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  1. 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

  2. HACE1-dependent protein degradation provides cardiac protection in response to haemodynamic stress

    NASA Astrophysics Data System (ADS)

    Zhang, Liyong; Chen, Xin; Sharma, Parveen; Moon, Mark; Sheftel, Alex D.; Dawood, Fayez; Nghiem, Mai P.; Wu, Jun; Li, Ren-Ke; Gramolini, Anthony O.; Sorensen, Poul H.; Penninger, Josef M.; Brumell, John H.; Liu, Peter P.

    2014-03-01

    The HECT E3 ubiquitin ligase HACE1 is a tumour suppressor known to regulate Rac1 activity under stress conditions. HACE1 is increased in the serum of patients with heart failure. Here we show that HACE1 protects the heart under pressure stress by controlling protein degradation. Hace1 deficiency in mice results in accelerated heart failure and increased mortality under haemodynamic stress. Hearts from Hace1-/- mice display abnormal cardiac hypertrophy, left ventricular dysfunction, accumulation of LC3, p62 and ubiquitinated proteins enriched for cytoskeletal species, indicating impaired autophagy. Our data suggest that HACE1 mediates p62-dependent selective autophagic turnover of ubiquitinated proteins by its ankyrin repeat domain through protein-protein interaction, which is independent of its E3 ligase activity. This would classify HACE1 as a dual-function E3 ligase. Our finding that HACE1 has a protective function in the heart in response to haemodynamic stress suggests that HACE1 may be a potential diagnostic and therapeutic target for heart disease.

  3. Oxidative stress induces mitochondrial dysfunction and a protective unfolded protein response in RPE cells.

    PubMed

    Cano, Marisol; Wang, Lei; Wan, Jun; Barnett, Bradley P; Ebrahimi, Katayoon; Qian, Jiang; Handa, James T

    2014-04-01

    How cells degenerate from oxidative stress in aging-related disease is incompletely understood. This study's intent was to identify key cytoprotective pathways activated by oxidative stress and determine the extent of their protection. Using an unbiased strategy with microarray analysis, we found that retinal pigmented epithelial (RPE) cells treated with cigarette smoke extract (CSE) had overrepresented genes involved in the antioxidant and unfolded protein response (UPR). Differentially expressed antioxidant genes were predominantly located in the cytoplasm, with no induction of genes that neutralize superoxide and H2O2 in the mitochondria, resulting in accumulation of superoxide and decreased ATP production. Simultaneously, CSE induced the UPR sensors IRE1α, p-PERK, and ATP6, including CHOP, which was cytoprotective because CHOP knockdown decreased cell viability. In mice given intravitreal CSE, the RPE had increased IRE1α and decreased ATP and developed epithelial-mesenchymal transition, as suggested by decreased LRAT abundance, altered ZO-1 immunolabeling, and dysmorphic cell shape. Mildly degenerated RPE from early age-related macular degeneration (AMD) samples had prominent IRE1α, but minimal mitochondrial TOM20 immunolabeling. Although oxidative stress is thought to induce an antioxidant response with cooperation between the mitochondria and the ER, herein we show that mitochondria become impaired sufficiently to induce epithelial-mesenchymal transition despite a protective UPR. With similar responses in early AMD samples, these results suggest that mitochondria are vulnerable to oxidative stress despite a protective UPR during the early phases of aging-related disease.

  4. The Mitochondrial Unfolded Protein Response Protects against Anoxia in Caenorhabditis elegans

    PubMed Central

    Peña, Salvador; Sherman, Teresa; Brookes, Paul S.; Nehrke, Keith

    2016-01-01

    The mitochondrial unfolded protein response (UPRmt) is a surveillance pathway that defends proteostasis in the “powerhouse” of the cell. Activation of the UPRmt protects against stresses imposed by reactive oxygen species, respiratory chain deficits, and pathologic bacteria. Consistent with the UPRmt’s role in adaption, we found that either its pharmacological or genetic activation by ethidium bromide (EtBr) or RNAi of the mitochondrial AAA-protease spg-7 was sufficient to reduce death in an anoxia-based Caenorhabditis elegans model of ischemia-reperfusion injury. The UPRmt-specific transcription factor atfs-1 was necessary for protection and atfs-1 gain-of-function (gf) mutants were endogenously protected from both death and dysfunction. Neurons exhibited less axonal degeneration following non-lethal anoxia-reperfusion (A-R) when the UPRmt was pre-activated, and consistent with the concept of mitochondrial stress leading to cell non-autonomous (ie. “remote”) effects, we found that restricted activation of the UPRmt in neurons decreased A-R death. However, expression of the atfs-1(gf) mutant in neurons, which resulted in a robust activation of a neuronal UPRmt, did not upregulate the UPRmt in distal tissues, nor did it protect the worms from A-R toxicity. These findings suggest that remote signaling requires additional component(s) acting downstream of de facto mitochondrial stress. PMID:27459203

  5. Mechanisms of protective immune responses induced by the Plasmodium falciparum circumsporozoite protein-based, self-assembling protein nanoparticle vaccine

    PubMed Central

    2013-01-01

    Background A lack of defined correlates of immunity for malaria, combined with the inability to induce long-lived sterile immune responses in a human host, demonstrate a need for improved understanding of potentially protective immune mechanisms for enhanced vaccine efficacy. Protective sterile immunity (>90%) against the Plasmodium falciparum circumsporozoite protein (CSP) has been achieved using a transgenically modified Plasmodium berghei sporozoite (Tg-Pb/PfCSP) and a self-assembling protein nanoparticle (SAPN) vaccine presenting CSP epitopes (PfCSP-SAPN). Here, several possible mechanisms involved in the independently protective humoral and cellular responses induced following SAPN immunization are described. Methods Inbred mice were vaccinated with PfCSP-SAPN in PBS. Serum antibodies were harvested and effects on P. falciparum sporozoites mobility and integrity were examined using phase contrast microscopy. The functionality of SAPN-induced antibodies on inhibition of sporozoite invasion and growth within primary human hepatocytes was also examined. The internal processing of SAPN by bone marrow-derived dendritic cells (BMDDC), using organelle-specific, fluorescent-tagged antibody or gold-encapsulated SAPN, was observed using confocal or electron microscopy, respectively. Results The results of this work demonstrate that PfCSP-SAPN induces epitope-specific antibody titers, predominantly of the Th2 isotype IgG1, and that serum antibodies from PfCSP-SAPN-immunized mice appear to target P. falciparum sporozoites via the classical pathway of complement. This results in sporozoite death as indicated by cessation of motility and the circumsporozoite precipitation reaction. Moreover, PfCSP-SAPN-induced antibodies are able to inhibit wild-type P. falciparum sporozoite invasion and growth within cultured primary human hepatocytes. In addition, the observation that PfCSP-SAPN are processed (and presented) to the immune system by dendritic cells in a slow and continuous

  6. Immunization with truncated envelope protein of Zika virus induces protective immune response in mice.

    PubMed

    Han, Jian-Feng; Qiu, Yang; Yu, Jiu-Yang; Wang, Hong-Jiang; Deng, Yong-Qiang; Li, Xiao-Feng; Zhao, Hui; Sun, Han-Xiao; Qin, Cheng-Feng

    2017-08-30

    The global spread of Zika virus (ZIKV) as well as its unexpected link to infant microcephaly have resulted in serious public health concerns. No antiviral drugs against ZIKV is currently available, and vaccine development is of high priority to prepare for potential ZIKV pandemic. In the present study, a truncated E protein with the N-terminal 90% region reserved (E90) from a contemporary ZIKV strain was cloned and expressed in Escherichia coli, purified by a Ni-NTA column, and characterized by Western blotting assays. Immunization with recombinant E90 induced robust ZIKV-specific humoral response in adult BALB/c mice. Passive transfer of the antisera from E90-immunized mice conferred full protection against lethal ZIKV challenge in a neonatal mice model. Our results indicate that recombinant ZIKV E90 described here represents as a promising ZIKV subunit vaccine that deserves further clinical development.

  7. The unfolded protein response has a protective role in yeast models of classic galactosemia

    PubMed Central

    De-Souza, Evandro A.; Pimentel, Felipe S. A.; Machado, Caio M.; Martins, Larissa S.; da-Silva, Wagner S.; Montero-Lomelí, Mónica; Masuda, Claudio A.

    2014-01-01

    Classic galactosemia is a human autosomal recessive disorder caused by mutations in the GALT gene (GAL7 in yeast), which encodes the enzyme galactose-1-phosphate uridyltransferase. Here we show that the unfolded protein response pathway is triggered by galactose in two yeast models of galactosemia: lithium-treated cells and the gal7Δ mutant. The synthesis of galactose-1-phosphate is essential to trigger the unfolded protein response under these conditions because the deletion of the galactokinase-encoding gene GAL1 completely abolishes unfolded protein response activation and galactose toxicity. Impairment of the unfolded protein response in both yeast models makes cells even more sensitive to galactose, unmasking its cytotoxic effect. These results indicate that endoplasmic reticulum stress is induced under galactosemic conditions and underscores the importance of the unfolded protein response pathway to cellular adaptation in these models of classic galactosemia. PMID:24077966

  8. The unfolded protein response has a protective role in yeast models of classic galactosemia.

    PubMed

    De-Souza, Evandro A; Pimentel, Felipe S A; Machado, Caio M; Martins, Larissa S; da-Silva, Wagner S; Montero-Lomelí, Mónica; Masuda, Claudio A

    2014-01-01

    Classic galactosemia is a human autosomal recessive disorder caused by mutations in the GALT gene (GAL7 in yeast), which encodes the enzyme galactose-1-phosphate uridyltransferase. Here we show that the unfolded protein response pathway is triggered by galactose in two yeast models of galactosemia: lithium-treated cells and the gal7Δ mutant. The synthesis of galactose-1-phosphate is essential to trigger the unfolded protein response under these conditions because the deletion of the galactokinase-encoding gene GAL1 completely abolishes unfolded protein response activation and galactose toxicity. Impairment of the unfolded protein response in both yeast models makes cells even more sensitive to galactose, unmasking its cytotoxic effect. These results indicate that endoplasmic reticulum stress is induced under galactosemic conditions and underscores the importance of the unfolded protein response pathway to cellular adaptation in these models of classic galactosemia.

  9. Induction of a protective response in mice by the dengue virus NS3 protein using DNA vaccines.

    PubMed

    Costa, Simone M; Yorio, Anna Paula; Gonçalves, Antônio J S; Vidale, Mariana M; Costa, Emmerson C B; Mohana-Borges, Ronaldo; Motta, Marcia A; Freire, Marcos S; Alves, Ada M B

    2011-01-01

    The dengue non-structural 3 (NS3) is a multifunctional protein, containing a serino-protease domain, located at the N-terminal portion, and helicase, NTPase and RTPase domains present in the C-terminal region. This protein is considered the main target for CD4+ and CD8+ T cell responses during dengue infection, which may be involved in protection. However, few studies have been undertaken evaluating the use of this protein as a protective antigen against dengue, as well as other flavivirus. In the present work, we investigate the protective efficacy of DNA vaccines based on the NS3 protein from DENV2. Different recombinant plasmids were constructed, encoding either the full-length NS3 protein or only its functional domains (protease and helicase), fused or not to a signal peptide (t-PA). The recombinant proteins were successfully expressed in transfected BHK-21 cells, and only plasmids encoding the t-PA signal sequence mediated protein secretion. Balb/c mice were immunized with the different DNA vaccines and challenged with a lethal dose of DENV2. Most animals immunized with plasmids encoding the full-length NS3 or the helicase domain survived challenge, regardless of the presence of the t-PA. However, some mice presented clinical signs of infection with high morbidity (hind leg paralysis and hunched posture), mainly in animal groups immunized with the DNA vaccines based on the helicase domain. On the other hand, inoculation with plasmids encoding the protease domain did not induce any protection, since mortality and morbidity rates in these mouse groups were similar to those detected in the control animals. The cellular immune response was analyzed by ELISPOT with a specific-CD8+ T cell NS3 peptide. Results revealed that the DNA vaccines based on the full-length protein induced the production of INF-γ, thus suggesting the involvement of this branch of the immune system in the protection.

  10. Immune response and protective profile elicited by a multi-epitope chimeric protein derived from Leptospira interrogans.

    PubMed

    Fernandes, Luis G V; Teixeira, Aline F; Filho, Antonio F S; Souza, Gisele O; Vasconcellos, Silvio A; Heinemann, Marcos B; Romero, Eliete C; Nascimento, Ana L T O

    2017-04-01

    Pathogenic Leptospira is the causative agent of leptospirosis, a widely disseminated disease of human and veterinary concern. The development of vaccines that elicit cross-protective immunity through multiple leptospiral serovars has long been pursued. The aim of this study was to develop a novel chimeric multi-epitope fusion antigen, containing sequences of previously studied outer membrane proteins (OMPs) of Leptospira. The chimeric protein was designed based on the amino acid sequences of the LigA, Mce, Lsa45, OmpL1, and LipL41 proteins, cloned into pAE vector, the protein expressed in Escherichia coli, and its immune response evaluated in the hamster infection model. The recombinant chimeric protein (rChi) was recognized by antibodies present in serum samples of confirmed cases of human leptospirosis and experimentally infected hamsters, demonstrating that the rChi protein participates in the immune response activation during infection. However, despite high antibody titers achieved when the rChi protein was administered with either Alhydrogel or Bordetella pertussis monophosphoryl lipid A (MPLA), only 50% of the hamsters were protected against infection. Although a complete characterization of the immune response elicited by rChi/adjuvant in hamsters is required, it is believed that the construction of chimeric genes is an important attempt towards the generation of an effective vaccine against leptospirosis. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  11. Alkaline stress response in Enterococcus faecalis: adaptation, cross-protection, and changes in protein synthesis.

    PubMed Central

    Flahaut, S; Hartke, A; Giard, J C; Auffray, Y

    1997-01-01

    The alkaline shock response in Enterococcus faecalis was studied in this work. Cells adapted to an optimum pH of 10.5 were tolerate to pH 11.9 conditions but acquired sensitivity to acid damage. An analysis of stress proteins revealed that 37 polypeptides were amplified. Two of these are DnaK and GroEL. The combined results show that bile salts and alkaline stress responses are closely related. PMID:9023964

  12. The mitochondrial unfolded protein response activator ATFS-1 protects cells from inhibition of the mevalonate pathway

    PubMed Central

    Rauthan, Manish; Ranji, Parmida; Aguilera Pradenas, Nataly; Pitot, Christophe; Pilon, Marc

    2013-01-01

    Statins are cholesterol-lowering drugs that inhibit 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, the rate-limiting enzyme in the synthesis of cholesterol via the mevalonate pathway. This pathway also produces coenzyme Q (a component of the respiratory chain), dolichols (important for protein glycosylation), and isoprenoids (lipid moieties responsible for the membrane association of small GTPases). We previously showed that the nematode Caenorhabditis elegans is useful to study the noncholesterol effects of statins because its mevalonate pathway lacks the sterol synthesis branch but retains all other branches. Here, from a screen of 150,000 mutagenized genomes, we isolated four C. elegans mutants resistant to statins by virtue of gain-of-function mutations within the first six amino acids of the protein ATFS-1, the key regulator of the mitochondrial unfolded protein response that includes activation of the chaperones HSP-6 and HSP-60. The atfs-1 gain-of-function mutants are also resistant to ibandronate, an inhibitor of an enzyme downstream of HMG-CoA reductase, and to gliotoxin, an inhibitor acting on a subbranch of the pathway important for protein prenylation, and showed improved mitochondrial function and protein prenylation in the presence of statins. Additionally, preinduction of the mitochondrial unfolded protein response in wild-type worms using ethidium bromide or paraquat triggered statin resistance, and similar observations were made in Schizosaccharomyces pombe and in a mammalian cell line. We conclude that statin resistance through maintenance of mitochondrial homeostasis is conserved across species, and that the cell-lethal effects of statins are caused primarily through impaired protein prenylation that results in mitochondria dysfunction. PMID:23530189

  13. A Computationally Designed Hemagglutinin Stem-Binding Protein Provides In Vivo Protection from Influenza Independent of a Host Immune Response

    PubMed Central

    Koday, Merika Treants; Nelson, Jorgen; Chevalier, Aaron; Koday, Michael; Kalinoski, Hannah; Stewart, Lance; Carter, Lauren; Nieusma, Travis; Lee, Peter S.; Ward, Andrew B.; Wilson, Ian A.; Dagley, Ashley; Smee, Donald F.; Baker, David; Fuller, Deborah Heydenburg

    2016-01-01

    Broadly neutralizing antibodies targeting a highly conserved region in the hemagglutinin (HA) stem protect against influenza infection. Here, we investigate the protective efficacy of a protein (HB36.6) computationally designed to bind with high affinity to the same region in the HA stem. We show that intranasal delivery of HB36.6 affords protection in mice lethally challenged with diverse strains of influenza independent of Fc-mediated effector functions or a host antiviral immune response. This designed protein prevents infection when given as a single dose of 6.0 mg/kg up to 48 hours before viral challenge and significantly reduces disease when administered as a daily therapeutic after challenge. A single dose of 10.0 mg/kg HB36.6 administered 1-day post-challenge resulted in substantially better protection than 10 doses of oseltamivir administered twice daily for 5 days. Thus, binding of HB36.6 to the influenza HA stem region alone, independent of a host response, is sufficient to reduce viral infection and replication in vivo. These studies demonstrate the potential of computationally designed binding proteins as a new class of antivirals for influenza. PMID:26845438

  14. Adjuvant-Enhanced Antibody Responses to Recombinant Proteins Correlates with Protection of Mice and Monkeys to Orthopoxvirus Challenges

    PubMed Central

    Fogg, Christiana N.; Americo, Jeffrey L.; Lustig, Shlomo; Huggins, John W.; Smith, Scott K.; Damon, Inger; Resch, Wolfgang; Earl, Patricia L.; Klinman, Dennis M.; Moss, Bernard

    2007-01-01

    Recombinant proteins are being evaluated as smallpox and monkeypox vaccines because of their perceived safety compared to live vaccinia virus. Previously, we demonstrated that three or more injections of a Ribi-type adjuvant with a combination of three proteins from the outer membranes of intracellular (L1 protein) and extracellular (A33 and B5 proteins) forms of vaccinia virus protected mice against a lethal intranasal challenge with vaccinia virus. Here, we compared several adjuvants and found that QS-21 and to a lesser extent alum plus CpG oligodeoxynucleotides accelerated and enhanced neutralizing antibody responses to a mixture of L1 and A33 proteins, provided the highest ratio of IgG2a to IgG1 isotype response, and protected mice against disease and death after only two immunizations three weeks apart. In addition, sera of monkeys immunized with recombinant vaccinia virus proteins and QS-21 neutralized monkeypox virus in vitro and reduced monkeypox virus load, skin lesions, and morbidity compared to the non-immunized group following challenge. PMID:17229505

  15. Recombinant Mycoplasma mycoides proteins elicit protective immune responses against contagious bovine pleuropneumonia.

    PubMed

    Nkando, Isabel; Perez-Casal, Jose; Mwirigi, Martin; Prysliak, Tracy; Townsend, Hugh; Berberov, Emil; Kuria, Joseph; Mugambi, John; Soi, Reuben; Liljander, Anne; Jores, Joerg; Gerdts, Volker; Potter, Andrew; Naessens, Jan; Wesonga, Hezron

    2016-03-01

    Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia (CBPP), a devastating respiratory disease mainly affecting cattle in sub-Saharan Africa. The current vaccines are based on live-attenuated Mmm strains and present problems with temperature stability, duration of immunity and adverse reactions, thus new vaccines are needed to overcome these issues. We used a reverse vaccinology approach to identify 66 Mmm potential vaccine candidates. The selection and grouping of the antigens was based on the presence of specific antibodies in sera from CBPP-positive animals. The antigens were used to immunize male Boran cattle (Bos indicus) followed by a challenge with the Mmm strain Afadé. Two of the groups immunized with five proteins each showed protection after the Mmm challenge (Groups A and C; P<0.05) and in one group (Group C) Mmm could not be cultured from lung specimens. A third group (Group N) showed a reduced number of animals with lesions and the cultures for Mmm were also negative. While immunization with some of the antigens conferred protection, others may have increased immune-related pathology. This is the first report that Mmm recombinant proteins have been successfully used to formulate a prototype vaccine and these results pave the way for the development of a novel commercial vaccine. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Outer Membrane Protein A (OmpA) of Shigella flexneri 2a, Induces Protective Immune Response in a Mouse Model

    PubMed Central

    Pore, Debasis; Mahata, Nibedita; Pal, Amit; Chakrabarti, Manoj K.

    2011-01-01

    Background In our earlier studies 34 kDa outer membrane protein (OMP) of Shigella flexneri 2a has been identified as an efficient immunostimulant. Key Results In the present study MALDI-TOF MS analysis of the purified 34 kDa OMP of Shigella flexneri 2a shows considerable sequence homology (Identity 65%) with the OmpA of S. flexneri 2a. By using the specific primers, the gene of interest has been amplified from S. flexneri 2a (N.Y-962/92) genomic DNA, cloned in pET100/D-TOPO® vector and expressed using induction with isopropyl thiogalactoside (IPTG) for the first time. Immunogenicity and protective efficacy of the recombinant OmpA has been evaluated in an intranasally immunized murine pulmonary model. The recombinant protein induces significantly enhanced protein specific IgG and IgA Abs in both mucosal and systemic compartments and IgA secreting cells in the systemic compartment (spleen). The mice immunized with OmpA have been protected completely from systemic challenge with a lethal dose of virulent S. flexneri 2a. Immunization with the protein causes mild polymorphonuclear neutrophil infiltration in the lung, without inducing the release of large amounts of proinflammatory cytokines. Conclusion These results suggest that the OmpA of S. flexneri 2a can be an efficacious mucosal immunogen inducing protective immune responses. Our findings also demonstrate that antibodies and Th1 immune response may be associated with the marked protective efficacy of immunized mice after intranasal shigellae infection. PMID:21818362

  17. The Rai (Shc C) adaptor protein regulates the neuronal stress response and protects against cerebral ischemia

    PubMed Central

    Troglio, Flavia; Echart, Cinara; Gobbi, Alberto; Pawson, Tony; Pelicci, Pier Giuseppe; De Simoni, Maria Grazia; Pelicci, Giuliana

    2004-01-01

    Rai (Shc C or N-Shc) is a neuron-specific member of the family of Shc-like adaptor proteins. Rai functions in the cytoplasmic propagation of Ret-dependent survival signals and regulates, in vivo, the number of sympathetic neurons. We report here a function of Rai, i.e., the regulation of the neuronal adaptive response to environmental stresses. We demonstrate that (i) primary cultures of cortical neurons from Rai-/- mice are more sensitive to apoptosis induced by hypoxia or oxidative stress; (ii) in Rai-/- mice, ischemia/reperfusion injury induces severe neurological deficits, increased apoptosis and size of the infarct area, and significantly higher mortality; and (iii) Rai functions as a stress-response gene that increases phosphatidylinositol 3-kinase activation and Akt phosphorylation after hypoxic or oxidation insults. These data suggest that Rai has a functional neuroprotective role in brain injury, with possible implications in the treatment of stroke. PMID:15494442

  18. Role of the Yersinia pestis Ail protein in preventing a protective polymorphonuclear leukocyte response during bubonic plague.

    PubMed

    Hinnebusch, B Joseph; Jarrett, Clayton O; Callison, Julie A; Gardner, Donald; Buchanan, Susan K; Plano, Gregory V

    2011-12-01

    The ability of Yersinia pestis to forestall the mammalian innate immune response is a fundamental aspect of plague pathogenesis. In this study, we examined the effect of Ail, a 17-kDa outer membrane protein that protects Y. pestis against complement-mediated lysis, on bubonic plague pathogenesis in mice and rats. The Y. pestis ail mutant was attenuated for virulence in both rodent models. The attenuation was greater in rats than in mice, which correlates with the ability of normal rat serum, but not mouse serum, to kill ail-negative Y. pestis in vitro. Intradermal infection with the ail mutant resulted in an atypical, subacute form of bubonic plague associated with extensive recruitment of polymorphonuclear leukocytes (PMN or neutrophils) to the site of infection in the draining lymph node and the formation of large purulent abscesses that contained the bacteria. Systemic spread and mortality were greatly attenuated, however, and a productive adaptive immune response was generated after high-dose challenge, as evidenced by high serum antibody levels against Y. pestis F1 antigen. The Y. pestis Ail protein is an important bubonic plague virulence factor that inhibits the innate immune response, in particular the recruitment of a protective PMN response to the infected lymph node.

  19. Role of the Yersinia pestis Ail Protein in Preventing a Protective Polymorphonuclear Leukocyte Response during Bubonic Plague▿

    PubMed Central

    Hinnebusch, B. Joseph; Jarrett, Clayton O.; Callison, Julie A.; Gardner, Donald; Buchanan, Susan K.; Plano, Gregory V.

    2011-01-01

    The ability of Yersinia pestis to forestall the mammalian innate immune response is a fundamental aspect of plague pathogenesis. In this study, we examined the effect of Ail, a 17-kDa outer membrane protein that protects Y. pestis against complement-mediated lysis, on bubonic plague pathogenesis in mice and rats. The Y. pestis ail mutant was attenuated for virulence in both rodent models. The attenuation was greater in rats than in mice, which correlates with the ability of normal rat serum, but not mouse serum, to kill ail-negative Y. pestis in vitro. Intradermal infection with the ail mutant resulted in an atypical, subacute form of bubonic plague associated with extensive recruitment of polymorphonuclear leukocytes (PMN or neutrophils) to the site of infection in the draining lymph node and the formation of large purulent abscesses that contained the bacteria. Systemic spread and mortality were greatly attenuated, however, and a productive adaptive immune response was generated after high-dose challenge, as evidenced by high serum antibody levels against Y. pestis F1 antigen. The Y. pestis Ail protein is an important bubonic plague virulence factor that inhibits the innate immune response, in particular the recruitment of a protective PMN response to the infected lymph node. PMID:21969002

  20. Protective Antibody and CD8+ T-Cell Responses to the Plasmodium falciparum Circumsporozoite Protein Induced by a Nanoparticle Vaccine

    PubMed Central

    Kaba, Stephen A.; McCoy, Margaret E.; Doll, Tais A. P. F.; Brando, Clara; Guo, Qin; Dasgupta, Debleena; Yang, Yongkun; Mittelholzer, Christian; Spaccapelo, Roberta; Crisanti, Andrea; Burkhard, Peter; Lanar, David E.

    2012-01-01

    Background The worldwide burden of malaria remains a major public health problem due, in part, to the lack of an effective vaccine against the Plasmodium falciparum parasite. An effective vaccine will most likely require the induction of antigen specific CD8+ and CD4+ T-cells as well as long-lasting antibody responses all working in concert to eliminate the infection. We report here the effective modification of a self-assembling protein nanoparticle (SAPN) vaccine previously proven effective in control of a P. berghei infection in a rodent model to now present B- and T-cell epitopes of the human malaria parasite P. falciparum in a platform capable of being used in human subjects. Methodology/Principal Findings To establish the basis for a SAPN-based vaccine, B- and CD8+ T-cell epitopes from the P. falciparum circumsporozoite protein (PfCSP) and the universal CD4 T-helper epitope PADRE were engineered into a versatile small protein (∼125 amino acids) that self-assembles into a spherical nanoparticle repetitively displaying the selected epitopes. P. falciparum epitope specific immune responses were evaluated in mice using a transgenic P. berghei malaria parasite of mice expressing the human malaria full-length P. falciparum circumsporozoite protein (Tg-Pb/PfCSP). We show that SAPN constructs, delivered in saline, can induce high-titer, long-lasting (1 year) protective antibody and poly-functional (IFNγ+, IL-2+) long-lived central memory CD8+ T-cells. Furthermore, we demonstrated that these Ab or CD8+ T–cells can independently provide sterile protection against a lethal challenge of the transgenic parasites. Conclusion The SAPN construct induces long-lasting antibody and cellular immune responses to epitope specific sequences of the P. falciparum circumsporozoite protein (PfCSP) and prevents infection in mice by a transgenic P. berghei parasite displaying the full length PfCSP. PMID:23144750

  1. The Respiratory Syncytial Virus G Protein Conserved Domain Induces a Persistent and Protective Antibody Response in Rodents

    PubMed Central

    Nguyen, Thien N.; Power, Ultan F.; Robert, Alain; Haeuw, Jean-François; Helffer, Katia; Perez, Amadeo; Asin, Miguel-Angel; Corvaia, Nathalie; Libon, Christine

    2012-01-01

    Respiratory syncytial virus (RSV) is an important cause of severe upper and lower respiratory disease in infants and in the elderly. There are 2 main RSV subtypes A and B. A recombinant vaccine was designed based on the central domain of the RSV-A attachment G protein which we had previously named G2Na (aa130–230). Here we evaluated immunogenicity, persistence of antibody (Ab) response and protective efficacy induced in rodents by: (i) G2Na fused to DT (Diphtheria toxin) fragments in cotton rats. DT fusion did not potentiate neutralizing Ab responses against RSV-A or cross-reactivity to RSV-B. (ii) G2Nb (aa130–230 of the RSV-B G protein) either fused to, or admixed with G2Na. G2Nb did not induce RSV-B-reactive Ab responses. (iii) G2Na at low doses. Two injections of 3 µg G2Na in Alum were sufficient to induce protective immune responses in mouse lungs, preventing RSV-A and greatly reducing RSV-B infections. In cotton rats, G2Na-induced RSV-reactive Ab and protective immunity against RSV-A challenge that persisted for at least 24 weeks. (iv) injecting RSV primed mice with a single dose of G2Na/Alum or G2Na/PLGA [poly(D,L-lactide-co-glycolide]. Despite the presence of pre-existing RSV-specific Abs, these formulations effectively boosted anti-RSV Ab titres and increased Ab titres persisted for at least 21 weeks. Affinity maturation of these Abs increased from day 28 to day 148. These data indicate that G2Na has potential as a component of an RSV vaccine formulation. PMID:22479601

  2. Immune responses and protective effect in mice vaccinated orally with surface sporozoite protein of Eimeria falciformis in ISCOMs.

    PubMed

    Kazanji, M; Laurent, F; Péry, P

    1994-07-01

    Immunostimulating complexes (ISCOMs) were built after treatment of a purified surface protein from Eimeria falciformis sporozoites with a palmitic acid derivation, leading to a high ratio (33-64%) of P27 incorporation in these cage-like structures. P27 kept its antigenicity after incorporation in ISCOMs, which induced, after iterative intubations by the oral route to groups of mice, a systemic IgG response, a local IgA response, and a local enhanced cellular response as demonstrated by lymphoproliferation of mesenteric lymph node cells upon in vitro stimulation with antigen. This immunization (120 micrograms in six oral doses at 2-day intervals) afforded mice a partial protection (60%) against a subsequent 400 oocyst challenge. The reduction in daily oocyst excretion was corroborated by significantly different weight losses between immunized and control mice on days 9 and 10 postinfection and the subsequent death of these control mice. These observations provide the first application of ISCOMs to parasitic intestinal diseases.

  3. Screening and Characterization of Drugs That Protect Corneal Endothelial Cells Against Unfolded Protein Response and Oxidative Stress

    PubMed Central

    Kim, Eun Chul; Toyono, Tetsuya; Berlinicke, Cynthia A.; Zack, Donald J.; Jurkunas, Ula; Usui, Tomohiko; Jun, Albert S.

    2017-01-01

    Purpose To screen for and characterize compounds that protect corneal endothelial cells against unfolded protein response (UPR) and oxidative stress. Methods Bovine corneal endothelial cells (BCECs) were treated for 48 hours with 640 compounds from a Food and Drug Administration (FDA)-approved drug library and then challenged with thapsigargin or H2O2 to induce UPR or oxidative stress, respectively. Cell viability was measured using the CellTiter-Glo survival assay. Selected “hits” were subjected to further dose-response testing, and their ability to modulate expression of UPR and oxidative stress markers was assessed by RT-PCR, Western blot, and measurement of protein carbonyl and 8-hydroxydeoxyguanosine (8-OHdG) adducts in immortalized human corneal endothelial cells (iHCECs). Results Forty-one drugs at 20 μM and 55 drugs at 100 μM increased survival of H2O2-challenged cells, and 8 drugs at 20 μM and 2 drugs at 100 μM increased survival of thapsigargin-challenged cells, compared with untreated control cells. Nicergoline, ergothioneine, nimesulide, oxotremorine, and mefenamic acid increased survival of both H2O2- and thapsigargin-challenged cells. Oxotremorine altered DNA damage inducible 3 (CHOP) gene expression, glucose-regulated protein 78 kDa (GRP78) and activating transcription factor 4 (ATF4) protein expression, and protein carbonyl and 8-OHdG levels. Mefenamic acid altered GRP78 protein expression and protein carbonyl and 8-OHdG levels. Conclusions Oxotremorine and mefenamic acid are potential survival factors for corneal endothelial cells under UPR and oxidative stress. The described assay can be further expanded to screen additional drugs for potential therapeutic effect in corneal endothelial diseases such as Fuchs' endothelial corneal dystrophy. PMID:28159976

  4. Connecting two-component regulatory systems by a protein that protects a response regulator from dephosphorylation by its cognate sensor

    PubMed Central

    Kato, Akinori; Groisman, Eduardo A.

    2004-01-01

    A fundamental question in signal transduction is how an organism integrates multiple signals into a cellular response. Here we report the mechanism by which the Salmonella PmrA/PmrB two-component system responds to the signal controlling the PhoP/PhoQ two-component system. We establish that the PhoP-activated PmrD protein binds to the phosphorylated form of the response regulator PmrA, preventing both its intrinsic dephosphorylation and that promoted by its cognate sensor kinase PmrB. This results in PmrA-mediated transcription because phosphorylated PmrA exhibits higher affinity for its target promoters than unphosphorylated PmrA. A PmrD-independent form of the PmrA protein was resistant to PmrB-catalyzed dephosphorylation and promoted transcription of PmrA-activated genes in the absence of inducing signals. This is the first example of a protein that enables a two-component system to respond to the signal governing a different two-component system by protecting the phosphorylated form of a response regulator. PMID:15371344

  5. DNA vaccine encoding Middle East respiratory syndrome coronavirus S1 protein induces protective immune responses in mice.

    PubMed

    Chi, Hang; Zheng, Xuexing; Wang, Xiwen; Wang, Chong; Wang, Hualei; Gai, Weiwei; Perlman, Stanley; Yang, Songtao; Zhao, Jincun; Xia, Xianzhu

    2017-04-11

    The Middle East respiratory syndrome coronavirus (MERS-CoV), is an emerging pathogen that continues to cause outbreaks in the Arabian peninsula and in travelers from this region, raising the concern that a global pandemic could occur. Here, we show that a DNA vaccine encoding the first 725 amino acids (S1) of MERS-CoV spike (S) protein induces antigen-specific humoral and cellular immune responses in mice. With three immunizations, high titers of neutralizing antibodies (up to 1: 10(4)) were generated without adjuvant. DNA vaccination with the MERS-CoV S1 gene markedly increased the frequencies of antigen-specific CD4(+) and CD8(+) T cells secreting IFN-γ and other cytokines. Both pcDNA3.1-S1 DNA vaccine immunization and passive transfer of immune serum from pcDNA3.1-S1 vaccinated mice protected Ad5-hDPP4-transduced mice from MERS-CoV challenge. These results demonstrate that a DNA vaccine encoding MERS-CoV S1 protein induces strong protective immune responses against MERS-CoV infection. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. The responsibility to protect.

    PubMed

    Deutscher, Matt

    2005-01-01

    The decision whether, if ever, to intervene in the affairs of a sovereign state with military force has become a critical issue of the post Cold War era. In 2000 the Canadian government launched the International Commission on Intervention and State Sovereignty (ICISS), which in 2001 published its findings in The Responsibility to Protect. The Commission found broad support for the notion of sovereignty not only as a right, but also a responsibility, the responsibility of a state to provide protection for its people. The primary responsibility for protecting citizens rests with states. But when states are unable or unwilling to provide this protection, or are themselves the perpetrators of atrocities, the Commission argues that the international community has a responsibility temporarily to step in, forcefully if necessary. The Commission resisted the temptation to identify human rights violations falling short of outright killing or ethnic cleansing. This eliminates the possibility of intervening on the basis of systematic oppression of human rights or intervening to remove a military dictatorship. The intention of the report was to provoke debate; to strengthen the role of the United Nations and ensure that such interventions were multilateral and meeting the wider needs of a region and not the interests of major powers. There is an ongoing need to ensure that the Security Council is effective and that resources match the political will. These debates must continue within a UN framework.

  7. Recombinant VP1 protein expressed in Pichia pastoris induces protective immune responses against EV71 in mice.

    PubMed

    Wang, Man; Jiang, Shuai; Wang, Yefu

    2013-01-04

    Human enterovirus 71 (EV71) is one of the major causative agents of hand, foot and mouth disease and is also associated with serious neurological diseases in children. Currently, there are no effective antiviral drugs or vaccines against EV71 infection. VP1, one of the major immunogenic capsid proteins of EV71, is widely considered to be the candidate antigen for an EV71 vaccine. In this study, VP1 of EV71 was expressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast Pichia pastoris, and purified by Ni-NTA affinity chromatography. Immunogenicity and vaccine efficacy of the recombinant VP1 were assessed in mouse models. The results showed that the recombinant VP1 could efficiently induce anti-VP1 antibodies in BALB/c mice, which were able to neutralize EV71 viruses in an in vitro neutralization assay. Passive protection of neonatal mice further confirmed the prophylactic efficacy of the antisera from VP1 vaccinated mice. Furthermore, VP1 vaccination induced strong lymphoproliferative and Th1 cytokine responses. Taken together, our study demonstrated that the yeast-expressed VP1 protein retained good immunogenicity and was a potent EV71 vaccine candidate.

  8. The Conserved G-Protein Coupled Receptor FSHR-1 Regulates Protective Host Responses to Infection and Oxidative Stress.

    PubMed

    Miller, Elizabeth V; Grandi, Leah N; Giannini, Jennifer A; Robinson, Joseph D; Powell, Jennifer R

    2015-01-01

    The innate immune system's ability to sense an infection is critical so that it can rapidly respond if pathogenic microorganisms threaten the host, but otherwise maintain a quiescent baseline state to avoid causing damage to the host or to commensal microorganisms. One important mechanism for discriminating between pathogenic and non-pathogenic bacteria is the recognition of cellular damage caused by a pathogen during the course of infection. In Caenorhabditis elegans, the conserved G-protein coupled receptor FSHR-1 is an important constituent of the innate immune response. FSHR-1 activates the expression of antimicrobial infection response genes in infected worms and delays accumulation of the ingested pathogen Pseudomonas aeruginosa. FSHR-1 is central not only to the worm's survival of infection by multiple pathogens, but also to the worm's survival of xenobiotic cadmium and oxidative stresses. Infected worms produce reactive oxygen species to fight off the pathogens; FSHR-1 is required at the site of infection for the expression of detoxifying genes that protect the host from collateral damage caused by this defense response. Finally, the FSHR-1 pathway is important for the ability of worms to discriminate pathogenic from benign bacteria and subsequently initiate an aversive learning program that promotes selective pathogen avoidance.

  9. Activation of unfolded protein response protects osteosarcoma cells from cisplatin-induced apoptosis through NF-κB pathway

    PubMed Central

    Yan, Mingming; Ni, Jiangdong; Song, Deye; Ding, Muliang; Huang, Jun

    2015-01-01

    The aim of this study was to uncover that unfolded protein response (UPR) contributed to the development of cisplatin resistance in osteosarcoma. MG-63 cells and SaOS-2 cells were exposed to cisplatin at presence or absence of 4-phenylbutyrayte (4-pba) and then analyzed by MTT assay and flow cytometry to determine the cell survival rates and apoptosis. Levels of glucose regulated protein 78KD (GRP78), C/EBP homologus protein (CHOP), cytoplasmic and nuclear NF-κB were detected by Western blot. Further, MG-63 cells and SaOS-2 cells were subjected to cisplatin with or without Bay 11-7082, a well-known inhibitor of NF-κB. After that, MTT assay and flow cytometry were used to determine the cell survival rates and apoptosis. Cisplatin and 4-PBA co-treatment significantly enhanced the cell apoptosis. Administration of cisplatin substantially increased the levels of GRP78 and CHOP. Moreover, mechanistic investigation uncovered that cisplatin promoted the levels of nuclear NF-κB whereas 4-PBA administration suppressed the cisplatin-induced accumulation of nuclear NF-κB level in osteosarcoma cells. Cisplatin combined with Bay 11-7082 obviously augmented MG-63 cells and SaOS-2 cells apoptosis when compared to that in osteosarcoma cells treated by cisplatin alone. Taken together, our data show that UPR protects osteosarcoma from cisplatin-mediated apoptosis through activation of NF-κB pathway. Therefore, targeting UPR may be a potential strategy to improve the osteosarcoma therapy. PMID:26617729

  10. The Drosophila telomere-capping protein Verrocchio binds single-stranded DNA and protects telomeres from DNA damage response.

    PubMed

    Cicconi, Alessandro; Micheli, Emanuela; Vernì, Fiammetta; Jackson, Alison; Gradilla, Ana Citlali; Cipressa, Francesca; Raimondo, Domenico; Bosso, Giuseppe; Wakefield, James G; Ciapponi, Laura; Cenci, Giovanni; Gatti, Maurizio; Cacchione, Stefano; Raffa, Grazia Daniela

    2017-04-07

    Drosophila telomeres are sequence-independent structures maintained by transposition to chromosome ends of three specialized retroelements rather than by telomerase activity. Fly telomeres are protected by the terminin complex that includes the HOAP, HipHop, Moi and Ver proteins. These are fast evolving, non-conserved proteins that localize and function exclusively at telomeres, protecting them from fusion events. We have previously suggested that terminin is the functional analogue of shelterin, the multi-protein complex that protects human telomeres. Here, we use electrophoretic mobility shift assay (EMSA) and atomic force microscopy (AFM) to show that Ver preferentially binds single-stranded DNA (ssDNA) with no sequence specificity. We also show that Moi and Ver form a complex in vivo. Although these two proteins are mutually dependent for their localization at telomeres, Moi neither binds ssDNA nor facilitates Ver binding to ssDNA. Consistent with these results, we found that Ver-depleted telomeres form RPA and γH2AX foci, like the human telomeres lacking the ssDNA-binding POT1 protein. Collectively, our findings suggest that Drosophila telomeres possess a ssDNA overhang like the other eukaryotes, and that the terminin complex is architecturally and functionally similar to shelterin. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Protective humoral response against pneumococcal infection in mice elicited by recombinant bacille Calmette-Guerin vaccines expressing pneumococcal surface protein A

    PubMed Central

    1994-01-01

    Pneumococcal surface protein A (PspA), a cell-surface protein present on all strains of pneumococci, has been shown to elicit protective antibody responses in mice in the absence of capsular polysaccharide. Whereas PspA is polymorphic, considerable cross-reactivity and cross- protection have been demonstrated among PspA proteins of pneumococci exhibiting different capsular and PspA serotypes. A gene segment encoding the nonrepetitive variable NH2-terminal portion of PspA has been cloned into three distinct recombinant Bacille Calmette-Guerin (rBCG) vectors, allowing for expression of PspA as a cytoplasmic or secreted protein, or a chimeric exported membrane-associated lipoprotein. All rBCG-PspA strains elicited comparable anti-PspA ELISA titers, ranging from 10(4) to 10(5) (reciprocal titers) in both BALB/c and C3H/HeJ mice. However, protective responses were observed only in animals immunized with the rBCG-PspA vaccines expressing PspA as a secreted protein or chimeric exported lipoprotein. In addition, anti- PspA immune sera elicited by the rBCG vaccines passively protected X- linked immunodeficient mice from lethal challenge with the highly virulent, encapsulated WU2 strain of Streptococcus pneumoniae and two additional virulent strains exhibiting heterologous PspA and capsular serotypes. These studies confirm previous PspA immunization studies showing cross-protection against heterologous serotypes of S. pneumoniae and demonstrate a potential for rBCG-based PspA vaccines to elicit protective humoral responses against pneumococcal disease in humans. PMID:7964500

  12. Recombinant gp90 protein expressed in Pichia pastoris induces a protective immune response against reticuloendotheliosis virus in chickens.

    PubMed

    Li, Kai; Gao, Honglei; Gao, Li; Qi, Xiaole; Gao, Yulong; Qin, Liting; Wang, Yongqiang; Wang, Xiaomei

    2012-03-16

    Reticuloendotheliosis virus (REV) causes an oncogenic, immunosuppressive and runting syndrome in multiple avian hosts worldwide. In this study, the gp90 protein of REV was secretory expressed in Pichia pastoris with high production level and good antigenicity. To fully utilize the expression potential of the P. pastoris expression system, a panel of Pichia clones carrying increasing copies of the gp90 expression cassette was created using an in vitro multimerization approach and the effects of gene dosage on gp90 expression were investigated. Results demonstrated that an increase in gp90 copy number can significantly improve the yields of gp90 protein. Following expression and scale-up, the gp90 protein production level could reach up to 400mg/L, and the protein could be detected by gp90-specific monoclonal antibody. Investigations of its vaccine efficacy demonstrated that the recombinant gp90 protein was able to induce sustained high levels of antibodies against REV as being detected by ELISA and virus neutralizing test. Furthermore, immunization of chickens with the recombinant gp90 vaccine fully protected the animals from viremia after REV infection. Overall, the yeast-expressed gp90 protein retains good immunogenicity and could be used as a potential subunit vaccine candidate for REV prevention.

  13. Evaluation of immune response to recombinant potential protective antigens of Mycoplasma hyopneumoniae delivered as cocktail DNA and/or recombinant protein vaccines in mice.

    PubMed

    Chen, Austen Y; Fry, Scott R; Daggard, Grant E; Mukkur, Trilochan K S

    2008-08-12

    Intramuscular immunization of mice with DNA cocktail vaccines, comprising potential protective antigens P36, P46, NrdF, and P97or P97R1 of Mycoplasma hyopneumoniae, induced strong Th1-polarized immune responses against each antigen, with only P46 eliciting a serum IgG response. Subcutaneous immunization with protein cocktail vaccines, surprisingly, induced both Th1-polarized immune response as well as antibody response whereas mice immunized with DNA cocktail vaccines followed by boosting with protein cocktail vaccines generated strong Th1-polarized and humoral immune responses. P97 was not recognized by serum antibodies from commercial bacterin-immunized mice indicating potential lack of expression of this important antigen in inactivated whole-cell vaccines.

  14. Depletion of the cereblon gene activates the unfolded protein response and protects cells from ER stress-induced cell death.

    PubMed

    Lee, Kwang Min; Yang, Seung-Joo; Park, Sojung; Choi, Yoo Duk; Shin, Hwa Kyoung; Pak, Jhang Ho; Park, Chul-Seung; Kim, Inki

    2015-02-27

    Previous studies showed that cereblon (CRBN) binds to various cellular target proteins, implying that CRBN regulates a wide range of cell responses. In this study, we found that deletion of the Crbn gene desensitized mouse embryonic fibroblast cells to various cell death-promoting stimuli, including endoplasmic reticulum stress inducers. Mechanistically, deletion of Crbn activates pathways involved in the unfolded protein response prior to ER stress induction. Loss of Crbn activated PKR-like ER kinase (PERK) with enhanced phosphorylation of eIF2α. Following ER stress induction, loss of Crbn delayed dephosphorylation of eIF2α, while reconstitution of Crbn reversed enhanced phosphorylation of PERK and eIF2α. Lastly, we found that activation of the PERK/eIF2α pathway following Crbn deletion is caused by activation of AMP-activated protein kinase (AMPK). We propose that CRBN plays a role in cellular stress signaling, including the unfolded protein response, by controlling the activity of AMPK.

  15. A novel recombinant BCG-expressing pro-apoptotic protein BAX enhances Th1 protective immune responses in mice.

    PubMed

    Li, Guanghua; Liu, Guoyuan; Song, Na; Kong, Cong; Huang, Qi; Su, Haibo; Bi, Aixiao; Luo, Liulin; Zhu, Lin; Xu, Ying; Wang, Honghai

    2015-08-01

    One-third of the world's population is infected with Mycobacterium tuberculosis (MTB). The protective efficacy of bacille Calmette Guérin (BCG) vaccine against tuberculosis (TB) in adults is highly controversial even though the BCG vaccine has been available for more than 90 years. Because BCG is effective against infantile tuberculosis meningitis and miliary tuberculosis in young children and provides cost-effective prevention from tuberculosis for developing countries, it would be desirable to modify the existing BCG vaccine to provide more comprehensive protection. In our study, we constructed a novel recombinant BCG strain expressing pro-apoptotic BAX (rBCG::BAX) and demonstrated that it significantly induced the apoptosis of macrophages infected with rBCG::BAX both in vitro and in vivo. In addition, it significantly enhanced Ag85B-specific IFN-γ enzyme-linked immunospot responses, IFN-γ secretion, IL-2 secretion and the ratio of Ag85B-specific IgG2b/IgG1, and it significantly decreased Ag85B-specific IL-4. Furthermore, it presumably facilitated antigen presentation by inducing a significant up-regulation in the expression of MHC-II and B7.1 (CD80) co-stimulatory molecules on macrophages. In conclusion, these results suggest that the rBCG::BAX strain elicited predominantly a Th1 protective immune responses and might be a potential tuberculosis vaccine candidate for further study.

  16. Protective immune response induced by co-immunization with the Trichinella spiralis recombinant Ts87 protein and a Ts87 DNA vaccine.

    PubMed

    Yang, Yaping; Yang, Xiaodi; Gu, Yuan; Wang, Yunyun; Zhao, Xi; Zhu, Xinping

    2013-05-20

    Ts87 is an immunodominant antigen that induces protective immunity against Trichinella spiralis larval challenge in mice. To determine if a combination of recombinant Ts87 protein and its coding DNA induces a stronger immune response in female C57BL/6 mice were immunized with 100 μg of recombinant Ts87 protein plus its coding DNA cloned in vector pVAX1, or the same amount of recombinant protein or DNA only. Mouse subclass IgG responses showed that both co-immunized and single-immunized mice produced a balanced IgG2a/IgG1 (Th1/Th2) response. T-cell proliferation in co-immunized animals was significantly higher than in single-immunized mice. Cytokine profiling in the co-immunization group showed a significant increase in the levels of IL-2, IL-4, IL-6 and IFN-γ in the splenocytes of mice upon stimulation with the recombinant Ts87 protein; however, the expression of IL-17 was down-regulated. Challenge results showed that mice immunized with the recombinant Ts87 protein and its coding DNA produced reduced the muscle larval burden to a greater extent (43.8%) than the groups immunized with only the protein (39.7%) or the DNA (9.7%). A better Th1/Th2 immune response and consequent protection induced by co-immunization with the recombinant Ts87 protein and its coding DNA may result from an adjuvant effect of DNA and a specific persistent expression of Ts87.

  17. Protective and disease-enhancing immune responses induced by recombinant modified vaccinia Ankara (MVA) expressing respiratory syncytial virus proteins.

    PubMed

    Olszewska, Wieslawa; Suezer, Yasemin; Sutter, Gerd; Openshaw, Peter J M

    2004-11-25

    Modified vaccinia Ankara (MVA) recombinants expressing single or multiple RSV surface proteins (F or G) are promising potential vaccines. We studied humoral and cellular responses induced by MVA-F and MVA-G in mice, comparing them to a formalin inactivated RSV preparation (FI-RSV) known to increase disease severity. MVA-F or MVA-G vaccination enhanced weight loss during RSV challenge, but did not show the lung eosinophilia seen after FI-RSV vaccination. FI-RSV induced a stronger total RSV IgG response than the MVA recombinants, but very little IgG2a. MVA recombinants induced cytokine responses biased towards IFNgamma and IL-12, while FI-RSV induced strong IL-4/5 responses in the lungs during RSV challenge. Thus, MVA vaccines induce a favourable immune profile in RSV disease but retain the potential to enhance disease.

  18. A novel fusion protein domain III-capsid from dengue-2, in a highly aggregated form, induces a functional immune response and protection in mice

    SciTech Connect

    Valdes, Iris; Bernardo, Lidice; Pavon, Alekis; Guzman, Maria G.

    2009-11-25

    Based on the immunogenicity of domain III from the Envelope protein of dengue virus as well as the proven protective capacity of the capsid antigen, we have designed a novel domain III-capsid chimeric protein with the goal of obtaining a molecule potentially able to induce both humoral and cell-mediated immunity (CMI). After expression of the recombinant gene in Escherichia coli, the domain III moiety retained its antigenicity as evaluated with anti-dengue sera. In order to explore alternatives for modulating the immunogenicity of the protein, it was mixed with oligodeoxynucleotides in order to obtain particulated aggregates and then immunologically evaluated in mice in comparison with non-aggregated controls. Although the humoral immune response induced by both forms of the protein was equivalent, the aggregated variant resulted in a much stronger CMI as measured by in vitro IFN-gamma secretion and protection experiments, mediated by CD4{sup +} and CD8{sup +} cells. The present work provides additional evidence in support for a crucial role of CMI in protection against dengue virus and describes a novel vaccine candidate against the disease based on a recombinant protein that can stimulate both arms of the acquired immune system.

  19. A novel fusion protein domain III-capsid from dengue-2, in a highly aggregated form, induces a functional immune response and protection in mice.

    PubMed

    Valdés, Iris; Bernardo, Lidice; Gil, Lázaro; Pavón, Alekis; Lazo, Laura; López, Carlos; Romero, Yaremis; Menendez, Ivón; Falcón, Viviana; Betancourt, Lázaro; Martín, Jorge; Chinea, Glay; Silva, Ricardo; Guzmán, María G; Guillén, Gerardo; Hermida, Lisset

    2009-11-25

    Based on the immunogenicity of domain III from the Envelope protein of dengue virus as well as the proven protective capacity of the capsid antigen, we have designed a novel domain III-capsid chimeric protein with the goal of obtaining a molecule potentially able to induce both humoral and cell-mediated immunity (CMI). After expression of the recombinant gene in Escherichia coli, the domain III moiety retained its antigenicity as evaluated with anti-dengue sera. In order to explore alternatives for modulating the immunogenicity of the protein, it was mixed with oligodeoxynucleotides in order to obtain particulated aggregates and then immunologically evaluated in mice in comparison with non-aggregated controls. Although the humoral immune response induced by both forms of the protein was equivalent, the aggregated variant resulted in a much stronger CMI as measured by in vitro IFN-gamma secretion and protection experiments, mediated by CD4(+) and CD8(+) cells. The present work provides additional evidence in support for a crucial role of CMI in protection against dengue virus and describes a novel vaccine candidate against the disease based on a recombinant protein that can stimulate both arms of the acquired immune system.

  20. Immunity to Sand Fly Salivary Protein LJM11 Modulates Host Response to Vector-Transmitted Leishmania Conferring Ulcer-Free Protection

    PubMed Central

    Gomes, Regis; Oliveira, Fabiano; Teixeira, Clarissa; Meneses, Claudio; Gilmore, Dana C; Elnaiem, Dia-Eldin; Kamhawi, Shaden; Valenzuela, Jesus G

    2012-01-01

    Leishmania vaccines that protect against needle challenge fail against the potency of a Leishmania-infected sand fly transmission. Here, we demonstrate that intradermal immunization of mice with 500 ng of the sand fly salivary recombinant protein LJM11 (rLJM11) from Lutzomyia longipalpis, in the absence of adjuvant, induces long-lasting immunity that results in ulcer-free protection against Leishmania major delivered by vector bites. This protection is antibody independent and abrogated by depletion of CD4+ T cells. Two weeks after challenge, early induction of IFN-γ specifically to rLJM11 correlates to diminished parasite replication in protected animals. At this time point, Leishmania-specific induction of IFN-γ in these mice is low in comparison with its high level in non-protected controls. We hypothesize that early control of parasites in a T-cell helper type 1 environment induced by immunity to LJM11 permits the slow development of Leishmania-specific immunity in the absence of open ulcers. Leishmania-specific immunity observed 5 weeks after infection in rLJM11-immunized mice shows a twofold increase over controls in the percentage of IFN-γ-producing CD4+ T cells. We propose LJM11 as an immunomodulator that drives an efficient and controlled protective immune response to a sand fly–transmitted Leishmania somewhat mimicking “leishmanization”-induced protective immunity but without its associated lesions. PMID:22739793

  1. Changes in the amount of lysine in protective proteins and immune cells after a systemic response to dead Escherichia coli: implications for the nutritional costs of immunity.

    PubMed

    Iseri, V J; Klasing, K C

    2014-11-01

    The nutritional demands of the immune system may result in tradeoffs with competing processes such as growth and reproduction. The magnitude of the nutritional costs of immunity is largely unknown. Thus, we examine the lysine content of the systemic components of the immune system in adult male chickens (Gallus gallus domesticus) in a healthy condition (maintenance) and following a robust Escherichia coli-specific immune response. Lysine was used as a metric, because it is found both in leukocytes and in protective proteins. The dynamics of subsets of leukocytes were monitored in primary and secondary immune tissues (thymus, bone marrow, and spleen) that would be expected to be involved in the response following iv injection of E. coli. The systemic immune system at maintenance has the same lysine content as 332 average-sized feathers, 16% of an egg, or 5.4% of a pectoralis muscle from an adult chicken. During the acute-phase response to E. coli, the additional lysine needed would equal 355 feathers, 17% of an egg, or 5.5% of a pectoralis muscle. The acute-phase proteins accounted for the greatest proportion of lysine in the immune system at maintenance and the proportion increased substantially during an acute-phase response. Hypertrophy of the liver required more lysine than all of the leukocytes and protective proteins that were produced during the acute-phase response. Size of the liver and levels of protein during the acute phase returned to normal during the time when the adaptive response began to utilize significant quantities of lysine. The catabolism would release a surfeit of lysine to provision the anabolic processes of the adaptive response, thus making proliferation of lymphocytes and production of immunoglobulins very cheap.

  2. A Combination of Recombinant Mycobacterium bovis BCG Strains Expressing Pneumococcal Proteins Induces Cellular and Humoral Immune Responses and Protects against Pneumococcal Colonization and Sepsis.

    PubMed

    Goulart, Cibelly; Rodriguez, Dunia; Kanno, Alex I; Converso, Thiago Rojas; Lu, Ying-Jie; Malley, Richard; Leite, Luciana C C

    2017-10-01

    Pneumococcal diseases remain a substantial cause of mortality in young children in developing countries. The development of potentially serotype-transcending vaccines has been extensively studied; ideally, such a vaccine should include antigens that are able to induce protection against colonization (likely mediated by interleukin-17A [IL-17A]) and invasive disease (likely mediated by antibody). The use of strong adjuvants or alternative delivery systems that are able to improve the immunological response of recombinant proteins has been proposed but poses potential safety and practical concerns in children. We have previously constructed a recombinant Mycobacterium bovis BCG strain expressing a pneumococcal surface protein A (PspA)-PdT fusion protein (rBCG PspA-PdT) that was able to induce an effective immune response and protection against sepsis in a prime-boost strategy. Here, we constructed two new rBCG strains expressing the pneumococcal proteins SP 0148 and SP 2108, which confer IL-17A-dependent protection against pneumococcal colonization in mouse models. Immunization of mice with rBCG 0148 or rBCG 2108 in a prime-boost strategy induced IL-17A and gamma interferon (IFN-γ) production. The combination of these rBCG strains with rBCG PspA-PdT (rBCG Mix), followed by a booster dose of the combined recombinant proteins (rMix) induced an IL-17A response against SP 0148 and SP 2108 and a humoral response characterized by increased levels of IgG2c against PspA and functional antibodies against pneumolysin. Furthermore, immunization with the rBCG Mix prime/rMix booster (rBCG Mix/rMix) provides protection against pneumococcal colonization and sepsis. These results suggest the use of combined rBCG strains as a potentially serotype-transcending pneumococcal vaccine in a prime-boost strategy, which could provide protection against pneumococcal colonization and sepsis. Copyright © 2017 American Society for Microbiology.

  3. Recombinant Bivalent Fusion Protein rVE Induces CD4+ and CD8+ T-Cell Mediated Memory Immune Response for Protection Against Yersinia enterocolitica Infection

    PubMed Central

    Singh, Amit K.; Kingston, Joseph J.; Gupta, Shishir K.; Batra, Harsh V.

    2015-01-01

    Studies investigating the correlates of immune protection against Yersinia infection have established that both humoral and cell mediated immune responses are required for the comprehensive protection. In our previous study, we established that the bivalent fusion protein (rVE) comprising immunologically active regions of Y. pestis LcrV (100–270 aa) and YopE (50–213 aa) proteins conferred complete passive and active protection against lethal Y. enterocolitica 8081 challenge. In the present study, cohort of BALB/c mice immunized with rVE or its component proteins rV, rE were assessed for cell mediated immune responses and memory immune protection against Y. enterocolitica 8081. rVE immunization resulted in extensive proliferation of both CD4 and CD8 T cell subsets; significantly high antibody titer with balanced IgG1: IgG2a/IgG2b isotypes (1:1 ratio) and up-regulation of both Th1 (TNF-α, IFN-γ, IL-2, and IL-12) and Th2 (IL-4) cytokines. On the other hand, rV immunization resulted in Th2 biased IgG response (11:1 ratio) and proliferation of CD4+ T-cell; rE group of mice exhibited considerably lower serum antibody titer with predominant Th1 response (1:3 ratio) and CD8+ T-cell proliferation. Comprehensive protection with superior survival (100%) was observed among rVE immunized mice when compared to the significantly lower survival rates among rE (37.5%) and rV (25%) groups when IP challenged with Y. enterocolitica 8081 after 120 days of immunization. Findings in this and our earlier studies define the bivalent fusion protein rVE as a potent candidate vaccine molecule with the capability to concurrently stimulate humoral and cell mediated immune responses and a proof of concept for developing efficient subunit vaccines against Gram negative facultative intracellular bacterial pathogens. PMID:26733956

  4. Recombinant Bivalent Fusion Protein rVE Induces CD4+ and CD8+ T-Cell Mediated Memory Immune Response for Protection Against Yersinia enterocolitica Infection.

    PubMed

    Singh, Amit K; Kingston, Joseph J; Gupta, Shishir K; Batra, Harsh V

    2015-01-01

    Studies investigating the correlates of immune protection against Yersinia infection have established that both humoral and cell mediated immune responses are required for the comprehensive protection. In our previous study, we established that the bivalent fusion protein (rVE) comprising immunologically active regions of Y. pestis LcrV (100-270 aa) and YopE (50-213 aa) proteins conferred complete passive and active protection against lethal Y. enterocolitica 8081 challenge. In the present study, cohort of BALB/c mice immunized with rVE or its component proteins rV, rE were assessed for cell mediated immune responses and memory immune protection against Y. enterocolitica 8081. rVE immunization resulted in extensive proliferation of both CD4 and CD8 T cell subsets; significantly high antibody titer with balanced IgG1: IgG2a/IgG2b isotypes (1:1 ratio) and up-regulation of both Th1 (TNF-α, IFN-γ, IL-2, and IL-12) and Th2 (IL-4) cytokines. On the other hand, rV immunization resulted in Th2 biased IgG response (11:1 ratio) and proliferation of CD4+ T-cell; rE group of mice exhibited considerably lower serum antibody titer with predominant Th1 response (1:3 ratio) and CD8+ T-cell proliferation. Comprehensive protection with superior survival (100%) was observed among rVE immunized mice when compared to the significantly lower survival rates among rE (37.5%) and rV (25%) groups when IP challenged with Y. enterocolitica 8081 after 120 days of immunization. Findings in this and our earlier studies define the bivalent fusion protein rVE as a potent candidate vaccine molecule with the capability to concurrently stimulate humoral and cell mediated immune responses and a proof of concept for developing efficient subunit vaccines against Gram negative facultative intracellular bacterial pathogens.

  5. Adsorption of recombinant poxvirus L1-protein to aluminum hydroxide/CpG vaccine adjuvants enhances immune responses and protection of mice from vaccinia virus challenge.

    PubMed

    Xiao, Yuhong; Zeng, Yuhong; Alexander, Edward; Mehta, Shyam; Joshi, Sangeeta B; Buchman, George W; Volkin, David B; Middaugh, C Russell; Isaacs, Stuart N

    2013-01-02

    The stockpiling of live vaccinia virus vaccines has enhanced biopreparedness against the intentional or accidental release of smallpox. Ongoing research on future generation smallpox vaccines is providing key insights into protective immune responses as well as important information about subunit-vaccine design strategies. For protein-based recombinant subunit vaccines, the formulation and stability of candidate antigens with different adjuvants are important factors to consider for vaccine design. In this work, a non-tagged secreted L1-protein, a target antigen on mature virus, was expressed using recombinant baculovirus technology and purified. To identify optimal formulation conditions for L1, a series of biophysical studies was performed over a range of pH and temperature conditions. The overall physical stability profile was summarized in an empirical phase diagram. Another critical question to address for development of an adjuvanted vaccine was if immunogenicity and protection could be affected by the interactions and binding of L1 to aluminum salts (Alhydrogel) with and without a second adjuvant, CpG. We thus designed a series of vaccine formulations with different binding interactions between the L1 and the two adjuvants, and then performed a series of vaccination-challenge experiments in mice including measurement of antibody responses and post-challenge weight loss and survival. We found that better humoral responses and protection were conferred with vaccine formulations when the L1-protein was adsorbed to Alhydrogel. These data demonstrate that designing vaccine formulation conditions to maximize antigen-adjuvant interactions is a key factor in smallpox subunit-vaccine immunogenicity and protection. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Adsorption of recombinant poxvirus L1-protein to aluminum hydroxide/CpG vaccine adjuvants enhances immune responses and protection of mice from vaccinia virus challenge

    PubMed Central

    Xiao, Yuhong; Zeng, Yuhong; Alexander, Edward; Mehta, Shyam; Joshi, Sangeeta B.; Buchman, George W.; Volkin, David B.; Middaugh, C. Russell; Isaacs, Stuart N.

    2012-01-01

    The stockpiling of live vaccinia virus vaccines has enhanced biopreparedness against the intentional or accidental release of smallpox. Ongoing research on future generation smallpox vaccines is providing key insights into protective immune responses as well as important information about subunit vaccine design strategies. For protein-based recombinant subunit vaccines, the formulation and stability of candidate antigens with different adjuvants are important factors to consider for vaccine design. In this work, a non-tagged secreted L1-protein, a target antigen on mature virus, was expressed using recombinant baculovirus technology and purified. To identify optimal formulation conditions for L1, a series of biophysical studies was performed over a range of pH and temperature conditions. The overall physical stability profile was summarized in an empirical phase diagram. Another critical question to address for development of an adjuvanted-vaccine was if immunogenicity and protection could be affected by the interactions and binding of L1 to aluminum salts (Alhydrogel) with and without a second adjuvant, CpG. We thus designed a series of vaccine formulations with different binding interactions between the L1 and the two adjuvants, and then performed a series of vaccination-challenge experiments in mice including measurement of antibody responses and post-challenge weight-loss and survival. We found that better humoral responses and protection were conferred with vaccine formulations when the L1-protein was adsorbed to Alhydrogel. These data demonstrate that designing vaccine formulation conditions to maximize antigen-adjuvant interactions is a key factor in smallpox subunit vaccine immunogenicity and protection. PMID:23153450

  7. Sestrin2 is induced by glucose starvation via the unfolded protein response and protects cells from non-canonical necroptotic cell death

    PubMed Central

    Ding, Boxiao; Parmigiani, Anita; Divakaruni, Ajit S.; Archer, Kellie; Murphy, Anne N.; Budanov, Andrei V.

    2016-01-01

    Sestrin2 is a member of a family of stress responsive proteins, which controls cell viability via antioxidant activity and regulation of the mammalian target of rapamycin protein kinase (mTOR). Sestrin2 is induced by different stress insults, which diminish ATP production and induce energetic stress in the cells. Glucose is a critical substrate for ATP production utilized via glycolysis and mitochondrial respiration as well as for glycosylation of newly synthesized proteins in the endoplasmic reticulum (ER) and Golgi. Thus, glucose starvation causes both energy deficiency and activation of ER stress followed by the unfolding protein response (UPR). Here, we show that UPR induces Sestrin2 via ATF4 and NRF2 transcription factors and demonstrate that Sestrin2 protects cells from glucose starvation-induced cell death. Sestrin2 inactivation sensitizes cells to necroptotic cell death that is associated with a decline in ATP levels and can be suppressed by Necrostatin 7. We propose that Sestrin2 protects cells from glucose starvation-induced cell death via regulation of mitochondrial homeostasis. PMID:26932729

  8. Alphavirus-based vaccines encoding nonstructural proteins of hepatitis C virus induce robust and protective T-cell responses.

    PubMed

    Ip, Peng Peng; Boerma, Annemarie; Regts, Joke; Meijerhof, Tjarko; Wilschut, Jan; Nijman, Hans W; Daemen, Toos

    2014-04-01

    An absolute prerequisite for a therapeutic vaccine against hepatitis C virus (HCV) infection is the potency to induce HCV-specific vigorous and broad-spectrum T-cell responses. Here, we generated three HCV vaccines based on a recombinant Semliki Forest virus (rSFV) vector expressing all- or a part of the conserved nonstructural proteins (nsPs) of HCV. We demonstrated that an rSFV vector was able to encode a transgene as large as 6.1 kb without affecting its vaccine immunogenicity. Prime-boost immunizations of mice with rSFV expressing all nsPs induced strong and long-lasting NS3-specific CD8(+) T-cell responses. The strength and functional heterogeneity of the T-cell response was similar to that induced with rSFV expressing only NS3/4A. Furthermore this leads to a significant growth delay and negative selection of HCV-expressing EL4 tumors in an in vivo mouse model. In general, as broad-spectrum T-cell responses are only seen in patients with resolved HCV infection, this rSFV-based vector, which expresses all nsPs, inducing robust T-cell activity has a potential for the treatment of HCV infections.

  9. Inter-Clade Protection Offered by Mw-Adjuvanted Recombinant HA, NP Proteins, and M2e Peptide Combination Vaccine in Mice Correlates with Cellular Immune Response

    PubMed Central

    Ingle, Nilesh B.; Virkar, Rashmi G.; Arankalle, Vidya A.

    2017-01-01

    We documented earlier that Mw (heat-killed suspension of Mycobacterium indicus pranii) adjuvant when used with conserved antigens, nucleoprotein (NP), and ectodomain of matrix (M2) protein (M2e) provided complete protection against homologous (clade 2.2) virus challenge in mice. The present study extends these observations to inter-clade challenge (clade 2.3.2.1) H5N1 virus and attempts to understand preliminary immunologic basis for the observed protection. Female BALB/c mice immunized with a single or two doses of vaccine formulations (clade 2.2 antigens) were challenged with 100LD50 homologous or heterologous (clade 2.3.2.1) virus. To understand the preliminary immunologic mechanism, we studied proportions of selected immune cell types, immune response gene expression, and Th1/Th2 cytokines induced by antigen-stimulated splenocytes from immunized mice, at different time points. Complete protection was conferred by Mw-HA, Mw-HA + NP, and Mw-HA + NP + M2e against homologous challenge. The protection correlated with IgG2a antibody titers indicating important role of Th1 response. Despite high inter-cladal antigenic differences, complete protection against the heterologous strain was achieved with Mw-HA + NP + M2e. Of note, a single dose with higher antigen concentrations (50 µg HA + 50 μg NP + 50 μg M2e) led to 80% protection against clade 2.3.2.1 strain. The protection conferred by Mw-HNM correlated with induction of IFN-γ, CD8+ T cytotoxic cells, and CD4+ T helper cells. Mw-adjuvanted HA + NP + M2e combination represents a promising vaccine candidate deserving further evaluation. PMID:28119689

  10. Protective Effects of Transforming Growth Factor β2 in Intestinal Epithelial Cells by Regulation of Proteins Associated with Stress and Endotoxin Responses

    PubMed Central

    Nguyen, Duc Ninh; Jiang, Pingping; Jacobsen, Susanne; Sangild, Per T.; Bendixen, Emøke; Chatterton, Dereck E. W.

    2015-01-01

    Transforming growth factor (TGF)-β2 is an important anti-inflammatory protein in milk and colostrum. TGF-β2 supplementation appears to reduce gut inflammatory diseases in early life, such as necrotizing enterocolitis (NEC) in young mice. However, the molecular mechanisms by which TGF-β2 protects immature intestinal epithelial cells (IECs) remain to be more clearly elucidated before interventions in infants can be considered. Porcine IECs PsIc1 were treated with TGF-β2 and/or lipopolysaccharide (LPS), and changes in the cellular proteome were subsequently analyzed using two-dimensional gel electrophoresis-MS and LC-MS-based proteomics. TGF-β2 alone induced the differential expression of 13 proteins and the majority of the identified proteins were associated with stress responses, TGF-β and Toll-like receptor 4 signaling cascades. In particular, a series of heat shock proteins had similar differential trends as previously shown in the intestine of NEC-resistant preterm pigs and young mice. Furthermore, LC-MS-based proteomics and Western blot analyses revealed 20 differentially expressed proteins following treatment with TGF-β2 in LPS-challenged IECs. Thirteen of these proteins were associated with stress response pathways, among which five proteins were altered by LPS and restored by TGF-β2, whereas six were differentially expressed only by TGF-β2 in LPS-challenged IECs. Based on previously reported biological functions, these patterns indicate the anti-stress and anti-inflammatory effects of TGF-β2 in IECs. We conclude that TGF-β2 of dietary or endogenous origin may regulate the IEC responses against LPS stimuli, thereby supporting cellular homeostasis and innate immunity in response to bacterial colonization, and the first enteral feeding in early life. PMID:25668313

  11. Effect of vaccination with carrier protein on response to meningococcal C conjugate vaccines and value of different immunoassays as predictors of protection.

    PubMed

    Burrage, Moya; Robinson, Andrew; Borrow, Ray; Andrews, Nick; Southern, Joanna; Findlow, Jamie; Martin, Sarah; Thornton, Carol; Goldblatt, David; Corbel, Michael; Sesardic, Dorothea; Cartwight, Keith; Richmond, Peter; Miller, Elizabeth

    2002-09-01

    In order to plan for the wide-scale introduction of meningococcal C conjugate (MCC) vaccine for United Kingdom children up to 18 years old, phase II trials were undertaken to investigate whether there was any interaction between MCC vaccines conjugated to tetanus toxoid (TT) or a derivative of diphtheria toxin (CRM(197)) and diphtheria-tetanus vaccines given for boosting at school entry or leaving. Children (n = 1,766) received a diphtheria-tetanus booster either 1 month before, 1 month after, or concurrently with one of three MCC vaccines conjugated to CRM(197) or TT. All of the MCC vaccines induced high antibody responses to the serogroup C polysaccharide that were indicative of protection. The immune response to the MCC-TT vaccine was reduced as a result of prior immunization with a tetanus-containing vaccine, but antibody levels were still well above the lower threshold for protection. Prior or simultaneous administration of a diphtheria-containing vaccine did not affect the response to MCC-CRM(197) vaccines. The immune responses to the carrier proteins were similar to those induced by a comparable dose of diphtheria or tetanus vaccine. The results also demonstrate that, for these conjugate vaccines in these age groups, both standard enzyme-linked immunosorbent assays and those that measure high-avidity antibodies to meningococcal C polysaccharide correlated equally well with assays that measure serum bactericidal antibodies, the established serological correlate of protection for MCC vaccines.

  12. The bovine viral diarrhea virus E2 protein formulated with a novel adjuvant induces strong, balanced immune responses and provides protection from viral challenge in cattle.

    PubMed

    Snider, Marlene; Garg, Ravendra; Brownlie, Robert; van den Hurk, Jan V; van Drunen Littel-van den Hurk, Sylvia

    2014-11-28

    Bovine viral diarrhea virus (BVDV) is still one of the most serious pathogens in cattle, meriting the development of improved vaccines. Recently, we developed a new adjuvant consisting of poly[di(sodium carboxylatoethylphenoxy)]-phosphazene (PCEP), either CpG ODN or poly(I:C), and an immune defense regulator (IDR) peptide. As this adjuvant has been shown to mediate the induction of robust, balanced immune responses, it was evaluated in an E2 subunit vaccine against BVDV in lambs and calves. The BVDV type 2 E2 protein was produced at high levels in a mammalian expression system and purified. When formulated with either CpG ODN or poly(I:C), together with IDR and PCEP, the E2 protein elicited high antibody titers and production of IFN-γ secreting cells in lambs. As the immune responses were stronger when poly(I:C) was used, the E2 protein with poly(I:C), IDR and PCEP was subsequently tested in cattle. Robust virus neutralizing antibodies as well as cell-mediated immune responses, including CD8(+) cytotoxic T cell (CTL) responses, were induced. The fact that CTL responses were demonstrated in calves vaccinated with an E2 protein subunit vaccine indicates that this adjuvant formulation promotes cross-presentation. Furthermore, upon challenge with a high dose of virulent BVDV-2, the vaccinated calves showed almost no temperature response, weight loss, leukopenia or virus replication, in contrast to the control animals, which had severe clinical disease. These data suggest that this E2 subunit formulation induces significant protection from BVDV-2 challenge, and thus is a promising BVDV vaccine candidate; in addition, the adjuvant platform has applications in bovine vaccines in general.

  13. Th1 stimulatory proteins of Leishmania donovani: comparative cellular and protective responses of rTriose phosphate isomerase, rProtein disulfide isomerase and rElongation factor-2 in combination with rHSP70 against visceral leishmaniasis.

    PubMed

    Jaiswal, Anil Kumar; Khare, Prashant; Joshi, Sumit; Kushawaha, Pramod Kumar; Sundar, Shyam; Dube, Anuradha

    2014-01-01

    In visceral leishmaniasis, the recovery from the disease is always associated with the generation of Th1-type of cellular responses. Based on this, we have previously identified several Th1-stimulatory proteins of Leishmania donovani -triose phosphate isomerase (TPI), protein disulfide isomerase (PDI) and elongation factor-2 (EL-2) etc. including heat shock protein 70 (HSP70) which induced Th1-type of cellular responses in both cured Leishmania patients/hamsters. Since, HSPs, being the logical targets for vaccines aimed at augmenting cellular immunity and can be early targets in the immune response against intracellular pathogens; they could be exploited as vaccine/adjuvant to induce long-term immunity more effectively. Therefore, in this study, we checked whether HSP70 can further enhance the immunogenicity and protective responses of the above said Th1-stimulatory proteins. Since, in most of the studies, immunogenicity of HSP70 of L. donovani was assessed in native condition, herein we generated recombinant HSP70 and tested its potential to stimulate immune responses in lymphocytes of cured Leishmania infected hamsters as well as in the peripheral blood mononuclear cells (PBMCs) of cured patients of VL either individually or in combination with above mentioned recombinant proteins. rLdHSP70 alone elicited strong cellular responses along with remarkable up-regulation of IFN-γ and IL-12 cytokines and extremely lower level of IL-4 and IL-10. Among the various combinations, rLdHSP70 + rLdPDI emerged as superior one augmenting improved cellular responses followed by rLdHSP70 + rLdEL-2. These combinations were further evaluated for its protective potential wherein rLdHSP70 + rLdPDI again conferred utmost protection (∼80%) followed by rLdHSP70 + rLdEL-2 (∼75%) and generated a strong cellular immune response with significant increase in the levels of iNOS transcript as well as IFN-γ and IL-12 cytokines which was further supported by the high level of IgG2 antibody

  14. Characterization of the antibody response elicited by immunization with pneumococcal surface protein A (PspA) as recombinant protein or DNA vaccine and analysis of protection against an intranasal lethal challenge with Streptococcus pneumoniae.

    PubMed

    Vadesilho, Cintia F M; Ferreira, Daniela M; Moreno, Adriana T; Chavez-Olortegui, Carlos; Machado de Avila, Ricardo A; Oliveira, Maria Leonor S; Ho, Paulo L; Miyaji, Eliane N

    2012-01-01

    Pneumococcal surface protein A (PspA) is an important candidate for a vaccine against pneumococcal infections. DNA vaccines expressing PspA were shown to protect mice against intraperitoneal and colonization challenge models in mice. We now show that a DNA vaccine expressing PspA from clade 4 (pSec-pspA4Pro) is also able to elicit protection against an intranasal lethal challenge model at levels similar to the recombinant protein PspA4Pro adjuvanted with alum. PspA4Pro + alum induced an IgG response characterized by a high IgG1/IgG2a ratio, leading to a lack of binding of anti-PspA IgG2a antibodies to intact pneumococci in vitro, which is in contrast to the response elicited by pSec-pspA4Pro. Epitopes recognized by the sera were mapped and antibodies induced by immunization with PspA4Pro + alum showed positive reaction with several synthetic peptides, mostly located in the first half of the protein. On the other hand, antibodies induced by the DNA vaccine showed reactivity with only two peptides. Though both strategies were protective against the intranasal lethal challenge model, the elicited humoral responses differ significantly, with the detection of important differences in the Fc (IgG1/IgG2a ratios) and Fab (recognized epitopes) regions of the induced antibodies.

  15. An in vivo protective response against toxic effects of the dermonecrotic protein from Loxosceles intermedia spider venom elicited by synthetic epitopes.

    PubMed

    Felicori, Liza; Fernandes, Paula B; Giusta, Mario S; Duarte, Clara G; Kalapothakis, Evanguedes; Nguyen, Christophe; Molina, Frank; Granier, Claude; Chávez-Olórtegui, Carlos

    2009-06-24

    Loxoscelism is a necrotic-hemolytic syndrome caused by bites of brown spiders belonging to the genus Loxosceles. Many approaches for the treatment of Loxosceles poisoning have already been proposed, among which administration of specific antivenom is thought to be the more specific. We have evaluated the use of peptides as immunogen to raise in rabbits an antibody response that could protect animals from a challenge by the Loxtox isoform LiD1, one of the main toxic component of Loxosceles intermedia venom. Six antigenic regions of LiD1 were mapped by using the SPOT method. The corresponding peptides were further chemically synthesized, mixed, and used as immunogens in rabbits. Control animal received recombinant LiD1 alone or together with peptides. We found that the rabbit antibody response to peptides was cross-reactive with LiD1, although only one peptide from the mix of six was immunogenic. The dermonecrotic, hemorrhagic and oedema forming activities induced by LiD1 in naïve rabbits were inhibited by 82%, 35% and 35% respectively, by preincubation of LiD1 with anti-peptide antibodies prepared from immunized rabbits. Animals that were immunized with peptides or LiD1r, were found to be protected from the dermonecrotic, hemorrhagic and oedema forming activities induced by a challenge with LiD1. The protection conferred by peptides was, however, lower than that provided by the peptide protein combination or by the full-length protein. These results encourage us in the utilization of synthetic peptides for therapeutic serum development or vaccination approaches.

  16. Immunological responses induced by a DNA vaccine expressing RON4 and by immunogenic recombinant protein RON4 failed to protect mice against chronic toxoplasmosis.

    PubMed

    Rashid, Imran; Hedhli, Dorsaf; Moiré, Nathalie; Pierre, Josette; Debierre-Grockiego, Françoise; Dimier-Poisson, Isabelle; Mévélec, Marie Noëlle

    2011-11-08

    The development of an effective vaccine against Toxoplasma gondii infection is an important issue due to the seriousness of the related public health problems, and the economic importance of this parasitic disease worldwide. Rhoptry neck proteins (RONs) are components of the moving junction macromolecular complex formed during invasion. The aim of this study was to evaluate the vaccine potential of RON4 using two vaccination strategies: DNA vaccination by the intramuscular route, and recombinant protein vaccination by the nasal route. We produced recombinant RON4 protein (RON4S2) using the Schneider insect cells expression system, and validated its antigenicity and immunogenicity. We also constructed optimized plasmids encoding full length RON4 (pRON4), or only the N-terminal (pNRON4), or the C-terminal part (pCRON4) of RON4. CBA/J mice immunized with pRON4, pNRON4 or pCRON4 plus a plasmid encoding the granulocyte-macrophage-colony-stimulating factor showed high IgG titers against rRON4S2. Mice immunized by the nasal route with rRON4S2 plus cholera toxin exhibited low levels of anti-RON4S2 IgG antibodies, and no intestinal IgA antibodies specific to RON4 were detected. Both DNA and protein vaccination generated a mixed Th1/Th2 response polarized towards the IgG1 antibody isotype. Both DNA and protein vaccination primed CD4+ T cells in vivo. In addition to the production of IFN-γ, and IL-2, Il-10 and IL-5 were also produced by the spleen cells of the immunized mice stimulated with RON4S2, suggesting that a mixed Th1/Th2 type immune response occurred in all the immunized groups. No cytokine was detectable in stimulated mesenteric lymph nodes from mice immunized by the nasal route. Immune responses were induced by both DNA and protein vaccination, but failed to protect the mice against a subsequent oral challenge with T. gondii cysts. In conclusion, strategies designed to enhance the immunogenicity and to redirect the cellular response towards a Th1 type response

  17. Protective immune responses against systemic candidiasis mediated by phage-displayed specific epitope of Candida albicans heat shock protein 90 in C57BL/6J mice.

    PubMed

    Wang, Guiyun; Sun, Meiyan; Fang, Jinbo; Yang, Qiong; Tong, Haibin; Wang, Li

    2006-08-28

    Specific epitope (DEPAGE) of Candida albicans heat shock protein 90 (SE-CA-HSP90) was successfully expressed on the surface of filamentous phage fd, fused to the major coat protein pVIII. Protective immune responses mediated by hybrid-phage expressing SE-CA-HSP90 in C57BL/6J mice were evaluated in this paper. The results showed that hybrid-phage particles induced the specific antibody response against SE-CA-HSP90, enhanced delayed-type hypersensitivity (DTH) response, natural killer (NK) cell activity and concanavalin A (ConA)-induced splenocyte proliferation. Furthermore, this study demonstrated that hybrid-phage-immunized mice had fewer colony forming unites (CFU) in the kidneys compared with wild-type phage-immunized mice and TE (1.0mM EDTA, 0.01M Tris-HCl, pH 8.0)-injected mice and showed statistically significant survival advantage over TE-injected group. In conclusion, the results suggest that the hybrid-phage displaying SE-CA-HSP90 may be considered as a potential candidate for producing vaccines against systemic candidiasis.

  18. The immunization-induced antibody response to the Anaplasma marginale major surface protein 2 and its association with protective immunity

    USDA-ARS?s Scientific Manuscript database

    Many vector-borne pathogens evade clearance via rapid variation in immunogenic surface expressed proteins. In the case of A. marginale, the generation of major surface protein 2 (Msp2) variants allows for immune escape and long-term pathogen persistence. In the experiments reported here, we pose t...

  19. Antibody Responses to a Novel Plasmodium falciparum Merozoite Surface Protein Vaccine Correlate with Protection against Experimental Malaria Infection in Aotus Monkeys

    PubMed Central

    Cavanagh, David R.; Kocken, Clemens H. M.; White, John H.; Cowan, Graeme J. M.; Samuel, Kay; Dubbeld, Martin A.; der Wel, Annemarie Voorberg-van; Thomas, Alan W.; McBride, Jana S.; Arnot, David E.

    2014-01-01

    The Block 2 region of the merozoite surface protein-1 (MSP-1) of Plasmodium falciparum has been identified as a target of protective immunity by a combination of seroepidemiology and parasite population genetics. Immunogenicity studies in small animals and Aotus monkeys were used to determine the efficacy of recombinant antigens derived from this region of MSP-1 as a potential vaccine antigen. Aotus lemurinus griseimembra monkeys were immunized three times with a recombinant antigen derived from the Block 2 region of MSP-1 of the monkey-adapted challenge strain, FVO of Plasmodium falciparum, using an adjuvant suitable for use in humans. Immunofluorescent antibody assays (IFA) against erythrocytes infected with P. falciparum using sera from the immunized monkeys showed that the MSP-1 Block 2 antigen induced significant antibody responses to whole malaria parasites. MSP-1 Block 2 antigen-specific enzyme-linked immunosorbent assays (ELISA) showed no significant differences in antibody titers between immunized animals. Immunized animals were challenged with the virulent P. falciparum FVO isolate and monitored for 21 days. Two out of four immunized animals were able to control their parasitaemia during the follow-up period, whereas two out of two controls developed fulminating parasitemia. Parasite-specific serum antibody titers measured by IFA were four-fold higher in protected animals than in unprotected animals. In addition, peptide-based epitope mapping of serum antibodies from immunized Aotus showed distinct differences in epitope specificities between protected and unprotected animals. PMID:24421900

  20. Evaluation of protective immune responses induced by DNA vaccines encoding Toxoplasma gondii surface antigen 1 (SAG1) and 14-3-3 protein in BALB/c mice.

    PubMed

    Meng, Min; He, Shenyi; Zhao, Guanghui; Bai, Yang; Zhou, Huaiyu; Cong, Hua; Lu, Gang; Zhao, Qunli; Zhu, Xing-Quan

    2012-11-26

    Toxoplasmosis, caused by an obligate intracellular protozoan parasite Toxoplasma gondii, has been a serious clinical and veterinary problem. Effective DNA vaccines against T. gondii can prevent and control the spread of toxoplasmosis, which is important for both human health and the farming industry. The T. gondii 14-3-3 protein has been proved to be antigenic and immunogenic and was a potential vaccine candidate against toxoplasmosis. In this study, we evaluated the immune responses induced by recombinant plasmids encoding T. gondii surface antigen 1 (SAG1) and 14-3-3 protein by immunizing BALB/c mice intramuscularly. In the present study, BALB/c mice were randomly divided into five groups, including three experimental groups (pSAG1, p14-3-3 and pSAG1/14-3-3) and two control groups (PBS and pBudCE4.1), and were immunized intramuscularly three times. The levels of IgG antibodies and cytokine production in mouse sera were determined by enzyme-linked immunosorbent assays (ELISA). Two weeks after the last immunization, all mice were challenged intraperitoneally (i.p.) with 1×10(4) tachyzoites of T. gondii and the survival time of mice was observed and recorded every day. Mice vaccinated with pSAG1, p14-3-3 or pSAG1/14-3-3 developed high levels of IgG2a and gamma interferon (IFN-γ) and low levels of interleukin-4 (IL-4) and interleukin-10 (IL-10) compared to control groups (PBS or pBudCE4.1), which suggested a modulated Th1 type immune response (P<0.05). After intraperitoneal challenge with 1×10(4) tachyzoites of T. gondii (RH strain), the survival time of mice in experimental groups was longer than control groups (P<0.05). Mouse immunized with pSAG1/14-3-3 induced a higher level of IgG antibody response and significantly prolonged the survival time when compared with pSAG1 or p14-3-3 (P<0.05). The study suggested that T. gondii 14-3-3 protein can induce effective immune responses in BALB/c mice and was a novel DNA vaccine candidate against toxoplasmosis, and the

  1. Brucella abortus Omp19 recombinant protein subcutaneously co-delivered with an antigen enhances antigen-specific T helper 1 memory responses and induces protection against parasite challenge.

    PubMed

    Coria, Lorena M; Ibañez, Andrés E; Pasquevich, Karina A; Cobiello, Paula L González; Frank, Fernanda M; Giambartolomei, Guillermo H; Cassataro, Juliana

    2016-01-20

    The discovery of effective adjuvants for many vaccines especially those with limited commercial appeal, such as vaccines to poverty-related diseases, is required. In this work, we demonstrated that subcutaneous co-administration of mice with the outer membrane protein U-Omp19 from Brucella spp. plus OVA as antigen (Ag) increases Ag-specific T cell proliferation and T helper (Th) 1 immune responses in vitro and in vivo. U-Omp19 treated dendritic cells promote IFN-γ production by specific CD4(+) T cells and increases T cell proliferation. U-Omp19 co-administration induces the production of Ag specific effector memory T cell populations (CD4(+) CD44(high) CD62L(low) T cells). Finally, subcutaneous co-administration of U-Omp19 with Trypanosoma cruzi Ags confers protection against virulent parasite challenge, reducing parasitemia and weight loss while increasing mice survival. These results indicate that the bacterial protein U-Omp19 when delivered subcutaneously could be a suitable component of vaccine formulations against infectious diseases requiring Th1 immune responses.

  2. Display of enterovirus 71 VP1 on baculovirus as a type II transmembrane protein elicits protective B and T cell responses in immunized mice.

    PubMed

    Kolpe, Annasaheb B; Kiener, Tanja K; Grotenbreg, Gijsbert M; Kwang, Jimmy

    2012-09-01

    Human enterovirus 71 (EV71) has become a major public health threat across Asia Pacific. The virus causes hand, foot, and mouth disease which can lead to neurological complications in young children. There are no specific antivirals or vaccines against EV71 infection. The major neutralizing epitope of EV71 is located in the carboxy-terminal half of the VP1 protein at amino acid positions 215-219 (Lim et al., 2012). To study the immunogenicity of VP1 we have developed a baculovirus vector which displays VP1 as a type II transmembrane protein, providing an accessible C-terminus. Immunization of mice with this recombinant baculovirus elicited neutralizing antibodies against heterologous EV71 in an in vitro microneutralization assay. Passive protection of neonatal mice confirmed the prophylactic efficacy of the antisera. Additionally, EV71 specific T cell responses were stimulated. Taken together, our results demonstrate that the display of VP1 as a type II transmembrane protein efficiently stimulated both humoral and cellular immunities.

  3. Evaluation of Protective Immune Responses Induced by Recombinant TrxLp and ENO2 Proteins against Toxoplasma gondii Infection in BALB/c Mice

    PubMed Central

    Wang, Meng; Yang, Xiao-Yu; Zhang, De-Lin

    2016-01-01

    Toxoplasma gondii is an obligate intracellular parasitic protozoan that can infect almost all species of warm-blooded animals. As any chemical-based drugs could not act against the tissue cyst stage of T. gondii, vaccination may be one of the ideal control strategies. In the present study, two new vaccine candidates, named TgENO2 and TgTrxLp, were purified from Escherichia coli with pET-30a(+) expression system and then were injected into BALB/c mice to evaluate the protective efficacy against acute and chronic toxoplasmosis. The results showed that both the recombinant proteins, either alone or in combination, could elicit strong humoral and cellular immune responses with a higher level of IgG antibodies, IFN-γ, IL-2, CD4+, and CD8+ T cells as compared to those in mice from control groups. After acute challenge with tachyzoites of the GJS strain, mice immunized with rTgTrxLp (8 ± 2.77 d), rTgENO2 (7.4 ± 1.81 d), and rTgTrxLp + rTgENO2 (8.38 ± 4.57 d) proteins showed significantly longer survival time than those that received Freund's adjuvant (6.78 ± 2.08 d) and PBS (6.38 ± 4.65 d) (χ2 = 9.687, df = 4, P = 0.046). The protective immunity of rTgTrxLp, rTgENO2, and rTgTrxLp + rTgENO2 proteins against chronic T. gondii infection showed 69.77%, 58.14%, and 20.93% brain cyst reduction as compared to mice that received PBS. The present study suggested that both TgENO2 and TgTrxLp were potential candidates for the development of multicomponent vaccines against toxoplasmosis. PMID:27803923

  4. Antibiotic resistance free plasmid DNA expressing LACK protein leads towards a protective Th1 response against Leishmania infantum infection.

    PubMed

    Ramos, I; Alonso, A; Peris, A; Marcen, J M; Abengozar, M A; Alcolea, P J; Castillo, J A; Larraga, V

    2009-11-12

    Canine visceral leishmaniasis is a serious public health concern in the Mediterranean basin since dogs are the main Leishmania infantum reservoir. However, there is not a vaccination method in veterinary use in this area, and therefore the development of a vaccine against this parasite is essential for the possible control of the disease. Previous reports have shown the efficacy of heterologous prime-boost vaccination with the pCIneo plasmid and the poxvirus VV (both Western Reserve and MVA strains) expressing L. infantum LACK antigen against canine leishmaniasis. As pCIneo-LACK plasmid contains antibiotic resistance genes, its use as a profilactic method is not recommended. Hence, the antibiotic resistance gene free pORT-LACK plasmid is a more suitable tool for its use as a vaccine. Here we report the protective and immunostimulatory effect of the prime-boost pORT-LACK/MVA-LACK vaccination tested in a canine experimental model. Vaccination induced a reduction in clinical signs and in parasite burden in the liver, an induction of the Leishmania-specific T cell activation, as well as an increase of the expression of Th1 type cytokines in PBMC and target organs.

  5. A Recombinant Trivalent Fusion Protein F1–LcrV–HSP70(II) Augments Humoral and Cellular Immune Responses and Imparts Full Protection against Yersinia pestis

    PubMed Central

    Verma, Shailendra K.; Batra, Lalit; Tuteja, Urmil

    2016-01-01

    Plague is one of the most dangerous infections in humans caused by Yersinia pestis, a Gram-negative bacterium. Despite of an overwhelming research success, no ideal vaccine against plague is available yet. It is well established that F1/LcrV based vaccine requires a strong cellular immune response for complete protection against plague. In our earlier study, we demonstrated that HSP70(II) of Mycobacterium tuberculosis modulates the humoral and cellular immunity of F1/LcrV vaccine candidates individually as well as in combinations in a mouse model. Here, we made two recombinant constructs caf1–lcrV and caf1–lcrV–hsp70(II). The caf1 and lcrV genes of Y. pestis and hsp70 domain II of M. tuberculosis were amplified by polymerase chain reaction. Both the recombinant constructs caf1–lcrV and caf1–lcrV–hsp70(II) were cloned in pET28a vector and expressed in Escherichia coli. The recombinant fusion proteins F1–LcrV and F1–LcrV–HSP70(II) were purified using Ni-NTA columns and formulated with alum to evaluate the humoral and cell mediated immune responses in mice. The protective efficacies of F1–LcrV and F1–LcrV–HSP70(II) were determined following challenge of immunized mice with 100 LD50 of Y. pestis through intraperitoneal route. Significant differences were noticed in the titers of IgG and it’s isotypes, i.e., IgG1, IgG2b, and IgG3 in anti- F1–LcrV–HSP70(II) sera in comparison to anti-F1–LcrV sera. Similarly, significant differences were also noticed in the expression levels of IL-2, IFN-γ and TNF-α in splenocytes of F1–LcrV–HSP(II) immunized mice in comparison to F1–LcrV. Both F1–LcrV and F1–LcrV–HSP70(II) provided 100% protection. Our research findings suggest that F1–LcrV fused with HSP70 domain II of M. tuberculosis significantly enhanced the humoral and cellular immune responses in mouse model. PMID:27458447

  6. A Recombinant Trivalent Fusion Protein F1-LcrV-HSP70(II) Augments Humoral and Cellular Immune Responses and Imparts Full Protection against Yersinia pestis.

    PubMed

    Verma, Shailendra K; Batra, Lalit; Tuteja, Urmil

    2016-01-01

    Plague is one of the most dangerous infections in humans caused by Yersinia pestis, a Gram-negative bacterium. Despite of an overwhelming research success, no ideal vaccine against plague is available yet. It is well established that F1/LcrV based vaccine requires a strong cellular immune response for complete protection against plague. In our earlier study, we demonstrated that HSP70(II) of Mycobacterium tuberculosis modulates the humoral and cellular immunity of F1/LcrV vaccine candidates individually as well as in combinations in a mouse model. Here, we made two recombinant constructs caf1-lcrV and caf1-lcrV-hsp70(II). The caf1 and lcrV genes of Y. pestis and hsp70 domain II of M. tuberculosis were amplified by polymerase chain reaction. Both the recombinant constructs caf1-lcrV and caf1-lcrV-hsp70(II) were cloned in pET28a vector and expressed in Escherichia coli. The recombinant fusion proteins F1-LcrV and F1-LcrV-HSP70(II) were purified using Ni-NTA columns and formulated with alum to evaluate the humoral and cell mediated immune responses in mice. The protective efficacies of F1-LcrV and F1-LcrV-HSP70(II) were determined following challenge of immunized mice with 100 LD50 of Y. pestis through intraperitoneal route. Significant differences were noticed in the titers of IgG and it's isotypes, i.e., IgG1, IgG2b, and IgG3 in anti- F1-LcrV-HSP70(II) sera in comparison to anti-F1-LcrV sera. Similarly, significant differences were also noticed in the expression levels of IL-2, IFN-γ and TNF-α in splenocytes of F1-LcrV-HSP(II) immunized mice in comparison to F1-LcrV. Both F1-LcrV and F1-LcrV-HSP70(II) provided 100% protection. Our research findings suggest that F1-LcrV fused with HSP70 domain II of M. tuberculosis significantly enhanced the humoral and cellular immune responses in mouse model.

  7. Aging promotes B-1b cell responses to native, but not protein-conjugated, pneumococcal polysaccharides: implications for vaccine protection in older adults.

    PubMed

    Haas, Karen M; Blevins, Maria W; High, Kevin P; Pang, Bing; Swords, W Edward; Yammani, Rama D

    2014-01-01

    The efficacy of different vaccines in protecting elderly individuals against Streptococcus pneumoniae infections is not clear. In the current study, aged mice (22-25 months old) exhibited significantly increased susceptibility to respiratory infection with serotype 3 S. pneumoniae relative to younger adult mice, regardless of whether mice were naive or immunized with native pneumococcal polysaccharide (PPS; Pneumovax23) or protein-PPS conjugate (Prevnar-13) vaccines. Nonetheless, Pneumovax-immunized aged mice developed limited bacteremia following respiratory challenge and exhibited significantly increased survival following systemic challenge relative to Prevnar-immune aged mice and young mice that had received either vaccine. This was explained by >10-fold increases in PPS-specific immunoglobulin G (IgG) levels in Pneumovax-immunized aged mice relative to other groups. Remarkably, PPS3-specific B-cell expansion, IgG switching, plasmablast differentiation, and spleen and bone marrow antibody-secreting cell frequencies were 10-fold higher in aged mice following Pneumovax immunization relative to young mice, due to significantly increased B-1b cell participation. In summary, this study highlights (1) the need to devise strategies to enhance respiratory immunity in aged populations, (2) the diverse responses young and aged populations generate to Pneumovax vs Prevnar vaccines, and (3) the potential value of exploiting B-1b cell responses in aged individuals for increased vaccine efficacy.

  8. Response of coccidiostat- versus vaccination-protected broilers to gelatin inclusion in high and low crude protein diets.

    PubMed

    Lehman, R; Moran, E T; Hess, J B

    2009-05-01

    Experimentation examined if Gly and Ser (Gly + Ser) and Pro from gelatin inclusion in low and high CP feeds would relieve impaired performance of broilers after vaccination for coccidiosis. Broilers in floor pens were fed 20-18-16% or 23-20-18% CP corn-soybean meal regimens from 0 to 8 wk of age such that 1 or more of the most limiting essential amino acid levels were similar across treatments and considered adequate respective of age. Gelatin was included in diets at 2%, which contributed Gly + Ser and Pro levels comparable to using 5% meat meal. Half of the chicks were spray-vaccinated with live oocysts before placement and received nonmedicated feed, whereas the other half received coccidiostat-medicated (salinomycin) feed to 6 wk of age. Vaccination adversely affected live performance in the first 3 wk, which could not be fully compensated to the level of birds receiving coccidiostat by the end of experimentation. Inclusion of gelatin mitigated vaccination response through the first 3 wk, while improving overall live performance of birds fed low CP diets. Gelatin had no effect on carcass composition, but the low CP regimen increased abdominal fat and reduced carcass and breast meat relative to high CP feeds. Vaccination also reduced overall carcass weight; however, part yields were not affected. Males were generally more sensitive to treatments than females. The conditionally essential amino acids Gly + Ser and Pro may be important factors in recovery from live anticoccidial vaccines.

  9. Transurethral instillation with fusion protein MrpH.FimH induces protective innate immune responses against uropathogenic Escherichia coli and Proteus mirabilis.

    PubMed

    Habibi, Mehri; Asadi Karam, Mohammad Reza; Bouzari, Saeid

    2016-06-01

    Urinary tract infections (UTIs) are among the most common infections in human. Innate immunity recognizes pathogen-associated molecular patterns (PAMPs) by Toll-like receptors (TLRs) to activate responses against pathogens. Recently, we demonstrated that MrpH.FimH fusion protein consisting of MrpH from Proteus mirabilis and FimH from Uropathogenic Escherichia coli (UPEC) results in the higher immunogenicity and protection, as compared with FimH and MrpH alone. In this study, we evaluated the innate immunity and adjuvant properties induced by fusion MrpH.FimH through in vitro and in vivo methods. FimH and MrpH.FimH were able to induce significantly higher IL-8 and IL-6 responses than untreated or MrpH alone in cell lines tested. The neutrophil count was significantly higher in the fusion group than other groups. After 6 h, IL-8 and IL-6 production reached a peak, with a significant decline at 24 h post-instillation in both bladder and kidney tissues. Mice instilled with the fusion and challenged with UPEC or P. mirabilis showed a significant decrease in the number of bacteria in bladder and kidney compared to control mice. The results of these studies demonstrate that the use of recombinant fusion protein encoding TLR-4 ligand represents an effective vaccination strategy that does not require the use of a commercial adjuvant. Furthermore, MrpH.FimH was presented as a promising vaccine candidate against UTIs caused by UPEC and P. mirabilis. © 2016 APMIS. Published by John Wiley & Sons Ltd.

  10. Pyridoxamine Protects Protein Backbone from Oxidative Fragmentation

    PubMed Central

    Chetyrkin, Sergei; Mathis, Missy; McDonald, W. Hayes; Shackelford, Xavier; Hudson, Billy; Voziyan, Paul

    2011-01-01

    Oxidative damage to proteins is one of the major pathogenic mechanisms in many chronic diseases. Therefore, inhibition of this oxidative damage can be an important part of therapeutic strategies. Pyridoxamine (PM), a prospective drug for treatment of diabetic nephropathy, has been previously shown to inhibit several oxidative and glycoxidative pathways, thus protecting amino acid side chains of the proteins from oxidative damage. Here, we demonstrated that PM can also protect protein backbone from fragmentation induced via different oxidative mechanisms including autoxidation of glucose. This protection was due to hydroxyl radical scavenging by PM and may contribute to PM therapeutic effects shown in clinical trials. PMID:21763683

  11. The budding yeast orthologue of Parkinson's disease-associated DJ-1 is a multi-stress response protein protecting cells against toxic glycolytic products.

    PubMed

    Natkańska, Urszula; Skoneczna, Adrianna; Sieńko, Marzena; Skoneczny, Marek

    2016-10-27

    Saccharomyces cerevisiae Hsp31p is a DJ-1/ThiJ/PfpI family protein that was previously shown to be important for survival in the stationary phase of growth and under oxidative stress. Recently, it was identified as a chaperone or as glutathione-independent glyoxalase. To elucidate the role played by this protein in budding yeast cells, we investigated its involvement in the protection against diverse environmental stresses. Our study revealed that HSP31 gene expression is controlled by multiple transcription factors, including Yap1p, Cad1p, Msn2p, Msn4p, Haa1p and Hsf1p. These transcription factors mediate the HSP31 promoter responses to oxidative, osmotic and thermal stresses, to potentially toxic products of glycolysis, such as methylglyoxal and acetic acid, and to the diauxic shift. We also demonstrated that the absence of the HSP31 gene sensitizes cells to these stressors. Overproduction of Hsp31p and its homologue Hsp32p rescued the sensitivity of glo1Δ cells to methylglyoxal. Hsp31p also reversed the increased sensitivity of the ald6Δ strain to acetic acid. Since Hsp31p glyoxalase III coexists in S. cerevisiae cells with thousand-fold more potent glyoxalase I/II system, its biological purpose requires substantiation. We postulate that S. cerevisiae Hsp31p may have broader substrate specificity than previously proposed and is able to eliminate various toxic products of glycolysis. Alternatively, Hsp31p might be effective under high concentration of exogenous methylglyoxal present in some natural environmental niches populated by budding yeast, when glyoxalase I/II system capacity is saturated.

  12. Immunization of a wild koala population with a recombinant Chlamydia pecorum Major Outer Membrane Protein (MOMP) or Polymorphic Membrane Protein (PMP) based vaccine: New insights into immune response, protection and clearance.

    PubMed

    Desclozeaux, Marion; Robbins, Amy; Jelocnik, Martina; Khan, Shahneaz Ali; Hanger, Jon; Gerdts, Volker; Potter, Andrew; Polkinghorne, Adam; Timms, Peter

    2017-01-01

    We assessed the effects of two different single-dose anti-Chlamydia pecorum (C. pecorum) vaccines (containing either Major Outer Membrane Protein (3MOMP) or Polymorphic Membrane Protein (Pmp) as antigens) on the immune response of a group of wild koalas. Both vaccines elicited a systemic humoral response as seen by the production of anti-chlamydial IgG antibodies in more than 90% of vaccinated koalas. A mucosal immune response was also observed, with an increase in Chlamydia-specific mucosal IgG and/or IgA antibodies in some koalas post-vaccination. Both vaccines elicited a cell-mediated immune response as measured by the production of the cytokines IFN-γ and IL-17 post-vaccination. To determine the level of protection provided by the vaccines under natural conditions we assessed C. pecorum infection loads and chlamydial disease status of all vaccinated koalas pre- and post-vaccination, compared to a non-vaccinated cohort from the same habitat. The MOMP vaccinated koalas that were infected on the day of vaccination showed significant clearance of their infection at 6 months post-vaccination. In contrast, the number of new infections in the PMP vaccine was similar to the control group, with some koalas progressing to disease. Genotyping of the ompA gene from the C. pecorum strains infecting the vaccinated animals, identified genetic variants of ompA-F genotype and a new genotype ompA-O. We found that those animals that were the least well protected became infected with strains of C. pecorum not covered by the vaccine. In conclusion, a single dose vaccine formulated with either recombinant PmpG or MOMP can elicit both cell-mediated and humoral (systemic and mucosal) immune responses, with the MOMP vaccine showing clearance of infection in all infected koalas. Although the capability of our vaccines to stimulate an adaptive response and be protective needs to be fully evaluated, this work illustrates the necessity to combine epitopes most relevant to a large panel of

  13. Immunization of a wild koala population with a recombinant Chlamydia pecorum Major Outer Membrane Protein (MOMP) or Polymorphic Membrane Protein (PMP) based vaccine: New insights into immune response, protection and clearance

    PubMed Central

    Robbins, Amy; Jelocnik, Martina; Khan, Shahneaz Ali; Hanger, Jon; Gerdts, Volker; Potter, Andrew; Polkinghorne, Adam; Timms, Peter

    2017-01-01

    We assessed the effects of two different single-dose anti-Chlamydia pecorum (C. pecorum) vaccines (containing either Major Outer Membrane Protein (3MOMP) or Polymorphic Membrane Protein (Pmp) as antigens) on the immune response of a group of wild koalas. Both vaccines elicited a systemic humoral response as seen by the production of anti-chlamydial IgG antibodies in more than 90% of vaccinated koalas. A mucosal immune response was also observed, with an increase in Chlamydia-specific mucosal IgG and/or IgA antibodies in some koalas post-vaccination. Both vaccines elicited a cell-mediated immune response as measured by the production of the cytokines IFN-γ and IL-17 post-vaccination. To determine the level of protection provided by the vaccines under natural conditions we assessed C. pecorum infection loads and chlamydial disease status of all vaccinated koalas pre- and post-vaccination, compared to a non-vaccinated cohort from the same habitat. The MOMP vaccinated koalas that were infected on the day of vaccination showed significant clearance of their infection at 6 months post-vaccination. In contrast, the number of new infections in the PMP vaccine was similar to the control group, with some koalas progressing to disease. Genotyping of the ompA gene from the C. pecorum strains infecting the vaccinated animals, identified genetic variants of ompA-F genotype and a new genotype ompA-O. We found that those animals that were the least well protected became infected with strains of C. pecorum not covered by the vaccine. In conclusion, a single dose vaccine formulated with either recombinant PmpG or MOMP can elicit both cell-mediated and humoral (systemic and mucosal) immune responses, with the MOMP vaccine showing clearance of infection in all infected koalas. Although the capability of our vaccines to stimulate an adaptive response and be protective needs to be fully evaluated, this work illustrates the necessity to combine epitopes most relevant to a large panel of

  14. Vaccination with heat-killed leishmania antigen or recombinant leishmanial protein and CpG oligodeoxynucleotides induces long-term memory CD4+ and CD8+ T cell responses and protection against leishmania major infection.

    PubMed

    Rhee, Elizabeth G; Mendez, Susana; Shah, Javeed A; Wu, Chang-you; Kirman, Joanna R; Turon, Tara N; Davey, Dylan F; Davis, Heather; Klinman, Dennis M; Coler, Rhea N; Sacks, David L; Seder, Robert A

    2002-06-17

    CpG oligodeoxynucleotides (ODN) have potent effects on innate and adaptive cellular immune responses. In this report, the ability of CpG ODN to confer long-term immunity and protection when used as a vaccine adjuvant with a clinical grade of leishmanial antigen, autoclaved Leishmania major (ALM), or a recombinant leishmanial protein was studied. In two different mouse models of L. major infection, vaccination with ALM plus CpG ODN was able to control infection and markedly reduce lesion development in susceptible BALB/c and resistant C57BL/6 (B6) mice, respectively, up to 12 wk after immunization. Moreover, B6 mice immunized with ALM plus CpG ODNs were still protected against infectious challenge even 6 mo after vaccination. In terms of immune correlates of protection, ALM plus CpG ODN-vaccinated mice displayed L. major-specific T helper cell 1 and CD8+ responses. In addition, complete protection was markedly abrogated in mice depleted of CD8+ T cells at the time of vaccination. Similarly, mice vaccinated with a recombinant leishmanial protein plus CpG ODN also had long-term protection that was dependent on CD8+ T cells in vivo. Together, these data demonstrate that CpG ODN, when used as a vaccine adjuvant with either a recombinant protein or heat-killed leishmanial antigen, can induce long-term protection against an intracellular infection in a CD8-dependent manner.

  15. Oral immunization with transgenic rice seeds expressing VP2 protein of infectious bursal disease virus induces protective immune responses in chickens.

    PubMed

    Wu, Jianxiang; Yu, Lian; Li, Long; Hu, Jinqiang; Zhou, Jiyong; Zhou, Xueping

    2007-09-01

    The expression of infectious bursal disease virus (IBDV) host-protective immunogen VP2 protein in rice seeds, its immunogenicity and protective capability in chickens were investigated. The VP2 cDNA of IBDV strain ZJ2000 was cloned downstream of the Gt1 promoter of the rice glutelin GluA-2 gene in the binary expression vector, pCambia1301-Gt1. Agrobacterium tumefaciens containing the recombinant vector was used to transform rice embryogenic calli, and 121 transgenic lines were obtained and grown to maturity in a greenhouse. The expression level of VP2 protein in transgenic rice seeds varied from 0.678% to 4.521% microg/mg of the total soluble seed protein. Specific pathogen-free chickens orally vaccinated with transgenic rice seeds expressing VP2 protein produced neutralizing antibodies against IBDV and were protected when challenged with a highly virulent IBDV strain, BC6/85. These results demonstrate that transgenic rice seeds expressing IBDV VP2 can be used as an effective, safe and inexpensive vaccine against IBDV.

  16. 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

  17. Urea/DTT solubilization of a recombinant Taenia ovis antigen, 45W, expressed as a GST fusion protein results in enhanced protective immune response to the 45W moiety.

    PubMed

    Rothel, J S; Wood, P R; Seow, H F; Lightowlers, M W

    1997-04-01

    Vaccination and challenge infection experiments were conducted in sheep using different forms of a recombinant protein (45W) from the cestode parasite Taenia ovis. 45W was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (45W-GST) and was produced as both soluble protein and insoluble inclusion bodies. Vaccination of animals with either the soluble or inclusion body derived protein resulted in the immune response being predominantly directed to the GST moiety of 45W-GST. Conversely, vaccination with 45W-GST which had been solubilized/treated with urea and dithiothreitol (DTT), elicited enhanced responses to the 45W moiety and significantly reduced responses to GST. Vaccination with all forms of 45W-GST protected sheep against experimental T. ovis infection. However, protection was highly correlated with anti-45W antibody levels and these were significantly higher in animals vaccinated with the urea/DTT treated form of 45W-GST. It is suggested that recombinant proteins expressed either with or without fusion partners may stimulate enhanced immune responses when incorporated in vaccine formulations in a denatured/reduced state.

  18. Foot-and-mouth disease virus-like particles produced by a SUMO fusion protein system in Escherichia coli induce potent protective immune responses in guinea pigs, swine and cattle

    PubMed Central

    2013-01-01

    Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals. The format of FMD virus-like particles (VLP) as a non-replicating particulate vaccine candidate is a promising alternative to conventional inactivated FMDV vaccines. In this study, we explored a prokaryotic system to express and assemble the FMD VLP and validated the potential of VLP as an FMDV vaccine candidate. VLP composed entirely of FMDV (Asia1/Jiangsu/China/2005) capsid proteins (VP0, VP1 and VP3) were simultaneously produced as SUMO fusion proteins by an improved SUMO fusion protein system in E. coli. Proteolytic removal of the SUMO moiety from the fusion proteins resulted in the assembly of VLP with size and shape resembling the authentic FMDV. Immunization of guinea pigs, swine and cattle with FMD VLP by intramuscular inoculation stimulated the FMDV-specific antibody response, neutralizing antibody response, T-cell proliferation response and secretion of cytokine IFN-γ. In addition, immunization with one dose of the VLP resulted in complete protection of these animals from homologous FMDV challenge. The 50% protection dose (PD50) of FMD VLP in cattle is up to 6.34. These results suggest that FMD VLP expressed in E. coli are an effective vaccine in guinea pigs, swine and cattle and support further development of these VLP as a vaccine candidate for protection against FMDV. PMID:23826638

  19. Foot-and-mouth disease virus-like particles produced by a SUMO fusion protein system in Escherichia coli induce potent protective immune responses in guinea pigs, swine and cattle.

    PubMed

    Guo, Hui-Chen; Sun, Shi-Qi; Jin, Ye; Yang, Shun-Li; Wei, Yan-Quan; Sun, De-Hui; Yin, Shuang-Hui; Ma, Jun-Wu; Liu, Zai-Xin; Guo, Jian-Hong; Luo, Jian-Xun; Yin, Hong; Liu, Xiang-Tao; Liu, Ding Xiang

    2013-07-04

    Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals. The format of FMD virus-like particles (VLP) as a non-replicating particulate vaccine candidate is a promising alternative to conventional inactivated FMDV vaccines. In this study, we explored a prokaryotic system to express and assemble the FMD VLP and validated the potential of VLP as an FMDV vaccine candidate. VLP composed entirely of FMDV (Asia1/Jiangsu/China/2005) capsid proteins (VP0, VP1 and VP3) were simultaneously produced as SUMO fusion proteins by an improved SUMO fusion protein system in E. coli. Proteolytic removal of the SUMO moiety from the fusion proteins resulted in the assembly of VLP with size and shape resembling the authentic FMDV. Immunization of guinea pigs, swine and cattle with FMD VLP by intramuscular inoculation stimulated the FMDV-specific antibody response, neutralizing antibody response, T-cell proliferation response and secretion of cytokine IFN-γ. In addition, immunization with one dose of the VLP resulted in complete protection of these animals from homologous FMDV challenge. The 50% protection dose (PD50) of FMD VLP in cattle is up to 6.34. These results suggest that FMD VLP expressed in E. coli are an effective vaccine in guinea pigs, swine and cattle and support further development of these VLP as a vaccine candidate for protection against FMDV.

  20. Vaccination with a Fusion Protein That Introduces HIV-1 Gag Antigen into a Multitrimer CD40L Construct Results in Enhanced CD8+ T Cell Responses and Protection from Viral Challenge by Vaccinia-Gag

    PubMed Central

    Gupta, Sachin; Termini, James M.; Raffa, Francesca N.; Williams, Cindi-Ann; Kornbluth, Richard S.

    2014-01-01

    CD40 ligand (CD40L, CD154) is a membrane protein that is important for the activation of dendritic cells (DCs) and DC-induced CD8+ T cell responses. To be active, CD40L must cluster CD40 receptors on responding cells. To produce a soluble form of CD40L that clusters CD40 receptors necessitates the use of a multitrimer construct. With this in mind, a tripartite fusion protein was made from surfactant protein D (SPD), HIV-1 Gag as a test antigen, and CD40L, where SPD serves as a scaffold for the multitrimer protein complex. This SPD-Gag-CD40L protein activated CD40-bearing cells and bone marrow-derived DCs in vitro. Compared to a plasmid for Gag antigen alone (pGag), DNA vaccination of mice with pSPD-Gag-CD40L induced an increased number of Gag-specific CD8+ T cells with increased avidity for major histocompatibility complex class I-restricted Gag peptide and improved vaccine-induced protection from challenge by vaccinia-Gag virus. The importance of the multitrimeric nature of the complex was shown using a plasmid lacking the N terminus of SPD that produced a single trimer fusion protein. This plasmid, pTrimer-Gag-CD40L, was only weakly active on CD40-bearing cells and did not elicit strong CD8+ T cell responses or improve protection from vaccinia-Gag challenge. An adenovirus 5 (Ad5) vaccine incorporating SPD-Gag-CD40L was much stronger than Ad5 expressing Gag alone (Ad5-Gag) and induced complete protection (i.e., sterilizing immunity) from vaccinia-Gag challenge. Overall, these results show the potential of a new vaccine design in which antigen is introduced into a construct that expresses a multitrimer soluble form of CD40L, leading to strongly protective CD8+ T cell responses. PMID:24227853

  1. Persistence of the protective immunity and kinetics of the isotype specific antibody response against the viral nucleocapsid protein after experimental Schmallenberg virus infection of sheep.

    PubMed

    Poskin, Antoine; Verite, Stephanie; Comtet, Loic; Van der Stede, Yves; Cay, Brigitte; De Regge, Nick

    2015-10-15

    Schmallenberg virus (SBV) is an Orthobunyavirus that induces abortion, stillbirths and congenital malformations in ruminants. SBV infection induces a long lasting seroconversion under natural conditions. The persistence of the protective immunity and the isotype specific antibody response upon SBV infection of sheep has however not been studied in detail. Five sheep were kept in BSL3 facilities for more than 16 months and subjected to repeated SBV infections. Blood was regularly sampled and organs were collected at euthanasia. The presence of SBV RNA in serum and organs was measured with quantitative real-time PCR. The appearance and persistence of neutralizing and SBV nucleoprotein (N) isotype specific antibodies was determined with virus neutralization tests (VNT) and ELISAs. The primo SBV infection protected ewes against clinical signs, viraemia and virus replication in organs upon challenge infections more than 15 months later. Production of neutralizing SBV specific antibodies was first detected around 6 days post primo-inoculation with VNT and correlated with the appearance of SBV-N specific IgM antibodies. These IgM antibodies remained present for 2 weeks. SBV-N specific IgG antibodies were first detected between 10 and 21 dpi and reached a plateau at 28 dpi. This plateau remained consistently high and no significant decrease in titre was found over a period of more than 1 year. Similar results were found for the neutralising antibody response. In conclusion, the SBV specific IgM response probably eliminates SBV from the blood and the protective immunity induced by SBV infection protects sheep against reinfection for at least 16 months.

  2. Induction of protective immune responses against EV71 in mice by baculovirus encoding a novel expression cassette for capsid protein VP1.

    PubMed

    Premanand, Balraj; Kiener, Tanja K; Meng, Tao; Tan, Yun Rui; Jia, Qiang; Chow, Vincent T K; Kwang, Jimmy

    2012-09-01

    EV71 is a major causative agent of hand, foot and mouth disease (HFMD) and is responsible for large outbreaks in various Asian Pacific countries. In the present study, we generated the recombinant baculovirus (Bac-VP1) encoding VP1 in a novel expression cassette. The transmembrane domain of hemagglutinin of the H3N2 influenza virus was included in the cassette as a minimal membrane anchor for VP1. The protective immunity of Bac-VP1 was investigated in a mouse model. The results showed that mice vaccinated with live Bac-VP1 had strong VP1 specific antibody responses. In an in vitro neutralization assay Bac-VP1 sera exhibited cross-neutralization against homologous and heterologous EV71 strains with a maximum titer of 1:512. Passive immunization studies confirmed that these sera were able to provide 100% protection against 5 MLD(50) of mouse adapted EV71 (B4 strain). This study revealed that baculovirus displaying VP1 with a HA transmembrane domain efficiently induced cross-neutralizing antibody responses in mice.

  3. Protein degradation and protection against misfolded or damaged proteins

    NASA Astrophysics Data System (ADS)

    Goldberg, Alfred L.

    2003-12-01

    The ultimate mechanism that cells use to ensure the quality of intracellular proteins is the selective destruction of misfolded or damaged polypeptides. In eukaryotic cells, the large ATP-dependent proteolytic machine, the 26S proteasome, prevents the accumulation of non-functional, potentially toxic proteins. This process is of particular importance in protecting cells against harsh conditions (for example, heat shock or oxidative stress) and in a variety of diseases (for example, cystic fibrosis and the major neurodegenerative diseases). A full understanding of the pathogenesis of the protein-folding diseases will require greater knowledge of how misfolded proteins are recognized and selectively degraded.

  4. Homeodomain interacting protein kinase 2 activation compromises endothelial cell response to laminar flow: protective role of p21(waf1,cip1,sdi1).

    PubMed

    Mattiussi, Stefania; Lazzari, Chiara; Truffa, Silvia; Antonini, Annalisa; Soddu, Silvia; Capogrossi, Maurizio C; Gaetano, Carlo

    2009-08-11

    In the cardiovascular system, laminar shear stress (SS) is one of the most important source of endothelial protecting signals. Physical and chemical agents, however, including ionising radiations and anticancer drugs, may injure endothelial cells determining an increase in oxidative stress and genotoxic damage. Whether the SS protective function remains intact in the presence of strong oxidants or DNA damage is currently unclear. To investigate this aspect a series of experiments were performed in which HUVEC were exposed to sub-lethal doses of the radio-mimetic compound Bleomycin (Bleo; 10 microg/ml) which generated free radicals (ROS) without significantly compromising cell survival. Remarkably, the application of a SS of 12 dyne/cm(2) did not protect endothelial cells but markedly accelerated apoptosis compared to controls kept in static culture and in the presence of Bleo. Experiments with the inducible nitric oxide synthase (iNOS) inhibitor GW274150 significantly reduced the SS-dependent apoptosis indicating that the production of NO was relevant for this effect. At molecular level, the ataxia-telangectasia-mutated (ATM) kinase, the homeodomain-interacting protein kinase-2 (HIPK2) and p53 were found activated along a pro-apoptotic signalling pathway while p21(waf1,cip1,sdi1) was prevented from its protective action. RNA interference experiments revealed that HIPK2 and p53 were both important for this process, however, only the forced expression p21(waf1,cip1,sdi1) fully restored the SS-dependent pro-survival function. This study provides the first evidence that, in the presence of genotoxic damage, laminar flow contributes to endothelial toxicity and death and identifies molecular targets potentially relevant in endothelial dysfunction and cardiovascular disease pathogenesis.

  5. Heat shock proteins and protection against ischemic injury.

    PubMed Central

    Dillmann, W H

    1999-01-01

    Heat shock proteins present a complex family of proteins exerting chaperone-like activities that are classified according to their molecular weight. We especially explored protective functions of inducible heat shock protein 70, the mitochondrial heat shock protein 60 and 10, and the small heat shock proteins HSP27 and alphaB-crystallin against ischemic, reoxygenation-mediated injury using transgenic animals and hearts under in vivo conditions and in isolated cardiac myocyte-derived cells using adenoviral vectors. We noted with great interest that differential protective effects are exerted by specific hsps. For example, alpha-B-crystallin and constitutive hsp70 markedly protect microtubular structure in cardiac myocytes from ischemia-induced injury. Inducible hsp70, hsp60 and hsp10 when coexpressed, and hsp27 and alphaB-crystallin have an overall protective effect against ischemic injury as determined by the release of enzymes like creatine kinase and LDH. We did not note inflammatory or immune responses elicited by the expression of hsps in transgenic animals and cardiac myocytes. The specific cell types in which hsps are expressed may contribute to the protective effect of hsps versus their inflammatory and immunogenic effects when expressed in other cell types. PMID:10231010

  6. The Bordetella pertussis Type III Secretion System Tip Complex Protein Bsp22 Is Not a Protective Antigen and Fails To Elicit Serum Antibody Responses during Infection of Humans and Mice

    PubMed Central

    Villarino Romero, Rodrigo; Bibova, Ilona; Cerny, Ondrej; Vecerek, Branislav; Wald, Tomas; Benada, Oldrich; Zavadilova, Jana; Sebo, Peter

    2013-01-01

    The type III secretion system (T3SS) of pathogenic bordetellae employs a self-associating tip complex protein Bsp22. This protein is immunogenic during infections by Bordetella bronchiseptica and could be used as a protective antigen to immunize mice against B. bronchiseptica challenge. Since low-passage clinical isolates of the human pathogen Bordetella pertussis produce a highly homologous Bsp22 protein (97% homology), we examined its vaccine and diagnostic potential. No Bsp22-specific antibodies were, however, detected in serum samples from 36 patients with clinically and serologically confirmed whooping cough disease (pertussis syndrome). Moreover, although the induction of Bsp22 secretion by the laboratory-adapted 18323 strain in the course of mice lung infection was observed, the B. pertussis 18323-infected mice did not mount any detectable serum antibody response against Bsp22. Furthermore, immunization with recombinant Bsp22 protein yielded induction of high Bsp22-specific serum antibody titers but did not protect mice against an intranasal challenge with B. pertussis 18323. Unlike for B. bronchiseptica, hence, the Bsp22 protein is nonimmunogenic, and/or the serum antibody response to it is suppressed, during B. pertussis infections of humans and mice. PMID:23690400

  7. A Single B-repeat of Staphylococcus epidermidis accumulation-associated protein induces protective immune responses in an experimental biomaterial-associated infection mouse model.

    PubMed

    Yan, Lin; Zhang, Lei; Ma, Hongyan; Chiu, David; Bryers, James D

    2014-09-01

    Nosocomial infections are the fourth leading cause of morbidity and mortality in the United States, resulting in 2 million infections and ∼100,000 deaths each year. More than 60% of these infections are associated with some type of biomedical device. Staphylococcus epidermidis is a commensal bacterium of the human skin and is the most common nosocomial pathogen infecting implanted medical devices, especially those in the cardiovasculature. S. epidermidis antibiotic resistance and biofilm formation on inert surfaces make these infections hard to treat. Accumulation-associated protein (Aap), a cell wall-anchored protein of S. epidermidis, is considered one of the most important proteins involved in the formation of S. epidermidis biofilm. A small recombinant protein vaccine comprising a single B-repeat domain (Brpt1.0) of S. epidermidis RP62A Aap was developed, and the vaccine's efficacy was evaluated in vitro with a biofilm inhibition assay and in vivo in a murine model of biomaterial-associated infection. A high IgG antibody response against S. epidermidis RP62A was detected in the sera of the mice after two subcutaneous immunizations with Brpt1.0 coadministered with Freund's adjuvant. Sera from Brpt1.0-immunized mice inhibited in vitro S. epidermidis RP62A biofilm formation in a dose-dependent pattern. After receiving two immunizations, each mouse was surgically implanted with a porous scaffold disk containing 5 × 10(6) CFU of S. epidermidis RP62A. Weight changes, inflammatory markers, and histological assay results after challenge with S. epidermidis indicated that the mice immunized with Brpt1.0 exhibited significantly higher resistance to S. epidermidis RP62A implant infection than the control mice. Day 8 postchallenge, there was a significantly lower number of bacteria in scaffold sections and surrounding tissues and a lower residual inflammatory response to the infected scaffold disks for the Brpt1.0-immunized mice than for of the ovalbumin (Ova

  8. HIV-1 Envelope Proteins and V1/V2 Domain Scaffolds with Mannose-5 to Improve the Magnitude and Quality of Protective Antibody Responses to HIV-1*

    PubMed Central

    Morales, Javier F.; Morin, Trevor J.; Yu, Bin; Tatsuno, Gwen P.; O'Rourke, Sara M.; Theolis, Richard; Mesa, Kathryn A.; Berman, Phillip W.

    2014-01-01

    Two lines of investigation have highlighted the importance of antibodies to the V1/V2 domain of gp120 in providing protection from HIV-1 infection. First, the recent RV144 HIV-1 vaccine trial documented a correlation between non-neutralizing antibodies to the V2 domain and protection. Second, multiple broadly neutralizing monoclonal antibodies to the V1/V2 domain (e.g. PG9) have been isolated from rare infected individuals, termed elite neutralizers. Interestingly, the binding of both types of antibodies appears to depend on the same cluster of amino acids (positions 167–171) adjacent to the junction of the B and C strands of the four-stranded V1/V2 domain β-sheet structure. However, the broadly neutralizing mAb, PG9, additionally depends on mannose-5 glycans at positions 156 and 160 for binding. Because the gp120 vaccine immunogens used in previous HIV-1 vaccine trials were enriched for complex sialic acid-containing glycans, and lacked the high mannose structures required for the binding of PG9-like mAbs, we wondered if these immunogens could be improved by limiting glycosylation to mannose-5 glycans. Here, we describe the PG9 binding activity of monomeric gp120s from multiple strains of HIV-1 produced with mannose-5 glycans. We also describe the properties of glycopeptide scaffolds from the V1/V2 domain also expressed with mannose-5 glycans. The V1/V2 scaffold from the A244 isolate was able to bind the PG9, CH01, and CH03 mAbs with high affinity provided that the proper glycans were present. We further show that immunization with A244 V1/V2 fragments alone, or in a prime/boost regimen with gp120, enhanced the antibody response to sequences in the V1/V2 domain associated with protection in the RV144 trial. PMID:24872420

  9. The lysine-rich motif of intrinsically disordered stress protein CDeT11-24 from Craterostigma plantagineum is responsible for phosphatidic acid binding and protection of enzymes from damaging effects caused by desiccation

    PubMed Central

    Röhrig, Horst

    2012-01-01

    The late embryogenesis abundant (LEA)-like protein CDeT11-24 is one of the major desiccation-related phosphoproteins of the resurrection plant Craterostigma plantagineum. In this study, it was shown that CDeT11-24 is mostly intrinsically disordered and protects two different enzymes, citrate synthase and lactate dehydrogenase, against damaging effects caused by desiccation. Lipid-binding assays revealed that CDeT11-24 is able to interact with phosphatidic acid, although electrostatic repulsion was expected due to the overall negative net charge of the protein under the tested physiological conditions. CDeT11-24 carries an N-terminal lysine-rich sequence, which is predicted to form an amphipathic α-helix. Analysis of the truncated CDeT11-24 protein identified this region to be responsible for both activities: enzyme protection and phosphatidic acid interaction. Possible functions of the CDeT11-24 protein are discussed in the context of desiccation tolerance. Abbreviations:ABAabscisic acid;CDcircular dichroism;CScitrate synthase;IDPintrinsically disordered protein;LDHlactate dehydrogenase;LEAlate embryogenesis abundant;PAphosphatidic acid;PCphosphatidylcholine;TFEtrifluoroethanol. PMID:22791833

  10. The mitochondrial UPR - protecting organelle protein homeostasis.

    PubMed

    Haynes, Cole M; Ron, David

    2010-11-15

    Mitochondria are required for numerous essential metabolic processes including the regulation of apoptosis; therefore, proper maintenance of the mitochondrial proteome is crucial. The protein-folding environment in mitochondria is challenged by organelle architecture, the presence of reactive oxygen species and the difficulties associated with assembly of the electron transport chain, which consists of components encoded by both the mitochondrial and the nuclear genomes. Mitochondria have dedicated molecular chaperones and proteases that promote proper protein folding, complex assembly and quality control. Work in cultured mammalian cells and Caenorhabditis elegans has yielded clues to the mechanisms linking perturbations in the protein-folding environment in the mitochondrial matrix to the expression of nuclear genes encoding mitochondrial proteins. Here, we review the current knowledge of this mitochondrial unfolded protein response (UPR(mt)), compare it with the better understood UPR of the endoplasmic reticulum and highlight its potential impact on development and disease.

  11. Intradermal Immunization of Leishmania donovani Centrin Knock-Out Parasites in Combination with Salivary Protein LJM19 from Sand Fly Vector Induces a Durable Protective Immune Response in Hamsters

    PubMed Central

    Fiuza, Jacqueline Araújo; Dey, Ranadhir; Davenport, Dwann; Abdeladhim, Maha; Meneses, Claudio; Oliveira, Fabiano; Kamhawi, Shaden; Valenzuela, Jesus G.; Gannavaram, Sreenivas; Nakhasi, Hira L.

    2016-01-01

    Background Visceral leishmaniasis (VL) is a neglected tropical disease and is fatal if untreated. There is no vaccine available against leishmaniasis. The majority of patients with cutaneous leishmaniasis (CL) or VL develop a long-term protective immunity after cure from infection, which indicates that development of an effective vaccine against leishmaniasis is possible. Such protection may also be achieved by immunization with live attenuated parasites that do not cause disease. We have previously reported a protective response in mice, hamsters and dogs with Leishmania donovani centrin gene knock-out parasites (LdCen-/-), a live attenuated parasite with a cell division specific centrin1 gene deletion. In this study we have explored the effects of salivary protein LJM19 as an adjuvant and intradermal (ID) route of immunization on the efficacy of LdCen-/- parasites as a vaccine against virulent L. donovani. Methodology/Principal Findings To explore the potential of a combination of LdCen-/- parasites and salivary protein LJM19 as vaccine antigens, LdCen-/- ID immunization followed by ID challenge with virulent L. donovani were performed in hamsters in a 9-month follow up study. We determined parasite burden (serial dilution), antibody production (ELISA) and cytokine expression (qPCR) in these animals. Compared to controls, animals immunized with LdCen-/- + LJM19 induced a strong antibody response, a reduction in spleen and liver parasite burden and a higher expression of pro-inflammatory cytokines after immunization and one month post-challenge. Additionally, a low parasite load in lymph nodes, spleen and liver, and a non-inflamed spleen was observed in immunized animals 9 months after the challenge infection. Conclusions Our results demonstrate that an ID vaccination using LdCen-/-parasites in combination with sand fly salivary protein LJM19 has the capability to confer long lasting protection against visceral leishmaniasis that is comparable to intravenous or

  12. Intradermal Immunization of Leishmania donovani Centrin Knock-Out Parasites in Combination with Salivary Protein LJM19 from Sand Fly Vector Induces a Durable Protective Immune Response in Hamsters.

    PubMed

    Fiuza, Jacqueline Araújo; Dey, Ranadhir; Davenport, Dwann; Abdeladhim, Maha; Meneses, Claudio; Oliveira, Fabiano; Kamhawi, Shaden; Valenzuela, Jesus G; Gannavaram, Sreenivas; Nakhasi, Hira L

    2016-01-01

    Visceral leishmaniasis (VL) is a neglected tropical disease and is fatal if untreated. There is no vaccine available against leishmaniasis. The majority of patients with cutaneous leishmaniasis (CL) or VL develop a long-term protective immunity after cure from infection, which indicates that development of an effective vaccine against leishmaniasis is possible. Such protection may also be achieved by immunization with live attenuated parasites that do not cause disease. We have previously reported a protective response in mice, hamsters and dogs with Leishmania donovani centrin gene knock-out parasites (LdCen-/-), a live attenuated parasite with a cell division specific centrin1 gene deletion. In this study we have explored the effects of salivary protein LJM19 as an adjuvant and intradermal (ID) route of immunization on the efficacy of LdCen-/- parasites as a vaccine against virulent L. donovani. To explore the potential of a combination of LdCen-/- parasites and salivary protein LJM19 as vaccine antigens, LdCen-/- ID immunization followed by ID challenge with virulent L. donovani were performed in hamsters in a 9-month follow up study. We determined parasite burden (serial dilution), antibody production (ELISA) and cytokine expression (qPCR) in these animals. Compared to controls, animals immunized with LdCen-/- + LJM19 induced a strong antibody response, a reduction in spleen and liver parasite burden and a higher expression of pro-inflammatory cytokines after immunization and one month post-challenge. Additionally, a low parasite load in lymph nodes, spleen and liver, and a non-inflamed spleen was observed in immunized animals 9 months after the challenge infection. Our results demonstrate that an ID vaccination using LdCen-/-parasites in combination with sand fly salivary protein LJM19 has the capability to confer long lasting protection against visceral leishmaniasis that is comparable to intravenous or intracardial immunization.

  13. 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.

  14. The lysine-rich motif of intrinsically disordered stress protein CDeT11-24 from Craterostigma plantagineum is responsible for phosphatidic acid binding and protection of enzymes from damaging effects caused by desiccation.

    PubMed

    Petersen, Jan; Eriksson, Sylvia K; Harryson, Pia; Pierog, Steffen; Colby, Thomas; Bartels, Dorothea; Röhrig, Horst

    2012-08-01

    The late embryogenesis abundant (LEA)-like protein CDeT11-24 is one of the major desiccation-related phosphoproteins of the resurrection plant Craterostigma plantagineum. In this study, it was shown that CDeT11-24 is mostly intrinsically disordered and protects two different enzymes, citrate synthase and lactate dehydrogenase, against damaging effects caused by desiccation. Lipid-binding assays revealed that CDeT11-24 is able to interact with phosphatidic acid, although electrostatic repulsion was expected due to the overall negative net charge of the protein under the tested physiological conditions. CDeT11-24 carries an N-terminal lysine-rich sequence, which is predicted to form an amphipathic α-helix. Analysis of the truncated CDeT11-24 protein identified this region to be responsible for both activities: enzyme protection and phosphatidic acid interaction. Possible functions of the CDeT11-24 protein are discussed in the context of desiccation tolerance.

  15. Administration of recombinant Reishi immunomodulatory protein (rLZ-8) diet enhances innate immune responses and elicits protection against nervous necrosis virus in grouper Epinephelus coioides.

    PubMed

    Kuan, Yen-Chou; Sheu, Fuu; Lee, Guo-Chi; Tsai, Ming-Wei; Hung, Chih-Liang; Nan, Fan-Hua

    2012-06-01

    Nervous necrosis virus (NNV) infection during larvae and juvenile stage in grouper (Epinephelus coioides) has caused severe economic losses in the aquaculture industry in Asia. The aims of this study were to evaluate the influence of recombinant Reishi protein, rLZ-8, on the innate immune responses and the viral resisting ability in fish. Groupers were fed with rLZ-8 supplemented diet (1.25-37.5 mg (rLZ-8)/kg(diet)), and the cytokine gene expression, innate immune responses, and survival rate after NNV challenge were examined. The fish fed with rLZ-8 diet showed 6- to 11-fold upregulated TNF-α and IL-1β gene expression, along with significant increased respiratory burst and phagocytic activity. Moreover, feeding the fish with 37.5 mg/kg rLZ-8 diet elicited significant improvement in post viral challenge survival rate (85.7%). These discoveries indicated that rLZ-8 could be utilized as an ant-pathogen immunostimulant, and provided a new candidate to fight against NNV infection in fish.

  16. CD4 T Cell Memory and Antibody Responses Directed against the Pneumococcal Histidine Triad Proteins PhtD and PhtE following Nasopharyngeal Colonization and Immunization and Their Role in Protection against Pneumococcal Colonization in Mice

    PubMed Central

    Khan, M. N.

    2013-01-01

    The present study was undertaken to understand the role of vaccine candidates PhtD and PhtE in pneumococcal nasopharyngeal (NP) colonization, their ability to induce CD4 T cell memory and antibody responses following primary NP colonization, and their contribution to protection against secondary pneumococcal colonization in mice. The study was also aimed at understanding the potential of immunization with PhtD and PhtE in eliciting qualitative CD4 T cell memory responses and protection against pneumococcal NP colonization in mice. PhtD and PhtE isogenic mutants in a TIGR4 background (TIGR4 ΔPhtD and TIGR4 ΔPhtE) were constructed and found to have a significantly reduced colonization density over time in the nasopharynges of mice compared to those of mice colonized with wild-type TIGR4. Mice with primary colonization by wild-type TIGR4, TIGR4 ΔPhtD, or TIGR4 ΔPhtE were protected against secondary colonization by wild-type TIGR4; nonetheless, the clearance of secondary colonization was slower in mice with primary colonization by either TIGR4 ΔPhtD or TIGR4 ΔPhtE than in mice with primary colonization by wild-type TIGR4. Colonization was found to be an immunizing event for PhtD and PhtE antigens (antibody response); however, we failed to detect any antigen (PhtD or PhtE)-specific CD4 T cell responses in any of the colonized groups of mice. Intranasal immunization with either PhtD or PhtE protein generated robust serum antibody and CD4 Th1-biased immune memory and conferred protection against pneumococcal colonization in mice. We conclude that PhtD and PhtE show promise as components in next-generation pneumococcal vaccine formulations. PMID:23897609

  17. Development of a DNA vaccine for chicken infectious anemia and its immunogenicity studies using high mobility group box 1 protein as a novel immunoadjuvant indicated induction of promising protective immune responses.

    PubMed

    Sawant, Pradeep Mahadev; Dhama, Kuldeep; Rawool, Deepak Bhiva; Wani, Mohd Yaqoob; Tiwari, Ruchi; Singh, Shambhu Dayal; Singh, Raj Kumar

    2015-01-03

    Chicken infectious anaemia (CIA) is an economically important and emerging poultry disease reported worldwide. Current CIA vaccines have limitations like, the inability of the virus to grow to high titres in embryos/cell cultures, possession of residual pathogenicity and a risk of reversion to virulence. In the present study, a DNA vaccine, encoding chicken infectious anaemia virus (CIAV) VP1 and VP2 genes, was developed and co-administered with truncated chicken high mobility group box 1 (HMGB1ΔC) protein in young chicks for the evaluation of vaccine immune response. CIAV VP1 and VP2 genes were cloned in pTARGET while HMGB1ΔC in PET32b vector. In vitro expression of these gene constructs was evaluated by Western blotting. Further, recombinant HMGB1ΔC was evaluated for its biological activity. The CIAV DNA vaccine administration in specific pathogen free chicks resulted in moderately protective ELISA antibody titres in the range of 4322.87 ± 359.72 to 8288.19 ± 136.38, increased CD8(+) cells, and a higher titre was observed by co-administration of novel adjuvant (HMGB1ΔC) and booster immunizations. The use of vaccine with adjuvant showed achieving antibody titres nearly 8500, titre considered as highly protective, which indicates that co-immunization of HMGB1ΔC may have a strong adjuvant activity on CIAV DNA vaccine induced immune responses. The able potential of HMGB1 protein holding strong adjuvant activity could be exploited further with trials with vaccines for other important pathogens for achieving the required protective immune responses.

  18. The Phage Shock Protein Response.

    PubMed

    Flores-Kim, Josué; Darwin, Andrew J

    2016-09-08

    The phage shock protein (Psp) system was identified as a response to phage infection in Escherichia coli, but rather than being a specific response to a phage, it detects and mitigates various problems that could increase inner-membrane (IM) permeability. Interest in the Psp system has increased significantly in recent years due to appreciation that Psp-like proteins are found in all three domains of life and because the bacterial Psp response has been linked to virulence and other important phenotypes. In this article, we summarize our current understanding of what the Psp system detects and how it detects it, how four core Psp proteins form a signal transduction cascade between the IM and the cytoplasm, and current ideas that explain how the Psp response keeps bacterial cells alive. Although recent studies have significantly improved our understanding of this system, it is an understanding that is still far from complete.

  19. Commercial Building Motor Protection Response Report

    SciTech Connect

    James, Daniel P.; Kueck, John

    2015-06-17

    When voltages recover, motors may immediately reenergize and reaccelerate, or delay for a few minutes, or stay stalled. The estimated motor response is given for both the voltage sag magnitude and voltage sag duration. These response estimates are based on experience and available test data. Good data is available for voltage sag response for many components such as relays and contactors, but little data is available for both voltage sag and recovery response. The tables in Appendix A include data from recent voltage sag and recovery tests performed by SCE and BPA on air conditioners and energy management systems. The response of the motor can vary greatly depending on the type of protection and control. The time duration for the voltage sag consists of those times that are of interest for bulk power system modelers.

  20. Nasal immunization with Lactococcus lactis expressing the pneumococcal protective protein A induces protective immunity in mice.

    PubMed

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

    2008-06-01

    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.

  1. Thermal response properties of protective clothing fabrics

    SciTech Connect

    Baitinger, W.F.

    1995-12-31

    In the industrial workplace, it becomes increasingly incumbent upon employers to require employees to use suitable protective equipment and to wear protective apparel. When workers may be subjected to accidental radiant, flame, or electric arc heat sources, work clothing should be used that does not become involved in burning. It is axiomatic that work clothing should not become a primary fuel source, adding to the level of heat exposure, since clothing is usually in intimate contact with the skin. Further, clothing should provide sufficient insulation to protect the skin from severe burn injury. If the worker receives such protection from clothing, action then may be taken to escape the confronted thermal hazard. Published laboratory test methods are used to measure flame resistance and thermal responses of flame resistant fabrics in protective clothing. The purpose of this article is to review these test methods, to discuss certain limitations in application, and to suggest how flame resistant cotton fabrics may be used to enhance worker safety.

  2. A prime/boost PfCS14K(M)/MVA-sPfCS(M) vaccination protocol generates robust CD8(+) T cell and antibody responses to Plasmodium falciparum circumsporozoite protein and protects mice against malaria.

    PubMed

    Vijayan, Aneesh; Mejías-Pérez, Ernesto; Espinosa, Diego A; Raman, Suresh C; Sorzano, Carlos Oscar S; Zavala, Fidel; Esteban, Mariano

    2017-03-15

    Vaccines against the pre-erythrocytic stages of malaria are appealing, since the parasite can be eliminated before disease onset and since they offer the unique possibility of targeting the parasite with both antibodies and T cells. Although the role CD8(+) T cells in pre-erythrocytic malaria stages is well documented, a highly effective T cell-inducing vaccine remains to be advanced. Here we report the development of a prime-boost immunization regimen with the Plasmodium falciparum circumsporozoite protein (PfCS) fused to the oligomer-forming vaccinia virus A27 protein and a modified vaccinia virus Ankara (MVA) vector expressing PfCS. This protocol induced polyfunctional CD8(+) T cells with effector memory phenotype and high PfCS antibody levels. These immune responses correlated with inhibition of liver stage parasitemia in 80% and sterile protection in 40% of mice challenged with a transgenic P. berghei parasite line that expressed PfCS. Our findings underscore the potential of T and B cell immunization strategies in improving protective effectiveness against malaria.

  3. Mosaic tetracycline resistance genes encoding ribosomal protection proteins

    PubMed Central

    Warburton, Philip J.; Amodeo, Nina; Roberts, Adam P.

    2016-01-01

    First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria. PMID:27494928

  4. Mosaic tetracycline resistance genes encoding ribosomal protection proteins.

    PubMed

    Warburton, Philip J; Amodeo, Nina; Roberts, Adam P

    2016-12-01

    First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria.

  5. BAD Modulates Counterregulatory Responses to Hypoglycemia and Protective Glucoprivic Feeding

    PubMed Central

    Osundiji, Mayowa A.; Godes, Marina L.; Evans, Mark L.; Danial, Nika N.

    2011-01-01

    Hypoglycemia or glucoprivation triggers protective hormonal counterregulatory and feeding responses to aid the restoration of normoglycemia. Increasing evidence suggests pertinent roles for the brain in sensing glucoprivation and mediating counterregulation, however, the precise nature of the metabolic signals and molecular mediators linking central glucose sensing to effector functions are not fully understood. Here, we demonstrate that protective hormonal and feeding responses to hypoglycemia are regulated by BAD, a BCL-2 family protein with dual functions in apoptosis and metabolism. BAD-deficient mice display impaired glycemic and hormonal counterregulatory responses to systemic glucoprivation induced by 2-deoxy-D-glucose. BAD is also required for proper counterregulatory responses to insulin-induced hypoglycemia as evident from significantly higher glucose infusion rates and lower plasma epinephrine levels during hyperinsulinemic hypoglycemic clamps. Importantly, RNA interference-mediated acute knockdown of Bad in the brain provided independent genetic evidence for its relevance in central glucose sensing and proper neurohumoral responses to glucoprivation. Moreover, BAD deficiency is associated with impaired glucoprivic feeding, suggesting that its role in adaptive responses to hypoglycemia extends beyond hormonal responses to regulation of feeding behavior. Together, these data indicate a previously unappreciated role for BAD in the control of central glucose sensing. PMID:22162752

  6. Clustered epitopes within the Gag-Pol fusion protein DNA vaccine enhance immune responses and protection against challenge with recombinant vaccinia viruses expressing HIV-1 Gag and Pol antigens.

    PubMed

    Bolesta, Elizabeth; Gzyl, Jaroslaw; Wierzbicki, Andrzej; Kmieciak, Dariusz; Kowalczyk, Aleksandra; Kaneko, Yutaro; Srinivasan, Alagarsamy; Kozbor, Danuta

    2005-02-20

    We have generated a codon-optimized hGagp17p24-Polp51 plasmid DNA expressing the human immunodeficiency virus type 1 (HIV-1) Gag-Pol fusion protein that consists of clusters of highly conserved cytotoxic T lymphocyte (CTL) epitopes presented by multiple MHC class I alleles. In the hGagp17p24-Polp51 construct, the ribosomal frameshift site had been deleted together with the potentially immunosuppressive Gag nucleocapsid (p15) as well as Pol protease (p10) and integrase (p31). Analyses of the magnitude and breadth of cellular responses demonstrated that immunization of HLA-A2/K(b) transgenic mice with the hGagp17p24-Polp51 construct induced 2- to 5-fold higher CD8+ T-cell responses to Gag p17-, p24-, and Pol reverse transcriptase (RT)-specific CTL epitopes than the full-length hGag-PolDeltaFsDeltaPr counterpart. The increases were correlated with higher protection against challenge with recombinant vaccinia viruses (rVVs) expressing gag and pol gene products. Consistent with the profile of Gag- and Pol-specific CD8+ T cell responses, an elevated level of type 1 cytokine production was noted in p24- and RT-stimulated splenocyte cultures established from hGagp17p24-Polp51-immunized mice compared to responses induced with the hGag-PolDeltaFsDeltaPr vaccine. Sera of mice immunized with the hGagp17p24-Polp51 vaccine also exhibited an increased titer of p24- and RT-specific IgG2 antibody responses. The results from our studies provide insights into approaches for boosting the breadth of Gag- and Pol-specific immune responses.

  7. Protective Immunogenicity of Group A Streptococcal M-Related Proteins

    PubMed Central

    Niedermeyer, Shannon E.; Agbaosi, Tina; Hysmith, Nicholas D.; Penfound, Thomas A.; Hohn, Claudia M.; Pullen, Matthew; Bright, Michael I.; Murrell, Daniel S.; Shenep, Lori E.; Courtney, Harry S.

    2015-01-01

    Many previous studies have focused on the surface M proteins of group A streptococci (GAS) as virulence determinants and protective antigens. However, the majority of GAS isolates express M-related protein (Mrp) in addition to M protein, and both have been shown to be required for optimal virulence. In the current study, we evaluated the protective immunogenicity of Mrp to determine its potential as a vaccine component that may broaden the coverage of M protein-based vaccines. Sequence analyses of 33 mrp genes indicated that there are three families of structurally related Mrps (MrpI, MrpII, and MrpIII). N-terminal peptides of Mrps were cloned, expressed, and purified from M type 2 (M2) (MrpI), M4 (MrpII), and M49 (MrpIII) GAS. Rabbit antisera against the Mrps reacted at high titers with the homologous Mrp, as determined by enzyme-linked immunosorbent assay, and promoted bactericidal activity against GAS emm types expressing Mrps within the same family. Mice passively immunized with rabbit antisera against MrpII were protected against challenge infections with M28 GAS. Assays for Mrp antibodies in serum samples from 281 pediatric subjects aged 2 to 16 indicated that the Mrp immune response correlated with increasing age of the subjects. Affinity-purified human Mrp antibodies promoted bactericidal activity against a number of GAS representing different emm types that expressed an Mrp within the same family but showed no activity against emm types expressing an Mrp from a different family. Our results indicate that Mrps have semiconserved N-terminal sequences that contain bactericidal epitopes which are immunogenic in humans. These findings may have direct implications for the development of GAS vaccines. PMID:25630406

  8. Novel influenza virus vectors expressing Brucella L7/L12 or Omp16 proteins in cattle induced a strong T-cell immune response, as well as high protectiveness against B. abortus infection.

    PubMed

    Tabynov, Kaissar; Kydyrbayev, Zhailaubay; Ryskeldinova, Sholpan; Yespembetov, Bolat; Zinina, Nadezhda; Assanzhanova, Nurika; Kozhamkulov, Yerken; Inkarbekov, Dulat; Gotskina, Tatyana; Sansyzbay, Abylai

    2014-04-11

    This paper presents the results of a study of the immunogenicity and protectiveness of new candidate vector vaccine against Brucella abortus - a bivalent vaccine formulation consisting of a mixture of recombinant influenza A subtype H5N1 or H1N1 (viral constructs vaccine formulation) viruses expressing Brucella ribosomal protein L7/L12 and Omp16, in cattle. To increase the effectiveness of the candidate vaccine, adjuvants such as Montanide Gel01 or chitosan were included in its composition. Immunization of cattle (heifers aged 1-1.5 years, 5 animals per group) with the viral constructs vaccine formulation only, or its combination with adjuvants Montanide Gel01 or chitosan, was conducted via the conjunctival method using cross prime (influenza virus subtype H5N1) and booster (influenza virus subtype H1N1) vaccination schedules at an interval of 28 days. Vaccine candidates were evaluated in comparison with the positive (B. abortus S19) and negative (PBS) controls. The viral constructs vaccine formulations, particularly in combination with Montanide Gel01 adjuvant promoted formation of IgG antibodies (with a predominance of antibodies of isotype IgG2a) against Brucella L7/L12 and Omp16 proteins in ELISA. Moreover, these vaccines in cattle induced a strong antigen-specific T-cell immune response, as indicated by a high number of CD4(+) and CD8(+) cells, as well as the concentration of IFN-γ, and most importantly provided a high level of protectiveness comparable to the commercial B. abortus S19 vaccine and superior to the B. abortus S19 vaccine in combination with Montanide Gel01 adjuvant. Based on these findings, we recommended the bivalent vaccine formulation containing the adjuvant Montanide Gel01 for practical use in cattle.

  9. A bicistronic DNA vaccine containing apical membrane antigen 1 and merozoite surface protein 4/5 can prime humoral and cellular immune responses and partially protect mice against virulent Plasmodium chabaudi adami DS malaria.

    PubMed

    Rainczuk, A; Scorza, T; Spithill, T W; Smooker, P M

    2004-10-01

    The ultimate malaria vaccine will require the delivery of multiple antigens from different stages of the complex malaria life cycle. In order to efficiently deliver multiple antigens with use of DNA vaccine technology, new antigen delivery systems must be assessed. This study utilized a bicistronic vector construct, containing an internal ribosome entry site, expressing a combination of malarial candidate antigens: merozoite surface protein 4/5 (MSP4/5) (fused to a monocyte chemotactic protein 3 chemoattractant sequence) and apical membrane antigen 1 (AMA-1) (fused to a tissue plasminogen activator secretion signal). Transfection of COS 7 cells with bicistronic plasmids resulted in production and secretion of both AMA-1 and MSP4/5 in vitro. Vaccination of BALB/c mice via intraepidermal gene gun and intramuscular routes against AMA-1 and MSP4/5 resulted in antibody production and significant in vitro proliferation of splenocytes stimulated by both AMA-1 and MSP4/5. Survival of BALB/c mice vaccinated with bicistronic constructs after lethal Plasmodium chabaudi adami DS erythrocytic-stage challenge was variable, although significant increases in survival and reductions in peak parasitemia were observed in several challenge trials when the vaccine was delivered by the intramuscular route. This study using a murine model demonstrates that the delivery of malarial antigens via bicistronic vectors is feasible. Further experimentation with bicistronic delivery systems is required for the optimization and refinement of DNA vaccines to effectively prime protective immune responses against malaria.

  10. Discriminating protective from non-protective Plasmodium-specific CD8+ T cell responses

    PubMed Central

    Doll, Katherine L.; Pewe, Lecia L.; Kurup, Samarchith P.; Harty, John T.

    2016-01-01

    Despite decades of research, malaria remains a global health crisis. Current subunit vaccine approaches do not provide efficient long-term, sterilizing immunity against Plasmodium infections in humans. Conversely, whole parasite vaccinations with their larger array of target antigens have conferred long lasting sterilizing protection to humans. Similar studies in rodent models of malaria reveal that CD8+ T cells play a critical role in liver-stage immunity after whole parasite vaccination. However, it is unknown whether all CD8+ T cell specificities elicited by whole parasite vaccination contribute to protection, an issue of great relevance for enhanced subunit vaccination. Here we show that robust CD8+ T cell responses of similar phenotype are mounted following prime-boost immunization against Plasmodium berghei GAP5041-48, S20318-325, TRAP130-138 or CSP252-260 protein-derived epitopes in mice, but only CSP252-260- and TRAP130-138-specific CD8+ T cells provide sterilizing immunity and reduce liver parasite burden following sporozoite challenge. Further, CD8+ T cells specific to sporozoite surface-expressed CSP and TRAP proteins, but not the intracellular GAP50 and S20 proteins, are efficiently recognized by sporozoite-infected hepatocytes in vitro. These results suggest that 1) protection-relevant antigenic targets, regardless of their immunogenic potential, must be efficiently presented by infected hepatocytes for CD8+ T cells to eliminate liver-stage Plasmodium infection and 2) proteins expressed on the surface of sporozoites may be good target antigens for protective CD8+ T cells. PMID:27084099

  11. 5-Aminoimidazole-4-carboxamide ribonucleoside-mediated adenosine monophosphate-activated protein kinase activation induces protective innate responses in bacterial endophthalmitis.

    PubMed

    Kumar, Ajay; Giri, Shailendra; Kumar, Ashok

    2016-12-01

    The retina is considered to be the most metabolically active tissue in the body. However, the link between energy metabolism and retinal inflammation, as incited by microbial infection such as endophthalmitis, remains unexplored. In this study, using a mouse model of Staphylococcus aureus (SA) endophthalmitis, we demonstrate that the activity (phosphorylation) of 5' adenosine monophosphate-activated protein kinase alpha (AMPKα), a cellular energy sensor and its endogenous substrate; acetyl-CoA carboxylase is down-regulated in the SA-infected retina. Intravitreal administration of an AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), restored AMPKα and acetyl-CoA carboxylase phosphorylation. AICAR treatment reduced both the bacterial burden and intraocular inflammation in SA-infected eyes by inhibiting NF-kB and MAP kinases (p38 and JNK) signalling. The anti-inflammatory effects of AICAR were diminished in eyes pretreated with AMPK inhibitor, Compound C. The bioenergetics (Seahorse) analysis of SA-infected microglia and bone marrow-derived macrophages revealed an increase in glycolysis, which was reinstated by AICAR treatment. AICAR also reduced the expression of SA-induced glycolytic genes, including hexokinase 2 and glucose transporter 1 in microglia, bone marrow-derived macrophages and the mouse retina. Interestingly, AICAR treatment enhanced the bacterial phagocytic and intracellular killing activities of cultured microglia, macrophages and neutrophils. Furthermore, AMPKα1 global knockout mice exhibited increased susceptibility towards SA endophthalmitis, as evidenced by increased inflammatory mediators and bacterial burden and reduced retinal function. Together, these findings provide the first evidence that AMPK activation promotes retinal innate defence in endophthalmitis by modulating energy metabolism and that it can be targeted therapeutically to treat ocular infections.

  12. Rabbit immunoglobulin responses to the flagella, somatic, and protective antigens of a highly protective strain of Clostridium chauvoei.

    PubMed

    Chandler, H M

    1975-07-01

    The immunoglobulin response of rabbits to the flagells (H), somatic (O), and protective antigens of a highly protective strain of Clostridium chauvoei was studied using antisera that had been fractionated by Sephadex G-200 chromatography. The H antigen elicited the characteristic agglutinin response to a protein antigen--early production of 19S globulin followed by persistent 7S globulin production. The O antigen stimulated a transient agglutinin response which was detected in both the 19S and 7S serum fractions. Protective antibody was assayed by passive protection tests in mice. Using these tests the protective activity of the rabbit sera was found to be confined exclusively to the 7S serum fractions. Purified immunoglobulin G, prepared by DEAE-cellulose chromatography of the above sera, was also tested and found to confer considerable passive protection on mice. It is considered that either the protective antigen fails to stimulate an immunoglobulin M response or that immunoglobulin M is relatively ineffective in conferring protection against infection in the mouse passive protection tests.

  13. Protein structure protection commits gene expression patterns.

    PubMed

    Chen, Jianping; Liang, Han; Fernández, Ariel

    2008-01-01

    Gene co-expressions often determine module-defining spatial and temporal concurrences of proteins. Yet, little effort has been devoted to tracing coordinating signals for expression correlations to the three-dimensional structures of gene products. We performed a global structure-based analysis of the yeast and human proteomes and contrasted this information against their respective transcriptome organizations obtained from comprehensive microarray data. We show that protein vulnerability quantifies dosage sensitivity for metabolic adaptation phases and tissue-specific patterns of mRNA expression, determining the extent of co-expression similarity of binding partners. The role of protein intrinsic disorder in transcriptome organization is also delineated by interrelating vulnerability, disorder propensity and co-expression patterns. Extremely vulnerable human proteins are shown to be subject to severe post-transcriptional regulation of their expression through significant micro-RNA targeting, making mRNA levels poor surrogates for protein-expression levels. By contrast, in yeast the expression of extremely under-wrapped proteins is likely regulated through protein aggregation. Thus, the 85 most vulnerable proteins in yeast include the five confirmed prions, while in human, the genes encoding extremely vulnerable proteins are predicted to be targeted by microRNAs. Hence, in both vastly different organisms protein vulnerability emerges as a structure-encoded signal for post-transcriptional regulation. Vulnerability of protein structure and the concurrent need to maintain structural integrity are shown to quantify dosage sensitivity, compelling gene expression patterns across tissue types and temporal adaptation phases in a quantifiable manner. Extremely vulnerable proteins impose additional constraints on gene expression: They are subject to high levels of regulation at the post-transcriptional level.

  14. Recombinant domains III of Tick-Borne Encephalitis Virus envelope protein in combination with dextran and CpGs induce immune response and partial protectiveness against TBE virus infection in mice.

    PubMed

    Ershova, Anna S; Gra, Olga A; Lyaschuk, Alexander M; Grunina, Tatyana M; Tkachuk, Artem P; Bartov, Mikhail S; Savina, Darya M; Sergienko, Olga V; Galushkina, Zoya M; Gudov, Vladimir P; Kozlovskaya, Liubov I; Kholodilov, Ivan S; Gmyl, Larissa V; Karganova, Galina G; Lunin, Vladimir G; Karyagina, Anna S; Gintsburg, Alexander L

    2016-10-07

    E protein of tick-borne encephalitis virus (TBEV) and other flaviviruses is located on the surface of the viral particle. Domain III of this protein seems to be a promising component of subunit vaccines for prophylaxis of TBE and kits for diagnostics of TBEV. Three variants of recombinant TBEV E protein domain III of European, Siberian and Far Eastern subtypes fused with dextran-binding domain of Leuconostoc citreum KM20 were expressed in E. coli and purified. The native structure of domain III was confirmed by ELISA antibody kit and sera of patients with tick-borne encephalitis. Immunogenic and protective properties of the preparation comprising these recombinant proteins immobilized on a dextran carrier with CpG oligonucleotides as an adjuvant were investigated on the mice model. All 3 variants of recombinant proteins immobilized on dextran demonstrate specific interaction with antibodies from the sera of TBE patients. Thus, constructed recombinant proteins seem to be promising for TBE diagnostics. The formulation comprising the 3 variants of recombinant antigens immobilized on dextran and CpG oligonucleotides, induces the production of neutralizing antibodies against TBEV of different subtypes and demonstrates partial protectivity against TBEV infection. Studied proteins interact with the sera of TBE patients, and, in combination with dextran and CPGs, demonstrate immunogenicity and limited protectivity on mice compared with reference "Tick-E-Vac" vaccine.

  15. Environmental Protection Agency Award Recipient Responsibilities

    EPA Pesticide Factsheets

    Itemized Award Phase information. Information about the Recipient's Responsibilities Upon Notification of the Award, The EPA Project Officer Responsibilities, and EPA Grant Specialists Responsibilities.

  16. DNA vaccines expressing pneumococcal surface protein A (PspA) elicit protection levels comparable to recombinant protein.

    PubMed

    Ferreira, Daniela M; Miyaji, Eliane N; Oliveira, Maria Leonor S; Darrieux, Michelle; Arêas, Ana Paula M; Ho, Paulo L; Leite, Luciana C C

    2006-04-01

    Pneumococcal surface protein A (PspA) is a promising candidate for the development of cost-effective vaccines against Streptococcus pneumoniae. In the present study, BALB/c mice were immunized with DNA vaccine vectors expressing the N-terminal region of PspA. Animals immunized with a vector expressing secreted PspA developed higher levels of antibody than mice immunized with the vector expressing the antigen in the cytosol. However, both immunogens elicited similar levels of protection against intraperitoneal challenge. Furthermore, immunization with exactly the same fragment in the form of a recombinant protein, with aluminium hydroxide as an adjuvant, elicited even higher antibody levels, but this increased humoral response did not correlate with enhanced protection. These results show that DNA vaccines expressing PspA are able to elicit protection levels comparable to recombinant protein, even though total anti-PspA IgG response is considerably lower.

  17. Cellular stress responses in protein misfolding diseases.

    PubMed

    Duennwald, Martin L

    2015-09-01

    Many human diseases, particularly neurodegenerative diseases, are associated with protein misfolding. Cellular protein quality control includes all processes that ensure proper protein folding and thus prevent the toxic consequences of protein misfolding. The heat shock response (HSR) and the unfolded protein response (UPR) are major stress response pathways within protein quality control that antagonize protein misfolding in the cytosol and the endoplasmic reticulum, respectively. Huntington's disease is an inherited neurodegenerative disease caused by the misfolding of an abnormally expanded polyglutamine (polyQ) region in the protein huntingtin (Htt), polyQHtt. Using Huntington's disease as a paradigm, I review here the central role of both the HSR and the UPR in defining the toxicity associated with polyQHtt in Huntington's disease. These findings may begin to unravel a previously unappreciated cooperation between different stress response pathways in cells expressing misfolded proteins and consequently in neurodegenerative diseases.

  18. Identification of polyvalent protective immunogens from outer membrane proteins in Vibrio parahaemolyticus to protect fish against bacterial infection.

    PubMed

    Peng, Bo; Ye, Jin-Zhou; Han, Yi; Zeng, Li; Zhang, Jian-Ying; Li, Hui

    2016-07-01

    Vaccination is one of the most effective and economic way to prevent infectious diseases in aquaculture. The development of effective vaccines, however, is still limited, especially for polyvalent vaccines, which are against multiple species. With this regard, identification of polyvalent protective immunogens, serving as polyvalent vaccines, became a key step in vaccine development. In the current study, 17 outer membrane proteins from Vibrio parahaemolyticus were identified as immunogens. Further, four of the 17 proteins including VP2309, VP0887, VPA0548 and VP1019 were characterized as efficiently protective immunogens against V. parahaemolyticus' infection through passive and active immunizations in zebrafish. Importantly, these four proteins showed cross-protective capability against infections by Aeromonas hydrophila or/and Pseudomonas fluorescens, which shared similar epitopes with V. parahaemolyticus in homology of these proteins. Further investigation showed that the expression level of the four protective immunogens elevated in response to fish plasma in a dose-dependent manner. These results indicate that the four protective immunogens are polyvalent vaccine candidates in aquaculture.

  19. Heat stress proteins and myocardial protection: experimental model or potential clinical tool?

    PubMed

    Gray, C C; Amrani, M; Yacoub, M H

    1999-05-01

    Heat stress proteins (hsp) are induced by a variety of stimuli including elevated temperature, ischaemia, hypoxia, pressure overload and some chemicals. They help to maintain the metabolic and structural integrity of the cell, as a protective response to external stresses. They are known to protect the myocardium from the damaging effects of ischaemia and reperfusion. The heat stress response results in accumulation of heat stress proteins. The beneficial effects associated with their expression include improved endothelial and mechanical recovery of the ischaemic heart. In addition, preservation of high energy phosphates and reduction in infarct size. It has also been shown that critical amounts of hsp70 are necessary to ensure protection of the myocardium. However, questions remain regarding the biochemical mechanisms underlying this protective effect. Alterations in the cell metabolism and chaperone function of cells expressing heat shock proteins, are thought to be responsible. Despite the obvious clinical benefits related to the heat stress response in a clinical setting, the application of this phenomena remains limited. Heat, both quantitatively and qualitatively is one of the best inducers of heat stress proteins. However, the effects of heat stress are nonspecific and intracellular damage is a common occurrence. The search for alternative stimuli, particularly within the fields of pharmacotherapy or genetic manipulation may offer more viable options, if the heat stress response is take its place as an established strategy for myocardial protection.

  20. A Translocated Bacterial Protein Protects Vascular Endothelial Cells from Apoptosis

    PubMed Central

    Schmid, Michael C; Scheidegger, Florine; Dehio, Michaela; Balmelle-Devaux, Nadège; Schulein, Ralf; Guye, Patrick; Chennakesava, Cuddapah S; Biedermann, Barbara; Dehio, Christoph

    2006-01-01

    The modulation of host cell apoptosis by bacterial pathogens is of critical importance for the outcome of the infection process. The capacity of Bartonella henselae and B. quintana to cause vascular tumor formation in immunocompromised patients is linked to the inhibition of vascular endothelial cell (EC) apoptosis. Here, we show that translocation of BepA, a type IV secretion (T4S) substrate, is necessary and sufficient to inhibit EC apoptosis. Ectopic expression in ECs allowed mapping of the anti-apoptotic activity of BepA to the Bep intracellular delivery domain, which, as part of the signal for T4S, is conserved in other T4S substrates. The anti-apoptotic activity appeared to be limited to BepA orthologs of B. henselae and B. quintana and correlated with (i) protein localization to the host cell plasma membrane, (ii) elevated levels of intracellular cyclic adenosine monophosphate (cAMP), and (iii) increased expression of cAMP-responsive genes. The pharmacological elevation of cAMP levels protected ECs from apoptosis, indicating that BepA mediates anti-apoptosis by heightening cAMP levels by a plasma membrane–associated mechanism. Finally, we demonstrate that BepA mediates protection of ECs against apoptosis triggered by cytotoxic T lymphocytes, suggesting a physiological context in which the anti-apoptotic activity of BepA contributes to tumor formation in the chronically infected vascular endothelium. PMID:17121462

  1. Structure-based design of broadly protective group a streptococcal M protein-based vaccines

    DOE PAGES

    Dale, James B.; Smeesters, Pierre R.; Courtney, Harry S.; ...

    2016-11-24

    Here, a major obstacle to the development of broadly protective M protein-based group A streptococcal (GAS) vaccines is the variability within the N-terminal epitopes that evoke potent bactericidal antibodies. The concept of M type-specific protective immune responses has recently been challenged based on the observation that multivalent M protein vaccines elicited cross-reactive bactericidal antibodies against a number of non-vaccine M types of GAS. Additionally, a new “cluster-based” typing system of 175 M proteins identified a limited number of clusters containing closely related M proteins. In the current study, we used the emm cluster typing system, in combination with computational structure-basedmore » peptide modeling, as a novel approach to the design of potentially broadly protective M protein-based vaccines.« less

  2. Structure-based design of broadly protective group a streptococcal M protein-based vaccines

    SciTech Connect

    Dale, James B.; Smeesters, Pierre R.; Courtney, Harry S.; Penfound, Thomas A.; Hohn, Claudia M.; Smith, Jeremy C.; Baudry, Jerome Y.

    2016-11-24

    Here, a major obstacle to the development of broadly protective M protein-based group A streptococcal (GAS) vaccines is the variability within the N-terminal epitopes that evoke potent bactericidal antibodies. The concept of M type-specific protective immune responses has recently been challenged based on the observation that multivalent M protein vaccines elicited cross-reactive bactericidal antibodies against a number of non-vaccine M types of GAS. Additionally, a new “cluster-based” typing system of 175 M proteins identified a limited number of clusters containing closely related M proteins. In the current study, we used the emm cluster typing system, in combination with computational structure-based peptide modeling, as a novel approach to the design of potentially broadly protective M protein-based vaccines.

  3. UVR8 mediated plant protective responses under low UV-B radiation leading to photosynthetic acclimation.

    PubMed

    Singh, Suruchi; Agrawal, S B; Agrawal, Madhoolika

    2014-08-01

    The UV-B photoreceptor UVR8 regulates the expression of several genes leading to acclimation responses in plants. Direct role of UVR8 in maintaining the photosynthesis is not defined but it is known to increase the expression of some chloroplastic proteins like SIG5 and ELIP. It provides indirect protection to photosynthesis by regulating the synthesis of secondary metabolites and photomorphogenesis. Signaling cascades controlled by UVR8 mediate many protective responses thus promotes plant acclimation against stress and secures its survival.

  4. Protective immune responses to fungal infections.

    PubMed

    Rivera, A

    2014-09-01

    The incidence of fungal infections has been on the rise over several decades. Fungal infections threaten animals, plants and humans alike and are thus of significant concern to scientists across disciplines. Over the last decade, significant advances on fungal immunology have lead to a better understanding of important mechanisms of host protection against fungi. In this article, I review recent advances of relevant mechanisms of immune-mediated protection to fungal infections.

  5. TpUB05, a Homologue of the Immunodominant Plasmodium falciparum Protein UB05, Is a Marker of Protective Immune Responses in Cattle Experimentally Vaccinated against East Coast Fever

    PubMed Central

    Dinga, Jerome Nyhalah; Wamalwa, Mark; Njimoh, Dieudonné Lemuh; Njahira, Moses N.; Djikeng, Appolinaire; Skilton, Rob; Titanji, Vincent Pryde Kehdingha; Pellé, Roger

    2015-01-01

    Introduction East Coast fever, a devastating disease of cattle, can be controlled partially by vaccination with live T. parva sporozoites. The antigens responsible for conferring immunity are not fully characterized. Recently it was shown that the P. falciparum immunodominant protein UB05 is highly conserved in T. parva, the causative agent of East Coast fever. The aim of the present investigation was to determine the role of the homologue TpUB05 in protective immunity to East Coast fever. Methods The cloning, sequencing and expression of TpUB05 were done according to standard protocols. Bioinformatics analysis of TpUB05 gene was carried out using algorithms found in the public domain. Polyclonal antiserum against recombinant TpUB05 were raised in rabbits and used for further analysis by Western blotting, ELISA, immunolocalization and in vitro infection neutralization assay. The ability of recombinant TpUB05 (r-TpUB05) to stimulate bovine PBMCs ex-vivo to produce IFN-γ or to proliferate was tested using ELISpot and [3H]-thymidine incorporation assays, respectively. Results All the 20 cattle immunised by the infection and treatment method (ITM) developed significantly higher levels of TpUB05 specific antibodies (p<0.0001) compared to the non-vaccinated ones. Similarly, r-TpUB05 highly stimulated bovine PMBCs from 8/12 (67%) of ITM-immunized cattle tested to produce IFN-γ and proliferate (p< 0.029) as compared to the 04 naїve cattle included as controls. Polyclonal TpUB05 antiserum raised against r-TpUB05 also marginally inhibited infection (p < 0.046) of bovine PBMCs by T. parva sporozoites. In further experiments RT-PCR showed that the TpUB05 gene is expressed by the parasite. This was confirmed by immunolocalization studies which revealed TpUB05 expression by schizonts and piroplasms. Bioinformatics analysis also revealed that this antigen possesses two transmembrane domains, a N-glycosylation site and several O-glycosylation sites. Conclusion It was concluded

  6. Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP).

    PubMed

    White, Carl; Nixon, Alex; Bradbury, Neil A

    2015-04-20

    The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

  7. Systems analysis of protective immune responses to RTS,S malaria vaccination in humans.

    PubMed

    Kazmin, Dmitri; Nakaya, Helder I; Lee, Eva K; Johnson, Matthew J; van der Most, Robbert; van den Berg, Robert A; Ballou, W Ripley; Jongert, Erik; Wille-Reece, Ulrike; Ockenhouse, Christian; Aderem, Alan; Zak, Daniel E; Sadoff, Jerald; Hendriks, Jenny; Wrammert, Jens; Ahmed, Rafi; Pulendran, Bali

    2017-02-28

    RTS,S is an advanced malaria vaccine candidate and confers significant protection against Plasmodium falciparum infection in humans. Little is known about the molecular mechanisms driving vaccine immunity. Here, we applied a systems biology approach to study immune responses in subjects receiving three consecutive immunizations with RTS,S (RRR), or in those receiving two immunizations of RTS,S/AS01 following a primary immunization with adenovirus 35 (Ad35) (ARR) vector expressing circumsporozoite protein. Subsequent controlled human malaria challenge (CHMI) of the vaccinees with Plasmodium-infected mosquitoes, 3 wk after the final immunization, resulted in ∼50% protection in both groups of vaccinees. Circumsporozoite protein (CSP)-specific antibody titers, prechallenge, were associated with protection in the RRR group. In contrast, ARR-induced lower antibody responses, and protection was associated with polyfunctional CD4(+) T-cell responses 2 wk after priming with Ad35. Molecular signatures of B and plasma cells detected in PBMCs were highly correlated with antibody titers prechallenge and protection in the RRR cohort. In contrast, early signatures of innate immunity and dendritic cell activation were highly associated with protection in the ARR cohort. For both vaccine regimens, natural killer (NK) cell signatures negatively correlated with and predicted protection. These results suggest that protective immunity against P. falciparum can be achieved via multiple mechanisms and highlight the utility of systems approaches in defining molecular correlates of protection to vaccination.

  8. Influence of adjuvants on protection induced by a recombinant fusion protein against malarial infection.

    PubMed

    Daly, T M; Long, C A

    1996-07-01

    Previously, we described a protective immune response induced by the carboxyl-terminal region of the merozoite surface protein-1 (MSP-1) from the rodent malarial parasite Plasmodium yoelii yoelii 17XL, expressed as a fusion protein and designated glutathione S-transferase (GST)-PYC2. We also demonstrated that the humoral response induced by GST-PYC2 was the primary mechanism by which immunized animals controlled their blood-stage infections. We have now examined the influence of several adjuvants on the immune response to the GST-PYC2 fusion protein. While alum, Freund's adjuvant, Ribi adjuvant system, and TiterMax were efficacious in eliciting a protective response with GST-PYC2 in BALB/c mice, saponin failed to induce protection, although significant levels of PYC2-specific antibodies were produced in all immunized animals. This protection depended on the mouse strain since immunization of Swiss Webster mice with GST-PYC2 in alum did not produce levels of PYC2-specific antibodies comparable to those in BALB/c mice nor did it induce any demonstrable level of protection against parasite challenge. Swiss Webster mice were protected, however, when immunized with GST-PYC2 in other adjuvants. Immunization with PYC2, isolated free of GST induced lower levels of antigen-specific antibody; only those animals given PYC2 in Freund's adjuvant demonstrated a significant degree of protection, suggesting the possibility (of additional cellular effector mechanisms. These findings demonstrate that adjuvant, host genotype, and the fine specificity of the response significantly influence the protection induced by the carboxyl terminus of MSP-1 in vivo and illustrate the need to consider these factors in evaluating MSP-1 as a vaccine component.

  9. Antibody response that protects against disseminated candidiasis.

    PubMed Central

    Han, Y; Cutler, J E

    1995-01-01

    We previously showed that surface mannans of Candida albicans function as adhesins during yeast cell attachment to mouse splenic marginal zone macrophages. The mannan adhesin fraction was encapsulated into liposomes and used to vaccinate mice over a 5- to 6-week period. Circulating agglutinins specific for the fraction correlated with increased resistance to disseminated candidiasis. Antiserum from vaccinated animals protected naive BALB/cByJ mice against C. albicans serotype A and B strains and Candida tropicalis. Antiserum also protected SCID mice against disseminated disease. The serum protective ability was stable at 56 degrees C, but this ability was adsorbed by C. albicans cells. The antiserum was divided into three fractions after separation by high-performance liquid chromatography. One fraction contained all of the agglutinin activity and transferred resistance to naive mice. A second fraction also transferred resistance. Two monoclonal antibodies (MAbs) specific for candidal surface determinants were obtained. MAb B6.1 is specific for a mannan epitope in the adhesin fraction, and MAb B6 is specific for a different epitope in the fraction. Both MAbs are immunoglobulin M, and both strongly agglutinate candidal cells, but only MAb B6.1 protected both normal and SCID mice against disseminated candidiasis. In one experiment, 10 normal mice were given MAb B6.1 and challenged with yeast cells. Six mice survived the 67-day observation period; 4 of the survivors were cured as evidenced by the lack of CFU in the kidney and spleen. Our studies show that antibodies against certain cell surface antigens of C. albicans help the host resist disseminated candidiasis. PMID:7790089

  10. How Egg Case Proteins Can Protect Cuttlefish Offspring?

    PubMed

    Cornet, Valérie; Henry, Joël; Goux, Didier; Duval, Emilie; Bernay, Benoit; Le Corguillé, Gildas; Corre, Erwan; Zatylny-Gaudin, Céline

    2015-01-01

    Sepia officinalis egg protection is ensured by a complex capsule produced by the female accessory genital glands and the ink bag. Our study is focused on the proteins constituting the main egg case. De novo transcriptomes from female genital glands provided essential databases for protein identification. A proteomic approach in SDS-PAGE coupled with MS unveiled a new egg case protein family: SepECPs, for Sepia officinalis Egg Case Proteins. N-glycosylation was demonstrated by PAS staining SDS-PAGE gels. These glycoproteins are mainly produced in the main nidamental glands. SepECPs share high sequence homology, especially in the signal peptide and the three cysteine-rich domains. SepECPs have a high number of cysteines, with conserved motifs involved in 3D-structure. SDS-PAGE showed that SepECPs could form dimers; this result was confirmed by TEM observations, which also revealed a protein network. This network is similar to the capsule network, and it associates these structural proteins with polysaccharides, melanin and bacteria to form a tight mesh. Its hardness and elasticity provide physical protection to the embryo. In addition, SepECPs also have bacteriostatic antimicrobial activity on GRAM- bacteria. By observing the SepECP / Vibrio aestuarianus complex in SEM, we demonstrated the ability of these proteins to agglomerate bacteria and thus inhibit their growth. These original proteins identified from the outer egg case ensure the survival of the species by providing physical and chemical protection to the embryos released in the environment without any maternal protection.

  11. How Egg Case Proteins Can Protect Cuttlefish Offspring?

    PubMed Central

    Cornet, Valérie; Henry, Joël; Goux, Didier; Duval, Emilie; Bernay, Benoit; Le Corguillé, Gildas; Corre, Erwan; Zatylny-Gaudin, Céline

    2015-01-01

    Sepia officinalis egg protection is ensured by a complex capsule produced by the female accessory genital glands and the ink bag. Our study is focused on the proteins constituting the main egg case. De novo transcriptomes from female genital glands provided essential databases for protein identification. A proteomic approach in SDS-PAGE coupled with MS unveiled a new egg case protein family: SepECPs, for Sepia officinalis Egg Case Proteins. N-glycosylation was demonstrated by PAS staining SDS-PAGE gels. These glycoproteins are mainly produced in the main nidamental glands. SepECPs share high sequence homology, especially in the signal peptide and the three cysteine-rich domains. SepECPs have a high number of cysteines, with conserved motifs involved in 3D-structure. SDS-PAGE showed that SepECPs could form dimers; this result was confirmed by TEM observations, which also revealed a protein network. This network is similar to the capsule network, and it associates these structural proteins with polysaccharides, melanin and bacteria to form a tight mesh. Its hardness and elasticity provide physical protection to the embryo. In addition, SepECPs also have bacteriostatic antimicrobial activity on GRAM- bacteria. By observing the SepECP / Vibrio aestuarianus complex in SEM, we demonstrated the ability of these proteins to agglomerate bacteria and thus inhibit their growth. These original proteins identified from the outer egg case ensure the survival of the species by providing physical and chemical protection to the embryos released in the environment without any maternal protection. PMID:26168161

  12. ABC-F Proteins Mediate Antibiotic Resistance through Ribosomal Protection

    PubMed Central

    Sharkey, Liam K. R.; Edwards, Thomas A.

    2016-01-01

    ABSTRACT Members of the ABC-F subfamily of ATP-binding cassette proteins mediate resistance to a broad array of clinically important antibiotic classes that target the ribosome of Gram-positive pathogens. The mechanism by which these proteins act has been a subject of long-standing controversy, with two competing hypotheses each having gained considerable support: antibiotic efflux versus ribosomal protection. Here, we report on studies employing a combination of bacteriological and biochemical techniques to unravel the mechanism of resistance of these proteins, and provide several lines of evidence that together offer clear support to the ribosomal protection hypothesis. Of particular note, we show that addition of purified ABC-F proteins to an in vitro translation assay prompts dose-dependent rescue of translation, and demonstrate that such proteins are capable of displacing antibiotic from the ribosome in vitro. To our knowledge, these experiments constitute the first direct evidence that ABC-F proteins mediate antibiotic resistance through ribosomal protection. PMID:27006457

  13. Attenuated vaccinia virus-circumsporozoite protein recombinants confer protection against rodent malaria.

    PubMed Central

    Lanar, D E; Tine, J A; de Taisne, C; Seguin, M C; Cox, W I; Winslow, J P; Ware, L A; Kauffman, E B; Gordon, D; Ballou, W R; Paoletti, E; Sadoff, J C

    1996-01-01

    NYVAC-based vaccinia virus recombinants expressing the circumsporozoite protein (CSP) were evaluated in the Plasmodium berghei rodent malaria model system. Immunization of mice with a NYVAC-based CSP recombinant elicited a high level of protection (60 to 100%). Protection did not correlate with CS repeat-specific antibody responses and was abrogated by in vivo CD8+ T-cell depletion. Protection was not enhanced by modification of the subcellular localization of CSP. These results suggest the potential of poxvirus-based vectors for the development of vaccine candidates for human malaria. PMID:8613376

  14. ABC-F Proteins Mediate Antibiotic Resistance through Ribosomal Protection.

    PubMed

    Sharkey, Liam K R; Edwards, Thomas A; O'Neill, Alex J

    2016-03-22

    Members of the ABC-F subfamily of ATP-binding cassette proteins mediate resistance to a broad array of clinically important antibiotic classes that target the ribosome of Gram-positive pathogens. The mechanism by which these proteins act has been a subject of long-standing controversy, with two competing hypotheses each having gained considerable support: antibiotic efflux versus ribosomal protection. Here, we report on studies employing a combination of bacteriological and biochemical techniques to unravel the mechanism of resistance of these proteins, and provide several lines of evidence that together offer clear support to the ribosomal protection hypothesis. Of particular note, we show that addition of purified ABC-F proteins to anin vitrotranslation assay prompts dose-dependent rescue of translation, and demonstrate that such proteins are capable of displacing antibiotic from the ribosomein vitro To our knowledge, these experiments constitute the first direct evidence that ABC-F proteins mediate antibiotic resistance through ribosomal protection.IMPORTANCEAntimicrobial resistance ranks among the greatest threats currently facing human health. Elucidation of the mechanisms by which microorganisms resist the effect of antibiotics is central to understanding the biology of this phenomenon and has the potential to inform the development of new drugs capable of blocking or circumventing resistance. Members of the ABC-F family, which includelsa(A),msr(A),optr(A), andvga(A), collectively yield resistance to a broader range of clinically significant antibiotic classes than any other family of resistance determinants, although their mechanism of action has been controversial since their discovery 25 years ago. Here we present the first direct evidence that proteins of the ABC-F family act to protect the bacterial ribosome from antibiotic-mediated inhibition. Copyright © 2016 Sharkey et al.

  15. A Federal Response: The President's Critical Infrastructure Protection Board.

    ERIC Educational Resources Information Center

    Schmidt, Howard

    2002-01-01

    Outlines the U.S. Critical Infrastructure Protection Board's purpose, budget, principles, and priorities. Describes the board's role in coordinating all federal activities related to protection of information systems and networks supporting critical infrastructures. Also discusses its responsibility in creating a policy and road map for government…

  16. A Federal Response: The President's Critical Infrastructure Protection Board.

    ERIC Educational Resources Information Center

    Schmidt, Howard

    2002-01-01

    Outlines the U.S. Critical Infrastructure Protection Board's purpose, budget, principles, and priorities. Describes the board's role in coordinating all federal activities related to protection of information systems and networks supporting critical infrastructures. Also discusses its responsibility in creating a policy and road map for government…

  17. Shigella outer membrane protein PSSP-1 is broadly protective against Shigella infection.

    PubMed

    Kim, Jae-Ouk; Rho, Semi; Kim, Su Hee; Kim, Heejoo; Song, Hyo Jin; Kim, Eun Jin; Kim, Ryang Yeo; Kim, Eun Hye; Sinha, Anuradha; Dey, Ayan; Yang, Jae Seung; Song, Man Ki; Nandy, Ranjan Kumar; Czerkinsky, Cecil; Kim, Dong Wook

    2015-04-01

    In developing countries, Shigella is a primary cause of diarrhea in infants and young children. Although antibiotic therapy is an effective treatment for shigellosis, therapeutic options are narrowing due to the emergence of antibiotic resistance. Thus, preventive vaccination could become the most efficacious approach for controlling shigellosis. We have identified several conserved protein antigens that are shared by multiple Shigella serotypes and species. Among these, one antigen induced cross-protection against experimental shigellosis, and we have named it pan-Shigella surface protein 1 (PSSP-1). PSSP-1-induced protection requires a mucosal administration route and coadministration of an adjuvant. When PSSP-1 was administered intranasally, it induced cross-protection against Shigella flexneri serotypes 2a, 5a, and 6, Shigella boydii, Shigella sonnei, and Shigella dysenteriae serotype 1. Intradermally administered PSSP-1 induced strong serum antibody responses but failed to induce protection in the mouse lung pneumonia model. In contrast, intranasal administration elicited efficient local and systemic antibody responses and production of interleukin 17A and gamma interferon. Interestingly, blood samples from patients with recent-onset shigellosis showed variable but significant mucosal antibody responses to other conserved Shigella protein antigens but not to PSSP-1. We suggest that PSSP-1 is a promising antigen for a broadly protective vaccine against Shigella.

  18. Shigella Outer Membrane Protein PSSP-1 Is Broadly Protective against Shigella Infection

    PubMed Central

    Rho, Semi; Kim, Su Hee; Kim, Heejoo; Song, Hyo Jin; Kim, Eun Jin; Kim, Ryang Yeo; Kim, Eun Hye; Sinha, Anuradha; Dey, Ayan; Yang, Jae Seung; Song, Man Ki; Nandy, Ranjan Kumar; Czerkinsky, Cecil

    2015-01-01

    In developing countries, Shigella is a primary cause of diarrhea in infants and young children. Although antibiotic therapy is an effective treatment for shigellosis, therapeutic options are narrowing due to the emergence of antibiotic resistance. Thus, preventive vaccination could become the most efficacious approach for controlling shigellosis. We have identified several conserved protein antigens that are shared by multiple Shigella serotypes and species. Among these, one antigen induced cross-protection against experimental shigellosis, and we have named it pan-Shigella surface protein 1 (PSSP-1). PSSP-1-induced protection requires a mucosal administration route and coadministration of an adjuvant. When PSSP-1 was administered intranasally, it induced cross-protection against Shigella flexneri serotypes 2a, 5a, and 6, Shigella boydii, Shigella sonnei, and Shigella dysenteriae serotype 1. Intradermally administered PSSP-1 induced strong serum antibody responses but failed to induce protection in the mouse lung pneumonia model. In contrast, intranasal administration elicited efficient local and systemic antibody responses and production of interleukin 17A and gamma interferon. Interestingly, blood samples from patients with recent-onset shigellosis showed variable but significant mucosal antibody responses to other conserved Shigella protein antigens but not to PSSP-1. We suggest that PSSP-1 is a promising antigen for a broadly protective vaccine against Shigella. PMID:25651919

  19. Protective host immune responses to Salmonella infection.

    PubMed

    Pham, Oanh H; McSorley, Stephen J

    2015-01-01

    Salmonella enterica serovars Typhi and Paratyphi are the causative agents of human typhoid fever. Current typhoid vaccines are ineffective and are not widely used in endemic areas. Greater understanding of host-pathogen interactions during Salmonella infection should facilitate the development of improved vaccines to combat typhoid and nontyphoidal Salmonellosis. This review will focus on our current understanding of Salmonella pathogenesis and the major host immune components that participate in immunity to Salmonella infection. In addition, recent findings regarding host immune mechanisms in response to Salmonella infection will be also discussed, providing a new perspective on the utility of improved tools to study the immune response to Salmonella infections.

  20. Protective host immune responses to Salmonella infection

    PubMed Central

    Pham, Oanh H; McSorley, Stephen J.

    2015-01-01

    Salmonella enterica serovars Typhi and Paratyphi are the causative agents of human typhoid fever. Current typhoid vaccines are ineffective and are not widely used in endemic areas. Greater understanding of host–pathogen interactions during Salmonella infection should facilitate the development of improved vaccines to combat typhoid and nontyphoidal Salmonellosis. This review will focus on our current understanding of Salmonella pathogenesis and the major host immune components that participate in immunity to Salmonella infection. In addition, recent findings regarding host immune mechanisms in response to Salmonella infection will be also discussed, providing a new perspective on the utility of improved tools to study the immune response to Salmonella infections. PMID:25598340

  1. ABC proteins protect the human body and maintain optimal health.

    PubMed

    Ueda, Kazumitsu

    2011-01-01

    Human MDR1, a multi-drug transporter gene, was isolated as the first of the eukaryote ATP Binding Cassette (ABC) proteins from a multidrug-resistant carcinoma cell line in 1986. To date, over 25 years, many ABC proteins have been found to play important physiological roles by transporting hydrophobic compounds. Defects in their functions cause various diseases, indicating that endogenous hydrophobic compounds, as well as water-soluble compounds, are properly transported by transmembrane proteins. MDR1 transports a large number of structurally unrelated drugs and is involved in their pharmacokinetics, and thus is a key factor in drug interaction. ABCA1, an ABC protein, eliminates excess cholesterol in peripheral cells by generating HDL. Because ABCA1 is a key molecule in cholesterol homeostasis, its function and expression are highly regulated. Eukaryote ABC proteins function on the body surface facing the outside and in organ pathways to adapt to the extracellular environment and protect the body to maintain optimal health.

  2. Theoretical tests of the mechanical protection strategy in protein nanomechanics.

    PubMed

    Chwastyk, Mateusz; Galera-Prat, Albert; Sikora, Mateusz; Gómez-Sicilia, Àngel; Carrión-Vázquez, Mariano; Cieplak, Marek

    2014-05-01

    We provide theoretical tests of a novel experimental technique to determine mechanostability of proteins based on stretching a mechanically protected protein by single-molecule force spectroscopy. This technique involves stretching a homogeneous or heterogeneous chain of reference proteins (single-molecule markers) in which one of them acts as host to the guest protein under study. The guest protein is grafted into the host through genetic engineering. It is expected that unraveling of the host precedes the unraveling of the guest removing ambiguities in the reading of the force-extension patterns of the guest protein. We study examples of such systems within a coarse-grained structure-based model. We consider systems with various ratios of mechanostability for the host and guest molecules and compare them to experimental results involving cohesin I as the guest molecule. For a comparison, we also study the force-displacement patterns in proteins that are linked in a serial fashion. We find that the mechanostability of the guest is similar to that of the isolated or serially linked protein. We also demonstrate that the ideal configuration of this strategy would be one in which the host is much more mechanostable than the single-molecule markers. We finally show that it is troublesome to use the highly stable cystine knot proteins as a host to graft a guest in stretching studies because this would involve a cleaving procedure. Copyright © 2014 Wiley Periodicals, Inc.

  3. Melatonin enhances thermotolerance by promoting cellular protein protection in tomato plants.

    PubMed

    Xu, Wen; Cai, Shu-Yu; Zhang, Yun; Wang, Yu; Ahammed, Golam Jalal; Xia, Xiao-Jian; Shi, Kai; Zhou, Yan-Hong; Yu, Jing-Quan; Reiter, Russel J; Zhou, Jie

    2016-11-01

    Melatonin is a pleiotropic signaling molecule that provides physiological protection against diverse environmental stresses in plants. Nonetheless, the mechanisms for melatonin-mediated thermotolerance remain largely unknown. Here, we report that endogenous melatonin levels increased with a rise in ambient temperature and that peaked at 40°C. Foliar pretreatment with an optimal dose of melatonin (10 μmol/L) or the overexpression of N-acetylserotonin methyltransferase (ASMT) gene effectively ameliorated heat-induced photoinhibition and electrolyte leakage in tomato plants. Both exogenous melatonin treatment and endogenous melatonin manipulation by overexpression of ASMT decreased the levels of insoluble and ubiquitinated proteins, but enhanced the expression of heat-shock proteins (HSPs) to refold denatured and unfolded proteins under heat stress. Meanwhile, melatonin also induced expression of several ATG genes and formation of autophagosomes to degrade aggregated proteins under the same stress. Proteomic profile analyses revealed that protein aggregates for a large number of biological processes accumulated in wild-type plants. However, exogenous melatonin treatment or overexpression of ASMT reduced the accumulation of aggregated proteins. Aggregation responsive proteins such as HSP70 and Rubisco activase were preferentially accumulated and ubiquitinated in wild-type plants under heat stress, while melatonin mitigated heat stress-induced accumulation and ubiquitination of aggregated proteins. These results suggest that melatonin promotes cellular protein protection through induction of HSPs and autophagy to refold or degrade denatured proteins under heat stress in tomato plants. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  4. [Protective immune response of guinea pigs against challenge with foot and mouth disease virus by immunization with foliar extracts from transgenic tomato plants expressing the FMDV structural protein VP1].

    PubMed

    Pan, Li; Zhang, Yong-Guang; Wang, Yong-Lu; Wang, Bao-Qin; Xie, Qing-Ge

    2006-10-01

    The plant constitutive expression vector pBin438/VP1 for VP1 gene of foot-and-mouth disease virus strain O/ China/99 was constructed. Mediated with Agrobacterium tumefaciens GV3101 harboring pBin438/VP1, VP1 gene was transferred into cotyledons of tomato. After selected by Kanamysin, sixty resistant lines were obtained. The integration and transcription of the VP1 gene in transformed plants was detected by PCR and RT-PCR. After being detected by sandwich-ELISA assays, about 40% transformed plants confirmed to express the recombinant protein. The leave extracts of two positive lines were respectively emulsified in Freund's adjuvant and guinea pigs were intramuscular inoculation at days 0, 15 and 30d. According to the sera antibody levels and the protection of the vaccinated guinea pigs against challenge with 100ID50 FMDV, probed into the immunogenicity of the target protein expressed in transgenic plants. Experimental results showed that the plant expression vector was successfully constructed. PCR and RT-PCR analyses confirmed VP1 gene was transformed into tomato plants and got expression at the transcription levels. The expressed VP1 protein of FMDV, which was identified by ELISA and Western blot, can be specifically recognized by polyclonal antibodies against FMDV. Indirect-ELISA antibody titers reached 1:64 twenty-one days after the third inoculation. In the challenge test, the protection against FMDV challenge in two groups was 80% and 40% respectively. The immunization test in guinea pigs indicated that the expression product of transgenic tomato plants had immunogenicity and could effectively induce the specific antibodies against FMDV.

  5. Cross-linking of anaplasma marginale outer membrane proteins enhances immunogenicity, but is not required for protection from challenge

    USDA-ARS?s Scientific Manuscript database

    Bacterial outer membrane proteins are the primary targets of a protective immune response. The specific characteristics of outer membrane-based immunogens, in terms of antigen content and context that are required for protective immunity remain unknown for a wide variety of bacterial pathogens. Usin...

  6. Identification of a protective protein from stationary-phase exoproteome of Brucella abortus.

    PubMed

    Jain, Shikha; Kumar, Subodh; Dohre, Sudhir; Afley, Prachiti; Sengupta, Nabonita; Alam, Syed I

    2014-02-01

    Brucellosis is a worldwide zoonotic disease. No Brucella vaccine is available for use in humans, and existing animal vaccines have limitations. To search the putative vaccine candidates, we studied the exoproteome of Brucella abortus NCTC 10093 using 2-DE-MS approach. Twenty-six proteins were identified using MALDI-TOF/TOF tandem mass spectrometry. Outer membrane protein 25, d-galactose periplasmic-binding protein, oligopeptide ABC transporter protein and isopropylmalate synthase were found to be the most abundant proteins. Most proteins (6, 23%) were predicted to be involved in amino acid transport and metabolism followed by carbohydrate transport and metabolism (4, 15%). Outer membrane protein 25, Omp2b porin and one hypothetical protein were predicted as outer membrane proteins. In addition, Omp28, Omp31 and one ribosomal protein (L9) were also identified. The ribosomal protein L9 was produced as a recombinant protein and was studied in mouse model for vaccine potential. It was found to be immunogenic in terms of generating serum antibody response and release of IFN-γ from mice spleen cells. Recombinant L9-immunized mice were protected against challenge with virulent B. abortus strain 544, suggesting usefulness of ribosomal protein L9 as a good vaccine candidate against brucellosis.

  7. Immunogenicity and protective efficacy of Clostridium difficile spore proteins.

    PubMed

    Ghose, Chandrabali; Eugenis, Ioannis; Edwards, Adrianne N; Sun, Xingmin; McBride, Shonna M; Ho, David D

    2016-02-01

    Clostridium difficile is a spore-forming, anaerobic, Gram-positive organism that is the leading cause of antibiotic-associated infectious diarrhea, commonly known as C. difficile infection (CDI). C. difficile spores play an important role in the pathogenesis of CDI. Spore proteins, especially those that are surface-bound may play an essential role in the germination, colonization and persistence of C. difficile in the human gut. In our current study, we report the identification of two surface-bound spore proteins, CdeC and CdeM that may be utilized as immunization candidates against C. difficile. These spore proteins are immunogenic in mice and are able to protect mice against challenge with C. difficile UK1, a clinically-relevant 027/B1/NAP1 strain. These spore proteins are also able to afford high levels of protection against challenge with C. difficile 630Δerm in golden Syrian hamsters. This unprecedented study shows the vaccination potential of C. difficile spore exosporium proteins.

  8. Transduced Tat-SAG fusion protein protects against oxidative stress and brain ischemic insult.

    PubMed

    Kim, Dae Won; Lee, Sun Hwa; Jeong, Min Seop; Sohn, Eun Jeong; Kim, Mi Jin; Jeong, Hoon Jae; An, Jae Jin; Jang, Sang Ho; Won, Moo Ho; Hwang, In Koo; Cho, Sung-Woo; Kang, Tae-Cheon; Lee, Kil Soo; Park, Jinseu; Yoo, Ki-Yeon; Eum, Won Sik; Choi, Soo Young

    2010-04-01

    Reactive oxygen species (ROS) have been implicated in the pathogenesis of ischemic brain injury. Sensitive to apoptosis gene (SAG) is a RING-finger protein that exhibits antioxidant activity against a variety of redox reagents. However, the protective effect of SAG in brain ischemic injury is unclear. Here, we investigated the protective effects of a Tat-SAG fusion protein against cell death and ischemic insult. When Tat-SAG fusion protein was added to the culture medium of astrocytes, it rapidly entered the cells and protected them against oxidative stress-induced cell death. Immunohistochemical analysis revealed that, when Tat-SAG fusion protein was intraperitoneally injected into gerbils, wild-type Tat-SAG prevented neuronal cell death in the CA1 region of the hippocampus in response to transient forebrain ischemia. In addition, wild-type Tat-SAG fusion protein decreased lipid peroxidation in the brain compared with mutant Tat-SAG- or vehicle-treated animals. Our results demonstrate that Tat-SAG fusion protein is a tool for the treatment of ischemic insult and it can be used in protein therapy for various disorders related to ROS, including stroke.

  9. The Nrf2–Keap1 cellular defense pathway and heat shock protein 70 (Hsp70) response. Role in protection against oxidative stress in early neonatal unilateral ureteral obstruction (UUO)

    PubMed Central

    Rinaldi Tosi, Martin E.; Bocanegra, Victoria; Manucha, Walter; Gil Lorenzo, Andrea

    2010-01-01

    Perturbation of renal tubular antioxidants and overproduction of reactive oxygen species may amplify the proinflammatory state of renal obstruction, culminating in oxidative stress and tubular loss. Here, we analyzed the heat shock protein 70 (Hsp70) response and the function and signal transduction of NF-E2-related protein 2 (Nrf2) transcription factor on oxidative stress modulation in obstruction. Rats were subjected to unilateral ureteral obstruction or sham operation and kidneys harvested at 5, 7, 10, and 14 days after obstruction. Hsp70 expression and Nrf2 activity and its downstream target gene products were assessed. After 10 and 14 days of obstruction, enhanced lipid peroxidation through higher thiobarbituric acid reactive substances levels and increased oxidative stress resulted in reduced total antioxidant activity and enhanced nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase activity were demonstrated. This was accompanied by decreased inducible Hsp70 expression and a progressive reduction of nuclear Nrf2 and its target gene products glutathione S-transferase A2 (GSTA2) and NADPH/quinone oxidoreductase 1 (NQO1), whereas the Nrf2 repressor Kelch-like ECH-associated protein-1 (Keap1) was upregulated. By contrast, on early obstruction for 7 days, lack of increased oxidative markers associated with higher inducible Hsp70 protein levels and a rapid nuclear accumulation of Nrf2, Keap1 downregulation, and mRNA induction of the identified Nrf2-dependent genes, NQO1 and GSTA2, were shown. For these results, we suggest that the magnitude of cytoprotection in early obstruction depends on the combined contribution of induced activation of Nrf2 upregulating its downstream gene products and Hsp70 response. Impaired ability to mount the biological response to the prevailing oxidative stress leading to renal injury was shown in prolonged obstruction. PMID:20734248

  10. Comparing Montanide ISA 720 and 50-V2 adjuvants formulated with LmSTI1 protein of Leishmania major indicated the potential cytokine patterns for induction of protective immune responses in BALB/c mice.

    PubMed

    Shokri, Mahdi; Roohvand, Farzin; Alimohammadian, Mohammad Hossein; Ebrahimirad, Mina; Ajdary, Soheila

    2016-08-01

    Adjuvants have a key role in subunit vaccine formulations to generate protective immune responses. Herein, we present results of a comparative study on mice immunized with E. coli-derived rLmSTI1 antigen formulated with Montanide ISA 720 (Ag-M720) and ISA 50-V2 (Ag-M50) adjuvants against Leishmania major (L. major). Groups of BALB/c mice were immunized with either Ag-M720 or Ag-M50 by 3 subcutaneous injections with 3-week intervals. Three weeks after the last injection mice were challenged by L. major promastigotes. Immune responses were evaluated before, 3 weeks, and 8 weeks after challenge. Results indicated lower parasite and lesion size in vaccinated mice (the lowest for Ag-M720 indicating the best protection) which correlated with higher IFN-γ induction in immunized groups (Ag-M720 and Ag-M50) compared to control (PBS/adjuvant alone) group. Immune assays showed comparable IFN-γ, total IgG, IgG1 and IgG2a levels for Ag-M720 and Ag-M50 immunized mice but higher induction of IL-4, IL-10 and IL-17 in Ag-M50 and the highest IL-10/IL-17 ratio in Ag-M720 group followed by Ag-M50 and control groups. Altogether, results indicated that lower induction of IL-4, IL-10 and IL-17 cytokines (and/or higher ratio of IL-10/IL-17) despite comparable IFN-γ might be the reason for the superior protection in Ag-M720 group. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Protective efficacy of oral immunization with heat-killed Shigella flexneri 2a in animal model: study of cross protection, immune response and antigenic recognition.

    PubMed

    Mukhopadhaya, A; Mahalanabis, D; Khanam, J; Chakrabarti, M K

    2003-06-20

    Oral immunization of rabbits with four doses of 10(11) heat-killed Shigella flexneri 2a showed 100% protection against challenge with virulent S. flexneri 2a. After orally immunizing Guinea pigs with four doses of heat-killed S. flexneri 2a 100% protection could be shown against ocular challenge with the same virulent S. flexneri 2a strain but this conferred no protection against challenge with Shigella dysenteriae type 1. In enzyme-linked immunosorbent assay (ELISA) and immunoblot experiments both whole cell lysate-envelope (WCL-E) fraction and outer membrane proteins (OMPs) were recognized by the antisera. Though protective mechanism in shigellosis is not established with certainty, outer membrane proteins (specially 38, 34, 23 and 20kDa proteins) may be the major antigens in the induction of protective immune responses as indicated by this observation.

  12. Hybrid cluster proteins and flavodiiron proteins afford protection to Desulfovibrio vulgaris upon macrophage infection.

    PubMed

    Figueiredo, Mafalda C O; Lobo, Susana A L; Sousa, Sara H; Pereira, Fábio P; Wall, Judy D; Nobre, Lígia S; Saraiva, Lígia M

    2013-06-01

    Desulfovibrio species are Gram-negative anaerobic sulfate-reducing bacteria that colonize the human gut. Recently, Desulfovibrio spp. have been implicated in gastrointestinal diseases and shown to stimulate the epithelial immune response, leading to increased production of inflammatory cytokines by macrophages. Activated macrophages are key cells of the immune system that impose nitrosative stress during phagocytosis. Hence, we have analyzed the in vitro and in vivo responses of Desulfovibrio vulgaris Hildenborough to nitric oxide (NO) and the role of the hybrid cluster proteins (HCP1 and HCP2) and rubredoxin oxygen oxidoreductases (ROO1 and ROO2) in NO protection. Among the four genes, hcp2 was the gene most highly induced by NO, and the hcp2 transposon mutant exhibited the lowest viability under conditions of NO stress. Studies in murine macrophages revealed that D. vulgaris survives incubation with these phagocytes and triggers NO production at levels similar to those stimulated by the cytokine gamma interferon (IFN-γ). Furthermore, D. vulgaris hcp and roo mutants exhibited reduced viability when incubated with macrophages, revealing that these gene products contribute to the survival of D. vulgaris during macrophage infection.

  13. Role of Humoral versus Cellular Responses Induced by a Protective Dengue Vaccine Candidate

    PubMed Central

    Zellweger, Raphaël M.; Miller, Robyn; Eddy, William E.; White, Laura J.; Johnston, Robert E.; Shresta, Sujan

    2013-01-01

    With 2.5 billion people at risk, dengue is a major emerging disease threat and an escalating public health problem worldwide. Dengue virus causes disease ranging from a self-limiting febrile illness (dengue fever) to the potentially fatal dengue hemorrhagic fever/dengue shock syndrome. Severe dengue disease is associated with sub-protective levels of antibody, which exacerbate disease upon re-infection. A dengue vaccine should generate protective immunity without increasing severity of disease. To date, the determinants of vaccine-mediated protection against dengue remain unclear, and additional correlates of protection are urgently needed. Here, mice were immunized with viral replicon particles expressing the dengue envelope protein ectodomain to assess the relative contribution of humoral versus cellular immunity to protection. Vaccination with viral replicon particles provided robust protection against dengue challenge. Vaccine-induced humoral responses had the potential to either protect from or exacerbate dengue disease upon challenge, whereas cellular immune responses were beneficial. This study explores the immunological basis of protection induced by a dengue vaccine and suggests that a safe and efficient vaccine against dengue should trigger both arms of the immune system. PMID:24204271

  14. Structure-based design of broadly protective group a streptococcal M protein-based vaccines.

    PubMed

    Dale, James B; Smeesters, Pierre R; Courtney, Harry S; Penfound, Thomas A; Hohn, Claudia M; Smith, Jeremy C; Baudry, Jerome Y

    2017-01-03

    A major obstacle to the development of broadly protective M protein-based group A streptococcal (GAS) vaccines is the variability within the N-terminal epitopes that evoke potent bactericidal antibodies. The concept of M type-specific protective immune responses has recently been challenged based on the observation that multivalent M protein vaccines elicited cross-reactive bactericidal antibodies against a number of non-vaccine M types of GAS. Additionally, a new "cluster-based" typing system of 175M proteins identified a limited number of clusters containing closely related M proteins. In the current study, we used the emm cluster typing system, in combination with computational structure-based peptide modeling, as a novel approach to the design of potentially broadly protective M protein-based vaccines. M protein sequences (AA 16-50) from the E4 cluster containing 17 emm types of GAS were analyzed using de novo 3-D structure prediction tools and the resulting structures subjected to chemical diversity analysis to identify sequences that were the most representative of the 3-D physicochemical properties of the M peptides in the cluster. Five peptides that spanned the range of physicochemical attributes of all 17 peptides were used to formulate synthetic and recombinant vaccines. Rabbit antisera were assayed for antibodies that cross-reacted with E4 peptides and whole bacteria by ELISA and for bactericidal activity against all E4GAS. The synthetic vaccine rabbit antisera reacted with all 17 E4M peptides and demonstrated bactericidal activity against 15/17 E4GAS. A recombinant hybrid vaccine containing the same E4 peptides also elicited antibodies that cross-reacted with all E4M peptides. Comprehensive studies using structure-based design may result in a broadly protective M peptide vaccine that will elicit cluster-specific and emm type-specific antibody responses against the majority of clinically relevant emm types of GAS. Copyright © 2016 Elsevier Ltd. All

  15. The specificity of protection against cationic antimicrobial peptides by lactoferrin binding protein B.

    PubMed

    Morgenthau, Ari; Partha, Sarathy K; Adamiak, Paul; Schryvers, Anthony B

    2014-10-01

    A variety of Gram-negative pathogens possess host-specific lactoferrin (Lf) receptors that mediate the acquisition of iron from host Lf. The integral membrane protein component of the receptor, lactoferrin binding protein A specifically binds host Lf and is required for acquisition of iron from Lf. In contrast, the role of the bi-lobed surface lipoprotein, lactoferrin binding protein B (LbpB), in Lf binding and iron acquisition is uncertain. A common feature of LbpBs from most species is the presence of clusters of negatively charged amino acids in the protein's C-terminal lobe. Recently it has been shown that the negatively charged regions from the Neisseria meningitidis LbpB are responsible for protecting against an 11 amino acid cationic antimicrobial peptide (CAP), lactoferricin (Lfcin), derived from human Lf. In this study we investigated whether the LbpB confers resistance to other CAPs since N. meningitidis is likely to encounter other CAPs from the host. LbpB provided protection against the cathelicidin derived peptide, cathelicidin related antimicrobial peptide (mCRAMP), but did not confer protection against Tritrp 1 or LL37 under our experimental conditions. When tested against a range of rationally designed synthetic peptides, LbpB was shown to protect against IDR-1002 and IDR-0018 but not against HH-2 or HHC10.

  16. Brain responses to high-protein diets.

    PubMed

    Journel, Marion; Chaumontet, Catherine; Darcel, Nicolas; Fromentin, Gilles; Tomé, Daniel

    2012-05-01

    Proteins are suspected to have a greater satiating effect than the other 2 macronutrients. After protein consumption, peptide hormones released from the gastrointestinal tract (mainly anorexigenic gut peptides such as cholecystokinin, glucagon peptide 1, and peptide YY) communicate information about the energy status to the brain. These hormones and vagal afferents control food intake by acting on brain regions involved in energy homeostasis such as the brainstem and the hypothalamus. In fact, a high-protein diet leads to greater activation than a normal-protein diet in the nucleus tractus solitarius and in the arcuate nucleus. More specifically, neural mechanisms triggered particularly by leucine consumption involve 2 cellular energy sensors: the mammalian target of rapamycin and AMP-activated protein kinase. In addition, reward and motivation aspects of eating behavior, controlled mainly by neurons present in limbic regions, play an important role in the reduced hedonic response of a high-protein diet. This review examines how metabolic signals emanating from the gastrointestinal tract after protein ingestion target the brain to control feeding, energy expenditure, and hormones. Understanding the functional roles of brain areas involved in the satiating effect of proteins and their interactions will demonstrate how homeostasis and reward are integrated with the signals from peripheral organs after protein consumption.

  17. Prostate cancer and the unfolded protein response

    PubMed Central

    Arnoldussen, Yke Jildouw; Saatcioglu, Fahri

    2016-01-01

    The endoplasmic reticulum (ER) is an essential organelle that contributes to several key cellular functions, including lipogenesis, gluconeogenesis, calcium storage, and organelle biogenesis. The ER also serves as the major site for protein folding and trafficking, especially in specialized secretory cells. Accumulation of misfolded proteins and failure of ER adaptive capacity activates the unfolded protein response (UPR) which has been implicated in several chronic diseases, including cancer. A number of recent studies have implicated UPR in prostate cancer (PCa) and greatly expanded our understanding of this key stress signaling pathway and its regulation in PCa. Here we summarize these developments and discuss their potential therapeutic implications. PMID:27303918

  18. Artemin protects cells and proteins against oxidative and salt stress.

    PubMed

    Takalloo, Zeinab; Sajedi, Reza H; Hosseinkhani, Saman; Moazzenzade, Taghi

    2017-02-01

    Artemin is an abundant thermostable protein in Artemia encysted embryos under environmental stresses. It is confirmed that high regulatory expression of artemin is relevant to stress resistance in this crustacean. Here, the protective role of artemin from Artemia urmiana has been investigated on survival of bacterial cells under salt and oxidative shocks. Also, for continuous monitoring of the effect of artemin in prevention of proteins aggregation/inactivation, co-expression of artemin and luciferase (as an intracellular reporter) in bacterial cells was performed. According to the results, residual activity of luciferase in artemin expressing E. coli cells exposing to different concentrations of H2O2 and NaCl was significantly higher than non-expressing cells. The luciferase activity was rapidly lost in control cells under salt treatments while in co-transformed cells, the activity was considerably retained at higher salt concentrations. Also, analysis from cell viability assays showed that artemin-expressing cells exhibited more resistance to both stress conditions. In the present study, we document for the first time that artemin can protect proteins and bacterial cells against oxidative and salt stress conditions. These results can declare the resistance property of this crustacean against harsh environmental conditions.

  19. Immunisation with proteins expressed during chronic murine melioidosis provides enhanced protection against disease.

    PubMed

    Champion, Olivia L; Gourlay, Louise J; Scott, Andrew E; Lassaux, Patricia; Conejero, Laura; Perletti, Lucia; Hemsley, Claudia; Prior, Joann; Bancroft, Gregory; Bolognesi, Martino; Titball, Richard W

    2016-03-29

    There is an urgent need for an effective vaccine against human disease caused by Burkholderia pseudomallei, and although a wide range of candidates have been tested in mice none provide high level protection. We considered this might reflect the inability of these vaccine candidates to protect against chronic disease. Using Q-RT PCR we have identified 6 genes which are expressed in bacteria colonising spleens and lungs of chronically infected mice. Three of the genes (BPSL1897, BPSL3369 and BPSL2287) have been expressed in Escherichia coli and the encoded proteins purified. We have also included BPSL2765, a protein known to induce immune responses associated with a reduced incidence of chronic/recurrent disease in humans. Immunisation of mice with a combination of these antigens resulted in the induction of antibody responses against all of the proteins. Compared with mice immunised with capsular polysaccharide or LolC protein, mice immunised with the combination of chronic stage antigens showed enhanced protection against experimental disease in mice. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Interferon responses in HIV infection: from protection to disease.

    PubMed

    Sivro, Aida; Su, Ruey-Chyi; Plummer, Francis A; Ball, T Blake

    2014-01-01

    Interferons, induced early during viral infections, represent important regulators of both innate and adaptive immune responses, and provide protective effects against a wide range of pathogens, including HIV. Several in vitro studies and some in vivo data from HIV-exposed seronegative cohorts indicate that interferons and interferon-mediated immune responses are crucial in preventing early HIV replication. Following establishment of HIV infection, the uncontrolled (aberrant) activation of the immune system, in part regulated by interferon levels, contributes to HIV-1-induced immune activation and disease progression. Modulation of interferon responses prior to and during HIV infection shows promise for development of novel therapeutics to prevent HIV transmission, clear HIV infection, and dampen chronic immune activation. In this review we discuss the role that interferons play in protection from HIV infection, acute infection, and their role in HIV pathogenesis and disease progression. Lastly, we review recent advances in modulating interferon responses for purposes of developing novel HIV therapeutic approaches.

  1. Cellular unfolded protein response against viruses used in gene therapy

    PubMed Central

    Sen, Dwaipayan; Balakrishnan, Balaji; Jayandharan, Giridhara R.

    2014-01-01

    Viruses are excellent vehicles for gene therapy due to their natural ability to infect and deliver the cargo to specific tissues with high efficiency. Although such vectors are usually “gutted” and are replication defective, they are subjected to clearance by the host cells by immune recognition and destruction. Unfolded protein response (UPR) is a naturally evolved cyto-protective signaling pathway which is triggered due to endoplasmic reticulum (ER) stress caused by accumulation of unfolded/misfolded proteins in its lumen. The UPR signaling consists of three signaling pathways, namely PKR-like ER kinase, activating transcription factor 6, and inositol-requiring protein-1. Once activated, UPR triggers the production of ER molecular chaperones and stress response proteins to help reduce the protein load within the ER. This occurs by degradation of the misfolded proteins and ensues in the arrest of protein translation machinery. If the burden of protein load in ER is beyond its processing capacity, UPR can activate pro-apoptotic pathways or autophagy leading to cell death. Viruses are naturally evolved in hijacking the host cellular translation machinery to generate a large amount of proteins. This phenomenon disrupts ER homeostasis and leads to ER stress. Alternatively, in the case of gutted vectors used in gene therapy, the excess load of recombinant vectors administered and encountered by the cell can trigger UPR. Thus, in the context of gene therapy, UPR becomes a major roadblock that can potentially trigger inflammatory responses against the vectors and reduce the efficiency of gene transfer. PMID:24904562

  2. The larval specific lymphatic filarial ALT-2: induction of protection using protein or DNA vaccination.

    PubMed

    Ramachandran, Sabarinathan; Kumar, Mishra Pankaj; Rami, Reddy Maryada Venkata; Chinnaiah, Harinath Basker; Nutman, Thomas; Kaliraj, Perumal; McCarthy, James

    2004-01-01

    Genes from the infective stage of lymphatic filarial parasites expressed at the time of host invasion have been identified as potential vaccine candidates. By screening an L3 cDNA library with sera from uninfected longstanding residents of an area endemic for onchocerciasis, so-called "endemic normals" (EN), we have cloned and characterized one such gene termed the abundant larval transcript two (ALT-2). The stage specificity of ALT-2 gene transcription and protein synthesis was confirmed by PCR using genespecific primers, and by western blot analysis of protein extracts from various stages of the parasite life cycle using specific antisera. Significant differences in antibody response to the recombinant ALT-2 were observed in endemic populations with differing clinical manifestations of lymphatic filariasis with an antibody response present in sera from 18 of 25 (72%) EN subjects compared to 9 of 25 (36%) with subclinical microfilaracmia (MF) and 14 of 25 (52%) of those with chronic lymphatic obstruction (CP) (P=0.01 for comparison of EN to CP or to MF). This differential responsiveness suggests that the protective immunity postulated to account for their uninfected status might be associated with a response to this protein. When the utility of ALT-2 as a vaccine candidate was tested in a murine model using either recombinant protein or a DNA vaccine construct, statistically significant protection was observed when compared to a control filarial gene product expressed across all stages of the parasite lifecycle (SXP-1; P=0.02 for protein and P=0.01 for the DNA vaccine) or compared to adjuvant alone. This level of protection indicates that this vaccine is a promising candidate for further development.

  3. Chlamydia trachomatis: Protective Adaptive Responses and Prospects for a Vaccine.

    PubMed

    Poston, Taylor B; Darville, Toni

    2016-04-01

    Chlamydia trachomatis is the most common cause of sexually transmitted bacterial infection globally. These infections translate to a significant public health burden, particularly women's healthcare costs due to serious disease sequelae such as pelvic inflammatory disease (PID), tubal factor infertility, chronic pelvic pain, and ectopic pregnancy. There is no evidence that natural immunity can provide complete, long-term protection necessary to prevent chronic pathology, making human vaccine development critical. Vaccine design will require careful consideration of protective versus pathological host-response mechanisms in concert with elucidation of optimal antigens and adjuvants. Evidence suggests that a Th1 response, facilitated by IFN-γ-producing CD4 T cells, will be instrumental in generating long-term, sterilizing immunity. Although the role of antibodies is not completely understood, they have exhibited a protective effect by enhancing chlamydial clearance. Future work will require investigation of broadly neutralizing antibodies and antibody-augmented cellular immunity to successfully design a vaccine that potently elicits both arms of the immune response. Sterilizing immunity is the ultimate goal. However, vaccine-induced partial immunity that prevents upper genital tract infection and inflammation would be cost-effective compared to current screening and treatment strategies. In this chapter, we examine evidence from animal and human studies demonstrating protective adaptive immune responses to Chlamydia and discuss future challenges and prospects for vaccine development.

  4. Vitamin D binding protein polymorphism protects against development of blastomycosis.

    PubMed

    Sainsbury, J P; Trajtman, A; Stalker, A T; Embil, J M; Keynan, Y

    2014-12-01

    Blastomycosis is an uncommon endemic fungal infection. It is presumed that in the endemic regions, the number of exposed individuals is significantly greater than those in whom clinical manifestations develop. We conducted a case-control study of individuals with clinical blastomycosis and controls with similar exposure but who did not develop disease. A genetic association was observed between the Gc-2 allele of vitamin D binding protein and reduced susceptibility to blastomycosis in a Canadian cohort. The Gc-2 allele can affect increased antimicrobial activity of macrophages. It may be possible to mimic this mechanism of protection by vitamin D supplementation.

  5. Characterization of culture supernatant proteins from Brucella abortus and its protection effects against murine brucellosis.

    PubMed

    Lee, Jin Ju; Lim, Jeong Ju; Kim, Dae Geun; Simborio, Hannah Leah; Kim, Dong Hyeok; Reyes, Alisha Wehdnesday Bernardo; Min, WonGi; Lee, Hu Jang; Kim, Dong Hee; Chang, Hong Hee; Kim, Suk

    2014-09-01

    In this study, we characterized the secreted proteins of Brucella abortus into the enriched media under the bacterial laboratory growth condition and investigated the pathogenic importance of culture supernatant (CS) proteins to B. abortus infection. CS proteins from stationary phase were concentrated and analyzed using 2D electrophoresis. In MALDI TOF/TOF analysis, more than 27 proteins including CuZn SOD, Dps, Tat, OMPs, Adh, LivF, Tuf, SucC, GroEL and DnaK were identified. Cytotoxic effects of CS proteins were found to increase in a dose-dependent manner in RAW 264.7 cells. Upon B. abortus challenge into phagocytes, however, CS proteins pre-treated cells exhibited lower bacterial uptake and intracellular replication compared to untreated cells. Immunization with CS proteins induced a strong humoral and cell mediated immune responses and exhibited significant higher degree of protection against virulence of B. abortus infection compared to mice immunized with Brucella broth protein (BBP). Taken together, these results indicate that B. abortus secreted a number of soluble immunogenic proteins under laboratory culture condition, which can promote antibody production resulted in enhancing host defense against to subsequently bacterial infection. Moreover, further analysis of CS proteins may help to understand the pathogenic mechanism of B. abortus infection and host-pathogen interaction.

  6. Conformational Nature of the Borrelia burgdorferi Decorin Binding Protein A Epitopes That Elicit Protective Antibodies

    PubMed Central

    Ulbrandt, Nancy D.; Cassatt, David R.; Patel, Nita K.; Roberts, William C.; Bachy, Christine M.; Fazenbaker, Christine A.; Hanson, Mark S.

    2001-01-01

    Decorin binding protein A (DbpA) has been shown by several laboratories to be a protective antigen for the prevention of experimental Borrelia burgdorferi infection in the mouse model of Lyme borreliosis. However, different recombinant forms of the antigen having either lipidated amino termini, approximating the natural secretion and posttranslational processing, or nonprocessed cytosolic forms have elicited disparate levels of protection in the mouse model. We have now used the unique functional properties of this molecule to investigate the structural requirements needed to elicit a protective immune response. Genetic and physicochemical alterations to DbpA showed that the ability to bind to the ligand decorin is indicative of a potent immunogen but is not conclusive. By mutating the two carboxy-terminal nonconserved cysteines of DbpA from B. burgdorferi strain N40, we have determined that the stability afforded by the putative disulfide bond is essential for the generation of protective antibodies. This mutated protein was more sensitive to thermal denaturation and proteolysis, suggesting that it is in a less ordered state. Immunization with DbpA that was thermally denatured and functionally inactivated stimulated an immune response that was not protective and lacked bactericidal antibodies. Antibodies against conformationally altered forms of DbpA also failed to kill heterologous B. garinii and B. afzelii strains. Additionally, nonsecreted recombinant forms of DbpAN40 were found to be inferior to secreted lipoprotein DbpAN40 in terms of functional activity and antigenic potency. These data suggest that elicitation of a bactericidal and protective immune response to DbpA requires a properly folded conformation for the production of functional antibodies. PMID:11447153

  7. Enhanced Protective Immunogenicity of Homodimeric Borrelia burgdorferi Outer Surface Protein C

    PubMed Central

    Edmondson, Diane G.; Prabhakaran, Sabitha; Ullmann, Amy J.; Piesman, Joe; Dolan, Marc; Probst, Christian; Radzimski, Christiane; Stöcker, Winfried

    2016-01-01

    ABSTRACT Lyme borreliosis is caused by tick-transmitted spirochetes of the Borrelia burgdorferi sensu lato group and is the most common vector-borne disease in the United States and Europe. Outer surface protein C (OspC) is a 23-kDa outer surface lipoprotein expressed during spirochete transmission from the tick to the vertebrate host. In a previous study, we found that immunization with a recombinant disulfide-bridged dimeric form of OspC (D-OspC) stimulates increased antibody responses relative to immunization with commonly employed monomeric OspC. Here, we report that mice immunized with dimeric OspC proteins also exhibited enhanced protection against infection with the cognate B. burgdorferi strain. Mice were protected by four immunizations containing as little as 100 ng of dimeric OspC, suggesting that this form of the protein can induce protective immunity within a dose range reasonable for a human or veterinary vaccine. In contrast, monomeric OspC was only partially protective at much higher doses. IgG subclass analysis revealed that D-OspC-immunized animals mainly possessed anti-OspC-IgG1. In contrast, infected animals develop anti-OspC restricted to the IgG3 isotype. A subset of antibodies generated by dimeric OspC immunization did not recognize the monomeric variant, indicating that unique epitopes exist on the dimeric form. Moreover, monoclonal antibodies that recognized only dimeric OspC protected mice from B. burgdorferi challenge, whereas another monoclonal that recognized both immunogens was not protective. These studies suggest that this dimeric OspC presents distinctive epitopes that generate antibodies protective against B. burgdorferi infection and could be a useful vaccine component. PMID:27733423

  8. Functional Analysis of Insect Molting Fluid Proteins on the Protection and Regulation of Ecdysis*

    PubMed Central

    Zhang, Jie; Lu, Anrui; Kong, Lulu; Zhang, Qiaoli; Ling, Erjun

    2014-01-01

    Molting fluid accumulates between the old and new cuticles during periodical ecdysis in Ecdysozoa. Natural defects in insect ecdysis are frequently associated with melanization (an immunity response) occurring primarily in molting fluids, suggesting that molting fluid may impact immunity as well as affect ecdysis. To address this hypothesis, proteomic analysis of molting fluids from Bombyx mori during three different types of ecdysis was performed. Many proteins were newly identified, including immunity-related proteins, in each molting fluid. Molting fluids inhibited the growth of bacteria in vitro. The entomopathogenic fungi Beauveria bassiana, which can escape immune responses in feeding larvae, is quickly recognized by larvae during ecdysis, followed by melanization in molting fluid and old cuticle. Fungal conidia germination was delayed, and no hyphae were detected in the hemocoels of pharate instar insects. Molting fluids protect the delicate pharate instar insects with extremely thin cuticles against microorganisms. To explore the function of molting fluids in ecdysis regulation, based on protein similarity, 32 genes were selected for analysis in ecdysis regulation through RNAi in Tribolium castaneum, a model commonly used to study integument development because RNAi is difficult to achieve in B. mori. We identified 24 molting proteins that affected ecdysis after knockdown, with different physiological functions, including old cuticle protein recycling, molting fluid pressure balance, detoxification, and signal detection and transfer of molting fluids. We report that insects secrete molting fluid for protection and regulation of ecdysis, which indicates a way to develop new pesticides through interrupting insect ecdysis in the future. PMID:25368323

  9. Functional analysis of insect molting fluid proteins on the protection and regulation of ecdysis.

    PubMed

    Zhang, Jie; Lu, Anrui; Kong, Lulu; Zhang, Qiaoli; Ling, Erjun

    2014-12-26

    Molting fluid accumulates between the old and new cuticles during periodical ecdysis in Ecdysozoa. Natural defects in insect ecdysis are frequently associated with melanization (an immunity response) occurring primarily in molting fluids, suggesting that molting fluid may impact immunity as well as affect ecdysis. To address this hypothesis, proteomic analysis of molting fluids from Bombyx mori during three different types of ecdysis was performed. Many proteins were newly identified, including immunity-related proteins, in each molting fluid. Molting fluids inhibited the growth of bacteria in vitro. The entomopathogenic fungi Beauveria bassiana, which can escape immune responses in feeding larvae, is quickly recognized by larvae during ecdysis, followed by melanization in molting fluid and old cuticle. Fungal conidia germination was delayed, and no hyphae were detected in the hemocoels of pharate instar insects. Molting fluids protect the delicate pharate instar insects with extremely thin cuticles against microorganisms. To explore the function of molting fluids in ecdysis regulation, based on protein similarity, 32 genes were selected for analysis in ecdysis regulation through RNAi in Tribolium castaneum, a model commonly used to study integument development because RNAi is difficult to achieve in B. mori. We identified 24 molting proteins that affected ecdysis after knockdown, with different physiological functions, including old cuticle protein recycling, molting fluid pressure balance, detoxification, and signal detection and transfer of molting fluids. We report that insects secrete molting fluid for protection and regulation of ecdysis, which indicates a way to develop new pesticides through interrupting insect ecdysis in the future.

  10. A thrombospondin-dependent pathway for a protective ER stress response

    PubMed Central

    Lynch, Jeffrey M.; Maillet, Marjorie; Vanhoutte, Davy; Schloemer, Aryn; Sargent, Michelle A.; Blair, N. Scott; Lynch, Kaari A.; Okada, Tetsuya; Aronow, Bruce J.; Osinska, Hanna; Prywes, Ron; Lorenz, John N.; Mori, Kazutoshi; Lawler, Jack; Robbins, Jeffrey; Molkentin, Jeffery D.

    2012-01-01

    SUMMARY Thrombospondin (Thbs) proteins are induced in sites of tissue damage or active remodeling. The endoplasmic reticulum (ER) stress response is also prominently induced with disease where it regulates protein production and resolution of misfolded proteins. Here we describe a novel function for Thbs’ as ER resident effectors of an adaptive ER stress response. Thbs4 cardiac-specific transgenic mice were protected from myocardial injury while Thbs4−/− mice were sensitized to cardiac maladaptation. Thbs induction produced a unique profile of adaptive ER stress response factors and expansion of the ER and downstream vesicles. The type-3 repeat domain in Thbs’ bind the ER luminal domain of activating transcription factor 6α (Atf6α) to promote its nuclear shuttling. Thbs4−/−mice failed to show activation of Atf6α and other ER stress response factors with injury, and Thbs4-mediated protection was lost when Atf6α was deleted. Hence, Thbs’ can function inside the cell during disease/remodeling to augment ER function and protect through a mechanism involving regulation of Atf6α. PMID:22682248

  11. C-reactive protein mediates protection from lipopolysaccharide through interactions with Fc gamma R.

    PubMed

    Mold, Carolyn; Rodriguez, Wilfredo; Rodic-Polic, Bojana; Du Clos, Terry W

    2002-12-15

    C-reactive protein (CRP) is a component of the acute phase response to infection, inflammation, and trauma. A major activity of acute phase proteins is to limit the inflammatory response. It has been demonstrated that CRP protects mice from lethal doses of LPS. In the mouse, CRP binds to the regulatory receptor, FcgammaRIIb, and to the gamma-chain-associated receptor, FcgammaRI. The goal ofthis study was to determine whether FcgammaRs are necessary for the protective effect of CRP. The ability of CRP to protect mice from a lethal dose of LPS was confirmed using injections of 500 and 250 micro g of CRP at 0 and 12 h. CRP treatment of FcgammaRIIb-deficient mice increased mortality after LPS challenge and increased serum levels of TNF and IL-12 in response to LPS. CRP did not protect FcR gamma-chain-deficient mice from LPS-induced mortality. Treatment of normal mice, but not gamma-chain-deficient mice, with CRP increased IL-10 levels following LPS injection. In vitro, in the presence of LPS, CRP enhanced IL-10 synthesis and inhibited IL-12 synthesis by bone marrow macrophages from normal, but not gamma-chain-deficient mice. The protective effect of CRP appears to be mediated by binding to FcgammaRI and FcgammaRII resulting in enhanced secretion of the anti-inflammatory cytokine IL-10 and the down-regulation of IL-12. These results suggest that CRP can alter the cytokine profile of mouse macrophages by acting through FcgammaR leading to a down-regulation of the inflammatory response.

  12. Drinking a hot blood meal elicits a protective heat shock response in mosquitoes.

    PubMed

    Benoit, Joshua B; Lopez-Martinez, Giancarlo; Patrick, Kevin R; Phillips, Zachary P; Krause, Tyler B; Denlinger, David L

    2011-05-10

    The mosquito's body temperature increases dramatically when it takes a blood meal from a warm-blooded, vertebrate host. By using the yellow fever mosquito, Aedes aegypti, we demonstrate that this boost in temperature following a blood meal prompts the synthesis of heat shock protein 70 (Hsp70). This response, elicited by the temperature of the blood meal, is most robust in the mosquito's midgut. When RNA interference is used to suppress expression of hsp70, protein digestion of the blood meal is impaired, leading to production of fewer eggs. We propose that Hsp70 protects the mosquito midgut from the temperature stress incurred by drinking a hot blood meal. Similar increases in hsp70 were documented immediately after blood feeding in two other mosquitoes (Culex pipiens and Anopheles gambiae) and the bed bug, Cimex lectularius, suggesting that this is a common protective response in blood-feeding arthropods.

  13. Yersinia pestis Yop secretion protein F: purification, characterization, and protective efficacy against bubonic plague.

    PubMed

    Swietnicki, Wieslaw; Powell, Bradford S; Goodin, Jeremy

    2005-07-01

    Yersinia pestis is a gram-negative human pathogen that uses a type III secretion system to deliver virulence factors into human hosts. The delivery is contact-dependent and it has been proposed that polymerization of Yop secretion protein F (YscF) is used to puncture mammalian cell membranes to facilitate delivery of Yersinia outer protein effectors into host cells. To evaluate the potential immunogenicity and protective efficacy of YscF against Y. pestis, we used a purified recombinant YscF protein as a potential vaccine candidate in a mouse subcutaneous infection model. YscF was expressed and purified from Escherichia coli by immobilized metal-ion affinity chromatography and protein identity was confirmed by ion trap mass spectrometry. The recombinant protein was highly alpha-helical and formed relatively stable aggregates under physiological conditions. The properties were consistent with behavior expected for the native YscF, suggesting that the antigen was properly folded. Ten mice were inoculated subcutaneously, administered booster injections after one month, and challenged with 130 LD(50) of wild type Y. pestis CO92. Six animals in the vaccinated group but none in the control group survived the challenge. The vaccinated animals produced high levels of specific antibodies against YscF as determined by Western blot. The data were statistically significant (P = 0.053 by two-tailed Fisher's test), suggesting that the YscF protein can provide a protective immune response against lethal plague challenge during subcutaneous plague infection.

  14. Arsenal of elevated defense proteins fails to protect tomato against Verticillium dahliae.

    PubMed

    Robb, Jane; Shittu, Hakeem; Soman, Kizhake V; Kurosky, Alexander; Nazar, Ross N

    2012-08-01

    Although the hypersensitive reaction in foliar plant diseases has been extensively described, little is clear regarding plant defense strategies in vascular wilt diseases affecting numerous economically important crops and trees. We have examined global genetic responses to Verticillium wilt in tomato (Lycopersicon esculentum Mill.) plants differing in Ve1 resistance alleles. Unexpectedly, mRNA analyses in the susceptible plant (Ve1-) based on the microarrays revealed a very heroic but unsuccessful systemic response involving many known plant defense genes. In contrast, the response is surprisingly low in plants expressing the Ve1+ R-gene and successfully resisting the pathogen. Similarly, whole-cell protein analyses, based on 2D gel electrophoresis and mass spectrometry, demonstrate large systemic increases in a variety of known plant defense proteins in the stems of susceptible plants but only modest changes in the resistant plant. Taken together, the results indicate that the large systemic increases in plant defense proteins do not protect the susceptible plant. Indeed, since a number of the highly elevated proteins are known to participate in the plant hypersensitive response as well as natural senescence, the results suggest that some or all of the disease symptoms, including ultimate plant death, actually may be the result of this exaggerated plant response.

  15. Trachoma: Protective and Pathogenic Ocular Immune Responses to Chlamydia trachomatis

    PubMed Central

    Hu, Victor H.; Holland, Martin J.; Burton, Matthew J.

    2013-01-01

    Trachoma, caused by Chlamydia trachomatis (Ct), is the leading infectious blinding disease worldwide. Chronic conjunctival inflammation develops in childhood and leads to eyelid scarring and blindness in adulthood. The immune response to Ct provides only partial protection against re-infection, which can be frequent. Moreover, the immune response is central to the development of scarring pathology, leading to loss of vision. Here we review the current literature on both protective and pathological immune responses in trachoma. The resolution of Ct infection in animal models is IFNγ-dependent, involving Th1 cells, but whether this is the case in human ocular infection still needs to be confirmed. An increasing number of studies indicate that innate immune responses arising from the epithelium and other innate immune cells, along with changes in matrix metalloproteinase activity, are important in the development of tissue damage and scarring. Current trachoma control measures, which are centred on repeated mass antibiotic treatment of populations, are logistically challenging and have the potential to drive antimicrobial resistance. A trachoma vaccine would offer significant advantages. However, limited understanding of the mechanisms of both protective immunity and immunopathology to Ct remain barriers to vaccine development. PMID:23457650

  16. Child protection and the conception of parental responsibility.

    PubMed

    Mass, Mili; van Nijnatten, Carolus

    2005-04-01

    The legal discourse on child protection that is characterized by the normalization-moralization paradigm focuses more on society's response to parental failure than on the predicament of the child. Findings from texts of legal discourse in Israel and in Holland portray an alliance between the respective legal systems and an epistemology of normality with regard to parenting that thereby turns normality into normalization. Both sets of texts are guided by an ontology of moral judgment that protects societal morale rather than the child. Morality is turned into moralization. To focus on the protection of the child, the article proposes a paradigm wherein the definition of morality is derived from concern for the other and relies on constructs that represent the evolving transaction between parent and child.

  17. Aggresomes: A Cellular Response to Misfolded Proteins

    PubMed Central

    Johnston, Jennifer A.; Ward, Cristina L.; Kopito, Ron R.

    1998-01-01

    Intracellular deposition of misfolded protein aggregates into ubiquitin-rich cytoplasmic inclusions is linked to the pathogenesis of many diseases. Why these aggregates form despite the existence of cellular machinery to recognize and degrade misfolded protein and how they are delivered to cytoplasmic inclusions are not known. We have investigated the intracellular fate of cystic fibrosis transmembrane conductance regulator (CFTR), an inefficiently folded integral membrane protein which is degraded by the cytoplasmic ubiquitin-proteasome pathway. Overexpression or inhibition of proteasome activity in transfected human embryonic kidney or Chinese hamster ovary cells led to the accumulation of stable, high molecular weight, detergent-insoluble, multiubiquitinated forms of CFTR. Using immunofluorescence and transmission electron microscopy with immunogold labeling, we demonstrate that undegraded CFTR molecules accumulate at a distinct pericentriolar structure which we have termed the aggresome. Aggresome formation is accompanied by redistribution of the intermediate filament protein vimentin to form a cage surrounding a pericentriolar core of aggregated, ubiquitinated protein. Disruption of microtubules blocks the formation of aggresomes. Similarly, inhibition of proteasome function also prevented the degradation of unassembled presenilin-1 molecules leading to their aggregation and deposition in aggresomes. These data lead us to propose that aggresome formation is a general response of cells which occurs when the capacity of the proteasome is exceeded by the production of aggregation-prone misfolded proteins. PMID:9864362

  18. Vaccination with Recombinant Microneme Proteins Confers Protection against Experimental Toxoplasmosis in Mice.

    PubMed

    Pinzan, Camila Figueiredo; Sardinha-Silva, Aline; Almeida, Fausto; Lai, Livia; Lopes, Carla Duque; Lourenço, Elaine Vicente; Panunto-Castelo, Ademilson; Matthews, Stephen; Roque-Barreira, Maria Cristina

    2015-01-01

    Toxoplasmosis, a zoonotic disease caused by Toxoplasma gondii, is an important public health problem and veterinary concern. Although there is no vaccine for human toxoplasmosis, many attempts have been made to develop one. Promising vaccine candidates utilize proteins, or their genes, from microneme organelle of T. gondii that are involved in the initial stages of host cell invasion by the parasite. In the present study, we used different recombinant microneme proteins (TgMIC1, TgMIC4, or TgMIC6) or combinations of these proteins (TgMIC1-4 and TgMIC1-4-6) to evaluate the immune response and protection against experimental toxoplasmosis in C57BL/6 mice. Vaccination with recombinant TgMIC1, TgMIC4, or TgMIC6 alone conferred partial protection, as demonstrated by reduced brain cyst burden and mortality rates after challenge. Immunization with TgMIC1-4 or TgMIC1-4-6 vaccines provided the most effective protection, since 70% and 80% of mice, respectively, survived to the acute phase of infection. In addition, these vaccinated mice, in comparison to non-vaccinated ones, showed reduced parasite burden by 59% and 68%, respectively. The protective effect was related to the cellular and humoral immune responses induced by vaccination and included the release of Th1 cytokines IFN-γ and IL-12, antigen-stimulated spleen cell proliferation, and production of antigen-specific serum antibodies. Our results demonstrate that microneme proteins are potential vaccines against T. gondii, since their inoculation prevents or decreases the deleterious effects of the infection.

  19. Feeding truncated heat shock protein 70s protect Artemia franciscana against virulent Vibrio campbellii challenge.

    PubMed

    Baruah, Kartik; Norouzitallab, Parisa; Shihao, Li; Sorgeloos, Patrick; Bossier, Peter

    2013-01-01

    The 70 kDa heat shock proteins (Hsp70s) are highly conserved in evolution, leading to striking similarities in structure and composition between eukaryotic Hsp70s and their homologs in prokaryotes. The eukaryotic Hsp70 like the DnaK (Escherichia coli equivalent Hsp70) protein, consist of three functionally distinct domains: an N-terminal 44-kDa ATPase portion, an 18-kDa peptide-binding domain and a C-terminal 10-kDa fragment. Previously, the amino acid sequence of eukaryotic (the brine shrimp Artemia franciscana) Hsp70 and DnaK proteins were shown to share a high degree of homology, particularly in the peptide-binding domain (59.6%, the putative innate immunity-activating portion) compared to the N-terminal ATPase (48.8%) and the C-terminal lid domains (19.4%). Next to this remarkable conservation, these proteins have been shown to generate protective immunity in Artemia against pathogenic Vibrio campbellii. This study, aimed to unravel the Vibrio-protective domain of Hsp70s in vivo, demonstrated that gnotobiotically cultured Artemia fed with recombinant C-terminal fragment (containing the conserved peptide binding domain) of Artemia Hsp70 or DnaK protein were well protected against subsequent Vibrio challenge. In addition, the prophenoloxidase (proPO) system, at both mRNA and protein activity levels, was also markedly induced by these truncated proteins, suggesting epitope(s) responsible for priming the proPO system and presumably other immune-related genes, consequently boosting Artemia survival upon challenge with V. campbellii, might be located within this conserved region of the peptide binding domain. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. The responsibility to protect: a new notion of state sovereignty.

    PubMed

    Ashford, Mary-Wynne

    2003-01-01

    The recent policies of the United States Administration threaten the entire structure of international law. Widespread concern in the US and elsewhere has been expressed in a different cluster of principles, in particular in the report of the Canadian-based Commission on Intervention and State Sovereignty. Its report states that the primary responsibility of a state is to protect its citizens; such protection includes prevention, reaction and rebuilding. The implications of this for military intervention are discussed; this must be a last resort after all non-military options have failed in response to the actual or probable large-scale loss of life and the objective to prevent further suffering. The action must be authorized by the United Nations Security Council or, if this fails to respond, the General Assembly. A further commission should examine how the international community should respond to states that refuse to comply with international law regarding weapons of mass destruction.

  1. Protection against virus infection in tobacco plants expressing the coat protein of grapevine fanleaf nepovirus.

    PubMed

    Bardonnet, N; Hans, F; Serghini, M A; Pinck, L

    1994-03-01

    Grapevine fanleaf nepovirus (GFLV) is responsible for the economically significant "court-noué" disease in vineyards. Its genome is made up of two single-stranded RNA molecules (RNA1 and RNA2) which direct the synthesis of polyproteins P1 and P2 respectively. A chimeric coat protein gene derived from the C-terminal part of P2 was constructed and subsequently introduced into a binary transformation vector. Transgenic Nicotiana benthamiana plants expressing the coat protein under the control of the CaMV 35S promoter were engineered by Agrobacterium tumefaciens-mediated transformation. Protection against infection with virions or viral RNA was tested in coat protein-expressing plants. A significant delay of systemic invasion was observed in transgenic plants inoculated with virus compared to control plants. This effect was also observed when plants were inoculated with viral RNA. No coat protein-mediated cross-protection was observed when transgenic plants were infected with arabis mosaic virus (ArMV), a closely related nepovirus also responsible for a "court-noué" disease.

  2. Redox-responsive self-healing for corrosion protection.

    PubMed

    Vimalanandan, Ashokanand; Lv, Li-Ping; Tran, The Hai; Landfester, Katharina; Crespy, Daniel; Rohwerder, Michael

    2013-12-23

    Raspberry-shaped redox-responsive capsules for storing corrosion inhibitors are introduced, targeted to solve the drawbacks of conducting-polymer-based coating systems for corrosion protection. These capsules synthesized via the miniemulsion technique have a remarkable release property upon reduction (onset of corrosion) and cease release upon reoxidation (passivation of the defect). The self-healing capability is demonstrated by application of these capsules as part of a composite coating on zinc.

  3. Thermal response of integral multicomponent composite thermal protection systems

    NASA Technical Reports Server (NTRS)

    Stewart, D. A.; Leiser, D. B.; Smith, M.; Kolodziej, P.

    1985-01-01

    Integral-multicomponent thermal-protection materials are discussed in terms of their thermal response to an arc-jet airstream. In-depth temperature measurements are compared with predictions from a one-dimensional, finite-difference code using calculated thermal conductivity values derived from an engineering model. The effect of composition, as well as the optical properties of the bonding material between components, on thermal response is discussed. The performance of these integral-multicomponent composite materials is compared with baseline Space Shuttle insulation.

  4. Melanoma and the Unfolded Protein Response

    PubMed Central

    Sykes, Erin K.; Mactier, Swetlana; Christopherson, Richard I.

    2016-01-01

    The UPR (unfolded protein response) has been identified as a key factor in the progression and metastasis of cancers, notably melanoma. Several mediators of the UPR are upregulated in cancers, e.g., high levels of GRP78 (glucose-regulator protein 78 kDa) correlate with progression and poor outcome in melanoma patients. The proliferative burden of cancer induces stress and activates several cellular stress responses. The UPR is a tightly orchestrated stress response that is activated upon the accumulation of unfolded proteins within the ER (endoplasmic reticulum). The UPR is designed to mediate two conflicting outcomtes, recovery and apoptosis. As a result, the UPR initiates a widespread signaling cascade to return the cell to homeostasis and failing to achieve cellular recovery, initiates UPR-induced apoptosis. There is evidence that ER stress and subsequently the UPR promote tumourigenesis and metastasis. The complete role of the UPR has yet to be defined. Understanding how the UPR allows for adaption to stress and thereby assists in cancer progression is important in defining an archetype of melanoma pathology. In addition, elucidation of the mechanisms of the UPR may lead to development of effective treatments of metastatic melanoma. PMID:26927180

  5. Interphotoreceptor Retinoid-Binding Protein Protects Retinoids from Photodegradation

    PubMed Central

    Gonzalez-Fernandez, Federico; Betts-Obregon, Brandi; Yust, Brian; Mimun, Joshua; Sung, Dongjin; Sardar, Dhiraj; Tsin, Andrew T.

    2015-01-01

    Retinol degrades rapidly in light into a variety of photoproducts. It is remarkable that visual cycle retinoids can evade photodegradation as they are exchanged between the photoreceptors, retinal pigment epithelium and Müller glia. Within the interphotoreceptor matrix, all-trans retinol, 11-cis retinol and retinal are bound by interphotoreceptor retinoid-binding protein (IRBP). Apart from its role in retinoid trafficking and targeting, could IRBP have a photoprotective function? HPLC was used to evaluate the ability of IRBP to protect all-trans and 11-cis retinols from photodegradation when exposed to incandescent light (0 to 8,842 μW/cm2); time periods of 0 – 60 min, and bIRBP: retinol molar ratios of 1:1 to 1:5. bIRBP afforded a significant prevention of both all-trans and 11-cis retinol to rapid photodegradation. The effect was significant over the entire light intensity range tested, and extended to the bIRBP: retinol ratio 1:5. In view of the continual exposure of the retina to light, and the high oxidative stress in the outer retina, our results suggest IRBP may have an important protective role in the visual cycle by reducing photodegradation of all-trans and 11-cis retinols. This role of IRBP is particularly relevant in the high flux conditions of the cone visual cycle. PMID:25565073

  6. Lack of protective efficacy in buffaloes vaccinated with Fasciola gigantica leucine aminopeptidase and peroxiredoxin recombinant proteins.

    PubMed

    Raina, O K; Nagar, Gaurav; Varghese, Anju; Prajitha, G; Alex, Asha; Maharana, B R; Joshi, P

    2011-06-01

    Gene coding for leucine aminopeptidase (LAP), a metalloprotease, was identified in the tropical liver fluke, Fasciola gigantica; that on sequence analysis showed a close homology (98.6%) with leucine aminopeptidase of the temperate liver fluke, Fasciola hepatica. The recombinant leucine aminopeptidase protein was expressed in Escherichia coli. F. gigantica peroxiredoxin, a hydrogen peroxide scavenger and an immunomodulating protein, was also cloned and expressed in E. coli. A vaccination trial in buffaloes was conducted with these two recombinant proteins, with 150 and 300 μg of leucine aminopeptidase and a cocktail of 150 μg each of recombinant leucine aminopeptidase and peroxiredoxin in three groups, respectively. Both Th1- and Th2-associated humoral immune responses were elicited to immunization with these antigens. A challenge study with 400 metacercariae did not show a significant protection in terms of reduction in the worm burden (8.4%) or anti-fecundity/embryonation effect in the immunized groups, as to the non-immunized control animals. Our observations in this buffalo vaccination trial are contrary to the earlier promise shown by leucine aminopeptidase of F. hepatica as a leading candidate vaccine molecule. Identification of leucine aminopeptidase gene and evaluation of the protein for its protective efficacy in buffaloes is the first scientific report on this protein in F. gigantica.

  7. Protein Degradation and the Stress Response

    PubMed Central

    Flick, Karin; Kaiser, Peter

    2012-01-01

    Environmental stresses are manifold and so are the responses they elicit. This is particularly true for higher eukaryotes where various tissues and cell types are differentially affected by the insult. Type and scope of the stress response can therefore differ greatly among cell types. Given the importance of the Ubiquitin Proteasome System (UPS) for most cellular processes, it comes as no surprise that the UPR plays a pivotal role in counteracting the effects of stressors. Here we outline contributions of the UPS to stress sensing, signaling, and response pathways. We make no claim to comprehensiveness but choose selected examples to illustrate concepts and mechanisms by which protein modification with ubiquitin and proteasomal degradation of key regulators ensures cellular integrity during stress situations. PMID:22414377

  8. Group 3 LEA Protein, ZmLEA3, Is Involved in Protection from Low Temperature Stress

    PubMed Central

    Liu, Yang; Liang, Jianan; Sun, Liping; Yang, Xinghong; Li, Dequan

    2016-01-01

    Late embryogenesis abundant (LEA) proteins are a family of small highly hydrophilic proteins that accumulate at the onset of seed desiccation and in response to adverse conditions such as drought, salinity, low temperature, or water deficit. In previous studies, we demonstrated that ZmLEA3 could enhance the transgenic tobacco tolerance to osmotic and oxidative stresses. Here, we demonstrated that the transcription of ZmLEA3 in the maize stems could be significantly induced by low temperature and osmotic stresses and by treatment with abscisic acid (ABA) and H2O2. Further study indicated that ZmLEA3 is a single copy gene in the maize genome. The ZmLEA3 protein could protect lactate dehydrogenase (LDH) activity at low temperatures. The overexpression of ZmLEA3 conferred tolerance to low-temperature stress to transgenic tobacco, yeast (GS115) and E. coli (BL21). PMID:27471509

  9. DDRprot: a database of DNA damage response-related proteins

    PubMed Central

    Andrés-León, Eduardo; Cases, Ildefonso; Arcas, Aida; Rojas, Ana M.

    2016-01-01

    The DNA Damage Response (DDR) signalling network is an essential system that protects the genome’s integrity. The DDRprot database presented here is a resource that integrates manually curated information on the human DDR network and its sub-pathways. For each particular DDR protein, we present detailed information about its function. If involved in post-translational modifications (PTMs) with each other, we depict the position of the modified residue/s in the three-dimensional structures, when resolved structures are available for the proteins. All this information is linked to the original publication from where it was obtained. Phylogenetic information is also shown, including time of emergence and conservation across 47 selected species, family trees and sequence alignments of homologues. The DDRprot database can be queried by different criteria: pathways, species, evolutionary age or involvement in (PTM). Sequence searches using hidden Markov models can be also used. Database URL: http://ddr.cbbio.es. PMID:27577567

  10. Cellular prion protein and NMDA receptor modulation: protecting against excitotoxicity

    PubMed Central

    Black, Stefanie A. G.; Stys, Peter K.; Zamponi, Gerald W.; Tsutsui, Shigeki

    2014-01-01

    Although it is well established that misfolding of the cellular prion protein (PrPC) into the β-sheet-rich, aggregated scrapie conformation (PrPSc) causes a variety of transmissible spongiform encephalopathies (TSEs), the physiological roles of PrPC are still incompletely understood. There is accumulating evidence describing the roles of PrPC in neurodegeneration and neuroinflammation. Recently, we identified a functional regulation of NMDA receptors by PrPC that involves formation of a physical protein complex between these proteins. Excessive NMDA receptor activity during conditions such as ischemia mediates enhanced Ca2+ entry into cells and contributes to excitotoxic neuronal death. In addition, NMDA receptors and/or PrPC play critical roles in neuroinflammation and glial cell toxicity. Inhibition of NMDA receptor activity protects against PrPSc-induced neuronal death. Moreover, in mice lacking PrPC, infarct size is increased after focal cerebral ischemia, and absence of PrPC increases susceptibility of neurons to NMDA receptor-dependent death. Recently, PrPC was found to be a receptor for oligomeric beta-amyloid (Aβ) peptides, suggesting a role for PrPC in Alzheimer's disease (AD). Our recent findings suggest that Aβ peptides enhance NMDA receptor current by perturbing the normal copper- and PrPC-dependent regulation of these receptors. Here, we review evidence highlighting a role for PrPC in preventing NMDA receptor-mediated excitotoxicity and inflammation. There is a need for more detailed molecular characterization of PrPC-mediated regulation of NMDA receptors, such as determining which NMDA receptor subunits mediate pathogenic effects upon loss of PrPC-mediated regulation and identifying PrPC binding site(s) on the receptor. This knowledge will allow development of novel therapeutic interventions for not only TSEs, but also for AD and other neurodegenerative disorders involving dysfunction of PrPC. PMID:25364752

  11. Thioredoxin-Interacting Protein Deficiency Protects against Diabetic Nephropathy.

    PubMed

    Shah, Anu; Xia, Ling; Masson, Elodie A Y; Gui, Chloe; Momen, Abdul; Shikatani, Eric A; Husain, Mansoor; Quaggin, Susan; John, Rohan; Fantus, I G

    2015-12-01

    Expression of thioredoxin-interacting protein (TxNIP), an endogenous inhibitor of the thiol oxidoreductase thioredoxin, is augmented by high glucose (HG) and promotes oxidative stress. We previously reported that TxNIP-deficient mesangial cells showed protection from HG-induced reactive oxygen species, mitogen-activated protein kinase phosphorylation, and collagen expression. Here, we investigated the potential role of TxNIP in the pathogenesis of diabetic nephropathy (DN) in vivo. Wild-type (WT) control, TxNIP(-/-), and TxNIP(+/-) mice were rendered equally diabetic with low-dose streptozotocin. In contrast to effects in WT mice, diabetes did not increase albuminuria, proteinuria, serum cystatin C, or serum creatinine levels in TxNIP(-/-) mice. Whereas morphometric studies of kidneys revealed a thickened glomerular basement membrane and effaced podocytes in the diabetic WT mice, these changes were absent in the diabetic TxNIP(-/-) mice. Immunohistochemical analysis revealed significant increases in the levels of glomerular TGF-β1, collagen IV, and fibrosis only in WT diabetic mice. Additionally, only WT diabetic mice showed significant increases in oxidative stress (nitrotyrosine, urinary 8-hydroxy-2-deoxy-guanosine) and inflammation (IL-1β mRNA, F4/80 immunohistochemistry). Expression levels of Nox4-encoded mRNA and protein increased only in the diabetic WT animals. A significant loss of podocytes, assessed by Wilms' tumor 1 and nephrin staining and urinary nephrin concentration, was found in diabetic WT but not TxNIP(-/-) mice. Furthermore, in cultured human podocytes exposed to HG, TxNIP knockdown with siRNA abolished the increased mitochondrial O2 (-) generation and apoptosis. These data indicate that TxNIP has a critical role in the progression of DN and may be a promising therapeutic target. Copyright © 2015 by the American Society of Nephrology.

  12. Protein response to ligation reactions in myoglobin

    SciTech Connect

    Causgrove, T.P.; Dyer, R.B.

    1992-01-01

    The protein response to the photodissociation, escape and subsequent rebinding of carbon monoxide in myoglobin is studied using time-resolved infrared (TRIR) spectroscopy. All phases of these reactions areinvestigated, from ultrafast phenomena (picoseconds) to relatively slow processes (milliseconds). Conformational changes in myoglobin (Mb) are detected by time-resolved absorption changes in the amide I band. On the hundreds of nanoseconds to milliseconds timescale, a real-time'' apparatus is used. This apparatus is based on a tunable diode laser operating in the region of 1650 cm[sup [minus]1] . The time course ofchanges in the amide I band are shown to follow the recombination of CO with photolyzed Mb. On the basis of the rise times of the amide I and FE-CO signals, it is concluded that protein motion is complete within 100 n. A time-resolved difference spectrum in the amide I region is generated from single wavelength transients taken throughout the amide I envelope. A static difference spectrum is also generated by subtracting FTIR spectra of carbonmonoxy and deoxy myoglobin. The two difference spectra are compared and are interpreted in terms of the three-dimensional structures of deoxy and carbonmonoxy Mb. Preliminary picosecond TRIR data are also given for the ultrafast response of the protein immediately following photodissociation of CO.

  13. Determinants of immunogenic response to protein therapeutics.

    PubMed

    Singh, Satish K; Cousens, Leslie P; Alvarez, David; Mahajan, Pramod B

    2012-09-01

    Protein therapeutics occupy a very significant position in the biopharmaceutical market. In addition to the preclinical, clinical and post marketing challenges common to other drugs, unwanted immunogenicity is known to affect efficacy and/or safety of most biotherapeutics. A standard set of immunogenicity risk factors are routinely used to inform monitoring strategies in clinical studies. A number of in-silico, in vivo and in vitro approaches have also been employed to predict immunogenicity of biotherapeutics, but with limited success. Emerging data also indicates the role of immune tolerance mechanisms and impact of several product-related factors on modulating host immune responses. Thus, a comprehensive discussion of the impact of innate and adaptive mechanisms and molecules involved in induction of host immune responses on immunogenicity of protein therapeutics is needed. A detailed understanding of these issues is essential in order to fully exploit the therapeutic potential of this class of drugs. This Roundtable Session was designed to provide a common platform for discussing basic immunobiological and pharmacological issues related to the role of biotherapeutic-associated risk factors, as well as host immune system in immunogenicity against protein therapeutics. The session included overview presentations from three speakers, followed by a panel discussion with audience participation.

  14. Protein response to ligation reactions in myoglobin

    NASA Astrophysics Data System (ADS)

    Causgrove, Timothy P.; Dyer, R. B.

    1993-05-01

    The protein response to the photodissociation, escape and subsequent rebinding of carbon monoxide in myoglobin is studied using time-resolved infrared (TRIR) spectroscopy. All phases of these reactions are investigated, from ultrafast phenomena (picoseconds) to relatively slow processes (milliseconds). Conformational changes in myoglobin (Mb) are detected by time-resolved infrared absorption changes in the amide I band. On the hundreds of nanoseconds to milliseconds timescale, a 'real-time' apparatus is used. This apparatus is based on a tunable diode laser operating in the region of 1650 cm-1. The time course of changes in the amide I band are shown to follow the recombination of CO with photolyzed Mb. On the basis of the rise times of the amide I and Fe-CO signals, it is concluded that protein motion is complete within 100 ns. A time-resolved difference spectrum in the amide I region is generated from single wavelength transients taken throughout the amide I envelope. A static difference spectrum is also generated by subtracting FTIR spectra of carbonmonoxy and deoxy myoglobin. The two difference spectra are compared and are interpreted in terms of the three-dimensional structures of deoxy and carbonmonoxy Mb. Preliminary picosecond TRIR data are also given for the ultrafast response of the protein immediately following photodissociation of CO.

  15. Protein response to ligation reactions in myoglobin

    SciTech Connect

    Causgrove, T.P.; Dyer, R.B.

    1992-12-31

    The protein response to the photodissociation, escape and subsequent rebinding of carbon monoxide in myoglobin is studied using time-resolved infrared (TRIR) spectroscopy. All phases of these reactions areinvestigated, from ultrafast phenomena (picoseconds) to relatively slow processes (milliseconds). Conformational changes in myoglobin (Mb) are detected by time-resolved absorption changes in the amide I band. On the hundreds of nanoseconds to milliseconds timescale, a ``real-time`` apparatus is used. This apparatus is based on a tunable diode laser operating in the region of 1650 cm{sup {minus}1} . The time course ofchanges in the amide I band are shown to follow the recombination of CO with photolyzed Mb. On the basis of the rise times of the amide I and FE-CO signals, it is concluded that protein motion is complete within 100 n. A time-resolved difference spectrum in the amide I region is generated from single wavelength transients taken throughout the amide I envelope. A static difference spectrum is also generated by subtracting FTIR spectra of carbonmonoxy and deoxy myoglobin. The two difference spectra are compared and are interpreted in terms of the three-dimensional structures of deoxy and carbonmonoxy Mb. Preliminary picosecond TRIR data are also given for the ultrafast response of the protein immediately following photodissociation of CO.

  16. Immune response to recombinant Escherichia coli Iss protein in poultry.

    PubMed

    Lynne, Aaron M; Foley, Steven L; Nolan, Lisa K

    2006-06-01

    Colibacillosis accounts for significant losses to the poultry industry, and control efforts are hampered by limited understanding of the mechanisms used by avian pathogenic Escherichia coli (APEC) to cause disease. We have found that the presence of the increased serum survival gene (iss) is strongly associated with APEC but not with commensal E. coli, making iss, and the protein it encodes (Iss), candidate targets of colibacillosis control procedures. To assess the potential of Iss to elicit a protective response in chickens against APEC challenge, Iss fusion proteins were produced and administered subcutaneously to four groups of 2-wk-old specific-pathogen-free leghorn chickens. At 4 wk postimmunization, birds were challenged with APEC from serogroups 02 and 078 via intramuscular injection. At 2 wk postchallenge, birds were necropsied, and lesions consistent with colibacillosis were scored. Also, sera were collected from the birds pre- and postimmunization, and antibody titers to Iss were determined. Immunized birds produced a humoral response to Iss, and they had significantly lower lesion scores than the unimmunized control birds following challenge with both APEC strains. Birds that received the smallest amount of immunogen had the lowest lesion scores. Although further study will be needed to confirm the value of Iss as an immunoprotective antigen, these preliminary data suggest that Iss may have the potential to elicit significant protection in birds against heterologous E. coli challenge.

  17. The Dose Window for Radiation-Induced Protective Adaptive Responses

    PubMed Central

    Mitchel, Ronald E. J.

    2009-01-01

    Adaptive responses to low doses of low LET radiation occur in all organisms thus far examined, from single cell lower eukaryotes to mammals. These responses reduce the deleterious consequences of DNA damaging events, including radiation-induced or spontaneous cancer and non-cancer diseases in mice. The adaptive response in mammalian cells and mammals operates within a certain window that can be defined by upper and lower dose thresholds, typically between about 1 and 100 mGy for a single low dose rate exposure. However, these thresholds for protection are not a fixed function of total dose, but also vary with dose rate, additional radiation or non-radiation stressors, tissue type and p53 functional status. Exposures above the upper threshold are generally detrimental, while exposures below the lower threshold may or may not increase either cancer or non-cancer disease risk. PMID:20585438

  18. Arthritis protective regulatory potential of self–heat shock protein cross-reactive T cells

    PubMed Central

    van Eden, Willem; Wendling, Uwe; Paul, Liesbeth; Prakken, Berent; van Kooten, Peter; van der Zee, Ruurd

    2000-01-01

    Immunization with heat shock proteins has protective effects in models of induced arthritis. Analysis has shown a reduced synovial inflammation in such protected animals. Adoptive transfer and immunization with selected T cell epitopes (synthetic peptides) have indicated the protection to be mediated by T cells directed to conserved hsp epitopes. This was shown first for mycobacterial hsp60 and later for mycobacterial hsp70. Fine specificity analysis showed that such T cells were cross-reactive with the homologous self hsp. Therefore protection by microbial hsp reactive T cells can be by cross-recognition of self hsp overexpressed in the inflamed tissue. Preimmunization with hsp leads to a relative expansion of such self hsp cross-responsive T cells. The regulatory nature of such T cells may originate from mucosal tolerance maintained by commensal flora derived hsp or from partial activation through recognition of self hsp as a partial agonist (Altered Peptide Ligand) or in the absence of proper costimulation. Recently, we reported the selective upregulation of B7.2 on microbial hsp60 specific T cells in response to self hsp60. Through a preferred interaction with CTLA-4 on proinflammatory T cells this may constitute an effector mechanism of regulation. Also, regulatory T cells produced IL10. PMID:11189451

  19. TLR2-targeted secreted proteins from Mycobacterium tuberculosis are protective as powdered pulmonary vaccines.

    PubMed

    Tyne, Anneliese S; Chan, John Gar Yan; Shanahan, Erin R; Atmosukarto, Ines; Chan, Hak-Kim; Britton, Warwick J; West, Nicholas P

    2013-09-13

    Despite considerable research efforts towards effective treatments, tuberculosis (TB) remains a staggering burden on global health. Suitably formulated sub-unit vaccines offer potential as safe and effective generators of protective immunity. The Mycobacterium tuberculosis antigens, cutinase-like proteins (Culp) 1 and 6 and MPT83, were conjugated directly to the novel adjuvant Lipokel (Lipotek Pty Ltd), a TLR2 ligand that delivers antigen to immune cells in a self-adjuvanting context. Protein-Lipokel complexes were formulated as dry powders for pulmonary delivery directly to the lungs of mice by intra-tracheal insufflation, leading to recruitment of neutrophils and antigen presenting cell populations to the lungs at 72 h, that persisted at 7 days post immunisation. Significant increases in the frequency of activated dendritic cells were observed in the mediastinal lymph node (MLN) at 1 and 4 weeks after homologous boosting with protein-Lipokel vaccine. This was associated with the increased recruitment of effector CD4(+) and CD8(+) T-lymphocytes to the MLN and systemic antigen-specific, IFN-γ producing T-lymphocyte and IgG responses. Notably, pulmonary immunisation with either Culp1-6-Lipokel or MPT83-Lipokel powder vaccines generated protective responses in the lungs against aerosol M. tuberculosis challenge. The successful combination of TLR2-targeting and dry powder vaccine formulation, together with important practical benefits, offers potential for pulmonary vaccination against M. tuberculosis.

  20. From protein denaturant to protectant: Comparative molecular dynamics study of alcohol/protein interactions

    NASA Astrophysics Data System (ADS)

    Shao, Qiang; Fan, Yubo; Yang, Lijiang; Qin Gao, Yi

    2012-03-01

    It is well known that alcohols can have strong effects on protein structures. For example, monohydric methanol and ethanol normally denature, whereas polyhydric glycol and glycerol protect, protein structures. In a recent combined theoretical and NMR experimental study, we showed that molecular dynamics simulations can be effectively used to understand the molecular mechanism of methanol denaturing protein. In this study, we used molecular dynamics simulations to investigate how alcohols with varied hydrophobicity and different numbers of hydrophilic groups (hydroxyl groups) exert effects on the structure of the model polypeptide, BBA5. First, we showed that methanol and trifluoroethanol (TFE) but not glycol or glycerol disrupt hydrophobic interactions. The latter two alcohols instead protect the assembly of the α- and β-domains of the polypeptide. Second, all four alcohols were shown to generally increase the stability of secondary structures, as revealed by the increased number of backbone hydrogen bonds formed in alcohol/water solutions compared to that in pure water, although individual hydrogen bonds can be weakened by certain alcohols, such as TFE. The two monohydric alcohols, methanol and TFE, display apparently different sequence-dependence in affecting the backbone hydrogen bond stability: methanol tends to enhance the stability of backbone hydrogen bonds of which the carbonyl groups are from polar residues, whereas TFE tends to stabilize those involving non-polar residues. These results demonstrated that subtle differences in the solution environment could have distinct consequences on protein structures.

  1. UV protection of euglenoids: computation of the electromagnetic response

    NASA Astrophysics Data System (ADS)

    Dolinko, Andrés.; Valencia, Claudio; Skigin, Diana C.; Inchaussandague, Marina E.; Tolivia, Analía.; Conforti, Visitación

    2015-06-01

    Euglenoids are a group of predominantly free-living unicellular microorganisms that mostly live in freshwater bodies but can also be found in marine and brackish waters. These organisms have a characteristic that distinguishes them form the other protists: they are covered by a surface pellicle formed by S-shaped overlapping bands which resemble a diffraction grating. These microorganisms have developed numerous protection mechanisms intended to avoid or reduce the damage produced by UV radiation, such as the production of pigments and the repair mechanisms in hours of darkness and during daylight. In a recent paper we have investigated the role played by the pellicle of Euglenoids in the protection of the cell against UV radiation, by means of an electromagnetic approach based on the approximation of the pellicle profile by a one-dimensional diffraction grating. This simplified model allowed us to confirm that under certain incidence conditions, the corrugation of the pellicle helps increase the UV reflection, and consequently, diminish the UV radiation that enters the cell. In order to analyze the electromagnetic response of the whole cell, we extend two different approaches to calculate the reflected response: a simulation method especially developed to deal with complex biological structures that permits the introduction of the scattering object via an electron microscopy image, and the integral method, which has been widely used to compute the electromagnetic response of finite structures. Numerical results of near and far fields are shown.

  2. Analysis of Unfolded Protein Response in Arabidopsis

    PubMed Central

    Chen, Yani; Brandizzi, Federica

    2014-01-01

    The unfolded protein response (UPR) is fundamental for development and adaption in eukaryotic cells. Arabidopsis has become one of the best model systems to uncover conserved mechanisms of the UPR in multicellular eukaryotes as well as organism-specific regulation of the UPR in plants. Monitoring the UPR in planta is an elemental approach to identifying regulatory components and to revealing molecular mechanisms of the plant UPR. In this chapter, we provide protocols for the induction and analyses of plant UPR at a molecular level in Arabidopsis. Three kinds of ER stress treatment methods and quantitation of the plant UPR activation are described here. PMID:23913037

  3. Inhibition of the plasma SCUBE1, a novel platelet adhesive protein, protects mice against thrombosis.

    PubMed

    Wu, Meng-Ying; Lin, Yuh-Charn; Liao, Wei-Ju; Tu, Cheng-Fen; Chen, Ming-Huei; Roffler, Steve R; Yang, Ruey-Bing

    2014-07-01

    Signal peptide-CUB-EGF domain-containing protein 1 (SCUBE1), a secreted and surface-exposed glycoprotein on activated platelets, promotes platelet-platelet interaction and supports platelet-matrix adhesion. Its plasma level is a biomarker of platelet activation in acute thrombotic diseases. However, the exact roles of plasma SCUBE1 in vivo remain undefined. We generated new mutant (Δ) mice lacking the soluble but retaining the membrane-bound form of SCUBE1. Plasma SCUBE1-depleted Δ/Δ mice showed normal hematologic and coagulant features and expression of major platelet receptors, but Δ/Δ platelet-rich plasma showed impaired platelet aggregation in response to ADP and collagen treatment. The addition of purified recombinant SCUBE1 protein restored the aggregation of platelets in Δ/Δ platelet-rich plasma and further enhanced platelet aggregation in +/+ platelet-rich plasma. Plasma deficiency of SCUBE1 diminished arterial thrombosis in mice and protected against lethal thromboembolism induced by collagen-epinephrine treatment. Last, antibodies directed against the epidermal growth factor-like repeats of SCUBE1, which are involved in trans-homophilic protein-protein interactions, protected mice against fatal thromboembolism without causing bleeding in vivo. We conclude that plasma SCUBE1 participates in platelet aggregation by bridging adjacent activated platelets in thrombosis. Blockade of soluble SCUBE1 might represent a novel antithrombotic strategy. © 2014 American Heart Association, Inc.

  4. Rapid outer-surface protein C DNA tattoo vaccination protects against Borrelia afzelii infection.

    PubMed

    Wagemakers, A; Mason, L M K; Oei, A; de Wever, B; van der Poll, T; Bins, A D; Hovius, J W R

    2014-12-01

    Borrelia afzelii is the predominant Borrelia species causing Lyme borreliosis in Europe. Currently there is no human vaccine against Lyme borreliosis, and most research focuses on recombinant protein vaccines against Borrelia burgdorferi sensu stricto. DNA tattooing is a novel vaccination method that can be applied in a rapid vaccination schedule. We vaccinated C3H/HeN mice with B. afzelii strain PKo OspC (outer-surface protein C) using a codon-optimized DNA vaccine tattoo and compared this with recombinant protein vaccination in a 0-2-4 week vaccination schedule. We also assessed protection by DNA tattoo in a 0-3-6 day schedule. DNA tattoo and recombinant OspC vaccination induced comparable total IgG responses, with a lower IgG1/IgG2a ratio after DNA tattoo. Two weeks after syringe-challenge with 5 × 10(5) B. afzelii spirochetes most vaccinated mice had negative B. afzelii tissue DNA loads and all were culture negative. Furthermore, DNA tattoo vaccination in a 0-3-6 day regimen also resulted in negative Borrelia loads and cultures after challenge. To conclude, DNA vaccination by tattoo was fully protective against B. afzelii challenge in mice in a rapid vaccination protocol, and induces a favorable humoral immunity compared to recombinant protein vaccination. Rapid DNA tattoo is a promising vaccination strategy against spirochetes.

  5. Extracellular proteins of Clostridium chauvoei are protective in a mouse model.

    PubMed

    Mattar, María A; Cortiñas, Teresa I; Stefanini, Ana M

    2007-06-01

    The anaerobic bacillus Clostridium chauvoei is the causative agent of blackleg, a lethal disease that has an important impact on the sheep and cattle industry worldwide. Immunity to C. chauvoei is considered to be mainly anticellular, and for this reason there is scarce information about the immunogenicity of extracellular proteins. In this work variations in protein profiles, immune response by ELISA and protective capacity of culture supernatants of three C. chauvoei strains, collected at different growth phases, are reported. Sera raised against extracellular antigens also recognised cellular antigens of the same molecular masses. Partially purified cell-free supernatants and those concentrated 10 times by ultrafiltration (C-CFS), obtained at the early stationary phase of growth, induced a strong immunoprotective response, even at low doses, that was more marked for C. chauvoei strain ATCC 10092 (p < or = 0.05). With C-CFS formulations, a clear relationship was observed between IgG titres, protective capacity and concentration of the antigen doses, indicating a specific immune response.

  6. Immunofluorescence Imaging of DNA Damage Response Proteins

    PubMed Central

    Bennett, Brian T.; Bewersdorf, Jörg; Knight, Kendall L.

    2013-01-01

    Immunofluorescence imaging has provided captivating visual evidence for numerous cellular events, from vesicular trafficking, organelle maturation and cell division to nuclear processes including the appearance of various proteins and chromatin components in distinct foci in response to DNA damaging agents. With the advent of new super-resolution microscope technologies such as 4Pi microscopy, standard immunofluorescence protocols deserve some reevaluation in order to take full advantage of these new technological accomplishments. Here we describe several methodological considerations that will help overcome some of the limitations that may result from the use of currently applied procedures, with particular attention paid to the analysis of possible colocalization of fluorescent signals. We conclude with an example of how application of optimized methods led to a breakthrough in super-resolution imaging of nuclear events occurring in response to DNA damage. PMID:19245833

  7. Protective immunity induced in Aotus monkeys by a recombinant SERA protein of Plasmodium falciparum: adjuvant effects on induction of protective immunity.

    PubMed Central

    Inselburg, J; Bathurst, I C; Kansopon, J; Barchfeld, G L; Barr, P J; Rossan, R N

    1993-01-01

    We report the results of vaccination trial 2 of Panamanian Aotus monkeys with a recombinant blood-stage antigen, SERA 1, of the malaria parasite Plasmodium falciparum. Monkeys were immunized with SERA 1, a 262-amino-acid fragment (amino acids 24 to 285) of the 989-amino-acid SERA protein produced by the Honduras 1 strain of the parasite. Immunization mixtures contained 100 micrograms of recombinant SERA 1 protein per dose mixed with one of five different adjuvants. The protein mixed with either Freund's adjuvant or MF75.2 adjuvant stimulated protective immunity. When other P. falciparum antigens were included in the SERA 1-Freund's adjuvant mixture, no protective immunity was observed, although high anti-SERA 1 antibody titers were produced. Three other adjuvants mixed with SERA 1 failed to induce a protective immune response. These results, their relationship to those reported previously in the first vaccination trial (trial 1), and their relationships to the quantitative measurement of anti-SERA 1 antibodies in enzyme-linked immunosorbent assays provided insights into the induction of a protective immune response in vaccinated monkeys. PMID:8478092

  8. Vaccines Displaying Mycobacterial Proteins on Biopolyester Beads Stimulate Cellular Immunity and Induce Protection against Tuberculosis

    PubMed Central

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

    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

  9. Moonlight-like proteins of the cell wall protect sessile cells of Candida from oxidative stress.

    PubMed

    Serrano-Fujarte, Isela; López-Romero, Everardo; Cuéllar-Cruz, Mayra

    2016-01-01

    Biofilms of Candida species are associated with high morbidity and hospital mortality. Candida forms biofilms by adhering to human host epithelium through cell wall proteins (CWP) and simultaneously neutralizing the reactive oxygen species (ROS) produced during the respiratory burst by phagocytic cells. The purpose of this paper is to identify the CWP of Candida albicans, Candida glabrata, Candida krusei and Candida parapsilosis expressed after exposure to different concentrations of H2O2 using a proteomic approach. CWP obtained from sessile cells, both treated and untreated with the oxidizing agent, were resolved by one and two-dimensional (2D-PAGE) gels and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Some of these proteins were identified and found to correspond to moonlighting CWP such as: (i) glycolytic enzymes, (ii) heat shock, (iii) OSR proteins, (iv) general metabolic enzymes and (v) highly conserved proteins, which are up- or down-regulated in the presence or absence of ROS. We also found that the expression of these CWP is different for each Candida species. Moreover, RT-PCR assays allowed us to demonstrate that transcription of the gene coding for Eno1, one of the moonlight-like CWP identified in response to the oxidant agent, is differentially regulated. To our knowledge this is the first demonstration that, in response to oxidative stress, each species of Candida, differentially regulates the expression of moonlighting CWP, which may protect the organism from the ROS generated during phagocytosis. Presumptively, these proteins allow the pathogen to adhere and form a biofilm, and eventually cause invasive candidiasis in the human host. We propose that, in addition to the antioxidant mechanisms present in Candida, the moonlighting CWP also confer protection to these pathogens from oxidative stress. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Electromagnetic response of the protective pellicle of different unicellular microalgae

    NASA Astrophysics Data System (ADS)

    Inchaussandague, Marina E.; Skigin, Diana C.; Tolivia, Analía.; Fuertes Vila, Isabel; Conforti, Visitación

    2014-03-01

    Euglenoids are unicellular aquatic organisms. These microalgae show a typical surface structure that distinguishes them from the other protists. Most cells are naked and bounded by a plasma membrane surrounded by a pellicle formed by overlapping bands. It is well known that all terrestrial and aquatic organisms are exposed to UV-A and UV-B radiation. This radiation is potentially harmful to life and since it can penetrate up to 12 meters in the water, it can reduce survival, growth and production of phytoplankton. However, the organisms have developed numerous protection mechanisms intended to reduce such damage, such as the production of pigments and other repair mechanisms. However, the possible protection that could provide the first barriers before entering into the cell has not been explored yet. In this paper we investigate, from an electromagnetic point of view, the role played by the pellicle of euglenoids in the protection of the cell against UV radiation. To do so, we investigate the electromagnetic response of different species that exhibit different behaviors against the UV radiation. We solve the diffraction problem by using the Chandezon Method and obtain the reflectance of the pellicle for the UV wavelengths. The results show that the corrugated pellicle could contribute to increase the reflectance, thus reducing the penetration of the UV radiation within the cell and therefore, minimizing the damage and increasing the survival of these organisms.

  11. 77 FR 15379 - Protecting Tenants at Foreclosure Act: Additional Guidance on Notification Responsibilities Under...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-15

    ... guidance on the notice, entitled ``Protecting Tenants at Foreclosure: Notice of Responsibilities Placed on... URBAN DEVELOPMENT Protecting Tenants at Foreclosure Act: Additional Guidance on Notification Responsibilities Under the Act With Respect to Occupied Conveyance AGENCY: Office of the Assistant Secretary...

  12. HDLs protect pancreatic β-cells against ER stress by restoring protein folding and trafficking.

    PubMed

    Pétremand, Jannick; Puyal, Julien; Chatton, Jean-Yves; Duprez, Jessica; Allagnat, Florent; Frias, Miguel; James, Richard W; Waeber, Gérard; Jonas, Jean-Christophe; Widmann, Christian

    2012-05-01

    Endoplasmic reticulum (ER) homeostasis alteration contributes to pancreatic β-cell dysfunction and death and favors the development of diabetes. In this study, we demonstrate that HDLs protect β-cells against ER stress induced by thapsigargin, cyclopiazonic acid, palmitate, insulin overexpression, and high glucose concentrations. ER stress marker induction and ER morphology disruption mediated by these stimuli were inhibited by HDLs. Using a temperature-sensitive viral glycoprotein folding mutant, we show that HDLs correct impaired protein trafficking and folding induced by thapsigargin and palmitate. The ability of HDLs to protect β-cells against ER stress was inhibited by brefeldin A, an ER to Golgi trafficking blocker. These results indicate that HDLs restore ER homeostasis in response to ER stress, which is required for their ability to promote β-cell survival. This study identifies a cellular mechanism mediating the beneficial effect of HDLs on β-cells against ER stress-inducing factors.

  13. HDLs Protect Pancreatic β-Cells Against ER Stress by Restoring Protein Folding and Trafficking

    PubMed Central

    Pétremand, Jannick; Puyal, Julien; Chatton, Jean-Yves; Duprez, Jessica; Allagnat, Florent; Frias, Miguel; James, Richard W.; Waeber, Gérard; Jonas, Jean-Christophe; Widmann, Christian

    2012-01-01

    Endoplasmic reticulum (ER) homeostasis alteration contributes to pancreatic β-cell dysfunction and death and favors the development of diabetes. In this study, we demonstrate that HDLs protect β-cells against ER stress induced by thapsigargin, cyclopiazonic acid, palmitate, insulin overexpression, and high glucose concentrations. ER stress marker induction and ER morphology disruption mediated by these stimuli were inhibited by HDLs. Using a temperature-sensitive viral glycoprotein folding mutant, we show that HDLs correct impaired protein trafficking and folding induced by thapsigargin and palmitate. The ability of HDLs to protect β-cells against ER stress was inhibited by brefeldin A, an ER to Golgi trafficking blocker. These results indicate that HDLs restore ER homeostasis in response to ER stress, which is required for their ability to promote β-cell survival. This study identifies a cellular mechanism mediating the beneficial effect of HDLs on β-cells against ER stress-inducing factors. PMID:22399686

  14. Novel protein-protein interaction family proteins involved in chloroplast movement response.

    PubMed

    Kodama, Yutaka; Suetsugu, Noriyuki; Wada, Masamitsu

    2011-04-01

    To optimize photosynthetic activity, chloroplasts change their intracellular location in response to ambient light conditions; chloroplasts move toward low intensity light to maximize light capture, and away from high intensity light to avoid photodamage. Although several proteins have been reported to be involved in the chloroplast photorelocation movement response, any physical interaction among them was not found so far. We recently found a physical interaction between two plant-specific coiled-coil proteins, WEB1 (Weak Chloroplast Movement under Blue Light 1) and PMI2 (Plastid Movement Impaired 2), that were identified to regulate chloroplast movement velocity. Since the both coiled-coil regions of WEB1 and PMI2 were classified into an uncharacterized protein family having DUF827 (DUF: Domain of Unknown Function) domain, it was the first report that DUF827 proteins could mediate protein-protein interaction. In this mini-review article, we discuss regarding molecular function of WEB1 and PMI2, and also define a novel protein family composed of WEB1, PMI2 and WEB1/PMI2-like proteins for protein-protein interaction in land plants.

  15. A Plasmodium Promiscuous T Cell Epitope Delivered within the Ad5 Hexon Protein Enhances the Protective Efficacy of a Protein Based Malaria Vaccine.

    PubMed

    Fonseca, Jairo Andres; Cabrera-Mora, Monica; Kashentseva, Elena A; Villegas, John Paul; Fernandez, Alejandra; Van Pelt, Amelia; Dmitriev, Igor P; Curiel, David T; Moreno, Alberto

    2016-01-01

    A malaria vaccine is a public health priority. In order to produce an effective vaccine, a multistage approach targeting both the blood and the liver stage infection is desirable. The vaccine candidates also need to induce balanced immune responses including antibodies, CD4+ and CD8+ T cells. Protein-based subunit vaccines like RTS,S are able to induce strong antibody response but poor cellular reactivity. Adenoviral vectors have been effective inducing protective CD8+ T cell responses in several models including malaria; nonetheless this vaccine platform exhibits a limited induction of humoral immune responses. Two approaches have been used to improve the humoral immunogenicity of recombinant adenovirus vectors, the use of heterologous prime-boost regimens with recombinant proteins or the genetic modification of the hypervariable regions (HVR) of the capsid protein hexon to express B cell epitopes of interest. In this study, we describe the development of capsid modified Ad5 vectors that express a promiscuous Plasmodium yoelii T helper epitope denominated PyT53 within the hexon HVR2 region. Several regimens were tested in mice to determine the relevance of the hexon modification in enhancing protective immune responses induced by the previously described protein-based multi-stage experimental vaccine PyCMP. A heterologous prime-boost immunization regime that combines a hexon modified vector with transgenic expression of PyCMP followed by protein immunizations resulted in the induction of robust antibody and cellular immune responses in comparison to a similar regimen that includes a vector with unmodified hexon. These differences in immunogenicity translated into a better protective efficacy against both the hepatic and red blood cell stages of P. yoelii. To our knowledge, this is the first time that a hexon modification is used to deliver a promiscuous T cell epitope. Our data support the use of such modification to enhance the immunogenicity and protective

  16. G Protein-coupled Estrogen Receptor Protects from Atherosclerosis

    PubMed Central

    Meyer, Matthias R.; Fredette, Natalie C.; Howard, Tamara A.; Hu, Chelin; Ramesh, Chinnasamy; Daniel, Christoph; Amann, Kerstin; Arterburn, Jeffrey B.; Barton, Matthias; Prossnitz, Eric R.

    2014-01-01

    Coronary atherosclerosis and myocardial infarction in postmenopausal women have been linked to inflammation and reduced nitric oxide (NO) formation. Natural estrogen exerts protective effects on both processes, yet also displays uterotrophic activity. Here, we used genetic and pharmacologic approaches to investigate the role of the G protein-coupled estrogen receptor (GPER) in atherosclerosis. In ovary-intact mice, deletion of gper increased atherosclerosis progression, total and LDL cholesterol levels and inflammation while reducing vascular NO bioactivity, effects that were in some cases aggravated by surgical menopause. In human endothelial cells, GPER was expressed on intracellular membranes and mediated eNOS activation and NO formation, partially accounting for estrogen-mediated effects. Chronic treatment with G-1, a synthetic, highly selective small molecule agonist of GPER, reduced postmenopausal atherosclerosis and inflammation without uterotrophic effects. In summary, this study reveals an atheroprotective function of GPER and introduces selective GPER activation as a novel therapeutic approach to inhibit postmenopausal atherosclerosis and inflammation in the absence of uterotrophic activity. PMID:25532911

  17. G protein-coupled estrogen receptor protects from atherosclerosis.

    PubMed

    Meyer, Matthias R; Fredette, Natalie C; Howard, Tamara A; Hu, Chelin; Ramesh, Chinnasamy; Daniel, Christoph; Amann, Kerstin; Arterburn, Jeffrey B; Barton, Matthias; Prossnitz, Eric R

    2014-12-23

    Coronary atherosclerosis and myocardial infarction in postmenopausal women have been linked to inflammation and reduced nitric oxide (NO) formation. Natural estrogen exerts protective effects on both processes, yet also displays uterotrophic activity. Here, we used genetic and pharmacologic approaches to investigate the role of the G protein-coupled estrogen receptor (GPER) in atherosclerosis. In ovary-intact mice, deletion of gper increased atherosclerosis progression, total and LDL cholesterol levels and inflammation while reducing vascular NO bioactivity, effects that were in some cases aggravated by surgical menopause. In human endothelial cells, GPER was expressed on intracellular membranes and mediated eNOS activation and NO formation, partially accounting for estrogen-mediated effects. Chronic treatment with G-1, a synthetic, highly selective small molecule agonist of GPER, reduced postmenopausal atherosclerosis and inflammation without uterotrophic effects. In summary, this study reveals an atheroprotective function of GPER and introduces selective GPER activation as a novel therapeutic approach to inhibit postmenopausal atherosclerosis and inflammation in the absence of uterotrophic activity.

  18. Unfolded protein response activation in cataracts.

    PubMed

    Torres-Bernal, Beatriz E; Torres-Bernal, Luis Fernando; Gutiérrez-Campos, Rafael R; Kershenobich Stalnikowitz, David D; Barba-Gallardo, Luis Fernando; Chayet, Arturo A; Ventura-Juárez, Javier

    2014-10-01

    To analyze the expression of 78 kDa glucose-regulated protein (GRP78) and activating transcription factor 6 (ATF6), 2 factors in the unfolded protein response (UPR), in age-related and diabetes-associated cataract. Universidad Autónoma de Aguascalientes, Aguascalientes, México. Experimental study. The qualitative and quantitative expression of GRP78 and ATF6 were measured in surgical samples from 11 senile cataracts, 9 diabetic-associated cataracts, and 3 normal lenses. Both proteins were detected by immunofluorescence and immunogold-conjugated antibodies. Quantitative morphometry was used to analyze the differences in GRP78 and ATF6 between samples. The Mann-Whitney test was used for statistical analysis. Scanning electron microscopy showed the characteristic organization of fibers in normal lenses with regular alignment and interdigitation between them. On the other hand, lenses from eyes with senile or diabetic cataract showed the same pattern of misalignment and disorganization of the fibers. Both proteins were detected through immunofluorescence in senile and diabetic cataracts, but not in normal lenses. Immunogold-conjugated antibodies and transmission electron microscopy showed that GRP78 and ATF6 grains were 30% higher and 35% higher, respectively, in diabetic cataracts than in senile cataracts (P<.05). These data show for the first time in humans that GRP78 and ATF6 are present in lens fibers of senile cataracts and diabetic cataracts, establishing that the UPR may be important in the process of cataractogenesis. No author has a financial or proprietary interest in any material or method mentioned. Copyright © 2014 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

  19. Immunization with Streptococcal Heme Binding Protein (Shp) Protects Mice Against Group A Streptococcus Infection.

    PubMed

    Zhang, Xiaolan; Song, Yingli; Li, Yuanmeng; Cai, Minghui; Meng, Yuan; Zhu, Hui

    2017-01-01

    Streptococcal heme binding protein (Shp) is a surface protein of the heme acquisition system that is an essential iron nutrient in Group A Streptococcus (GAS). Here, we tested whether Shp immunization protects mice from subcutaneous infection. Mice were immunized subcutaneously with recombinant Shp and then challenged with GAS. The protective effects against GAS challenge were evaluated two weeks after the last immunization. Immunization with Shp elicited a robust IgG response, resulting in high anti-Shp IgG titers in the serum. Immunized mice had a higher survival rate and smaller skin lesions than adjuvant control mice. Furthermore, immunized mice had lower GAS numbers at the skin lesions and in the liver, spleen and lung. Histological analysis with Gram staining showed that GAS invaded the surrounding area of the inoculation sites in the skin in control mice, but not in immunized mice. Thus, Shp immunization enhances GAS clearance and reduces GAS skin invasion and systemic dissemination. These findings indicate that Shp is a protective antigen.

  20. Normal Cellular Prion Protein Protects against Manganese-induced Oxidative Stress and Apoptotic Cell Death

    PubMed Central

    Choi, Christopher J.; Anantharam, Vellareddy; Saetveit, Nathan J.; Houk, Robert. S.; Kanthasamy, Arthi; Kanthasamy, Anumantha G.

    2012-01-01

    The normal prion protein is abundantly expressed in the CNS, but its biological function remains unclear. The prion protein has octapeptide repeat regions that bind to several divalent metals, suggesting that the prion proteins may alter the toxic effect of environmental neurotoxic metals. In the present study, we systematically examined whether prion protein modifies the neurotoxicity of manganese (Mn) by comparing the effect of Mn on mouse neural cells expressing prion protein (PrPC -cells) and prion-knockout (PrPKO -cells). Exposure to Mn (10 μM-1 mM) for 24 hr produced a dose-dependent cytotoxic response in both PrPC -cells and PrPKO -cells. Interestingly, PrPC -cells (EC50 117.6μM) were more resistant to Mn-induced cytotoxicity, as compared to PrPKO -cells (EC50 59.9μM), suggesting a protective role for PrPC against Mn neurotoxicity. Analysis of intracellular Mn levels showed less Mn accumulation in PrPC -cells as compared to PrPKO -cells. Furthermore, Mn-induced mitochondrial depolarization and ROS generation were significantly attenuated in PrPC -cells as compared to PrPKO -cells. Measurement of antioxidant status revealed similar basal levels of glutathione (GSH) in PrPC -cells and PrPKO -cells; however, Mn treatment caused greater depletion of GSH in PrPKO -cells. Mn-induced mitochondrial depolarization and ROS production were followed by time- and dose-dependent activation of the apoptotic cell death cascade involving caspase-9 and -3. Notably, DNA fragmentation induced by both Mn treatment and oxidative stress-inducer hydrogen peroxide (100μM) was significantly suppressed in PrPC -cells as compared to PrPKO -cells. Together, these results demonstrate that prion protein interferes with divalent metal Mn uptake and protects against Mn-induced oxidative stress and apoptotic cell death. PMID:17483122

  1. Acidic chitinase primes the protective immune response to gastrointestinal nematodes.

    PubMed

    Vannella, Kevin M; Ramalingam, Thirumalai R; Hart, Kevin M; de Queiroz Prado, Rafael; Sciurba, Joshua; Barron, Luke; Borthwick, Lee A; Smith, Allen D; Mentink-Kane, Margaret; White, Sandra; Thompson, Robert W; Cheever, Allen W; Bock, Kevin; Moore, Ian; Fitz, Lori J; Urban, Joseph F; Wynn, Thomas A

    2016-05-01

    Acidic mammalian chitinase (AMCase) is known to be induced by allergens and helminths, yet its role in immunity is unclear. Using AMCase-deficient mice, we show that AMCase deficiency reduced the number of group 2 innate lymphoid cells during allergen challenge but was not required for establishment of type 2 inflammation in the lung in response to allergens or helminths. In contrast, AMCase-deficient mice showed a profound defect in type 2 immunity following infection with the chitin-containing gastrointestinal nematodes Nippostrongylus brasiliensis and Heligmosomoides polygyrus bakeri. The impaired immunity was associated with reduced mucus production and decreased intestinal expression of the signature type 2 response genes Il13, Chil3, Retnlb, and Clca1. CD103(+) dendritic cells, which regulate T cell homing, were also reduced in mesenteric lymph nodes of infected AMCase-deficient mice. Thus, AMCase functions as a critical initiator of protective type 2 responses to intestinal nematodes but is largely dispensable for allergic responses in the lung.

  2. Montanide ISA™ 201 adjuvanted FMD vaccine induces improved immune responses and protection in cattle.

    PubMed

    Dar, Pervaiz; Kalaivanan, Ramya; Sied, Nuru; Mamo, Bedaso; Kishore, Subodh; Suryanarayana, V V S; Kondabattula, Ganesh

    2013-07-18

    Despite significant advancements in modern vaccinology, inactivated whole virus vaccines for foot-and-mouth disease (FMD) remain the mainstay for prophylactic and emergency uses. Many efforts are currently devoted to improve the immune responses and protective efficacy of these vaccines. Adjuvants, which are often used to potentiate immune responses, provide an excellent mean to improve the efficacy of FMD vaccines. This study aimed to evaluate three oil adjuvants namely: Montanide ISA-201, ISA-206 (SEPPIC, France) and GAHOL (an in-house developed oil-adjuvant) for adjuvant potential in inactivated FMD vaccine. Groups of cattle (n=6) were immunized once intramuscularly with monovalent FMDV 'O' vaccine formulated in these adjuvants, and humoral (serum neutralizing antibody, IgG1 and IgG2) and cellular (lymphoproliferation) responses were measured. Montanide ISA-201 adjuvanted vaccine induced earlier and higher neutralizing antibody responses as compared to the two other adjuvants. All the adjuvants induced mainly serum IgG1 isotype antibody responses against FMDV. However, Montanide ISA-201 induced relatively higher IgG2 responses than the other two adjuvants. Lymphoproliferative responses to recall FMDV antigen were relatively higher with Montanide ISA-201, although not always statistically significant. On homologous FMDV challenge at 30 days post-vaccination, 100% (6/6) of the cattle immunized with Montanide-201 adjuvanted vaccine were protected, which was superior to those immunized with ISA-206 (66.6%, 4/6) or GAHOL adjuvanted vaccine (50%, 3/6). Virus replication following challenge infection, as determined by presence of the viral genome in oropharynx and non-structural protein serology, was lowest with Montanide ISA-201 adjuvant. Collectively, these results indicate that the Montanide ISA-201 adjuvanted FMD vaccine induces enhanced immune responses and protective efficacy in cattle. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. 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

  4. The Stress Granule RNA-Binding Protein TIAR-1 Protects Female Germ Cells from Heat Shock in Caenorhabditis elegans.

    PubMed

    Huelgas-Morales, Gabriela; Silva-García, Carlos Giovanni; Salinas, Laura S; Greenstein, David; Navarro, Rosa E

    2016-04-07

    In response to stressful conditions, eukaryotic cells launch an arsenal of regulatory programs to protect the proteome. One major protective response involves the arrest of protein translation and the formation of stress granules, cytoplasmic ribonucleoprotein complexes containing the conserved RNA-binding proteins TIA-1 and TIAR. The stress granule response is thought to preserve mRNA for translation when conditions improve. For cells of the germline-the immortal cell lineage required for sexual reproduction-protection from stress is critically important for perpetuation of the species, yet how stress granule regulatory mechanisms are deployed in animal reproduction is incompletely understood. Here, we show that the stress granule protein TIAR-1 protects the Caenorhabditis elegans germline from the adverse effects of heat shock. Animals containing strong loss-of-function mutations in tiar-1 exhibit significantly reduced fertility compared to the wild type following heat shock. Analysis of a heat-shock protein promoter indicates that tiar-1 mutants display an impaired heat-shock response. We observed that TIAR-1 was associated with granules in the gonad core and oocytes during several stressful conditions. Both gonad core and oocyte granules are dynamic structures that depend on translation; protein synthesis inhibitors altered their formation. Nonetheless, tiar-1 was required for the formation of gonad core granules only. Interestingly, the gonad core granules did not seem to be needed for the germ cells to develop viable embryos after heat shock. This suggests that TIAR-1 is able to protect the germline from heat stress independently of these structures.

  5. The Stress Granule RNA-Binding Protein TIAR-1 Protects Female Germ Cells from Heat Shock in Caenorhabditis elegans

    PubMed Central

    Huelgas-Morales, Gabriela; Silva-García, Carlos Giovanni; Salinas, Laura S.; Greenstein, David; Navarro, Rosa E.

    2016-01-01

    In response to stressful conditions, eukaryotic cells launch an arsenal of regulatory programs to protect the proteome. One major protective response involves the arrest of protein translation and the formation of stress granules, cytoplasmic ribonucleoprotein complexes containing the conserved RNA-binding proteins TIA-1 and TIAR. The stress granule response is thought to preserve mRNA for translation when conditions improve. For cells of the germline—the immortal cell lineage required for sexual reproduction—protection from stress is critically important for perpetuation of the species, yet how stress granule regulatory mechanisms are deployed in animal reproduction is incompletely understood. Here, we show that the stress granule protein TIAR-1 protects the Caenorhabditis elegans germline from the adverse effects of heat shock. Animals containing strong loss-of-function mutations in tiar-1 exhibit significantly reduced fertility compared to the wild type following heat shock. Analysis of a heat-shock protein promoter indicates that tiar-1 mutants display an impaired heat-shock response. We observed that TIAR-1 was associated with granules in the gonad core and oocytes during several stressful conditions. Both gonad core and oocyte granules are dynamic structures that depend on translation; protein synthesis inhibitors altered their formation. Nonetheless, tiar-1 was required for the formation of gonad core granules only. Interestingly, the gonad core granules did not seem to be needed for the germ cells to develop viable embryos after heat shock. This suggests that TIAR-1 is able to protect the germline from heat stress independently of these structures. PMID:26865701

  6. Association between Interferon Response and Protective Efficacy of NS1-Truncated Mutants as Influenza Vaccine Candidates in Chickens

    PubMed Central

    Jang, Hyesun; Ngunjiri, John M.; Lee, Chang-Won

    2016-01-01

    Influenza virus mutants that encode C-terminally truncated NS1 proteins (NS1-truncated mutants) are attractive candidates for avian live attenuated influenza vaccine (LAIV) development because they are both attenuated and immunogenic in chickens. We previously showed that a high protective efficacy of NS1-truncated LAIV in chickens corresponds with induction of high levels of type I interferon (IFN) responses in chicken embryonic fibroblast cells. In this study, we investigated the relationship between induction of IFN and IFN-stimulated gene responses in vivo and the immunogenicity and protective efficacy of NS1-truncated LAIV. Our data demonstrates that accelerated antibody induction and protective efficacy of NS1-truncated LAIV correlates well with upregulation of IFN-stimulated genes. Further, through oral administration of recombinant chicken IFN alpha in drinking water, we provide direct evidence that type I IFN can promote rapid induction of adaptive immune responses and protective efficacy of influenza vaccine in chickens. PMID:27257989

  7. Barrier-protective effects of activated protein C in human alveolar epithelial cells.

    PubMed

    Puig, Ferranda; Fuster, Gemma; Adda, Mélanie; Blanch, Lluís; Farre, Ramon; Navajas, Daniel; Artigas, Antonio

    2013-01-01

    Acute lung injury (ALI) is a clinical manifestation of respiratory failure, caused by lung inflammation and the disruption of the alveolar-capillary barrier. Preservation of the physical integrity of the alveolar epithelial monolayer is of critical importance to prevent alveolar edema. Barrier integrity depends largely on the balance between physical forces on cell-cell and cell-matrix contacts, and this balance might be affected by alterations in the coagulation cascade in patients with ALI. We aimed to study the effects of activated protein C (APC) on mechanical tension and barrier integrity in human alveolar epithelial cells (A549) exposed to thrombin. Cells were pretreated for 3 h with APC (50 µg/ml) or vehicle (control). Subsequently, thrombin (50 nM) or medium was added to the cell culture. APC significantly reduced thrombin-induced cell monolayer permeability, cell stiffening, and cell contraction, measured by electrical impedance, optical magnetic twisting cytometry, and traction microscopy, respectively, suggesting a barrier-protective response. The dynamics of the barrier integrity was also assessed by western blotting and immunofluorescence analysis of the tight junction ZO-1. Thrombin resulted in more elongated ZO-1 aggregates at cell-cell interface areas and induced an increase in ZO-1 membrane protein content. APC attenuated the length of these ZO-1 aggregates and reduced the ZO-1 membrane protein levels induced by thrombin. In conclusion, pretreatment with APC reduced the disruption of barrier integrity induced by thrombin, thus contributing to alveolar epithelial barrier protection.

  8. Multiple antioxidant proteins protect Chlorobaculum tepidum against oxygen and reactive oxygen species.

    PubMed

    Li, Hui; Jubelirer, Sara; Garcia Costas, Amaya M; Frigaard, Niels-Ulrik; Bryant, Donald A

    2009-11-01

    The genome of the green sulfur bacterium Chlorobaculum (Cba.) tepidum, a strictly anaerobic photolithoautotroph, is predicted to encode more than ten genes whose products are potentially involved in protection from reactive oxygen species and an oxidative stress response. The encoded proteins include cytochrome bd quinol oxidase, NADH oxidase, rubredoxin oxygen oxidoreductase, several thiol peroxidases, alkyl hydroperoxide reductase, superoxide dismutase, methionine sulfoxide reductase, and rubrerythrin. To test the physiological functions of some of these proteins, ten genes were insertionally inactivated. Wild-type Cba. tepidum cells were very sensitive to oxygen in the light but were remarkably resistant to oxygen in the dark. When wild-type and mutant cells were subjected to air for various times under dark or light condition, significant decreases in viability were detected in most of the mutants relative to wild type. Treatments with hydrogen peroxide (H(2)O(2)), tert-butyl hydroperoxide (t-BOOH) and methyl viologen resulted in more severe effects in most of the mutants than in the wild type. The results demonstrated that these putative antioxidant proteins combine to form an effective defense against oxygen and reactive oxygen species. Reverse-transcriptase polymerase chain reaction studies showed that the genes with functions in oxidative stress protection were constitutively transcribed under anoxic growth conditions.

  9. Immunization with individual proteins of the Lrp/AsnC family induces protection against Brucella melitensis 16M challenges in mice

    PubMed Central

    Wang, Xinhui; An, Chang; Yang, Mingjuan; Li, Xinran; Ke, Yuehua; Lei, Shuangshuang; Xu, Xiaoyang; Yu, Jiuxuan; Ren, Hang; Du, Xinying; Wang, Zhoujia; Qiu, Yefeng; Liu, Bo; Chen, Zeliang

    2015-01-01

    Brucellosis is one of the most common zoonoses worldwide. Subunit vaccines are promising for the prevention of human brucellosis. In our previous protective antigen screening studies, we identified a new protective antigen, BMEI0357, which belongs to the Lrp/asnC protein family, a conserved transcriptional regulator in bacteria that is absent in eukaryotes. In the present study, the Brucella genome annotation was screened and a total of six proteins were identified as members of the Lrp/AsnC family. Lrp/AsnC proteins have two domains that are conserved among the family members. However, sequence similarities between these proteins ranged from 9 to 50%, indicating high sequence heterogeneity. To test whether proteins of this family have similar characteristics, all six proteins were cloned and expressed in Escherichia coli. The recombinant proteins were purified and their protective efficacy was evaluated in BALB/c mice challenged with Brucella melitensis 16M. The results show that all six Lrp/AsnC proteins could induce a protective immune response against Brucella melitensis 16M. Antibodies against the Lrp/AsnC proteins were detected in the immunized mice. However, levels of antibodies against these proteins were relatively variable in human brucellosis sera. Taken together, our results show that these six proteins of the Lrp/AsnC family in Brucella could induce protective immune responses in mice. PMID:26579099

  10. Evaluation of the protective immunity of the Legionella pneumophila recombinant protein FlaA/MompS/PilE in an A/J mouse model.

    PubMed

    Xu, Ying; Guan, Wang; Xu, Jia-nan; Cao, De-ping; Yang, Bin-bin; Chen, Da-li; Chen, Jian-ping

    2011-05-23

    To investigate the protect effects of the recombinant protein FlaA/MompS/PilE against Legionella pneumophila (L. pneumophila), the coding sequences of the three proteins were optimized by DNA Star software firstly, cloned, expressed by Escherichia coli BL21, and purified. To give an enhanced the immunological response, the proteins were linked together with (Linker) or without a linker insert (NLinker) and were purified from E. coli BL21. The A/J mouse model was used to determine the level of the induction of protective immunity from the purified proteins. Our results showed that the IgG titer, which was measured by ELISA, was increased after the administration of the five proteins. Compared to the administration of the individual proteins, the chimeric Linker and NLinker proteins displayed lasting immunity to a lethal dose of L. pneumophila challenge. The Linker protein protected the A/J mouse against a higher dose of L. pneumonia compared to the other proteins used in this study, as it contained a more effective immunogen. The work presented here demonstrates that the bioinformatics software, DNA Star, is a valid tool to analyse the epitopes of proteins and was useful in the optimization of proteins that could induce the protective immune response to L. pneumophila. The cross-immunity of recombinant proteins, such as the Linker and the NLinker chimera, have higher generates a greater immune than the single proteins.

  11. Eliminating a Region of Respiratory Syncytial Virus Attachment Protein Allows Induction of Protective Immunity without Vaccine-enhanced Lung Eosinophilia

    PubMed Central

    Sparer, Tim E.; Matthews, Stephen; Hussell, Tracy; Rae, Aaron J.; Garcia-Barreno, Blanca; Melero, Jose A.; Openshaw, Peter J.M.

    1998-01-01

    In a murine model of respiratory syncytial virus disease, prior sensitization to the attachment glycoprotein (G) leads to pulmonary eosinophilia and enhanced illness. Three different approaches were taken to dissect the region of G responsible for enhanced disease and protection against challenge. First, mutant viruses, containing frameshifts that altered the COOH terminus of the G protein, were used to challenge mice sensitized by scarification with recombinant vaccinia virus (rVV) expressing wild-type G. Second, cDNA expressing these mutated G proteins were expressed by rVV and used to vaccinate mice before challenge with wild-type respiratory syncytial virus (RSV). These studies identified residues 193–205 to be responsible for G-induced weight loss and lung eosinophilia and showed that this region was not was not necessary for induction of protective immunity. Third, mice were sensitized using an rVV that expressed only amino acids 124–203 of the G protein. Upon RSV challenge, mice sensitized with this rVV developed enhanced weight loss and eosinophilia. This is the first time that a region within RSV (amino acids 193–203) has been shown to be responsible for induction of lung eosinophilia and disease enhancement. Moreover, we now show that it is possible to induce protective immunity with an altered G protein without inducing a pathological response. PMID:9607931

  12. Immune Responses and Protection of Aotus Monkeys Immunized with Irradiated Plasmodium vivax Sporozoites

    PubMed Central

    Jordán-Villegas, Alejandro; Perdomo, Anilza Bonelo; Epstein, Judith E.; López, Jesús; Castellanos, Alejandro; Manzano, María R.; Hernández, Miguel A.; Soto, Liliana; Méndez, Fabián; Richie, Thomas L.; Hoffman, Stephen L.; Arévalo-Herrera, Myriam; Herrera, Sócrates

    2011-01-01

    A non-human primate model for the induction of protective immunity against the pre-erythrocytic stages of Plasmodium vivax malaria using radiation-attenuated P. vivax sporozoites may help to characterize protective immune mechanisms and identify novel malaria vaccine candidates. Immune responses and protective efficacy induced by vaccination with irradiated P. vivax sporozoites were evaluated in malaria-naive Aotus monkeys. Three groups of six monkeys received two, five, or ten intravenous inoculations, respectively, of 100,000 irradiated P. vivax sporozoites; control groups received either 10 doses of uninfected salivary gland extract or no inoculations. Immunization resulted in the production low levels of antibodies that specifically recognized P. vivax sporozoites and the circumsporozoite protein. Additionally, immunization induced low levels of antigen-specific IFN-γ responses. Intravenous challenge with viable sporozoites resulted in partial protection in a dose-dependent manner. These findings suggest that the Aotus monkey model may be able to play a role in preclinical development of P. vivax pre-erythrocytic stage vaccines. PMID:21292877

  13. Monocyte cytokine synthesis in response to extracellular cell stress proteins suggests these proteins exhibit network behaviour.

    PubMed

    Kaiser, Frank; Steptoe, Andrew; Thompson, Stephen; Henderson, Brian

    2014-01-01

    Human peripheral blood monocytes were exposed to single or pairs of cell stress proteins (CSPs), specifically Hsp10, Hsp27, Hsp60 and Hsp70-the former two having anti-inflammatory actions while the latter pair being assumed to be pro-inflammatory in activity. This study was to test if these proteins exhibited any network behaviour. To control for possible lipopolysaccharide contamination, polymyxin B was used. Surprisingly, at concentrations higher than 1 μg/ml, polymyxin B itself could induce cytokine synthesis. A number of commercial sources of the molecular chaperones were tested, and marked variations in monocyte cytokine synthesis were found. All four CSPs stimulated the same profile of IL-1/IL-6 synthesis and IL-10/TNF-α synthesis although the kinetics of production of these two pairs of cytokines were very different. A key question was whether extracellular molecular chaperones exhibited network behaviour. To test this, monocytes were cultured with suboptimal concentrations of single CSP and pairs of CSP to look for additive, synergistic or antagonistic cell responses. The major finding was that pairs of molecular chaperones, including chaperones thought to stimulate monocyte cytokine synthesis, could produce significant antagonistic cellular responses. This demonstrates that extracellular CSPs constitute an additional potent layer within the complex cytokine network and furthermore suggests that monocytes have evolved to dampen their immune responses upon exposure to extracellular networks of CSPs-perhaps as a mechanism for protecting cells against detrimental cellular stress responses.

  14. Acute phase proteins response in hunting dogs.

    PubMed

    Casella, Stefania; Fazio, Francesco; Russo, Carmelo; Giudice, Elisabetta; Piccione, Giuseppe

    2013-09-01

    Haptoglobin (Hp), serum amyloid A (SAA), C-reactive protein (CRP), and white blood cells (WBC) were assessed in 20 dogs divided into 2 groups. The dogs of group A were not subjected to hunting exercise (control group), while the dogs of group B were subjected to hunting exercise for 3 hr (experimental group). Blood samples were collected from each animal before hunting (T0), immediately after 3 hr of hunting (T1), and after 1 hr of recovery (T2). The general linear model (GLM) repeated measures procedure showed a significant difference between the 2 groups (P < 0.0001) and a significant rise (P < 0.0001) in concentration of Hp, SAA, and CRP after hunting exercise, with a consequent decline during recovery period in group B. These parameters could be considered valid and easily obtainable biomarkers in relation to hunting stress in dogs. Additional studies will continue to elucidate the magnitude and the time of response of other acute phase proteins.

  15. The Unfolded Protein Response and Gastrointestinal Disease

    PubMed Central

    Kaser, Arthur; Adolph, Timon Erik; Blumberg, Richard S

    2013-01-01

    As the inner lining of the gastrointestinal tract, the intestinal epithelium serves an essential role in innate immune function at the interface between the host and microbiota. Given the unique environmental challenges and thus physiologic secretory functions of this surface, it is exquisitely sensitive to perturbations that affect its capacity to resolve endoplasmic reticulum (ER) stress. Genetic deletion of factors involved in the unfolded protein response (UPR), which functions in the resolution of ER stress that arises from misfolded proteins, result in spontaneous intestinal inflammation closely mimicking human inflammatory bowel disease (IBD). This is demonstrated by observations wherein deletion of genes such as Xbp1 and Agr2 profoundly affects the intestinal epithelium and results in spontaneous intestinal inflammation. Moreover, both genes, along with others (e.g. ORDML3) represent genetic risk factors for human IBD, both Crohn’s disease and ulcerative colitis. Here we review the current mechanistic understanding for how unresolved ER stress can lead to intestinal inflammation, and highlight the findings that implicate ER stress as a genetically affected biological pathway in IBD. We further discuss environmental and microbial factors that might impact on the epithelium’s capacity to resolve ER stress, and which may constitute exogenous factors that may precipitate disease in genetically susceptible individuals. PMID:23588234

  16. HDAC6 controls major cell response pathways to cytotoxic accumulation of protein aggregates

    PubMed Central

    Boyault, Cyril; Zhang, Yu; Fritah, Sabrina; Caron, Cécile; Gilquin, Benoit; Kwon, So Hee; Garrido, Carmen; Yao, Tso-Pang; Vourc’h, Claire; Matthias, Patrick; Khochbin, Saadi

    2007-01-01

    A cellular defense mechanism counteracts the deleterious effects of misfolded protein accumulation by eliciting a stress response. The cytoplasmic deacetylase HDAC6 (histone deacetylase 6) was previously shown to be a key element in this response by coordinating the clearance of protein aggregates through aggresome formation and their autophagic degradation. Here, for the first time, we demonstrate that HDAC6 is involved in another crucial cell response to the accumulation of ubiquitinated protein aggregates, and unravel its molecular basis. Indeed, our data show that HDAC6 senses ubiquitinated cellular aggregates and consequently induces the expression of major cellular chaperones by triggering the dissociation of a repressive HDAC6/HSF1 (heat-shock factor 1)/HSP90 (heat-shock protein 90) complex and a subsequent HSF1 activation. HDAC6 therefore appears as a master regulator of the cell protective response to cytotoxic protein aggregate formation. PMID:17785525

  17. Platelets protect lung from injury induced by systemic inflammatory response

    PubMed Central

    Luo, Shuhua; Wang, Yabo; An, Qi; Chen, Hao; Zhao, Junfei; Zhang, Jie; Meng, Wentong; Du, Lei

    2017-01-01

    Systemic inflammatory responses can severely injure lungs, prompting efforts to explore how to attenuate such injury. Here we explored whether platelets can help attenuate lung injury in mice resulting from extracorporeal circulation (ECC)-induced systemic inflammatory responses. Mice were subjected to ECC for 30 min, then treated with phosphate-buffered saline, platelets, the GPIIb/IIIa inhibitor Tirofiban, or the combination of platelets and Tirofiban. Blood and lung tissues were harvested 60 min later, and lung injury and inflammatory status were assessed. As expected, ECC caused systemic inflammation and pulmonary dysfunction, and platelet transfusion resulted in significantly milder lung injury and higher lung function. It also led to greater numbers of circulating platelet-leukocyte aggregates and greater platelet accumulation in the lung. Platelet transfusion was associated with higher production of transforming growth factor-β and as well as lower levels of tumour necrosis factor-α and neutrophil elastase in plasma and lung. None of these platelet effects was observed in the presence of Tirofiban. Our results suggest that, at least under certain conditions, platelets can protect lung from injury induced by systemic inflammatory responses. PMID:28155889

  18. The Unfolded Protein Response Supports Plant Development and Defense as well as Responses to Abiotic Stress

    PubMed Central

    Bao, Yan; Howell, Stephen H.

    2017-01-01

    The unfolded protein response (UPR) is a stress response conserved in eukaryotic organisms and activated by the accumulation of misfolded proteins in the endoplasmic reticulum (ER). Adverse environmental conditions disrupt protein folding in the ER and trigger the UPR. Recently, it was found that the UPR can be elicited in the course of plant development and defense. During vegetative plant development, the UPR is involved in normal root growth and development, the effect of which can be largely attributed to the influence of the UPR on plant hormone biology. The UPR also functions in plant reproductive development by protecting male gametophyte development from heat stress. In terms of defense, the UPR has been implicated in virus and microbial defense. Viral defense represents a double edge sword in that various virus infections activate the UPR, however, in a number of cases, the UPR actually supports viral infections. The UPR also plays a role in plant immunity to bacterial infections, again through the action of plant hormones in regulating basal immunity responses. PMID:28360918

  19. Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge

    PubMed Central

    Konduru, Krishnamurthy; Shurtleff, Amy C.; Bradfute, Steven B.; Nakamura, Siham; Bavari, Sina; Kaplan, Gerardo

    2016-01-01

    Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105−106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deleted EBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-length GP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc, and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. Our data further support

  20. Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge

    DOE PAGES

    Konduru, Krishnamurthy; Shurtleff, Amy C.; Bradfute, Steven B.; ...

    2016-09-13

    Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulatedmore » with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105–106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deletedEBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-lengthGP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc,and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. In conclusion, our

  1. Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge

    SciTech Connect

    Konduru, Krishnamurthy; Shurtleff, Amy C.; Bradfute, Steven B.; Nakamura, Siham; Bavari, Sina; Kaplan, Gerardo; Tsuji, Moriya

    2016-09-13

    Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105–106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deletedEBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-lengthGP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc,and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with

  2. 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

  3. 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.

  4. Smart responsive phosphorescent materials for data recording and security protection.

    PubMed

    Sun, Huibin; Liu, Shujuan; Lin, Wenpeng; Zhang, Kenneth Yin; Lv, Wen; Huang, Xiao; Huo, Fengwei; Yang, Huiran; Jenkins, Gareth; Zhao, Qiang; Huang, Wei

    2014-04-07

    Smart luminescent materials that are responsive to external stimuli have received considerable interest. Here we report ionic iridium (III) complexes simultaneously exhibiting mechanochromic, vapochromic and electrochromic phosphorescence. These complexes share the same phosphorescent iridium (III) cation with a N-H moiety in the N^N ligand and contain different anions, including hexafluorophosphate, tetrafluoroborate, iodide, bromide and chloride. The anionic counterions cause a variation in the emission colours of the complexes from yellow to green by forming hydrogen bonds with the N-H proton. The electronic effect of the N-H moiety is sensitive towards mechanical grinding, solvent vapour and electric field, resulting in mechanochromic, vapochromic and electrochromic phosphorescence. On the basis of these findings, we construct a data-recording device and demonstrate data encryption and decryption via fluorescence lifetime imaging and time-gated luminescence imaging techniques. Our results suggest that rationally designed phosphorescent complexes may be promising candidates for advanced data recording and security protection.

  5. Glycaemic Response to Quality Protein Maize Grits

    PubMed Central

    Panlasigui, Leonora N.; Bayaga, Cecile L. T.; Barrios, Erniel B.; Cochon, Kim L.

    2010-01-01

    Background. Carbohydrates have varied rates of digestion and absorption that induces different hormonal and metabolic responses in the body. Given the abundance of carbohydrate sources in the Philippines, the determination of the glycaemic index (GI) of local foods may prove beneficial in promoting health and decreasing the risk of diabetes in the country. Methods. The GI of Quality Protein Maize (QPM) grits, milled rice, and the mixture of these two food items were determined in ten female subjects. Using a randomized crossover design, the control bread and three test foods were given on separate occasions after an overnight fast. Blood samples were collected through finger prick at time intervals of 0, 15, 30, 45, 60, 90, and 120 min and analyzed for glucose concentrations. Results. The computed incremental area under the glucose response curve (IAUC) varies significantly across test foods (P < .0379) with the pure QPM grits yielding the lowest IAUC relative to the control by 46.38. Resulting GI values of the test foods (bootstrapped) were 80.36 (SEM 14.24), 119.78 (SEM 18.81), and 93.17 (SEM 27.27) for pure QPM grits, milled rice, and rice-QPM grits mixture, respectively. Conclusion. Pure QPM corn grits has a lower glycaemic response compared to milled rice and the rice-corn grits mixture, which may be related in part to differences in their dietary fibre composition and physicochemical characteristics. Pure QPM corn grits may be a more health beneficial food for diabetic and hyperlipidemic individuals. PMID:20862364

  6. Cellular prion protein protects from inflammatory and neuropathic pain.

    PubMed

    Gadotti, Vinicius M; Zamponi, Gerald W

    2011-08-16

    Cellular prion protein (PrPC) inhibits N-Methyl-D-Aspartate (NMDA) receptors. Since NMDA receptors play an important role in the transmission of pain signals in the dorsal horn of spinal cord, we thus wanted to determine if PrPC null mice show a reduced threshold for various pain behaviours.We compared nociceptive thresholds between wild type and PrPC null mice in models of inflammatory and neuropathic pain, in the presence and the absence of a NMDA receptor antagonist. 2-3 months old male PrPC null mice exhibited an MK-801 sensitive decrease in the paw withdrawal threshold in response both mechanical and thermal stimuli. PrPC null mice also exhibited significantly longer licking/biting time during both the first and second phases of formalin-induced inflammation of the paw, which was again prevented by treatment of the mice with MK-801, and responded more strongly to glutamate injection into the paw. Compared to wild type animals, PrPC null mice also exhibited a significantly greater nociceptive response (licking/biting) after intrathecal injection of NMDA. Sciatic nerve ligation resulted in MK-801 sensitive neuropathic pain in wild-type mice, but did not further augment the basal increase in pain behaviour observed in the null mice, suggesting that mice lacking PrPC may already be in a state of tonic central sensitization. Altogether, our data indicate that PrPC exerts a critical role in modulating nociceptive transmission at the spinal cord level, and fit with the concept of NMDA receptor hyperfunction in the absence of PrPC.

  7. Dichotomy of protective cellular immune responses to human visceral leishmaniasis.

    PubMed

    Khalil, E A G; Ayed, N B; Musa, A M; Ibrahim, M E; Mukhtar, M M; Zijlstra, E E; Elhassan, I M; Smith, P G; Kieny, P M; Ghalib, H W; Zicker, F; Modabber, F; Elhassan, A M

    2005-05-01

    Healing/protective responses in human visceral leishmaniasis (VL) are associated with stimulation/production of Th1 cytokines, such as interferon IFN-gamma, and conversion in the leishmanin skin test (LST). Such responses were studied for 90 days in 44 adult healthy volunteers from VL non-endemic areas, with no past history of VL/cutaneous leishmaniasis (CL) and LST non-reactivity following injection with one of four doses of Alum-precipitated autoclaved Leishmania major (Alum/ALM) +/- bacille Calmette-Guerin (BCG), a VL candidate vaccine. The vaccine was well tolerated with minimal localized side-effects and without an increase in antileishmanial antibodies or interleukin (IL)-5. Five volunteers (5/44; 11.4%) had significant IFN-gamma production by peripheral blood mononuclear cells (PBMCs) in response to Leishmania antigens in their prevaccination samples (P = 0.001) but were LST non-reactive. On day 45, more than half the volunteers (26/44; 59.0%) had significantly high LST indurations (mean 9.2 +/- 2.7 mm) and high IFN-gamma levels (mean 1008 +/- 395; median 1247 pg/ml). Five volunteers had significant L. donovani antigen-induced IFN-gamma production (mean 873 +/- 290; median 902; P = 0.001), but were non-reactive in LST. An additional five volunteers (5/44; 11.4%) had low IFN-gamma levels (mean 110 +/- 124 pg/ml; median 80) and were non-reactive in LST (induration = 00 mm). The remaining eight volunteers had low IFN-gamma levels, but significant LST induration (mean 10 +/- 2.9 mm; median 11). By day 90 the majority of volunteers (27/44; 61.4%) had significant LST induration (mean 10.8 +/- 9.9 mm; P < 0.001), but low levels of L. donovani antigen-induced IFN-gamma (mean 66.0 +/- 62 pg/ml; P > 0.05). Eleven volunteers (11/44; 25%) had significantly high levels of IFN-gamma and LST induration, while five volunteers had low levels of IFN-gamma (<100 pg/ml) and no LST reactivity (00 mm). One volunteer was lost to follow-up. In conclusion, it is hypothesized that

  8. Iron regulatory protein-1 protects against mitoferrin-1-deficient porphyria.

    PubMed

    Chung, Jacky; Anderson, Sheila A; Gwynn, Babette; Deck, Kathryn M; Chen, Michael J; Langer, Nathaniel B; Shaw, George C; Huston, Nicholas C; Boyer, Leah F; Datta, Sumon; Paradkar, Prasad N; Li, Liangtao; Wei, Zong; Lambert, Amy J; Sahr, Kenneth; Wittig, Johannes G; Chen, Wen; Lu, Wange; Galy, Bruno; Schlaeger, Thorsten M; Hentze, Matthias W; Ward, Diane M; Kaplan, Jerry; Eisenstein, Richard S; Peters, Luanne L; Paw, Barry H

    2014-03-14

    Mitochondrial iron is essential for the biosynthesis of heme and iron-sulfur ([Fe-S]) clusters in mammalian cells. In developing erythrocytes, iron is imported into the mitochondria by MFRN1 (mitoferrin-1, SLC25A37). Although loss of MFRN1 in zebrafish and mice leads to profound anemia, mutant animals showed no overt signs of porphyria, suggesting that mitochondrial iron deficiency does not result in an accumulation of protoporphyrins. Here, we developed a gene trap model to provide in vitro and in vivo evidence that iron regulatory protein-1 (IRP1) inhibits protoporphyrin accumulation. Mfrn1(+/gt);Irp1(-/-) erythroid cells exhibit a significant increase in protoporphyrin levels. IRP1 attenuates protoporphyrin biosynthesis by binding to the 5'-iron response element (IRE) of alas2 mRNA, inhibiting its translation. Ectopic expression of alas2 harboring a mutant IRE, preventing IRP1 binding, in Mfrn1(gt/gt) cells mimics Irp1 deficiency. Together, our data support a model whereby impaired mitochondrial [Fe-S] cluster biogenesis in Mfrn1(gt/gt) cells results in elevated IRP1 RNA-binding that attenuates ALAS2 mRNA translation and protoporphyrin accumulation.

  9. Conformational Epitopes Recognized by Protective Anti-Neisserial Surface Protein A Antibodies

    PubMed Central

    Hou, Victor C.; Moe, Gregory R.; Raad, Zyde; Wuorimaa, Tomi; Granoff, Dan M.

    2003-01-01

    NspA is a conserved membrane protein that elicits protective antibody responses in mice against Neisseria meningitidis. A recent crystallographic study showed that NspA adopts an eight-stranded β-barrel structure when reconstituted in detergent. In order to define the segments of NspA-containing epitopes recognized by protective murine anti-NspA antibodies, we studied the binding of two bactericidal and protective anti-NspA monoclonal antibodies (MAbs), AL12 and 14C7. Neither MAb binds to overlapping synthetic peptides (10-mers, 12-mers, and cyclic 12-mers) corresponding to the entire mature sequence of NspA, or to denatured recombinant NspA (rNspA), although binding to the protein can be restored by refolding in liposomes. Based on the ability of the two MAbs to bind to Escherichia coli microvesicles prepared from a set of rNspA variants created by site-specific mutagenesis, the most important contacts between the MAbs and NspA appear to be located within the LGG segment of loop 3. The conformation of loop 2 also appears to be an important determinant, as particular combinations of residues in this segment resulted in loss of antibody binding. Thus, the two anti-NspA MAbs recognize discontinuous conformational epitopes that result from the close proximity of loops 2 and 3 in the three-dimensional structure of NspA. The data suggest that optimally immunogenic vaccines using rNspA will require formulations that permit proper folding of the protein. PMID:14638771

  10. ABCB10 depletion reduces unfolded protein response in mitochondria.

    PubMed

    Yano, Masato

    2017-04-29

    Mitochondria have many functions, including ATP generation. The electron transport chain (ETC) and the coupled ATP synthase generate ATP by consuming oxygen. Reactive oxygen species (ROS) are also produced by ETC, and ROS damage deoxyribonucleic acids, membrane lipids and proteins. Recent analysis indicate that mitochondrial unfolded protein response (UPR(mt)), which enhances expression of mitochondrial chaperones and proteases to remove damaged proteins, is activated when damaged proteins accumulate in the mitochondria. In Caenorhabditis elegans, HAF-1, a putative ortholog of human ABCB10, plays an essential role in signal transduction from mitochondria to nuclei to enhance UPR(mt). Therefore, it is possible that ABCB10 has a role similar to that of HAF-1. However, it has not been reported whether ABCB10 is a factor in the signal transduction pathway to enhance UPR(mt). In this study, ABCB10 was depleted in HepG2 cells using small interfering RNA (siRNA), and the effect was examined. ABCB10 depletion upregulated ROS and the expression of ROS-detoxifying enzymes (SOD2, GSTA1, and GSTA2), and SESN3, a protein induced by ROS to protect the cell from oxidative stress. In addition, ABCB10 depletion significantly decreased expression of UPR(mt)-related mitochondrial chaperones (HSPD1 and DNAJA3), and a mitochondrial protease (LONP1). However, the putative activity of ABCB10 to export peptides from mitochondria was not lost by ABCB10 depletion. Altogether, these data suggest that ABCB10 is involved in UPR(mt) signaling pathway similar to that of HAF-1, although ABCB10 probably does not participate in peptide export from mitochondria. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Protective Capacity of the Human Anamnestic Antibody Response during Acute Dengue Virus Infection

    PubMed Central

    Xu, Meihui; Züst, Roland; Toh, Ying Xiu; Pfaff, Jennifer M.; Kahle, Kristen M.; Davidson, Edgar; Doranz, Benjamin J.; Velumani, Sumathy; Tukijan, Farhana; Wang, Cheng-I

    2016-01-01

    ABSTRACT Half of the world's population is exposed to the risk of dengue virus infection. Although a vaccine for dengue virus is now available in a few countries, its reported overall efficacy of about 60% is not ideal. Protective immune correlates following natural dengue virus infection remain undefined, which makes it difficult to predict the efficacy of new vaccines. In this study, we address the protective capacity of dengue virus-specific antibodies that are produced by plasmablasts a few days after natural secondary infection. Among a panel of 18 dengue virus-reactive human monoclonal antibodies, four groups of antibodies were identified based on their binding properties. While antibodies targeting the fusion loop of the glycoprotein of dengue virus dominated the antibody response, two smaller groups of antibodies bound to previously undescribed epitopes in domain II of the E protein. The latter, largely serotype-cross-reactive antibodies, demonstrated increased stability of binding at pH 5. These antibodies possessed weak to moderate neutralization capacity in vitro but were the most efficacious in promoting the survival of infected mice. Our data suggest that the cross-reactive anamnestic antibody response has a protective capacity despite moderate neutralization in vitro and a moderate decrease of viremia in vivo. IMPORTANCE Antibodies can protect from symptomatic dengue virus infection. However, it is not easy to assess which classes of antibodies provide protection because in vitro assays are not always predictive of in vivo protection. During a repeat infection, dengue virus-specific immune memory cells are reactivated and large amounts of antibodies are produced. By studying antibodies cloned from patients with heterologous secondary infection, we tested the protective value of the serotype-cross-reactive “recall” or “anamnestic” response. We found that results from in vitro neutralization assays did not always correlate with the ability of

  12. Addressing membrane protein topology using the fluorescence protease protection (FPP) assay.

    PubMed

    Lorenz, Holger; Hailey, Dale W; Lippincott-Schwartz, Jennifer

    2008-01-01

    Determining a protein's correct topological distribution within the cell is essential for understanding the proper functioning of many proteins. Here, we describe a fluorescence-based technique, termed FPP for fluorescence protease protection, to determine protein topology in living cells. The FPP assay uses the restricted proteolytic digestibility of green fluorescent protein-tagged membrane proteins to reveal their intramembrane orientation. Membrane protein topology can be assessed using this technique for proteins residing in organelles as diverse as the Golgi apparatus, the endoplasmic reticulum (ER), peroxisomes, mitochondria, and autophagosomes. To illustrate the technique, we describe its use for deciphering the topology of a membrane protein in the ER.

  13. Maresin 1 Mitigates Inflammatory Response and Protects Mice from Sepsis

    PubMed Central

    Li, Ruidong; Wang, Yaxin; Ma, Zhijun; Ma, Muyuan; Wang, Di; Xie, Gengchen; Yin, Yuping

    2016-01-01

    Sepsis, frequently caused by infection of bacteria, is considered as an uncontrollable systematic inflammation response syndrome (SIRS). Maresin 1 (Mar1) is a new proresolving mediator with potent anti-inflammatory effect in several animal models. However, its effect in sepsis is still not investigated. To address this question, we developed sepsis model in BALB/c mice by cecal ligation and puncture (CLP) with or without Mar1 treatment. Our data showed that Mar1 markedly improved survival rate and decreased the levels of proinflammatory cytokines in CLP mice such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β). Furthermore, Mar1 reduced serum level of lipopolysaccharide (LPS) and enhanced the bacteria clearance in mice sepsis model. Moreover, Mar1 attenuated lung injury and decreased level of alanine transaminase (ALT), aspartate transaminase (AST), creatinine (Cre), and blood urea nitrogen (BUN) in serum in mice after CLP surgery. Treatment with Mar1 inhibited activation of nuclear factor kappa B (NF-κb) pathway. In conclusion, Mar1 exhibited protective effect in sepsis by reducing LPS, bacteria burden in serum, inhibiting inflammation response, and improving vital organ function. The possible mechanism is partly involved in inhibition of NF-κb activation. PMID:28042205

  14. S-Bacillithiolation Protects Conserved and Essential Proteins Against Hypochlorite Stress in Firmicutes Bacteria

    PubMed Central

    Chi, Bui Khanh; Roberts, Alexandra A.; Huyen, Tran Thi Thanh; Bäsell, Katrin; Becher, Dörte; Albrecht, Dirk; Hamilton, Chris J.

    2013-01-01

    Abstract Aims: Protein S-bacillithiolations are mixed disulfides between protein thiols and the bacillithiol (BSH) redox buffer that occur in response to NaOCl in Bacillus subtilis. We used BSH-specific immunoblots, shotgun liquid chromatography (LC)–tandem mass spectrometry (MS/MS) analysis and redox proteomics to characterize the S-bacillithiolomes of B. subtilis, B. megaterium, B. pumilus, B. amyloliquefaciens, and Staphylococcus carnosus and also measured the BSH/oxidized bacillithiol disulfide (BSSB) redox ratio after NaOCl stress. Results: In total, 54 proteins with characteristic S-bacillithiolation (SSB) sites were identified, including 29 unique proteins and eight proteins conserved in two or more of these bacteria. The methionine synthase MetE is the most abundant S-bacillithiolated protein in Bacillus species after NaOCl exposure. Further, S-bacillithiolated proteins include the translation elongation factor EF-Tu and aminoacyl-tRNA synthetases (ThrS), the DnaK and GrpE chaperones, the two-Cys peroxiredoxin YkuU, the ferredoxin–NADP+ oxidoreductase YumC, the inorganic pyrophosphatase PpaC, the inosine-5′-monophosphate dehydrogenase GuaB, proteins involved in thiamine biosynthesis (ThiG and ThiM), queuosine biosynthesis (QueF), biosynthesis of aromatic amino acids (AroA and AroE), serine (SerA), branched-chain amino acids (YwaA), and homocysteine (LuxS and MetI). The thioredoxin-like proteins, YphP and YtxJ, are S-bacillithiolated at their active sites, suggesting a function in the de-bacillithiolation process. S-bacillithiolation is accompanied by a two-fold increase in the BSSB level and a decrease in the BSH/BSSB redox ratio in B. subtilis. Innovation: Many essential and conserved proteins, including the dominant MetE, were identified in the S-bacillithiolome of different Bacillus species and S. carnosus using shotgun-LC-MS/MS analyses. Conclusion: S-bacillithiolation is a widespread redox control mechanism among Firmicutes bacteria that protects

  15. Protective responses induced by herbicide safeners in wheat.

    PubMed

    Taylor, Victoria L; Cummins, Ian; Brazier-Hicks, Melissa; Edwards, Robert

    2013-04-01

    Safeners are agrochemicals which enhance tolerance to herbicides in cereals including wheat (Triticum aestivum L.) by elevating the expression of xenobiotic detoxifying enzymes, such as glutathione transferases (GSTs). When wheat plants were spray-treated with three safener chemistries, namely cloquintocet mexyl, mefenpyr diethyl and fenchlorazole ethyl, an apparently identical subset of GSTs derived from the tau, phi and lambda classes accumulated in the foliage. Treatment with the closely related mefenpyr diethyl and fenchlorazole ethyl enhanced seedling shoot growth, but this effect was not determined with the chemically unrelated cloquintocet mexyl. Focussing on cloquintocet mexyl, treatments were found to only give a transient induction of GSTs, with the period of elevation being dose dependent. Examining the role of safener metabolism in controlling these responses, it was determined that cloquintocet mexyl was rapidly hydrolysed to the respective carboxylic acid. Studies with cloquintocet showed that the acid was equally effective at inducing GSTs as the ester and appeared to be the active safener. Studies on the tissue induction of GSTs showed that whilst phi and tau class enzymes were induced in all tissues, the induction of the lambda enzymes was restricted to the meristems. To test the potential protective effects of cloquintocet mexyl in wheat on chemicals other than herbicides, seeds were pre-soaked in safeners prior to sowing on soil containing oil and a range of heavy metals. Whilst untreated seeds were unable to germinate on the contaminated soil, safener treatments resulted in seedlings briefly growing before succumbing to the pollutants. Our results show that safeners exert a range of protective and growth promoting activities in wheat that extend beyond enhancing tolerance to herbicides.

  16. Protective responses induced by herbicide safeners in wheat

    PubMed Central

    Taylor, Victoria L.; Cummins, Ian; Brazier-Hicks, Melissa; Edwards, Robert

    2013-01-01

    Safeners are agrochemicals which enhance tolerance to herbicides in cereals including wheat (Triticum aestivum L.) by elevating the expression of xenobiotic detoxifying enzymes, such as glutathione transferases (GSTs). When wheat plants were spray-treated with three safener chemistries, namely cloquintocet mexyl, mefenpyr diethyl and fenchlorazole ethyl, an apparently identical subset of GSTs derived from the tau, phi and lambda classes accumulated in the foliage. Treatment with the closely related mefenpyr diethyl and fenchlorazole ethyl enhanced seedling shoot growth, but this effect was not determined with the chemically unrelated cloquintocet mexyl. Focussing on cloquintocet mexyl, treatments were found to only give a transient induction of GSTs, with the period of elevation being dose dependent. Examining the role of safener metabolism in controlling these responses, it was determined that cloquintocet mexyl was rapidly hydrolysed to the respective carboxylic acid. Studies with cloquintocet showed that the acid was equally effective at inducing GSTs as the ester and appeared to be the active safener. Studies on the tissue induction of GSTs showed that whilst phi and tau class enzymes were induced in all tissues, the induction of the lambda enzymes was restricted to the meristems. To test the potential protective effects of cloquintocet mexyl in wheat on chemicals other than herbicides, seeds were pre-soaked in safeners prior to sowing on soil containing oil and a range of heavy metals. Whilst untreated seeds were unable to germinate on the contaminated soil, safener treatments resulted in seedlings briefly growing before succumbing to the pollutants. Our results show that safeners exert a range of protective and growth promoting activities in wheat that extend beyond enhancing tolerance to herbicides. PMID:23564986

  17. Stress proteins and glial cell functions during chronic aluminium exposures: protective role of curcumin.

    PubMed

    Sood, Pooja Khanna; Nahar, Uma; Nehru, Bimla

    2012-03-01

    Involved in the ongoing debate is the speculation that aluminium is somehow toxic for neurons. Glial cells cope up to protect neurons from this toxic insult by maintaining the glutathione homeostasis. Of late newer and newer roles of glial cells have been depicted. The present work looks into the other regulatory mechanisms that show the glial cells response to pro-oxidant effects of aluminium exposure. In the present investigation we have evaluated the inflammatory responses of the glial cells as well as HSP70-induction during aluminium exposure. Further, the protective role of curcumin is also evaluated. Aluminium was administered by oral gavage at a dose level of 100 mg/kg b.wt/day for a period of 8 weeks. Curcumin was administered i.p. at a dose of 50 mg/kg b.wt./day on alternate days. Enhanced gene and protein expression of HSP70 in the glial fractions of the aluminium exposed animals as compared to the corresponding neuronal population. Aluminium exposure resulted in a significant increase in the NF-κB and TNF-α expression suggesting inflammatory responses. In the conjunctive treatment group of aluminium and curcumin exposure marked reduction in the gene and protein expression of NF-κB and TNF-α was observed. This was further reflected in histopathological studies showing no evidence of inflammation in conjunctive group as compared to aluminium treatment. From the present study, it can be concluded that curcumin has a potential anti-inflammatory action and can be exploited in other toxicological conditions also.

  18. Immunity to Staphylococcus aureus Secreted Proteins Protects Rabbits from Serious Illnesses

    PubMed Central

    Spaulding, Adam. R.; Lin, Ying-Chi; Merriman, Joseph A.; Brosnahan, Amanda J.; Peterson, Marnie L.; Schlievert, Patrick M.

    2012-01-01

    Staphylococcus aureus causes significant illnesses throughout the world, including toxic shock syndrome (TSS), pneumonia, and infective endocarditis. Major contributors to S. aureus illnesses are secreted virulence factors it produces, including superantigens and cytolysins. This study investigates the use of superantigens and cytolysins as staphylococcal vaccine candidates. Importantly, 20% of humans and 50% of rabbits in our TSS model cannot generate antibody responses to native superantigens. We generated three TSST-1 mutants; G31S/S32P, H135A, and Q136A. All rabbits administered these TSST-1 toxoids generated strong antibody responses (titers>10,000) that neutralized native TSST-1 in TSS models, both in vitro and in vivo. These TSST-1 mutants lacked detectable residual toxicity. Additionally, the TSST-1 mutants exhibited intrinsic adjuvant activity, increasing antibody responses to a second staphylococcal antigen (β-toxin). This effect may be due to TSST-1 mutants binding to the immune co-stimulatory molecule CD40. The superantigens TSST-1 and SEC and the cytolysin α-toxin are known to contribute to staphylococcal pneumonia. Immunization of rabbits against these secreted toxins provided complete protection from highly lethal challenge with a USA200 S. aureus strain producing all three exotoxins; USA200 strains are common causes of staphylococcal infections. The same three exotoxins plus the cytolysins β-toxin and γ-toxin contribute to infective endocarditis and sepsis caused by USA200 strains. Immunization against these five exotoxins protected rabbits from infective endocarditis and lethal sepsis. These data suggest that immunization against toxoid proteins of S. aureus exotoxins protects from serious illnesses, and concurrently superantigen toxoid mutants provide endogenous adjuvant activity. PMID:22691432

  19. Update on maximal anabolic response to dietary protein.

    PubMed

    Kim, Il-Young; Deutz, Nicolaas E P; Wolfe, Robert R

    2017-06-01

    The anabolic response to dietary protein can be defined as the difference between protein synthesis and breakdown, or the net protein balance, in response to ingestion of protein alone or a mixed meal containing protein. Others have concluded that a maximal anabolic response can be achieved with ingestion of 20-35 g of a high quality protein, leading to the formulation of a popular concept that the maximal anabolic response can be achieved by distributing the total protein intake evenly throughout the day, rather than eating a majority of dietary protein with dinner. However, this concept was based entirely on the measurement of muscle protein synthesis and thus ignored the potential contributions of suppression of protein breakdown to the anabolic response, as well as the possibility that tissues and organs other than muscle may also play a role in the anabolic response. In this review we discuss the factors comprising the total anabolic response, discuss relevant methodological issues, derive a theoretical maximal anabolic response based on current literature values, and interpret recent papers addressing the issue of maximal anabolic response as well as meal distribution of dietary protein. We conclude that it is not likely that there is a practical limit to the maximal anabolic response to a single meal, and the most efficient way in which to maximize the total anabolic response over a 24-h period is to increase dietary protein at breakfast and lunch without reducing protein intake with dinner. Copyright © 2017 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

  20. Efficacy of heterologous and homologous heat shock protein 70s as protective agents to Artemia franciscana challenged with Vibrio campbellii.

    PubMed

    Baruah, Kartik; Ranjan, Jayant; Sorgeloos, Patrick; Bossier, Peter

    2010-11-01

    The Hsp70 class of heat shock proteins (Hsps) has been implicated at multiple points in the immune response of both vertebrates and invertebrates. This class of chaperones is highly conserved in both sequence and structure, from prokaryotes to higher eukaryotes. In view of their high degree of homology, it was assumed that these Hsp70 proteins derived either from the prokaryotes or eukaryotes would have similar functions, especially in relation to their protective ability in a challenge assay. To verify this, we compared two evolutionary diverse Hsp70s, Artemia Hsp70 and Escherichia coli Hsp70 equivalent DnaK (each overproduced in E.coli), for their ability to protect Artemia against Vibrio challenge. Results showed that Artemia fed with E. coli producing Artemia Hsp70 or DnaK proteins, as assessed by immune-probing in western blots, survived better in a Vibrio challenge assay. The observed effects could be due to enhancement of the Artemia immune system as phenoloxidase activity was found to be increased by these proteins. These two Hsp70 proteins exhibit a high degree of homology, particularly in the peptide-binding domain (the putative innate immunity-activating portion) with 59.6% identity, indicating that the observed protective capacity of homologous or heterologous Hsp70 proteins might reside within this peptide-binding domain.

  1. Immunization with Pneumococcal Surface Protein K of Nonencapsulated Streptococcus pneumoniae Provides Protection in a Mouse Model of Colonization.

    PubMed

    Keller, Lance E; Luo, Xiao; Thornton, Justin A; Seo, Keun-Seok; Moon, Bo Youn; Robinson, D Ashley; McDaniel, Larry S

    2015-11-01

    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.

  2. Protective activity of the CnaBE3 domain conserved among Staphylococcus aureus Sdr proteins.

    PubMed

    Becherelli, Marco; Prachi, Prachi; Viciani, Elisa; Biagini, Massimiliano; Fiaschi, Luigi; Chiarot, Emiliano; Nosari, Sarah; Brettoni, Cecilia; Marchi, Sara; Biancucci, Marco; Fontana, Maria Rita; Montagnani, Francesca; Bagnoli, Fabio; Barocchi, Michèle A; Manetti, Andrea G O

    2013-01-01

    Staphylococcus aureus is an opportunistic pathogen, commensal of the human skin and nares, but also responsible for invasive nosocomial as well as community acquired infections. Staphylococcus aureus adheres to the host tissues by means of surface adhesins, such as SdrC, SdrD, and SdrE proteins. The Sdr family of proteins together with a functional A domain, contain respectively two, three or five repeated sequences called B motifs which comprise the CnaB domains. SdrD and SdrE proteins were reported to be protective in animal models against invasive diseases or lethal challenge with human clinical S. aureus isolates. In this study we identified a 126 amino acid sequence containing a CnaB domain, conserved among the three Sdr proteins. The three fragments defined here as CnaBC2, D5 and E3 domains even though belonging to phylogenetically distinct strains, displayed high sequence similarity. Based on the sequence conservation data, we selected the CnaBE3 domain for further analysis and characterization. Polyclonal antibodies raised against the recombinant CnaBE3 domain recognized SdrE, SdrC and SdrD proteins of different S. aureus lineages. Moreover, we demonstrated that the CnaBE3 domain was expressed in vivo during S. aureus infections, and that immunization of this domain alone significantly reduces the bacterial load in mice challenged with S. aureus. Furthermore, we show that the reduction of bacteria by CnaBE3 vaccination is due to functional antibodies. Finally, we demonstrated that the region of the SdrE protein containing the CnaBE3 domain was resistant to trypsin digestion, a characteristic often associated with the presence of an isopeptide bond.

  3. Balancing Immune Protection and Immune Pathology by CD8+ T-Cell Responses to Influenza Infection

    PubMed Central

    Duan, Susu; Thomas, Paul G.

    2016-01-01

    Influenza A virus (IAV) is a significant human pathogen causing annual epidemics and periodic pandemics. CD8+ cytotoxic T lymphocyte (CTL)-mediated immunity contributes to the clearance of virus-infected cells, and CTL immunity targeting the conserved internal proteins of IAVs is a key protection mechanism when neutralizing antibodies are absent during heterosubtypic IAV infection. However, CTL infiltration into the airways, its cytotoxicity, and the effects of produced proinflammatory cytokines can cause severe lung tissue injury, thereby contributing to immunopathology. Studies have discovered complicated and exquisite stimulatory and inhibitory mechanisms that regulate CTL magnitude and effector activities during IAV infection. Here, we review the state of knowledge on the roles of IAV-specific CTLs in immune protection and immunopathology during IAV infection in animal models, highlighting the key findings of various requirements and constraints regulating the balance of immune protection and pathology involved in CTL immunity. We also discuss the evidence of cross-reactive CTL immunity as a positive correlate of cross-subtype protection during secondary IAV infection in both animal and human studies. We argue that the effects of CTL immunity on protection and immunopathology depend on multiple layers of host and viral factors, including complex host mechanisms to regulate CTL magnitude and effector activity, the pathogenic nature of the IAV, the innate response milieu, and the host historical immune context of influenza infection. Future efforts are needed to further understand these key host and viral factors, especially to differentiate those that constrain optimally effective CTL antiviral immunity from those necessary to restrain CTL-mediated non-specific immunopathology in the various contexts of IAV infection, in order to develop better vaccination and therapeutic strategies for modifying protective CTL immunity. PMID:26904022

  4. Recent findings about the Yersinia enterocolitica phage shock protein response.

    PubMed

    Yamaguchi, Saori; Darwin, Andrew J

    2012-02-01

    The phage shock protein (Psp) system is a conserved extracytoplasmic stress response in bacteria that is essential for virulence of the human pathogen Yersinia enterocolitica. This article summarizes some recent findings about Y. enterocolitica Psp system function. Increased psp gene expression requires the transcription factor PspF, but under non-inducing conditions PspF is inhibited by an interaction with another protein, PspA, in the cytoplasm. A Psp-inducing stimulus causes PspA to relocate to the cytoplasmic membrane, freeing PspF to induce psp gene expression. This PspA relocation requires the integral cytoplasmic membrane proteins, PspB and PspC, which might sense an inducing trigger and sequester PspA by direct interaction. The subsequent induction of psp gene expression increases the PspA concentration, which also allows it to contact the membrane directly, perhaps for its physiological function. Mutational analysis of the PspB and PspC proteins has revealed that they both positively and negatively regulate psp gene expression and has also identified PspC domains associated with each function. We also compare the contrasting physiological roles of the Psp system in the virulence of Y. enterocolitica and Salmonella enterica sv. Typhimurium (S. Typhimurium). In S. Typhimurium, PspA maintains the proton motive force, which provides the energy needed to drive ion importers required for survival within macrophages. In contrast, in the extracellular pathogen Y. enterocolitica, PspB and PspC, but not PspA, are the Psp components needed for virulence. PspBC protect Y. enterocolitica from damage caused by the secretin component of its type 3 secretion system, an essential virulence factor.

  5. Recent Findings about the Yersinia enterocolitica Phage Shock Protein Response

    PubMed Central

    Yamaguchi, Saori; Darwin, Andrew J.

    2012-01-01

    The phage shock protein (Psp) system is a conserved extracytoplasmic stress response in bacteria that is essential for virulence of the human pathogen Yersinia enterocolitica. This article summarizes some recent findings about Y. enterocolitica Psp system function. Increased psp gene expression requires the transcription factor PspF, but under non-inducing conditions PspF is inhibited by an interaction with another protein, PspA, in the cytoplasm. A Psp-inducing stimulus causes PspA to relocate to the cytoplasmic membrane, freeing PspF to induce psp gene expression. This PspA relocation requires the integral cytoplasmic membrane proteins, PspB and PspC, which might sense an inducing trigger and sequester PspA by direct interaction. The subsequent induction of psp gene expression increases the PspA concentration, which also allows it to contact the membrane directly, perhaps for its physiological function. Mutational analysis of the PspB and PspC proteins has revealed that they both positively and negatively regulate psp gene expression and has also identified PspC domains associated with each function. We also compare the contrasting physiological roles of the Psp system in the virulence of Y. enterocolitica and Salmonella enterica sv. Typhimurium (S. Typhimurium). In S. Typhimurium, PspA maintains the proton motive force, which provides the energy needed to drive ion importers required for survival within macrophages. In contrast, in the extracellular pathogen Y. enterocolitica, PspB and PspC, but not PspA, are the Psp components needed for virulence. PspBC protect Y. enterocolitica from damage caused by the secretin component of its type 3 secretion system, an essential virulence factor. PMID:22367931

  6. Potential protective immunogenicity of tetanus toxoid, diphtheria toxoid and Cross Reacting Material 197 (CRM197) when used as carrier proteins in glycoconjugates.

    PubMed

    Bröker, Michael

    2016-03-03

    When tetanus toxoid (TT), diphtheria toxoid (DT) or Cross Reacting Material 197 (CRM197), a non-toxic diphtheria toxin mutant protein, are used as carrier proteins in glycoconjugate vaccines, these carriers induce a protein specific antibody response as measured by in vitro assays. Here, it was evaluated whether or not glycoconjugates based on TT, DT or CRM197 can induce a protective immune response as measured by potency tests according to the European Pharmacopoeia. It could be shown, that the conjugate carriers TT and DT can induce a protective immune response against a lethal challenge by toxins in animals, while glycoconjugates based on CRM197 failed to induce a protective immune response. Opportunities for new applications of glycoconjugates are discussed.

  7. Vaccination with multimeric L2 fusion protein and L1 VLP or Capsomeres to broaden protection against HPV infection

    PubMed Central

    Jagu, Subhashini; Kwak, Kihyuck; Garcea, Robert L; Roden, Richard BS

    2010-01-01

    Immunization with L1 as pentavalent capsomeres or virus-like particles (VLPs) generates high and long-lived titers of neutralizing antibodies and protection primarily against the human papillomavirus (HPV) type from which the vaccine was derived. Conversely, vaccination with L2 minor capsid protein derived from multiple HPV types induces lower titer, but more broadly neutralizing and protective antibody responses. We combined the advantages of each protective antigen by immunization with titrated doses of multi-type L2 with either L1 capsomeres or VLP. We observed no significant interference between the L1 and L2 antibody response upon co-administration of L1 vaccines with multi-type L2 vaccines. PMID:20434552

  8. Recombinant Phage Elicits Protective Immune Response against Systemic S. globosa Infection in Mouse Model

    PubMed Central

    Chen, Feng; Jiang, Rihua; Wang, Yicun; Zhu, Mingji; Zhang, Xu; Dong, Shuai; Shi, Hongxi; Wang, Li

    2017-01-01

    Sporothrix globosa is a type of fungus that typically infects immunocompromised patients. Its prevention continues to pose a challenge. A 70-KDa glycoprotein (Gp70) of Sporothrix has been previously reported to protect host against infection from this fungus. Here, we displayed an epitope peptide (kpvqhalltplgldr) of Gp70 on the major coat protein (pIII), and investigated its efficiency as a vaccine for preventing S. globosa infection. The recombinant phage and the heat-killed S. globosa were used to immunize mice separately. In this study, we evaluated the humoral and cellular immune responses in the mice and demonstrated that recombinant phage could induce mice to produce a stronger immune response and generate antibodies to inhibit S. globosa infection. Furthermore, immunization with recombinant phage could increase the survival rate of S. globosa infection in mice. All these results together indicated that recombinant phages displaying kpvqhalltplgldr are a potential vaccine candidate against S. globosa infection. PMID:28165018

  9. α/β-hydrolase domain containing protein 15 (ABHD15)--an adipogenic protein protecting from apoptosis.

    PubMed

    Walenta, Evelyn; Pessentheiner, Ariane R; Pelzmann, Helmut J; Deutsch, Alexander; Goeritzer, Madeleine; Kratky, Dagmar; Hackl, Hubert; Oh, Da Young; Prokesch, Andreas; Bogner-Strauss, Juliane G

    2013-01-01

    Our knowledge about adipocyte metabolism and development is steadily growing, yet many players are still undefined. Here, we show that α/β-hydrolase domain containing protein 15 (Abhd15) is a direct and functional target gene of peroxisome proliferator-activated receptor gamma (PPARγ), the master regulator of adipogenesis. In line, Abhd15 is mainly expressed in brown and white adipose tissue and strongly upregulated during adipogenesis in various murine and human cell lines. Stable knockdown of Abhd15 in 3T3-L1 cells evokes a striking differentiation defect, as evidenced by low lipid accumulation and decreased expression of adipocyte marker genes. In preconfluent cells, knockdown of Abhd15 leads to impaired proliferation, which is caused by apoptosis, as we see an increased SubG1 peak, caspase 3/7 activity, and BAX protein expression as well as a reduction in anti-apoptotic BCL-2 protein. Furthermore, apoptosis-inducing amounts of palmitic acid evoke a massive increase of Abhd15 expression, proposing an apoptosis-protecting role for ABHD15. On the other hand, in mature adipocytes physiological (i.e. non-apoptotic) concentrations of palmitic acid down-regulate Abhd15 expression. Accordingly, we found that the expression of Abhd15 in adipose tissue is reduced in physiological situations with high free fatty acid levels, like high-fat diet, fasting, and aging as well as in genetically obese mice. Collectively, our results position ABHD15 as an essential component in the development of adipocytes as well as in apoptosis, thereby connecting two substantial factors in the regulation of adipocyte number and size. Together with its intricate regulation by free fatty acids, ABHD15 might be an intriguing new target in obesity and diabetes research.

  10. SALIVARY ANTIMICROBIAL PROTEIN RESPONSE TO PROLONGED RUNNING

    PubMed Central

    Kuennen, M.; Gourley, C.; Schneider, S.; Dokladny, K.; Moseley, P.

    2013-01-01

    Introduction Prolonged exercise may compromise immunity through a reduction of salivary antimicrobial proteins (AMPs). Salivary IgA (IgA) has been extensively studied, but little is known about the effect of acute, prolonged exercise on AMPs including lysozyme (Lys) and lactoferrin (Lac). Objective To determine the effect of a 50-km trail race on salivary cortisol (Cort), IgA, Lys, and Lac. Methods 14 subjects: (6 females, 8 males) completed a 50km ultramarathon. Saliva was collected pre, immediately after (post) and 1.5 hrs post race (+1.5). Results Lac concentration was higher at +1.5 hrs post race compared to post exercise (p < 0.05). Lys was unaffected by the race (p > 0.05). IgA concentration, secretion rate, and IgA/Osm were lower +1.5 hrs post compared to pre race (p < 0.05). Cort concentration was higher at post compared to +1.5 (p < 0.05), but was unaltered from pre race levels. Subjects finished in 7.81±1.2 hrs. Saliva flow rate did not differ between time points. Saliva Osm increased at post (p < 0.05) compared to pre race. Conclusions The intensity could have been too low to alter Lys and Lac secretion rates and thus, may not be as sensitive as IgA to changes in response to prolonged running. Results expand our understanding of the mucosal immune system and may have implications for predicting illness after prolonged running. PMID:24744458

  11. The Immune Response to Sand Fly Salivary Proteins and Its Influence on Leishmania Immunity

    PubMed Central

    Gomes, Regis; Oliveira, Fabiano

    2012-01-01

    Leishmaniasis is a vector-borne disease transmitted by bites of phlebotomine sand flies. During Leishmania transmission, sand fly saliva is co-inoculated with parasites into the skin of the mammalian host. Sand fly saliva consists of roughly thirty different salivary proteins, many with known roles linked to blood feeding facilitation. Apart from the anti-hemostatic capacity of saliva, several sand fly salivary proteins have been shown to be immunogenic. Immunization with a single salivary protein or exposure to uninfected bites was shown to result in a protective immune response against leishmaniasis. Antibodies to saliva were not required for this protection. A strong body of evidence points to the role for saliva-specific T cells producing IFN-γ in the form of a delayed-type hypersensitivity reaction at the bite site as the main protective response. Herein, we review the immunity to sand fly salivary proteins in the context of its vector–parasite–host combinations and their vaccine potential, as well as some recent advances to shed light on the mechanism of how an immune response to sand fly saliva protects against leishmaniasis. PMID:22593758

  12. Protein A Suppresses Immune Responses during Staphylococcus aureus Bloodstream Infection in Guinea Pigs

    PubMed Central

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

    2015-01-01

    ABSTRACT   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. Importance  Staphylococcus aureus is the leading cause of soft tissue and bloodstream infections; however, a vaccine with clinical efficacy is not available. Using mice to model staphylococcal infection, earlier work identified protective antigens; however, corresponding human clinical trials did not reach their endpoints. We show that B cell receptor (IgM) cross-linking by protein A is an important immune evasion strategy of S. aureus that can be monitored in a guinea pig model of bloodstream infection. Further, immunization with nontoxigenic protein A enables infected guinea pigs to elicit antibody responses that are protective against S. aureus. Thus, the guinea pig model may support preclinical development of staphylococcal vaccines. PMID:25564466

  13. Spliced X-box binding protein 1 couples the unfolded protein response to hexosamine biosynthetic pathway.

    PubMed

    Wang, Zhao V; Deng, Yingfeng; Gao, Ningguo; Pedrozo, Zully; Li, Dan L; Morales, Cyndi R; Criollo, Alfredo; Luo, Xiang; Tan, Wei; Jiang, Nan; Lehrman, Mark A; Rothermel, Beverly A; Lee, Ann-Hwee; Lavandero, Sergio; Mammen, Pradeep P A; Ferdous, Anwarul; Gillette, Thomas G; Scherer, Philipp E; Hill, Joseph A

    2014-03-13

    The hexosamine biosynthetic pathway (HBP) generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Spliced X-box Binding Protein 1 Couples the Unfolded Protein Response to Hexosamine Biosynthetic Pathway

    PubMed Central

    Wang, Zhao V.; Deng, Yingfeng; Gao, Ningguo; Pedrozo, Zully; Li, Dan L.; Morales, Cyndi R.; Criollo, Alfredo; Luo, Xiang; Tan, Wei; Jiang, Nan; Lehrman, Mark A.; Rothermel, Beverly A.; Lee, Ann-Hwee; Lavandero, Sergio; Mammen, Pradeep P.A.; Ferdous, Anwarul; Gillette, Thomas G.; Scherer, Philipp E.; Hill, Joseph A.

    2014-01-01

    SUMMARY The hexosamine biosynthetic pathway (HBP) generates UDP-GlcNAc (uridine diphosphate N-acetylglucosamine) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis, by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress. PMID:24630721

  15. Protective role for heat shock protein-reactive alpha beta T cells in murine yersiniosis.

    PubMed Central

    Noll, A; Roggenkamp, A; Heesemann, J; Autenrieth, I B

    1994-01-01

    To investigate the role of heat shock proteins (HSP) of Yersinia enterocolitica for the host immune response against this pathogen, we cloned and expressed a 60-kDa HSP of Y. enterocolitica serotype O8. A fragment of Y. enterocolitica O8 HSP60 encoded by amino acids 90 to 286 was sequenced and showed more than 90% homology with HSP60 of Y. enterocolitica O3 and GroEL of Escherichia coli and 59% homology with HSP65 of Mycobacterium bovis. The arthritogenic T-cell epitope of mycobacterial HSP65 (amino acid residues 180 to 188) was not found on Yersinia HSP60. To determine whether Yersinia HSP60 is an immunodominant antigen, the immune responses of Yersinia-infected C57BL/6 mice were analyzed. Yersinia-infected mice evolved a significant serum antibody and splenic T-cell response against Yersinia HSP60. CD4+ alpha beta T-cell clones which were generated from splenic T cells isolated from either Yersinia-infected or Yersinia HSP60-immunized mice, recognized both heat-killed Yersinia serotypes O3 and O8 as well as recombinant Yersinia HSP60 but not heat-killed Yersinia pseudotuberculosis, Salmonella typhimurium, or recombinant HSP65 of Mycobacterium bovis. The adoptive transfer of HSP60-reactive T-cell clones mediated significant protection against a lethal infection with Y. enterocolitica O8. These results indicate that HSP60 of Y. enterocolitica is an immunodominant antigen which is recognized by both antibodies and CD4+ alpha beta T cells. Moreover, this is the first report providing direct evidence that microbial HSP may elicit a protective immune response which is not associated with autoimmunity. Images PMID:7911784

  16. Dexamethasone protection of rat intestinal epithelial cells against oxidant injury is mediated by induction of heat shock protein 72.

    PubMed Central

    Urayama, S; Musch, M W; Retsky, J; Madonna, M B; Straus, D; Chang, E B

    1998-01-01

    Although the therapeutic actions of glucocorticoids are largely attributed to their anti-inflammatory and immunosuppressive effects, they have been implicated in enhancing tissue and cellular protection. In this study, we demonstrate that dexamethasone significantly enhances viability of IEC-18 rat small intestinal cells against oxidant-induced stress in a dose-dependent fashion. This protective action is mediated by induction of hsp72, the major inducible heat shock protein in intestinal epithelial cells. Dexamethasone stimulates a time- and dose-dependent response in hsp72 protein expression that parallels its effects on cell viability. Furthermore, the induction of hsp72 is tissue dependent, as nonintestinal epithelioid HeLa cells show differential induction of hsp72 expression in response to the same dexamethasone treatment. Antisense hsp72 cDNA transfection of IEC-18 cells abolishes the dexamethasone-induced hsp72 response, without significantly affecting constitutive expression of its homologue, hsc73. Dexamethasone treatment also significantly induces hsp72 protein expression in rat intestinal mucosal cells in vivo. These data demonstrate that glucocorticoids protect intestinal epithelial cells against oxidant-induced stress by inducing hsp72. PMID:9819372

  17. Topical CpG Adjuvantation of a Protein-Based Vaccine Induces Protective Immunity to Listeria monocytogenes

    PubMed Central

    Cheng, Wing Ki; Wee, Kathleen; Kollmann, Tobias R.

    2014-01-01

    Robust CD8+ T cell responses are essential for immune protection against intracellular pathogens. Using parenteral administration of ovalbumin (OVA) protein as a model antigen, the effect of the Toll-like receptor 9 (TLR9) agonist, CpG oligodeoxynucleotide (ODN) 1826, as an adjuvant delivered either topically, subcutaneously, or intramuscularly on antigen-specific CD8+ T cell responses in a mouse model was evaluated. Topical CpG adjuvant increased the frequency of OVA-specific CD8+ T cells in the peripheral blood and in the spleen. The more effective strategy to administer topical CpG adjuvant to enhance CD8+ T cell responses was single-dose administration at the time of antigen injection with a prime-boost regimen. Topical CpG adjuvant conferred both rapid and long-lasting protection against systemic challenge with recombinant Listeria monocytogenes expressing the cytotoxic T lymphocyte (CTL) epitope of OVA257–264 (strain Lm-OVA) in a TLR9-dependent manner. Topical CpG adjuvant induced a higher proportion of CD8+ effector memory T cells than parenteral administration of the adjuvant. Although traditional vaccination strategies involve coformulation of antigen and adjuvant, split administration using topical adjuvant is effective and has advantages of safety and flexibility. Split administration of topical CpG ODN 1826 with parenteral protein antigen is superior to other administration strategies in enhancing both acute and memory protective CD8+ T cell immune responses to subcutaneous protein vaccines. This vaccination strategy induces rapid and persistent protective immune responses against the intracellular organism L. monocytogenes. PMID:24391136

  18. A protein-truncating R179X variant in RNF186 confers protection against ulcerative colitis

    PubMed Central

    Rivas, Manuel A.; Graham, Daniel; Sulem, Patrick; Stevens, Christine; Desch, A. Nicole; Goyette, Philippe; Gudbjartsson, Daniel; Jonsdottir, Ingileif; Thorsteinsdottir, Unnur; Degenhardt, Frauke; Mucha, Sören; Kurki, Mitja I.; Li, Dalin; D'Amato, Mauro; Annese, Vito; Vermeire, Severine; Weersma, Rinse K.; Halfvarson, Jonas; Paavola-Sakki, Paulina; Lappalainen, Maarit; Lek, Monkol; Cummings, Beryl; Tukiainen, Taru; Haritunians, Talin; Halme, Leena; Koskinen, Lotta L. E.; Ananthakrishnan, Ashwin N.; Luo, Yang; Heap, Graham A.; Visschedijk, Marijn C.; Barrett, J; de Lange, K; Edwards, C; Hart, A; Hawkey, C; Jostins, L; Kennedy, N; Lamb, C; Lee, J; Lees, C; Mansfield, J; Mathew, C; Mowatt, C; Newman, W; Nimmo, E; Parkes, M; Pollard, M; Prescott, N; Randall, J; Rice, D; Satsangi, J; Simmons, A; Tremelling, M; Uhlig, H; Wilson, D; Abraham, C; Achkar, J.P; Bitton, A; Boucher, G; Croitoru, K; Fleshner, P; Glas, J; Kugathasan, S; Limbergen, J.V; Milgrom, R; Proctor, D; Regueiro, M; Schumm, P.L; Sharma, Y; Stempak, J.M; Targan, S.R; Wang, M.H; MacArthur, Daniel G.; Neale, Benjamin M.; Ahmad, Tariq; Anderson, Carl A.; Brant, Steven R.; Duerr, Richard H.; Silverberg, Mark S.; Cho, Judy H; Palotie, Aarno; Saavalainen, Päivi; Kontula, Kimmo; Färkkilä, Martti; McGovern, Dermot P. B.; Franke, Andre; Stefansson, Kari; Rioux, John D.; Xavier, Ramnik J.; Daly, Mark J.; Barrett, J.; de Lane, K.; Edwards, C.; Hart, A.; Hawkey, C.; Jostins, L.; Kennedy, N.; Lamb, C.; Lee, J.; Lees, C.; Mansfield, J.; Mathew, C.; Mowatt, C.; Newman, B.; Nimmo, E.; Parkes, M.; Pollard, M.; Prescott, N.; Randall, J.; Rice, D.; Satsangi, J.; Simmons, A.; Tremelling, M.; Uhlig, H.; Wilson, D.; Abraham, C.; Achkar, J. P.; Bitton, A.; Boucher, G.; Croitoru, K.; Fleshner, P.; Glas, J.; Kugathasan, S.; Limbergen, J. V.; Milgrom, R.; Proctor, D.; Regueiro, M.; Schumm, P. L.; Sharma, Y.; Stempak, J. M.; Targan, S. R.; Wang, M. H.

    2016-01-01

    Protein-truncating variants protective against human disease provide in vivo validation of therapeutic targets. Here we used targeted sequencing to conduct a search for protein-truncating variants conferring protection against inflammatory bowel disease exploiting knowledge of common variants associated with the same disease. Through replication genotyping and imputation we found that a predicted protein-truncating variant (rs36095412, p.R179X, genotyped in 11,148 ulcerative colitis patients and 295,446 controls, MAF=up to 0.78%) in RNF186, a single-exon ring finger E3 ligase with strong colonic expression, protects against ulcerative colitis (overall P=6.89 × 10−7, odds ratio=0.30). We further demonstrate that the truncated protein exhibits reduced expression and altered subcellular localization, suggesting the protective mechanism may reside in the loss of an interaction or function via mislocalization and/or loss of an essential transmembrane domain. PMID:27503255

  19. Aldose Reductase Inhibitor Protects against Hyperglycemic Stress by Activating Nrf2-Dependent Antioxidant Proteins

    PubMed Central

    Shukla, Kirtikar; Pal, Pabitra Bikash; Sonowal, Himangshu; Srivastava, Satish K.

    2017-01-01

    We have shown earlier that pretreatment of cultured cells with aldose reductase (AR) inhibitors prevents hyperglycemia-induced mitogenic and proinflammatory responses. However, the effects of AR inhibitors on Nrf2-mediated anti-inflammatory responses have not been elucidated yet. We have investigated how AR inhibitor fidarestat protects high glucose- (HG-) induced cell viability changes by increasing the expression of Nrf2 and its dependent phase II antioxidant enzymes. Fidarestat pretreatment prevents HG (25 mM)-induced Thp1 monocyte viability. Further, treatment of Thp1 monocytes with fidarestat caused a time-dependent increase in the expression as well as the DNA-binding activity of Nrf2. In addition, fidarestat augmented the HG-induced Nrf2 expression and activity and also upregulated the expression of Nrf2-dependent proteins such as hemeoxygenase-1 (HO1) and NQO1 in Thp1 cells. Similarly, treatment with AR inhibitor also induced the expression of Nrf2 and HO1 in STZ-induced diabetic mice heart and kidney tissues. Further, AR inhibition increased the HG-induced expression of antioxidant enzymes such as SOD and catalase and activation of AMPK-α1 in Thp1 cells. Our results thus suggest that pretreatment with AR inhibitor prepares the monocytes against hyperglycemic stress by overexpressing the Nrf2-dependent antioxidative proteins. PMID:28740855

  20. Nuclear protein 1 promotes pancreatic cancer development and protects cells from stress by inhibiting apoptosis

    PubMed Central

    Hamidi, Tewfik; Algül, Hana; Cano, Carla Eliana; Sandi, Maria José; Molejon, Maria Inés; Riemann, Marc; Calvo, Ezequiel Luis; Lomberk, Gwen; Dagorn, Jean-Charles; Weih, Falk; Urrutia, Raul; Schmid, Roland Michael; Iovanna, Juan Lucio

    2012-01-01

    Pancreatic ductal adenocarcinoma (PDAC) has the lowest survival rate of all cancers and shows remarkable resistance to cell stress. Nuclear protein 1 (Nupr1), which mediates stress response in the pancreas, is frequently upregulated in pancreatic cancer. Here, we report that Nupr1 plays an essential role in pancreatic tumorigenesis. In a mouse model of pancreatic cancer with constitutively expressed oncogenic KrasG12D, we found that loss of Nupr1 protected from the development of pancreatic intraepithelial neoplasias (PanINs). Further, in cultured pancreatic cells, nutrient deprivation activated Nupr1 expression, which we found to be required for cell survival. We found that Nupr1 protected cells from stress-induced death by inhibiting apoptosis through a pathway dependent on transcription factor RelB and immediate early response 3 (IER3). NUPR1, RELB, and IER3 proteins were coexpressed in mouse PanINs from KrasG12D-expressing pancreas. Moreover, pancreas-specific deletion of Relb in a KrasG12D background resulted in delayed in PanIN development associated with a lack of IER3 expression. Thus, efficient PanIN formation was dependent on the expression of Nupr1 and Relb, with likely involvement of IER3. Finally, in patients with PDAC, expression of NUPR1, RELB, and IER3 was significantly correlated with a poor prognosis. Cumulatively, these results reveal a NUPR1/RELB/IER3 stress-related pathway that is required for oncogenic KrasG12D-dependent transformation of the pancreas. PMID:22565310

  1. Putative function of hypothetical proteins expressed by Clostridium perfringens type A strains and their protective efficacy in mouse model.

    PubMed

    Alam, Syed Imteyaz; Dwivedi, Pratistha

    2016-10-01

    The whole genome sequencing and annotation of Clostridium perfringens strains revealed several genes coding for proteins of unknown function with no significant similarities to genes in other organisms. Our previous studies clearly demonstrated that hypothetical proteins CPF_2500, CPF_1441, CPF_0876, CPF_0093, CPF_2002, CPF_2314, CPF_1179, CPF_1132, CPF_2853, CPF_0552, CPF_2032, CPF_0438, CPF_1440, CPF_2918, CPF_0656, and CPF_2364 are genuine proteins of C. perfringens expressed in high abundance. This study explored the putative role of these hypothetical proteins using bioinformatic tools and evaluated their potential as putative candidates for prophylaxis. Apart from a group of eight hypothetical proteins (HPs), a putative function was predicted for the rest of the hypothetical proteins using one or more of the algorithms used. The phylogenetic analysis did not suggest an evidence of a horizontal gene transfer event except for HP CPF_0876. HP CPF_2918 is an abundant extracellular protein, unique to C. perfringens species with maximum strain coverage and did not show any significant match in the database. CPF_2918 was cloned, recombinant protein was purified to near homogeneity, and probing with mouse anti-CPF_2918 serum revealed surface localization of the protein in C. perfringens ATCC13124 cultures. The purified recombinant CPF_2918 protein induced antibody production, a mixed Th1 and Th2 kind of response, and provided partial protection to immunized mice in direct C. perfringens challenge. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Protective contributions against invasive Streptococcus pneumoniae pneumonia of antibody and Th17-cell responses to nasopharyngeal colonisation.

    PubMed

    Cohen, Jonathan M; Khandavilli, Suneeta; Camberlein, Emilie; Hyams, Catherine; Baxendale, Helen E; Brown, Jeremy S

    2011-01-01

    The nasopharyngeal commensal bacteria Streptococcus pneumoniae is also a frequent cause of serious infections. Nasopharyngeal colonisation with S. pneumoniae inhibits subsequent re-colonisation by inducing Th17-cell adaptive responses, whereas vaccination prevents invasive infections by inducing antibodies to S. pneumoniae capsular polysaccharides. In contrast, protection against invasive infection after nasopharyngeal colonisation with mutant S. pneumoniae strains was associated with antibody responses to protein antigens. The role of colonisation-induced Th17-cell responses during subsequent invasive infections is unknown. Using mouse models, we show that previous colonisation with S. pneumoniae protects against subsequent lethal pneumonia mainly by preventing bacteraemia with a more modest effect on local control of infection within the lung. Previous colonisation resulted in CD4-dependent increased levels of Th17-cell cytokines during subsequent infectious challenge. However, mice depleted of CD4 cells prior to challenge remained protected against bacteraemia, whereas no protection was seen in antibody deficient mice and similar protection could be achieved through passive transfer of serum. Serum from colonised mice but not antibody deficient mice promoted phagocytosis of S. pneumoniae, and previously colonised mice were able to rapidly clear S. pneumoniae from the blood after intravenous inoculation. Thus, despite priming for a Th17-cell response during subsequent infection, the protective effects of prior colonisation in this model was not dependent on CD4 cells but on rapid clearance of bacteria from the blood by antibody-mediated phagocytosis. These data suggest that whilst nasopharyngeal colonisation induces a range of immune responses, the effective protective responses depend upon the site of subsequent infection.

  3. Protective Contributions against Invasive Streptococcus pneumoniae Pneumonia of Antibody and Th17-Cell Responses to Nasopharyngeal Colonisation

    PubMed Central

    Cohen, Jonathan M.; Khandavilli, Suneeta; Camberlein, Emilie; Hyams, Catherine; Baxendale, Helen E.; Brown, Jeremy S.

    2011-01-01

    The nasopharyngeal commensal bacteria Streptococcus pneumoniae is also a frequent cause of serious infections. Nasopharyngeal colonisation with S. pneumoniae inhibits subsequent re-colonisation by inducing Th17-cell adaptive responses, whereas vaccination prevents invasive infections by inducing antibodies to S. pneumoniae capsular polysaccharides. In contrast, protection against invasive infection after nasopharyngeal colonisation with mutant S. pneumoniae strains was associated with antibody responses to protein antigens. The role of colonisation-induced Th17-cell responses during subsequent invasive infections is unknown. Using mouse models, we show that previous colonisation with S. pneumoniae protects against subsequent lethal pneumonia mainly by preventing bacteraemia with a more modest effect on local control of infection within the lung. Previous colonisation resulted in CD4-dependent increased levels of Th17-cell cytokines during subsequent infectious challenge. However, mice depleted of CD4 cells prior to challenge remained protected against bacteraemia, whereas no protection was seen in antibody deficient mice and similar protection could be achieved through passive transfer of serum. Serum from colonised mice but not antibody deficient mice promoted phagocytosis of S. pneumoniae, and previously colonised mice were able to rapidly clear S. pneumoniae from the blood after intravenous inoculation. Thus, despite priming for a Th17-cell response during subsequent infection, the protective effects of prior colonisation in this model was not dependent on CD4 cells but on rapid clearance of bacteria from the blood by antibody-mediated phagocytosis. These data suggest that whilst nasopharyngeal colonisation induces a range of immune responses, the effective protective responses depend upon the site of subsequent infection. PMID:22003400

  4. Pediatric Response to a Large-Scale Child Protection Intervention

    ERIC Educational Resources Information Center

    Lukefahr, James L.; Kellogg, Nancy D.; Anderst, James D.; Gavril, Amy R.; Wehner, Karl K.

    2011-01-01

    Objective: In a rural area of the US state of Texas, in April 2008, the Texas Department of Family and Protective Services (DFPS) responded to evidence of widespread child abuse in an isolated religious compound by removing 463 individuals into state custody. This mass child protection intervention is the largest such action that has ever occurred…

  5. Pediatric Response to a Large-Scale Child Protection Intervention

    ERIC Educational Resources Information Center

    Lukefahr, James L.; Kellogg, Nancy D.; Anderst, James D.; Gavril, Amy R.; Wehner, Karl K.

    2011-01-01

    Objective: In a rural area of the US state of Texas, in April 2008, the Texas Department of Family and Protective Services (DFPS) responded to evidence of widespread child abuse in an isolated religious compound by removing 463 individuals into state custody. This mass child protection intervention is the largest such action that has ever occurred…

  6. Protein aggregation as a mechanism of adaptive cellular responses.

    PubMed

    Saarikangas, Juha; Barral, Yves

    2016-11-01

    Coalescence of proteins into different types of intracellular bodies has surfaced as a widespread adaptive mechanism to re-organize cells and cellular functions in response to specific cues. These structures, composed of proteins or protein-mRNA-complexes, regulate cellular processes through modulating enzymatic activities, gene expression or shielding macromolecules from damage. Accordingly, such bodies are associated with a wide-range of processes, including meiosis, memory-encoding, host-pathogen interactions, cancer, stress responses, as well as protein quality control, DNA replication stress and aneuploidy. Importantly, these distinct coalescence responses are controlled, and in many cases regulated by chaperone proteins. While cells can tolerate and proficiently coordinate numerous distinct types of protein bodies, some of them are also intimately linked to diseases or the adverse effects of aging. Several protein bodies that differ in composition, packing, dynamics, size, and localization were originally discovered in budding yeast. Here, we provide a concise and comparative review of their nature and nomenclature.

  7. Protective responses to sublytic complement in the retinal pigment epithelium

    PubMed Central

    Tan, Li Xuan; Toops, Kimberly A.; Lakkaraju, Aparna

    2016-01-01

    The retinal pigment epithelium (RPE) is a key site of injury in inherited and age-related macular degenerations. Abnormal activation of the complement system is a feature of these blinding diseases, yet how the RPE combats complement attack is poorly understood. The complement cascade terminates in the cell-surface assembly of membrane attack complexes (MACs), which promote inflammation by causing aberrant signal transduction. Here, we investigated mechanisms crucial for limiting MAC assembly and preserving cellular integrity in the RPE and asked how these are compromised in models of macular degeneration. Using polarized primary RPE and the pigmented Abca4−/− Stargardt disease mouse model, we provide evidence for two protective responses occurring within minutes of complement attack, which are essential for maintaining mitochondrial health in the RPE. First, accelerated recycling of the membrane-bound complement regulator CD59 to the RPE cell surface inhibits MAC formation. Second, fusion of lysosomes with the RPE plasma membrane immediately after complement attack limits sustained elevations in intracellular calcium and prevents mitochondrial injury. Cholesterol accumulation in the RPE, induced by vitamin A dimers or oxidized LDL, inhibits these defense mechanisms by activating acid sphingomyelinase (ASMase), which increases tubulin acetylation and derails organelle traffic. Defective CD59 recycling and lysosome exocytosis after complement attack lead to mitochondrial fragmentation and oxidative stress in the RPE. Drugs that stimulate cholesterol efflux or inhibit ASMase restore both these critical safeguards in the RPE and avert complement-induced mitochondrial injury in vitro and in Abca4−/− mice, indicating that they could be effective therapeutic approaches for macular degenerations. PMID:27432952

  8. Immunogenicity and protective efficacy of recombinant S1 domain of the porcine epidemic diarrhea virus spike protein.

    PubMed

    Oh, Jongsuk; Lee, Kyung-Won; Choi, Hwan-Won; Lee, Changhee

    2014-11-01

    Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of swine. Acute PEDV outbreaks have continually emerged in most swine-producing Asian countries and, recently, in the United States, causing significant economic losses in the pig industry. The spike (S) protein of PEDV is a type 1 transmembrane envelope glycoprotein and consists of the S1 and S2 domains, which are responsible for virus binding and fusion, respectively. Since the S1 domain is involved in a specific high-affinity interaction with the cellular receptor and induction of neutralizing antibody in the natural host, it is a primary target for the development of effective vaccines against PEDV. In this study, a codon-optimized PEDV S1 gene containing amino acid residues 25-738 was synthesized based on a multiple alignment of the S amino acid sequences of PEDV field isolates and used to establish a stable porcine cell line constitutively expressing the PEDV S1 protein. The purified recombinant S1 protein was found to mediate highly potent antibody responses in immunized rabbits. The antibodies strongly recognized the recombinant S1 protein from cell lysates and supernatants of S1-expressing cells, whereas they bound weakly to the authentic S protein of PEDV vaccine strain SM98-1. Furthermore, a serum neutralization test revealed that the rabbit antisera completely inhibit infection of the PEDV vaccine strain at a serum dilution of 1:16. We then tested the ability of vaccination with the recombinant S1 protein to protect piglets against PEDV. Late-term pregnant sows were inoculated intramuscularly with the purified S1 protein, and the outcome was investigated in passively immunized suckling piglets after a virulent PEDV challenge. The results showed that vaccination with S1 protein efficiently protected neonatal piglets against PEDV. Our data suggest that the recombinant S1 protein shows potential as an effective and safe subunit vaccine for PED prevention.

  9. Genetic and antigenic diversity of the surface protective antigen proteins of Erysipelothrix rhusiopathiae.

    PubMed

    To, Ho; Nagai, Shinya

    2007-07-01

    The surface protective antigen (Spa) protein of Erysipelothrix rhusiopathiae has been shown to be highly immunogenic and is a potential candidate for a new vaccine against erysipelas. In this study, we cloned and sequenced spa genes from all E. rhusiopathiae serovar reference strains as well as from a serovar 18 strain which was not classified as any species in the genus Erysipelothrix. Sequence analysis revealed that the Spa proteins could be classified into three molecular species, including SpaA, which was previously found in serovars 1a and 2, and the newly designated SpaB and SpaC proteins. The SpaA protein is produced by E. rhusiopathiae serovars 1a, 1b, 2, 5, 8, 9, 12, 15, 16, 17, and N, the SpaB protein is produced by E. rhusiopathiae serovars 4, 6, 11, 19, and 21, and the SpaC protein is produced only by serovar 18. The amino acid sequence similarity was high among members of each Spa type (96 to 99%) but low between different Spa types ( approximately 60%). The greatest diversity in Spa proteins was found in the N-terminal half of the molecule (50 to 57% similarity), which was shown to be involved in immunoprotection. Coinciding with this, immunoblot analysis revealed that rabbit antisera specific to each Spa reacted strongly with the homologous Spa protein but weakly with heterologous Spa proteins. A mouse cross-protection study showed that the three recombinant Spa (rSpa) proteins elicited complete protection against challenge with homologous strains but that the level of protection against challenge with heterologous strains varied depending on the rSpa protein used for immunization. Our study is the first to demonstrate sequence and antigenic diversity in Spa proteins and to indicate that rSpaC may be the most promising antigen for use as a vaccine component because of its broad cross-protectiveness.

  10. [Immunogenic and protective characteristics of recombinant Lassa virus NP protein].

    PubMed

    Ignat'ev, G M

    2002-01-01

    Recombinant fragment of Lassa virus (strain Josiah) nucleocapsid protein (corresponding to amino acid residues 141 + 569) constructed by Dr. Jan ter Meulen (Tropenmedizine, Hamburg) was used for immunizing CBA/calac mice. The preparation was injected intraperitoneally twice with 2-week interval in a dose of 10 micrograms. The parameters of the resultant specific humoral and cell-mediated immunity were comparable to those in reference animals immunized with inactivated Lassa virus. Challenge with Lassa virus (10,000 PFU) resulted in 100% death of the reference animals, while of 15 animals immunized with the recombinant NP protein 8 survived.

  11. Endoplasmic Reticulum Stress Sensing in the Unfolded Protein Response

    PubMed Central

    Gardner, Brooke M.; Pincus, David; Gotthardt, Katja; Gallagher, Ciara M.; Walter, Peter

    2013-01-01

    Secretory and transmembrane proteins enter the endoplasmic reticulum (ER) as unfolded proteins and exit as either folded proteins in transit to their target organelles or as misfolded proteins targeted for degradation. The unfolded protein response (UPR) maintains the protein-folding homeostasis within the ER, ensuring that the protein-folding capacity of the ER meets the load of client proteins. Activation of the UPR depends on three ER stress sensor proteins, Ire1, PERK, and ATF6. Although the consequences of activation are well understood, how these sensors detect ER stress remains unclear. Recent evidence suggests that yeast Ire1 directly binds to unfolded proteins, which induces its oligomerization and activation. BiP dissociation from Ire1 regulates this oligomeric equilibrium, ultimately modulating Ire1’s sensitivity and duration of activation. The mechanistic principles of ER stress sensing are the focus of this review. PMID:23388626

  12. Engineering of chaperone systems and of the unfolded protein response

    PubMed Central

    Khan, Saeed U.

    2008-01-01

    Production of recombinant proteins in mammalian cells is a successful technology that delivers protein pharmaceuticals for therapies and for diagnosis of human disorders. Cost effective production of protein biopharmaceuticals requires extensive optimization through cell and fermentation process engineering at the upstream and chemical engineering of purification processes at the downstream side of the production process. The majority of protein pharmaceuticals are secreted proteins. Accumulating evidence suggests that the folding and processing of these proteins in the endoplasmic reticulum (ER) is a general rate- and yield limiting step for their production. We will summarize our knowledge of protein folding in the ER and of signal transduction pathways activated by accumulation of unfolded proteins in the ER, collectively called the unfolded protein response (UPR). On the basis of this knowledge we will evaluate engineering approaches to increase cell specific productivities through engineering of the ER-resident protein folding machinery and of the UPR. PMID:19003179

  13. Protein Contribution to Plant Salinity Response and Tolerance Acquisition

    PubMed Central

    Kosová, Klára; Prášil, Ilja T.; Vítámvás, Pavel

    2013-01-01

    The review is focused on plant proteome response to salinity with respect to physiological aspects of plant salt stress response. The attention is paid to both osmotic and ionic effects of salinity stress on plants with respect to several protein functional groups. Therefore, the role of individual proteins involved in signalling, changes in gene expression, protein biosynthesis and degradation and the resulting changes in protein relative abundance in proteins involved in energy metabolism, redox metabolism, stressand defence-related proteins, osmolyte metabolism, phytohormone, lipid and secondary metabolism, mechanical stress-related proteins as well as protein posttranslational modifications are discussed. Differences between salt-sensitive (glycophytes) and salt-tolerant (halophytes) plants are analysed with respect to differential salinity tolerance. In conclusion, contribution of proteomic studies to understanding plant salinity tolerance is summarised and discussed. PMID:23531537

  14. Serologic response to cell wall mannoproteins and proteins of Candida albicans.

    PubMed

    Martínez, J P; Gil, M L; López-Ribot, J L; Chaffin, W L

    1998-01-01

    The cell wall of Candida albicans not only is the structure in which many biological functions essential for the fungal cells reside but also is a significant source of candidal antigens. The major cell wall components that elicit a response from the host immune system are proteins and glycoproteins, the latter being predominantly mannoproteins. Both the carbohydrate and protein moieties are able to trigger immune responses. Although cell-mediated immunity is often considered to be the most important line of defense against candidiasis, cell wall protein and glycoprotein components also elicit a potent humoral response from the host that may include some protective antibodies. Proteins and glycoproteins exposed at the most external layers of the wall structure are involved in several types of interactions of fungal cells with the exocellular environment. Thus, coating of fungal cells with host antibodies has the potential to influence profoundly the host-parasite interaction by affecting antibody-mediated functions such as opsonin-enhanced phagocytosis and blocking the binding activity of fungal adhesins for host ligands. In this review, the various members of the protein and glycoprotein fraction of the C. albicans cell wall that elicit an antibody response in vivo are examined. Although a number of proteins have been shown to stimulate an antibody response, for some of these species the response is not universal. On the other hand, some of the studies demonstrate that certain cell wall antigens and anti-cell wall antibodies may be the basis for developing specific and sensitive serologic tests for the diagnosis of candidasis, particularly the disseminated form. In addition, recent studies have focused on the potential for antibodies to cell wall protein determinants to protect the host against infection. Hence, a better understanding of the humoral response to cell wall antigens of C. albicans may provide the basis for the development of (i) effective procedures

  15. Atomic level description of the protecting effect of osmolytes against thermal denaturation of proteins

    NASA Astrophysics Data System (ADS)

    Pieraccini, Stefano; Burgi, Luigi; Genoni, Alessandro; Benedusi, Anna; Sironi, Maurizio

    2007-04-01

    The protecting effect of the osmolyte molecule taurine against thermal denaturation of the protein Chimotripsin Inhibitor 2 was modelled using Molecular Dynamics simulations. The protein was simulated in denaturing conditions at different taurine concentrations. Analysis of the molecular details of its behaviour shows that the protective effect of the osmolyte is concentration dependent. Moreover, the influence of taurine on the solvent structure was studied. A concentration dependent ordering effect of taurine on water molecules emerges from solvent structure analysis and is well correlated to the protecting effect observed. Based on these observations an interpretation of the osmoprotective effect is proposed.

  16. 20 CFR 670.220 - Are we responsible for the protection and maintenance of center facilities?

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... maintenance of center facilities? 670.220 Section 670.220 Employees' Benefits EMPLOYMENT AND TRAINING... and Protection and Maintenance of Facilities § 670.220 Are we responsible for the protection and maintenance of center facilities? (a) Yes, the Secretary establishes procedures for the protection...

  17. Electroporation of a multivalent DNA vaccine cocktail elicits a protective immune response against anthrax and plague.

    PubMed

    Albrecht, Mark T; Livingston, Brian D; Pesce, John T; Bell, Matt G; Hannaman, Drew; Keane-Myers, Andrea M

    2012-07-06

    Electroporation of DNA vaccines represents a platform technology well positioned for the development of multivalent biodefense vaccines. To evaluate this hypothesis, three vaccine constructs were produced using codon-optimized genes encoding Bacillus anthracis Protective Antigen (PA), and the Yersinia pestis genes LcrV and F1, cloned into pVAX1. A/J mice were immunized on a prime-boost schedule with these constructs using the electroporation-based TriGrid Delivery System. Immunization with the individual pDNA vaccines elicited higher levels of antigen-specific IgG than when used in combination. DNA vaccine effectiveness was proven, the pVAX-PA titers were toxin neutralizing and fully protective against a lethal B. anthracis spore challenge when administered alone or co-formulated with the plague pDNA vaccines. LcrV and F1 pVAX vaccines against plague were synergistic, resulting in 100% survival, but less protective individually and when co-formulated with pVAX-PA. These DNA vaccine responses were Th1/Th2 balanced with high levels of IFN-γ and IL-4 in splenocyte recall assays, contrary to complimentary protein Alum vaccinations displaying a Th2 bias with increased IL-4 and low levels of IFN-γ. These results demonstrate the feasibility of electroporation to deliver and maintain the overall efficacy of an anthrax-plague DNA vaccine cocktail whose individual components have qualitative immunological differences when combined.

  18. Invasive Escherichia coli vaccines expressing Brucella melitensis outer membrane proteins 31 or 16 or periplasmic protein BP26 confer protection in mice challenged with B. melitensis.

    PubMed

    Gupta, V K; Radhakrishnan, G; Harms, J; Splitter, G

    2012-06-08

    Because of the serious economic and medical consequences of brucellosis, efforts are to prevent infection of domestic animals through vaccines. Many disadvantages are associated with the current Brucella melitensis Rev.1 vaccine prompting development of alternative vaccines and delivery. Escherichia coli (DH5α) was engineered to express a plasmid containing the inv gene from Yersinia pseudotuberculosis and the hly gene from Listeria monocytogenes. These recombinant invasive E. coli expressing B. melitensis outer membrane proteins (Omp31 or 16) or the periplasmic protein BP26 were evaluated for protection of mice against virulent B. melitensis. Importantly, these invasive E. coli vaccines induced significant protection against B. melitensis challenged mice. Invasive E. coli may be an ideal vaccine platform with natural adjuvant properties for application against B. melitensis since the E. coli delivery system is non-pathogenic and can deliver antigens to antigen-presenting cells promoting cellular immune responses.

  19. HSF transcription factor family, heat shock response, and protein intrinsic disorder.

    PubMed

    Westerheide, Sandy D; Raynes, Rachel; Powell, Chase; Xue, Bin; Uversky, Vladimir N

    2012-02-01

    Intrinsically disordered proteins are highly abundant in all kingdoms of life, and several protein functional classes, such as transcription factors, transcriptional regulators, hub and scaffold proteins, signaling proteins, and chaperones are especially enriched in intrinsic disorder. One of the unique cellular reactions to protein damaging stress is the so-called heat shock response that results in the upregulation of heat shock proteins including molecular chaperones. This molecular protective mechanism is conserved from prokaryotes to eukaryotes and allows an organism to respond to various proteotoxic stressors, such as heat shock, oxidative stress, exposure to heavy metals, and drugs. The heat shock response- related proteins can be expressed during normal conditions (e.g., during the cell growth and development) or can be induced by various pathological conditions, such as infection, inflammation, and protein conformation diseases. The initiation of the heat shock response is manifested by the activation of the heat shock transcription factors HSF 1, part of a family of related HSF transcription factors. This review analyzes the abundance and functional roles of intrinsic disorder in various heat shock transcription factors and clearly shows that the heat shock response requires HSF flexibility to be more efficient. © 2012 Bentham Science Publishers

  20. Heat shock (stress response) proteins and renal ischemia/reperfusion injury.

    PubMed

    Kelly, Katherine J

    2005-01-01

    Acute renal failure occurs frequently, may be increasing, carries an unacceptably high mortality, yet there is no specific treatment. The induction of stress response (heat shock) proteins (HSPs) is a highly conserved response that protects many cell types from diverse physiological and environmental stressors. HSP families of different sizes function as molecular chaperones that facilitate the folding of enzymes and other proteins into functional conformations. After injury, HSPs are believed to facilitate the restoration of normal function by assisting in the refolding of denatured proteins and degradation of irreparably damaged proteins and toxic metabolites, limitation of aggregation of damaged peptides and aiding appropriate folding of newly synthesized essential polypeptides. HSPs may also regulate apoptosis and immune functions. We have demonstrated protection from the functional deficits and histological evidence of experimental ischemic renal injury with heat stress 6 but not 48 h prior to ischemia. Limitation of the induction of HSPs (either with a short period of hyperthermia or pharmacologically) attenuated the protection observed. Other investigators have demonstrated a correlation between the levels of HSP25 and renal ischemic preconditioning in the mouse. Several pharmacological agents have been shown to increase HSP expression. Enhancement of these endogenous protective mechanisms has potential benefit in human disease.

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

    PubMed

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

    2015-06-01

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

  2. A novel bacterial Water Hypersensitivity-like protein shows in vivo protection against cold and freeze damage.

    PubMed

    Anderson, Dominique; Ferreras, Eloy; Trindade, Marla; Cowan, Don

    2015-08-01

    Metagenomic library screening, by functional or sequence analysis, has become an established method for the identification of novel genes and gene products, including genetic elements implicated in microbial stress response and adaptation. We have identified, using a sequence-based approach, a fosmid clone from an Antarctic desert soil metagenome library containing a novel gene which codes for a protein homologous to a Water Hypersensitivity domain (WHy). The WHy domain is typically found as a component of specific LEA (Late Embryogenesis Abundant) proteins, particularly the LEA-14 (LEA-8) variants, which occur widely in plants, nematodes, bacteria and archaea and which are typically induced by exposure to stress conditions. The novel WHy-like protein (165 amino acid, 18.6 kDa) exhibits a largely invariant NPN motif at the N-terminus and has high sequence identity to genes identified in Pseudomonas genomes. Expression of this protein in Escherichia coli significantly protected the recombinant host against cold and freeze stress.

  3. Subdominant outer membrane antigens in anaplasma marginale: conservation, antigenicity, and protective capacity using recombinant protein

    USDA-ARS?s Scientific Manuscript database

    Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP) extracts or a well- defined surface protein complex reproducibly induce protective immunity. However, there are seve...

  4. Protective effect of black tea on integral membrane proteins in rat liver.

    PubMed

    Szachowicz-Petelska, Barbara; Skrzydlewska, Elżbieta; Figaszewski, Zbigniew

    2013-01-01

    Ethanol intoxication is accompanied by oxidative stress formation. Consequently, it leads to disturbances in cellular metabolism that can alter the structure and function of cell membrane components. Black tea displays antioxidant properties, protects membrane phospholipids and may protect integral membrane proteins. In the present study, we examined whether black tea induces changes in the liver integral membrane proteins of 12-months old rats chronically intoxicated with ethanol. To estimate qualitatively and quantitatively the levels of the liver integral membrane proteins, the proteins were selectively hydrolyzed by trypsin, the obtained peptides were resolved by HPLC and the levels of specific amino acids within the individual peptides were determined. All of the obtained peptides contained phenylalanine (Phe), cysteine (Cys) and lysine (Lys). Compared to the control group, rats in the ethanol intoxication group showed decreased liver levels of integral membrane proteins as well as fewer trypsin-hydrolyzed peptides and amino acids in the hydrolyzed peptides. Administration of black tea to ethanol-intoxicated rats partially protected proteins against the structural changes caused by ethanol. Black tea prevented decreases in the levels of cysteine (in about 90% of cases), lysine (in about 60% of cases), phenylalanine (in about 70% of cases) and examined peptides (in about 60% of cases). The liver protein level was higher (by about 18%) in rats who received black tea and ethanol than in those who received ethanol alone. In conclusion, black tea partially protects the composition and level of rat liver cell integral membrane proteins against changes caused by ethanol intoxication.

  5. Humoral immune response in breeding hens and protective immunity provided by administration of purified Salmonella Gallinarum porins.

    PubMed

    Gómez-Verduzco, G; Téllez, G; Quintana, A L; Isibasi, A; Ortiz-Navarrete, V

    2010-03-01

    The current studies were undertaken to assess the ability of humoral immune response in breeding hens to provide protective maternal antibody in the progeny. A highly purified outer membrane protein, 34 kDa, was isolated from a virulent strain of Salmonella Gallinarum. Cross-reactivity was observed between this protein and Salmonella Typhi porins; thus we consider this outer membrane protein as a Salmonella Gallinarum porin. To evaluate passive immunity against Salmonella Gallinarum, 200 broiler breeder hens were immunized with either 10 microg of Salmonella Gallinarum porins, 30 microg of Salmonella Gallinarum porins, or PBS without porins as a control group. Anti-Salmonella Gallinarum porin antibodies were detected in broiler breeder serum and in fertile eggs (P < 0.05). Consequently, chickens from immunized broiler breeder hens were protected between 53 to 70% against challenges of 20 to 500 half-maximal lethal dose of Salmonella Gallinarum (P < 0.001) when compared with control hens that were injected with PBS. These results suggest that Salmonella Gallinarum porins, as those of other Salmonella species, participate in the induction of the passive protective immunity, and the humoral immune response may be one of the mechanisms involved in the establishment of this protection.

  6. Clonogenicity of human leukemic cells protected from cell-lethal agents by heat shock protein 70

    PubMed Central

    Bases, Robert

    2005-01-01

    Pretreatment of human leukemia THP-1 cells with heat shock protein Hsp70 (Hsp70) protected them from the cell-lethal effects of the topoisomerase II inhibitor, lucanthone and from ionizing radiation. Cell viability was scored in clonogenic assays of single cells grown in liquid medium containing 0.5% methyl cellulose. Colonies were observed and rapidly scored after staining with the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide. The frequency of abasic sites in the deoxyribonucleic acid (DNA) of THP-1 cells was reduced when these cells were treated with Hsp70. Hsp70 is presumed to have protected the cells by promoting repair of cell DNA, in agreement with previous studies that showed that Hsp70 enhanced base excision repair by purified enzymes. The shoulders of radiation dose-response curves were enhanced by pretreatment of cells with Hsp70 and, importantly, were reduced when cells were transfected with ribonucleic acid designed to silence Hsp70. Hsp70 influenced repair of sublethal damage after radiation. PMID:15832946

  7. Relative induction of heat shock protein in coronary endothelial cells and cardiomyocytes: implications for myocardial protection.

    PubMed

    Amrani, M; Latif, N; Morrison, K; Gray, C C; Jayakumar, J; Corbett, J; Goodwin, A T; Dunn, M J; Yacoub, M H

    1998-01-01

    Induction of the 70 kd heat shock protein in the heart is known to exert a protective effect against postischemic mechanical and endothelial dysfunction. However, the exact site of induction and the mechanisms involved remain unknown. The aim of this study was to investigate the relative capacity of endothelial and myocardial cells to express the 70 kd heat shock protein in response to heat stress, as well as their significance. (1) Postischemic recovery of cardiac mechanical and endothelial function was studied in isolated rat hearts with and without endothelial denudation with saponin. (2) Semiquantitative determination of induction of 70 kd heat shock protein by Western immunoblotting was performed in the whole cardiac homogenate, in isolated cardiac myocytes, and in coronary endothelial cells. (3) Immunocytochemistry was used to visualize the distribution of induction of 70 kd heat shock protein in both cell types. Postischemic recovery (percent preischemic value +/- standard error of the mean) of cardiac output in hearts from heat-stressed animals was significantly improved (66.7 +/- 6.9 vs 44.5 +/- 4.5 in the control group, p < 0.01). In heat-stressed hearts treated with saponin no improvement in the recovery of cardiac output was noted (44.7 +/- 6.9 in heat-stressed hearts vs 38.0 +/- 4.0 in heat-stressed, saponin-treated hearts, p = not significant). Endothelial function (as assessed by the vasodilatory response to the endothelium-dependent vasodilator 5-hydroxytryptamine) improved from 31.0 +/- 5.2 in the control group to 65.8 +/- 7.1 in heat-stressed hearts (p < 0.02 vs control) and dropped to -1.9 +/- 3.8 in heat-stressed hearts treated with saponin. Immunocytochemistry showed that only sections of hearts from heat-treated rats showed a strong specific reaction with heat shock protein antibody. The positive staining was seen in endothelial cells. Induction of 70 kd heat shock protein content in the whole cardiac homogenate from heat-stressed rats as

  8. Intein-mediated backbone cyclization of VP1 protein enhanced protection of CVB3-induced viral myocarditis

    PubMed Central

    Qi, Xingmei; Xiong, Sidong

    2017-01-01

    CVB3 is a common human pathogen to be highly lethal to newborns and causes viral myocarditis and pancreatitis in adults. However, there is no vaccine available for clinical use. CVB3 capsid protein VP1 is an immunodominant structural protein, containing several B- and T-cell epitopes. However, immunization of mice with VP1 protein is ineffective. Cyclization of peptide is commonly used to improve their in vivo stability and biological activity. Here, we designed and synthesizd cyclic VP1 protein by using engineered split Rma DnaB intein and the cyclization efficiency was 100% in E. coli. As a result, the cyclic VP1 was significantly more stable against irreversible aggregation upon heating and against carboxypeptidase in vitro and the degradation rate was more slowly in vivo. Compared with linear VP1, immunization mice with circular VP1 significantly increased CVB3-specific serum IgG level and augmented CVB3-specific cellular immune responses, consequently afforded better protection against CVB3-induced viral myocarditis. The cyclic VP1 may be a novel candidate protein vaccine for preventing CVB3 infection and similar approaches could be employed to a variety of protein vaccines to enhance their protection effect. PMID:28148910

  9. Protein modification caused by a high dose of gamma irradiation in cryo-sterilized plasma: protective effects of ascorbate.

    PubMed

    Zbikowska, Halina M; Nowak, Pawel; Wachowicz, Barbara

    2006-02-01

    Gamma irradiation is a method of pathogen inactivation in plasma derivatives currently under development. Gamma rays inactivate all known blood-borne viruses. However, the virucidally effective dose of radiation may affect the integrity and function of plasma proteins. Biological activity recoveries of the therapeutic products were shown to be significantly improved by lowering the irradiation temperature and by the addition of antioxidants; the mechanisms responsible for this have not been elucidated yet. Here we sterilized human plasma by gamma irradiation (50 kGy), on dry ice, in the presence (or absence) of ascorbate. The subsequent protein oxidation was quantified by a ferric-xylenol orange (hydroperoxides) and by DNPH-coupled assays (carbonyls). We demonstrated for the first time that irradiation of frozen plasma (without saturation with oxygen) resulted in the generation of protein hydroperoxides, the yield of which was dramatically decreased when plasma was irradiated in the presence of either sodium azide or ascorbate. In irradiated plasma the concentration of protein carbonyls was twofold higher than in nonirradiated control. Ascorbate significantly inhibited protein carbonylation. We concluded that freezing of plasma during irradiation does not provide the complete protection against protein carbonylation and hydroperoxide generation. Addition of ascorbate and some nontoxic metabolic inhibitors might be useful as protecting stabilizers.

  10. Coherence Dynamics in Photosynthesis: Protein Protection of Excitonic Coherence

    NASA Astrophysics Data System (ADS)

    Lee, Hohjai; Cheng, Yuan-Chung; Fleming, Graham R.

    2007-06-01

    The role of quantum coherence in promoting the efficiency of the initial stages of photosynthesis is an open and intriguing question. We performed a two-color photon echo experiment on a bacterial reaction center that enabled direct visualization of the coherence dynamics in the reaction center. The data revealed long-lasting coherence between two electronic states that are formed by mixing of the bacteriopheophytin and accessory bacteriochlorophyll excited states. This coherence can only be explained by strong correlation between the protein-induced fluctuations in the transition energy of neighboring chromophores. Our results suggest that correlated protein environments preserve electronic coherence in photosynthetic complexes and allow the excitation to move coherently in space, enabling highly efficient energy harvesting and trapping in photosynthesis.

  11. Coherence dynamics in photosynthesis: protein protection of excitonic coherence.

    PubMed

    Lee, Hohjai; Cheng, Yuan-Chung; Fleming, Graham R

    2007-06-08

    The role of quantum coherence in promoting the efficiency of the initial stages of photosynthesis is an open and intriguing question. We performed a two-color photon echo experiment on a bacterial reaction center that enabled direct visualization of the coherence dynamics in the reaction center. The data revealed long-lasting coherence between two electronic states that are formed by mixing of the bacteriopheophytin and accessory bacteriochlorophyll excited states. This coherence can only be explained by strong correlation between the protein-induced fluctuations in the transition energy of neighboring chromophores. Our results suggest that correlated protein environments preserve electronic coherence in photosynthetic complexes and allow the excitation to move coherently in space, enabling highly efficient energy harvesting and trapping in photosynthesis.

  12. Protective effect of taraxasterol against rheumatoid arthritis by the modulation of inflammatory responses in mice.

    PubMed

    Jiang, Shu-Hua; Ping, Li-Feng; Sun, Feng-Yan; Wang, Xiao-Lei; Sun, Zhi-Juan

    2016-12-01

    Taraxasterol is an effective component of dandelion that has anti-inflammatory effects in vivo and in vitro. The present study was performed to explore whether taraxasterol exhibits a protective effect against rheumatoid arthritis through the modulation of inflammatory responses in mice. Eight-week-old CCR9-deficient mice were injected with a collagen II monoclonal antibody cocktail to create a rheumatoid arthritis model. In the experimental group, arthritic model mice were treated with 10 mg/kg taraxasterol once per day for 5 days. Treatment with taraxasterol significantly increased the pain thresholds and reduced the clinical arthritic scores of the mice in the experimental group compared with those of the model group. Furthermore, treatment with taraxasterol significantly suppressed tumor necrosis factor-α, interleukin (IL)-1β, IL-6 and nuclear factor-κB protein expression levels compared with those in the rheumatoid arthritis model mice. Taraxasterol treatment also significantly reduced nitric oxide, prostaglandin E2 and cyclooxygenase-2 levels compared with those in the rheumatoid arthritis model group. These observations indicate that the protective effect of taraxasterol against rheumatoid arthritis is mediated via the modulation of inflammatory responses in mice.

  13. Protective effect of taraxasterol against rheumatoid arthritis by the modulation of inflammatory responses in mice

    PubMed Central

    Jiang, Shu-Hua; Ping, Li-Feng; Sun, Feng-Yan; Wang, Xiao-Lei; Sun, Zhi-Juan

    2016-01-01

    Taraxasterol is an effective component of dandelion that has anti-inflammatory effects in vivo and in vitro. The present study was performed to explore whether taraxasterol exhibits a protective effect against rheumatoid arthritis through the modulation of inflammatory responses in mice. Eight-week-old CCR9-deficient mice were injected with a collagen II monoclonal antibody cocktail to create a rheumatoid arthritis model. In the experimental group, arthritic model mice were treated with 10 mg/kg taraxasterol once per day for 5 days. Treatment with taraxasterol significantly increased the pain thresholds and reduced the clinical arthritic scores of the mice in the experimental group compared with those of the model group. Furthermore, treatment with taraxasterol significantly suppressed tumor necrosis factor-α, interleukin (IL)-1β, IL-6 and nuclear factor-κB protein expression levels compared with those in the rheumatoid arthritis model mice. Taraxasterol treatment also significantly reduced nitric oxide, prostaglandin E2 and cyclooxygenase-2 levels compared with those in the rheumatoid arthritis model group. These observations indicate that the protective effect of taraxasterol against rheumatoid arthritis is mediated via the modulation of inflammatory responses in mice. PMID:28101182

  14. Antibodies are not required to a protective immune response against dengue virus elicited in a mouse encephalitis model.

    PubMed

    Amorim, Jaime Henrique; dos Santos Alves, Rúbens Prince; Bizerra, Raíza; Araújo Pereira, Sara; Ramos Pereira, Lennon; Nascimento Fabris, Denicar Lina; Santos, Robert Andreata; Romano, Camila Malta; de Souza Ferreira, Luís Carlos

    2016-01-01

    Generating neutralizing antibodies have been considered a prerequisite to control dengue virus (DENV) infection. However, T lymphocytes have also been shown to be important in a protective immune state. In order to investigate the contribution of both humoral and cellular immune responses in DENV immunity, we used an experimental model in which a non-lethal DENV2 strain (ACS46) is used to intracranially prime Balb/C mice which develop protective immunity against a lethal DENV2 strain (JHA1). Primed mice generated envelope-specific antibodies and CD8(+) T cell responses targeting mainly non-structural proteins. Immune sera from protected mice did not confer passive protection to naïve mice challenged with the JHA1 strain. In contrast, depletion of CD4(+) and CD8(+) T lymphocytes significantly reduced survival of ACS46-primed mice challenged with the JHA1 strain. Collectively, results presented in this study show that a cellular immune response targeting non-structural proteins are a promising way in vaccine development against dengue.

  15. From Humanitarian Intervention to the Responsibility to Protect: From Kosovo to Libya and Beyond

    DTIC Science & Technology

    2011-12-01

    10 Noam Chomsky (statement, United Nations General Assembly Thematic Dialogue on the Responsibility to Protect, New York, NY, 23 July 2009). 11... Chomsky , Noam . Statement. United Nations General Assembly Thematic Dialogue on the Responsibility to Protect. New York, NY, 23 July 2009. Cody

  16. 10 CFR 35.24 - Authority and responsibilities for the radiation protection program.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 1 2013-01-01 2013-01-01 false Authority and responsibilities for the radiation... MATERIAL General Administrative Requirements § 35.24 Authority and responsibilities for the radiation protection program. (a) In addition to the radiation protection program requirements of § 20.1101 of this...

  17. 10 CFR 35.24 - Authority and responsibilities for the radiation protection program.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 1 2012-01-01 2012-01-01 false Authority and responsibilities for the radiation... MATERIAL General Administrative Requirements § 35.24 Authority and responsibilities for the radiation protection program. (a) In addition to the radiation protection program requirements of § 20.1101 of this...

  18. 10 CFR 35.24 - Authority and responsibilities for the radiation protection program.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 1 2010-01-01 2010-01-01 false Authority and responsibilities for the radiation... MATERIAL General Administrative Requirements § 35.24 Authority and responsibilities for the radiation protection program. (a) In addition to the radiation protection program requirements of § 20.1101 of this...

  19. 10 CFR 35.24 - Authority and responsibilities for the radiation protection program.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 1 2011-01-01 2011-01-01 false Authority and responsibilities for the radiation... MATERIAL General Administrative Requirements § 35.24 Authority and responsibilities for the radiation protection program. (a) In addition to the radiation protection program requirements of § 20.1101 of this...

  20. 10 CFR 35.24 - Authority and responsibilities for the radiation protection program.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 1 2014-01-01 2014-01-01 false Authority and responsibilities for the radiation... MATERIAL General Administrative Requirements § 35.24 Authority and responsibilities for the radiation protection program. (a) In addition to the radiation protection program requirements of § 20.1101 of this...

  1. Rabbit hemorrhagic disease virus capsid, a versatile platform for foreign B-cell epitope display inducing protective humoral immune responses.

    PubMed

    Moreno, Noelia; Mena, Ignacio; Angulo, Iván; Gómez, Yolanda; Crisci, Elisa; Montoya, María; Castón, José R; Blanco, Esther; Bárcena, Juan

    2016-08-23

    Virus-like particles (VLPs), comprised of viral structural proteins devoid of genetic material, are tunable nanoparticles that can be chemically or genetically engineered, to be used as platforms for multimeric display of foreign antigens. Here, we report the engineering of chimeric VLPs, derived from rabbit hemorrhagic disease virus (RHDV) for presentation of foreign B-cell antigens to the immune system. The RHDV capsid comprises 180 copies of a single capsid subunit (VP60). To evaluate the ability of chimeric RHDV VLPs to elicit protective humoral responses against foreign antigens, we tested two B-cell epitopes: a novel neutralizing B-cell epitope, derived from feline calicivirus capsid protein, and a well characterized B-cell epitope from the extracellular domain of influenza A virus M2 protein (M2e). We generated sets of chimeric RHDV VLPs by insertion of the foreign B-cell epitopes at three different locations within VP60 protein (which involved different levels of surface accessibility) and in different copy numbers per site. The immunogenic potential of the chimeric VLPs was analyzed in the mouse model. The results presented here indicated that chimeric RHDV VLPs elicit potent protective humoral responses against displayed foreign B-cell epitopes, demonstrated by both, in vitro neutralization and in vivo protection against a lethal challenge.

  2. Rabbit hemorrhagic disease virus capsid, a versatile platform for foreign B-cell epitope display inducing protective humoral immune responses

    PubMed Central

    Moreno, Noelia; Mena, Ignacio; Angulo, Iván; Gómez, Yolanda; Crisci, Elisa; Montoya, María; Castón, José R.; Blanco, Esther; Bárcena, Juan

    2016-01-01

    Virus-like particles (VLPs), comprised of viral structural proteins devoid of genetic material, are tunable nanoparticles that can be chemically or genetically engineered, to be used as platforms for multimeric display of foreign antigens. Here, we report the engineering of chimeric VLPs, derived from rabbit hemorrhagic disease virus (RHDV) for presentation of foreign B-cell antigens to the immune system. The RHDV capsid comprises 180 copies of a single capsid subunit (VP60). To evaluate the ability of chimeric RHDV VLPs to elicit protective humoral responses against foreign antigens, we tested two B-cell epitopes: a novel neutralizing B-cell epitope, derived from feline calicivirus capsid protein, and a well characterized B-cell epitope from the extracellular domain of influenza A virus M2 protein (M2e). We generated sets of chimeric RHDV VLPs by insertion of the foreign B-cell epitopes at three different locations within VP60 protein (which involved different levels of surface accessibility) and in different copy numbers per site. The immunogenic potential of the chimeric VLPs was analyzed in the mouse model. The results presented here indicated that chimeric RHDV VLPs elicit potent protective humoral responses against displayed foreign B-cell epitopes, demonstrated by both, in vitro neutralization and in vivo protection against a lethal challenge. PMID:27549017

  3. Protein stability in stored decellularized heart valve scaffolds and diffusion kinetics of protective molecules.

    PubMed

    Wang, Shangping; Oldenhof, Harriëtte; Dai, Xiaolei; Haverich, Axel; Hilfiker, Andres; Harder, Michael; Wolkers, Willem F

    2014-02-01

    Decellularized tissues can be used as matrix implants. The aims of this study were to investigate protein stability and solvent accessibility in decellularized pulmonary heart valve tissues. Protein denaturation profiles of tissues were studied by differential scanning calorimetry. Protein solvent accessibility of tissue exposed to D2O, and diffusion kinetics of various protective molecules were studied by Fourier transform infrared spectroscopy. Little changes were observed in the protein denaturation temperature during storage, at either 5 or 40°C. Glycerol was found to stabilize proteins; it increased the protein denaturation temperature. The stabilizing effect of glycerol disappeared after washing the sample with saline solution. Hydrogen-to-deuterium exchange rates of protein amide groups were fastest in leaflet tissue, followed by artery and muscle tissue. Diffusion of glycerol was found to be fastest in muscle tissue, followed by artery and leaflet tissue. Diffusion coefficients were derived and used to estimate the time needed to reach saturation. Fixation of tissue with glutaraldehyde had little effects on exchange and diffusion rates. Diffusion rates decreased with increasing molecular size. Proteins in decellularized heart valve tissue are stable during storage. Glycerol increases protein stability in a reversible manner. Solvent accessibility studies of protein amide groups provide an additional tool to study proteins in tissues. Diffusion coefficients can be derived to simulate diffusion kinetics of protective molecules in tissues. This study provides novel tools to evaluate protein stability and solvent accessibility in tissues, which can be used to develop biopreservation strategies.

  4. Human protein status modulates brain reward responses to food cues.

    PubMed

    Griffioen-Roose, Sanne; Smeets, Paul Am; van den Heuvel, Emmy; Boesveldt, Sanne; Finlayson, Graham; de Graaf, Cees

    2014-07-01

    Protein is indispensable in the human diet, and its intake appears tightly regulated. The role of sensory attributes of foods in protein intake regulation is far from clear. We investigated the effect of human protein status on neural responses to different food cues with the use of functional magnetic resonance imaging (fMRI). The food cues varied by taste category (sweet compared with savory) and protein content (low compared with high). In addition, food preferences and intakes were measured. We used a randomized crossover design whereby 23 healthy women [mean ± SD age: 22 ± 2 y; mean ± SD body mass index (in kg/m(2)): 22.5 ± 1.8] followed two 16-d fully controlled dietary interventions involving consumption of either a low-protein diet (0.6 g protein · kg body weight(-1) · d(-1), ~7% of energy derived from protein, approximately half the normal protein intake) or a high-protein diet (2.2 g protein · kg body weight(-1) · d(-1), ~25% of energy, approximately twice the normal intake). On the last day of the interventions, blood oxygen level-dependent (BOLD) responses to odor and visual food cues were measured by using fMRI. The 2 interventions were followed by a 1-d ad libitum phase, during which a large array of food items was available and preference and intake were measured. When exposed to food cues (relative to the control condition), the BOLD response was higher in reward-related areas (orbitofrontal cortex, striatum) in a low-protein state than in a high-protein state. Specifically, BOLD was higher in the inferior orbitofrontal cortex in response to savory food cues. In contrast, the protein content of the food cues did not modulate the BOLD response. A low protein state also increased preferences for savory food cues and increased protein intake in the ad libitum phase as compared with a high-protein state. Protein status modulates brain responses in reward regions to savory food cues. These novel findings suggest that dietary protein status

  5. Novel protein targets of the humoral immune response to Listeria monocytogenes infection in rabbits.

    PubMed

    Yu, Wei Ling; Dan, Hanhong; Lin, Min

    2007-07-01

    The role of the humoral immune response in protective immunity against listerial infection has been overlooked and is essentially unknown. This study aimed to discover the protein targets of Listeria monocytogenes that elicit an antibody response following infection in a rabbit model. A genomic expression library for L. monocytogenes was constructed and differentially screened to identify genes encoding proteins that reacted with antiserum from rabbits infected with live L. monocytogenes serotype 4b (RalphaL), but not with that from animals immunized with heat-killed bacteria (RalphaK). Thirty-one clones expressing proteins that reacted exclusively with RalphaL were identified and sequenced. Sequence analysis, together with Western blot analysis of the proteins expressed from positive clones, led to the identification of eight L. monocytogenes proteins as targets of humoral immune responses during listerial infection: three internalin members (InlA, InlD and InlC2) and five novel proteins of unknown function (designated IspA, IspB, IspC, IspD and IspE, respectively). Exhibition of humoral immune responses to these proteins in actively infected rabbits but not in animals receiving heat-killed L. monocytogenes suggested that they were induced or significantly upregulated in vivo during infection and thus are important in Listeria pathogenesis. With the exception of antibodies to InlA, this is the first demonstration of antibodies to the other seven proteins in infected hosts. These immunogenic proteins may be useful candidates for elucidation of the role of antibodies in protective immunity in the context of listerial infection, as well as potential targets for serodiagnostic reagents and vaccine and drug development.

  6. The transcriptional response of Escherichia coli to recombinant protein insolubility.

    PubMed

    Smith, Harold E

    2007-03-01

    Bacterial production of recombinant proteins offers several advantages over alternative expression methods and remains the system of choice for many structural genomics projects. However, a large percentage of targets accumulate as insoluble inclusion bodies rather than soluble protein, creating a significant bottleneck in the protein production pipeline. Numerous strategies have been reported that can improve in vivo protein solubility, but most do not scale easily for high-throughput expression screening. To understand better the host cell response to the accumulation of insoluble protein, we determined genome-wide changes in bacterial gene expression upon induction of either soluble or insoluble target proteins. By comparing transcriptional profiles for multiple examples from the soluble or insoluble class, we identified a pattern of gene expression that correlates strongly with protein solubility. Direct targets of the sigma32 heat shock sigma factor, which includes genes involved in protein folding and degradation, were highly expressed in response to induction of insoluble protein. This same group of genes was also upregulated by insoluble protein accumulation under a different growth regime, indicating that sigma32-mediated gene expression is a general response to protein insolubility. This knowledge provides a starting point for the rational design of growth parameters and host strains with improved protein solubility characteristics. Summary Problems with protein solubility are frequently encountered when recombinant proteins are expressed in E. coli. The bacterial host responds to this problem by increasing expression of the protein folding machinery via the heat shock sigma factor sigma32. Manipulation of the sigma32 regulon might provide a general mechanism for improving recombinant protein solubility.

  7. Responses of proteins to different ionic environment are linearly interrelated.

    PubMed

    Ferreira, Luisa A; Madeira, Pedro P; Uversky, Alexey V; Uversky, Vladimir N; Zaslavsky, Boris Y

    2015-03-27

    Protein partitioning in aqueous two-phase systems (ATPS) is widely used as a convenient, inexpensive, and readily scaled-up separation technique. Protein partition behavior in ATPS is known to be readily manipulated by ionic composition. However, the available data on the effects of salts and buffer concentrations on protein partitioning are very limited. To fill this gap, partitioning of 15 proteins was examined in dextran-poly(ethylene glycol) ATPSs with different salt additives (Na2SO4, NaClO4, NaSCN, CsCl) in 0.11 M sodium phosphate buffer, pH 7.4. This analysis reveals that there is a linear relationship between the logarithms of the protein partition coefficients determined in the presence of different salts. This relationship suggests that the protein response to ionic environment is determined by the protein structure and type and concentrations of the ions present. Analysis of the differences between protein structures (described in terms of proteins responses to different salts) and that of cytochrome c chosen as a reference showed that the peculiarities of the protein surface structure and B-factor used as a measure of the protein flexibility are the determining parameters. Our results provide better insight into the use of different salts in manipulating protein partitioning in aqueous two-phase systems. These data also demonstrate that the protein responses to different ionic environments are interrelated and are determined by the structural peculiarities of protein surface. It is suggested that changes in ionic microenvironment of proteins may regulate protein transport and behavior in biological systems. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Identifying the Role of E2 Domains on Alphavirus Neutralization and Protective Immune Responses.

    PubMed

    Weger-Lucarelli, James; Aliota, Matthew T; Kamlangdee, Attapon; Osorio, Jorge E

    2015-01-01

    Chikungunya virus (CHIKV) and other alphaviruses are the etiologic agents of numerous diseases in both humans and animals. Despite this, the viral mediators of protective immunity against alphaviruses are poorly understood, highlighted by the lack of a licensed human vaccine for any member of this virus genus. The alphavirus E2, the receptor-binding envelope protein, is considered to be the predominant target of the protective host immune response. Although envelope protein domains have been studied for vaccine and neutralization in flaviviruses, their role in alphaviruses is less characterized. Here, we describe the role of the alphavirus E2 domains in neutralization and protection through the use of chimeric viruses. Four chimeric viruses were constructed in which individual E2 domains of CHIKV were replaced with the corresponding domain from Semliki Forest virus (SFV) (ΔDomA/ΔDomB/ΔDomC/ ΔDomA+B). Vaccination studies in mice (both live and inactivated virus) revealed that domain B was the primary determinant of neutralization. Neutralization studies with CHIKV immune serum from humans were consistent with mouse studies, as ΔDomB was poorly neutralized. Using chimeric viruses, it was determined that the alphavirus E2 domain B was the critical target of neutralizing antibodies in both mice and humans. Therefore, chimeric viruses may have more relevance for vaccine discovery than peptide-based approaches, which only detect linear epitopes. This study provides new insight into the role of alphavirus E2 domains on neutralization determinants and may be useful for the design of novel therapeutic technologies.

  9. The phage-shock-protein response.

    PubMed

    Darwin, Andrew J

    2005-08-01

    The phage-shock-protein (Psp) system responds to extracytoplasmic stress that may reduce the energy status of the cell. It is conserved in many different bacteria and has been linked to several important phenotypes. Escherichia coli psp mutants have defects in maintenance of the proton-motive force, protein export by the sec and tat pathways, survival in stationary phase at alkaline pH, and biofilm formation. Yersinia enterocolitica psp mutants cannot grow when the secretin component of a type III secretion system is mislocalized, and have a severe virulence defect in animals. A Salmonella enterica psp mutation exacerbates some phenotypes of an rpoE null mutant and the psp genes of S. enterica and Shigella flexneri are highly induced during macrophage infection. PspA, the most abundant of the Psp proteins, is required for most of the phenotypes associated with the Psp system. Therefore, PspA is probably an effector that may play a role in maintaining cytoplasmic membrane integrity and/or the proton-motive force. However, PspA is not required for the ability to tolerate secretin mislocalization, which suggests an important physiological role for other Psp proteins. This article summarizes our current understanding of the Psp system: inducing signals, the underlying signal transduction mechanisms, the physiological roles it may play, and a genomic analysis of its conservation.

  10. Increased Immunogenicity and Protective Efficacy of Influenza M2e Fused to a Tetramerizing Protein

    PubMed Central

    Andersson, Anne-Marie Carola; Håkansson, Kjell O.; Jensen, Benjamin Anderschou Holbech; Christensen, Dennis; Andersen, Peter; Thomsen, Allan Randrup; Christensen, Jan Pravsgaard

    2012-01-01

    The ectodomain of the matrix 2 protein (M2e) of influenza A virus represents an attractive target for developing a universal influenza A vaccine, with its sequence being highly conserved amongst human variants of this virus. With the aim of targeting conformational epitopes presumably shared by diverse influenza A viruses, a vaccine (M2e-NSP4) was constructed linking M2e (in its consensus sequence) to the rotavirus fragment NSP498–135; due to its coiled-coil region this fragment is known to form tetramers in aqueous solution and in this manner we hoped to mimick the natural configuration of M2e as presented in membranes. M2e-NSP4 was then evaluated side-by-side with synthetic M2e peptide for its immunogenicity and protective efficacy in a murine influenza challenge model. Here we demonstrate that M2e fused to the tetramerizing protein induces an accelerated, augmented and more broadly reactive antibody response than does M2e peptide as measured in two different assays. Most importantly, vaccination with M2e-NSP4 caused a significant decrease in lung virus load early after challenge with influenza A virus and maintained its efficacy against a lethal challenge even at very low vaccine doses. Based on the results presented in this study M2e-NSP4 merits further investigation as a candidate for or as a component of a universal influenza A vaccine. PMID:23049700

  11. Protective benefits of AMP-activated protein kinase in hepatic ischemia-reperfusion injury

    PubMed Central

    Zhang, Min; Yang, Dan; Gong, Xianqiong; Ge, Pu; Dai, Jie; Lin, Ling; Zhang, Li

    2017-01-01

    Hepatic ischemia-reperfusion injury (HIRI) is a major cause of hepatic failure and death after liver trauma, haemorrhagic shock, resection surgery and liver transplantation. AMP-activated protein kinase (AMPK) is an energy sensitive kinase that plays crucial roles in the regulation of metabolic homeostasis. In HIRI, ischemia induces the decline of ATP and the increased ratio of AMP/ATP, which promotes the phosphorylation and activation of AMPK. Three AMPK kinases, liver kinase B1 (LKB1), Ca2+/calmodulin-depedent protein kinase kinase β (CaMKKβ) and TGF-β-activated kinase-1 (TAK1), are main upstream kinases for the phosphorylation of AMPK. In addition to the changed AMP/ATP ratio, the activated CaMKKβ by increased intracelluar Ca2+ and the overproduction of reactive oxygen species (ROS) are also involved in the activation of AMPK during HIRI. The activated AMPK might provide protective benefits in HIRI via prevention of energy decline, inhibition of inflammatory response, suppression of hepatocyte apoptosis and attenuation of oxidative stress. Thus, AMPK might become a novel target for the pharmacological intervention of HIRI. PMID:28386315

  12. Proteomic Analysis of Pure Human Airway Gland Mucus Reveals a Large Component of Protective Proteins

    PubMed Central

    Joo, Nam Soo; Evans, Idil Apak T.; Cho, Hyung-Ju; Park, Il-Ho; Engelhardt, John F.; Wine, Jeffrey J.

    2015-01-01

    Airway submucosal glands contribute to innate immunity and protect the lungs by secreting mucus, which is required for mucociliary clearance and which also contains antimicrobial, anti-inflammatory, anti-proteolytic and anti-oxidant proteins. We stimulated glands in tracheal trimmings from three lung donors and collected droplets of uncontaminated mucus as they formed at the gland orifices under an oil layer. We analyzed the mucus using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analysis identified 5486 peptides and 441 proteins from across the 3 samples (269–319 proteins per subject). We focused on 269 proteins common to at least 2 0f 3 subjects, of which 102 (38%) had protective or innate immunity functions. While many of these have long been known to play such roles, for many others their cellular protective functions have only recently been appreciated in addition to their well-studied biologic functions (e.g. annexins, apolipoproteins, gelsolin, hemoglobin, histones, keratins, and lumican). A minority of the identified proteins are known to be secreted via conventional exocytosis, suggesting that glandular secretion occurs via multiple mechanisms. Two of the observed protective proteins, major vault protein and prohibitin, have not been observed in fluid from human epithelial cultures or in fluid from nasal or bronchoalveolar lavage. Further proteomic analysis of pure gland mucus may help clarify how healthy airways maintain a sterile environment. PMID:25706550

  13. Oxidative stress response induced by atrazine in Palaemonetes argentinus: the protective effect of vitamin E.

    PubMed

    Griboff, Julieta; Morales, David; Bertrand, Lidwina; Bonansea, Rocío Inés; Monferrán, Magdalena Victoria; Asis, Ramón; Wunderlin, Daniel Alberto; Amé, María Valeria

    2014-10-01

    The widespread contamination and persistence of the herbicide atrazine residues in the environment resulted in the exposure of non-target organisms. The present study was undertaken to investigate the effect of atrazine in the response of oxidative stress biomarkers in the freshwater shrimp Palaemonetes argentinus and the protective effect of vitamin-E against atrazine-induced toxicity. Therefore, two batches of P. argentinus were fed for 21 days with a commercial food enriched in proteins (D1) or with D2, composed of D1 enriched with vitamin-E (6.8 and 16.0mg% of vitamin-E, respectively). Subsequently, half of the individuals of each group were exposed to atrazine (0.4mgL(-1)) for 24h and the others remained as controls. Atrazine promoted oxidative stress response in P. argentinus fed with D1 as indicated by enhanced H2O2 content and induction of superoxide dismutase, glutathione-S-transferases and glutathione reductase. This antioxidant activity would prevent the increment of thiobarbituric acid reactive substances in the shrimp tissues. P. argentinus fed with D2 reversed the response of the biomarkers measured. However, the activation of antioxidants response had an energetic cost, which was revealed by a decrease in lipids storage in shrimps. These results show the modulatory effect of vit-E on oxidative stress and its potential use as an effective antioxidant to be applied in chemoprotection strategies during aquaculture.

  14. Identification and Cloning of a Cryptococcal Deacetylase That Produces Protective Immune Responses

    PubMed Central

    Biondo, Carmelo; Beninati, Concetta; Delfino, Demetrio; Oggioni, Marco; Mancuso, Giuseppe; Midiri, Angelina; Bombaci, Mauro; Tomaselli, Giuseppe; Teti, Giuseppe

    2002-01-01

    Cell-mediated immunity plays a crucial role in host defenses against Cryptococcus (Filobasidiella) neoformans. Therefore, the identification of cryptococcal antigens capable of producing T-cell-mediated responses, such as delayed-type hypersensitivity (DTH) reactions, may be useful in the development of immune-based strategies to control cryptococcosis. In order to characterize DTH-producing antigens, culture supernatants from the unencapsulated Cap-67 strain were separated by anion-exchange chromatography. After further fractionation by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a purified protein with an apparent molecular mass of 25 kDa was found to produce DTH, as evidenced by increased footpad swelling in mice immunized with culture supernatants, relative to unimmunized mice. The 20-amino-acid N-terminal sequence of the 25-kDa protein was used to search data of the C. neoformans Genome Project. Based on the genomic DNA sequence, a DNA probe was used to screen a λ cDNA library prepared from strain B3501. Clones were isolated containing the full-length gene (d25), which showed homology with a number of polysaccharide deacetylases from fungi and bacteria. The recombinant d25 protein expressed in Escherichia coli was similar to the natural one in DTH-producing activity. Moreover, immunization with either the natural or the recombinant protein prolonged survival and decreased fungal burden in mice challenged with the highly virulent C. neoformans strain H99. In conclusion, we have described the first cryptococcal gene whose product, a 25-kDa extracellular polysaccharide deacetylase, has been shown to induce protective immunity responses. PMID:11953374

  15. Taurine protects cisplatin induced cardiotoxicity by modulating inflammatory and endoplasmic reticulum stress responses.

    PubMed

    Chowdhury, Sayantani; Sinha, Krishnendu; Banerjee, Sharmistha; Sil, Parames C

    2016-11-12

    Oxidative stress, ER stress, inflammation, and apoptosis results in the pathogenesis of cisplatin-induced cardiotoxicity. The present study was designed to investigate the signaling mechanisms involved in the ameliorating effect of taurine, a conditionally essential amino acid, against cisplatin-mediated cardiac ER stress dependent apoptotic death and inflammation. Mice were simultaneously treated with taurine (150 mg kg(-1) body wt, i.p.) and cisplatin (10 mg kg(-1) body wt, i.p.) for a week. Cisplatin exposure significantly altered serum creatine kinase and troponin T levels. In addition, histological studies revealed disintegration in the normal radiation pattern of cardiac muscle fibers. However, taurine administration could abate such adverse effects of cisplatin. Taurine administration significantly mitigated the reactive oxygen species production, alleviated the overexpression of nuclear factor-κB (NF-κB), and inhibited the elevation of proinflammatoy cytokines, adhesion molecules, and chemokines. Cisplatin exposure resulted in the unfolded protein response (UPR)-regulated CCAAT/enhancer binding protein (CHOP) up-regulation, induction of GRP78: a marker of ER stress and eIF2α signaling. Increase in calpain-1 expression level, activation of caspase-12 and caspase-3, cleavage of the PARP protein as well as the inhibition of antiapoptotic protein Bcl-2 were reflected on cisplatin-triggered apoptosis. Taurine could, however, combat against such cisplatin induced cardiac-abnormalities. The above mentioned findings suggest that taurine plays a beneficial role in providing protection against cisplatin-induced cardiac damage by modulating inflammatory responses and ER stress. © 2016 BioFactors, 42(6):647-664, 2016.

  16. The protective immune response against infectious bronchitis virus induced by multi-epitope based peptide vaccines.

    PubMed

    Yang, Tai; Wang, Hong-Ning; Wang, Xue; Tang, Jun-Ni; Lu, Dan; Zhang, Yun-Fei; Guo, Zi-Cheng; Li, Yu-Ling; Gao, Rong; Kang, Run-Min

    2009-07-01

    Peptide vaccine was found to be an effective and powerful approach to a variety of pathogens. To explore multi-epitope based peptide vaccines against infectious bronchitis virus (IBV), the immunogenic peptides were fused to the 3' terminal of glutathione S transferase gene (GST) and expressed in Escherichia coli. ELISA and Western blot analysis showed that the purified fusion proteins had excellent immune activity with chicken anti-IBV serum. During the vaccination course, the candidate peptide vaccines induced strong humoral and cellular response, and provided up to 80.0% immune protection, while all non-immunized chickens in the negative control group manifested obvious typical symptoms and died after virus challenge. Our finding provides a new way to develop multi-epitope based peptide vaccine against IBV.

  17. XBP1, Unfolded Protein Response, and Endocrine Responsiveness

    DTIC Science & Technology

    2012-05-01

    induced gene, being rapidly induced in response to E2-stimulation (40, 41). Expression of XBP1 is a key component in the molecular classification... rapidly induced in breast cancer cells following 17b-estradiol (E2) stimulation (37, 38). Upregulation of XBP1 by activation of the UPR or by a UPR...recent study into the role of Mcl-1 in autophagy reported that, in response to glucose deprivation and hypoxia, Mcl-1 is rapidly degraded and

  18. Proteomic analysis of membrane proteins of vero cells: exploration of potential proteins responsible for virus entry.

    PubMed

    Guo, Donghua; Zhu, Qinghe; Zhang, Hong; Sun, Dongbo

    2014-01-01

    Vero cells are highly susceptible to many viruses in humans and animals, and its membrane proteins (MPs) are responsible for virus entry. In our study, the MP proteome of the Vero cells was investigated using a shotgun LC-MS/MS approach. Six hundred twenty-seven proteins, including a total of 1839 peptides, were identified in MP samples of the Vero cells. In 627 proteins, 307 proteins (48.96%) were annotated in terms of biological process of gene ontology (GO) categories; 356 proteins (56.78%) were annotated in terms of molecular function of GO categories; 414 proteins (66.03%) were annotated in terms of cellular components of GO categories. Of 627 identified proteins, seventeen proteins had been revealed to be virus receptor proteins. The resulting protein lists and highlighted proteins may provide valuable information to increase understanding of virus infection of Vero cells.

  19. Recombinant BCG prime and PPE protein boost provides potent protection against acute Mycobacterium tuberculosis infection in mice.

    PubMed

    Yang, Enzhuo; Gu, Jin; Wang, Feifei; Wang, Honghai; Shen, Hongbo; Chen, Zheng W

    2016-04-01

    Since BCG, the only vaccine widely used against tuberculosis (TB) in the world, provides varied protective efficacy and may not be effective for inducing long-term cellular immunity, it is in an urgent need to develop more effective vaccines and more potent immune strategies against TB. Prime-boost is proven to be a good strategy by inducing long-term protection. In this study, we tested the protective effect against Mycobacterium tuberculosis (Mtb) challenge of prime-boost strategy by recombinant BCG (rBCG) expressing PPE protein Rv3425 fused with Ag85B and Rv3425. Results showed that the prime-boost strategy could significantly increase the protective efficiency against Mtb infection, characterized by reduction of bacterial load in lung and spleen, attenuation of tuberculosis lesions in lung tissues. Importantly, we found that Rv3425 boost, superior to Ag85B boost, provided better protection against Mtb infection. Further research proved that rBCG prime-Rv3425 boost could obviously increase the expansion of lymphocytes, significantly induce IL-2 production by lymphocytes upon PPD stimulation, and inhibit IL-6 production at an early stage. It implied that rBCG prime-Rv3425 boost opted to induce Th1 immune response and provided a long-term protection against TB. These results implicated that rBCG prime-Rv3425 boost is a potent and promising strategy to prevent acute Mtb infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. 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.

  1. 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.

  2. Immunization of N terminus of enterovirus 71 VP4 elicits cross-protective antibody responses

    PubMed Central

    2013-01-01

    Background Enterovirus 71 (EV71) is major cause of hand, foot and mouth disease. Large epidemics of EV71 infection have been recently reported in the Asian-Pacific region. Currently, no vaccine is available to prevent EV71 infection. Results The peptide (VP4N20) consisting of the first 20 amino acids at the N-terminal of VP4 of EV71 genotype C4 were fused to hepatitis B core (HBcAg) protein. Expression of fusion proteins in E. coli resulted in the formation of chimeric virus-like particles (VLPs). Mice immunized with the chimeric VLPs elicited anti-VP4N20 antibody response. In vitro microneutralization experiments showed that anti-chimeric VLPs sera were able to neutralize not only EV71 of genotype C4 but also EV71 of genotype A. Neonatal mice model confirmed the neutralizing ability of anti-chimeric VLPs sera. Eiptope mapping led to the identification of a “core sequence” responsible for antibody recognition within the peptide. Conclusions Immunization of chimeric VLPs is able to elicit antibodies displaying a broad neutralizing activity against different genotypes of EV71 in vitro. The “core sequence” of EV71-VP4 is highly conserved across EV71 genotypes. The chimeric VLPs have a great potential to be a novel vaccine candidate with a broad cross-protection against different EV71 genotypes. PMID:24320792

  3. ULTRAVIOLET PROTECTIVE COMPOUNDS AS A RESPONSE TO ULTRAVIOLET RADIATION EXPOSURE

    EPA Science Inventory

    Life on Earth has evolved adaptations to many environmental stresses over the epochs. One consistent stress has been exposure to ultraviolet radiation. In response to UVR organisms have adapted myriad responses; behavioral, morphological and physiological. Behaviorally, some orga...

  4. ULTRAVIOLET PROTECTIVE COMPOUNDS AS A RESPONSE TO ULTRAVIOLET RADIATION EXPOSURE

    EPA Science Inventory

    Life on Earth has evolved adaptations to many environmental stresses over the epochs. One consistent stress has been exposure to ultraviolet radiation. In response to UVR organisms have adapted myriad responses; behavioral, morphological and physiological. Behaviorally, some orga...

  5. Expression of heat shock protein genes in insect stress responses

    USDA-ARS?s Scientific Manuscript database

    The heat shock proteins (HSPs) that are abundantly expressed in insects are important modulators of insect survival. Expression of HSP genes in insects is not only developmentally regulated, but also induced by various stressors in order to confer protection against such stressors. The expression o...

  6. FK506 protects against various immune responses and secondary degeneration following cerebral ischemia.

    PubMed

    Brecht, Stephan; Waetzig, Vicki; Hidding, Ute; Hanisch, Uwe-Karsten; Walther, Michael; Herdegen, Thomas; Neiss, Wolfram F

    2009-12-01

    The immunosuppressant FK506 (1 mg/kg, i.p.) reduces the infarct size following 90 min occlusion of the middle cerebral artery (MCAo) in adult rat brain. Here we have investigated the effect of FK506 on cerebral immune cells that are considered to contribute to neurodegeneration. FK506 substantially attenuated the response of resident and peripheral immune cells following transient ischemia. Between 24 hr and 5 days after MCAo, FK506 reduced the T-cell infiltration in the infarct area as well as the presence of activated and/or phagocytic OX-18, OX-42, GSA-IB4, Iba1, and ED1 positive microglia/macrophages. FK506 also lowered the protein levels of TNFalpha and IL-2 in ischemic brain areas. Repetitive application of FK506 over 20 days attenuated the activation of microglia in the substantia nigra (SN), an area of secondary degeneration. Importantly, FK506 conferred also lasting protection of the neurons of SN; these neurons degenerate by withdrawal of neurotrophic factors from the striatum that undergoes necrotic death as part of the ischemic core. To understand the molecular basis of FK506 effects in cerebral immune cells, we determined in primary postnatal day 0/1 (P0/P1) microglia (i) the expression of the FK506 binding proteins FKBP12, FKBP52, and FKPB65 and (ii) that FK506 (1-100 ng/mL) lowered the number of resting or lipopolysaccharide stimulated microglia as well as we induced the lipopolysaccharide release of TNFalpha in a dose-dependent manner. In summary, FK506 confers rescue of brain tissue following cerebral ischemia not only by neuronal protection, but also by suppression of microglial activation and peripheral immune responses.

  7. Pediatric response to a large-scale child protection intervention.

    PubMed

    Lukefahr, James L; Kellogg, Nancy D; Anderst, James D; Gavril, Amy R; Wehner, Karl K

    2011-08-01

    In a rural area of the US state of Texas, in April 2008, the Texas Department of Family and Protective Services (DFPS) responded to evidence of widespread child abuse in an isolated religious compound by removing 463 individuals into state custody. This mass child protection intervention is the largest such action that has ever occurred in the United States. The objective of this paper is to characterize the burdens placed on the area's community resources, healthcare providers, and legal system, the limitations encountered by the forensic and public health professionals, and how these might be minimized in future large-scale child protection interventions. Drawing on publicly available information, this article describes the child abuse investigation, legal outcomes, experiences of pediatric healthcare providers directly affected by the mass removal, and the roles of regional child abuse pediatric specialists. Because the compound's residents refused to cooperate with the investigation and the population of the compound was eight times higher than expected, law enforcement and child protection resources were insufficient to conduct standard child abuse investigations. Local medical and public health resources were also quickly overwhelmed. Consulting child abuse pediatricians were asked to recommend laboratory and radiologic studies that could assist in identifying signs of child abuse, but the lack of cooperation from patients and parents, inadequate medical histories, and limited physical examinations precluded full implementation of the recommendations. Although most children in danger of abuse were removed from the high-risk environment for several months and some suspected abusers were found guilty in criminal trials, the overall success of the child protection intervention was reduced by the limitations imposed by insufficient resources and lack of cooperation from the compound's residents. Recommendations for community and child abuse pediatricians who may

  8. Borrelia burgdorferi-pulsed dendritic cells induce a protective immune response against tick-transmitted spirochetes.

    PubMed Central

    Mbow, M L; Zeidner, N; Panella, N; Titus, R G; Piesman, J

    1997-01-01

    Borrelia burgdorferi-pulsed dendritic cells and epidermal cells were able to initiate the production of anti-outer surface protein A (OspA) antibody in vitro with normal T and B cells from either BALB/c or C3H/HeJ mice. Inhibition of anti-B. burgdorferi antibody production was observed after 3 days, but not after 2 days, of exposure of the antigen-presenting cells to tumor necrosis factor alpha +/- granulocyte-macrophage colony-stimulating factor. Furthermore, splenic dendritic cells pulsed in vitro with live B. burgdorferi spirochetes and then adoptively transferred into naive syngeneic mice mediated a protective immune response against tick-transmitted spirochetes. This protection appeared not to be due to killing of spirochetes in the feeding ticks, since ticks fed to repletion on B. burgdorferi-pulsed dendritic cell-sensitized mice still harbored live spirochetes. Western blot analysis of the sera collected from dendritic cell-sensitized mice demonstrated that the mice responded to a limited set of B. burgdorferi antigens, including OspA, -B, and -C compared to control groups that either had received unpulsed dendritic cells or were not treated. Finally, mice in the early stage of B. burgdorferi infection were able to develop anti-OspA antibody following injection with B. burgdorferi-pulsed dendritic cells. Our results demonstrate for the first time that adoptive transfer of B. burgdorferi-pulsed dendritic cells induces a protective immune response against tick-transmitted B. burgdorferi and stimulates the production of antibodies specific for a limited set of B. burgdorferi antigens in vivo. PMID:9234802

  9. Protein-surface interactions on stimuli-responsive polymeric biomaterials.

    PubMed

    Cross, Michael C; Toomey, Ryan G; Gallant, Nathan D

    2016-03-04

    Responsive surfaces: a review of the dependence of protein adsorption on the reversible volume phase transition in stimuli-responsive polymers. Specifically addressed are a widely studied subset: thermoresponsive polymers. Findings are also generalizable to other materials which undergo a similarly reversible volume phase transition. As of 2015, over 100,000 articles have been published on stimuli-responsive polymers and many more on protein-biomaterial interactions. Significantly, fewer than 100 of these have focused specifically on protein interactions with stimuli-responsive polymers. These report a clear trend of increased protein adsorption in the collapsed state compared to the swollen state. This control over protein interactions makes stimuli-responsive polymers highly useful in biomedical applications such as wound repair scaffolds, on-demand drug delivery, and antifouling surfaces. Outstanding questions are whether the protein adsorption is reversible with the volume phase transition and whether there is a time-dependence. A clear understanding of protein interactions with stimuli-responsive polymers will advance theoretical models, experimental results, and biomedical applications.

  10. Kinetic response of a photoperturbed allosteric protein.

    PubMed

    Buchli, Brigitte; Waldauer, Steven A; Walser, Reto; Donten, Mateusz L; Pfister, Rolf; Blöchliger, Nicolas; Steiner, Sandra; Caflisch, Amedeo; Zerbe, Oliver; Hamm, Peter

    2013-07-16

    By covalently linking an azobenzene photoswitch across the binding groove of a PDZ domain, a conformational transition, similar to the one occurring upon ligand binding to the unmodified domain, can be initiated on a picosecond timescale by a laser pulse. The protein structures have been characterized in the two photoswitch states through NMR spectroscopy and the transition between them through ultrafast IR spectroscopy and molecular dynamics simulations. The binding groove opens on a 100-ns timescale in a highly nonexponential manner, and the molecular dynamics simulations suggest that the process is governed by the rearrangement of the water network on the protein surface. We propose this rearrangement of the water network to be another possible mechanism of allostery.

  11. Protective Efficacy in Sheep of Adenovirus-Vectored Vaccines against Bluetongue Virus Is Associated with Specific T Cell Responses

    PubMed Central

    Martín, Verónica; Pascual, Elena; Avia, Miguel; Peña, Lourdes; Valcárcel, Félix; Sevilla, Noemí

    2015-01-01

    Bluetongue virus (BTV) is an economically important Orbivirus of the Reoviridae family that causes a hemorrhagic disease in ruminants. Its control has been achieved by inactivated-vaccines that have proven to protect against homologous BTV challenge although unable to induce long-term immunity. Therefore, a more efficient control strategy needs to be developed. Recombinant adenovirus vectors are lead vaccine candidates for protection of several diseases, mainly because of their potency to induce potent T cell immunity. Here we report the induction of humoral and T-cell mediated responses able to protect animals against BTV challenge by recombinant replication-defective human adenovirus serotype 5 (Ad5) expressing either VP7, VP2 or NS3 BTV proteins. First we used the IFNAR(-/-) mouse model system to establish a proof of principle, and afterwards we assayed the protective efficacy in sheep, the natural host of BTV. Mice were completely protected against BTV challenge, developing humoral and BTV-specific CD8+- and CD4+-T cell responses by vaccination with the different rAd5. Sheep vaccinated with Ad5-BTV-VP2 and Ad5-BTV-VP7 or only with Ad5-BTV-VP7 and challenged with BTV showed mild disease symptoms and reduced viremia. This partial protection was achieved in the absence of neutralizing antibodies but strong BTV-specific CD8+ T cell responses in those sheep vaccinated with Ad5-BTV-VP7. These data indicate that rAd5 is a suitable vaccine vector to induce T cell immunity during BTV vaccination and provide new data regarding the relevance of T cell responses in protection during BTV infection. PMID:26619062

  12. Regulating Secretory Proteostasis through the Unfolded Protein Response: From Function to Therapy.

    PubMed

    Plate, Lars; Wiseman, R Luke

    2017-10-01

    Imbalances in secretory proteostasis induced by genetic, environmental, or aging-related insults are pathologically associated with etiologically diverse protein misfolding diseases. To protect the secretory proteome from these insults, organisms evolved stress-responsive signaling pathways that regulate the composition and activity of biologic pathways involved in secretory proteostasis maintenance. The most prominent of these is the endoplasmic reticulum (ER) unfolded protein response (UPR), which functions to regulate ER proteostasis in response to ER stress. While the signaling mechanisms involved in UPR activation are well defined, the impact of UPR activation on secretory proteostasis is only now becoming clear. Here, we highlight recent reports defining how activation of select UPR signaling pathways influences proteostasis within the ER and downstream secretory environments. Furthermore, we describe recent evidence that highlights the therapeutic potential for targeting UPR signaling pathways to correct pathologic disruption in secretory proteostasis associated with diverse types of protein misfolding diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. A new recombinant SsnA protein combined with aluminum hydroxide protects mouse against Streptococcus suis.

    PubMed

    Gómez-Gascón, Lidia; Cardoso-Toset, Fernando; Amarilla, Paola Shyrley; Tarradas, Carmen; Carrasco, Librado; Olaya-Abril, Alfonso; Jiménez-Munguía, Irene; Rodríguez-Ortega, Manuel J; Luque, Inmaculada

    2014-12-05

    An experimental challenge in a mouse model was used to select the most effective adjuvant in a vaccine formulation with the surface-anchored DNA-nuclease (SsnA). We used a protocol based on clinical, histopathological, bacterial kinetics and immune response against S. suis serotype 2 in infected animals. The three adjuvants used, aluminum hydroxide (ALOH), incomplete Freund's adjuvant (FIA), oil-in-water adjuvant (OW) showed a protective effect against death by S. suis serotype 2 in this mouse model, although aluminum hydroxide revealed as the best option. Subsequently, in a second experimental assay, we showed that a recombinant SsnA protein combined with ALOH as adjuvant allowed a significant decrease of clinical and lesional findings in animals, faster reduction of the bacteria from organs and a highest humoral response against S. suis after 3 days post-infection. The results show that this combination (rSsnA+AlOH) could be a good vaccine formulation against S. suis, although further studies are necessary to evaluate their use for swine and human species. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Prion protein participates in the protection of mice from lipopolysaccharide infection by regulating the inflammatory process.

    PubMed

    Liu, Jin; Zhao, Deming; Liu, Chunfa; Ding, Tianjian; Yang, Lifeng; Yin, Xiaomin; Zhou, Xiangmei

    2015-01-01

    Despite the overwhelming evidence of the involvement of prion protein (PrP) in prion disease pathogenesis, the normal functions of this cell surface glycoprotein remain unclear. Previously, we showed that PrP may have a dual regulatory role by regulating the opposite poles of pro-inflammation and anti-inflammation as well as tissue repair in activated microglia. In the present work, we compared the mRNA expression of inflammation-related cytokines (TNF-α, IL-1β, IL-6, NOS2, and IL-10) and IL-4-related alternative activation markers (Arg1 and Mrc1) after lipopolysaccharide (LPS) challenge in the brain and spleen and examined peripheral leukocyte recovery and LPS-induced mortality in PrP knockout mice (PrP(-/-)) and wild-type (WT) mice. During the acute phase, WT mice exhibited higher levels of pro-inflammatory cytokines in the brain and spleen than in PrP(-/-) mice, while PrP(-/-) mice sustained higher levels of pro-inflammatory cytokines and lower levels of anti-inflammatory cytokines, Arg1, and Mrc1 during the later phase. PrP(-/-) mice also exhibited a slower peripheral leukocyte recovery process and higher mortality in response to LPS-induced septic shock. These results suggest that the PrP may participate in the protection of mice from LPS infection by regulating the process of inflammatory response.

  15. Prion proteins with pathogenic and protective mutations show similar structure and dynamics

    PubMed Central

    Bae, Sung-Hun; Legname, Giuseppe; Serban, Ana; Prusiner, Stanley B.; Wright, Peter E.; Dyson, H. Jane

    2009-01-01

    Conformational change in the prion protein (PrP) is thought to be responsible for a group of rare but fatal neurodegenerative diseases of humans and other animals, including Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. However, little is known about the mechanism by which normal cellular PrPs initiate and propagate the conformational change. Here, we studied backbone dynamics of the inherited pathogenic mutants (P101L and H186R), protective mutants (Q167R and Q218K), and wild type mouse PrP(89−230) at pH 5.5 and 3.5. Mutations result in minor chemical shift changes around the mutation sites except that H186R induces large chemical shift changes at distal regions. At lowered pH values, the C-terminal half of the second helix is significantly disordered for the wild type and all mutant proteins, while other parts of the protein are essentially unaffected. This destabilization is accompanied by protonation of the partially exposed histidine H186 in the second helix of the wild type protein. This region in the mutant protein H186R is disordered even at pH 5.5. The wild type and mutant proteins have similar μs conformational exchange near the two β-strands and have similar ns internal motions in several regions including the C-terminal half of the second helix, but only wild type and P101L have extensive ns internal motions throughout the helices. These motions mostly disappear at lower pH. Our findings raise the possibility that the pathogenic or dominant negative mutations exert their effects on some non-native intermediate form such as PrP* after conversion of cellular PrP (PrPC) into the pathogenic isoform PrPSc has been initiated; additionally, formation of PrPSc might begin within the C-terminal folded region rather than in the disordered N-terminal region. PMID:19618915

  16. Vaccination with a single CD4 T cell peptide epitope from a Salmonella type III-secreted effector protein provides protection against lethal infection.

    PubMed

    Kurtz, Jonathan R; Petersen, Hailey E; Frederick, Daniel R; Morici, Lisa A; McLachlan, James B

    2014-06-01

    Salmonella infections affect millions worldwide and remain a significant cause of morbidity and mortality. It is known from mouse studies that CD4 T cells are essential mediators of immunity against Salmonella infection, yet it is not clear whether targeting CD4 T cell responses directly with peptide vaccines against Salmonella can be effective in combating infection. Additionally, it is not known whether T cell responses elicited against Salmonella secreted effector proteins can provide protective immunity against infection. In this study, we investigated both of these possibilities using prime-boost immunization of susceptible mice with a single CD4 T cell peptide epitope from Salmonella secreted effector protein I (SseI), a component of the Salmonella type III secretion system. This immunization conferred significant protection against lethal oral infection, equivalent to that conferred by whole heat-killed Salmonella bacteria. Surprisingly, a well-characterized T cell epitope from the flagellar protein FliC afforded no protection compared to immunization with an irrelevant control peptide. The protective response appeared to be most associated with polyfunctional CD4 T cells raised against the SseI peptide, since no antibodies were produced against any of the peptides and very little CD8 T cell response was observed. Overall, this study demonstrates that eliciting CD4 T cell responses against components of the Salmonella type III secretion system can contribute to protection against infection and should be considered in the design of future Salmonella subunit vaccines.

  17. Gut Microbiota Elicits a Protective Immune Response against Malaria Transmission

    PubMed Central

    Yilmaz, Bahtiyar; Portugal, Silvia; Tran, Tuan M.; Gozzelino, Raffaella; Ramos, Susana; Gomes, Joana; Regalado, Ana; Cowan, Peter J.; d’Apice, Anthony J.F.; Chong, Anita S.; Doumbo, Ogobara K.; Traore, Boubacar; Crompton, Peter D.; Silveira, Henrique; Soares, Miguel P.

    2014-01-01

    Summary Glycosylation processes are under high natural selection pressure, presumably because these can modulate resistance to infection. Here, we asked whether inactivation of the UDP-galactose:β-galactoside-α1-3-galactosyltransferase (α1,3GT) gene, which ablated the expression of the Galα1-3Galβ1-4GlcNAc-R (α-gal) glycan and allowed for the production of anti-α-gal antibodies (Abs) in humans, confers protection against Plasmodium spp. infection, the causative agent of malaria and a major driving force in human evolution. We demonstrate that both Plasmodium spp. and the human gut pathobiont E. coli O86:B7 express α-gal and that anti-α-gal Abs are associated with protection against malaria transmission in humans as well as in α1,3GT-deficient mice, which produce protective anti-α-gal Abs when colonized by E. coli O86:B7. Anti-α-gal Abs target Plasmodium sporozoites for complement-mediated cytotoxicity in the skin, immediately after inoculation by Anopheles mosquitoes. Vaccination against α-gal confers sterile protection against malaria in mice, suggesting that a similar approach may reduce malaria transmission in humans. PaperFlick PMID:25480293

  18. Overload protection: avoidance response to heavy plantar surface loading.

    PubMed

    Robbins, S E; Hanna, A M; Gouw, G J

    1988-02-01

    Current footwear which are designed for use in running are examples of intentional biomechanical model integration into device design. The inadequacy of this footwear in protecting against injury is postulated to be due to fixation on inadequate models of locomotory biomechanics that do not provide for feedback control; in particular, an hypothesized plantar surface sensory-mediated feedback control system, which imparts overload protection during locomotion. A heuristic approach was used to identify the hypothesized system. A random series of loads (0 to 164 kg) was applied to the knee flexed at 90 degrees. In this testing system, plantar surface avoidance behavior was the difference between the sum of the leg weight and the load applied to the knee, and the load measured at the plantar surface; this was produced by activation of hip flexors. Significant avoidance behavior was found in all of the subjects (P less than 0.001). On all surfaces tested, including modern athletic footwear (P less than 0.001), its magnitude increased directly in relation to the load applied to the knee (P less than 0.001). There were significant differences in avoidance behavior in relation to the weight-bearing surfaces tested (P less than 0.05). With the identification of a feedback control system which would serve to moderate loading during locomotion, an explanation is provided as to why current athletic footwear do not protect and may be injurious; thus allowing the design of footwear which may be truly protective.

  19. Gut microbiota elicits a protective immune response against malaria transmission.

    PubMed

    Yilmaz, Bahtiyar; Portugal, Silvia; Tran, Tuan M; Gozzelino, Raffaella; Ramos, Susana; Gomes, Joana; Regalado, Ana; Cowan, Peter J; d'Apice, Anthony J F; Chong, Anita S; Doumbo, Ogobara K; Traore, Boubacar; Crompton, Peter D; Silveira, Henrique; Soares, Miguel P

    2014-12-04

    Glycosylation processes are under high natural selection pressure, presumably because these can modulate resistance to infection. Here, we asked whether inactivation of the UDP-galactose:β-galactoside-α1-3-galactosyltransferase (α1,3GT) gene, which ablated the expression of the Galα1-3Galβ1-4GlcNAc-R (α-gal) glycan and allowed for the production of anti-α-gal antibodies (Abs) in humans, confers protection against Plasmodium spp. infection, the causative agent of malaria and a major driving force in human evolution. We demonstrate that both Plasmodium spp. and the human gut pathobiont E. coli O86:B7 express α-gal and that anti-α-gal Abs are associated with protection against malaria transmission in humans as well as in α1,3GT-deficient mice, which produce protective anti-α-gal Abs when colonized by E. coli O86:B7. Anti-α-gal Abs target Plasmodium sporozoites for complement-mediated cytotoxicity in the skin, immediately after inoculation by Anopheles mosquitoes. Vaccination against α-gal confers sterile protection against malaria in mice, suggesting that a similar approach may reduce malaria transmission in humans.

  20. Vaccination with recombinant flagellar proteins FlgJ and FliN induce protection against Brucella abortus 544 infection in BALB/c mice.

    PubMed

    Li, Xianbo; Xu, Jie; Xie, Yongfei; Qiu, Yefeng; Fu, Simei; Yuan, Xitong; Ke, Yuehua; Yu, Shuang; Du, Xinying; Cui, Mingquan; Chen, Yanfen; Wang, Tongkun; Wang, Zhoujia; Yu, Yaqing; Huang, Kehe; Huang, Liuyu; Peng, Guangneng; Chen, Zeliang; Wang, Yufei

    2012-12-28

    Brucella has been considered as a non-motile, facultative intracellular pathogenic bacterium. However, the genome sequences of different Brucella species reveal the presence of the flagellar genes needed for the construction of a functional flagellum. Due to its roles in the interaction between pathogen and host, we hypothesized that some of the flagellar proteins might induce protective immune responses and these proteins will be good subunit vaccine candidates. This study was conducted to screening of protective antigens among these flagellar proteins. Firstly, according to the putative functional roles, a total of 30 flagellar genes of Brucella abortus were selected for in vitro expression. 15 of these flagellar genes were successfully expressed as his-tagged recombinant proteins in Escherichia coli ER2566. Then, these proteins were purified and used to analyze their T cell immunity induction activity by an in vitro gamma interferon (IFN-γ) assay. Five of the flagellar proteins could stimulate significantly higher levels of IFN-γ secretion in splenocytes from S19 immunized mice, indicating their T cell induction activity. Finally, immunogenicity and protection activity of these 5 flagellar proteins were evaluated in BALB/c mice. Results showed that immunization with FlgJ (BAB1_0260) or FliN (BAB2_0122) plus adjuvant could provide protection against B. abortus 544 infection. Furthermore, mice immunized with FlgJ and FliN developed a vigorous immunoglobulin G response, and in vitro stimulation of their splenocytes with immunizing proteins induced the secretion of IFN-γ. Altogether, these data suggest that flagellar proteins FlgJ and FliN are protective antigens that could produce humoral and cell-mediated responses in mice and candidates for use in future studies of vaccination against brucellosis.

  1. Preclinical evaluation of bacterially produced RSV-G protein vaccine: Strong protection against RSV challenge in cotton rat model

    PubMed Central

    Fuentes, Sandra; Klenow, Laura; Golding, Hana; Khurana, Surender

    2017-01-01

    In current study, we evaluated the safety and protective efficacy of recombinant unglycosylated RSV G protein ectodomain produced in E. coli (in presence and absence of oil-in-water adjuvant) in a preclinical RSV susceptible cotton rat challenge model compared to formaldehyde inactivated RSV (FI-RSV) and live RSV experimental infection. The adjuvanted G protein vaccine induced robust neutralization antibody responses comparable to those generated by live RSV infection. Importantly, adjuvanted G protein significantly reduced viral loads in both the lungs and nose at early time points following viral challenge. Antibody kinetics determined by Surface Plasmon Resonance showed that adjuvanted G generated 10-fold higher G-binding antibodies compared to non-adjvuanted G vaccine and live RSV infection, which correlated strongly with both neutralization titers and viral load titers in the nose and lungs post-viral challenge. Antibody diversity analysis revealed immunodominant antigenic sites in the N- and C-termini of the RSV-G protein, that were boosted >10-fold by adjuvant and inversely correlated with viral load titers. Enhanced lung pathology was observed only in animals vaccinated with FI-RSV, but not in animals vaccinated with unadjuvanted or adjuvanted RSV-G vaccine after viral challenge. The bacterially produced unglycosylated G protein could be developed as a protective vaccine against RSV disease. PMID:28186208

  2. Preclinical evaluation of bacterially produced RSV-G protein vaccine: Strong protection against RSV challenge in cotton rat model.

    PubMed

    Fuentes, Sandra; Klenow, Laura; Golding, Hana; Khurana, Surender

    2017-02-10

    In current study, we evaluated the safety and protective efficacy of recombinant unglycosylated RSV G protein ectodomain produced in E. coli (in presence and absence of oil-in-water adjuvant) in a preclinical RSV susceptible cotton rat challenge model compared to formaldehyde inactivated RSV (FI-RSV) and live RSV experimental infection. The adjuvanted G protein vaccine induced robust neutralization antibody responses comparable to those generated by live RSV infection. Importantly, adjuvanted G protein significantly reduced viral loads in both the lungs and nose at early time points following viral challenge. Antibody kinetics determined by Surface Plasmon Resonance showed that adjuvanted G generated 10-fold higher G-binding antibodies compared to non-adjvuanted G vaccine and live RSV infection, which correlated strongly with both neutralization titers and viral load titers in the nose and lungs post-viral challenge. Antibody diversity analysis revealed immunodominant antigenic sites in the N- and C-termini of the RSV-G protein, that were boosted >10-fold by adjuvant and inversely correlated with viral load titers. Enhanced lung pathology was observed only in animals vaccinated with FI-RSV, but not in animals vaccinated with unadjuvanted or adjuvanted RSV-G vaccine after viral challenge. The bacterially produced unglycosylated G protein could be developed as a protective vaccine against RSV disease.

  3. Reticulon-4A (Nogo-A) redistributes protein disulfide isomerase to protect mice from SOD1-dependent amyotrophic lateral sclerosis

    PubMed Central

    Yang, Yvonne S.; Harel, Noam Y.; Strittmatter, Stephen M.

    2009-01-01

    Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease inherited in a small subset of patients. The SOD1(G93A) transgenic mouse models this subset of patients, and studies of this strain have suggested that endoplasmic reticulum (ER) stress and deficits in ER chaperone function are contributors to ALS pathophysiology. Here, we demonstrate that the reticulon family of proteins is a novel regulator of the ER chaperone protein disulfide isomerase (PDI), and that through PDI, reticulon-4A (Nogo-A) can protect mice against the neurodegeneration that characterizes ALS. We show that overexpressing reticulon protein induces a punctate redistribution of PDI intracellularly, both in vitro and in vivo. Conversely, reduction of endogenous NogoA expression causes a more homogeneous expression pattern in vivo. These effects occur without induction of the unfolded protein response. To examine the effect of PDI redistribution on ALS disease progression, we conducted survival and behavior studies of SOD1(G93A) mice. Deletion of a single copy of the NogoA,B gene accelerates disease onset and progression, while deletion of both copies further worsens disease. We conclude that NogoA contributes to the proper function of the ER resident chaperone PDI, and is protective against ALS-like neurodegeneration. Our results provide a novel intracellular role for reticulon proteins and support the hypothesis that modulation of PDI function is a potential therapeutic approach to ALS. PMID:19889996

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

    DOE PAGES

    Kim, Hwan Keun; Falugi, Fabiana; Thomer, Lena; ...

    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

  5. ATF4 Protein Deficiency Protects against High Fructose-induced Hypertriglyceridemia in Mice*

    PubMed Central

    Xiao, Guozhi; Zhang, Ting; Yu, Shibing; Lee, Sojin; Calabuig-Navarro, Virtu; Yamauchi, Jun; Ringquist, Steven; Dong, H. Henry

    2013-01-01

    Hypertriglyceridemia is the most common lipid disorder in obesity and type 2 diabetes. It results from increased production and/or decreased clearance of triglyceride-rich lipoproteins. To better understand the pathophysiology of hypertriglyceridemia, we studied hepatic regulation of triglyceride metabolism by the activating transcription factor 4 (ATF4), a member of the basic leucine zipper-containing protein subfamily. We determined the effect of ATF4 on hepatic lipid metabolism in Atf4−/− mice fed regular chow or provided with free access to fructose drinking water. ATF4 depletion preferentially attenuated hepatic lipogenesis without affecting hepatic triglyceride production and fatty acid oxidation. This effect prevented excessive fat accumulation in the liver of Atf4−/− mice, when compared with wild-type littermates. To gain insight into the underlying mechanism, we showed that ATF4 depletion resulted in a significant reduction in hepatic expression of peroxisome proliferator-activated receptor-γ, a nuclear receptor that acts to promote lipogenesis in the liver. This effect was accompanied by a significant reduction in hepatic expression of sterol regulatory element-binding protein 1c (SREBP-1c), acetyl-CoA carboxylase, and fatty-acid synthase, three key functions in the lipogenic pathway in Atf4−/− mice. Of particular significance, we found that Atf4−/− mice, as opposed to wild-type littermates, were protected against the development of steatosis and hypertriglyceridemia in response to high fructose feeding. These data demonstrate that ATF4 plays a critical role in regulating hepatic lipid metabolism in response to nutritional cues. PMID:23888053

  6. Cytomegalovirus downregulates IRE1 to repress the unfolded protein response.

    PubMed

    Stahl, Sebastian; Burkhart, Julia M; Hinte, Florian; Tirosh, Boaz; Mohr, Hermine; Zahedi, René P; Sickmann, Albert; Ruzsics, Zsolt; Budt, Matthias; Brune, Wolfram

    2013-01-01

    During viral infection, a massive demand for viral glycoproteins can overwhelm the capacity of the protein folding and quality control machinery, leading to an accumulation of unfolded proteins in the endoplasmic reticulum (ER). To restore ER homeostasis, cells initiate the unfolded protein response (UPR) by activating three ER-to-nucleus signaling pathways, of which the inositol-requiring enzyme 1 (IRE1)-dependent pathway is the most conserved. To reduce ER stress, the UPR decreases protein synthesis, increases degradation of unfolded proteins, and upregulates chaperone expression to enhance protein folding. Cytomegaloviruses, as other viral pathogens, modulate the UPR to their own advantage. However, the molecular mechanisms and the viral proteins responsible for UPR modulation remained to be identified. In this study, we investigated the modulation of IRE1 signaling by murine cytomegalovirus (MCMV) and found that IRE1-mediated mRNA splicing and expression of the X-box binding protein 1 (XBP1) is repressed in infected cells. By affinity purification, we identified the viral M50 protein as an IRE1-interacting protein. M50 expression in transfected or MCMV-infected cells induced a substantial downregulation of IRE1 protein levels. The N-terminal conserved region of M50 was found to be required for interaction with and downregulation of IRE1. Moreover, UL50, the human cytomegalovirus (HCMV) homolog of M50, affected IRE1 in the same way. Thus we concluded that IRE1 downregulation represents a previously undescribed viral strategy to curb the UPR.

  7. Cytomegalovirus Downregulates IRE1 to Repress the Unfolded Protein Response

    PubMed Central

    Stahl, Sebastian; Burkhart, Julia M.; Hinte, Florian; Tirosh, Boaz; Mohr, Hermine; Zahedi, René P.; Sickmann, Albert; Ruzsics, Zsolt; Budt, Matthias; Brune, Wolfram

    2013-01-01

    During viral infection, a massive demand for viral glycoproteins can overwhelm the capacity of the protein folding and quality control machinery, leading to an accumulation of unfolded proteins in the endoplasmic reticulum (ER). To restore ER homeostasis, cells initiate the unfolded protein response (UPR) by activating three ER-to-nucleus signaling pathways, of which the inositol-requiring enzyme 1 (IRE1)-dependent pathway is the most conserved. To reduce ER stress, the UPR decreases protein synthesis, increases degradation of unfolded proteins, and upregulates chaperone expression to enhance protein folding. Cytomegaloviruses, as other viral pathogens, modulate the UPR to their own advantage. However, the molecular mechanisms and the viral proteins responsible for UPR modulation remained to be identified. In this study, we investigated the modulation of IRE1 signaling by murine cytomegalovirus (MCMV) and found that IRE1-mediated mRNA splicing and expression of the X-box binding protein 1 (XBP1) is repressed in infected cells. By affinity purification, we identified the viral M50 protein as an IRE1-interacting protein. M50 expression in transfected or MCMV-infected cells induced a substantial downregulation of IRE1 protein levels. The N-terminal conserved region of M50 was found to be required for interaction with and downregulation of IRE1. Moreover, UL50, the human cytomegalovirus (HCMV) homolog of M50, affected IRE1 in the same way. Thus we concluded that IRE1 downregulation represents a previously undescribed viral strategy to curb the UPR. PMID:23950715

  8. Nanoparticles modulate surfactant protein A and D mediated protection against influenza A infection in vitro

    PubMed Central

    McKenzie, Zofi; Kendall, Michaela; Mackay, Rose-Marie; Tetley, Teresa D.; Morgan, Cliff; Griffiths, Mark; Clark, Howard W.; Madsen, Jens

    2015-01-01

    Numerous epidemiological and toxicological studies have indicated that respiratory infections are exacerbated following enhanced exposure to airborne particulates. Surfactant protein A (SP-A) and SP-D form an important part of the innate immune response in the lung and can interact with nanoparticles to modulate the cellular uptake of these particles. We hypothesize that this interaction will also affect the ability of these proteins to combat infections. TT1, A549 and differentiated THP-1 cells, representing the predominant cell types found in the alveolus namely alveolar type I (ATI) epithelial cells, ATII cells and macrophages, were used to examine the effect of two model nanoparticles, 100 nm amine modified (A-PS) and unmodified polystyrene (U-PS), on the ability of SP-A and SP-D to neutralize influenza A infections in vitro. Pre-incubation of low concentrations of U-PS with SP-A resulted in a reduction of SP-A anti-influenza activity in A549 cells, whereas at higher concentrations there was an increase in SP-A antiviral activity. This differential pattern of U-PS concentration on surfactant protein mediated protection against IAV was also shown with SP-D in TT1 cells. On the other hand, low concentrations of A-PS particles resulted in a reduction of SP-A activity in TT1 cells and a reduction in SP-D activity in A549 cells. These results indicate that nanoparticles can modulate the ability of SP-A and SP-D to combat viral challenges. Furthermore, the nanoparticle concentration, surface chemistry and cell type under investigation are important factors in determining the extent of these modulations. PMID:25533100

  9. Hypothesis: NDL proteins function in stress responses by regulating microtubule organization

    PubMed Central

    Khatri, Nisha; Mudgil, Yashwanti

    2015-01-01

    N-MYC DOWNREGULATED-LIKE proteins (NDL), members of the alpha/beta hydrolase superfamily were recently rediscovered as interactors of G-protein signaling in Arabidopsis thaliana. Although the precise molecular function of NDL proteins is still elusive, in animals these proteins play protective role in hypoxia and expression is induced by hypoxia and nickel, indicating role in stress. Homology of NDL1 with animal counterpart N-MYC DOWNREGULATED GENE (NDRG) suggests similar functions in animals and plants. It is well established that stress responses leads to the microtubule depolymerization and reorganization which is crucial for stress tolerance. NDRG is a microtubule-associated protein which mediates the microtubule organization in animals by causing acetylation and increases the stability of α-tubulin. As NDL1 is highly homologous to NDRG, involvement of NDL1 in the microtubule organization during plant stress can also be expected. Discovery of interaction of NDL with protein kinesin light chain- related 1, enodomembrane family protein 70, syntaxin-23, tubulin alpha-2 chain, as a part of G protein interactome initiative encourages us to postulate microtubule stabilizing functions for NDL family in plants. Our search for NDL interactors in G protein interactome also predicts the role of NDL proteins in abiotic stress tolerance management. Based on published report in animals and predicted interacting partners for NDL in G protein interactome lead us to hypothesize involvement of NDL in the microtubule organization during abiotic stress management in plants. PMID:26583023

  10. Immediate induction of heat shock proteins is not protective against cryopreservation in normal human fibroblasts.

    PubMed

    Park, S J; Choi, H R; Nam, K M; Na, J I; Huh, C H; Park, K C

    2013-01-01

    Heat shock proteins (HSPs) were first identified as proteins whose synthesis was enhanced by stresses, such as increased temperature. HSPs can protect cells from various cytotoxic factors by stabilizing proteins. Thus, it could be hypothesized that heat induced HSPs can provide protective effects against cryopreservation-induced cell death. The aim of this study was to determine whether induction of HSPs can increase the cell viability of normal human fibroblasts after cryopreservation. Cytotoxic effects of heat treatment were tested and the induction of HSPs was assessed by examining time-dependent HSP expression. A cell counting method using fluorescence microscopy was used to determine the viability of cells. In addition, the effects of geranylgeranylacetone were evaluated in terms of HSP expression and cytoskeleton changes. The results of this study showed that immediate induction of HSPs does not protect normal human fibroblasts against cryopreservation-induced cell death possibly by inducing cytoskeleton changes.

  11. Vaccination with TAT-antigen fusion protein induces protective, CD8(+) T cell-mediated immunity against Leishmania major.

    PubMed

    Kronenberg, Katharina; Brosch, Sven; Butsch, Florian; Tada, Yayoi; Shibagaki, Naotaka; Udey, Mark C; von Stebut, Esther

    2010-11-01

    In murine leishmaniasis, healing is mediated by IFN-γ-producing CD4(+) and CD8(+) T cells. Thus, an efficacious vaccine should induce Th1 and Tc1 cells. Dendritic cells (DCs) pulsed with exogenous proteins primarily induce strong CD4-dependent immunity; induction of CD8 responses has proven to be difficult. We evaluated the immunogenicity of fusion proteins comprising the protein transduction domain of HIV-1 TAT and the Leishmania antigen LACK (Leishmania homolog of receptors for activated C kinase), as TAT-fusion proteins facilitate major histocompatibility complex class I-dependent antigen presentation. In vitro, TAT-LACK-pulsed DCs induced stronger proliferation of Leishmania-specific CD8(+) T cells compared with DCs incubated with LACK alone. Vaccination with TAT-LACK-pulsed DCs or fusion proteins plus adjuvant in vivo significantly improved disease outcome in Leishmania major-infected mice and was superior to vaccination with DCs treated with LACK alone. Vaccination with DC+TAT-LACK resulted in stronger proliferation of CD8(+) T cells when compared with immunization with DC+LACK. Upon depletion of CD4(+) or CD8(+) T cells, TAT-LACK-mediated protection was lost. TAT-LACK-pulsed IL-12p40-deficient DCs did not promote protection in vivo. In summary, these data show that TAT-fusion proteins are superior in activating Leishmania-specific Tc1 cells when compared with antigen alone and suggest that IL-12-dependent preferential induction of antigen-specific CD8(+) cells promotes significant protection against this important human pathogen.

  12. Tomato QM-like protein protects Saccharomyces cerevisiae cells against oxidative stress by regulating intracellular proline levels.

    PubMed

    Chen, Changbin; Wanduragala, Srimevan; Becker, Donald F; Dickman, Martin B

    2006-06-01

    Exogenous proline can protect cells of Saccharomyces cerevisiae from oxidative stress. We altered intracellular proline levels by overexpressing the proline dehydrogenase gene (PUT1) of S. cerevisiae. Put1p performs the first enzymatic step of proline degradation in S. cerevisiae. Overexpression of Put1p results in low proline levels and hypersensitivity to oxidants, such as hydrogen peroxide and paraquat. A put1-disrupted yeast mutant deficient in Put1p activity has increased protection from oxidative stress and increased proline levels. Following a conditional life/death screen in yeast, we identified a tomato (Lycopersicon esculentum) gene encoding a QM-like protein (tQM) and found that stable expression of tQM in the Put1p-overexpressing strain conferred protection against oxidative damage from H2O2, paraquat, and heat. This protection was correlated with reactive oxygen species (ROS) reduction and increased proline accumulation. A yeast two-hybrid system assay was used to show that tQM physically interacts with Put1p in yeast, suggesting that tQM is directly involved in modulating proline levels. tQM also can rescue yeast from the lethality mediated by the mammalian proapoptotic protein Bax, through the inhibition of ROS generation. Our results suggest that tQM is a component of various stress response pathways and may function in proline-mediated stress tolerance in plants.

  13. Tomato QM-Like Protein Protects Saccharomyces cerevisiae Cells against Oxidative Stress by Regulating Intracellular Proline Levels

    PubMed Central

    Chen, Changbin; Wanduragala, Srimevan; Becker, Donald F.; Dickman, Martin B.

    2006-01-01

    Exogenous proline can protect cells of Saccharomyces cerevisiae from oxidative stress. We altered intracellular proline levels by overexpressing the proline dehydrogenase gene (PUT1) of S. cerevisiae. Put1p performs the first enzymatic step of proline degradation in S. cerevisiae. Overexpression of Put1p results in low proline levels and hypersensitivity to oxidants, such as hydrogen peroxide and paraquat. A put1-disrupted yeast mutant deficient in Put1p activity has increased protection from oxidative stress and increased proline levels. Following a conditional life/death screen in yeast, we identified a tomato (Lycopersicon esculentum) gene encoding a QM-like protein (tQM) and found that stable expression of tQM in the Put1p-overexpressing strain conferred protection against oxidative damage from H2O2, paraquat, and heat. This protection was correlated with reactive oxygen species (ROS) reduction and increased proline accumulation. A yeast two-hybrid system assay was used to show that tQM physically interacts with Put1p in yeast, suggesting that tQM is directly involved in modulating proline levels. tQM also can rescue yeast from the lethality mediated by the mammalian proapoptotic protein Bax, through the inhibition of ROS generation. Our results suggest that tQM is a component of various stress response pathways and may function in proline-mediated stress tolerance in plants. PMID:16751508

  14. Interleukin-37 suppresses the inflammatory response to protect cardiac function in old endotoxemic mice.

    PubMed

    Li, Jilin; Zhai, Yufeng; Ao, Lihua; Hui, Haipeng; Fullerton, David A; Dinarello, Charles A; Meng, Xianzhong

    2017-07-01

    Myocardial inflammatory responses to endotoxemia are enhanced in old mice, which results in worse cardiac dysfunction. Anti-inflammatory cytokine interleukin (IL)-37 has a broad effect on innate immunoresponses. We hypothesized that IL-37 suppresses myocardial inflammatory responses to protect cardiac function during endotoxemia in old mice. Old (20-24month) wild-type (WT), and IL-37 transgenic (IL-37tg) mice were treated with lipopolysaccharide (LPS, 0.5mg/kg, iv) or normal saline (0.1ml/mouse, iv). Six hours later, left ventricle (LV) function was assessed using a pressure-volume microcatheter. Levels of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 in plasma and myocardial tissue, as well as mononuclear cell density in the myocardium, were examined. Cardiac microvascular endothelial cells isolated from WT and IL-37tg mice were treated with LPS (0.2µg/ml) for 0.5-24h. Nuclear factor-kappa B (NF-κB) p65 phosphorylation was examined by immunoblotting, and MCP-1 levels in cell culture supernatant was determined using enzyme-linked immunosorbent assay. LV dysfunction in old WT endotoxemic mice was accompanied by up-regulated MCP-1, myocardial accumulation of mononuclear cells and production of TNF-α, IL-1β and IL-6. Expression of IL-37 suppressed myocardial inflammatory responses to endotoxemia in old mice, resulting in improved LV function. Treatment of old WT endotoxemic mice with recombinant IL-37 also improved LV function. In vitro experiments revealed that cardiac microvascular endothelial cells from IL-37tg mice had attenuated NF-κB activation and MCP-1 production following LPS stimulation. In conclusion, IL-37 is potent to suppress myocardial inflammation and protects against cardiac dysfunction during endotoxemia in old mice. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. The Immune Response in Measles: Virus Control, Clearance and Protective Immunity

    PubMed Central

    Griffin, Diane E.

    2016-01-01

    Measles is an acute systemic viral infection with immune system interactions that play essential roles in multiple stages of infection and disease. Measles virus (MeV) infection does not induce type 1 interferons, but leads to production of cytokines and chemokines associated with nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) signaling and activation of the NACHT, LRR and PYD domains-containing protein (NLRP3) inflammasome. This restricted response allows extensive virus replication and spread during a clinically silent latent period of 10–14 days. The first appearance of the disease is a 2–3 day prodrome of fever, runny nose, cough, and conjunctivitis that is followed by a characteristic maculopapular rash that spreads from the face and trunk to the extremities. The rash is a manifestation of the MeV-specific type 1 CD4+ and CD8+ T cell adaptive immune response with lymphocyte infiltration into tissue sites of MeV replication and coincides with clearance of infectious virus. However, clearance of viral RNA from blood and tissues occurs over weeks to months after resolution of the rash and is associated with a period of immunosuppression. However, during viral RNA clearance, MeV-specific antibody also matures in type and avidity and T cell functions evolve from type 1 to type 2 and 17 responses that promote B cell development. Recovery is associated with sustained levels of neutralizing antibody and life-long protective immunity. PMID:27754341

  16. The Immune Response in Measles: Virus Control, Clearance and Protective Immunity.

    PubMed

    Griffin, Diane E

    2016-10-12

    Measles is an acute systemic viral infection with immune system interactions that play essential roles in multiple stages of infection and disease. Measles virus (MeV) infection does not induce type 1 interferons, but leads to production of cytokines and chemokines associated with nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) signaling and activation of the NACHT, LRR and PYD domains-containing protein (NLRP3) inflammasome. This restricted response allows extensive virus replication and spread during a clinically silent latent period of 10-14 days. The first appearance of the disease is a 2-3 day prodrome of fever, runny nose, cough, and conjunctivitis that is followed by a characteristic maculopapular rash that spreads from the face and trunk to the extremities. The rash is a manifestation of the MeV-specific type 1 CD4⁺ and CD8⁺ T cell adaptive immune response with lymphocyte infiltration into tissue sites of MeV replication and coincides with clearance of infectious virus. However, clearance of viral RNA from blood and tissues occurs over weeks to months after resolution of the rash and is associated with a period of immunosuppression. However, during viral RNA clearance, MeV-specific antibody also matures in type and avidity and T cell functions evolve from type 1 to type 2 and 17 responses that promote B cell development. Recovery is associated with sustained levels of neutralizing antibody and life-long protective immunity.

  17. Enhanced protective antibody to a mutant meningococcal factor H-binding protein with low-factor H binding

    PubMed Central

    Granoff, Dan M.; Giuntini, Serena; Gowans, Flor A.; Lujan, Eduardo; Sharkey, Kelsey; Beernink, Peter T.

    2016-01-01

    Meningococcal factor H-binding protein (FHbp) is an antigen in 2 serogroup B meningococcal vaccines. FHbp specifically binds human and some nonhuman primate complement FH. To investigate the effect of binding of FH to FHbp on protective antibody responses, we immunized infant rhesus macaques with either a control recombinant FHbp antigen that bound macaque FH or a mutant antigen with 2 amino acid substitutions and >250-fold lower affinity for FH. The mutant antigen elicited 3-fold higher serum IgG anti-FHbp titers and up to 15-fold higher serum bactericidal titers than the control FHbp vaccine. When comparing sera with similar IgG anti-FHbp titers, the antibodies elicited by the mutant antigen gave greater deposition of complement component C4b on live meningococci (classical complement pathway) and inhibited binding of FH, while the anti-FHbp antibodies elicited by the control vaccine enhanced FH binding. Thus, the mutant FHbp vaccine elicited an anti-FHbp antibody repertoire directed at FHbp epitopes within the FH binding site, which resulted in greater protective activity than the antibodies elicited by the control vaccine, which targeted FHbp epitopes outside of the FH combining site. Binding of a host protein to a vaccine antigen impairs protective antibody responses, which can be overcome with low-binding mutant antigens. PMID:27668287

  18. Cross Protection against Influenza A Virus by Yeast-Expressed Heterologous Tandem Repeat M2 Extracellular Proteins

    PubMed Central

    Lee, Yu-Na; Kim, Min-Chul; Lee, Young-Tae; Hwang, Hye Suk; Lee, Jongsang; Kim, Cheol; Kang, Sang-Moo

    2015-01-01

    The influenza M2 ectodomain (M2e) is well conserved across human influenza A subtypes, but there are few residue changes among avian and swine origin influenza A viruses. We expressed a tandem repeat construct of heterologous M2e sequences (M2e5x) derived from human, swine, and avian origin influenza A viruses using the yeast expression system. Intramuscular immunization of mice with AS04-adjuvanted M2e5x protein vaccines was effective in inducing M2e-specific antibodies reactive to M2e peptide and native M2 proteins on the infected cells with human, swine, or avian influenza virus, mucosal and systemic memory cellular immune responses, and cross-protection against H3N2 virus. Importantly, M2e5x immune sera were found to confer protection against different subtypes of H1N1 and H5N1 influenza A viruses in naïve mice. Also, M2e5x-immune complexes of virus-infected cells stimulated macrophages to secrete cytokines via Fc receptors, indicating a possible mechanism of protection. The present study provides evidence that M2e5x proteins produced in yeast cells could be developed as a potential universal influenza vaccine. PMID:26366729

  19. Response of surge protection devices to fast rising pulses

    NASA Technical Reports Server (NTRS)

    Mindel, I. N.

    1980-01-01

    Two types of lightning protection modules incorporating leadless (pill type) Zener like devices were evaluated with regard to their ability to suppress EMP induced transients. Two series of tests were performed to evaluate the ability of these modules to react to fast rate of rise ( 1Kv/ns) transients, and the attenuation introduced and the ability to limit damped sinusoid pulses which may be induced due to an EMP resulting from a nuclear detonation.

  20. Operationalizing the Impossible: The Responsibility to Protect Movement

    DTIC Science & Technology

    2015-05-23

    the first body of thought regarding the moral and just use of military force to restore order and protect humanity.35 Medieval Europe would give way to...rights and a governing body that could facilitate the debate regarding any military action for humanitarian purposes. In fact, some scholars have gone...keep in mind because of the distinct connection between the strategic, operational, and tactical levels of war as presented in US military doctrine

  1. Response of surge protection devices to fast rising pulses

    NASA Technical Reports Server (NTRS)

    Mindel, I. N.

    1980-01-01

    Two types of lightning protection modules incorporating leadless (pill type) Zener like devices were evaluated with regard to their ability to suppress EMP induced transients. Two series of tests were performed to evaluate the ability of these modules to react to fast rate of rise ( 1Kv/ns) transients, and the attenuation introduced and the ability to limit damped sinusoid pulses which may be induced due to an EMP resulting from a nuclear detonation.

  2. Biofilm Matrix Exoproteins Induce a Protective Immune Response against Staphylococcus aureus Biofilm Infection

    PubMed Central

    Gil, Carmen; Solano, Cristina; Burgui, Saioa; Latasa, Cristina; García, Begoña; Toledo-Arana, Alejandro

    2014-01-01

    The Staphylococcus aureus biofilm mode of growth is associated with several chronic infections that are very difficult to treat due to the recalcitrant nature of biofilms to clearance by antimicrobials. Accordingly, there is an increasing interest in preventing the formation of S. aureus biofilms and developing efficient antibiofilm vaccines. Given the fact that during a biofilm-associated infection, the first primary interface between the host and the bacteria is the self-produced extracellular matrix, in this study we analyzed the potential of extracellular proteins found in the biofilm matrix to induce a protective immune response against S. aureus infections. By using proteomic approaches, we characterized the exoproteomes of exopolysaccharide-based and protein-based biofilm matrices produced by two clinical S. aureus strains. Remarkably, results showed that independently of the nature of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. Intradermal administration of an exoproteome extract of an exopolysaccharide-dependent biofilm induced a humoral immune response and elicited the production of interleukin 10 (IL-10) and IL-17 in mice. Antibodies against such an extract promoted opsonophagocytosis and killing of S. aureus. Immunization with the biofilm matrix exoproteome significantly reduced the number of bacterial cells inside a biofilm and on the surrounding tissue, using an in vivo model of mesh-associated biofilm infection. Furthermore, immunized mice also showed limited organ colonization by bacteria released from the matrix at the dispersive stage of the biofilm cycle. Altogether, these data illustrate the potential of biofilm matrix exoproteins as a promising candidate multivalent vaccine against S. aureus biofilm-associated infections. PMID:24343648

  3. Stimuli-Responsive Nanomaterials for Therapeutic Protein Delivery

    PubMed Central

    Lu, Yue; Sun, Wujin; Gu, Zhen

    2014-01-01

    Protein therapeutics have emerged as a significant role in treatment of a broad spectrum of diseases, including cancer, metabolic disorders and autoimmune diseases. The efficacy of protein therapeutics, however, is limited by their instability, immunogenicity and short half-life. In order to overcome these barriers, tremendous efforts have recently been made in developing controlled protein delivery systems. Stimuli-triggered release is an appealing and promising approach for protein delivery and has made protein delivery with both spatiotemporal- and dosage-controlled manners possible. This review surveys recent advances in controlled protein delivery of proteins or peptides using stimuli-responsive nanomaterials. Strategies utilizing both physiological and external stimuli are introduced and discussed. PMID:25151983

  4. Knockdown of Ice-Binding Proteins in Brachypodium distachyon Demonstrates Their Role in Freeze Protection.

    PubMed

    Bredow, Melissa; Vanderbeld, Barbara; Walker, Virginia K

    2016-01-01

    Sub-zero temperatures pose a major threat to the survival of cold-climate perennials. Some of these freeze-tolerant plants produce ice-binding proteins (IBPs) that offer frost protection by restricting ice crystal growth and preventing expansion-induced lysis of the plasma membranes. Despite the extensive in vitro characterization of such proteins, the importance of IBPs in the freezing stress response has not been investigated. Using the freeze-tolerant grass and model crop, Brachypodium distachyon, we characterized putative IBPs (BdIRIs) and generated the first 'IBP-knockdowns'. Seven IBP sequences were identified and expressed in Escherichia coli, with all of the recombinant proteins demonstrating moderate to high levels of ice-recrystallization inhibition (IRI) activity, low levels of thermal hysteresis (TH) activity (0.03-0.09°C at 1 mg/mL) and apparent adsorption to ice primary prism planes. Following plant cold acclimation, IBPs purified from wild-type B. distachyon cell lysates similarly showed high levels of IRI activity, hexagonal ice-shaping, and low levels of TH activity (0.15°C at 0.5 mg/mL total protein). The transfer of a microRNA construct to wild-type plants resulted in the attenuation of IBP activity. The resulting knockdown mutant plants had reduced ability to restrict ice-crystal growth and a 63% reduction in TH activity. Additionally, all transgenic lines were significantly more vulnerable to electrolyte leakage after freezing to -10°C, showing a 13-22% increase in released ions compared to wild-type. IBP-knockdown lines also demonstrated a significant decrease in viability following freezing to -8°C, with some lines showing only two-thirds the survival seen in control lines. These results underscore the vital role IBPs play in the development of a freeze-tolerant phenotype and suggests that expression of these proteins in frost-susceptible plants could be valuable for the production of more winter-hardy crops.

  5. Knockdown of Ice-Binding Proteins in Brachypodium distachyon Demonstrates Their Role in Freeze Protection

    PubMed Central

    Bredow, Melissa; Vanderbeld, Barbara; Walker, Virginia K.

    2016-01-01

    Sub-zero temperatures pose a major threat to the survival of cold-climate perennials. Some of these freeze-tolerant plants produce ice-binding proteins (IBPs) that offer frost protection by restricting ice crystal growth and preventing expansion-induced lysis of the plasma membranes. Despite the extensive in vitro characterization of such proteins, the importance of IBPs in the freezing stress response has not been investigated. Using the freeze-tolerant grass and model crop, Brachypodium distachyon, we characterized putative IBPs (BdIRIs) and generated the first ‘IBP-knockdowns’. Seven IBP sequences were identified and expressed in Escherichia coli, with all of the recombinant proteins demonstrating moderate to high levels of ice-recrystallization inhibition (IRI) activity, low levels of thermal hysteresis (TH) activity (0.03−0.09°C at 1 mg/mL) and apparent adsorption to ice primary prism planes. Following plant cold acclimation, IBPs purified from wild-type B. distachyon cell lysates similarly showed high levels of IRI activity, hexagonal ice-shaping, and low levels of TH activity (0.15°C at 0.5 mg/mL total protein). The transfer of a microRNA construct to wild-type plants resulted in the attenuation of IBP activity. The resulting knockdown mutant plants had reduced ability to restrict ice-crystal growth and a 63% reduction in TH activity. Additionally, all transgenic lines were significantly more vulnerable to electrolyte leakage after freezing to −10°C, showing a 13−22% increase in released ions compared to wild-type. IBP-knockdown lines also demonstrated a significant decrease in viability following freezing to −8°C, with some lines showing only two-thirds the survival seen in control lines. These results underscore the vital role IBPs play in the development of a freeze-tolerant phenotype and suggests that expression of these proteins in frost-susceptible plants could be valuable for the production of more winter-hardy crops. PMID:27959937

  6. Defining the humoral immune response to infectious agents using high-density protein microarrays

    PubMed Central

    Vigil, Adam; Davies, D Huw; Felgner, Philip L

    2010-01-01

    A major component of the adaptive immune response to infection is the generation of protective and long-lasting humoral immunity. Traditional approaches to understanding the host’s humoral immune response are unable to provide an integrated understanding of the antibody repertoire generated in response to infection. By studying multiple antigenic responses in parallel, we can learn more about the breadth and dynamics of the antibody response to infection. Measurement of antibody production following vaccination is also a gauge for efficacy, as generation of antibodies can protect from future infections and limit disease. Protein microarrays are well suited to identify, quantify and compare individual antigenic responses following exposure to infectious agents. This technology can be applied to the development of improved serodiagnostic tests, discovery of subunit vaccine antigen candidates, epidemiologic research and vaccine development, as well as providing novel insights into infectious disease and the immune system. In this review, we will discuss the use of protein microarrays as a powerful tool to define the humoral immune response to bacteria and viruses. PMID:20143947

  7. Immunogenicity and protective role of antigenic regions from five outer membrane proteins of Flavobacterium columnare in grass carp Ctenopharyngodon idella

    NASA Astrophysics Data System (ADS)

    Luo, Zhang; Liu, Zhixin; Fu, Jianping; Zhang, Qiusheng; Huang, Bei; Nie, Pin

    2016-11-01

    Flavobacterium columnare causes columnaris disease in freshwater fish. In the present study, the antigenic regions of five outer membrane proteins (OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 kDa, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purified recombinant protein was used to vaccinate the grass carp, Ctenopharyngodon idella. Following vaccination of the fish their IgM antibody levels were examined, as was the expression of IgM, IgD and IgZ immunoglobulin genes and other genes such as MHC Iα and MHC IIβ, which are also involved in adaptive immunity. Interleukin genes ( IL), including IL-1β, IL-8 and IL-10, and type I and type II interferon ( IFN) genes were also examined. At 3 and 4 weeks post-vaccination (wpv), significant increases in IgM antibody levels were observed in the fish vaccinated with the recombinant fusion protein, and an increase in the expression levels of IgM, IgD and IgZ genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fish. At four wpv, the fish were challenged with F. columnare, and the vaccinated fish showed a good level of protection against this pathogen, with 39% relative percent survival (RPS) compared with the control group. It can be concluded, therefore, that the five OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fish and protection against F. columnare.

  8. Responses of the Protein-Deficient Rabbit to Staphylococcal Bacteremia

    PubMed Central

    Bhuyan, U. N.; Ramalingaswami, V.

    1972-01-01

    Staphylococcal bacteremia was induced in two groups of rabbits that were pairfed high protein and protein deficient diets. Marked deviations in response were observed as a result of protein deficiency: a) blood clearance of S aureus was delayed; b) persistence and multiplication of bacteria in blood and tissues were pronounced; c) neutrophilic response was poor and transitory; d) mortality was very high and occurred very early and e) focal necrotizing lesions, rather than well-formed abscesses, were found in organs. ImagesFig 1Fig 2Fig 3Fig 4 PMID:4634740

  9. Pathogenesis-related protein 1b1 (PR1b1) is a major tomato fruit protein responsive to chilling temperature and upregulated in high polyamine transgenic genotypes

    USDA-ARS?s Scientific Manuscript database

    Plants execute an array of mechanisms in response to stress which include upregulation of defense-related proteins and changes in specific metabolites. A group of commonly found metabolites implicated in protection against stresses such as chilling stress constitute ubiquitous biogenic amines calle...

  10. Overexpressed heat shock protein 70 protects cells against DNA damage caused by ultraviolet C in a dose-dependent manner

    PubMed Central

    Niu, Piye; Liu, Lin; Gong, Zhiyong; Tan, Hao; Wang, Feng; Yuan, Jing; Feng, Youmei; Wei, Qingyi; Tanguay, Robert M; Wu, Tangchun

    2006-01-01

    Heat shock protein 70 (Hsp70) comprises proteins that have been reported to protect cells, tissues, and organisms against damage from a wide variety of stressful stimuli; however, little is known about whether Hsp70 protects against DNA damage. In this study, we investigated the relationship between Hsp70 expression and the levels of ultraviolet C (UVC)–induced DNA damage in A549 cells with normal, inhibited, and overexpressed Hsp70 levels. Hsp70 expression was inhibited by treatment with quercetin or overexpressed by transfection of plasmids harboring the hsp70 gene. The level of DNA damage was assessed by the comet assay. The results showed that the levels of DNA damage (shown as the percentage of comet cells) in A549 cells increased in all cells after exposure to an incident dose of 0, 10, 20, 40, and 80 J/m2 whether Hsp70 was inhibited or overexpressed. This response was dose dependent: a protection against UVC-induced DNA damage in cells with overexpressed Hsp70 was observed at UVC dose 20 J/ m2 with a maximum at 40 J/m2 when compared with cells with normal Hsp70 levels and in quercetin-treated cells. This differential protection disappeared at 80 J/m2. These results suggest that overexpressed Hsp70 might play a role in protecting A549 cells from DNA damage caused by UVC irradiation, with a threshold of protection from at UVC irradiation-induced DNA damage by Hsp70. The detailed mechanism how Hsp70 is involved in DNA damage and possible DNA repair warrants further investigation. PMID:16817322

  11. Yersinia ruckeri lipopolysaccharide is necessary and sufficient for eliciting a protective immune response in rainbow trout (Oncorhynchus mykiss, Walbaum).

    PubMed

    Welch, Timothy J; LaPatra, Scott

    2016-02-01

    Enteric redmouth disease (ERM), caused by Yersinia ruckeri, has been controlled successfully using immersion-applied bacterin vaccines for several decades. While the host response to vaccination and the mechanism of protection of this vaccine have been elucidated, the bacterial components eliciting protection have remained unclear. Here we show that highly purified serotype O1 Y. ruckeri lipopolysaccharide (LPS) is sufficient to induce a protective response to experimental challenge in rainbow trout (Oncorhynchus mykiss). Dose response experiments demonstrated that Y. ruckeri LPS at doses of 1 ng/fish and above resulted in essentially complete protection and doses as low as 0.01 ng/fish (1.38 ng/kg) resulted in significant protection, thus demonstrating the extremely high potency of this immunogen. Analysis of the Y. ruckeri genome identified a cluster of putative O-antigen biosynthetic genes specific to serotype O1 strains. This cluster primarily consisted of genes encoding proteins predicted to function in the biosynthesis of legionamic acid, a nonulosonic acid known to be part of the O-polysaccharide repeat of O1 Y. ruckeri. Mutation of the nab2 gene, a nonulosonic acid biosynthesis gene (nab gene), resulted in production of severely truncated forms of LPS. Vaccination with bacterin vaccines derived from the nab2 mutant and its wild type parent strain demonstrated that LPS is a required component of the whole-cell bacterin vaccine and suggests that LPS is the only cellular component contributing to the protective response elicited by this vaccine. We speculate that the exceptionally high potency of Y. ruckeri LPS accounts for the unusual success of this vaccine when delivered by immersion. Published by Elsevier Ltd.

  12. Molecular dissection of the human antibody response to the structural repeat epitope of Plasmodium falciparum sporozoite from a protected donor

    PubMed Central

    Chappel, Jonathan A; Rogers, William O; Hoffman, Stephen L; Kang, Angray S

    2004-01-01

    Background The circumsporozoite surface protein is the primary target of human antibodies against Plasmodium falciparum sporozoites, these antibodies are predominantly directed to the major repetitive epitope (Asn-Pro-Asn-Ala)n, (NPNA)n. In individuals immunized by the bites of irradiated Anopheles mosquitoes carrying P. falciparum sporozoites in their salivary glands, the anti-repeat response dominates and is thought by many to play a role in protective immunity. Methods The antibody repertoire from a protected individual immunized by the bites of irradiated P. falciparum infected Anopheles stephensi was recapitulated in a phage display library. Following affinity based selection against (NPNA)3 antibody fragments that recognized the PfCSP repeat epitope were rescued. Results Analysis of selected antibody fragments implied the response was restricted to a single antibody fragment consisting of VH3 and VκI families for heavy and light chain respectively with moderate affinity for the ligand. Conclusion The dissection of the protective antibody response against the repeat epitope revealed that the response was apparently restricted to a single VH/VL pairing (PfNPNA-1). The affinity for the ligand was in the μM range. If anti-repeat antibodies are involved in the protective immunity elicited by exposure to radiation attenuated P. falciparum sporozoites, then high circulating levels of antibodies against the repeat region may be more important than intrinsic high affinity for protection. The ability to attain and sustain high levels of anti-(NPNA)n will be one of the key determinants of efficacy for a vaccine that relies upon anti-PfCSP repeat antibodies as the primary mechanism of protective immunity against P. falciparum. PMID:15283866

  13. Protective effects of leucine against lipopolysaccharide-induced inflammatory response in Labeo rohita fingerlings.

    PubMed

    Giri, Sib Sankar; Sen, Shib Sankar; Jun, Jin Woo; Sukumaran, Venkatachalam; Park, Se Chang

    2016-05-01

    The present study investigated the protective effects of leucine against lipopolysaccharide (LPS)-induced inflammatory responses in Labeo rohita (rohu) in vivo and in vitro. Primary hepatocytes, isolated from the hepatopancreas, were exposed to different concentrations of LPS for 24 h to induce an inflammatory response, and the protective effects of leucine against LPS-induced inflammation were studied. Finally, we investigated the efficiency of dietary leucine supplementation in attenuating an immune challenge induced by LPS in vivo. Exposure of cells to 10-25 μg mL(-1) of LPS for 24 h resulted in a significant production of nitric oxide and release of lactate dehydrogenase to the medium, whereas cell viability and protein content were reduced (p < 0.05). LPS exposure (10 μg mL(-1)) increased mRNA levels of the pro-inflammatory cytokines TNF-α, IL-1β and IL-8 in vitro (p < 0.05). However, pretreatment with leucine prevented the LPS-induced upregulation of TNF-α, IL-1β and IL-8 mRNAs by downregulating TLR4, MyD88, NF-κBp65, and MAPKp38 mRNA expression. Interestingly, mRNA expression of the anti-inflammatory cytokine, IL-10, which was increased by LPS treatment, was further enhanced (p < 0.05) by leucine pretreatment. The enhanced expression of IL-10 might inhibit the production of other pro-inflammatory cytokines. It was found that leucine pretreatment attenuated the excessive activation of LPS-induced TLR4-MyD88 signaling as manifested by lower level of TLR4, MyD88, MAPKp38, NF-κBp65 and increased level of IκB-α protein in leucine pre-treatment group. In vivo experiments demonstrated that leucine pre-supplementation could protect fish against LPS-induced inflammation through an attenuation of TLR4-MyD88 signaling pathway. Taken together, we propose that leucine pre-supplementation decreases LPS-induced immune damage in rohu by enhancing the expression of IL-10 and by regulating the TLR4-MyD88 signaling pathways.

  14. Functional Mapping of Protective Domains and Epitopes in the Rotavirus VP6 Protein

    PubMed Central

    Choi, Anthony H. C.; Basu, Mitali; McNeal, Monica M.; Flint, Jason; VanCott, John L.; Clements, John D.; Ward, Richard L.

    2000-01-01

    The purpose of this study was to determine which regions of the VP6 protein of the murine rotavirus strain EDIM are able to elicit protection against rotavirus shedding in the adult mouse model following intranasal (i.n.) immunization with fragments of VP6 and a subsequent oral EDIM challenge. In the initial experiment, the first (fragment AB), middle (BC), or last (CD) part of VP6 that was genetically fused to maltose-binding protein (MBP) and expressed in Escherichia coli was examined. Mice (BALB/c) immunized with two 9-μg doses of each of the chimeras and 10 μg of the mucosal adjuvant LT(R192G) were found to be protected against EDIM shedding (80, 92, and nearly 100% reduction, respectively; P ≤ 0.01) following challenge. Because CD produced almost complete protection, we prepared four E. coli-expressed, MBP-fused chimeras containing overlapping fragments of the CD region (i.e., CD1, CD2, CD3, and CD4) whose lengths ranged from 61 to 67 amino acid residues. Following i.n. immunization, CD1, CD2, and CD4 induced significant (P ≤ 0.004) protection (88, 84, and 92% reduction, respectively). In addition, 11 peptides (18 to 30 residues) of the CD region with between 0 and 13 overlapping amino acids were synthesized. Two 50-μg doses of each peptide with LT(R192G) were administered i.n. to BALB/c mice. Five peptides were found to elicit significant (P ≤ 0.02) protection. Moreover, a 14-amino-acid region within peptide 6 containing a putative CD4+ T-cell epitope was found to confer nearly complete protection, suggesting a protective role for CD4+ T cells. Mice that were protected by fragments BC and CD1 and four of the five protective synthetic peptides did not develop measurable rotavirus antibodies in serum or stool, implying that protection induced by these domains was not dependent on antibody. Together, these observations suggest that multiple regions of VP6 can stimulate protection, a region of VP6 as small as 14 amino acids containing a CD4+ T

  15. Protein-protected luminescent noble metal quantum clusters: an emerging trend in atomic cluster nanoscience

    PubMed Central

    Xavier, Paulrajpillai Lourdu; Chaudhari, Kamalesh; Baksi, Ananya; Pradeep, Thalappil

    2012-01-01

    Noble metal quantum clusters (NMQCs) are the missing link between isolated noble metal atoms and nanoparticles. NMQCs are sub-nanometer core sized clusters composed of a group of atoms, most often luminescent in the visible region, and possess intriguing photo-physical and chemical properties. A trend is observed in the use of ligands, ranging from phosphines to functional proteins, for the synthesis of NMQCs in the liquid phase. In this review, we briefly overview recent advancements in the synthesis of protein protected NMQCs with special emphasis on their structural and photo-physical properties. In view of the protein protection, coupled with direct synthesis and easy functionalization, this hybrid QC-protein system is expected to have numerous optical and bioimaging applications in the future, pointers in this direction are visible in the literature. PMID:22312454

  16. Protein-protected luminescent noble metal quantum clusters: an emerging trend in atomic cluster nanoscience.

    PubMed

    Xavier, Paulrajpillai Lourdu; Chaudhari, Kamalesh; Baksi, Ananya; Pradeep, Thalappil

    2012-01-01

    Noble metal quantum clusters (NMQCs) are the missing link between isolated noble metal atoms and nanoparticles. NMQCs are sub-nanometer core sized clusters composed of a group of atoms, most often luminescent in the visible region, and possess intriguing photo-physical and chemical properties. A trend is observed in the use of ligands, ranging from phosphines to functional proteins, for the synthesis of NMQCs in the liquid phase. In this review, we briefly overview recent advancements in the synthesis of protein protected NMQCs with special emphasis on their structural and photo-physical properties. In view of the protein protection, coupled with direct synthesis and easy functionalization, this hybrid QC-protein system is expected to have numerous optical and bioimaging applications in the future, pointers in this direction are visible in the literature.

  17. Stimuli responsive smart-gels realized via modular protein design

    PubMed Central

    Grove, Tijana Z.; Osuji, Chinedum O.; Forster, Jason D.; Dufresne, Eric R.; Regan, Lynne

    2012-01-01

    Smart-gels have a variety of applications including tissue engineering and controlled drug delivery. Here we present a modular, bottom-up approach that permits the creation of protein-based smart-gels with encoded morphology, functionality, and responsiveness to external stimuli. The properties of these gels are encoded by the proteins from which they are synthesized. In particular, the strength and density of the network of intermolecular cross-links are specified by the interactions of the gel’s constituent protein modules with their cognate peptide ligands. Thus, these gels exhibit stimuli-responsive assembly and disassembly, dissolving (or gelling) under conditions that weaken (or strengthen) the protein-peptide interaction. We further demonstrate that such gels can encapsulate and release both proteins and small molecules and that their rheological properties are well suited for biomedical applications. PMID:20860358

  18. Targeting unfolded protein response signaling pathways to ameliorate protein misfolding diseases.

    PubMed

    Ryno, Lisa M; Wiseman, R Luke; Kelly, Jeffery W

    2013-06-01

    Protein homeostasis (or proteostasis) within the endoplasmic reticulum (ER) is regulated by the unfolded protein response (UPR). The UPR consists of three integrated signaling pathways activated by the accumulation of misfolded proteins within the ER lumen. Activation of the UPR alters ER proteostasis through translational attenuation of new protein synthesis and transcriptional remodeling of ER proteostasis pathways, providing a mechanism to adapt ER proteostasis in response to cellular stress. The capacity of the UPR to alter ER proteostasis suggests that exogenous manipulation of UPR signaling pathways offers therapeutic promise to alter the fate of pathologic proteins associated with human protein misfolding diseases. Here, we discuss the therapeutic potential of exogenous UPR activation to treat human disease and highlight specific small molecule approaches for regulating UPR signaling that could be beneficial to treat protein misfolding diseases. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. A DNA vaccine encoding the E protein of West Nile virus is protective and can be boosted by recombinant domain DIII.

    PubMed

    Schneeweiss, Anne; Chabierski, Stefan; Salomo, Mathias; Delaroque, Nicolas; Al-Robaiy, Samiya; Grunwald, Thomas; Bürki, Kurt; Liebert, Uwe G; Ulbert, Sebastian

    2011-08-26

    West Nile Virus (WNV) is an emerging pathogenic flavivirus with increasing distribution worldwide. Birds are the natural host of the virus, but also mammals, including humans, can be infected. In some cases, a WNV infection can be associated with severe neurological symptoms. All currently available WNV vaccines are in the veterinary sector, and there is a need to develop safe and effective immunization technologies, which can also be used in humans. An alternative to current vaccination methods is DNA immunization. Most current DNA vaccine candidates against flaviviruses simultaneously express the viral envelope (E) and membrane (prM) proteins, which leads to the formation of virus-like particles. Here we generated a DNA plasmid, which expresses only the E-protein ectodomain. Vaccination of mice stimulated anti-WNV T-cell responses and neutralizing antibodies that were higher than those obtained after immunizing with a recombinant protein previously shown to be a protective WNV vaccine. A single dose of the plasmid was sufficient to protect animals from a lethal challenge with the virus. Moreover, immunogenicity could be boosted when DNA injection was followed by immunization with recombinant domain DIII of the E-protein. This resulted in significantly enhanced neutralizing antibody titers and a more prominent cellular immune response. The results suggest that the WNV E-protein is sufficient as a protective antigen in DNA vaccines and that protection can be significantly improved by adding a recombinant protein boost to the DNA prime.

  20. Protective Properties of Vaccinia Virus-Based Vaccines: Skin Scarification Promotes a Nonspecific Immune Response That Protects against Orthopoxvirus Disease

    PubMed Central

    Rice, Amanda D.; Adams, Mathew M.; Lindsey, Scott F.; Swetnam, Daniele M.; Manning, Brandi R.; Smith, Andrew J.; Burrage, Andrew M.; Wallace, Greg; MacNeill, Amy L.

    2014-01-01

    ABSTRACT The process of vaccination introduced by Jenner generated immunity against smallpox and ultimately led to the eradication of the disease. Procedurally, in modern times, the virus is introduced into patients via a process called scarification, performed with a bifurcated needle containing a small amount of virus. What was unappreciated was the role that scarification itself plays in generating protective immunity. In rabbits, protection from lethal disease is induced by intradermal injection of vaccinia virus, whereas a protective response occurs within the first 2 min after scarification with or without virus, suggesting that the scarification process itself is a major contributor to immunoprotection. IMPORTANCE These results show the importance of local nonspecific immunity in controlling poxvirus infections and indicate that the process of scarification should be critically considered during the development of vaccination protocols for other infectious agents. PMID:24760885

  1. Protective properties of vaccinia virus-based vaccines: skin scarification promotes a nonspecific immune response that protects against orthopoxvirus disease.

    PubMed

    Rice, Amanda D; Adams, Mathew M; Lindsey, Scott F; Swetnam, Daniele M; Manning, Brandi R; Smith, Andrew J; Burrage, Andrew M; Wallace, Greg; MacNeill, Amy L; Moyer, Richard W

    2014-07-01

    The process of vaccination introduced by Jenner generated immunity against smallpox and ultimately led to the eradication of the disease. Procedurally, in modern times, the virus is introduced into patients via a process called scarification, performed with a bifurcated needle containing a small amount of virus. What was unappreciated was the role that scarification itself plays in generating protective immunity. In rabbits, protection from lethal disease is induced by intradermal injection of vaccinia virus, whereas a protective response occurs within the first 2 min after scarification with or without virus, suggesting that the scarification process itself is a major contributor to immunoprotection. importance: These results show the importance of local nonspecific immunity in controlling poxvirus infections and indicate that the process of scarification should be critically considered during the development of vaccination protocols for other infectious agents. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  2. Difenoconazole Petition and Response (Data Protection Expires - October 29, 2024)

    EPA Pesticide Factsheets

    FIFRA exclusive use provisions for minor use registrations provide incentives to pesticide companies to register minor uses of pesticides. View petitions for exclusive use periods or extensions and EPA responses.

  3. Difenoconazole Petition and Response (Data Protection Expires December 18, 2019)

    EPA Pesticide Factsheets

    FIFRA exclusive use provisions for minor use registrations provide incentives to pesticide companies to register minor uses of pesticides. View petitions for exclusive use periods or extensions and EPA responses.

  4. RNA-Processing Protein TDP-43 Regulates FOXO-Dependent Protein Quality Control in Stress Response

    PubMed Central

    Zhang, Tao; Baldie, Gerard; Periz, Goran; Wang, Jiou

    2014-01-01

    Protein homeostasis is critical for cell survival and functions during stress and is regulated at both RNA and protein levels. However, how the cell integrates RNA-processing programs with post-translational protein quality control systems is unknown. Transactive response DNA-binding protein (TARDBP/TDP-43) is an RNA-processing protein that is involved in the pathogenesis of major neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we report a conserved role for TDP-43, from C. elegans to mammals, in the regulation of protein clearance via activation of FOXO transcription factors. In response to proteotoxic insults, TDP-43 redistributes from the nucleus to the cytoplasm, promoting nuclear translocation of FOXOs and relieving an inhibition of FOXO activity in the nucleus. The interaction between TDP-43 and the FOXO pathway in mammalian cells is mediated by their competitive binding to 14-3-3 proteins. Consistent with FOXO-dependent protein quality control, TDP-43 regulates the levels of misfolded proteins. Therefore, TDP-43 mediates stress responses and couples the regulation of RNA metabolism and protein quality control in a FOXO-dependent manner. The results suggest that compromising the function of TDP-43 in regulating protein homeostasis may contribute to the pathogenesis of related neurodegenerative diseases. PMID:25329970

  5. RNA-processing protein TDP-43 regulates FOXO-dependent protein quality control in stress response.

    PubMed

    Zhang, Tao; Baldie, Gerard; Periz, Goran; Wang, Jiou

    2014-10-01

    Protein homeostasis is critical for cell survival and functions during stress and is regulated at both RNA and protein levels. However, how the cell integrates RNA-processing programs with post-translational protein quality control systems is unknown. Transactive response DNA-binding protein (TARDBP/TDP-43) is an RNA-processing protein that is involved in the pathogenesis of major neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we report a conserved role for TDP-43, from C. elegans to mammals, in the regulation of protein clearance via activation of FOXO transcription factors. In response to proteotoxic insults, TDP-43 redistributes from the nucleus to the cytoplasm, promoting nuclear translocation of FOXOs and relieving an inhibition of FOXO activity in the nucleus. The interaction between TDP-43 and the FOXO pathway in mammalian cells is mediated by their competitive binding to 14-3-3 proteins. Consistent with FOXO-dependent protein quality control, TDP-43 regulates the levels of misfolded proteins. Therefore, TDP-43 mediates stress responses and couples the regulation of RNA metabolism and protein quality control in a FOXO-dependent manner. The results suggest that compromising the function of TDP-43 in regulating protein homeostasis may contribute to the pathogenesis of related neurodegenerative diseases.

  6. C/EBP homologous protein-induced macrophage apoptosis protects mice from steatohepatitis.

    PubMed

    Malhi, Harmeet; Kropp, Erin M; Clavo, Vinna F; Kobrossi, Christina R; Han, JaeSeok; Mauer, Amy S; Yong, Jing; Kaufman, Randal J

    2013-06-28

    Nonalcoholic fatty liver disease is a heterogeneous disorder characterized by liver steatosis; inflammation and fibrosis are features of the progressive form nonalcoholic steatohepatitis. The endoplasmic reticulum stress response is postulated to play a role in the pathogenesis of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. In particular, C/EBP homologous protein (CHOP) is undetectable under normal conditions but is induced by cellular stress, including endoplasmic reticulum stress. Chop wild type (Chop(+/+)) and knock-out (Chop(-/-)) mice were used in these studies to elucidate the role of CHOP in the pathogenesis of fatty liver disease. Paradoxically, Chop(-/-) mice developed greater liver injury, inflammation, and fibrosis than Chop(+/+) mice, with greater macrophage activation. Primary, bone marrow-derived, and peritoneal macrophages from Chop(+/+) and Chop(-/-) were challenged with palmitic acid, an abundant saturated free fatty acid in plasma and liver lipids. Where palmitic acid treatment activated Chop(+/+) and Chop(-/-) macrophages, Chop(-/-) macrophages were resistant to its lipotoxicity. Chop(-/-) mice were sensitized to liver injury in a second model of dietary steatohepatitis using the methionine-choline-deficient diet. Analysis of bone marrow chimeras between Chop(-/-) and Chop(+/+) mice demonstrated that Chop in macrophages protects from liver injury and inflammation when fed the methionine-choline-deficient diet. We conclude that Chop deletion has a proinflammatory effect in fatty liver injury apparently due to decreased cell death of activated macrophages, resulting in their net accumulation in the liver. Thus, macrophage CHOP plays a key role in protecting the liver from steatohepatitis likely by limiting macrophage survival during lipotoxicity.

  7. C/EBP Homologous Protein-induced Macrophage Apoptosis Protects Mice from Steatohepatitis*

    PubMed Central

    Malhi, Harmeet; Kropp, Erin M.; Clavo, Vinna F.; Kobrossi, Christina R.; Han, JaeSeok; Mauer, Amy S.; Yong, Jing; Kaufman, Randal J.

    2013-01-01

    Nonalcoholic fatty liver disease is a heterogeneous disorder characterized by liver steatosis; inflammation and fibrosis are features of the progressive form nonalcoholic steatohepatitis. The endoplasmic reticulum stress response is postulated to play a role in the pathogenesis of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. In particular, C/EBP homologous protein (CHOP) is undetectable under normal conditions but is induced by cellular stress, including endoplasmic reticulum stress. Chop wild type (Chop+/+) and knock-out (Chop−/−) mice were used in these studies to elucidate the role of CHOP in the pathogenesis of fatty liver disease. Paradoxically, Chop−/− mice developed greater liver injury, inflammation, and fibrosis than Chop+/+ mice, with greater macrophage activation. Primary, bone marrow-derived, and peritoneal macrophages from Chop+/+ and Chop−/− were challenged with palmitic acid, an abundant saturated free fatty acid in plasma and liver lipids. Where palmitic acid treatment activated Chop+/+ and Chop−/− macrophages, Chop−/− macrophages were resistant to its lipotoxicity. Chop−/− mice were sensitized to liver injury in a second model of dietary steatohepatitis using the methionine-choline-deficient diet. Analysis of bone marrow chimeras between Chop−/− and Chop+/+ mice demonstrated that Chop in macrophages protects from liver injury and inflammation when fed the methionine-choline-deficient diet. We conclude that Chop deletion has a proinflammatory effect in fatty liver injury apparently due to decreased cell death of activated macrophages, resulting in their net accumulation in the liver. Thus, macrophage CHOP plays a key role in protecting the liver from steatohepatitis likely by limiting macrophage survival during lipotoxicity. PMID:23720735

  8. The responsibility to protect and the root causes of conflicts.

    PubMed

    von Horn, Helge

    2005-01-01

    The increase in humanitarian crises due to states failing to observe the human rights of their citizens led to the establishment of the International Commission on Intervention and State Sovereignty. Whilst all agree with the need for concerted action to protect innocent civilians from human rights abuses the focus must be on prevention; the use of military force creates further problems. An analysis of failed states, such as Rwanda, shows how the actions of the international community, especially the most powerful states, contribute to conditions creating the abuse and how appropriate actions by the external players could have prevented these abuses.

  9. Transcription Factor Functional Protein-Protein Interactions in Plant Defense Responses

    PubMed Central

    Alves, Murilo S.; Dadalto, Silvana P.; Gonçalves, Amanda B.; de Souza, Gilza B.; Barros, Vanessa A.; Fietto, Luciano G.

    2014-01-01

    Responses to biotic stress in plants lead to dramatic reprogramming of gene expression, favoring stress responses at the expense of normal cellular functions. Transcription factors are master regulators of gene expression at the transcriptional level, and controlling the activity of these factors alters the transcriptome of the plant, leading to metabolic and phenotypic changes in response to stress. The functional analysis of interactions between transcription factors and other proteins is very important for elucidating the role of these transcriptional regulators in different signaling cascades. In this review, we present an overview of protein-protein interactions for the six major families of transcription factors involved in plant defense: basic leucine zipper containing domain proteins (bZIP), amino-acid sequence WRKYGQK (WRKY), myelocytomatosis related proteins (MYC), myeloblastosis related proteins (MYB), APETALA2/ ETHYLENE-RESPONSIVE ELEMENT BINDING FACTORS (AP2/EREBP) and no apical meristem (NAM), Arabidopsis transcription activation factor (ATAF), and cup-shaped cotyledon (CUC) (NAC). We describe the interaction partners of these transcription factors as molecular responses during pathogen attack and the key components of signal transduction pathways that take place during plant defense responses. These interactions determine the activation or repression of response pathways and are crucial to understanding the regulatory networks that modulate plant defense responses. PMID:28250372

  10. AMP-18 protects barrier function of colonic epithelial cells: role of tight junction proteins

    PubMed Central

    Walsh-Reitz, Margaret M.; Huang, Erick F.; Musch, Mark W.; Chang, Eugene B.; Martin, Terence E.; Kartha, Sreedharan; Toback, F. Gary

    2005-01-01

    AMP-18, a novel gastric antrum mucosal protein, and a synthetic peptide of amino acids 77-97, have mitogenic and motogenic properties for epithelial cells. The possibility that AMP-18 is also protective was evaluated in the colonic mucosa of mice and monolayer cultures of human colonic epithelial Caco2/bbe (C2) cells. Administration of AMP peptide to mice with dextran sulfate sodium (DSS)-induced colonic injury delayed the onset of bloody diarrhea, and reduced weight loss. Treatment of C2 cells with AMP peptide protected monolayers against decreases in transepithelial electrical resistance (TER) induced by the oxidant monochloramine, indomethacin, or DSS. A molecular mechanism for these barrier-protective effects was sought by asking if AMP peptide acted on specific tight junction (TJ) proteins. Immunoblots of detergent-insoluble fractions of C2 cells treated with AMP peptide exhibited increased accumulation of specific TJ proteins. Occludin immunoreactivity was also increased in detergent-insoluble fractions obtained from colonic mucosal cells of mice injected with AMP peptide. Laser scanning confocal microscopy (CF) supported the capacity of AMP peptide to enhance accumulation of occludin and ZO-1 in TJ domains of C2 cell monolayers, and together with immunoblot analysis showed that the peptide protected against loss of these TJ proteins following oxidant injury. AMP peptide also protected against a fall in TER during disruption of actin filaments by cytochalasin D, and stabilized perijunctional actin during oxidant injury when assessed by CF. These findings suggest that AMP-18 could protect the intestinal mucosal barrier by acting on specific TJ proteins and stabilizing perijunctional actin. PMID:15961882

  11. Recombinant Yellow Fever Viruses Elicit CD8+ T Cell Responses and Protective Immunity against Trypanosoma cruzi

    PubMed Central

    Nogueira, Raquel Tayar; Nogueira, Alanderson Rocha; Pereira, Mirian Claudia Souza; Rodrigues, Maurício Martins; Neves, Patrícia Cristina da Costa; Galler, Ricardo; Bonaldo, Myrna Cristina

    2013-01-01

    Chagas’ disease is a major public health problem affecting nearly 10 million in Latin America. Despite several experimental vaccines have shown to be immunogenic and protective in mouse models, there is not a current vaccine being licensed for humans or in clinical trial against T. cruzi infection. Towards this goal, we used the backbone of Yellow Fever (YF) 17D virus, one of the most effective and well-established human vaccines, to express an immunogenic fragment derived from T. cruzi Amastigote Surface Protein 2 (ASP-2). The cDNA sequence of an ASP-2 fragment was inserted between E and NS1 genes of YF 17D virus through the construction of a recombinant heterologous cassette. The replication ability and genetic stability of recombinant YF virus (YF17D/ENS1/Tc) was confirmed for at least six passages in Vero cells. Immunogenicity studies showed that YF17D/ENS1/Tc virus elicited neutralizing antibodies and gamma interferon (IFN-γ) producing-cells against the YF virus. Also, it was able to prime a CD8+ T cell directed against the transgenic T. cruzi epitope (TEWETGQI) which expanded significantly as measured by T cell-specific production of IFN-γ before and after T. cruzi challenge. However, most important for the purposes of vaccine development was the fact that a more efficient protective response could be seen in mice challenged after vaccination with the YF viral formulation consisting of YF17D/ENS1/Tc and a YF17D recombinant virus expressing the TEWETGQI epitope at the NS2B-3 junction. The superior protective immunity observed might be due to an earlier priming of epitope-specific IFN-γ-producing T CD8+ cells induced by vaccination with this viral formulation. Our results suggest that the use of viral formulations consisting of a mixture of recombinant YF 17D viruses may be a promising strategy to elicit protective immune responses against pathogens, in general. PMID:23527169

  12. Recombinant yellow fever viruses elicit CD8+ T cell responses and protective immunity against Trypanosoma cruzi.

    PubMed

    Nogueira, Raquel Tayar; Nogueira, Alanderson Rocha; Pereira, Mirian Claudia Souza; Rodrigues, Maurício Martins; Neves, Patrícia Cristina da Costa; Galler, Ricardo; Bonaldo, Myrna Cristina

    2013-01-01

    Chagas' disease is a major public health problem affecting nearly 10 million in Latin America. Despite several experimental vaccines have shown to be immunogenic and protective in mouse models, there is not a current vaccine being licensed for humans or in clinical trial against T. cruzi infection. Towards this goal, we used the backbone of Yellow Fever (YF) 17D virus, one of the most effective and well-established human vaccines, to express an immunogenic fragment derived from T. cruzi Amastigote Surface Protein 2 (ASP-2). The cDNA sequence of an ASP-2 fragment was inserted between E and NS1 genes of YF 17D virus through the construction of a recombinant heterologous cassette. The replication ability and genetic stability of recombinant YF virus (YF17D/ENS1/Tc) was confirmed for at least six passages in Vero cells. Immunogenicity studies showed that YF17D/ENS1/Tc virus elicited neutralizing antibodies and gamma interferon (IFN-γ) producing-cells against the YF virus. Also, it was able to prime a CD8(+) T cell directed against the transgenic T. cruzi epitope (TEWETGQI) which expanded significantly as measured by T cell-specific production of IFN-γ before and after T. cruzi challenge. However, most important for the purposes of vaccine development was the fact that a more efficient protective response could be seen in mice challenged after vaccination with the YF viral formulation consisting of YF17D/ENS1/Tc and a YF17D recombinant virus expressing the TEWETGQI epitope at the NS2B-3 junction. The superior protective immunity observed might be due to an earlier priming of epitope-specific IFN-γ-producing T CD8(+) cells induced by vaccination with this viral formulation. Our results suggest that the use of viral formulations consisting of a mixture of recombinant YF 17D viruses may be a promising strategy to elicit protective immune responses against pathogens, in general.

  13. Emerging functions of the unfolded protein response in immunity

    PubMed Central

    Janssens, Sophie; Pulendran, Bali; Lambrecht, Bart N.

    2015-01-01

    The unfolded protein response (UPR) has traditionally been viewed as an adaptive response triggered upon accumulation of unfolded proteins in the endoplasmic reticulum (ER), aimed at restoring ER function. The UPR can also be an anticipatory response that is activated well before the disruption of protein homeostasis. UPR signaling intersects at many levels with the innate and adaptive immune response. In some immune cell types like dendritic cells and B cells, particular UPR sensors appear constitutively active in the absence of traditional UPR gene program induction, necessary for antigen presentation and immunoglobulin synthesis. The UPR also influences Toll-like receptor signaling and NF-κB activation, and some pathogens subvert the UPR. This review summarizes these emerging non-canonical functions of the UPR in immunity. PMID:25232821

  14. Extracellular proteins of Cryptococcus neoformans and host antibody response.

    PubMed Central

    Chen, L C; Pirofski, L A; Casadevall, A

    1997-01-01

    Proteins secreted by the fungal pathogen Cryptococcus neoformans may be involved in invasion and could be useful in vaccine design. Despite the medical importance of this fungus, little is known about its extracellular proteins or the immune response to these antigens. To study C. neoformans extracellular proteins, 12 strains were metabolically radiolabeled and protein supernatants were analyzed. Both strain- and growth condition-dependent differences were observed. Enzymatic assays of filtered culture supernatants revealed butyrate esterase and caprylate esterase lipase activity for 11 of 12 strains, as well as acid phosphatase, naphthol-AS-BI-phosphohydrolase, and beta-glucosidase activities in some strains. Serum from infected rodents immunoprecipitated several secreted proteins, consistent with in vivo expression and development of an antibody response. For strain 24067, two immunodominant species, of approximately 75 and 30 kDa, were recognized. The relative intensity of the autoradiographic bands depended on the route of infection for both rats and mice. In summary, our results indicate that (i) there are multiple proteins in C. neoformans culture supernatants, (ii) there are strain differences in supernatant protein profiles, (iii) there are differences in supernatant protein profile depending on the growth conditions, (iv) there are several new extracellular and/or cell-associated enzymatic activities, and (v) antibodies to several supernatant proteins are made in the course of infection. PMID:9199426

  15. Protective antibody responses elicited by a meningococcal outer membrane vesicle vaccine with overexpressed genome-derived neisserial antigen 1870.

    PubMed

    Hou, Victor C; Koeberling, Oliver; Welsch, Jo Anne; Granoff, Dan M

    2005-08-15

    Background. Meningococcal outer membrane vesicle (OMV) vaccines are efficacious in humans but have serosubtype-specific serum bactericidal antibody responses directed at the porin protein PorA and the potential for immune selection of PorA-escape mutants.Methods. We prepared an OMV vaccine from a Neisseria meningitidis strain engineered to overexpress genome-derived neisserial antigen (GNA) 1870, a lipoprotein discovered by genome mining that is being investigated for use in a vaccine.Results. Mice immunized with the modified GNA1870-OMV vaccine developed broader serum bactericidal antibody responses than control mice immunized with a recombinant GNA1870 protein vaccine or an OMV vaccine prepared from wild-type N. meningitidis or a combination of vaccines prepared from wild-type N. meningitidis and recombinant protein. Antiserum from mice immunized with the modified GNA1870-OMV vaccine also elicited greater deposition of human C3 complement on the surface of live N. meningitidis bacteria and greater passive protective activity against meningococcal bacteremia in infant rats. A N. meningitidis mutant with decreased expression of PorA was more susceptible to bactericidal activity of anti-GNA1870 antibodies.Conclusions. The modified GNA1870-OMV vaccine elicits broader protection against meningococcal disease than recombinant GNA1870 protein or conventional OMV vaccines and also has less risk of selection of PorA-escape mutants than a conventional OMV vaccine.

  16. Protective Antibody Responses Elicited by a Meningococcal Outer Membrane Vesicle Vaccine with Overexpressed Genome-Derived Neisserial Antigen 1870

    PubMed Central

    Hou, Victor C.; Koeberling, Oliver; Welsch, Jo Anne; Granoff, Dan M.

    2008-01-01

    Background Meningococcal outer membrane vesicle (OMV) vaccines are efficacious in humans but have serosubtype-specific serum bactericidal antibody responses directed at the porin protein PorA and the potential for immune selection of PorA-escape mutants. Methods We prepared an OMV vaccine from a Neisseria meningitidis strain engineered to overexpress genome-derived neisserial antigen (GNA) 1870, a lipoprotein discovered by genome mining that is being investigated for use in a vaccine. Results Mice immunized with the modified GNA1870-OMV vaccine developed broader serum bactericidal antibody responses than control mice immunized with a recombinant GNA1870 protein vaccine or an OMV vaccine prepared from wild-type N. meningitidis or a combination of vaccines prepared from wild-type N. meningitidis and recombinant protein. Antiserum from mice immunized with the modified GNA1870-OMV vaccine also elicited greater deposition of human C3 complement on the surface of live N. meningitidis bacteria and greater passive protective activity against meningococcal bacteremia in infant rats. A N. meningitidis mutant with decreased expression of PorA was more susceptible to bactericidal activity of anti-GNA1870 antibodies. Conclusions The modified GNA1870-OMV vaccine elicits broader protection against meningococcal disease than recombinant GNA1870 protein or conventional OMV vaccines and also has less risk of selection of PorA-escape mutants than a conventional OMV vaccine. PMID:16028126

  17. Serum Response Factor Protects Retinal Ganglion Cells Against High-Glucose Damage.

    PubMed

    Cao, Yan; Wang, Liang; Zhao, Junhong; Zhang, Hongbing; Tian, Ying; Liang, Houcheng; Ma, Qiang

    2016-06-01

    Serum response factor (SRF), which encodes the MADS-box family of related proteins, is a common transcription factor related to the expression of genes associated with cell survival. However, SRF's role in retinal ganglion cells (RGCs) after high-glucose injury remains unclear. In this study, we investigate the protective role of SRF after high-glucose injury and its underlying mechanism. The in vitro RGC model subjected to high glucose was established by employing a 50 mmol/L glucose culture environment. As detected by real-time quantitative PCR and Western blot, SRF was significantly upregulated in RGCs treated with high glucose. Overexpression of SRF significantly promoted survival among RGCs exposed to high glucose and inhibited RGC apoptosis. Knockdown of SRF exerted an inverse effect. Moreover, SRF upregulation enhanced expression of an antioxidant protein, nuclear factor erythroid 2-related factor (Nrf2), via control of the Fos-related antigen 1 (Fra-1). SRF upregulation also affected RGC survival after high-glucose treatment. Our findings showed that overexpression of SRF promoted survival of RGCs after high-glucose injury by regulating Fra-1 and Nrf2.

  18. Glycyrrhizin protects against porcine endotoxemia through modulation of systemic inflammatory response

    PubMed Central

    2013-01-01

    Introduction Glycyrrhizin (GL) was recently found to suppress high-mobility group box 1 (HMGB1)-induced injury by binding directly to it. However, the effect of GL on HMGB1 expression in endotoxemia as well as its underlying molecular mechanism remained unclear. Methods Twenty-one pigs were divided into four groups: sham group (n = 3), control group (n = 6), ethyl pyruvate group (n = 6) and glycyrrhizin group (n = 6). Pigs were anesthetized, mechanically ventilated, monitored and given a continuous intravenous infusion of lipopolysaccharide (LPS). Twelve hours after the start of the LPS infusion, ethyl pyruvate (30 mg/kg/hr) or glycyrrhizin (1 mg/kg/hr) was administered for 12 hours. Systemic and pulmonary hemodynamics, oxygen exchange, and metabolic status were measured. The concentrations of cytokines in serum and the corresponding gene and protein expressions in tissue samples from liver, lungs, kidneys, small intestine and lymph nodes were measured. Results GL maintained the stability of systemic hemodynamics and improved pulmonary oxygen exchange and metabolic status. GL also attenuated organ injury and decreased the serum levels of HMGB1 and other pro-inflammatory cytokines by inhibiting their gene and protein expression. Conclusions GL improved systemic hemodynamics and protected vital organs against porcine endotoxemia through modulation of the systemic inflammatory response. By reducing the serum level and gene expression of HMGB1 and other pro-inflammatory cytokines, GL may become a potential agent for the treatment of sepsis. PMID:23497622

  19. 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.

  20. The Unfolded Protein Response in Homeostasis and Modulation of Mammalian Immune Cells.

    PubMed

    Martins, Ana Sofia; Alves, Inês; Helguero, Luisa; Domingues, Maria Rosário; Neves, Bruno Miguel

    2016-11-01

    The endoplasmic reticulum (ER) plays important roles in eukaryotic protein folding and lipid biosynthesis. Several exogenous and endogenous cellular sources of stress can perturb ER homeostasis leading to the accumulation of unfolded proteins in the lumen. Unfolded protein accumulation triggers a signal-transduction cascade known as the unfolded protein response (UPR), an adaptive mechanism which aims to protect cells from protein aggregates and to restore ER functions. Further to this protective mechanism, in immune cells, UPR molecular effectors have been shown to participate in a wide range of biological processes such as cell differentiation, survival and immunoglobulin and cytokine production. Recent findings also highlight the involvement of the UPR machinery in the maturational program and antigen presentation capacities of dendritic cells. UPR is therefore a key element in immune system homeostasis with direct implications on both adaptive and innate immune responses. The present review summarizes the knowledge on the emerging roles of UPR signaling cascades in mammalian immune cells as well as the consequences of their dysregulation in relation to the pathogenesis of several diseases.

  1. Aging Is Accompanied by a Blunted Muscle Protein Synthetic Response to Protein Ingestion

    PubMed Central

    Wall, Benjamin Toby; Gorissen, Stefan H.; Pennings, Bart; Koopman, René; Groen, Bart B. L.; Verdijk, Lex B.; van Loon, Luc J. C.

    2015-01-01

    Purpose Progressive loss of skeletal muscle mass with aging (sarcopenia) forms a global health concern. It has been suggested that an impaired capacity to increase muscle protein synthesis rates in response to protein intake is a key contributor to sarcopenia. We assessed whether differences in post-absorptive and/or post-prandial muscle protein synthesis rates exist between large cohorts of healthy young and older men. Procedures We performed a cross-sectional, retrospective study comparing in vivo post-absorptive muscle protein synthesis rates determined with stable isotope methodologies between 34 healthy young (22±1 y) and 72 older (75±1 y) men, and post-prandial muscle protein synthesis rates between 35 healthy young (22±1 y) and 40 older (74±1 y) men. Findings Post-absorptive muscle protein synthesis rates did not differ significantly between the young and older group. Post-prandial muscle protein synthesis rates were 16% lower in the older subjects when compared with the young. Muscle protein synthesis rates were >3 fold more responsive to dietary protein ingestion in the young. Irrespective of age, there was a strong negative correlation between post-absorptive muscle protein synthesis rates and the increase in muscle protein synthesis rate following protein ingestion. Conclusions Aging is associated with the development of muscle anabolic inflexibility which represents a key physiological mechanism underpinning sarcopenia. PMID:26536130

  2. Zinc finger antiviral protein inhibits coxsackievirus B3 virus replication and protects against viral myocarditis.

    PubMed

    Li, Min; Yan, Kepeng; Wei, Lin; Yang, Jie; Lu, Chenyu; Xiong, Fei; Zheng, Chunfu; Xu, Wei

    2015-11-01

    The host Zinc finger antiviral protein (ZAP) has been reported exhibiting antiviral activity against positive-stranded RNA viruses (Togaviridae), negative-stranded RNA viruses (Filoviridae) and retroviruses (Retroviridae). However, whether ZAP restricts the infection of enterovirus and the development of enterovirus mediated disease remains unknown. Here, we reported the antiviral properties of ZAP against coxsackievirus B3 (CVB3), a single-stranded RNA virus of the Enterovirus genus within the Picornaviridae as a major causative agent of viral myocarditis (VMC). We found that the expression of ZAP was significantly induced after CVB3 infection in heart tissues of VMC mice. ZAP potently inhibited CVB3 replication in cells after infection, while overexpression of ZAP in mice significantly increased the resistance to CVB3 replication and viral myocarditis by significantly reducing cardiac inflammatory cytokine production. The ZAP-responsive elements (ZREs) were mapped to the 3'UTR and 5'UTR of viral RNA. Taken together, ZAP confers resistance to CVB3 infection via directly targeting viral RNA and protects mice from acute myocarditis by suppressing viral replication and cardiac inflammatory cytokine production. Our finding further expands ZAP's range of viral targets, and suggests ZAP as a potential therapeutic target for viral myocarditis caused by CVB3.

  3. Bioinformatics and variability in drug response: a protein structural perspective

    PubMed Central

    Lahti, Jennifer L.; Tang, Grace W.; Capriotti, Emidio; Liu, Tianyun; Altman, Russ B.

    2012-01-01

    Marketed drugs frequently perform worse in clinical practice than in the clinical trials on which their approval is based. Many therapeutic compounds are ineffective for a large subpopulation of patients to whom they are prescribed; worse, a significant fraction of patients experience adverse effects more severe than anticipated. The unacceptable risk–benefit profile for many drugs mandates a paradigm shift towards personalized medicine. However, prior to adoption of patient-specific approaches, it is useful to understand the molecular details underlying variable drug response among diverse patient populations. Over the past decade, progress in structural genomics led to an explosion of available three-dimensional structures of drug target proteins while efforts in pharmacogenetics offered insights into polymorphisms correlated with differential therapeutic outcomes. Together these advances provide the opportunity to examine how altered protein structures arising from genetic differences affect protein–drug interactions and, ultimately, drug response. In this review, we first summarize structural characteristics of protein targets and common mechanisms of drug interactions. Next, we describe the impact of coding mutations on protein structures and drug response. Finally, we highlight tools for analysing protein structures and protein–drug interactions and discuss their application for understanding altered drug responses associated with protein structural variants. PMID:22552919

  4. DNA vaccines encoding viral envelope proteins confer protective immunity against WSSV in black tiger shrimp.

    PubMed

    Rout, Namita; Kumar, Sudhir; Jaganmohan, Shanmugam; Murugan, Vadivel

    2007-04-12

    White Spot Syndrome Virus (WSSV) is a major cause of mortality in shrimp and poses a huge threat to aquaculture industry. Till now no comprehensive or individual strategy has been established to combat white spot disease. Previous efforts by other investigators have given insight of protein vaccination and its efficacy to protect shrimp against WSSV infection. In this study, we have explored the protective efficacy of DNA vaccination and tissue distribution of the immunised recombinant plasmid in black tiger shrimp (Penaeus monodon). Four recombinant constructs were generated by inserting four genes encoding the WSSV structural proteins VP15, VP28, VP35 and VP281 individually into DNA vaccine vector pVAX1. Expression of these proteins from the recombinant plasmids was confirmed in vitro in CHO cell lines. For vaccination experiments, shrimp were immunised with these DNA constructs and later challenged with WSSV. A significant level of protection was offered by the plasmids encoding VP28 or VP281 till 7 weeks whereas protein vaccination failed to protect vaccinated shrimp after 3 weeks of first immunisation. In addition, our tissue distribution study revealed the persistence of immunised DNA at least upto 2 months in the injected shrimp muscle. Thus, our results suggest that DNA vaccination strategy will have potential utility against WSSV infection in shrimp cultivation.

  5. The 90-kDa Heat Shock Protein Hsp90 Protects Tubulin against Thermal Denaturation*

    PubMed Central

    Weis, Felix; Moullintraffort, Laura; Heichette, Claire; Chrétien, Denis; Garnier, Cyrille

    2010-01-01

    Hsp90 and tubulin are among the most abundant proteins in the cytosol of eukaryotic cells. Although Hsp90 plays key roles in maintaining its client proteins in their active state, tubulin is essential for fundamental processes such as cell morphogenesis and division. Several studies have suggested a possible connection between Hsp90 and the microtubule cytoskeleton. Because tubulin is a labile protein in its soluble form, we investigated whether Hsp90 protects it against thermal denaturation. Both proteins were purified from porcine brain, and their interaction was characterized in vitro by using spectrophotometry, sedimentation assays, video-enhanced differential interference contrast light microscopy, and native polyacrylamide gel electrophoresis. Our results show that Hsp90 protects tubulin against thermal denaturation and keeps it in a state compatible with microtubule polymerization. We demonstrate that Hsp90 cannot resolve tubulin aggregates but that it likely binds early unfolding intermediates, preventing their aggregation. Protection was maximal at a stoichiometry of two molecules of Hsp90 for one of tubulin. This protection does not require ATP binding and hydrolysis by Hsp90, but it is counteracted by geldanamycin, a specific inhibitor of Hsp90. PMID:20110359

  6. Meeting Report: Structural Determination of Environmentally Responsive Proteins

    PubMed Central

    Reinlib, Leslie

    2005-01-01

    The three-dimensional structure of gene products continues to be a missing lynchpin between linear genome sequences and our understanding of the normal and abnormal function of proteins and pathways. Enhanced activity in this area is likely to lead to better understanding of how discrete changes in molecular patterns and conformation underlie functional changes in protein complexes and, with it, sensitivity of an individual to an exposure. The National Institute of Environmental Health Sciences convened a workshop of experts in structural determination and environmental health to solicit advice for future research in structural resolution relative to environmentally responsive proteins and pathways. The highest priorities recommended by the workshop were to support studies of structure, analysis, control, and design of conformational and functional states at molecular resolution for environmentally responsive molecules and complexes; promote understanding of dynamics, kinetics, and ligand responses; investigate the mechanisms and steps in posttranslational modifications, protein partnering, impact of genetic polymorphisms on structure/function, and ligand interactions; and encourage integrated experimental and computational approaches. The workshop participants also saw value in improving the throughput and purity of protein samples and macromolecular assemblies; developing optimal processes for design, production, and assembly of macromolecular complexes; encouraging studies on protein–protein and macromolecular interactions; and examining assemblies of individual proteins and their functions in pathways of interest for environmental health. PMID:16263521

  7. Responsive block copolymer photonics triggered by protein-polyelectrolyte coacervation.

    PubMed

    Fan, Yin; Tang, Shengchang; Thomas, Edwin L; Olsen, Bradley D

    2014-11-25

    Ionic interactions between proteins and polyelectrolytes are demonstrated as a method to trigger responsive transitions in block copolymer (BCP) photonic gels containing one neutral hydrophobic block and one cationic hydrophilic block. Poly(2-vinylpyridine) (P2VP) blocks in lamellar poly(styrene-b-2-vinylpyridine) block copolymer thin films are quaternized with primary bromides to yield swollen gels that show strong reflectivity peaks in the visible range; exposure to aqueous solutions of various proteins alters the swelling ratios of the quaternized P2VP (QP2VP) gel layers in the PS-QP2VP materials due to the ionic interactions between proteins and the polyelectrolyte. Parameters such as charge density, hydrophobicity, and cross-link density of the QP2VP gel layers as well as the charge and size of the proteins play significant roles on the photonic responses of the BCP gels. Differences in the size and pH-dependent charge of proteins provide a basis for fingerprinting proteins based on their temporal and equilibrium photonic response. The results demonstrate that the BCP gels and their photonic effect provide a robust and visually interpretable method to differentiate different proteins.

  8. Eimeria tenella heat shock protein 70 enhances protection of recombinant microneme protein MIC2 subunit antigen vaccination against E. tenella challenge.

    PubMed

    Zhang, Lei; Ma, Liping; Liu, Renqiang; Zhang, Yunfei; Zhang, Shouping; Hu, Chunmei; Song, Meng; Cai, Jianping; Wang, Ming

    2012-09-10

    Heat shock proteins have been reported to stimulate the immune system via innate receptors. Our study found that the novel immunopotentiator, Eimeria tenella (E. tenella) heat shock protein 70 (HSP70), enhanced protective immunity elicited by E. tenella antigen microneme protein 2 (EtMIC2) against avian coccidiosis. It demonstrated that the expression of TLR2 and TLR4 were strongly upregulated in EtHSP70 and EtMIC2 plus EtHSP70 stimulated chicken embryo fibroblasts (CEF) compared with untreated controls and EtMIC2 alone. In addition, the same treatment induced high levels of interleukin (IL)-12 and interferon (IFN)-γ that are critical cytokines of innate immunity. In vivo experiments involved using broiler chickens subcutaneously immunized with EtMIC2 alone or EtMIC2 plus EtHSP70 at 7 and 14 days post-hatch, which were then orally challenged with live E. tenella at 7 days following secondary immunization. Body weight gains, cecal lesion scores, fecal oocyst shedding, serum antibody responses against MIC2, and intestinal cytokine transcript levels were assessed as measures of protective immunity. Chickens immunized with EtMIC2 plus EtHSP70 showed increased body weight gains, decreased oocyst shedding, increased serum antibody responses, and high levels of IL-12, IFN-γ, and IL-17 compared with the EtMIC2 only or control groups. Moreover, chickens immunized with EtHSP70 alone showed significantly protective effect against E. tenella infection. In summary, this study provides the first evidence of the immunoenhancing activities of EtHSP70 in poultry.

  9. Immune Responses and Protection of Aotus Monkeys Immunized with Irradiated Plasmodium vivax Sporozoites

    DTIC Science & Technology

    2011-01-01

    responses and protective efficacy induced by vacci- nation with irradiated P vivax sporozoites were evaluated in malaria-naive Aotus monkeys. Three groups...received either 10 doses of uninfected salivary gland extract or no inoculations. Immunization resulted in the production low levels of antibodies that...responses. Intravenous challenge with viable sporozoites resulted in partial protection in a dose-dependent manner.l11ese findings suggest that the

  10. Protection from heat-induced protein migration and DNA repair inhibition by cycloheximide.

    PubMed

    Armour, E P; Lee, Y J; Corry, P M; Borrelli, M J

    1988-12-15

    The mechanism by which Cycloheximide (CHM) protects cells from heat induced killing has been investigated. Cycloheximide (10 micrograms/ml) added for 2 hr before and during a 3 hour heating at 43 degrees C prevented a 40% increase of heat-induced protein accumulation in the nucleus and protected cells (0.0001 vs. 0.15 surviving fraction) from heat-induced killing. Heat-induced DNA repair inhibition was also suppressed when cells were treated with CHM in the above manner. This combination of results suggests that protein accumulation in the nucleus and inhibition of DNA repair are related and these events are associated with CHM protection from heat induced cell killing.

  11. LKTA and PlpE small fragments fusion protein protect against Mannheimia haemolytica challenge.

    PubMed

    Guzmán-Brambila, Carolina; Quintero-Fabián, Saray; González-Castillo, Celia; de Obeso-Fernández del Valle, Álvaro; Flores-Samaniego, Beatriz; de la Mora, Germán; Rojas-Mayorquín, Argelia E; Ortuño-Sahagún, Daniel

    2012-12-01

    Bovine respiratory disease (BRD) complex is a major cause of economic losses for the cattle backgrounding and feedlot industries. Mannheimia haemolytica is considered the most important pathogen associated with this disease. Vaccines against M. haemolytica have been prepared and used for many decades, but traditional bacterins have failed to demonstrate effective protection and their use has often exacerbated disease in vaccinated animals. Thus, the BRD complex continues to exert a strong adverse effect on the health and wellbeing of stocker and feeder cattle. Therefore, generation of recombinant proteins has been helpful in formulating enhanced vaccines against M. haemolytica, which could confer better protection against BRD. In the present study, we formulated a vaccine preparation enriched with recombinant small fragments of leukotoxin A (LKTA) and outer-membrane lipoprotein (PlpE) proteins, and demonstrated its ability to generate high antibody titers in rabbits and sheep, which protected against M. haemolytica bacterial challenge in mice.

  12. E3 Ubiquitin Ligase CHIP and NBR1-Mediated Selective Autophagy Protect Additively against Proteotoxicity in Plant Stress Responses

    PubMed Central

    Qi, Jingxia; Chi, Yingjin; Fan, Baofang; Yu, Jing-Quan; Chen, Zhixiang

    2014-01-01

    Plant stress responses require both protective measures that reduce or restore stress-inflicted damage to cellular structures and mechanisms that efficiently remove damaged and toxic macromolecules, such as misfolded and damaged proteins. We have recently reported that NBR1, the first identified plant autophagy adaptor with a ubiquitin-association domain, plays a critical role in plant stress tolerance by targeting stress-induced, ubiquitinated protein aggregates for degradation by autophagy. Here we report a comprehensive genetic analysis of CHIP, a chaperone-associated E3 ubiquitin ligase from Arabidopsis thaliana implicated in mediating degradation of nonnative proteins by 26S proteasomes. We isolated two chip knockout mutants and discovered that they had the same phenotypes as the nbr1 mutants with compromised tolerance to heat, oxidative and salt stresses and increased accumulation of insoluble proteins under heat stress. To determine their functional interactions, we generated chip nbr1 double mutants and found them to be further compromised in stress tolerance and in clearance of stress-induced protein aggregates, indicating additive roles of CHIP and NBR1. Furthermore, stress-induced protein aggregates were still ubiquitinated in the chip mutants. Through proteomic profiling, we systemically identified heat-induced protein aggregates in the chip and nbr1 single and double mutants. These experiments revealed that highly aggregate-prone proteins such as Rubisco activase and catalases preferentially accumulated in the nbr1 mutant while a number of light-harvesting complex proteins accumulated at high levels in the chip mutant after a relatively short period of heat stress. With extended heat stress, aggregates for a large number of intracellular proteins accumulated in both chip and nbr1 mutants and, to a greater extent, in the chip nbr1 double mutant. Based on these results, we propose that CHIP and NBR1 mediate two distinct but complementary anti

  13. Humoral and Cellular Immunity to Plasmodium falciparum Merozoite Surface Protein 1 and Protection From Infection With Blood-Stage Parasites

    PubMed Central

    Moormann, Ann M.; Sumba, Peter Odada; Chelimo, Kiprotich; Fang, Hua; Tisch, Daniel J.; Dent, Arlene E.; John, Chandy C.; Long, Carole A.; Vulule, John; Kazura, James W.

    2013-01-01

    Background. Acquired immunity to malaria develops with increasing age and repeated infections. Understanding immune correlates of protection from malaria would facilitate vaccine development and identification of biomarkers that reflect changes in susceptibility resulting from ongoing malaria control efforts. Methods. The relationship between immunoglobulin G (IgG) antibody and both interferon γ (IFN-γ) and interleukin 10 (IL-10) responses to the 42-kD C-terminal fragment of Plasmodium falciparum merozoite surface protein 1 (MSP142) and the risk of (re)infection were examined following drug-mediated clearance of parasitemia in 94 adults and 95 children in an area of holoendemicity of western Kenya. Results. Positive IFN-γ enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunosorbent spot assay (ELISPOT) responses to MSP142 3D7 were associated with delayed time to (re)infection, whereas high-titer IgG antibodies to MSP142 3D7 or FVO alleles were not independently predictive of the risk of (re)infection. When IFN-γ and IL-10 responses were both present, the protective effect of IFN-γ was abrogated. A Cox proportional hazard model including IFN-γ, IL-10, MSP142 3D7 IgG antibody responses, hemoglobin S genotype, age, and infection status at baseline showed that the time to blood-stage infection correlated positively with IFN-γ responses and negatively with IL-10 responses, younger age, and asymptomatic parasitemia. Conclusions. Evaluating combined allele-specific cellular and humoral immunity elicited by malaria provides a more informative measure of protection relative to evaluation of either measure