua; Zhang, Zhiyong; Gatto, Louis A.; Hawgood, Samuel; Poulain, Francis R.; Cooney, Robert N.; Wang, Guirong

    2015-01-01

    To investigate the effects of surfactant proteins A and D (SP-A, SP-D) in urinary tract infection (UTI), SP-A and SP-D double knockout (SP-A/D KO) and wild type (WT) C57BL/6 female mice were infected with uropathogenic Escherichia coli by intravesical inoculation. Compared with WT mice SP-A/D KO mice showed increased susceptibility to UTI as evidenced by higher bacterial CFU, more infiltrating neutrophils and severe pathological changes. Keratinocyte-derived chemokine increased in the kidney of WT mice but not in SP-A/D KO mice 24 h post-infection. Compared to control, level of IL-17 was elevated in the kidney of infected WT and SP-A/D KO mice and the level of IL-17 was higher in the infected SP-A/D KO mice than infected WT mice 24 and 48 h post-infection. Basal level of p38 MAPK phosphorylation in SP-A/D KO mice was higher compared to WT mice. Phosphorylated-p38 level was elevated in the kidney of WT mice post-infection but not in SP-A/D KO mice. Furthermore, in vitro growth of uropathogenic E. coli was inhibited by SP-A and SP-D. We conclude that SP-A and SP-D function as mediators of innate immunity by inhibiting bacterial growth and modulating renal inflammation in part by regulating p38 MAPK-related pathway in murine UTI. PMID:26511057

  19. The Encapsulation of Hemagglutinin in Protein Bodies Achieves a Stronger Immune Response in Mice than the Soluble Antigen

    PubMed Central

    Hofbauer, Anna; Melnik, Stanislav; Tschofen, Marc; Arcalis, Elsa; Phan, Hoang T.; Gresch, Ulrike; Lampel, Johannes; Conrad, Udo; Stoger, Eva

    2016-01-01

    Zein is a water-insoluble polymer from maize seeds that has been widely used to produce carrier particles for the delivery of therapeutic molecules. We encapsulated a recombinant model vaccine antigen in newly formed zein bodies in planta by generating a fusion construct comprising the ectodomain of hemagglutinin subtype 5 and the N-terminal part of γ-zein. The chimeric protein was transiently produced in tobacco leaves, and H5-containing protein bodies (PBs) were used to immunize mice. An immune response was achieved in all mice treated with H5-zein, even at low doses. The fusion to zein markedly enhanced the IgG response compared the soluble H5 control, and the effect was similar to a commercial adjuvant. The co-administration of adjuvants with the H5-zein bodies did not enhance the immune response any further, suggesting that the zein portion itself mediates an adjuvant effect. While the zein portion used to induce protein body formation was only weakly immunogenic, our results indicate that zein-induced PBs are promising production and delivery vehicles for subunit vaccines. PMID:26909090

  20. A role for plasma cell targeting agents in immune tolerance induction in autoimmune disease and antibody responses to therapeutic proteins.

    PubMed

    Rosenberg, A S; Pariser, A R; Diamond, B; Yao, L; Turka, L A; Lacana, E; Kishnani, P S

    2016-04-01

    Antibody responses to life saving therapeutic protein products, such as enzyme replacement therapies (ERT) in the setting of lysosomal storage diseases, have nullified product efficacy and caused clinical deterioration and death despite treatment with immune-suppressive therapies. Moreover, in some autoimmune diseases, pathology is mediated by a robust antibody response to endogenous proteins such as is the case in pulmonary alveolar proteinosis, mediated by antibodies to Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF). In this work, we make the case that in such settings, when the antibody response is high titered, sustained, and refractory to immune suppressive treatments, the antibody response is mediated by long-lived plasma cells which are relatively unperturbed by immune suppressants including rituximab. However, long-lived plasma cells can be targeted by proteasome inhibitors such as bortezomib. Recent reports of successful reversal of antibody responses with bortezomib in the settings of ERT and Thrombotic Thrombocytopenic Purpura (TTP) argue that the safety and efficacy of such plasma cell targeting agents should be evaluated in larger scale clinical trials to delineate the risks and benefits of such therapies in the settings of antibody-mediated adverse effects to therapeutic proteins and autoantibody mediated pathology. PMID:26928739

  1. Immune Response Elicited by DNA Vaccination Using Lactococcus lactis Is Modified by the Production of Surface Exposed Pathogenic Protein

    PubMed Central

    Pontes, Daniela; Azevedo, Marcela; Innocentin, Silvia; Blugeon, Sébastien; Lefévre, François; Azevedo, Vasco; Miyoshi, Anderson; Courtin, Pascal; Chapot-Chartier, Marie-Pierre; Langella, Philippe; Chatel, Jean-Marc

    2014-01-01

    In this study, we compared immune responses elicited by DNA immunization using Lactococcus lactis or L. lactis expressing the Staphylococcus aureus invasin Fibronectin Binding Protein A (FnBPA) at its surface. Both strains carried pValac:BLG, a plasmid containing the cDNA of Beta-Lactoglobulin (BLG), and were designated LL-BLG and LL-FnBPA+ BLG respectively. A TH2 immune response characterized by the secretion of IL-4 and IL-5 in medium of BLG reactivated splenocytes was detected after either oral or intranasal administration of LL-FnBPA+ BLG. In contrast, intranasal administration of LL-BLG elicited a TH1 immune response. After BLG sensitization, mice previously intranasally administered with LL-BLG showed a significantly lower concentration of BLG-specific IgE than the mice non-administered. Altenatively administration of LL-FnBPA+ BLG didn't modify the BLG-specific IgE concentration obtained after sensitization, thus confirming the TH2 orientation of the immune response. To determine if the TH2-skewed immune response obtained with LL-FnBpA+ BLG was FnBPA-specific or not, mice received another L. lactis strain producing a mutated form of the Listeria monocytogenes invasin Internalin A intranasally, allowing thus the binding to murine E-cadherin, and containing pValac:BLG (LL-mInlA+ BLG). As with LL-FnBPA+ BLG, LL-mInlA+ BLG was not able to elicit a TH1 immune response. Furthermore, we observed that these difference were not due to the peptidoglycan composition of the cell wall as LL-FnBPA+ BLG, LL-mInlA+ BLG and LL-BLG strains shared a similar composition. DNA vaccination using LL-BLG elicited a pro-inflammatory TH1 immune response while using LL-FnBPA+ BLG or LL-mInlA+ BLG elicited an anti-inflammatory TH2 immune response. PMID:24465412

  2. Synergistic effect of detergents and aluminium phosphate on the humoral immune response to bacterial and viral membrane proteins.

    PubMed

    Teerlink, T; Beuvery, E C; Evenberg, D; van Wezel, T L

    1987-12-01

    The influence of detergents on the immunogenic activity of the major outer membrane protein of Neisseria gonorrhoeae was investigated. Most detergents tested were found to enhance the immune response. This effect was synergistic with the adjuvant activity of AlPO4. The combination of detergent and AlPO4 showed a stronger adjuvant activity than Freund's complete adjuvant. The adjuvant effect was only observed with protein preparations with very low lipopolysaccharide content. The immunostimulating effect of detergents was also observed with meningococcal group C polysaccharide conjugated to a Haemophilus influenzae type b outer membrane protein and with the fusion protein of measles virus. The influence of some detergent parameters (critical micelle concentration, hydrophile-lipophile balance, charge) was investigated.

  3. An unbiased nuclear proteomics approach reveals novel nuclear protein components that participates in MAMP-triggered immunity

    PubMed Central

    Fakih, Zainab; Ahmed, Md Bulbul; Letanneur, Claire; Germain, Hugo

    2016-01-01

    ABSTRACT (MAMP)-triggered immunity (MTI) is the first layer of molecular defense encountered by pathogens. Genetic screens have contributed to our knowledge of MTI, but are limited to phenotype-causing mutations. Here we attempt to identify novel factors involved in the early event leading to plant MTI by comparing the nuclear proteomes of two Arabidopsis genotypes treated with chitosan. Our approach revealed that following chitosan treatment, cerk1 plants had many nuclear accumulating proteins in common, but also some unique ones, when compared with Col-0 plants. Analysis of the identified proteins revealed a nuclear accumulation of DNA-modifying enzymes, RNA-binding proteins and ribosomal proteins. Our results demonstrate that nuclear proteomic is a valid, phenotype-independent approach to uncover factor involved in cellular processes. PMID:27177187

  4. Receptor-like kinase SOBIR1/EVR interacts with receptor-like proteins in plant immunity against fungal infection.

    PubMed

    Liebrand, Thomas W H; van den Berg, Grardy C M; Zhang, Zhao; Smit, Patrick; Cordewener, Jan H G; America, Antoine H P; America, Antione H P; Sklenar, Jan; Jones, Alexandra M E; Tameling, Wladimir I L; Robatzek, Silke; Thomma, Bart P H J; Joosten, Matthieu H A J

    2013-06-11

    The plant immune system is activated by microbial patterns that are detected as nonself molecules. Such patterns are recognized by immune receptors that are cytoplasmic or localized at the plasma membrane. Cell surface receptors are represented by receptor-like kinases (RLKs) that frequently contain extracellular leucine-rich repeats and an intracellular kinase domain for activation of downstream signaling, as well as receptor-like proteins (RLPs) that lack this signaling domain. It is therefore hypothesized that RLKs are required for RLPs to activate downstream signaling. The RLPs Cf-4 and Ve1 of tomato (Solanum lycopersicum) mediate resistance to the fungal pathogens Cladosporium fulvum and Verticillium dahliae, respectively. Despite their importance, the mechanism by which these immune receptors mediate downstream signaling upon recognition of their matching ligand, Avr4 and Ave1, remained enigmatic. Here we show that the tomato ortholog of the Arabidopsis thaliana RLK Suppressor Of BIR1-1/Evershed (SOBIR1/EVR) and its close homolog S. lycopersicum (Sl)SOBIR1-like interact in planta with both Cf-4 and Ve1 and are required for the Cf-4- and Ve1-mediated hypersensitive response and immunity. Tomato SOBIR1/EVR interacts with most of the tested RLPs, but not with the RLKs FLS2, SERK1, SERK3a, BAK1, and CLV1. SOBIR1/EVR is required for stability of the Cf-4 and Ve1 receptors, supporting our observation that these RLPs are present in a complex with SOBIR1/EVR in planta. We show that SOBIR1/EVR is essential for RLP-mediated immunity and propose that the protein functions as a regulatory RLK of this type of cell-surface receptors.

  5. Intestinal transport of sulfanilic acid in rats immunized with protein-sulfanilic acid conjugate.

    PubMed

    Yamamoto, A; Kawaratani, T; Kawashima, K; Hashida, M; Sezaki, H

    1990-07-01

    Intestinal transport of sulfanilic acid was examined by means of an in vitro everted sac technique in rats immunized with a bovine gamma-globulin-sulfanilic acid conjugate. At a low concentration of sulfanilic acid, the intestinal transport of sulfanilic acid was decreased in rats immunized with bovine gamma-globulin-sulfanilic acid conjugate. This phenomenon was dose dependent and antigen specific, since there was no difference in the transport of sulfanilic acid at a high concentration and of an unrelated hapten. These results suggested that parenteral immunization impaired not only the intestinal transport of macromolecular antigens, as previously shown, but also the transport of the low molecular weight hapten, sulfanilic acid.

  6. Sulfated galactans from Gracilaria fisheri bind to shrimp haemocyte membrane proteins and stimulate the expression of immune genes.

    PubMed

    Rudtanatip, Tawut; Withyachumnarnkul, Boonsirm; Wongprasert, Kanokpan

    2015-11-01

    Previous studies demonstrated that sulfated galactans (SG) from Gracilaria fisheri (G. fisheri) exhibit immunostimulant activity in shrimp. The present study was conducted to test the hypothesis that SG stimulates signaling molecules of the immune response of shrimp by binding to receptors on the host cell membrane. Accordingly, we evaluated the ability of SG to bind to shrimp haemocytes and showed that SG bound to the shrimp haemocyte membrane (SHM), potentially to specific receptors. Furthermore, this binding was associated with an activation of immune response genes of shrimp. Data from confocal laser scanning micrographs revealed that FITC-labeled SG bound to haemocytes. Far western blot analysis demonstrated that SHM peptides, with molecular sizes of 13, 14, 15, 17, and 25 kDa, were associated with SG. Peptide sequence analysis of the isolated bands using LC-MS/MS and NCBI blast search revealed the identity of the 13, 14, and 17 kDa peptides as lipopolysaccharide and β-1,3-glucan binding protein (LGBP). SG induced the expression of immune related genes and downstream signaling mediators of LGBP including IMD, IKKs, NF-κB, antimicrobial peptides (crustin and PEN-4), the antiviral immunity (dicer), and proPO system (proPO-I and proPO-II). A LGBP neutralizing assay with anti-LGBP antibody indicated a decrease in SG-induced expression of LGBP downstream signaling mediators and the immune related genes. In conclusion, this study demonstrated that the SG-stimulated immune activity in haemocytes is mediated, in part, through the LGBP, and IMD-NF-κB pathway.

  7. Chikungunya virus induces IPS-1-dependent innate immune activation and protein kinase R-independent translational shutoff.

    PubMed

    White, Laura K; Sali, Tina; Alvarado, David; Gatti, Evelina; Pierre, Philippe; Streblow, Daniel; Defilippis, Victor R

    2011-01-01

    Chikungunya virus (CHIKV) is an arthritogenic mosquito-transmitted alphavirus that is undergoing reemergence in areas around the Indian Ocean. Despite the current and potential danger posed by this virus, we know surprisingly little about the induction and evasion of CHIKV-associated antiviral immune responses. With this in mind we investigated innate immune reactions to CHIKV in human fibroblasts, a demonstrable in vivo target of virus replication and spread. We show that CHIKV infection leads to activation of the transcription factor interferon regulatory factor 3 (IRF3) and subsequent transcription of IRF3-dependent antiviral genes, including beta interferon (IFN-β). IRF3 activation occurs by way of a virus-induced innate immune signaling pathway that includes the adaptor molecule interferon promoter stimulator 1 (IPS-1). Despite strong transcriptional upregulation of these genes, however, translation of the corresponding proteins is not observed. We further demonstrate that translation of cellular (but not viral) genes is blocked during infection and that although CHIKV is found to trigger inactivation of the translational molecule eukaryotic initiation factor subunit 2α by way of the double-stranded RNA sensor protein kinase R, this response is not required for the block to protein synthesis. Furthermore, overall diminution of cellular RNA synthesis is also observed in the presence of CHIKV and transcription of IRF3-dependent antiviral genes appears specifically blocked late in infection. We hypothesize that the observed absence of IFN-β and antiviral proteins during infection results from an evasion mechanism exhibited by CHIKV that is dependent on widespread shutoff of cellular protein synthesis and a targeted block to late synthesis of antiviral mRNA transcripts.

  8. Modulation of immune response to Toxoplasma gondii in sheep by immunization with a DNA vaccine encoding ROP1 antigen as a fusion protein with ovine CD154.

    PubMed

    Hiszczyńska-Sawicka, Elżbieta; Li, Hong; Xu, Janet Boyu; Holec-Gąsior, Lucyna; Kur, Józef; Sedcole, Richard; Bickerstaffe, Roy; Stankiewicz, Mirosław

    2011-12-29

    CD154 is a cell surface molecule expressed by activated T cells. CD40 and CD154 interaction is critically important in regulating humoral and cell-mediated immune responses. In this study we have investigated whether a DNA vaccine encoding rhoptry protein 1 (ROP1) of Toxoplasma gondii, and encoding ovine CD154 induces an enhanced ROP1-specific immune response in sheep. Two groups of twelve animals received two intramuscular injections, of a DNA plasmid encoding T. gondii ROP1 antigen (group 1) or an ROP1 antigen fused to ovine CD154 (group 2). There were two control groups of sheep. One was injected with an empty vector (group 3) and the other received no injections at all (group 4). The injection of the plasmid containing ROP1 (group 1) at weeks 0 and 4 induced a significant IgG2 response at week 2 which was amplified at week 4 after the booster injection and persisted to week 8 compared to the control animals in groups 3 and 4. For IgG1, significant differences from the control animals were only observed from week 5 onwards. The fusion of CD154 and ROP1 elicited significant IgG1 and IgG2 responses from week 1 which were amplified from weeks 5 to 8 compared to the control animals in groups 3 and 4. The IgG1 response was significantly higher in group 2 animals receiving pROP1-CD154 compared to group 1 receiving pROP1 only. There was no significant difference in IgG2 responses between groups 1 and 2. Significant differences in IFN-γ levels were only observed in treatment group 1 at week 2 and treatment group 2 at weeks 1 and 2 compared to the control animals. The results demonstrated that an intramuscular injection of pROP1-CD154 gene to sheep significantly enhanced their immune response and induced a mixed Th1/Th2 response while the intramuscular injection of pROP1 only induced a Th1-specific immune response.

  9. The Fundamental Role of NOX Family Proteins in Plant Immunity and Their Regulation

    PubMed Central

    Wang, Ya-Jing; Wei, Xiao-Yong; Jing, Xiu-Qing; Chang, Yan-Li; Hu, Chun-Hong; Wang, Xiang; Chen, Kun-Ming

    2016-01-01

    NADPH oxidases (NOXs), also known as respiratory burst oxidase homologs (RBOHs), are the major source of reactive oxygen species (ROS), and are involved in many important processes in plants such as regulation of acclimatory signaling and programmed cell death (PCD). Increasing evidence shows that NOXs play crucial roles in plant immunity and their functions in plant immune responses are not as separate individuals but with other signal molecules such as kinases, Rac/Rop small GTPases and hormones, mediating a series of signal transmissions. In a similar way, NOX-mediated signaling also participates in abiotic stress response of plants. We summarized here the complex role and regulation mechanism of NOXs in mediating plant immune response, and the viewpoint that abiotic stress response of plants may be a kind of special plant immunity is also proposed. PMID:27240354

  10. Analysis of Globodera rostochiensis effectors reveals conserved functions of SPRYSEC proteins in suppressing and eliciting plant immune responses

    PubMed Central

    Ali, Shawkat; Magne, Maxime; Chen, Shiyan; Obradovic, Natasa; Jamshaid, Lubna; Wang, Xiaohong; Bélair, Guy; Moffett, Peter

    2015-01-01

    Potato cyst nematodes (PCNs), including Globodera rostochiensis (Woll.), are important pests of potato. Plant parasitic nematodes produce multiple effector proteins, secreted from their stylets, to successfully infect their hosts. These include proteins delivered to the apoplast and to the host cytoplasm. A number of effectors from G. rostochiensis predicted to be delivered to the host cytoplasm have been identified, including several belonging to the secreted SPRY domain (SPRYSEC) family. SPRYSEC proteins are unique to members of the genus Globodera and have been implicated in both the induction and the repression of host defense responses. We have tested the properties of six different G. rostochiensis SPRYSEC proteins by expressing them in Nicotiana benthamiana and N. tabacum. We have found that all SPRYSEC proteins tested are able to suppress defense responses induced by NB-LRR proteins as well as cell death induced by elicitors, suggesting that defense repression is a common characteristic of members of this effector protein family. At the same time, GrSPRYSEC-15 elicited a defense responses in N. tabacum, which was found to be resistant to a virus expressing GrSPRYSEC-15. These results suggest that SPRYSEC proteins may possess characteristics that allow them to be recognized by the plant immune system. PMID:26322064

  11. Analysis of Globodera rostochiensis effectors reveals conserved functions of SPRYSEC proteins in suppressing and eliciting plant immune responses.

    PubMed

    Ali, Shawkat; Magne, Maxime; Chen, Shiyan; Obradovic, Natasa; Jamshaid, Lubna; Wang, Xiaohong; Bélair, Guy; Moffett, Peter

    2015-01-01

    Potato cyst nematodes (PCNs), including Globodera rostochiensis (Woll.), are important pests of potato. Plant parasitic nematodes produce multiple effector proteins, secreted from their stylets, to successfully infect their hosts. These include proteins delivered to the apoplast and to the host cytoplasm. A number of effectors from G. rostochiensis predicted to be delivered to the host cytoplasm have been identified, including several belonging to the secreted SPRY domain (SPRYSEC) family. SPRYSEC proteins are unique to members of the genus Globodera and have been implicated in both the induction and the repression of host defense responses. We have tested the properties of six different G. rostochiensis SPRYSEC proteins by expressing them in Nicotiana benthamiana and N. tabacum. We have found that all SPRYSEC proteins tested are able to suppress defense responses induced by NB-LRR proteins as well as cell death induced by elicitors, suggesting that defense repression is a common characteristic of members of this effector protein family. At the same time, GrSPRYSEC-15 elicited a defense responses in N. tabacum, which was found to be resistant to a virus expressing GrSPRYSEC-15. These results suggest that SPRYSEC proteins may possess characteristics that allow them to be recognized by the plant immune system.

  12. Effects of fish protein hydrolysate on growth performance and humoral immune response in large yellow croaker (Pseudosciaena crocea R.)* §

    PubMed Central

    Tang, Hong-gang; Wu, Tian-xing; Zhao, Zhan-yu; Pan, Xiao-dong

    2008-01-01

    We investigated the effects of fish protein hydrolysate (FPH) on growth performance and humoral immune response of the large yellow croaker (Pseudosciaena crocea R.). One thousand and two hundred large yellow croakers [initial average weight: (162.75±23.85) g] were divided into four groups and reared in floating sea cages (3 m×3 m×3 m). The animals were fed with 4 diets: basal diet only (control) or diets supplemented with 5%, 10% and 15% (w/w) FPH. The results show that dietary FPH levels significantly influenced the growth and immunity of the large yellow croaker. Compared with the control group, total weight gain (TWG) in all treatment groups, relative weight gain (RWG) and specific growth rate (SGR) in fish fed with diets supplemented with 10% and 15% FPH were significantly increased (P<0.05). Similar results were observed in immune parameters [lysozyme activity, serum complements, immunoglobulin M (IgM)]. Lysozyme activity, complement C4 and IgM were also significantly increased (P<0.05) in fish fed with diets supplemented with 10% and 15% FPH, while complement C3 level was significantly increased (P<0.05) in all treatment groups. In general, with the supplementation of FPH, particularly at dose of 10%, the growth performance and immunity of the large yellow croaker can be improved effectively. PMID:18763300

  13. Inhibitors of apoptosis proteins (IAPs) are required for effective T-cell expansion/survival during antiviral immunity in mice.

    PubMed

    Gentle, Ian E; Moelter, Isabel; Lechler, Nadja; Bambach, Sarah; Vucikuja, Smiljka; Häcker, Georg; Aichele, Peter

    2014-01-30

    Inhibitors of apoptosis proteins (IAPs) were originally described as regulating apoptosis by direct binding to caspases. More recently, IAPs have been identified as important modulators of canonical and noncanonical nuclear factor κB signaling via their ubiquitin-E3 ligase activity. IAPs are therefore, not only gatekeepers of cell death, but are probably also involved in the regulation of inflammation, as well as innate and adaptive immunity. In this study, we analyzed the role of IAPs in T-cell immunity during lymphocytic choriomeningitis virus (LCMV) infection by pharmacological targeting with an IAP antagonist/second mitochondria-derived activator of caspase-mimetic. Expansion of virus-specific CD8 T cells was drastically reduced in LCMV-infected mice exposed to IAP antagonists. Accordingly, virus control was substantially impaired, indicated by high virus titres in the spleen and the spread of LCMV to peripheral organs. The profound negative effect of IAP antagonists on T-cell immunity was partially linked to tumor necrosis factor-mediated cell death of activated T cells and required inhibition of X-linked inhibitor of apoptosis, as well as cellular IAP-1. Thus, IAPs play an important role in T-cell expansion and survival in the context of a highly inflammatory environment such as a virus infection, indicating that IAP antagonists may interfere with immune responses.

  14. The Salmonella Effector Protein SopA Modulates Innate Immune Responses by Targeting TRIM E3 Ligase Family Members

    PubMed Central

    Kamanova, Jana; Sun, Hui; Lara-Tejero, Maria; Galán, Jorge E.

    2016-01-01

    Salmonella Typhimurium stimulates inflammatory responses in the intestinal epithelium, which are essential for its ability to replicate within the intestinal tract. Stimulation of these responses is strictly dependent on the activity of a type III secretion system encoded within its pathogenicity island 1, which through the delivery of effector proteins, triggers signaling pathways leading to inflammation. One of these effectors is SopA, a HECT-type E3 ligase, which is required for the efficient stimulation of inflammation in an animal model of Salmonella Typhimurium infection. We show here that SopA contributes to the stimulation of innate immune responses by targeting two host E3 ubiquitin ligases, TRIM56 and TRIM65. We also found that TRIM65 interacts with the innate immune receptor MDA5 enhancing its ability to stimulate interferon-β signaling. Therefore, by targeting TRIM56 and TRIM65, SopA can stimulate signaling through two innate immune receptors, RIG-I and MDA5. These findings describe a Salmonella mechanism to modulate inflammatory responses by directly targeting innate immune signaling mechanisms. PMID:27058235

  15. Major cytoplasmic membrane protein of Legionella pneumophila, a genus common antigen and member of the hsp 60 family of heat shock proteins, induces protective immunity in a guinea pig model of Legionnaires' disease.

    PubMed Central

    Blander, S J; Horwitz, M A

    1993-01-01

    We have examined the capacity of the major cytoplasmic membrane protein (MCMP) of Legionella pneumophila, a genus common antigen and member of the hsp 60 family of heat shock proteins, to induce protective immunity in a guinea pig model of Legionnaires' disease. We purified MCMP to homogeneity from L. pneumophila by buffer extraction, ion-exchange chromatography, and molecular sieve chromatography. Guinea pigs immunized with MCMP developed a strong cell-mediated immune response to the immunogen manifest by marked cutaneous delayed-type hypersensitivity. Guinea pigs immunized with MCMP and then challenged with a lethal aerosol dose of L. pneumophila exhibited a high level of protective immunity. Altogether, in four independent experiments, 55 of 64 (86%) animals immunized three times with 0.6-40 micrograms MCMP including 11 of 11 (100%) animals immunized three times with 40 micrograms MCMP survived aerosol challenge with L. pneumophila compared with 1 of 29 (3%) sham-immunized control animals (P < 0.0001, Cochran-Mantel-Haenszel X2 statistic for pooled data). To our knowledge, MCMP is the first member of the hsp 60 family of proteins shown to induce protective immunity to a microbial pathogen. MCMP has potential as a vaccine against Legionnaires' disease. Since MCMP is a genus common antigen, vaccination with a combination of MCMPs derived from different Legionella species has the potential of inducing protective immunity against all the major Legionella species causing human disease. Images PMID:8432872

  16. Different genome stability proteins underpin primed and naïve adaptation in E. coli CRISPR-Cas immunity

    PubMed Central

    Ivančić-Baće, Ivana; Cass, Simon D; Wearne, Stephen J; Bolt, Edward L

    2015-01-01

    CRISPR-Cas is a prokaryotic immune system built from capture and integration of invader DNA into CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci, termed ‘Adaptation’, which is dependent on Cas1 and Cas2 proteins. In Escherichia coli, Cascade-Cas3 degrades invader DNA to effect immunity, termed ‘Interference’. Adaptation can interact with interference (‘primed’), or is independent of it (‘naïve’). We demonstrate that primed adaptation requires the RecG helicase and PriA protein to be present. Genetic analysis of mutant phenotypes suggests that RecG is needed to dissipate R-loops at blocked replication forks. Additionally, we identify that DNA polymerase I is important for both primed and naive adaptation, and that RecB is needed for naïve adaptation. Purified Cas1-Cas2 protein shows specificity for binding to and nicking forked DNA within single strand gaps, and collapsing forks into DNA duplexes. The data suggest that different genome stability systems interact with primed or naïve adaptation when responding to blocked or collapsed invader DNA replication. In this model, RecG and Cas3 proteins respond to invader DNA replication forks that are blocked by Cascade interference, enabling DNA capture. RecBCD targets DNA ends at collapsed forks, enabling DNA capture without interference. DNA polymerase I is proposed to fill DNA gaps during spacer integration. PMID:26578567

  17. Evaluation of recombinant P23 protein as a vaccine for passive immunization of newborn calves against Cryptosporidium parvum.

    PubMed

    Askari, N; Shayan, P; Mokhber-Dezfouli, M R; Ebrahimzadeh, E; Lotfollahzadeh, S; Rostami, A; Amininia, N; Ragh, M J

    2016-05-01

    Cryptosporidiosis is a zoonotic protozoan disease that affects the gastrointestinal tract of animals and humans. Diarrhoea as the most important indication of the infection leads to high economic losses in livestock industries and is a life threatening infection in immunocompromised individuals. In the absence of the effective drugs, vaccine has an effective role in the prevention of infection. For this purpose we developed a vaccine utilizing recombinant P23 protein and immunized pregnant cows four times from 70 days to parturition every 2 weeks. After parturition, each calf received his dam colostrum and challenged with 1 × 10(7) Cryptosporidium parvum oocysts at 12 h of age. Results showed that in contrast with the control group, the antibody titre in the sera and first milking colostra of the immunized cows significantly increased and calves fed hyperimmune colostrum did not show cryptosporidiosis signs. Moreover, enriched colostrum not only reduced significantly the amount of oocyst excretion but also delayed its onset. Our study showed that recombinant P23 protein could be used for passive immunization of newborn calves against Cryptosporidium parvum. PMID:27012710

  18. IRE1/bZIP60-Mediated Unfolded Protein Response Plays Distinct Roles in Plant Immunity and Abiotic Stress Responses

    PubMed Central

    Blanco, Francisca; Boatwright, Jon Lucas; Moreno, Ignacio; Jordan, Melissa R.; Chen, Yani; Brandizzi, Federica; Dong, Xinnian

    2012-01-01

    Endoplasmic reticulum (ER)-mediated protein secretion and quality control have been shown to play an important role in immune responses in both animals and plants. In mammals, the ER membrane-located IRE1 kinase/endoribonuclease, a key regulator of unfolded protein response (UPR), is required for plasma cell development to accommodate massive secretion of immunoglobulins. Plant cells can secrete the so-called pathogenesis-related (PR) proteins with antimicrobial activities upon pathogen challenge. However, whether IRE1 plays any role in plant immunity is not known. Arabidopsis thaliana has two copies of IRE1, IRE1a and IRE1b. Here, we show that both IRE1a and IRE1b are transcriptionally induced during chemically-induced ER stress, bacterial pathogen infection and treatment with the immune signal salicylic acid (SA). However, we found that IRE1a plays a predominant role in the secretion of PR proteins upon SA treatment. Consequently, the ire1a mutant plants show enhanced susceptibility to a bacterial pathogen and are deficient in establishing systemic acquired resistance (SAR), whereas ire1b is unaffected in these responses. We further demonstrate that the immune deficiency in ire1a is due to a defect in SA- and pathogen-triggered, IRE1-mediated cytoplasmic splicing of the bZIP60 mRNA, which encodes a transcription factor involved in the expression of UPR-responsive genes. Consistently, IRE1a is preferentially required for bZIP60 splicing upon pathogen infection, while IRE1b plays a major role in bZIP60 processing upon Tunicamycin (Tm)-induced stress. We also show that SA-dependent induction of UPR-responsive genes is altered in the bzip60 mutant resulting in a moderate susceptibility to a bacterial pathogen. These results indicate that the IRE1/bZIP60 branch of UPR is a part of the plant response to pathogens for which the two Arabidopsis IRE1 isoforms play only partially overlapping roles and that IRE1 has both bZIP60-dependent and bZIP60-independent functions in

  19. Dysfunction of constitutive and inducible ubiquitin-proteasome system in amyotrophic lateral sclerosis: implication for protein aggregation and immune response.

    PubMed

    Bendotti, Caterina; Marino, Marianna; Cheroni, Cristina; Fontana, Elena; Crippa, Valeria; Poletti, Angelo; De Biasi, Silvia

    2012-05-01

    The ubiquitin-proteasome system (UPS) is the major intracellular proteolytic mechanism controlling the degradation of misfolded/abnormal proteins. A common hallmark in amyotrophic lateral sclerosis (ALS) and in other neurodegenerative disorders is the accumulation of misfolded/abnormal proteins into the damaged neurons, leading to the formation of cellular inclusions that are mostly ubiquitin-positive. Although proteolysis is a complex mechanism requiring the participation of different pathways, the abundant accumulation of ubiquitinated proteins strongly suggests an important contribution of UPS to these neuropathological features. The use of cellular and animal models of ALS, particularly those expressing mutant SOD1, the gene mutation most represented in familiar ALS, has provided significant evidence for a role of UPS in protein inclusions formation and motor neuron death. This review will specifically discuss this piece of evidence and provide suggestions of potential strategies for therapeutic intervention. We will also discuss the finding that, unlike the constitutive proteasome subunits, the inducible subunits are overexpressed early during disease progression in SOD1 mice models of ALS. These subunits form the immunoproteasome and generate peptides for the major histocompatibility complex class I molecules, suggesting a role of this system in the immune responses associated with the pathological features of ALS. Since recent discoveries indicate that innate and adaptive immunity may influence the disease process, in this review we will also provide evidence of a possible connection between immune-inflammatory reactions and UPS function, in the attempt to better understand the etiopathology of ALS and to identify appropriate targets for novel treatment strategies of this devastating disease. PMID:22033150

  20. A novel homolog of protein tyrosine kinase Fyn identified in Lampetra japonica with roles in the immune response.

    PubMed

    Zhang, Qiong; Song, Xueying; Su, Peng; Li, Ranran; Liu, Chang; Gou, Meng; Wang, Hao; Liu, Xin; Li, Qingwei

    2016-04-01

    The non-receptor protein tyrosine kinase (nrPTK) Fyn, a member of the avian sarcoma virus transforming gene (Src) kinase family, plays a very significant role in cell growth, survival, apoptosis, tumor formation and immune response. In this study, a homolog of nrPTK Fyn was identified for the first time in the lamprey, Lampetra japonica and was named "Lja-Fyn". The cDNA fragment of lamprey lja-fyn contains a 1611-bp open reading frame, which encodes a protein of 537 amino acids. Multiple sequence alignment analysis showed that it shares four conserved domains (Src homology (SH) 4, SH3, SH2 and protein kinases catalytic domains) and a variable unique domain with vertebrates Fyn molecules. Though Lja-Fyn has high sequence similarity with typical Fyn and Yes molecules of jawed vertebrates, the identities among Lja-Fyn and typical Fyn molecules in unique domain are relatively higher than that among Lja-Fyn and typical Yes molecules. The result indicates that Lja-Fyn is a homolog of Fyn rather than Yes. The phylogenetic analysis showed that Fyn, Yes and Src molecules are grouped into three distinct phylogenetic clusters, and Lja-Fyn is grouped as a single branch in Fyn cluster. The real-time quantitative PCR assay revealed the wide distribution of the lja-fyn mRNA in lamprey immune related tissues. After stimulation with mixed antigens, the levels of lja-fyn mRNA were obviously up-regulated in the gill and lymphocyte-like cells, and the similar results were got by western blot analysis of Lja-Fyn protein expression. These results indicated that nrPTK Lja-Fyn was likely to be involved in immune response. Furthermore, our present findings also provide the necessary information for understanding the distinction between lamprey Lja-Fyn and other members of jawed vertebrates in Src family. PMID:26743129

  1. The Pseudomonas syringae effector protein HopZ1a suppresses effector-triggered immunity.

    PubMed

    Macho, Alberto P; Guevara, Carlos M; Tornero, Pablo; Ruiz-Albert, Javier; Beuzón, Carmen R

    2010-09-01

    *The Pseudomonas syringae pv syringae type III effector HopZ1a is a member of the HopZ effector family of cysteine-proteases that triggers immunity in Arabidopsis. This immunity is dependent on HopZ1a cysteine-protease activity, and independent of known resistance genes. We have previously shown that HopZ1a-triggered immunity is partially additive to that triggered by AvrRpt2. These partially additive effects could be caused by at least two mechanisms: their signalling pathways share a common element(s), or one effector interferes with the response triggered by the other. *Here, we investigate the molecular basis for the partially additive effect displayed by AvrRpt2- and HopZ1a-triggered immunities, by analysing competitive indices, hypersensitive response and symptom induction, PR-1 accumulation, expression of PR genes, and systemic acquired resistance (SAR) induction. *Partially additive effects between these defence responses require HopZ1a cysteine-protease activity, and also take place between HopZ1a and AvrRps4 or AvrRpm1-triggered responses. We establish that HopZ1a-triggered immunity is independent of salicylic acid (SA), EDS1, jasmonic acid (JA) and ethylene (ET)-dependent pathways, and show that HopZ1a suppresses the induction of PR-1 and PR-5 associated with P. syringae pv tomato (Pto)-triggered effector-triggered immunity (ETI)-like defences, AvrRpt2-triggered immunity, and Pto or Pto (avrRpt2) activation of SAR, and that suppression requires HopZ1a cysteine-protease activity. *Our results indicate that HopZ1a triggers an unusual resistance independent of known pathways and suppresses SA and EDS1-dependent resistance.

  2. The channel domain of colicin A is inhibited by its immunity protein through direct interaction in the Escherichia coli inner membrane.

    PubMed Central

    Espesset, D; Duché, D; Baty, D; Géli, V

    1996-01-01

    A bacterial signal sequence was fused to the colicin A pore-forming domain: the exported pore-forming domain was highly cytotoxic. We thus introduced a cysteine-residue pair in the fusion protein which has been shown to form a disulfide bond in the natural colicin A pore-forming domain between alpha-helices 5 and 6. Formation of the disulfide bond prevented the cytotoxic activity of the fusion protein, presumably by preventing the membrane insertion of helices 5 and 6. However, the cytotoxicity of the disulfide-linked pore-forming domain was reactivated by adding dithiothreitol into the culture medium. We were then able to co-produce the immunity protein with the disulfide linked pore-forming domain, by using a co-immunoprecipitation procedure, in order to show that they interact. We showed both proteins to be co-localized in the Escherichia coli inner membrane and subsequently co-immunoprecipitated them. The interaction required a functional immunity protein. The immunity protein also interacted with a mutant form of the pore-forming domain carrying a mutation located in the voltage-gated region: this mutant was devoid of pore-forming activity but still inserted into the membrane. Our results indicate that the immunity protein interacts with the membrane-anchored channel domain; the interaction requires a functional membrane-inserted immunity protein but does not require the channel to be in the open state. Images PMID:8665842

  3. Culture and the Immune System: Cultural Consonance in Social Support and C-reactive Protein in Urban Brazil.

    PubMed

    Dressler, William W; Balieiro, Mauro C; Ribeiro, Rosane P; Dos Santos, José Ernesto

    2016-06-01

    In this article, we examine the distribution of a marker of immune system stimulation-C-reactive protein-in urban Brazil. Social relationships are associated with immunostimulation, and we argue that cultural dimensions of social support, assessed by cultural consonance, are important in this process. Cultural consonance is the degree to which individuals, in their own beliefs and behaviors, approximate shared cultural models. A measure of cultural consonance in social support, based on a cultural consensus analysis regarding sources and patterns of social support in Brazil, was developed. In a survey of 258 persons, the association of cultural consonance in social support and C-reactive protein was examined, controlling for age, sex, body mass index, low-density lipoprotein cholesterol, depressive symptoms, and a social network index. Lower cultural consonance in social support was associated with higher C-reactive protein. Implications of these results for future research are discussed.

  4. Proteins of Leishmania (Viannia) shawi confer protection associated with Th1 immune response and memory generation

    PubMed Central

    2012-01-01

    Background Leishmania (Viannia) shawi parasite was first characterized in 1989. Recently the protective effects of soluble leishmanial antigen (SLA) from L. (V.) shawi promastigotes were demonstrated using BALB/c mice, the susceptibility model for this parasite. In order to identify protective fractions, SLA was fractionated by reverse phase HPLC and five antigenic fractions were obtained. Methods F1 fraction was purified from L. (V.) shawi parasite extract by reverse phase HPLC. BALB/c mice were immunized once a week for two consecutive weeks by subcutaneous routes in the rump, using 25 μg of F1. After 1 and 16 weeks of last immunization, groups were challenged in the footpad with L. (V.) shawi promastigotes. After 2 months, those same mice were sacrificed and parasite burden, cellular and humoral immune responses were evaluated. Results The F1 fraction induced a high degree of protection associated with an increase in IFN-γ, a decrease in IL-4, increased cell proliferation and activation of CD8+T lymphocytes. Long-term protection was acquired in F1-immunized mice, associated with increased CD4+ central memory T lymphocytes and activation of both CD4+ and CD8+ T cells. In addition, F1-immunized groups showed an increase in IgG2a levels. Conclusions The inductor capability of antigens to generate memory lymphocytes that can proliferate and secrete beneficial cytokines upon infection could be an important factor in the development of vaccine candidates against American Tegumentary Leishmaniasis. PMID:22463817

  5. Sublingual immunization with the phosphate-binding-protein (PstS) reduces oral colonization by Streptococcus mutans.

    PubMed

    Ferreira, E L; Batista, M T; Cavalcante, R C M; Pegos, V R; Passos, H M; Silva, D A; Balan, A; Ferreira, L C S; Ferreira, R C C

    2016-10-01

    Bacterial ATP-binding cassette (ABC) transporters play a crucial role in the physiology and pathogenicity of different bacterial species. Components of ABC transporters have also been tested as target antigens for the development of vaccines against different bacterial species, such as those belonging to the Streptococcus genus. Streptococcus mutans is the etiological agent of dental caries, and previous studies have demonstrated that deletion of the gene encoding PstS, the substrate-binding component of the phosphate uptake system (Pst), reduced the adherence of the bacteria to abiotic surfaces. In the current study, we generated a recombinant form of the S. mutans PstS protein (rPstS) with preserved structural features, and we evaluated the induction of antibody responses in mice after sublingual mucosal immunization with a formulation containing the recombinant protein and an adjuvant derived from the heat-labile toxin from enterotoxigenic Escherichia coli strains. Mice immunized with rPstS exhibited systemic and secreted antibody responses, measured by the number of immunoglobulin A-secreting cells in draining lymph nodes. Serum antibodies raised in mice immunized with rPstS interfered with the adhesion of bacteria to the oral cavity of naive mice challenged with S. mutans. Similarly, mice actively immunized with rPstS were partially protected from oral colonization after challenge with the S. mutans NG8 strain. Therefore, our results indicate that S. mutans PstS is a potential target antigen capable of inducing specific and protective antibody responses after sublingual administration. Overall, these observations raise interesting perspectives for the development of vaccines to prevent dental caries. PMID:26462737

  6. Immunization with FSHβ fusion protein antigen prevents bone loss in a rat ovariectomy-induced osteoporosis model

    SciTech Connect

    Geng, Wenxin; Yan, Xingrong; Du, Huicong; Cui, Jihong; Li, Liwen Chen, Fulin

    2013-05-03

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

  7. Enhancement of Clearance of Bacteria from Murine Lungs by Immunization with Detoxified Lipooligosaccharide from Moraxella catarrhalis Conjugated to Proteins

    PubMed Central

    Hu, Wei-Gang; Chen, Jing; Battey, James F.; Gu, Xin-Xing

    2000-01-01

    Moraxella catarrhalis strain 25238 detoxified lipooligosaccharide (dLOS)-protein conjugates induced a significant rise of bactericidal anti-LOS antibodies in animals. This study reports the effect of active or passive immunization with the conjugates or their antiserum on pulmonary clearance of M. catarrhalis in an aerosol challenge mouse model. Mice were injected subcutaneously with dLOS-tetanus toxoid (dLOS-TT), dLOS–high-molecular-weight proteins (dLOS-HMP) from nontypeable Haemophilus influenzae (NTHi), or nonconjugated materials in Ribi adjuvant and then challenged with M. catarrhalis strain 25238 or O35E or NTHi strain 12. Immunization with dLOS-TT or dLOS-HMP generated a significant rise of serum anti-LOS immunoglobulin G and 68% and 35 to 41% reductions of bacteria in lungs compared with the control (P < 0.01) following challenge with homologous strain 25238 and heterologous strain O35E, respectively. Serum anti-LOS antibody levels correlated with its bactericidal titers against M. catarrhalis and bacterial CFU in lungs. Additionally, immunization with dLOS-HMP generated a 54% reduction of NTHi strain 12 compared with the control (P < 0.01). Passive immunization with a rabbit antiserum against dLOS-TT conferred a significant reduction of strain 25238 CFU in lungs in a dose- and time-dependent pattern compared with preimmune serum-treated mice. Kinetic examination of lung tissue sections demonstrated that antiserum-treated mice initiated and offset inflammatory responses more rapidly than preimmune serum-treated mice. These data indicate that LOS antibodies (whether active or passive) play a major role in the enhancement of pulmonary clearance of different test strains of M. catarrhalis in mice. In addition, dLOS-HMP is a potential candidate for a bivalent vaccine against M. catarrhalis and NTHi infections. PMID:10948114

  8. Protective Humoral Immunity Elicited by a Needle-Free Malaria Vaccine Comprised of a Chimeric Plasmodium falciparum Circumsporozoite Protein and a Toll-Like Receptor 5 Agonist, Flagellin

    PubMed Central

    Carapau, Daniel; Mitchell, Robert; Nacer, Adéla; Shaw, Alan; Othoro, Caroline; Frevert, Ute

    2013-01-01

    Immunization with Plasmodium sporozoites can elicit high levels of sterile immunity, and neutralizing antibodies from protected hosts are known to target the repeat region of the circumsporozoite (CS) protein on the parasite surface. CS-based subunit vaccines have been hampered by suboptimal immunogenicity and the requirement for strong adjuvants to elicit effective humoral immunity. Pathogen-associated molecular patterns (PAMPs) that signal through Toll-like receptors (TLRs) can function as potent adjuvants for innate and adaptive immunity. We examined the immunogenicity of recombinant proteins containing a TLR5 agonist, flagellin, and either full-length or selected epitopes of the Plasmodium falciparum CS protein. Mice immunized with either of the flagellin-modified CS constructs, administered intranasally (i.n.) or subcutaneously (s.c.), developed similar levels of malaria-specific IgG1 antibody and interleukin-5 (IL-5)-producing T cells. Importantly, immunization via the i.n. but not the s.c. route elicited sporozoite neutralizing antibodies capable of inhibiting >90% of sporozoite invasion in vitro and in vivo, as measured using a transgenic rodent parasite expressing P. falciparum CS repeats. These findings demonstrate that functional sporozoite neutralizing antibody can be elicited by i.n. immunization with a flagellin-modified P. falciparum CS protein and raise the potential of a scalable, safe, needle-free vaccine for the 40% of the world's population at risk of malaria. PMID:24042110

  9. Staphylococcal Protein A Promotes Colonization and Immune Evasion of the Epidemic Healthcare-Associated MRSA ST239

    PubMed Central

    Hong, Xufen; Qin, Juanxiu; Li, Tianming; Dai, Yingxin; Wang, Yanan; Liu, Qian; He, Lei; Lu, Huiying; Gao, Qianqian; Lin, Yong; Li, Min

    2016-01-01

    The highly successful epidemic of healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) ST239 is a growing concern worldwide, due to its progressive adaptation to the highly selective environment of the healthcare system. HA-MRSA ST239 display the reduced virulence and successfully colonize in hospital settings, while the emergent community-associated MRSA (CA-MRSA) maintain full virulence and cause infections in the community environment. Our aim was to investigate what enables S. aureus ST239 to be highly adaptive under hospital circumstances and gradually progress to a series of widespread invasive infections. We found that spa expression of HA-MRSA ST239 is much higher than that of CA-SA ST398. And we discovered that the highly production of staphylococcal protein A (SpA), having no concern with spa gene structure, enhances nasal colonization and cell adhesion in ST239. S. aureus ST239 defends against the adaptive immune response by resisting phagocytosis and inducing apoptosis of B cells through expression of surface-anchored and released protein A, facilitating its dissemination within the circulatory system to other organs. Protein A also plays another key role in subverting the host immune response through its ability to induce early shedding of TNF-α receptor 1 (TNFR1) from phagocytic cells. The increased levels of soluble TNFR1 present during experimental S. aureus ST239 infection may neutralize circulating TNF-α and impair the host inflammatory response. Protein A is also a virulence factor, as tested in our bacteremia model in mice, contributing to the durative tissue damage of abscess formation sites in ST239 infection. These functions of protein A eventually benefit to widespread infections of S. aureus ST239. We draw the conclusion that Staphylococcal Protein A may be a crucial determinant in the colonization and immune evasion of ST239 infections, contributing to persistent spread in the hospital settings. These results

  10. Staphylococcal Protein A Promotes Colonization and Immune Evasion of the Epidemic Healthcare-Associated MRSA ST239.

    PubMed

    Hong, Xufen; Qin, Juanxiu; Li, Tianming; Dai, Yingxin; Wang, Yanan; Liu, Qian; He, Lei; Lu, Huiying; Gao, Qianqian; Lin, Yong; Li, Min

    2016-01-01

    The highly successful epidemic of healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) ST239 is a growing concern worldwide, due to its progressive adaptation to the highly selective environment of the healthcare system. HA-MRSA ST239 display the reduced virulence and successfully colonize in hospital settings, while the emergent community-associated MRSA (CA-MRSA) maintain full virulence and cause infections in the community environment. Our aim was to investigate what enables S. aureus ST239 to be highly adaptive under hospital circumstances and gradually progress to a series of widespread invasive infections. We found that spa expression of HA-MRSA ST239 is much higher than that of CA-SA ST398. And we discovered that the highly production of staphylococcal protein A (SpA), having no concern with spa gene structure, enhances nasal colonization and cell adhesion in ST239. S. aureus ST239 defends against the adaptive immune response by resisting phagocytosis and inducing apoptosis of B cells through expression of surface-anchored and released protein A, facilitating its dissemination within the circulatory system to other organs. Protein A also plays another key role in subverting the host immune response through its ability to induce early shedding of TNF-α receptor 1 (TNFR1) from phagocytic cells. The increased levels of soluble TNFR1 present during experimental S. aureus ST239 infection may neutralize circulating TNF-α and impair the host inflammatory response. Protein A is also a virulence factor, as tested in our bacteremia model in mice, contributing to the durative tissue damage of abscess formation sites in ST239 infection. These functions of protein A eventually benefit to widespread infections of S. aureus ST239. We draw the conclusion that Staphylococcal Protein A may be a crucial determinant in the colonization and immune evasion of ST239 infections, contributing to persistent spread in the hospital settings. These results

  11. Oral immunization with an attenuated vaccine strain of Salmonella typhimurium expressing the serine-rich Entamoeba histolytica protein induces an antiamebic immune response and protects gerbils from amebic liver abscess.

    PubMed Central

    Zhang, T; Stanley, S L

    1996-01-01

    Attenuated salmonellae represent attractive candidates for the delivery of foreign antigens by oral vaccination. In this report, we describe the high-level expression of a recombinant fusion protein containing the serine-rich Entamoeba histolytica protein (SREHP), a protective antigen derived from virulent amebae, and a bacterially derived maltose-binding protein (MBP) in an attenuated strain of Salmonella typhimurium. Mice and gerbils immunized with S. typhimurium expressing SREHP-MBP produced mucosal immunoglobulin A antiamebic antibodies and serum immunoglobulin G antiamebic antibodies. Gerbils vaccinated with S typhimurium SREHP-MBP were protected against amebic liver abscess, the most common extraintestinal complication of amebiasis. Our findings indicate that the induction of mucosal and immune responses to the amebic SREHP antigen is dependent on the level of SREHP-MBP expression in S. typhimurium and establish that oral vaccination with SREHP can produce protective immunity to invasive amebiasis. PMID:8613356

  12. Tomato 14-3-3 protein TFT7 interacts with a MAP kinase kinase to regulate immunity-associated programmed cell death mediated by diverse disease resistance proteins.

    PubMed

    Oh, Chang-Sik; Martin, Gregory B

    2011-04-22

    Programmed cell death (PCD) associated with immunity is triggered when a plant disease resistance (R) protein recognizes a corresponding pathogen virulence protein. In tomato, detection by the host Pto kinase of the Pseudomonas syringae proteins AvrPto or AvrPtoB causes localized PCD. Previously, we reported that both MAPKKKα (mitogen-activated protein kinase kinase kinase) and the tomato 14-3-3 protein 7 (TFT7) positively regulate Pto-mediated PCD in tomato and Nicotiana benthamiana. In addition, in contrast to MAPKKKα, TFT7 is required for PCD mediated by four other R proteins. Here we investigate why TFT7 is required for PCD induced by diverse R proteins in plants. We discovered that a MAPKK, SlMKK2, which acts downstream of SlMAPKKKα, also interacts with TFT7 in plant cells. Gene silencing experiments revealed that the orthologous genes of both SlMKK2 and TFT7 in N. benthamiana are required for PCD mediated by the same set of R proteins. SlMKK2 and its orthologs contain a 14-3-3 binding site in their N terminus, and Thr(33) in this site is required for interaction with TFT7 in vivo. Like the structurally similar human 14-3-3ε protein, TFT7 forms a homodimer in vivo. Because TFT7 interacts with both SlMAPKKKα and SlMKK2 and also forms a homodimer, we propose that TFT7 may coordinately recruit these client proteins for efficient signal transfer, leading to PCD induction. PMID:21378171

  13. Antimicrobial proteins from snake venoms: direct bacterial damage and activation of innate immunity against Staphylococcus aureus skin infection.

    PubMed

    Samy, R P; Stiles, B G; Gopalakrishnakone, P; Chow, V T K

    2011-01-01

    The innate immune system is the first line of defense against microbial diseases. Antimicrobial proteins produced by snake venoms have recently attracted significant attention due to their relevance to bacterial infection and potential development into new therapeutic agents. Staphylococcus aureus is one of the major human pathogens causing a variety of infections involving pneumonia, toxic shock syndrome, and skin lesions. With the recent emergence of methicillin (MRSA) and vancomycin (VRSA) resistance, S. aureus infection is a serious clinical problem that will have a grave socio-economic impact in the near future. Although S. aureus susceptibility to innate antimicrobial peptides has been reported recently, the protective effect of snake venom phospholipase A₂ (svPLA₂) proteins on the skin from S. aureus infection has been understudied. This review details the protective function of svPLA₂s derived from venoms against skin infections caused by S. aureus. We have demonstrated in vivo that local application of svPLA₂ provides complete clearance of S. aureus within 2 weeks after treatment compared to fusidic acid ointment (FAO). In vitro experiments also demonstrate that svPLA₂ proteins have inhibitory (bacteriostatic) and killing (bactericidal) effects on S. aureus in a dose-dependant manner. The mechanism of bacterial membrane damage and perturbation was clearly evidenced by electron microscopic studies. In summary, svPLA₂s from Viperidae and Elapidae snakes are novel molecules that can activate important mechanisms of innate immunity in animals to endow them with protection against skin infection caused by S. aureus.

  14. Effect of conjugation methodology, carrier protein, and adjuvants on the immune response to Staphylococcus aureus capsular polysaccharides.

    PubMed

    Fattom, A; Li, X; Cho, Y H; Burns, A; Hawwari, A; Shepherd, S E; Coughlin, R; Winston, S; Naso, R

    1995-10-01

    Conjugate vaccines were prepared with S. aureus type 8 capsular polysaccharide (CP) using three carrier proteins: Pseudomonas aeruginosa exotoxin A (ETA), a non-toxic recombinant ETA (rEPA), and diphtheria toxoid (DTd). Adipic acid dihydrazide (ADH) or N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) was used as a spacer to link the CP to carrier protein. All conjugates gave a high immune response with a boost after the second immunization. Conjugates prepared with ADH gave higher antibody titers than conjugates prepared with SPDP. IgG1 was the primary subclass elicited by all conjugates regardless of the carrier protein or the conjugation method used to prepare the vaccines. The non-immunogenic CP and the conjugates were formulated with either monophosphoryl lipid A (MPL), QS21, or in Novasomes and evaluated in mice. While the adjuvants failed to improve the immunogenicity of the nonconjugated CP, a more than fivefold increase in the antibody levels was observed when these adjuvants were used with the conjugates. Significant rises in IgG2b and IgG3 were observed with all formulations. The enhancement of the immunogenicity and the IgG subclass shift, as seen with some adjuvants, may prove to be important in immunocompromised patients. PMID:8585282

  15. Outer membrane vesicles induce immune responses to virulence proteins and protect against colonization by enterotoxigenic Escherichia coli.

    PubMed

    Roy, Koushik; Hamilton, David J; Munson, George P; Fleckenstein, James M

    2011-11-01

    Enterotoxigenic Escherichia coli (ETEC) strains are a heterogeneous group of pathogens that produce heat-labile (LT) and/or heat-stable (ST) enterotoxins. Collectively, these pathogens are responsible for hundreds of thousands of deaths annually in developing countries, particularly in children under the age of 5 years. The heterogeneity of previously investigated molecular targets and the lack of complete sustained protection afforded by antitoxin immunity have impeded progress to date toward a broadly protective vaccine. Many pathogens, including ETEC, have the capacity to form outer membrane vesicles (OMV), which often contain one or more virulence proteins. Prompted by recent studies that identified several immunogenic virulence proteins in outer membrane vesicles of ETEC, we sought to examine the immunogenicity and protective efficacy of these structures in a murine model of infection. Here we demonstrate that immunization with OMV impairs ETEC colonization of the small intestine and stimulates antibodies that recognize the heat-labile toxin and two additional putative virulence proteins, the EtpA adhesin and CexE. Similar to earlier studies with EtpA, vaccination with LT alone also inhibited intestinal colonization. Together, these findings suggest that OMV could be exploited to deliver protective antigens relevant to development of ETEC vaccines. PMID:21900530

  16. Cellular immune responses induced in vitro by Ehrlichia ruminantium secreted proteins and identification of vaccine candidate peptides.

    PubMed

    Thema, Nontobeko; Pretorius, Alri; Tshilwane, Selaelo I; Liebenberg, Junita; Steyn, Helena; Van Kleef, Mirinda

    2016-01-01

    Secreted proteins are reported to induce cell-mediated immunity characterised by the production of interferon-gamma (IFN)-γ. In this study three open reading frames (ORFs) (Erum8060, Erum7760, Erum5000) encoding secreted proteins were selected from the Ehrlichia ruminantium (Welgevonden) genome sequence using bioinformatics tools to determine whether they induce a cellular immune response in vitro with mononuclear cells from needle and tick infected animals. The whole recombinant protein of the three ORFs as well as four adjacent fragments of the Erum5000 protein (Erum5000A, Erum5000B, Erum5000C, Erum5000D) were successfully expressed in a bacterial expression system which was confirmed by immunoblots using anti-His antibodies and sheep sera. These recombinant proteins were assayed with immune sheep and cattle peripheral blood mononuclear cells (PBMCs), spleen and lymph node (LN) cells to determine whether they induce recall cellular immune responses in vitro. Significant proliferative responses and IFN-γ production were evident for all recombinant proteins, especially Erum5000A, in both ruminant species tested. Thus overlapping peptides spanning Erum5000A were synthesised and peptides that induce proliferation of memory CD4+ and CD8+ T cells and production of IFN-γ were identified. These results illustrate that a Th1 type immune response was elicited and these recombinant proteins and peptides may therefore be promising candidates for development of a heartwater vaccine. PMID:27608502

  17. Shaping of adaptive immune responses to soluble proteins by TLR agonists: a role for IFN-alpha/beta.

    PubMed

    Durand, Vanessa; Wong, Simon Yc; Tough, David F; Le Bon, Agnes

    2004-12-01

    Toll-like receptors (TLR) are believed to play a major role in the recognition of invading organisms, although their ability to shape immune responses is not completely understood. Our aim was to investigate in vivo the effect of different TLR stimuli on the generation of antibody responses and the induction of CD8+ T-cell cross-priming after immunization with soluble protein antigens. While all TLR agonists tested elicited the production of immunomodulatory cytokines, marked differences were observed in their ability to stimulate antigen-specific immune responses. Zymosan, poly(I:C) and CpG DNA, which signal through TLR2/6, 3 and 9, respectively, were found to strongly induce the production of IgG2a antibodies, whereas R-848 (TLR7) and LPS (TLR4) did so much more weakly. In contrast, LPS, poly(I:C) and CpG DNA, but not zymosan, induced functional CD8+ T-cell responses against OVA; peptidoglycan (TLR2/?) and R-848 were also ineffective in stimulating cross-priming. Experiments using IFN-alpha/beta R-deficient mice showed that the induction of cross-priming by LPS and poly(I:C) was abrogated in the absence of IFN-alpha/beta signalling, and induction by CpG DNA was greatly reduced. Overall, our results identify LPS as another TLR agonist that is able to generate functional cross-priming against a soluble protein antigen. In addition, our results demonstrate that the ability of TLR stimuli to initiate CD8+ T-cell responses against soluble protein antigens is largely dependent on the IFN-alpha/beta signalling pathway. PMID:15550117

  18. Generation of cellular immune responses to HCV NS5 protein through in vivo activation of dendritic cells

    PubMed Central

    Wintermeyer, P.; Gehring, S.; Eken, A.; Wands, J. R.

    2014-01-01

    SUMMARY Chronic hepatitis C (HCV) infection is a substantial medical problem that leads to progressive liver disease, cirrhosis, and hepatocellular carcinoma (HCC). The aim of this study was to achieve sustained cellular immune responses in vivo to a HCV nonstructural protein using dendritic cell (DC)-based immunization approach. We targeted the HCV NS5 protein to DCs in vivo by injecting microparticles loaded with this antigen. The DC population was expanded in BALB/C mice (H-2d) by hydrodynamic injection of a plasmid pUMVC3-hFLex expressing the secreted portion of the human Fms-like tyrosine kinase receptor-3 ligand (hFlt3). Mice were subsequently injected with microparticles coated with HCV NS5 protein via the tail vein. Cellular immune responses were determined with respect to secretion of INFγ and IL2 by CD4+ cells and cytotoxic T-lymphocyte (CTL) assays in vitro; inhibition of tumour cell growth was employed for the assessment of CD8+ generated activity in vivo. We found that Flt3L treatment expanded the DC population in the spleen to 43%, and such cells displayed a striking upregulation of CD86 as well as CD80 and CD40 co-stimulating molecules. Viral antigen-specific TH1 cytokine secretion by splenocytes was generated, and CTL activity against syngeneic NS5 expressing myeloma target cells was observed. In addition, these cells inhibited tumour growth indicating that NS5-specific robust CTL activity was operative in vivo. Thus, the capability of activating DCs in vivo using the methods described is valuable as a therapeutic vaccine strategy for chronic HCV infection. PMID:20002303

  19. Apoplastic venom allergen-like proteins of cyst nematodes modulate the activation of basal plant innate immunity by cell surface receptors.

    PubMed

    Lozano-Torres, Jose L; Wilbers, Ruud H P; Warmerdam, Sonja; Finkers-Tomczak, Anna; Diaz-Granados, Amalia; van Schaik, Casper C; Helder, Johannes; Bakker, Jaap; Goverse, Aska; Schots, Arjen; Smant, Geert

    2014-12-01

    Despite causing considerable damage to host tissue during the onset of parasitism, nematodes establish remarkably persistent infections in both animals and plants. It is thought that an elaborate repertoire of effector proteins in nematode secretions suppresses damage-triggered immune responses of the host. However, the nature and mode of action of most immunomodulatory compounds in nematode secretions are not well understood. Here, we show that venom allergen-like proteins of plant-parasitic nematodes selectively suppress host immunity mediated by surface-localized immune receptors. Venom allergen-like proteins are uniquely conserved in secretions of all animal- and plant-parasitic nematodes studied to date, but their role during the onset of parasitism has thus far remained elusive. Knocking-down the expression of the venom allergen-like protein Gr-VAP1 severely hampered the infectivity of the potato cyst nematode Globodera rostochiensis. By contrast, heterologous expression of Gr-VAP1 and two other venom allergen-like proteins from the beet cyst nematode Heterodera schachtii in plants resulted in the loss of basal immunity to multiple unrelated pathogens. The modulation of basal immunity by ectopic venom allergen-like proteins in Arabidopsis thaliana involved extracellular protease-based host defenses and non-photochemical quenching in chloroplasts. Non-photochemical quenching regulates the initiation of the defense-related programmed cell death, the onset of which was commonly suppressed by venom allergen-like proteins from G. rostochiensis, H. schachtii, and the root-knot nematode Meloidogyne incognita. Surprisingly, these venom allergen-like proteins only affected the programmed cell death mediated by surface-localized immune receptors. Furthermore, the delivery of venom allergen-like proteins into host tissue coincides with the enzymatic breakdown of plant cell walls by migratory nematodes. We, therefore, conclude that parasitic nematodes most likely utilize

  20. Apoplastic Venom Allergen-like Proteins of Cyst Nematodes Modulate the Activation of Basal Plant Innate Immunity by Cell Surface Receptors

    PubMed Central

    Lozano-Torres, Jose L.; Wilbers, Ruud H. P.; Warmerdam, Sonja; Finkers-Tomczak, Anna; Diaz-Granados, Amalia; van Schaik, Casper C.; Helder, Johannes; Bakker, Jaap; Goverse, Aska; Schots, Arjen; Smant, Geert

    2014-01-01

    Despite causing considerable damage to host tissue during the onset of parasitism, nematodes establish remarkably persistent infections in both animals and plants. It is thought that an elaborate repertoire of effector proteins in nematode secretions suppresses damage-triggered immune responses of the host. However, the nature and mode of action of most immunomodulatory compounds in nematode secretions are not well understood. Here, we show that venom allergen-like proteins of plant-parasitic nematodes selectively suppress host immunity mediated by surface-localized immune receptors. Venom allergen-like proteins are uniquely conserved in secretions of all animal- and plant-parasitic nematodes studied to date, but their role during the onset of parasitism has thus far remained elusive. Knocking-down the expression of the venom allergen-like protein Gr-VAP1 severely hampered the infectivity of the potato cyst nematode Globodera rostochiensis. By contrast, heterologous expression of Gr-VAP1 and two other venom allergen-like proteins from the beet cyst nematode Heterodera schachtii in plants resulted in the loss of basal immunity to multiple unrelated pathogens. The modulation of basal immunity by ectopic venom allergen-like proteins in Arabidopsis thaliana involved extracellular protease-based host defenses and non-photochemical quenching in chloroplasts. Non-photochemical quenching regulates the initiation of the defense-related programmed cell death, the onset of which was commonly suppressed by venom allergen-like proteins from G. rostochiensis, H. schachtii, and the root-knot nematode Meloidogyne incognita. Surprisingly, these venom allergen-like proteins only affected the programmed cell death mediated by surface-localized immune receptors. Furthermore, the delivery of venom allergen-like proteins into host tissue coincides with the enzymatic breakdown of plant cell walls by migratory nematodes. We, therefore, conclude that parasitic nematodes most likely utilize

  1. The Ankyrin-Repeat Transmembrane Protein BDA1 Functions Downstream of the Receptor-Like Protein SNC2 to Regulate Plant Immunity1[C][OA

    PubMed Central

    Yang, Yuanai; Zhang, Yaxi; Ding, Pingtao; Johnson, Kaeli; Li, Xin; Zhang, Yuelin

    2012-01-01

    Plants utilize a large number of immune receptors to recognize pathogens and activate defense responses. A small number of these receptors belong to the receptor-like protein family. Previously, we showed that a gain-of-function mutation in the receptor-like protein SNC2 (for Suppressor of NPR1, Constitutive2) leads to constitutive activation of defense responses in snc2-1D mutant plants. To identify defense signaling components downstream of SNC2, we carried out a suppressor screen in the snc2-1D mutant background of Arabidopsis (Arabidopsis thaliana). Map-based cloning of one of the suppressor genes, BDA1 (for bian da; “becoming big” in Chinese), showed that it encodes a protein with amino-terminal ankyrin repeats and carboxyl-terminal transmembrane domains. Loss-of-function mutations in BDA1 suppress the dwarf morphology and constitutive defense responses in snc2-1D npr1-1 (for nonexpressor of pathogenesis-related genes1,1) and also result in enhanced susceptibility to bacterial pathogens. In contrast, a gain-of-function allele of bda1 isolated from a separate genetic screen to search for mutants with enhanced pathogen resistance was found to constitutively activate cell death and defense responses. These data suggest that BDA1 is a critical signaling component that functions downstream of SNC2 to regulate plant immunity. PMID:22740615

  2. Novel Bat Influenza Virus NS1 Proteins Bind Double-Stranded RNA and Antagonize Host Innate Immunity.

    PubMed

    Turkington, Hannah L; Juozapaitis, Mindaugas; Kerry, Philip S; Aydillo, Teresa; Ayllon, Juan; García-Sastre, Adolfo; Schwemmle, Martin; Hale, Benjamin G

    2015-10-01

    We demonstrate that novel bat HL17NL10 and HL18NL11 influenza virus NS1 proteins are effective interferon antagonists but do not block general host gene expression. Solving the RNA-binding domain structures revealed the canonical NS1 symmetrical homodimer, and RNA binding required conserved basic residues in this domain. Interferon antagonism was strictly dependent on RNA binding, and chimeric bat influenza viruses expressing NS1s defective in this activity were highly attenuated in interferon-competent cells but not in cells unable to establish antiviral immunity.

  3. Novel Bat Influenza Virus NS1 Proteins Bind Double-Stranded RNA and Antagonize Host Innate Immunity

    PubMed Central

    Turkington, Hannah L.; Juozapaitis, Mindaugas; Kerry, Philip S.; Aydillo, Teresa; Ayllon, Juan; García-Sastre, Adolfo

    2015-01-01

    We demonstrate that novel bat HL17NL10 and HL18NL11 influenza virus NS1 proteins are effective interferon antagonists but do not block general host gene expression. Solving the RNA-binding domain structures revealed the canonical NS1 symmetrical homodimer, and RNA binding required conserved basic residues in this domain. Interferon antagonism was strictly dependent on RNA binding, and chimeric bat influenza viruses expressing NS1s defective in this activity were highly attenuated in interferon-competent cells but not in cells unable to establish antiviral immunity. PMID:26246567

  4. Reduction of Factor VIII Inhibitor Titers During Immune Tolerance Induction With Recombinant Factor VIII-Fc Fusion Protein.

    PubMed

    Groomes, Charles L; Gianferante, David M; Crouch, Gary D; Parekh, Dina S; Scott, David W; Lieuw, Kenneth

    2016-05-01

    The development of inhibitors toward factor VIII (FVIII) is a common and serious complication of hemophilia A (HA) therapy. Patients with hemophilia who develop inhibitors often undergo time- and resource-intensive immune tolerance induction (ITI) protocols. We report a 15-month-old male with severe HA and a high-titer inhibitor that occurred while receiving prophylactic treatment with recombinant FVIII (rFVIII), in whom significant inhibitor titer reduction was achieved with thrice weekly infusions of a new, prolonged half-life rFVIII-Fc fusion protein product (trade name Eloctate). Further studies are warranted to explore the potential of Eloctate in ITI protocols. PMID:26739399

  5. NMR resonance assignments of the lantibiotic immunity protein NisI from Lactococcus lactis.

    PubMed

    Hacker, Carolin; Christ, Nina Alexandra; Duchardt-Ferner, Elke; Korn, Sophie; Berninger, Lucija; Kötter, Peter; Entian, Karl-Dieter; Wöhnert, Jens

    2015-10-01

    The lantibiotic nisin is a small antimicrobial peptide which acts against a wide range of Gram-positive bacteria. Nisin-producing Lactococcus lactis strains express four genes for self-protection against their own antimicrobial compound. This immunity system consists of the lipoprotein NisI and the ABC transporter NisFEG. NisI is attached to the outside of the cytoplasmic membrane via a covalently linked diacylglycerol anchor. Both the lipoprotein and the ABC transporter are needed for full immunity but the exact immunity mechanism is still unclear. To gain insights into the highly specific immunity mechanism of nisin producing strains on a structural level we present here the backbone resonance assignment of NisI (25.8 kDa) as well as the virtually complete (1)H,(15)N,(13)C chemical shift assignments for the isolated 12.7 kDa N-terminal and 14.6 kDa C-terminal domains of NisI.

  6. NMR resonance assignments of the lantibiotic immunity protein NisI from Lactococcus lactis.

    PubMed

    Hacker, Carolin; Christ, Nina Alexandra; Duchardt-Ferner, Elke; Korn, Sophie; Berninger, Lucija; Kötter, Peter; Entian, Karl-Dieter; Wöhnert, Jens

    2015-10-01

    The lantibiotic nisin is a small antimicrobial peptide which acts against a wide range of Gram-positive bacteria. Nisin-producing Lactococcus lactis strains express four genes for self-protection against their own antimicrobial compound. This immunity system consists of the lipoprotein NisI and the ABC transporter NisFEG. NisI is attached to the outside of the cytoplasmic membrane via a covalently linked diacylglycerol anchor. Both the lipoprotein and the ABC transporter are needed for full immunity but the exact immunity mechanism is still unclear. To gain insights into the highly specific immunity mechanism of nisin producing strains on a structural level we present here the backbone resonance assignment of NisI (25.8 kDa) as well as the virtually complete (1)H,(15)N,(13)C chemical shift assignments for the isolated 12.7 kDa N-terminal and 14.6 kDa C-terminal domains of NisI. PMID:25613223

  7. Interleukin-17-induced protein lipocalin 2 is dispensable for immunity to oral candidiasis.

    PubMed

    Ferreira, Maria Carolina; Whibley, Natasha; Mamo, Anna J; Siebenlist, Ulrich; Chan, Yvonne R; Gaffen, Sarah L

    2014-03-01

    Oropharyngeal candidiasis (OPC; thrush) is an opportunistic fungal infection caused by the commensal microbe Candida albicans. Immunity to OPC is strongly dependent on CD4+ T cells, particularly those of the Th17 subset. Interleukin-17 (IL-17) deficiency in mice or humans leads to chronic mucocutaneous candidiasis, but the specific downstream mechanisms of IL-17-mediated host defense remain unclear. Lipocalin 2 (Lcn2; 24p3; neutrophil gelatinase-associated lipocalin [NGAL]) is an antimicrobial host defense factor produced in response to inflammatory cytokines, particularly IL-17. Lcn2 plays a key role in preventing iron acquisition by bacteria that use catecholate-type siderophores, and lipocalin 2(-/-) mice are highly susceptible to infection by Escherichia coli and Klebsiella pneumoniae. The role of Lcn2 in mediating immunity to fungi is poorly defined. Accordingly, in this study, we evaluated the role of Lcn2 in immunity to oral infection with C. albicans. Lcn2 is strongly upregulated following oral infection with C. albicans, and its expression is almost entirely abrogated in mice with defective IL-17 signaling (IL-17RA(-/-) or Act1(-/-) mice). However, Lcn2(-/-) mice were completely resistant to OPC, comparably to wild-type (WT) mice. Moreover, Lcn2 deficiency mediated protection from OPC induced by steroid immunosuppression. Therefore, despite its potent regulation during C. albicans infection, Lcn2 is not required for immunity to mucosal candidiasis.

  8. A single vertebrate DNA virus protein disarms invertebrate immunity to RNA virus infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-host interactions drive a remarkable diversity of immune responses and countermeasures. While investigating virus-invertebrate host interactions we found that two RNA viruses with broad host ranges, vesicular stomatitis virus (VSV) and Sindbis virus (SINV), were unable to infect certain Lepido...

  9. Interleukin-17-Induced Protein Lipocalin 2 Is Dispensable for Immunity to Oral Candidiasis

    PubMed Central

    Ferreira, Maria Carolina; Whibley, Natasha; Mamo, Anna J.; Siebenlist, Ulrich; Chan, Yvonne R.

    2014-01-01

    Oropharyngeal candidiasis (OPC; thrush) is an opportunistic fungal infection caused by the commensal microbe Candida albicans. Immunity to OPC is strongly dependent on CD4+ T cells, particularly those of the Th17 subset. Interleukin-17 (IL-17) deficiency in mice or humans leads to chronic mucocutaneous candidiasis, but the specific downstream mechanisms of IL-17-mediated host defense remain unclear. Lipocalin 2 (Lcn2; 24p3; neutrophil gelatinase-associated lipocalin [NGAL]) is an antimicrobial host defense factor produced in response to inflammatory cytokines, particularly IL-17. Lcn2 plays a key role in preventing iron acquisition by bacteria that use catecholate-type siderophores, and lipocalin 2−/− mice are highly susceptible to infection by Escherichia coli and Klebsiella pneumoniae. The role of Lcn2 in mediating immunity to fungi is poorly defined. Accordingly, in this study, we evaluated the role of Lcn2 in immunity to oral infection with C. albicans. Lcn2 is strongly upregulated following oral infection with C. albicans, and its expression is almost entirely abrogated in mice with defective IL-17 signaling (IL-17RA−/− or Act1−/− mice). However, Lcn2−/− mice were completely resistant to OPC, comparably to wild-type (WT) mice. Moreover, Lcn2 deficiency mediated protection from OPC induced by steroid immunosuppression. Therefore, despite its potent regulation during C. albicans infection, Lcn2 is not required for immunity to mucosal candidiasis. PMID:24343647

  10. Intramammary Immunization of Pregnant Mice with Staphylococcal Protein A Reduces the Post-Challenge Mammary Gland Bacterial Load but Not Pathology

    PubMed Central

    Gogoi-Tiwari, Jully; Williams, Vincent; Waryah, Charlene Babra; Mathavan, Sangeetha; Tiwari, Harish Kumar; Costantino, Paul; Mukkur, Trilochan

    2016-01-01

    Protein A, encoded by the spa gene, is one of the major immune evading MSCRAMM of S. aureus, demonstrated to be prevalent in a significant percentage of clinical bovine mastitis isolates in Australia. Given its’ reported significance in biofilm formation and the superior performance of S. aureus biofilm versus planktonic vaccine in the mouse mastitis model, it was of interest to determine the immunogenicity and protective potential of Protein A as a potential vaccine candidate against bovine mastitis using the mouse mastitis model. Pregnant Balb/c mice were immunised with Protein A emulsified in an alum-based adjuvant by subcutaneous (s/c) or intramammary (i/mam) routes. While humoral immune response of mice post-immunization were determined using indirect ELISA, cell-mediated immune response was assessed by estimation of interferon-gamma (IFN-γ) produced by protein A-stimulated splenocyte supernatants. Protective potential of Protein A against experimental mastitis was determined by challenge of immunized versus sham-vaccinated mice by i/mam route, based upon manifestation of clinical symptoms, total bacterial load and histopathological damage to mammary glands. Significantly (p<0.05) higher levels of IgG1 isotype were produced in mice immunized by the s/c route. In contrast, significantly higher levels of the antibody isotype IgG2a were produced in mice immunized by the i/mam route (p<0.05). There was significant reduction (p<0.05) in bacterial loads of the mammary glands of mice immunized by Protein A regardless of the route of immunization, with medium level of clinical symptoms observed up to day 3 post-challenge. However, Protein A vaccine failed to protect immunized mice post-challenge with biofilm producing encapsulated S. aureus via i/mam route, regardless of the route of immunization, as measured by the level of mammary tissue damage. It was concluded that, Protein A in its’ native state was apparently not a suitable candidate for inclusion in a cell

  11. Intramammary Immunization of Pregnant Mice with Staphylococcal Protein A Reduces the Post-Challenge Mammary Gland Bacterial Load but Not Pathology.

    PubMed

    Gogoi-Tiwari, Jully; Williams, Vincent; Waryah, Charlene Babra; Mathavan, Sangeetha; Tiwari, Harish Kumar; Costantino, Paul; Mukkur, Trilochan

    2016-01-01

    Protein A, encoded by the spa gene, is one of the major immune evading MSCRAMM of S. aureus, demonstrated to be prevalent in a significant percentage of clinical bovine mastitis isolates in Australia. Given its' reported significance in biofilm formation and the superior performance of S. aureus biofilm versus planktonic vaccine in the mouse mastitis model, it was of interest to determine the immunogenicity and protective potential of Protein A as a potential vaccine candidate against bovine mastitis using the mouse mastitis model. Pregnant Balb/c mice were immunised with Protein A emulsified in an alum-based adjuvant by subcutaneous (s/c) or intramammary (i/mam) routes. While humoral immune response of mice post-immunization were determined using indirect ELISA, cell-mediated immune response was assessed by estimation of interferon-gamma (IFN-γ) produced by protein A-stimulated splenocyte supernatants. Protective potential of Protein A against experimental mastitis was determined by challenge of immunized versus sham-vaccinated mice by i/mam route, based upon manifestation of clinical symptoms, total bacterial load and histopathological damage to mammary glands. Significantly (p<0.05) higher levels of IgG1 isotype were produced in mice immunized by the s/c route. In contrast, significantly higher levels of the antibody isotype IgG2a were produced in mice immunized by the i/mam route (p<0.05). There was significant reduction (p<0.05) in bacterial loads of the mammary glands of mice immunized by Protein A regardless of the route of immunization, with medium level of clinical symptoms observed up to day 3 post-challenge. However, Protein A vaccine failed to protect immunized mice post-challenge with biofilm producing encapsulated S. aureus via i/mam route, regardless of the route of immunization, as measured by the level of mammary tissue damage. It was concluded that, Protein A in its' native state was apparently not a suitable candidate for inclusion in a cell

  12. Intramammary Immunization of Pregnant Mice with Staphylococcal Protein A Reduces the Post-Challenge Mammary Gland Bacterial Load but Not Pathology.

    PubMed

    Gogoi-Tiwari, Jully; Williams, Vincent; Waryah, Charlene Babra; Mathavan, Sangeetha; Tiwari, Harish Kumar; Costantino, Paul; Mukkur, Trilochan

    2016-01-01

    Protein A, encoded by the spa gene, is one of the major immune evading MSCRAMM of S. aureus, demonstrated to be prevalent in a significant percentage of clinical bovine mastitis isolates in Australia. Given its' reported significance in biofilm formation and the superior performance of S. aureus biofilm versus planktonic vaccine in the mouse mastitis model, it was of interest to determine the immunogenicity and protective potential of Protein A as a potential vaccine candidate against bovine mastitis using the mouse mastitis model. Pregnant Balb/c mice were immunised with Protein A emulsified in an alum-based adjuvant by subcutaneous (s/c) or intramammary (i/mam) routes. While humoral immune response of mice post-immunization were determined using indirect ELISA, cell-mediated immune response was assessed by estimation of interferon-gamma (IFN-γ) produced by protein A-stimulated splenocyte supernatants. Protective potential of Protein A against experimental mastitis was determined by challenge of immunized versus sham-vaccinated mice by i/mam route, based upon manifestation of clinical symptoms, total bacterial load and histopathological damage to mammary glands. Significantly (p<0.05) higher levels of IgG1 isotype were produced in mice immunized by the s/c route. In contrast, significantly higher levels of the antibody isotype IgG2a were produced in mice immunized by the i/mam route (p<0.05). There was significant reduction (p<0.05) in bacterial loads of the mammary glands of mice immunized by Protein A regardless of the route of immunization, with medium level of clinical symptoms observed up to day 3 post-challenge. However, Protein A vaccine failed to protect immunized mice post-challenge with biofilm producing encapsulated S. aureus via i/mam route, regardless of the route of immunization, as measured by the level of mammary tissue damage. It was concluded that, Protein A in its' native state was apparently not a suitable candidate for inclusion in a cell

  13. Proteins encoded in genomic regions associated with immune-mediated disease physically interact and suggest underlying biology.

    PubMed

    Rossin, Elizabeth J; Lage, Kasper; Raychaudhuri, Soumya; Xavier, Ramnik J; Tatar, Diana; Benita, Yair; Cotsapas, Chris; Daly, Mark J

    2011-01-01

    Genome-wide association studies (GWAS) have defined over 150 genomic regions unequivocally containing variation predisposing to immune-mediated disease. Inferring disease biology from these observations, however, hinges on our ability to discover the molecular processes being perturbed by these risk variants. It has previously been observed that different genes harboring causal mutations for the same Mendelian disease often physically interact. We sought to evaluate the degree to which this is true of genes within strongly associated loci in complex disease. Using sets of loci defined in rheumatoid arthritis (RA) and Crohn's disease (CD) GWAS, we build protein-protein interaction (PPI) networks for genes within associated loci and find abundant physical interactions between protein products of associated genes. We apply multiple permutation approaches to show that these networks are more densely connected than chance expectation. To confirm biological relevance, we show that the components of the networks tend to be expressed in similar tissues relevant to the phenotypes in question, suggesting the network indicates common underlying processes perturbed by risk loci. Furthermore, we show that the RA and CD networks have predictive power by demonstrating that proteins in these networks, not encoded in the confirmed list of disease associated loci, are significantly enriched for association to the phenotypes in question in extended GWAS analysis. Finally, we test our method in 3 non-immune traits to assess its applicability to complex traits in general. We find that genes in loci associated to height and lipid levels assemble into significantly connected networks but did not detect excess connectivity among Type 2 Diabetes (T2D) loci beyond chance. Taken together, our results constitute evidence that, for many of the complex diseases studied here, common genetic associations implicate regions encoding proteins that physically interact in a preferential manner, in

  14. Using hydroxyl radical footprinting to explore the free energy landscape of protein folding

    PubMed Central

    Calabrese, Antonio N.; Ault, James R.; Radford, Sheena E.; Ashcroft, Alison E.

    2015-01-01

    Characterisation of the conformational states adopted during protein folding, including globally unfolded/disordered structures and partially folded intermediate species, is vital to gain fundamental insights into how a protein folds. In this work we employ fast photochemical oxidation of proteins (FPOP) to map the structural changes that occur in the folding of the four-helical bacterial immunity protein, Im7. Oxidative footprinting coupled with mass spectrometry (MS) is used to probe changes in the solvent accessibility of amino acid side-chains concurrent with the folding process, by quantifying the degree of oxidation experienced by the wild-type protein relative to a kinetically trapped, three-helical folding intermediate and an unfolded variant that lacks secondary structure. Analysis of the unfolded variant by FPOP–MS shows oxidative modifications consistent with the species adopting a solution conformation with a high degree of solvent accessibility. The folding intermediate, by contrast, experiences increased levels of oxidation relative to the wild-type, native protein only in regions destabilised by the amino acid substitutions introduced. The results demonstrate the utility of FPOP–MS to characterise protein variants in different conformational states and to provide insights into protein folding mechanisms that are complementary to measurements such as hydrogen/deuterium exchange labelling and Φ-value analysis. PMID:25746386

  15. Mucosal and systemic immune responses to plasmid protein pgp3 in patients with genital and ocular Chlamydia trachomatis infection

    PubMed Central

    GHAEM-MAGHAMI, S; RATTI, G; GHAEM-MAGHAMI, M; COMANDUCCI, M; HAY, P E; BAILEY, R L; MABEY, D C W; WHITTLE, H C; WARD, M E; LEWIS, D J M

    2003-01-01

    The circulating and cervical B cell responses to Chlamydia trachomatis plasmid protein pgp3 were characterized in children and adults with ocular or genital chlamydial infection using the enzyme-linked immunospot assay (ELISPOT) and ELISA. No pgp3-specific ASCs were detected in healthy controls, but predominantly IgA ASCs were detected in UK adults with uncomplicated cervicitis or urethritis (P = 0·03, 0·019). In patients with extragenital complications or pelvic inflammatory disease a mixed response with more IgG and IgM ASCs was evident, suggesting a breach of mucosal immune compartmentalization with more extensive infection. In women with chlamydial cervicitis, ASCs secreting predominantly IgA, but also IgG, to pgp3 were present in cervix at presentation, with a frequency 30–50 times higher than blood. Cervical ASC numbers, especially IgG, fell markedly six weeks after antibiotic treatment. We detected principally IgA pgp3-specific antibody secreting cells (ASCs) in children resident in a Gambian endemic area, with a trend towards suppression of IgA responses during intense trachomatous inflammation (P = 0·06), as previously reported for other chlamydial antigens, and in keeping with the findings in genital disease. These data provide a rationale for further studies of immune responses to pgp3 in humans and animal models of chlamydia-induced disease, and its potential use in diagnostic assays and protective immunization strategies. PMID:12780690

  16. Maternal immunization of mice with group B streptococcal type III polysaccharide-beta C protein conjugate elicits protective antibody to multiple serotypes.

    PubMed Central

    Madoff, L C; Paoletti, L C; Tai, J Y; Kasper, D L

    1994-01-01

    Group B streptococcal infection is a major cause of neonatal mortality. Antibody to the capsular polysaccharide protects against invasive neonatal disease, but immunization with capsular polysaccharides fails to elicit protective antibody in many recipients. Conjugation of the polysaccharide to tetanus toxoid has been shown to increase immune response to the polysaccharide. In animal models, C proteins of group B streptococci are also protective determinants. We examined the ability of the beta C protein to serve in the dual role of carrier for the polysaccharide and protective immunogen. Type III polysaccharide was covalently coupled to beta C protein by reductive amination. Immunization of rabbits with the polysaccharide-protein conjugate elicited high titers of antibody to both components, and the serum induced opsonophagocytic killing of type III, Ia/C, and Ib/C strains of group B streptococci. Female mice were immunized with the conjugate vaccine and then bred; 93% of neonatal pups born to these dams vaccinated with conjugate survived type III group B streptococcal challenge and 76% survived type Ia/C challenge, compared with 3% and 8% survival, respectively, in controls (P < 0.001). The beta C protein acted as an effective carrier for the type III polysaccharide while simultaneously induced protective immunity against beta C protein--containing strains of group B streptococci. Images PMID:7518832

  17. A novel tetravalent formulation combining the four aggregated domain III-capsid proteins from dengue viruses induces a functional immune response in mice and monkeys.

    PubMed

    Suzarte, Edith; Gil, Lázaro; Valdés, Iris; Marcos, Ernesto; Lazo, Laura; Izquierdo, Alienys; García, Angélica; López, Lázaro; Álvarez, Maylin; Pérez, Yusleydis; Castro, Jorge; Romero, Yaremis; Guzmán, María G; Guillén, Gerardo; Hermida, Lisset

    2015-08-01

    Our group developed a subunit vaccine candidate against dengue virus based on two different viral regions: the domain III of the envelope protein and the capsid protein. The novel chimeric protein from dengue-2 virus [domain III-capsid (DIIIC-2)], when presented as aggregated incorporating oligodeoxynucleotides, induced anti-viral and neutralizing antibodies, a cellular immune response and conferred significant protection to mice and monkeys. The remaining constructs were already obtained and properly characterized. Based on this evidence, this work was aimed at assessing the immune response in mice of the chimeric proteins DIIIC of each serotype, as monovalent and tetravalent formulations. Here, we demonstrated the immunogenicity of each protein in terms of humoral and cell-mediated immunity, without antigen competition on the mixture forming the formulation tetra DIIIC. Accordingly, significant protection was afforded as measured by the limited viral load in the mouse encephalitis model. The assessment of the tetravalent formulation in non-human primates was also conducted. In this animal model, it was demonstrated that the formulation induced neutralizing antibodies and memory cell-mediated immune response with IFN-γ-secreting and cytotoxic capacity, regardless the route of immunization used. Taken together, we can assert that the tetravalent formulation of DIIIC proteins constitutes a promising vaccine candidate against dengue virus, and propose it for further efficacy experiments in monkeys or in the dengue human infection model, as it has been recently proposed.

  18. Differential Immune Responses in Mice Immunized with Recombinant Neutralizing Epitope Protein of Hepatitis E Virus Formulated with Liposome and Alum Adjuvants.

    PubMed

    Joshi, Shivali Shirish; Arankalle, Vidya Avinash

    2016-01-01

    In the developing countries, Hepatitis E virus (HEV) is a predominant cause of sporadic acute hepatitis in adults and waterborne epidemics leading to high mortality in pregnant women. Vaccine development mainly focuses on the structural capsid protein open-reading-frame-2 (ORF-2) of the virus. We successfully evaluated liposome-adjuvanted recombinant neutralizing epitope protein (rNEp), a part of ORF-2, 458-607aa, in mice and rhesus macaques. We compared immune response to adjuvants alone, rNEp alone, or adjuvanted with liposome (lipo-rNEp)/alum (al-rNEp) in mice following intramuscular administration of two doses of 5 μg each. IgG anti-HEV titers (enzyme-linked immunosorbent assay), immunophenotyping (flow cytometry, CD3(+)CD4(+), CD3(+)CD8(+), CD11c(+), CD11b(+), CD19(+) cells; costimulatory markers CD80, CD86, MHC-I, MHC-II, and early activation marker CD69), and levels of Th1/Th2 cytokines (IL-2/IFN-γ/IL-4/IL-5 and additionally IL-1β/IL-6/IL-10/TNF for early time points) were determined at early (4/12/24-h postdose-1) and later time points (2 weeks post-both doses). IgG anti-HEV titers were higher in the lipo-rNEp group than al-rNEp post-both doses (p < 0.05). At early time points, cell type proportions were comparable at the site of injection; IL-Iβ levels increased in lipo-rNEp, 24 h, while IL-6 levels rose in lipo-rNEp/al-rNEp/alum-alone groups, 4 h, compared to controls. In the draining lymph nodes (DLNs), CD11c(+)CD86(+) cells increased at 24 h in liposome-alone/lipo-rNEp groups. A rise in the CD11c(+)CD69(+) cells was noted in the lipo-rNEp group compared to other groups (p < 0.05). Cytokine levels in the spleen/sera remained unchanged in all the groups (p > 0.05). At 2 weeks postdose-2, CD11c(+)MHC-II(+)/CD11b(+)MHC-II(+) cells increased in the spleen in the lipo-rNEp and al-rNEp groups, respectively. In the DLNs, CD19(+)MHC-II(+) cells increased in rNEp/al-rNEp/lipo-rNEp groups post-both doses and CD11c(+)CD86(+) cells in the lipo

  19. Phosphorylation of the Plant Immune Regulator RPM1-INTERACTING PROTEIN4 Enhances Plant Plasma Membrane H+-ATPase Activity and Inhibits Flagellin-Triggered Immune Responses in Arabidopsis[OPEN

    PubMed Central

    Lee, DongHyuk; Bourdais, Gildas; Yu, Gang; Robatzek, Silke; Coaker, Gitta

    2015-01-01

    The Pseudomonas syringae effector AvrB targets multiple host proteins during infection, including the plant immune regulator RPM1-INTERACTING PROTEIN4 (RIN4) and RPM1-INDUCED PROTEIN KINASE (RIPK). In the presence of AvrB, RIPK phosphorylates RIN4 at Thr-21, Ser-160, and Thr-166, leading to activation of the immune receptor RPM1. Here, we investigated the role of RIN4 phosphorylation in susceptible Arabidopsis thaliana genotypes. Using circular dichroism spectroscopy, we show that RIN4 is a disordered protein and phosphorylation affects protein flexibility. RIN4 T21D/S160D/T166D phosphomimetic mutants exhibited enhanced disease susceptibility upon surface inoculation with P. syringae, wider stomatal apertures, and enhanced plasma membrane H+-ATPase activity. The plasma membrane H+-ATPase AHA1 is highly expressed in guard cells, and its activation can induce stomatal opening. The ripk knockout also exhibited a strong defect in pathogen-induced stomatal opening. The basal level of RIN4 Thr-166 phosphorylation decreased in response to immune perception of bacterial flagellin. RIN4 Thr166D lines exhibited reduced flagellin-triggered immune responses. Flagellin perception did not lower RIN4 Thr-166 phosphorylation in the presence of strong ectopic expression of AvrB. Taken together, these results indicate that the AvrB effector targets RIN4 in order to enhance pathogen entry on the leaf surface as well as dampen responses to conserved microbial features. PMID:26198070

  20. Two endoplasmic reticulum proteins (calnexin and calreticulin) are involved in innate immunity in Chinese mitten crab (Eriocheir sinensis)

    PubMed Central

    Huang, Ying; Hui, Kaimin; Jin, Min; Yin, Shaowu; Wang, Wen; Ren, Qian

    2016-01-01

    Calnexin (Cnx) and calreticulin (Crt), which are important chaperones in the endoplasmic reticulum (ER), participate in the folding and quality control of client proteins. Cnx and Crt identified from Chinese mitten crab (Eriocheir sinensis) are designated as EsCnx and EsCrt, respectively. EsCnx and EsCrt are expressed in the hemocyte, hepatopancrea, gill, and intestine at the mRNA and protein level. Immunofluorescence analysis indicated that EsCnx and EsCRT are located in the ER. Moreover, the mRNA and protein expression levels of EsCnx and EsCrt were altered by challenge with lipopolysaccharides (LPS), peptidoglycans (PGN), Staphyloccocus aureus, and Vibrio parahaemolyticus. Recombinant EsCnx and EsCrt (rEsCnx and rEsCrt, respectively) proteins can bind to various Gram-positive and Gram-negative bacteria, as well as to different polysaccharides (LPS and PGN). rEsCnx and rEsCrt assisted in the clearance of V. parahaemolyticus in vivo, and the clearance efficiency was impaired after silencing of EsCnx and EsCrt. Our results suggest that the two ER proteins are involved in anti-bacterial immunity in E. sinensis. PMID:27279413

  1. Serum and salivary antibody responses in rats orally immunized with Streptococcus mutans carbohydrate protein conjugate associated with liposomes.

    PubMed Central

    Wachsmann, D; Klein, J P; Scholler, M; Ogier, J; Ackermans, F; Frank, R M

    1986-01-01

    In this study we describe the preparation of a Streptococcus mutans vaccine consisting of a purified polysaccharide antigen, derived from S. mutans OMZ175 serotype f, covalently coupled through reductive amination to a previously isolated 74,000-molecular-weight (74K) cell wall protein which interacts with saliva proteins (74K-SR). We also investigated the local and systemic immune response to the poly-74K-SR conjugate after oral administration of the conjugate associated with liposomes. Intragastric administration of liposome-associated poly-74K-SR conjugate in rats produced a local immunoglobulin A (IgA) response directed against the polysaccharide and the cell surface protein, whereas liposome-associated polysaccharide was unable to induce any detectable local IgA response. The antigenicity of the polysaccharide in the conjugate was not affected by the coupling reaction, while that of the cell surface protein was reduced. We showed that the immunogenicity of S. mutans polysaccharide could be improved by chemical coupling with a carrier cell surface protein. If such a conjugate were orally administered with liposomes it could constitute a potential vaccine against dental caries. Images PMID:3699888

  2. Two endoplasmic reticulum proteins (calnexin and calreticulin) are involved in innate immunity in Chinese mitten crab (Eriocheir sinensis).

    PubMed

    Huang, Ying; Hui, Kaimin; Jin, Min; Yin, Shaowu; Wang, Wen; Ren, Qian

    2016-01-01

    Calnexin (Cnx) and calreticulin (Crt), which are important chaperones in the endoplasmic reticulum (ER), participate in the folding and quality control of client proteins. Cnx and Crt identified from Chinese mitten crab (Eriocheir sinensis) are designated as EsCnx and EsCrt, respectively. EsCnx and EsCrt are expressed in the hemocyte, hepatopancrea, gill, and intestine at the mRNA and protein level. Immunofluorescence analysis indicated that EsCnx and EsCRT are located in the ER. Moreover, the mRNA and protein expression levels of EsCnx and EsCrt were altered by challenge with lipopolysaccharides (LPS), peptidoglycans (PGN), Staphyloccocus aureus, and Vibrio parahaemolyticus. Recombinant EsCnx and EsCrt (rEsCnx and rEsCrt, respectively) proteins can bind to various Gram-positive and Gram-negative bacteria, as well as to different polysaccharides (LPS and PGN). rEsCnx and rEsCrt assisted in the clearance of V. parahaemolyticus in vivo, and the clearance efficiency was impaired after silencing of EsCnx and EsCrt. Our results suggest that the two ER proteins are involved in anti-bacterial immunity in E. sinensis. PMID:27279413

  3. A Phytophthora sojae effector suppresses endoplasmic reticulum stress-mediated immunity by stabilizing plant Binding immunoglobulin Proteins

    PubMed Central

    Jing, Maofeng; Guo, Baodian; Li, Haiyang; Yang, Bo; Wang, Haonan; Kong, Guanghui; Zhao, Yao; Xu, Huawei; Wang, Yan; Ye, Wenwu; Dong, Suomeng; Qiao, Yongli; Tyler, Brett M.; Ma, Wenbo; Wang, Yuanchao

    2016-01-01

    Phytophthora pathogens secrete an array of specific effector proteins to manipulate host innate immunity to promote pathogen colonization. However, little is known about the host targets of effectors and the specific mechanisms by which effectors increase susceptibility. Here we report that the soybean pathogen Phytophthora sojae uses an essential effector PsAvh262 to stabilize endoplasmic reticulum (ER)-luminal binding immunoglobulin proteins (BiPs), which act as negative regulators of plant resistance to Phytophthora. By stabilizing BiPs, PsAvh262 suppresses ER stress-triggered cell death and facilitates Phytophthora infection. The direct targeting of ER stress regulators may represent a common mechanism of host manipulation by microbes. PMID:27256489

  4. [THE SCREENING OF DIAGNOSTIC POTENTIAL OF NATIVE PROTEIN FRACTIONS OF MYCOBACTERIUM TUBERCULOSIS USING TECHNIQUE OF IMMUNE BLOTTING].

    PubMed

    Tsibulkin, A P; Khaertinova, I M; Urazov, N G; Khaertinov, K S

    2016-02-01

    The technique of immune blotting was used to analyze surface proteins obtained from cells M Tuberculosis exposed to partialmode of delipidization. At that, there were applied serums of patients with tuberculosis of lungs; HIV agents and patients with concomitant infection tuberculosis-AIDS and also HIV-negative patients without clinical signs of disease of lungs and with chronic diseases of lungs of other etiology The fractions oflow-molecular antigens with molecular mass 6.5-30 kilodaltons became diagnostically significant. To this fraction of antigens reacted serums of all patients with tuberculosis of lungs and serums of 91% of patients with concomitant tuberculosis-AIDS infection. The antigens of protein fractions with high (70-100 kilodaltons) and interim (30-69 kilodaltons) molecular mass became diagnostically insignificant. PMID:27455562

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

    NASA Astrophysics Data System (ADS)

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

    2000-07-01

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

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

    PubMed Central

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

    2000-01-01

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

  7. Glucose Availability and AMP-Activated Protein Kinase Link Energy Metabolism and Innate Immunity in the Bovine Endometrium.

    PubMed

    Turner, Matthew L; Cronin, James G; Noleto, Pablo G; Sheldon, I Martin

    2016-01-01

    Defences against the bacteria that usually infect the endometrium of postpartum cattle are impaired when there is metabolic energy stress, leading to endometritis and infertility. The endometrial response to bacteria depends on innate immunity, with recognition of pathogen-associated molecular patterns stimulating inflammation, characterised by secretion of interleukin (IL)-1β, IL-6 and IL-8. How metabolic stress impacts tissue responses to pathogens is unclear, but integration of energy metabolism and innate immunity means that stressing one system might affect the other. Here we tested the hypothesis that homeostatic pathways integrate energy metabolism and innate immunity in bovine endometrial tissue. Glucose deprivation reduced the secretion of IL-1β, IL-6 and IL-8 from ex vivo organ cultures of bovine endometrium challenged with the pathogen-associated molecular patterns lipopolysaccharide and bacterial lipopeptide. Endometrial inflammatory responses to lipopolysaccharide were also reduced by small molecules that activate or inhibit the intracellular sensor of energy, AMP-activated protein kinase (AMPK). However, inhibition of mammalian target of rapamycin, which is a more global metabolic sensor than AMPK, had little effect on inflammation. Similarly, endometrial inflammatory responses to lipopolysaccharide were not affected by insulin-like growth factor-1, which is an endocrine regulator of metabolism. Interestingly, the inflammatory responses to lipopolysaccharide increased endometrial glucose consumption and induced the Warburg effect, which could exacerbate deficits in glucose availability in the tissue. In conclusion, metabolic energy stress perturbed inflammatory responses to pathogen-associated molecular patterns in bovine endometrial tissue, and the most fundamental regulators of cellular energy, glucose availability and AMPK, had the greatest impact on innate immunity. PMID:26974839

  8. Glucose Availability and AMP-Activated Protein Kinase Link Energy Metabolism and Innate Immunity in the Bovine Endometrium

    PubMed Central

    Turner, Matthew L.; Cronin, James G.; Noleto, Pablo G.; Sheldon, I. Martin

    2016-01-01

    Defences against the bacteria that usually infect the endometrium of postpartum cattle are impaired when there is metabolic energy stress, leading to endometritis and infertility. The endometrial response to bacteria depends on innate immunity, with recognition of pathogen-associated molecular patterns stimulating inflammation, characterised by secretion of interleukin (IL)-1β, IL-6 and IL-8. How metabolic stress impacts tissue responses to pathogens is unclear, but integration of energy metabolism and innate immunity means that stressing one system might affect the other. Here we tested the hypothesis that homeostatic pathways integrate energy metabolism and innate immunity in bovine endometrial tissue. Glucose deprivation reduced the secretion of IL-1β, IL-6 and IL-8 from ex vivo organ cultures of bovine endometrium challenged with the pathogen-associated molecular patterns lipopolysaccharide and bacterial lipopeptide. Endometrial inflammatory responses to lipopolysaccharide were also reduced by small molecules that activate or inhibit the intracellular sensor of energy, AMP-activated protein kinase (AMPK). However, inhibition of mammalian target of rapamycin, which is a more global metabolic sensor than AMPK, had little effect on inflammation. Similarly, endometrial inflammatory responses to lipopolysaccharide were not affected by insulin-like growth factor-1, which is an endocrine regulator of metabolism. Interestingly, the inflammatory responses to lipopolysaccharide increased endometrial glucose consumption and induced the Warburg effect, which could exacerbate deficits in glucose availability in the tissue. In conclusion, metabolic energy stress perturbed inflammatory responses to pathogen-associated molecular patterns in bovine endometrial tissue, and the most fundamental regulators of cellular energy, glucose availability and AMPK, had the greatest impact on innate immunity. PMID:26974839

  9. Polymeric hepatitis C virus non-structural protein 5A nanocapsules induce intrahepatic antigen-specific immune responses.

    PubMed

    Fichter, Michael; Piradashvili, Keti; Pietrzak-Nguyen, Anette; Pretsch, Leah; Kuhn, Gabor; Strand, Susanne; Knuf, Markus; Zepp, Fred; Wurm, Frederik R; Mailänder, Volker; Landfester, Katharina; Gehring, Stephan

    2016-11-01

    Targeting antigen combined with adjuvants to hepatic antigen-presenting cells (APCs) is essential for the induction of intrahepatic T cellular immunity controlling and resolving viral infections of the liver. Intravenous injection of antigen-loaded nanoparticles is a promising approach for the delivery of antigens to liver APCs. Accordingly, polymeric nanocapsules (NCs) synthesized exclusively of hepatitis C virus non-structural protein 5A (NS5A) and the adjuvant monophosphoryl lipid A (MPLA) adsorbed to the nanocapsule surface were developed. Aim of the present study was the evaluation of the in vitro and in vivo behavior of MPLA-functionalized NS5A-NCs regarding the interaction with liver dendritic cells (DCs) and the potential to induce intrahepatic immune responses in a mouse model. Maturation of DCs was significantly increased by application of NS5A+MPLA-NCs compared to non-functionalized NS5A-NCs promoting a vigorous expression of CD40, CD80, CD86 and a strong secretion of the Th1-related cytokine IL-12. NS5A-NCs were preferentially deposited in DCs and Kupffer cells residing in the liver after intravenous administration. Immunization with NS5A-NCs induced intrahepatic antigen-specific CD4(+) T cellular immune responses determined by the secretion of IFNγ and IL-2. Furthermore, supplementation with MPLA induced significant levels of NS5A-specific antibodies. The application of polymeric nanocapsules synthesized exclusively out of antigen avoids the risk of unintended side effects caused by additional carrier substances. Functionalization with adjuvants like MPLA and the efficient targeting to liver-resident APCs inherits the potential for application of antigen nanocapsules in further vaccination approaches against pathogens affecting the liver. PMID:27614817

  10. Immune reactivity to heat shock protein 70 expressed in the kidney is cause of salt-sensitive hypertension

    PubMed Central

    Pons, Héctor; Ferrebuz, Atilio; Quiroz, Yasmir; Romero-Vasquez, Freddy; Parra, Gustavo; Johnson, Richard J.

    2013-01-01

    Hypertension affects one-third of the adult population of the world. The causes of hypertension are incompletely understood, but relative impairment of sodium excretion is central to its pathogenesis. Immune cell infiltration in the kidney is a constant finding in hypertension that in association with local angiotensin and oxidants causes a defect in sodium excretion. However, it is unclear if the T cell influx into the kidney responds to nonspecific chemokine cues or is due to antigen-driven immune attraction. We found that T cells in experimentally induced salt-driven hypertension present a CD4 clonal response to heat shock protein 70 (HSP70) that is overexpressed in the kidney. We used a highly preserved amino acid sequence within the HSP molecule to induce immune tolerance associated with the generation of IL-10 producing regulatory T cells. Immune tolerant rats to HSP70 developed minimal renal inflammation and were protected from the development of salt-sensitive hypertension. Adoptive transfer of T lymphocytes isolated from spleen of tolerized rats also reversed hypertension. HSP70 gene delivery to the renal vein of the kidneys of rats sensitized to HSP70 caused an increment in blood pressure in response to a high-salt diet. The HSP70 peptide used in this work induces a strong proliferative response in peripheral blood lymphocytes of patients with essential hypertension. These studies provide evidence that autoimmunity plays a role in salt-sensitive hypertension and identifies HSP70 expressed in the kidney as one key antigen. These findings raise the possibility of novel approaches to the treatment of this condition. PMID:23097471

  11. Oral Immunization with Recombinant Vaccinia Virus Prime and Intramuscular Protein Boost Provides Protection against Intrarectal Simian-Human Immunodeficiency Virus Challenge in Macaques

    PubMed Central

    Thippeshappa, Rajesh; Tian, Baoping; Cleveland, Brad; Guo, Wenjin; Polacino, Patricia

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) acquisition occurs predominantly through mucosal transmission. We hypothesized that greater mucosal immune responses and protective efficacy against mucosal HIV-1 infection may be achieved by prime-boost immunization at mucosal sites. We used a macaque model to determine the safety, immunogenicity, and protective efficacy of orally delivered, replication-competent but attenuated recombinant vaccinia viruses expressing full-length HIV-1 SF162 envelope (Env) or simian immunodeficiency virus (SIV) Gag-Pol proteins. We examined the dose and route that are suitable for oral immunization with recombinant vaccinia viruses. We showed that sublingual inoculation of two vaccinia virus-naive pigtailed macaques with 5 × 108 PFU of recombinant vaccinia viruses was safe. However, sublingual inoculation with a higher dose or tonsillar inoculation resulted in secondary oral lesions, indicating the need to optimize the dose and route for oral immunization with replication-competent vaccinia virus vectors. Oral priming alone elicited antibody responses to vaccinia virus and to the SF162 Env protein. Intramuscular immunization with the SF162 gp120 protein at either 20 or 21 weeks postpriming resulted in a significant boost in antibody responses in both systemic and mucosal compartments. Furthermore, we showed that immune responses induced by recombinant vaccinia virus priming and intramuscular protein boosting provided protection against intrarectal challenge with the simian-human immunodeficiency virus SHIV-SF162-P4. PMID:26718849

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

    PubMed Central

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

    1994-01-01

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

  13. Mucosal immunization with PsaA protein, using chitosan as a delivery system, increases protection against acute otitis media and invasive infection by Streptococcus pneumoniae.

    PubMed

    Xu, J-H; Dai, W-J; Chen, B; Fan, X-Y

    2015-03-01

    As infection with Streptococcus pneumoniae (mainly via the mucosal route) is a leading cause of acute otitis media, sinus and bacterial pneumonia, the mucosal immunity plays an important role in the prevention of pneumococcal diseases. Therefore, intranasal vaccination may be an effective immunization strategy, but requires appropriate mucosal vaccine delivery systems. In this work, chitosan was used as a mucosal delivery system to form chitosan-PsaA nanoparticles based on ionotropic gelation methods and used to immunize BALB/c mice intranasally. Compared to mice immunized with naked PsaA, levels of IFN-γ, IL-17A and IL-4 in spleen lymphocytes, the systemic (IgG in serum) and mucosal (IgA in mucosal lavage) specific antibodies were enhanced significantly in mice inoculated with chitosan-PsaA. Furthermore, increased protection against acute otitis media following middle ear challenge with pneumococcus serotype 14, and improved survival following intraperitoneal challenge with pneumococcus serotype 3 or serotype 14, was found in the mice immunized with chitosan-PsaA nanoparticles. Thus, intranasal immunization with chitosan-PsaA can successfully induce mucosal and systemic immune responses and increase protection against pneumococcal acute otitis media and invasive infections. Hence, intranasal immunization with PsaA protein, based on chitosan as a delivery system, is an efficient immunization strategy for preventing pneumococcal infections.

  14. A protein with characteristics of factor H is present on rodent platelets and functions as the immune adherence receptor.

    PubMed

    Alexander, J J; Hack, B K; Cunningham, P N; Quigg, R J

    2001-08-24

    Complement-coated particles interact with specific immune adherence receptors (IAR). In primates, this function is served by complement receptor 1 (CR1) on erythrocytes. In contrast, rodent platelets bear IAR distinct from CR1, the identity of which was studied here. A 150-kDa C3b-binding protein was isolated from rat platelets, which had immunochemical and biochemical identity to plasma factor H. Immunofluorescence microscopy and flow cytometry demonstrated that factor H was present on the surface of rat and mouse platelets, which could be removed by treatment with neuraminidase. Sheep erythrocytes bearing C3b underwent immune adherence with rat and mouse platelets, which was blocked with anti-factor H F(ab')(2) antibodies, but not with antibodies binding to the complement regulator, Crry, on the platelet surface. By reverse transcription-polymerase chain reaction using rat platelet RNA and primers designed from mouse factor H, a 472-base pair product was generated that was identical in sequence to that produced from rat liver RNA. The translated protein product was 85% similar to mouse liver factor H. The 3'-nucleotide sequence from platelets predicted a soluble factor H protein. By Northern analysis, liver and platelets had identically sized factor H mRNA. Thus, rat and mouse platelets have a membrane protein with characteristics of factor H that is linked via sialic acid residues and functions as the IAR. Whether platelet factor H is acquired by passive adsorption from sera and/or is produced by platelets remains to be determined.

  15. Identification of adhesin-like protein ALP41 from Spiroplasma eriocheiris and induction immune response of Eriocheir sinensis.

    PubMed

    Meng, Qingguo; Li, Wenjie; Liang, Tingming; Jiang, Xuejiao; Gu, Wei; Wang, Wen

    2010-10-01

    Spiroplasma eriocheiris is a causative agent of the tremor disease (TD) of Chinese mitten crab Eriocheir sinensis which is a novel pathogen of aquatic animals found in recent years. A gene, adhesin-like protein (ALP41), of S. eriocheiris from E. sinensis was identified and its characteristics were analyzed in present paper. The role of this pathogen's host-binding protein in promoting immune responses was characterized through analyzing the interaction between S. eriocheiris and E. sinensis. The full-length DNA of ALP41 is 1074 bp and encodes 357 amino acid residues. The theoretical molecular weight and isoelectric point for the ALP41 are 40.94 kDa and 4.79, respectively. Since UGA is read as a tryptophan codon and not as a termination signal in most Mollicute species, the ALP41 gene was site-mutated from TGA to TGG and transcribed in Escherichia coli to full expression; the titer of rabbits anti-ALP41 serum was about 1:6000. A specific immunoreactive band was identified when rabbits anti-S. eriocheiris serum was opposed to the recombinant protein. The ALP41 band was detected using anti-ALP41 serum and the total proteins of S. eriocheiris. Realtime-PCR was used for detection of expression levels of the immune genes in E. sinensis. Among the examined genes, the mRNA expression of anti-lipopolysaccharide factor (ALF), prophenoloxidase (proPO), peroxiredoxin 6 (Prx6) and pacifastin light chain (PLC) in E. sinensis were significantly induced after ALP41 treatment. PMID:20538062

  16. Proteins Encoded in Genomic Regions Associated with Immune-Mediated Disease Physically Interact and Suggest Underlying Biology

    PubMed Central

    Rossin, Elizabeth J.; Lage, Kasper; Raychaudhuri, Soumya; Xavier, Ramnik J.; Tatar, Diana; Benita, Yair

    2011-01-01

    Genome-wide association studies (GWAS) have defined over 150 genomic regions unequivocally containing variation predisposing to immune-mediated disease. Inferring disease biology from these observations, however, hinges on our ability to discover the molecular processes being perturbed by these risk variants. It has previously been observed that different genes harboring causal mutations for the same Mendelian disease often physically interact. We sought to evaluate the degree to which this is true of genes within strongly associated loci in complex disease. Using sets of loci defined in rheumatoid arthritis (RA) and Crohn's disease (CD) GWAS, we build protein–protein interaction (PPI) networks for genes within associated loci and find abundant physical interactions between protein products of associated genes. We apply multiple permutation approaches to show that these networks are more densely connected than chance expectation. To confirm biological relevance, we show that the components of the networks tend to be expressed in similar tissues relevant to the phenotypes in question, suggesting the network indicates common underlying processes perturbed by risk loci. Furthermore, we show that the RA and CD networks have predictive power by demonstrating that proteins in these networks, not encoded in the confirmed list of disease associated loci, are significantly enriched for association to the phenotypes in question in extended GWAS analysis. Finally, we test our method in 3 non-immune traits to assess its applicability to complex traits in general. We find that genes in loci associated to height and lipid levels assemble into significantly connected networks but did not detect excess connectivity among Type 2 Diabetes (T2D) loci beyond chance. Taken together, our results constitute evidence that, for many of the complex diseases studied here, common genetic associations implicate regions encoding proteins that physically interact in a preferential manner, in

  17. Immunization of Mice with Recombinant Protein CobB or AsnC Confers Protection against Brucella abortus Infection

    PubMed Central

    Du, Xinying; Yu, Shuang; Bai, Yaoxia; Chen, Yanfen; Wang, Tongkun; Wang, Zhoujia; Yu, Yaqing; Peng, Guangneng; Huang, Kehe; Huang, Liuyu; Wang, Yufei; Chen, Zeliang

    2012-01-01

    Due to drawbacks of live attenuated vaccines, much more attention has been focused on screening of Brucella protective antigens as subunit vaccine candidates. Brucella is a facultative intracellular bacterium and cell mediated immunity plays essential roles for protection against Brucella infection. Identification of Brucella antigens that present T-cell epitopes to the host could enable development of such vaccines. In this study, 45 proven or putative pathogenesis-associated factors of Brucella were selected according to currently available data. After expressed and purified, 35 proteins were qualified for analysis of their abilities to stimulate T-cell responses in vitro. Then, an in vitro gamma interferon (IFN-γ) assay was used to identify potential T-cell antigens from B. abortus. In total, 7 individual proteins that stimulated strong IFN-γ responses in splenocytes from mice immunized with B. abortus live vaccine S19 were identified. The protective efficiencies of these 7 recombinant proteins were further evaluated. Mice given BAB1_1316 (CobB) or BAB1_1688 (AsnC) plus adjuvant could provide protection against virulent B. abortus infection, similarly with the known protective antigen Cu-Zn SOD and the license vaccine S19. In addition, CobB and AsnC could induce strong antibodies responses in BALB/c mice. Altogether, the present study showed that CobB or AsnC protein could be useful antigen candidates for the development of subunit vaccines against brucellosis with adequate immunogenicity and protection efficacy. PMID:22383953

  18. Goat immune response to capripox vaccine expressing the hemagglutinin protein of peste des petits ruminants.

    PubMed

    Diallo, A; Minet, C; Berhe, G; Le Goff, C; Black, D N; Fleming, M; Barrett, T; Grillet, C; Libeau, G

    2002-10-01

    Sheep-pox and capripox are contagious diseases of domestic small ruminants for which the causal agent is a poxvirus classified into the Capripoxvirus genus. Viruses of this group have a host range specific to sheep, goats, cattle, and possibly buffalo. Thus, they are clearly indicated as vectors for the development of recombinant vaccines for peste des petits ruminants (PPR). Here we report the immune response of goats inoculated with a recombinant capripox-PPR hemagglutinin.

  19. Effects of immunization against androstenedione or bone morphogenetic protein 15 (BMP15) on reproductive performance in sheep.

    PubMed

    Juengel, J L; Proctor, L E; Wearne, K; Olliver, D; Hudson, N L; Jensen, D; Davis, G H; Johnstone, P D; McNatty, K P

    2013-12-01

    Partial neutralization of bone morphogenetic protein 15 (BMP15) bioactivity by immunization is known to increase ovulation rate in sheep. However, it remains uncertain whether BMP15 vaccination would be a suitable procedure for increasing lambing rate. The aim of this study was to compare the efficacy of a BMP15 vaccination treatment on lamb production to that of commercially-available androstenedione-based vaccines that are used for this purpose. Ewes were immunized for 3 yr against androstenedione, BMP15, or no antigen (control). Vaccination with androstenedione or BMP15 altered (P < 0.05) ovulation rate as well as litter size at midpregnancy, birth, and weaning compared with controls. No differences were detected in the proportions of ewes conceiving in the first cycle or partial failure of multiple ovulations. Both gender and litter size affected birth weight of the lamb (P < 0.05), but no effect of treatment was found. Growth rate was significantly affected (P < 0.05) by gender, birth weight, and the number of lambs raised, but not treatment. In conclusion, immunization against either androstenedione or BMP15 increased ovulation rate. Androstenedione vaccination also increased the number of lambs weaned (P < 0.05). Bone morphogenetic protein 15 vaccination altered the pattern of the number of lambs weaned, but no increase in lamb production was observed as more ewes produced zero or three lambs. Overall, androstenedione or BMP15 vaccination did not significantly affect embryo or fetal survival or lamb performance independently of the effects of these treatments on ovulation rate.

  20. PBL13 Is a Serine/Threonine Protein Kinase That Negatively Regulates Arabidopsis Immune Responses1[OPEN

    PubMed Central

    2015-01-01

    Receptor-like cytoplasmic kinases (RLCKs) are a subset of plant receptor-like kinases lacking both extracellular and transmembrane domains. Some of the 46 members in the Arabidopsis (Arabidopsis thaliana) RLCK subfamily VII have been linked to plant innate immunity; however, most remain uncharacterized. Thus, multiple subfamily VII members are expected to be involved in plant immune signaling. Here, we investigate the role of AvrPphB SUSCEPTIBLE1-LIKE13 (PBL13), a subfamily VII RLCK with unique domain architecture. Unlike other characterized RLCKs, PBL13 transfer DNA insertion lines exhibit enhanced disease resistance after inoculation with virulent Pseudomonas syringae. The pbl13-2 knockout also exhibits elevated basal-level expression of the PATHOGENESIS-RELATED GENE1 defense marker gene, enhanced reactive oxygen species (ROS) burst in response to perception of bacterial microbial patterns, and accelerated flagellin-induced activation of mitogen-activated protein kinases. Recombinant PBL13 is an active kinase, and its primary autophosphorylated sites map to a 15-amino acid repeat motif unique to PBL13. Complementation of pbl13-2 with PBL13-3xFLAG converts the enhanced resistance and elevated ROS phenotypes back to wild-type levels. In contrast, kinase-dead PBL13K111A-3xFLAG was unable to rescue pbl13-2 disease phenotypes. Consistent with the enhanced ROS burst in the pbl13-2 knockout, PBL13 is able to associate with the nicotinamide adenine dinucleotide phosphate, reduced oxidase RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN D (RBOHD) by split-luciferase complementation assay, and this association is disrupted by flagellin treatment. We conclude that the PBL13 kinase negatively regulates plant innate immunity to pathogenic bacteria and can associate with RBOHD before pathogen perception. These data are consistent with the hypothesis that PBL13 acts to prevent inappropriate activation of defense responses in the absence of pathogen challenge. PMID:26432875

  1. Immune Surveillance and Therapy of Lymphomas Driven by Epstein-Barr -Virus Protein LMP1 in a Mouse Model

    PubMed Central

    Zhang, Baochun; Kracker, Sven; Yasuda, Tomoharu; Casola, Stefano; Vanneman, Matthew; Hömig-Hölzel, Cornelia; Wang, Zhe; Derudder, Emmanuel; Li, Shuang; Chakraborty, Tirtha; Cotter, Shane E.; Koyama, Shohei; Currie, Treeve; Freeman, Gordon J.; Kutok, Jeffery L.; Rodig, Scott J.; Dranoff, Glenn; Rajewsky, Klaus

    2012-01-01

    SUMMARY B cells infected by Epstein-Barr-Virus (EBV), a transforming virus endemic in humans, are rapidly cleared by the immune system, but some cells harboring the virus persist for life. Under conditions of immunosuppression EBV can spread from these cells and cause life threatening pathologies. We have generated mice expressing the transforming EBV latent membrane protein 1 (LMP1), mimicking a constitutively active CD40 coreceptor, specifically in B cells. Like human EBV infected cells, LMP1+ B cells were efficiently eliminated by T cells, and breaking immune surveillance resulted in rapid, fatal lymphoproliferation and lymphomagenesis. The lymphoma cells expressed ligands for a natural killer (NK) cell receptor, NKG2D, and could be targeted by an NKG2D-Fc fusion protein. These experiments indicate a central role for LMP1 in the surveillance and transformation of EBV infected B cells in vivo, establish a pre-clinical model for B cell lymphomagenesis in immunosuppressed patients, and validate a novel therapeutic approach. PMID:22341446

  2. Immune Activation in the Female Genital Tract: Expression Profiles of Soluble Proteins in Women at High Risk for HIV Infection.

    PubMed

    Francis, Suzanna C; Hou, Yanwen; Baisley, Kathy; van de Wijgert, Janneke; Watson-Jones, Deborah; Ao, Trong T; Herrera, Carolina; Maganja, Kaballa; Andreasen, Aura; Kapiga, Saidi; Coulton, Gary R; Hayes, Richard J; Shattock, Robin J

    2016-01-01

    Soluble cervicovaginal biomarkers of inflammation, immune activation and risk of HIV acquisition are needed to reliably assess the safety of new biomedical prevention strategies including vaccines and microbicides. However, a fuller understanding of expression profiles in women at high risk for HIV infection is crucial to the effective use of these potential biomarkers in Phase 3 trial settings. We have measured 45 soluble proteins and peptides in cervicovaginal lavage samples from 100 HIV negative women at high risk for HIV infection. Women were followed over one menstrual cycle to investigate modulation by hormonal contraception, menstrual cycle phase, recent sexual exposure and intravaginal practices. Women using injectable DMPA had increased concentration of several soluble proteins of the innate and adaptive immune system, including IL-1α, IL-1β, IL-2, MIP-1β, IP-10, IL-8, TGF-β, HBD4, IgA, IgG1, and IgG2. Women using combined oral contraceptives had a similar signature. There were differences in concentrations among samples from post-ovulation compared to pre-ovulation, notably increased immunoglobulins. Increased prostate-specific antigen, indicative of recent sexual exposure, was correlated with increased IL-6, MCP-1, and SLPI, and decreased GM-CSF and HBD3. The identified signature profiles may prove critical in evaluating the potential safety and impact on risk of HIV acquisition of different biomedical intervention strategies.

  3. Absorption of PCB126 by upper airways impairs G protein-coupled receptor-mediated immune response

    NASA Astrophysics Data System (ADS)

    Shimada, Ana Lúcia B.; Cruz, Wesley S.; Loiola, Rodrigo A.; Drewes, Carine C.; Dörr, Fabiane; Figueiredo, Natália G.; Pinto, Ernani; Farsky, Sandra H. P.

    2015-10-01

    PCB126 is a dioxin-like polychlorinated biphenyl (PCB) environmental pollutant with a significant impact on human health, as it bioaccumulates and causes severe toxicity. PCB126-induced immune toxicity has been described, although the mechanisms have not been fully elucidated. In this study, an in vivo protocol of PCB126 intoxication into male Wistar rats by intranasal route was used, which has not yet been described. The intoxication was characterised by PCB126 accumulation in the lungs and liver, and enhanced aryl hydrocarbon receptor expression in the liver, lungs, kidneys, and adipose tissues. Moreover, an innate immune deficiency was characterised by impairment of adhesion receptors on blood leukocytes and by reduced blood neutrophil locomotion and oxidative burst activation elicited by ex vivo G protein-coupled receptor (GPCR) activation. Specificity of PCB126 actions on the GPCR pathway was shown by normal burst oxidative activation evoked by Toll-like receptor 4 and protein kinase C direct activation. Moreover, in vivo PCB180 intoxication did not alter adhesion receptors on blood leukocytes either blood neutrophil locomotion, and only partially reduced the GPCR-induced burst oxidative activation on neutrophils. Therefore, a novel mechanism of in vivo PCB126 toxicity is described which impairs a pivotal inflammatory pathway to the host defence against infections.

  4. Immune Activation in the Female Genital Tract: Expression Profiles of Soluble Proteins in Women at High Risk for HIV Infection

    PubMed Central

    Francis, Suzanna C.; Hou, Yanwen; Baisley, Kathy; van de Wijgert, Janneke; Watson-Jones, Deborah; Ao, Trong T.; Herrera, Carolina; Maganja, Kaballa; Andreasen, Aura; Kapiga, Saidi; Coulton, Gary R.; Hayes, Richard J.; Shattock, Robin J.

    2016-01-01

    Soluble cervicovaginal biomarkers of inflammation, immune activation and risk of HIV acquisition are needed to reliably assess the safety of new biomedical prevention strategies including vaccines and microbicides. However, a fuller understanding of expression profiles in women at high risk for HIV infection is crucial to the effective use of these potential biomarkers in Phase 3 trial settings. We have measured 45 soluble proteins and peptides in cervicovaginal lavage samples from 100 HIV negative women at high risk for HIV infection. Women were followed over one menstrual cycle to investigate modulation by hormonal contraception, menstrual cycle phase, recent sexual exposure and intravaginal practices. Women using injectable DMPA had increased concentration of several soluble proteins of the innate and adaptive immune system, including IL-1α, IL-1β, IL-2, MIP-1β, IP-10, IL-8, TGF-β, HBD4, IgA, IgG1, and IgG2. Women using combined oral contraceptives had a similar signature. There were differences in concentrations among samples from post-ovulation compared to pre-ovulation, notably increased immunoglobulins. Increased prostate-specific antigen, indicative of recent sexual exposure, was correlated with increased IL-6, MCP-1, and SLPI, and decreased GM-CSF and HBD3. The identified signature profiles may prove critical in evaluating the potential safety and impact on risk of HIV acquisition of different biomedical intervention strategies. PMID:26814891

  5. Aptamer-conjugated live human immune cell based biosensors for the accurate detection of C-reactive protein

    NASA Astrophysics Data System (ADS)

    Hwang, Jangsun; Seo, Youngmin; Jo, Yeonho; Son, Jaewoo; Choi, Jonghoon

    2016-10-01

    C-reactive protein (CRP) is a pentameric protein that is present in the bloodstream during inflammatory events, e.g., liver failure, leukemia, and/or bacterial infection. The level of CRP indicates the progress and prognosis of certain diseases; it is therefore necessary to measure CRP levels in the blood accurately. The normal concentration of CRP is reported to be 1–3 mg/L. Inflammatory events increase the level of CRP by up to 500 times; accordingly, CRP is a biomarker of acute inflammatory disease. In this study, we demonstrated the preparation of DNA aptamer-conjugated peripheral blood mononuclear cells (Apt-PBMCs) that specifically capture human CRP. Live PBMCs functionalized with aptamers could detect different levels of human CRP by producing immune complexes with reporter antibody. The binding behavior of Apt-PBMCs toward highly concentrated CRP sites was also investigated. The immune responses of Apt-PBMCs were evaluated by measuring TNF-alpha secretion after stimulating the PBMCs with lipopolysaccharides. In summary, engineered Apt-PBMCs have potential applications as live cell based biosensors and for in vitro tracing of CRP secretion sites.

  6. Aptamer-conjugated live human immune cell based biosensors for the accurate detection of C-reactive protein

    PubMed Central

    Hwang, Jangsun; Seo, Youngmin; Jo, Yeonho; Son, Jaewoo; Choi, Jonghoon

    2016-01-01

    C-reactive protein (CRP) is a pentameric protein that is present in the bloodstream during inflammatory events, e.g., liver failure, leukemia, and/or bacterial infection. The level of CRP indicates the progress and prognosis of certain diseases; it is therefore necessary to measure CRP levels in the blood accurately. The normal concentration of CRP is reported to be 1–3 mg/L. Inflammatory events increase the level of CRP by up to 500 times; accordingly, CRP is a biomarker of acute inflammatory disease. In this study, we demonstrated the preparation of DNA aptamer-conjugated peripheral blood mononuclear cells (Apt-PBMCs) that specifically capture human CRP. Live PBMCs functionalized with aptamers could detect different levels of human CRP by producing immune complexes with reporter antibody. The binding behavior of Apt-PBMCs toward highly concentrated CRP sites was also investigated. The immune responses of Apt-PBMCs were evaluated by measuring TNF-alpha secretion after stimulating the PBMCs with lipopolysaccharides. In summary, engineered Apt-PBMCs have potential applications as live cell based biosensors and for in vitro tracing of CRP secretion sites. PMID:27708384

  7. Immune Activation in the Female Genital Tract: Expression Profiles of Soluble Proteins in Women at High Risk for HIV Infection.

    PubMed

    Francis, Suzanna C; Hou, Yanwen; Baisley, Kathy; van de Wijgert, Janneke; Watson-Jones, Deborah; Ao, Trong T; Herrera, Carolina; Maganja, Kaballa; Andreasen, Aura; Kapiga, Saidi; Coulton, Gary R; Hayes, Richard J; Shattock, Robin J

    2016-01-01

    Soluble cervicovaginal biomarkers of inflammation, immune activation and risk of HIV acquisition are needed to reliably assess the safety of new biomedical prevention strategies including vaccines and microbicides. However, a fuller understanding of expression profiles in women at high risk for HIV infection is crucial to the effective use of these potential biomarkers in Phase 3 trial settings. We have measured 45 soluble proteins and peptides in cervicovaginal lavage samples from 100 HIV negative women at high risk for HIV infection. Women were followed over one menstrual cycle to investigate modulation by hormonal contraception, menstrual cycle phase, recent sexual exposure and intravaginal practices. Women using injectable DMPA had increased concentration of several soluble proteins of the innate and adaptive immune system, including IL-1α, IL-1β, IL-2, MIP-1β, IP-10, IL-8, TGF-β, HBD4, IgA, IgG1, and IgG2. Women using combined oral contraceptives had a similar signature. There were differences in concentrations among samples from post-ovulation compared to pre-ovulation, notably increased immunoglobulins. Increased prostate-specific antigen, indicative of recent sexual exposure, was correlated with increased IL-6, MCP-1, and SLPI, and decreased GM-CSF and HBD3. The identified signature profiles may prove critical in evaluating the potential safety and impact on risk of HIV acquisition of different biomedical intervention strategies. PMID:26814891

  8. Enhanced antitumor immunity of nanoliposome-encapsulated heat shock protein 70 peptide complex derived from dendritic tumor fusion cells.

    PubMed

    Zhang, Yunfei; Luo, Wen; Wang, Yucai; Chen, Jun; Liu, Yunyan; Zhang, Yong

    2015-06-01

    Tumor-derived heat shock proteins peptide complex (HSP.PC-Tu) has been regarded as a promising antitumor agent. However, inadequate immunogenicity and low bioavailability limit the clinical uses of this agent. In a previous study, we first produced an improved HSP70.PC-based vaccine purified from dendritic cell (DC)-tumor fusion cells (HSP70.PC-Fc) which had increased immunogenicity due to enhanced antigenic tumor peptides compared to HSP70.PC-Tu. In order to increase the bioavailability of HSP70.PC-Fc, the peptide complex was encapsulated with nanoliposomes (NL-HSP70.PC-Fc) in this study. After encapsulation, the tumor immunogenicity was observed using various assays. It was demonstrated that the NL-HSP70.PC-Fc has acceptable stability. The in vivo antitumor immune response was increased with regard to T-cell activation, CTL response and tumor therapy efficiency compared to that of HSP70.PC-Fc. In addition, it was shown that DC maturation was improved by NL-HSP70.PC-Fc, which added to the antitumor immunity. The results obtained for NL-HSP70.PC-Fc, which improved immunogenicity and increases the bioavailability of HSP70.PC, may represent superior heat shock proteins (HSPs)-based tumor vaccines. Such vaccines deserve further investigation and may provide a preclinical rationale to translate findings into early phase trials for patients with breast tumors.

  9. Modulation of apoptosis and immune signaling pathways by the Hantaan virus nucleocapsid protein

    SciTech Connect

    Ontiveros, Steven J.; Li Qianjun; Jonsson, Colleen B.

    2010-06-05

    Herein, we show a direct relationship between the Hantaan virus (HTNV) nucleocapsid (N) protein and the modulation of apoptosis. We observed an increase in caspase-7 and -8, but not -9 in cells expressing HTNV N protein mutants lacking amino acids 270-330. Similar results were observed for the New World hantavirus, Andes virus. Nuclear factor kappa B (NF-kappaB) was sequestered in the cytoplasm after tumor necrosis factor receptor (TNFR) stimulation in cells expressing HTNV N protein. Further, TNFR stimulated cells expressing HTNV N protein inhibited caspase activation. In contrast, cells expressing N protein truncations lacking the region from amino acids 270-330 were unable to inhibit nuclear import of NF-kappaB and the mutants also triggered caspase activity. These results suggest that the HTNV circumvents host antiviral signaling and apoptotic response mediated by the TNFR pathway through host interactions with the N protein.

  10. Four C1q domain-containing proteins involved in the innate immune response in Hyriopsis cumingii.

    PubMed

    Zhao, Ling-Ling; Jin, Min; Li, Xin-Cang; Ren, Qian; Lan, Jiang-Feng

    2016-08-01

    C1q is a key subcomponent of the complement C1 complex. This subcomponent contains a globular C1q (gC1q) domain with remarkable ligand binding properties. C1q domain-containing (C1qDC) proteins are composed of all proteins with a gC1q domain. C1qDC proteins exist in many invertebrates and recognize non-self-ligands. In our study, four C1qDC genes, namely, HcC1qDC1-HcC1qDC4, were identified from Hyriopsis cumingii. HcC1qDC1-HcC1qDC4 encode a protein of 224, 204, 305, and 332 amino acids, respectively. All C1qDC proteins consist of a gC1q domain at the C terminal. In addition to the gC1q domain, a coiled-coil region is found in HcC1qDC4. Multiple alignments and phylogenetic tree analysis revealed that the C1qDC proteins highly differ from one another. Tissue distribution analysis demonstrated that HcC1qDC1-HcC1qDC4 are widely distributed in hemocytes, hepatopancreas, gills, mantle, and foot. These C1qDC genes are regulated by bacteria to varying degrees. These recombinant HcC1qDC proteins exhibit a binding activity against different bacterial species. Our results may suggest the roles of HcC1qDC genes in anti-bacterial immune defense. PMID:27288256

  11. Optimal dietary protein/lipid ratio for improved immune status of a newly cultivated Mediterranean fish species, the shi drum Umbrina cirrosa, L.

    PubMed

    Henry, Morgane; Fountoulaki, Eleni

    2014-04-01

    Shi drum is a new fish species recently cultivated in Greek fish farms. Its feeding requirements and health status must be determined in order to optimize its culture conditions. The present study looked at the effects of different dietary protein levels (47 or 52%) in combination with 3 lipid levels (10, 15 or 20%) on the immune system of juvenile shi drums. Most immune parameters assessed were affected by the dietary treatment. The ceruloplasmin activity, the whole blood respiratory burst activity, the serum myeloperoxidase activity, the antibacterial activity of serum lysozyme and the serum antiprotease activity all varied with the dietary protein/lipid ratio. In view of these results, the lowest dietary lipid level tested (10%) was optimal for the immune system of shi drum but a clear determination of the optimal dietary protein levels was not obtained as 47% reduced stress but also increased the ceruloplasmin response of the fish. A dietary lipid level of 10% lipids was recommended for optimal immune status of the fish. Dietary protein level of 47% seemed to give better immune responses but further study is needed to refine protein requirements.

  12. Molecular characterization of a lipid-modified virulence-associated protein of Rhodococcus equi and its potential in protective immunity.

    PubMed

    Tan, C; Prescott, J F; Patterson, M C; Nicholson, V M

    1995-01-01

    Virulent strains of Rhodococcus equi produce plasmid-mediated 15- and 17-kDa proteins, which are thermoregulated and apparently surface-expressed. We demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that R. equi produce three antigenically-related virulence-associated proteins, a diffuse 18-22-kDa, a 17.5-kDa and a 15-kDa protein. Phase partitioning of whole cells of R. equi strain 103 with Triton X-114 (TX-114) and labelling with [3H]-labelled palmitic acid showed that the two higher molecular weight proteins are hydrophobic and lipid modified. The 15-kDa protein did not partition into TX-114 and was not lipid modified. Cloning and expression of a fragment of the R. equi virulence plasmid in Escherichia coli showed that the three proteins were expressed from a single gene. Sequence analysis of this gene (designated vapA) revealed a 570-bp open reading frame encoding a polypeptide of 189 amino acids with a calculated molecular mass of 19,175 Da. The mature, nonlipid modified protein had a calculated mass of 16,246 Da. The 17.5- and 18-22-kDa forms of the protein are therefore due to lipid modification. No significant sequence homology of the vapA gene with other reported nucleotide sequences were found. Opsonization of virulent R. equi with an IgG1 mouse monoclonal antibody (MAb103) to the VapA protein significantly enhanced uptake in the murine macrophage cell line IC-21. Intraperitoneal injection of mice with Mab103 enhanced initial clearance from the liver of mice challenged intravenously with R. equi. Immunization of mice with the lipid-modified VapA purified by SDS-PAGE fractionation or with acetone precipitated VapA protein following TX-114 extraction resulted in significantly enhanced clearance from the liver and spleen following intravenous challenge. The VapA protein of R. equi appears therefore to be a protective immunogen. PMID:7704843

  13. High dietary protein exacerbates hypertension and renal damage in Dahl SS rats by increasing infiltrating immune cells in the kidney.

    PubMed

    De Miguel, Carmen; Lund, Hayley; Mattson, David L

    2011-02-01

    The present study evaluated the influence and mechanism of action of dietary protein intake in Dahl SS hypertension and renal disease. Rats were fed isocaloric diets with low (6%), normal (18%), or high (30%) amounts of protein and 0.4% NaCl from 5 to 12 weeks of age; the NaCl content of the diets was then increased to 4.0% NaCl from 12 to 15 weeks of age. Rats fed the high-protein diet developed the highest mean arterial blood pressure and urine albumin-to-creatinine ratio when fed the 4.0% NaCl diet (153 ± 7 mm Hg and 8.0 ± 2.4, respectively) compared to rats fed normal protein (132 ± 3 mm Hg, 1.2 ± 0.3) or low-protein (132 ± 6 mm Hg, 0.3 ± 0.1) diets. Significantly greater numbers of infiltrating T lymphocytes were observed in kidneys of SS rats fed the high-protein diet (18.9 ± 3 × 10⁵ cells) than in rats fed the low-protein diet (9.1 ± 3 × 10⁵ cells). Furthermore, treatment of SS rats fed the high-protein diet with the immunosuppressant agent mycophenolate mofetil (20 mg/kg per day, ip) significantly reduced the number of infiltrating T cells in the kidneys (from 18.9 ± 2.7 to 10.6 ± 2.0 × 10⁵ cells) while decreasing blood pressure (from 133 ± 3 to 113 ± 4 mm Hg) and the albumin/creatinine ratio (from 10.9 ± 2.3 to 5.4 ± 1.2). These results demonstrate that restriction of protein intake protects the Dahl SS rats from hypertension and kidney disease and indicates that infiltrating immune cells play a pathological role in Dahl SS rats fed a high-protein diet. Moreover, the results show that hypertension in Dahl SS rats is sensitive to both NaCl and protein intake.

  14. Multiple myeloma cell lines and primary tumors proteoma: protein biosynthesis and immune system as potential therapeutic targets

    PubMed Central

    Mazzotti, Diego Robles; Evangelista, Adriane Feijó; Braga, Walter Moisés Tobias; de Lourdes Chauffaille, Maria; Leme, Adriana Franco Paes; Colleoni, Gisele Wally Braga

    2015-01-01

    Despite great advance in multiple myeloma (MM) treatment since 2000s, it is still an incurable disease and novel therapies are welcome. Therefore, the purpose of this study was to explore MM plasma cells' (MM-PC) proteome, in comparison with their normal counterparts (derived from palatine tonsils of normal donors, ND-PC), in order to find potential therapeutic targets expressed on the surface of these cells. We also aimed to evaluate the proteome of MM cell lines with different genetic alterations, to confirm findings obtained with primary tumor cells. Bone marrow (BM) samples from eight new cases of MM and palatine tonsils from seven unmatched controls were submitted to PC separation and, in addition to two MM cell lines (U266, RPMI-8226), were submitted to protein extraction for mass spectrometry analyses. A total of 81 proteins were differentially expressed between MM-PC and ND-PC - 72 upregulated and nine downregulated; U266 vs. RPMI 8226 cell lines presented 61 differentially expressed proteins - 51 upregulated and 10 downregulated. On primary tumors, bioinformatics analyses highlighted upregulation of protein biosynthesis machinery, as well as downregulation of immune response components, such as MHC class I and II, and complement receptors. We also provided comprehensive information about U266 and RPMI-8226 cell lines' proteome and could confirm some patients' findings. PMID:26807199

  15. Multiple myeloma cell lines and primary tumors proteoma: protein biosynthesis and immune system as potential therapeutic targets.

    PubMed

    Fernando, Rodrigo Carlini; de Carvalho, Fabricio; Mazzotti, Diego Robles; Evangelista, Adriane Feijó; Braga, Walter Moisés Tobias; de Lourdes Chauffaille, Maria; Paes Leme, Adriana Franco; Colleoni, Gisele Wally Braga

    2015-11-01

    Despite great advance in multiple myeloma (MM) treatment since 2000s, it is still an incurable disease and novel therapies are welcome. Therefore, the purpose of this study was to explore MM plasma cells' (MM-PC) proteome, in comparison with their normal counterparts (derived from palatine tonsils of normal donors, ND-PC), in order to find potential therapeutic targets expressed on the surface of these cells. We also aimed to evaluate the proteome of MM cell lines with different genetic alterations, to confirm findings obtained with primary tumor cells. Bone marrow (BM) samples from eight new cases of MM and palatine tonsils from seven unmatched controls were submitted to PC separation and, in addition to two MM cell lines (U266, RPMI-8226), were submitted to protein extraction for mass spectrometry analyses. A total of 81 proteins were differentially expressed between MM-PC and ND-PC - 72 upregulated and nine downregulated; U266 vs. RPMI 8226 cell lines presented 61 differentially expressed proteins - 51 upregulated and 10 downregulated. On primary tumors, bioinformatics analyses highlighted upregulation of protein biosynthesis machinery, as well as downregulation of immune response components, such as MHC class I and II, and complement receptors. We also provided comprehensive information about U266 and RPMI-8226 cell lines' proteome and could confirm some patients' findings. PMID:26807199

  16. PfEMP1 - A Parasite Protein Family of Key Importance in Plasmodium falciparum Malaria Immunity and Pathogenesis.

    PubMed

    Hviid, Lars; Jensen, Anja T R

    2015-04-01

    Plasmodium falciparum causes the most severe form of malaria and is responsible for essentially all malaria-related deaths. The accumulation in various tissues of erythrocytes infected by mature P. falciparum parasites can lead to circulatory disturbances and inflammation, and is thought to be a central element in the pathogenesis of the disease. It is mediated by the interaction of parasite ligands on the erythrocyte surface and a range of host receptor molecules in many organs and tissues. Among several proteins and protein families implicated in this process, the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of high-molecular weight and highly variable antigens appears to be the most prominent. In this chapter, we aim to provide a systematic overview of the current knowledge about these proteins, their structure, their function, how they are presented on the erythrocyte surface, and how the var genes encoding them are regulated. The role of PfEMP1 in the pathogenesis of malaria, PfEMP1-specific immune responses, and the prospect of PfEMP1-specific vaccination against malaria are also covered briefly.

  17. A heat shock protein 90 β isoform involved in immune response to bacteria challenge and heat shock from Miichthys miiuy.

    PubMed

    Wei, Tao; Gao, Yunhang; Wang, Rixin; Xu, Tianjun

    2013-08-01

    Heat shock protein 90 (HSP90) is highly conserved molecular chaperone that plays a critical role in cellular stress response. In this study, we reported the identification and functional analysis of a heat shock protein 90 gene from miiuy croaker (designated Mimi-HSP90). Mimi-HSP90 contained five conserved HSP90 protein family signatures and shared 89.6%-99.5% similarity with other known HSP90 β isoform. Homology analysis and structure comparison further indicated that Mimi-HSP90 should be β isoform member of the HSP90 family. The molecular evolutionary analysis showed that HSP90 was under an overall strong purifying select pressure among fish species. Mimi-HSP90 gene was constitutively expressed in ten examined tissues, and the expression level of liver was higher than in other tissues. The expression level of Mimi-HSP90 gene under bacterial infection and heat shock were analyzed by real-time quantitative RT-PCR, resulted in significant changes in liver, spleen, and kidney tissues. The purified recombinant pET-HSP90 protein was used to produce the polyclonal antibody in mice. The specificity of the antibody was determined by Western blot analysis. All results suggested that Mimi-HSP90 was involved in thermal stress and immune response in miiuy croaker. PMID:23684810

  18. Super-resolution microscopy reveals protein spatial reorganization in early innate immune responses.

    SciTech Connect

    Carson, Bryan D.; Aaron, Jesse S.; Timlin, Jerilyn Ann

    2010-10-01

    Over the past decade optical approaches were introduced that effectively break the diffraction barrier. Of particular note were introductions of Stimulated Emission/Depletion (STED) microscopy, Photo-Activated Localization Microscopy (PALM), and the closely related Stochastic Optical Reconstruction Microscopy (STORM). STORM represents an attractive method for researchers, as it does not require highly specialized optical setups, can be implemented using commercially available dyes, and is more easily amenable to multicolor imaging. We implemented a simultaneous dual-color, direct-STORM imaging system through the use of an objective-based TIRF microscope and filter-based image splitter. This system allows for excitation and detection of two fluorophors simultaneously, via projection of each fluorophor's signal onto separate regions of a detector. We imaged the sub-resolution organization of the TLR4 receptor, a key mediator of innate immune response, after challenge with lipopolysaccharide (LPS), a bacteria-specific antigen. While distinct forms of LPS have evolved among various bacteria, only some LPS variations (such as that derived from E. coli) typically result in significant cellular immune response. Others (such as from the plague bacteria Y. pestis) do not, despite affinity to TLR4. We will show that challenge with LPS antigens produces a statistically significant increase in TLR4 receptor clusters on the cell membrane, presumably due to recruitment of receptors to lipid rafts. These changes, however, are only detectable below the diffraction limit and are not evident using conventional imaging methods. Furthermore, we will compare the spatiotemporal behavior of TLR4 receptors in response to different LPS chemotypes in order to elucidate possible routes by which pathogens such as Y. pestis are able to circumvent the innate immune system. Finally, we will exploit the dual-color STORM capabilities to simultaneously image LPS and TLR4 receptors in the cellular

  19. Immune response

    MedlinePlus

    Innate immunity; Humoral immunity; Cellular immunity; Immunity; Inflammatory response; Acquired (adaptive) immunity ... and usually does not react against them. INNATE IMMUNITY Innate, or nonspecific, immunity is the defense system ...

  20. Initiating protease with modular domains interacts with β-glucan recognition protein to trigger innate immune response in insects.

    PubMed

    Takahashi, Daisuke; Garcia, Brandon L; Kanost, Michael R

    2015-11-10

    The autoactivation of an initiating serine protease upon binding of pattern recognition proteins to pathogen surfaces is a crucial step in eliciting insect immune responses such as the activation of Toll and prophenoloxidase pathways. However, the molecular mechanisms responsible for autoactivation of the initiating protease remains poorly understood. Here, we investigated the molecular basis for the autoactivation of hemolymph protease 14 (HP14), an initiating protease in hemolymph of Manduca sexta, upon the binding of β-1,3-glucan by its recognition protein, βGRP2. Biochemical analysis using HP14 zymogen (proHP14), βGRP2, and the recombinant proteins as truncated forms showed that the amino-terminal modular low-density lipoprotein receptor class A (LA) domains within HP14 are required for proHP14 autoactivation that is stimulated by its interaction with βGRP2. Consistent with this result, recombinant LA domains inhibit the activation of proHP14 and prophenoloxidase, likely by competing with the interaction between βGRP2 and LA domains within proHP14. Using surface plasmon resonance, we demonstrated that immobilized LA domains directly interact with βGRP2 in a calcium-dependent manner and that high-affinity interaction requires the C-terminal glucanase-like domain of βGRP2. Importantly, the affinity of LA domains for βGRP2 increases nearly 100-fold in the presence of β-1,3-glucan. Taken together, these results present the first experimental evidence to our knowledge that LA domains of an insect modular protease and glucanase-like domains of a βGRP mediate their interaction, and that this binding is essential for the protease autoactivation. Thus, our study provides important insight into the molecular basis underlying the initiation of protease cascade in insect immune responses. PMID:26504233

  1. Initiating protease with modular domains interacts with β-glucan recognition protein to trigger innate immune response in insects

    PubMed Central

    Takahashi, Daisuke; Garcia, Brandon L.; Kanost, Michael R.

    2015-01-01

    The autoactivation of an initiating serine protease upon binding of pattern recognition proteins to pathogen surfaces is a crucial step in eliciting insect immune responses such as the activation of Toll and prophenoloxidase pathways. However, the molecular mechanisms responsible for autoactivation of the initiating protease remains poorly understood. Here, we investigated the molecular basis for the autoactivation of hemolymph protease 14 (HP14), an initiating protease in hemolymph of Manduca sexta, upon the binding of β-1,3-glucan by its recognition protein, βGRP2. Biochemical analysis using HP14 zymogen (proHP14), βGRP2, and the recombinant proteins as truncated forms showed that the amino-terminal modular low-density lipoprotein receptor class A (LA) domains within HP14 are required for proHP14 autoactivation that is stimulated by its interaction with βGRP2. Consistent with this result, recombinant LA domains inhibit the activation of proHP14 and prophenoloxidase, likely by competing with the interaction between βGRP2 and LA domains within proHP14. Using surface plasmon resonance, we demonstrated that immobilized LA domains directly interact with βGRP2 in a calcium-dependent manner and that high-affinity interaction requires the C-terminal glucanase-like domain of βGRP2. Importantly, the affinity of LA domains for βGRP2 increases nearly 100-fold in the presence of β-1,3-glucan. Taken together, these results present the first experimental evidence to our knowledge that LA domains of an insect modular protease and glucanase-like domains of a βGRP mediate their interaction, and that this binding is essential for the protease autoactivation. Thus, our study provides important insight into the molecular basis underlying the initiation of protease cascade in insect immune responses. PMID:26504233

  2. Partial protection against genital reinfection by immunization of guinea-pigs with isolated outer-membrane proteins of the chlamydial agent of guinea-pig inclusion conjunctivitis.

    PubMed

    Batteiger, B E; Rank, R G; Bavoil, P M; Soderberg, L S

    1993-12-01

    Because partial protection against reinfection is induced by experimental infection in the guinea-pig model of genital chlamydial infection, we sought to induce immunity by immunization. Female guinea-pigs were immunized subcutaneously with the major outer-membrane protein (MOMP) and the 61 kDa cysteine-rich outer-membrane protein (61 kDa) of the agent of guinea-pig inclusion conjunctivitis (GPIC) eluted from SDS-polyacrylamide gels (SDS-MOMP, SDS-61 kDa). Post-immunization sera and secretions contained antibodies to the SDS-purified proteins at high titre as measured by immunoblotting, whereas enzyme immunoassays (EIA) using whole elementary bodies as antigen showed significantly lower titres (P < 0.001). Likewise, blastogenic responses of peripheral mononuclear cells to GPIC elementary bodies were weak. Animals immunized with SDS-MOMP and SDS-61 kDa were fully susceptible to intravaginal challenge, as were control animals immunized with buffer without protein. Another group of animals were immunized with material prepared by extraction of chlamydial outer-membrane complexes with octyl beta-D-glucopyranoside (OGP) and dithiothreitol, which consisted largely of MOMP (OGP-MOMP). In contrast to the SDS-MOMP group, sera and secretions in the OGP-MOMP group showed high titres in EIA, and high titre antibodies to MOMP by immunoblot; however, most animals also had antibodies to 61 kDa, 72 kDa and ca. 84 kDa outer-membrane proteins. OGP-MOMP animals were partially protected against genital challenge as evidenced by low inclusion scores compared to control animals, although duration of infection measured by culture isolation was similar to controls.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Cyclic di-GMP sensing via the innate immune signaling protein STING.

    PubMed

    Yin, Qian; Tian, Yuan; Kabaleeswaran, Venkataraman; Jiang, Xiaomo; Tu, Daqi; Eck, Michael J; Chen, Zhijian J; Wu, Hao

    2012-06-29

    Detection of foreign materials is the first step of successful immune responses. Stimulator of interferon genes (STING) was shown to directly bind cyclic diguanylate monophosphate (c-di-GMP), a bacterial second messenger, and to elicit strong interferon responses. Here we elucidate the structural features of the cytosolic c-di-GMP binding domain (CBD) of STING and its complex with c-di-GMP. The CBD exhibits an α + β fold and is a dimer in the crystal and in solution. Surprisingly, one c-di-GMP molecule binds to the central crevice of a STING dimer, using a series of stacking and hydrogen bonding interactions. We show that STING is autoinhibited by an intramolecular interaction between the CBD and the C-terminal tail (CTT) and that c-di-GMP releases STING from this autoinhibition by displacing the CTT. The structures provide a remarkable example of pathogen-host interactions in which a unique microbial molecule directly engages the innate immune system.

  4. Host cell polarity proteins participate in innate immunity to Pseudomonas aeruginosa infection