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Sample records for bacteriocin-like immunity protein

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

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

    2009-12-17

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

  2. Description of two Enterococcus strains isolated from traditional Peruvian artisanal-produced cheeses with a bacteriocin-like inhibitory activity

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    Aguilar Galvez A.

    2009-01-01

    Full Text Available The aim of this work was to isolate and to characterize strains of lactic acid bacteria (LAB with bacteriocin-like inhibitory activity from 27 traditional cheeses artisanal-produced obtained from different Peruvian regions. Twenty Gram+ and catalasenegative strains among 2,277 isolates exhibited bacteriocin-like inhibitory activity against Listeria monocytogenes CWBIB2232 as target strain. No change in inhibitory activity was observed after organic acid neutralization and treatment with catalase of the cell-free supernatant (CFS. The proteinic nature of the antimicrobial activity was confirmed for the twenty LAB strains by proteolytic digestion of the CFS. Two strains, CWBI-B1431 and CWBI-B1430, with the best antimicrobial activity were selected for further researches. These strains were taxonomically identified by phenotypic and genotypic analyses as Enterococcus mundtii (CWBI-B1431 and Enterococcus faecium (CWBI-B1430. The two strains were sensitive to vancomycin (MIC 2 μg.ml-1 and showed absence of haemolysis.

  3. Characterization of a bacteriocin-like substance produced from a novel isolated strain of Bacillus subtilis SLYY-3

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    Li, Junfeng; Li, Hongfang; Zhang, Yuanyuan; Duan, Xiaohui; Liu, Jie

    2014-12-01

    In the present research, the strain SLYY-3 was isolated from sediments of Jiaozhou Bay, Qingdao, China. The strain SLYY-3, which produced a bacteriocin-like substance (BLS), was characterized to be a strain of Bacillus subtillis by biochemical profiling and 16S rDNA sequence analysis. It is the first time to report that Bacillus subtilis from Jiaozhou Bay sediments could produce a BLS. The BLS of B. subtillis SLYY-3 exhibited strong inhibitory activity against gram-positive bacteria (including Staphylococcus aureus and B. subtillis) and some fungi (including Penicillium glaucum, Aspergillus niger and Aspergillus flavus). The antimicrobial activity was detected from culture in the exponential growth phase and reached its maximum when culture entered into stationary growth phase. It was thermo-tolerant even when being kept at 100°C for 60 min without losing any activity and stable over a wide pH range from 1.0 to 12.0 while being inactivated by proteolytic enzyme and trypsin, indicating the proteinaceous nature of the BLS. The BLS was purified by precipitation with hydrochloric acid (HCl) and gel filteration (Sephadex G-100). SDS-PAGE analysis of the extracellular peptides of SLYY-3 revealed a bacteriocin-like protein with a molecular mass of 66 kDa. Altogether, these characteristics indicate the potential of the BLS for food industry as a protection against pathogenic and spoilage microorganisms.

  4. Diverse Ecological Strategies Are Encoded by Streptococcus pneumoniae Bacteriocin-Like Peptides.

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    Miller, Eric L; Abrudan, Monica I; Roberts, Ian S; Rozen, Daniel E

    2016-04-13

    The opportunistic pathogen Streptococcus pneumoniae is commonly carried asymptomatically in the human nasopharynx. Due to high rates of cocolonization with other pneumococcus strains, intraspecific competitive interactions partly determine the carriage duration of strains and thereby their potential to cause disease. These interactions may be mediated by bacteriocins, such as the type IIb bacteriocins encoded by the blp (bacteriocin-like peptide) locus. To understand blp diversity and evolution, we undertook a bioinformatic analysis of 4,418 pneumococcal genomes, including 168 newly sequenced genomes. We describe immense variation at all levels of genomic organization: Gene presence/absence, gene order, and allelic diversity. If we make the extreme and naive hypothesis that assumes all genes in this operon can assort randomly, this variation could lead to 10(15) distinct bacteriocin-related phenotypes, each potentially representing a unique ecological strategy; however, we provide several explanations for why this extreme is not realized. Although rarefaction analysis indicates that the number of unique strategies is not saturated, even after sampling thousands of genomes, we show that the variation is neither unbounded nor random. We delimit three bacteriocin groups, which contain group-specific bacteriocins, immunity genes, and blp operon gene order, and argue that this organization places a constraint on realized ecological strategies. We additionally show that ecological strategy diversity is significantly constrained by pneumococcal phylogeny and clonal structure. By examining patterns of association between alleles within the blp operon, we show that bacteriocin genes, which were believed to function in pairs, can be found with a broad diversity of partner alleles and immunity genes; this overall lack of allelic fidelity likely contributes to the fluid structure of this operon. Our results clarify the diversity of antagonistic ecological strategies in the

  5. In vitro evaluation of bacteriocin-like inhibitory substances produced by lactic acid bacteria isolated during traditional Sicilian cheese making

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    Giusi Macaluso

    2016-02-01

    Full Text Available Bacteriocins are antimicrobial proteins produced by bacteria that inhibit the growth of other bacteria with a bactericidal or bacteriostatic mode of action. Many lactic acid bacteria (LAB produce a high diversity of different bacteriocins. Bacteriocinogenic LAB are generally recognised as safe (GRAS and useful to control the frequent development of pathogens and spoilage microorganisms. For this reason they are commonly used as starter cultures in food fermentations. In this study, the authors describe the results of a screening on 699 LAB isolated from wooden vat surfaces, raw milk and traditional Sicilian cheeses, for the production of bacteriocin-like inhibitory substances, by comparing two alternative methods. The antagonistic activity of LAB and its proteinaceous nature were evaluated using the spot-on-the-lawn and the well-diffusion assay (WDA and the sensitivity to proteolytic (proteinase K, protease B and trypsin, amylolytic (α-amylase and lipolytic (lipase enzymes. The indicator strains used were: Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Salmonella enteritidis. A total of 223 strains (belonging to the species Enterococcus spp., Lactobacillus spp., Pediococcus spp., Streptococcus spp., Leuconostoc spp. and Lactococcus lactis were found to inhibit the growth of Listeria monocytogenes by using the spot-on-the-lawn method; only 37 of these were confirmed by using the WDA. The direct addition of bacteriocin-producing cultures into dairy products can be a more practical and economic option for the improvement of the safety and quality of the final product.

  6. In Vitro Evaluation of Bacteriocin-Like Inhibitory Substances Produced by Lactic Acid Bacteria Isolated During Traditional Sicilian Cheese Making

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    Macaluso, Giusi; Fiorenza, Gerlando; Gaglio, Raimondo; Mancuso, Isabella

    2016-01-01

    Bacteriocins are antimicrobial proteins produced by bacteria that inhibit the growth of other bacteria with a bactericidal or bacteriostatic mode of action. Many lactic acid bacteria (LAB) produce a high diversity of different bacteriocins. Bacteriocinogenic LAB are generally recognised as safe (GRAS) and useful to control the frequent development of pathogens and spoilage microorganisms. For this reason they are commonly used as starter cultures in food fermentations. In this study, the authors describe the results of a screening on 699 LAB isolated from wooden vat surfaces, raw milk and traditional Sicilian cheeses, for the production of bacteriocin-like inhibitory substances, by comparing two alternative methods. The antagonistic activity of LAB and its proteinaceous nature were evaluated using the spot-on-the-lawn and the well-diffusion assay (WDA) and the sensitivity to proteolytic (proteinase K, protease B and trypsin), amylolytic (a-amylase) and lipolytic (lipase) enzymes. The indicator strains used were: Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Salmonella enteritidis. A total of 223 strains (belonging to the species Enterococcus spp., Lactobacillus spp., Pediococcus spp., Streptococcus spp., Leuconostoc spp. and Lactococcus lactis) were found to inhibit the growth of Listeria monocytogenes by using the spot-on-the-lawn method; only 37 of these were confirmed by using the WDA. The direct addition of bacteriocin-producing cultures into dairy products can be a more practical and economic option for the improvement of the safety and quality of the final product.

  7. Evidence for production of a bacteriocin-like substance by Staphylococcus pseudintermedius, inhibitory to Staphylococcus aureus from foods.

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    Pinto, Taiz Siqueira; de Oliveira, Cybelle Pereira; da Costa, Ana Carolina Vieira; Lima, Catiana Oliveira; Barreto, Humberto Medeiros; de Souza, Evandro Leite; Siqueira-Junior, José Pinto

    2013-01-01

    This study assessed the production of a bacteriocin-like substance by Staphylococcus pseudintermedius S28, and evaluates its inhibitory effect against isolates of S. aureus from foods. All indicator isolates were sensitive to the substance produced from S. pseudintermedius S28, showing growth inhibition zones ranging from 14.2 to 28.3 mm. The inhibitory substance has no effect against the producer strain. The inhibitory substance was affected by proteolytic enzymes, while glycolytic and lipolytic enzymes had no effect, suggesting that the active substance could be considered as a bacteriocin-like substance. From these results, S. pseudintermedius S28 could be an interesting producer of a bacteriocin-like substance capable of strongly inhibiting S. aureus.

  8. Antibacterial activity of bacteriocin-like substance P34 on Listeria monocytogenes in chicken sausage.

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    Sant'Anna, Voltaire; Quadros, Deoni A F; Motta, Amanda S; Brandelli, Adriano

    2013-12-01

    The antimicrobial activity of the bacteriocin-like substance (BLS) P34 against Listeria monocytogenes was investigated in chicken sausage. The BLS was applied to chicken sausages (256 AU g(-1)) previously inoculated with a suspension of 10(2) cfu g(-1) of L. monocytogenes. BLS P34 inhibited the indicator microorganism in situ in all incubation times for up to 10 days at 5 °C. The effectiveness of BLS P34 was increased when it was added in combination with nisin. The bacteriocin was also tested in natural eatable natural bovine wrapping (salty semi-dried tripe) against the same indicator microorganism, also showing inhibitory capability in vitro. BLS P34 showed potential to control L. monocytogenes in refrigerated meat products.

  9. Antibacterial activity of bacteriocin-like substance P34 on Listeria monocytogenes in chicken sausage

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    Voltaire Sant'Anna

    2013-12-01

    Full Text Available The antimicrobial activity of the bacteriocin-like substance (BLS P34 against Listeria monocytogenes was investigated in chicken sausage. The BLS was applied to chicken sausages (256 AU g-1 previously inoculated with a suspension of 10² cfu g-1 of L. monocytogenes. BLS P34 inhibited the indicator microorganism in situ in all incubation times for up to 10 days at 5 °C. The effectiveness of BLS P34 was increased when it was added in combination with nisin. The bacteriocin was also tested in natural eatable natural bovine wrapping (salty semi-dried tripe against the same indicator microorganism, also showing inhibitory capability in vitro. BLS P34 showed potential to control L. monocytogenes in refrigerated meat products.

  10. Bacteriocin-like substance from Bacillus amyloliquefaciens shows remarkable inhibition of Acanthamoeba polyphaga.

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    Benitez, Lisianne Brittes; Caumo, Karin; Brandelli, Adriano; Rott, Marilise Brittes

    2011-03-01

    The effectiveness of a bacteriocin-like substance (BLS) produced by Bacillus amyloliquefaciens was tested against Acanthamoeba polyphaga strains, and its cytotoxic potential on Vero cells was investigated. Amebicidal activity of the purified BLS was tested by plate bioassays with concentrations ranging from 12.5 to 6,400 AU mL(-1). Damage to A. pholyphaga cells was monitored using an inverted microscope and counted in a Fuchs-Rosenthal chamber after 24, 48, and 72 h. According to the results obtained, the BLS showed remarkable amebicidal and amebostatic effect on A. polyphaga and showed no cytotoxicity on the Vero cells. These results may have great relevance in the development of new acanthamoebicidal compounds.

  11. Antibacterial activity of bacteriocin-like substance P34 on Listeria monocytogenes in chicken sausage

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    Sant’Anna, Voltaire; Quadros, Deoni A.F.; Motta, Amanda S.; Brandelli, Adriano

    2013-01-01

    The antimicrobial activity of the bacteriocin-like substance (BLS) P34 against Listeria monocytogenes was investigated in chicken sausage. The BLS was applied to chicken sausages (256 AU g−1) previously inoculated with a suspension of 102 cfu g−1 of L. monocytogenes. BLS P34 inhibited the indicator microorganism in situ in all incubation times for up to 10 days at 5 °C. The effectiveness of BLS P34 was increased when it was added in combination with nisin. The bacteriocin was also tested in natural eatable natural bovine wrapping (salty semi-dried tripe) against the same indicator microorganism, also showing inhibitory capability in vitro. BLS P34 showed potential to control L. monocytogenes in refrigerated meat products. PMID:24688506

  12. Characterization of Bacteriocin like inhibitory substance produced by a new Strain Brevibacillus borstelensis AG1 Isolated from 'Marcha'

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    Nivedita Sharma

    2014-09-01

    Full Text Available In the present study, a bacterium isolated from Marcha- a herbal cake used as traditional starter culture to ferment local wine in North East India, was evaluated for bacteriocin like inhibitory substance production and was tested against six food borne/spoilage causing pathogens viz. Listeria monocytogenes MTCC 839, Bacillus subtilis MTCC 121, Clostridium perfringens MTCC 450, Staphylococcus aureus, Lactobacillus plantarum and Leuconostoc mesenteroides MTCC 107 by using bit/disc method followed by well diffusion method. The bacterial isolate was identified as Brevibacillus borstelensis on the basis of phenotypic, biochemical and molecular characteristics using 16Sr RNA gene technique. Bacteriocin like inhibitory substance produced by Brevibacillus borstelensis AG1 was purified by gel exclusion chromatography. The molecular mass of the Brevibacillus borstelensis AG1 was found to be 12 kDa. Purified bacteriocin like inhibitory substance of Brevibacillus borstelensis was further characterized by studying the effect of temperature, pH, proteolytic enzyme and stability. Bacteriocin like inhibitory substance was found to be thermostable upto 100 °C, active at neutral pH, sensitive to trypsin, and partially stable till third week of storage thus showing a bright prospective to be used as a potential food biopreservative.

  13. Characterization of Bacteriocin like inhibitory substance produced by a new Strain Brevibacillus borstelensis AG1 Isolated from 'Marcha'.

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    Sharma, Nivedita; Gupta, Anupama; Gautam, Neha

    2014-01-01

    In the present study, a bacterium isolated from Marcha- a herbal cake used as traditional starter culture to ferment local wine in North East India, was evaluated for bacteriocin like inhibitory substance production and was tested against six food borne/spoilage causing pathogens viz. Listeria monocytogenes MTCC 839, Bacillus subtilis MTCC 121, Clostridium perfringens MTCC 450, Staphylococcus aureus, Lactobacillus plantarum and Leuconostoc mesenteroides MTCC 107 by using bit/disc method followed by well diffusion method. The bacterial isolate was identified as Brevibacillus borstelensis on the basis of phenotypic, biochemical and molecular characteristics using 16Sr RNA gene technique. Bacteriocin like inhibitory substance produced by Brevibacillus borstelensis AG1 was purified by gel exclusion chromatography. The molecular mass of the Brevibacillus borstelensis AG1 was found to be 12 kDa. Purified bacteriocin like inhibitory substance of Brevibacillus borstelensis was further characterized by studying the effect of temperature, pH, proteolytic enzyme and stability. Bacteriocin like inhibitory substance was found to be thermostable upto 100 °C, active at neutral pH, sensitive to trypsin, and partially stable till third week of storage thus showing a bright prospective to be used as a potential food biopreservative.

  14. Characterization of Bacteriocin like inhibitory substance produced by a new Strain Brevibacillus borstelensis AG1 Isolated from ‘Marcha’

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    Sharma, Nivedita; Gupta, Anupama; Gautam, Neha

    2014-01-01

    In the present study, a bacterium isolated from Marcha- a herbal cake used as traditional starter culture to ferment local wine in North East India, was evaluated for bacteriocin like inhibitory substance production and was tested against six food borne/spoilage causing pathogens viz. Listeria monocytogenes MTCC 839, Bacillus subtilis MTCC 121, Clostridium perfringens MTCC 450, Staphylococcus aureus, Lactobacillus plantarum and Leuconostoc mesenteroides MTCC 107 by using bit/disc method followed by well diffusion method. The bacterial isolate was identified as Brevibacillus borstelensis on the basis of phenotypic, biochemical and molecular characteristics using 16Sr RNA gene technique. Bacteriocin like inhibitory substance produced by Brevibacillus borstelensis AG1 was purified by gel exclusion chromatography. The molecular mass of the Brevibacillus borstelensis AG1 was found to be 12 kDa. Purified bacteriocin like inhibitory substance of Brevibacillus borstelensis was further characterized by studying the effect of temperature, pH, proteolytic enzyme and stability. Bacteriocin like inhibitory substance was found to be thermostable upto 100 °C, active at neutral pH, sensitive to trypsin, and partially stable till third week of storage thus showing a bright prospective to be used as a potential food biopreservative. PMID:25477937

  15. Isolation and biochemical characterisation of a bacteriocin-like substance produced by Bacillus amyloliquefaciens An6.

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    Ayed, Hanen Ben; Maalej, Hana; Hmidet, Noomen; Nasri, Moncef

    2015-12-01

    This study focuses on the isolation and characterisation of a peptide with bacteriocin-like properties from Bacillus amyloliquefaciens An6. Incubation conditions were optimised, and the effects of the incubation period and of carbon and nitrogen sources were investigated. The produced bacteriocin was partially purified with ammonium sulphate precipitation, dialysis and ultrafiltration and was then biochemically characterised. Maximum bacteriocin production was achieved after 48h of incubation in a culture medium containing 20g/L starch and 10g/L yeast extract, with an initial pH 8.0 at 30°C under continuous agitation at 200rpm. The bacteriocin was sequentially purified and its molecular weight was determined to be 11kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The bacteriocin was relatively heat-resistant and was not sensitive to acid and alkaline conditions (pH 4.0-10.0). Its inhibitory activity was sensitive to proteinase K but was resistant to the proteolytic action of alcalase, trypsin, chymotrypsin and pepsin. In conclusion, bacteriocin An6, owing its wide spectrum of activity as well as its high tolerance to acidic and alkaline pH values, temperature and proteases shows great potential for use as a food biopreservative.

  16. Production of bacteriocin-like inhibitory substance by Bifidobacterium lactis in skim milk supplemented with additives.

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    Martinez, Fabio Andres Castillo; Domínguez, José Manuel; Converti, Attilio; Oliveira, Ricardo Pinheiro de Souza

    2015-08-01

    Bacteriocins are natural compounds used as food biopreservatives instead of chemical preservatives. Bifidobacterium animalis subsp. lactis (Bifid. lactis) was shown to produce a bacteriocin-like inhibitory substance (BLIS) able to inhibit the growth of Listeria monocytogenes selected as an indicator microorganism. To enhance this production by the strain Bifid. lactis BL 04, skim milk (SM) was used as a fermentation medium either in the presence or in the absence of yeast extract, Tween 80 or inulin as stimulating additives, and the results in terms of bacterial growth and BLIS production were compared with those obtained in a traditional high cost complex medium such as Man, Rogosa and Sharpe (MRS). To this purpose, all the cultivations were carried out in flasks at 200 rpm under anaerobic conditions ensured by a nitrogen flowrate of 1.0 L/min for 48 h, and BLIS production was quantified by means of a modified agar diffusion assay at low values of both temperature and concentration of List. monocytogenes. Although all these ingredients were shown to exert positive influence on BLIS production in both media, yeast extract and SM were by far the best ingredient and the best medium, respectively, allowing for a BLIS production at the late exponential phase of 2000 AU/ml.

  17. TSST-1, enterotoxin and bacteriocin-like substance production by Staphylococcus aureus isolated from foods

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    S.A. Carvalho

    2013-10-01

    Full Text Available The production of Toxic Shock Syndrome Toxin-1 (TSST-1, enterotoxins and bacteriocin-like substances was evaluated in 95 strains of Staphylococcus aureus recovered from raw bovine milk (n=31 and from food samples involved in staphylococcal food poisoning (n=64. Enterotoxigenicity tests with the membrane over agar associated to optimal sensibility plate assays were performed and showed that 96.77% of strains recovered from milk and 95.31% from food samples produced enterotoxins A, B, C, D or TSST-1. Reference strains S. epidermidis, Bacillus cereus, Listeria monocytogenes, Lactobacillus casei, Pseudomonas aeruginosa, S. aureus, Salmonella Typhimurium, Escherichia coli, Enterococcus faecalis and Bacteroides fragilis were used as indicator bacteria in the antagonistic assays, the first five being sensitive to antagonistic substances. Brain heart infusion agar, in pH values ranging from 5.0 to 7.0 in aerobic atmosphere showed to be the optimum condition for antagonistic activity as evaluated with the best producer strains against the most sensitive indicator bacterium, L. monocytogenes. Sensitivity to enzymes confirmed the proteinaceous nature of these substances. Neither bacteriophage activity nor fatty acids were detected and the antagonistic activity was not due to residual chloroform. Results did not establish a positive correlation between the bacteriocinogenic profile and toxigenicity in the tested S. aureus strains.

  18. Antimicrobial activity and partial characterization of bacteriocin-like inhibitory substances produced by Lactobacillus spp. isolated from artisanal Mexican cheese.

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    Heredia-Castro, Priscilia Y; Méndez-Romero, José I; Hernández-Mendoza, Adrián; Acedo-Félix, Evelia; González-Córdova, Aarón F; Vallejo-Cordoba, Belinda

    2015-12-01

    Lactobacillus spp. from Mexican Cocido cheese were shown to produce bacteriocin-like substances (BLS) active against Staphylococcus aureus,Listeria innocua,Escherichia coli, andSalmonella typhimurium by using the disk diffusion method. Crude extracts of Lactobacillus fermentum showed strong inhibitory activity against Staph. aureus, L. innocua, E. coli, and Salmonella cholerae. Complete inactivation of antimicrobial activity was observed after treatment of crude extracts with proteinase K, pronase, papain, trypsin, and lysozyme, confirming their proteinaceous nature. However, antimicrobial activity was partly lost for some of the crude extracts when treated with α-amylase, indicating that carbohydrate moieties were involved. The antimicrobial activity of the crude extracts was stable at 65°C for 30min over a wide pH range (2-8), and addition of potassium chloride, sodium citrate, ethanol, and butanol did not affect antibacterial activity. However, antimicrobial activity was lost after heating at 121°C for 15min, addition of methanol or Tween 80. Fourteen out of 18 Lactobacillus spp. showed antimicrobial activity against different test microorganisms, and 12 presented bacteriocin-like substances. Generation time and growth rate parameters indicated that the antimicrobial activity of crude extracts from 3 different strains was effective against the 4 indicator microorganisms. One of the crude extracts showed inhibition not only against gram-positive but also against gram-negative bacteria. Bacteriocin-like substances produced by this specific Lactobacillus strain showed potential for application as a food biopreservative.

  19. Screening of marine bacteria with bacteriocin-like activities and probiotic potential for ornate spiny lobster (Panulirus ornatus) juveniles.

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    Nguyen, Van Duy; Pham, Thu Thuy; Nguyen, Thi Hai Thanh; Nguyen, Thi Thanh Xuan; Hoj, Lone

    2014-09-01

    Bacteriocins are ribosomally synthesized antimicrobial peptides, which have been found in diverse bacterial species of terrestrial origins and some from the sea. New bacteriocins with new characteristics, new origins and new applications are likely still awaiting discovery. The present study screened bacteria isolated from marine animals of interest to the aquaculture industry for antimicrobial and bacteriocin-like activities in order to uncover biodiversity of bacteriocin producers, and explore the potential application in aquaculture. In total, 24 of 100 screened isolates showed antimicrobial activities and 7 of these exerted bacteriocin-like activities. Sequencing of 16S rRNA genes identified the isolates as members of the six genera Proteus, Providencia, Klebsiella, Alcaligenes, Bacillus and Enterococcus. In some cases, further analysis of housekeeping genes, rpoB for Proteus and recA for Klebsiella, as well as biochemical tests was necessary for identification to species level, and some of the Proteus isolates may represent novel species. The seven bacteriocinogenic isolates showed a wide antimicrobial spectrum against foodborne and animal pathogens, which opens the way to their potential use as marine drugs and probiotics in food, aquaculture, livestock and clinical settings. As a case study, the protective effect of shortlisted bacteriocinogenic isolates were tested in aquaculture-raised spiny lobster (Panulirus ornatus) juveniles. A single-strain (Bacillus pumilus B3.10.2B) and a three-strain (B. pumilus B3.10.2B, Bacillus cereus D9, Lactobacillus plantarum T13) probiotic preparation were added to the feed of Panulirus ornatus juveniles, which were subsequently challenged with the pathogen Vibrio owensii DY05. Juveniles in the probiotic treatments displayed increased growth and reduced feed conversion rates after 60 days, and increased survival rate after pathogen challenge relative to the control. This study represents the first evidence of bacteriocin

  20. Aqueous two-phase flotation for primary recovery of bacteriocin-like inhibitory substance (BLIS) from Pediococcus acidilactici Kp10.

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    Md Sidek, Nurul Lyana; Tan, Joo Shun; Abbasiliasi, Sahar; Wong, Fadzlie Wong Faizal; Mustafa, Shuhaimi; Ariff, Arbakariya B

    2016-08-01

    An aqueous two-phase flotation (ATPF) system based on polyethylene glycol (PEG) and sodium citrate (NaNO3C6H5O7·2H2O) was considered for primary recovery of bacteriocin-like inhibitory substance (BLIS) from Pediococcus acidilactici Kp10. The effects of ATPF parameters namely phase composition, tie-line length (TLL), volume ratio between the two phases (VR), amount of crude load (CL), pH, nitrogen gas flow rate (FR) and flotation time (FT) on the performance of recovery were evaluated. BLIS was mainly concentrated into the upper PEG-rich phase in all systems tested so far. The optimum conditions for BLIS purification, which composed of PEG 8000/sodium citrate, were: TLL of 42.6, VR of 0.4, CL of 22% (w/w), pH 7, average FT of 30min and FR of 20mL/min. BLIS was partially purified up to 5.9-fold with a separation efficiency of 99% under this optimal conditions. A maximum yield of BLIS activity of about 70.3% was recovered in the PEG phase. The BLIS from the top phase was successfully recovered with a single band in SDS-gel with molecular weight of about 10-15kDa. ATPF was found to be an effective technique for the recovery of BLIS from the fermentation broth of P. acidilactici Kp10.

  1. The role of environmental factors and medium composition on bacteriocin-like inhibitory substances (BLIS) production by Enterococcus mundtii strains.

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    Settanni, Luca; Valmorri, Sara; Suzzi, Giovanna; Corsetti, Aldo

    2008-08-01

    Bacteriocin-like inhibitory substances (BLIS)-producers Enterococcus mundtii WGWT1-1A, WGW11.2, WGJ20.1, WGJ40.2 and WGK53 from raw material origin were subjected to a study for the characterization of antimicrobial compound production under several growth conditions, including different cultivation media, growth temperatures, pHs, different concentrations and sources of nitrogen compounds, carbohydrates and other nutritional factors, and in the presence of different percentages of ethanol and NaCl. The five E. mundtii strains showed different behaviors. However, in all cases, MRS and sour dough bacteria (SDB) were found as the optimal media for BLIS production. In general, the higher BLIS production was observed with pH in the range 6.0-8.0 and, except 45 degrees C, the temperature did not show a defining effect. Low or no BLIS activity was detected after growth without nitrogen sources and carbohydrates. Absence of Tween 80, triammoniun citrate, K2HPO4, MgSO4 and MnSO4 did not affect BLIS activity levels. Except for a strain (WGWT1-1A), ethanol did not play a negative role in BLIS expression, while NaCl determined decrease of BLIS activity, proportional with concentration. The above strains did not contain plasmids, hence, BLIS expression is encoded by chromosomal DNA.

  2. Identification and partial characterization of a bacteriocin-like inhibitory substance (BLIS) from Lb. Bulgaricus K41 isolated from indigenous yogurts.

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    Zaeim, Davood; Soleimanian-Zad, Sabihe; Sheikh-Zeinoddin, Mahmoud

    2014-01-01

    Forty-two strains of Lactobacillus bulgaricus isolated from locally made yogurts were examined and compared for bacteriocin producing ability using spot on lawn assay which improved by taking photo and image processing. Lb. bulgaricus K41 exhibited the highest inhibition level against indicators. K41 Bacteriocin-like inhibitory substance is sensitive to proteolytic enzymes (proteinase K, pepsin, and trypsin) but α-amylase makes slight reduction in its activity and it is resistant to lipase. This antibacterial peptide is extremely heat-stable (121 °C for 15 min) and remains active over a wide pH range (pH = 2 to 10); also nonionic detergents (Tween-20, Tween-80, and Triton X100) showed no effect on its activity. The inhibitory spectrum is against Gram-positive bacteria (except Staphylococcus aureus) with extremely antilisterial activity and it is almost ineffective against Gram-negative bacteria. The mode of its action was identified as bactericidal against Listeria monocytogenes. The properties of K41 bacteriocin-like inhibitory substance add to its safety as a biopreservative produced by a generally recognized as safe (GRAS) bacterium suggesting it can be used in hurdle technology for ready-to-eat foods as one of the main sources of Listeria contaminations.

  3. Heat Shock Protein and Innate Immunity

    Institute of Scientific and Technical Information of China (English)

    Min-FuTsan; BaochongGao

    2004-01-01

    In addition to serving as molecular chaperones, heat shock proteins (HSPs) have been implicated in autoimmune diseases, antigen presentation and tumor immunity. Extensive work in the last 10 years has also suggested that HSPs such as Hsp60, Hsp70, Hsp90 and gp96, may be potent activators of the innate immune system capable of inducing the production of pro-inflammatory cytokines by the monocyte-macrophage system, and the activation and maturation of dendritic cells via the Toll-like receptor 2 and 4 signal transduction pathways. However, recent evidence suggests that the reported cytokine effects of HSPs may be a result of the contaminating bacterial cell-wall products. This concise review summarizes the current controversy over the role of HSPs in innate immunity. Cellular & Molecular Immunology.

  4. Heat Shock Protein and Innate Immunity

    Institute of Scientific and Technical Information of China (English)

    Min-Fu Tsan; Baochong Gao

    2004-01-01

    In addition to serving as molecular chaperones, heat shock proteins (HSPs) have been implicated in autoimmune diseases, antigen presentation and tumor immunity. Extensive work in the last 10 years has also suggested that HSPs such as Hsp60, Hsp70, Hsp90 and gp96, may be potent activators of the innate immune system capable of inducing the production of pro-inflammatory cytokines by the monocyte-macrophage system, and the activation and maturation of dendritic cells via the Toll-like receptor 2 and 4 signal transduction pathways. However, recent evidence suggests that the reported cytokine effects of HSPs may be a result of the contaminating bacterial cell-wall products. This concise review summarizes the current controversy over the role of HSPs in innate immunity.

  5. Functional Classification of Immune Regulatory Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Rubinstein, Rotem [Albert Einstein College of Medicine, Bronx, NY (United States); Ramagopal, Udupi A. [Albert Einstein College of Medicine, Bronx, NY (United States); Nathenson, Stanley G. [Albert Einstein College of Medicine, Bronx, NY (United States); Almo, Steven C. [Albert Einstein College of Medicine, Bronx, NY (United States); Fiser, Andras [Albert Einstein College of Medicine, Bronx, NY (United States)

    2013-05-01

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

  6. In vivo protein synthesis determinations in human immune cells

    OpenAIRE

    Januszkiewicz, Anna

    2005-01-01

    Intact immune responses are essential for defeating severe infections in individual patients. Insufficient function of the immune system contributes to a poor prognosis in these patients, in particular the ICU patients. Nevertheless, the immune system function is not easily monitored and evaluated. The ongoing metabolic activity of immune competent cells is reflected by their in vivo protein synthesis rate. The aim of this thesis was to apply in vivo protein synthesis measur...

  7. Anti-Listeria monocytogenes bacteriocin-like inhibitory substances from Enterococcus faecium UQ31 isolated from artisan Mexican-style cheese.

    Science.gov (United States)

    Alvarado, C; García-Almendárez, B E; Martin, S E; Regalado, C

    2005-08-01

    Artisan fresh Mexican-style cheeses are commonly made from raw milk that provides not only rich flavors, but also a diversity of associated lactic acid bacteria (LAB) strains. Enterococcus faecium UQ31 was isolated from panela cheese and produced bacteriocin-like inhibitory substances (BLIS) with a strong anti-Listeria activity. A modified pH-mediated adsorption-desorption purification process resulted in (after SDS-PAGE) two bands showing antimicrobial activities, where most of the activity corresponded to the band with an estimated molecular weight of 7.5 kDa. The BLIS produced by E. faecium UQ31 were heat resistant, stable at ambient storage conditions, and active in the pH range 5--9. The BLIS antimicrobial activities were detected during logarithmic growth phase and remained constant until the end of incubation time (19 h). These BLIS showed a wide anti-Listeria monocytogenes spectra. The E. faecium UQ31 strain or their BLIS represent a promising potential as antimicrobial food preservatives.

  8. Isolation of Pediococcus acidilactici Kp10 with ability to secrete bacteriocin-like inhibitory substance from milk products for applications in food industry

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    Abbasiliasi Sahar

    2012-11-01

    Full Text Available Abstract Background Lactic acid bacteria (LAB can be isolated from traditional milk products. LAB that secrete substances that inhibit pathogenic bacteria and are resistant to acid, bile, and pepsin but not vancomycin may have potential in food applications. Results LAB isolated from a range of traditional fermented products were screened for the production of bacteriocin-like inhibitory substances. A total of 222 LAB strains were isolated from fermented milk products in the form of fresh curds, dried curds, and ghara (a traditional flavor enhancer prepared from whey, and fermented cocoa bean. Eleven LAB isolates that produced antimicrobial substances were identified as Lactococcus lactis, Lactobacillus plantarum, and Pediococcus acidilactici strains by biochemical methods and 16S rDNA gene sequencing. Of these, the cell-free supernatant of Kp10 (P. acidilactici most strongly inhibited Listeria monocytogenes. Further analysis identified the antimicrobial substance produced by Kp10 as proteinaceous in nature and active over a wide pH range. Kp10 (P. acidilactici was found to be catalase-negative, able to produce β-galactosidase, resistant to bile salts (0.3% and acidic conditions (pH 3, and susceptible to most antibiotics. Conclusion Traditionally prepared fermented milk products are good sources of LAB with characteristics suitable for industrial applications. The isolate Kp10 (P. acidilactici shows potential for the production of probiotic and functional foods.

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

    Science.gov (United States)

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

    2016-10-01

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

  10. Differential Protein Network Analysis of the Immune Cell Lineage

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    Trevor Clancy

    2014-01-01

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

  11. Inducible immune proteins in the dampwood termite Zootermopsis angusticollis

    Science.gov (United States)

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

    2007-01-01

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

  12. The immune response to sand fly salivary proteins and its influence on Leishmania immunity

    Directory of Open Access Journals (Sweden)

    Regis eGomes

    2012-05-01

    Full Text Available 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 upon multiple contacts with a mammalian host. Immunization with single immunogenic salivary proteins or exposure to uninfected bites can produce protective immune responses against leishmaniasis. These sand fly salivary proteins induce cellular immune responses and/or antibodies. Antibodies to saliva are not required for protection in a mouse model against leishmaniasis. 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 immunity to sand fly salivary proteins in the context of its vector-parasite-host combinations and 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.

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

    Science.gov (United States)

    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.

  14. Heat Shock Proteins: Stimulators of Innate and Acquired Immunity

    Directory of Open Access Journals (Sweden)

    Camilo A. Colaco

    2013-01-01

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

  15. Polyclonal Antibody Production for Membrane Proteins via Genetic Immunization.

    Science.gov (United States)

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

    2016-02-24

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

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

    Science.gov (United States)

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

    2009-03-23

    Dermanyssus gallinae is the most significant ectoparasite of European poultry egg laying production systems due to high costs of control and associated production losses as well as adverse effects on bird welfare. In this study, soluble proteins were extracted from unfed D. gallinae (DGE) using a urea-based detergent and ultra-filtration, passed through a 0.22 microm filter and blended aseptically with adjuvant. One group of laying hens was immunized with DGE and adjuvant (Montanide ISA 50 V) whilst another group (Control) received physiological saline and adjuvant. All birds were immunized on two occasions, 21 days apart. Antibody response to immunization was determined by ELISA and western blotting using immunoglobulins (Igs) extracted from egg yolk. DGE immunization of hens resulted in a significant (Pgallinae can be used to stimulate a protective immune response in laying hens. Further work is needed to identify other proteins of interest that could confer higher protection against D. gallinae, as well as optimization of the vaccination and in vitro testing protocol.

  17. 枯草芽孢杆菌MA139类细菌素抑菌活性的研究%Studies on the Antibacterial Activity of Bacteriocin-like Inhibitory Substances (BLIS) Produced from Bacillus subtilis MA139

    Institute of Scientific and Technical Information of China (English)

    胡婷; 陆文清

    2012-01-01

    为测定枯草芽孢杆菌MA139产生的类细菌素的抑菌活性.本试验采用管碟法对枯草芽孢杆菌MA139产生的类细菌素对多株革兰氏阳性菌、革兰氏阴性菌和真菌的抑菌活性进行测定.结果表明:类细菌素不但对芽孢杆菌属的其他菌种有抗菌活性,对病原菌和霉菌也有拮抗作用;不同指示菌对类细菌素的敏感性不一样,其中金黄色葡萄球菌对其最敏感.该类细菌素具有广谱抗菌作用,有作为畜禽饲料添加剂的潜力.%One experiment was conducted to determine the antibacterial activity of bacteriocin -like inhibitory substances (BLIS) produced from Bacillus subtilis MA139. The bacteriocin-like inhibitory substances were screened for antibacterial activity against indicator strains using agar spot assay. The results showed that they had broad spectrum of antagonistic activity against indicator strains, and Staphylococcus aureus showed greatest sensitivity to the BLIS. In conclusion, BOS from Bacillus subtilis MA139 had broad spectrum of antagonistic activity against various species of bacterial and fungal pathogens and could potentially be used as feed additives.

  18. Protein bio-corona: critical issue in immune nanotoxicology.

    Science.gov (United States)

    Neagu, Monica; Piperigkou, Zoi; Karamanou, Konstantina; Engin, Ayse Basak; Docea, Anca Oana; Constantin, Carolina; Negrei, Carolina; Nikitovic, Dragana; Tsatsakis, Aristidis

    2017-03-01

    With the expansion of the nanomedicine field, the knowledge focusing on the behavior of nanoparticles in the biological milieu has rapidly escalated. Upon introduction to a complex biological system, nanomaterials dynamically interact with all the encountered biomolecules and form the protein "bio-corona." The decoration with these surface biomolecules endows nanoparticles with new properties. The present review will address updates of the protein bio-corona characteristics as influenced by nanoparticle's physicochemical properties and by the particularities of the encountered biological milieu. Undeniably, bio-corona generation influences the efficacy of the nanodrug and guides the actions of innate and adaptive immunity. Exploiting the dynamic process of protein bio-corona development in combination with the new engineered horizons of drugs linked to nanoparticles could lead to innovative functional nanotherapies. Therefore, bio-medical nanotechnologies should focus on the interactions of nanoparticles with the immune system for both safety and efficacy reasons.

  19. Cross-serotype immunity induced by immunization with a conserved rhinovirus capsid protein.

    Directory of Open Access Journals (Sweden)

    Nicholas Glanville

    Full Text Available Human rhinovirus (RV infections are the principle cause of common colds and precipitate asthma and COPD exacerbations. There is currently no RV vaccine, largely due to the existence of ∼150 strains. We aimed to define highly conserved areas of the RV proteome and test their usefulness as candidate antigens for a broadly cross-reactive vaccine, using a mouse infection model. Regions of the VP0 (VP4+VP2 capsid protein were identified as having high homology across RVs. Immunization with a recombinant VP0 combined with a Th1 promoting adjuvant induced systemic, antigen specific, cross-serotype, cellular and humoral immune responses. Similar cross-reactive responses were observed in the lungs of immunized mice after infection with heterologous RV strains. Immunization enhanced the generation of heterosubtypic neutralizing antibodies and lung memory T cells, and caused more rapid virus clearance. Conserved domains of the RV capsid therefore induce cross-reactive immune responses and represent candidates for a subunit RV vaccine.

  20. Human immune cell targeting of protein nanoparticles - caveospheres

    Science.gov (United States)

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

    2016-04-01

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

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

    Science.gov (United States)

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

    2009-01-01

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

  2. INDUCTION OF ANTIVIRAL IMMUNE-RESPONSES BY IMMUNIZATION WITH RECOMBINANT-DNA ENCODED AVIAN CORONAVIRUS NUCLEOCAPSID PROTEIN

    NARCIS (Netherlands)

    BOOTS, AMH; BENAISSATROUW, BJ; HESSELINK, W; RIJKE, E; SCHRIER, C; HENSEN, EJ; Boots, Annemieke

    1992-01-01

    Immune responses to the infectious bronchitis virus (IBV) nucleocapsid protein were studied using a recombinant-DNA expression product. In mice, a lymphocyte proliferative response and a delayed-type hypersensitivity reaction to IBV were induced upon immunization with this nucleocapsid protein. Next

  3. RNA-binding protein Lin28 in cancer and immunity.

    Science.gov (United States)

    Jiang, Shuai; Baltimore, David

    2016-05-28

    The highly conserved RNA-binding protein, Lin28, is involved in many biological processes, including development, reprogramming, pluripotency, and metabolism. Importantly, Lin28 functions as an oncogene, promoting tumor progression and metastasis in various human cancers. Lin28 can regulate gene expression either by directly binding to mRNAs or by blocking microRNA biogenesis, and the underlying mechanisms include Let-7-dependent and Let-7-independent modes of action. Recent evidence shows that Lin28 also plays a fundamental role in immunity. The roles of Lin28 in disease are complex and require characterization of its physiological functions in cancer and immunological contexts. Here we review emerging information on the role of Lin28 in cancer and immunity and the molecular mechanisms it uses. We discuss our present knowledge of the system and highlight remaining mysteries related to the functions of this small RNA-binding protein. This knowledge may lead to Lin28 becoming a diagnostic marker for cancer or immune-related diseases and a possible therapeutic target.

  4. Bacteriocin-like activity of oral Fusobacterium nucleatum isolated from human and non-human primates Atividade semelhante a bacteriocina de Fusobacterium nucleatum orais isolados de primatas humanos e não-humanos

    Directory of Open Access Journals (Sweden)

    Elerson Gaetti-Jardim Júnior

    1999-12-01

    Full Text Available Fusobacterium nucleatum is indigenous of the human oral cavity and has been involved in different infectious processes. The production of bacteriocin-like substances may be important in regulation of bacterial microbiota in oral cavity. The ability to produce bacteriocin-like substances by 80 oral F. nucleatum isolates obtained from periodontal patients, healthy individuals and Cebus apella monkeys, was examinated. 17.5% of all tested isolates showed auto-antagonism and 78.8% iso- or hetero-antagonism. No isolate from monkey was capable to produce auto-inhibition. In this study, the antagonistic substances production was variable in all tested isolates. Most of the F. nucleatum showed antagonistic activity against tested reference strains. These data suggest a possible participation of these substances on the oral microbial ecology in humans and animals. However, the role of bacteriocins in regulating dental plaque microbiota in vivo is discussed.Fusobacterium nucleatum é indígena da cavidade oral humana e tem sido envolvido em diferentes processos infecciosos. A produção de substâncias semelhantes a bacteriocinas pode ser importante na regulação da microbiota bacteriana da cavidade oral. A capacidade de produzir substâncias tipo bacteriocina de 80 isolados de F. nucleatum orais, obtidos de pacientes com doença periodontal, indivíduos sadios e macaco Cebus apella, foi avaliada. 17,5% de todos os isolados mostrou auto-antagonismo e 78,8% iso- ou hetero-antagonismo. Nenhum isolado de macaco foi capaz de produzir auto-inibição. Neste estudo, a produção de substâncias antagonístas foi variável em todos os isolados testados. A maioria dos F. nucleatum mostrou atividade antagonísta para as cepas de referência testadas. Esses dados sugerem a possível participação dessas substâncias sobre a ecologia microbiana em humanos e animais. Entretanto, o papel das bacteriocinas na regulação da microbiota da placa dental in vivo

  5. Alphacoronavirus protein 7 modulates host innate immune response.

    Science.gov (United States)

    Cruz, Jazmina L G; Becares, Martina; Sola, Isabel; Oliveros, Juan Carlos; Enjuanes, Luis; Zúñiga, Sonia

    2013-09-01

    Innate immune response is the first line of antiviral defense resulting, in most cases, in pathogen clearance with minimal clinical consequences. Viruses have developed diverse strategies to subvert host defense mechanisms and increase their survival. In the transmissible gastroenteritis virus (TGEV) as a model, we previously reported that accessory gene 7 counteracts the host antiviral response by associating with the catalytic subunit of protein phosphatase 1 (PP1c). In the present work, the effect of the absence of gene 7 on the host cell, during infection, was further analyzed by transcriptomic analysis. The pattern of gene expression of cells infected with a recombinant mutant TGEV, lacking gene 7 expression (rTGEV-Δ7), was compared to that of cells infected with the parental virus (rTGEV-wt). Genes involved in the immune response, the interferon response, and inflammation were upregulated during TGEV infection in the absence of gene 7. An exacerbated innate immune response during infection with rTGEV-Δ7 virus was observed both in vitro and in vivo. An increase in macrophage recruitment and activation in lung tissues infected with rTGEV-Δ7 virus was observed compared to cells infected with the parental virus. In summary, the absence of protein 7 both in vitro and in vivo led to increased proinflammatory responses and acute tissue damage after infection. In a porcine animal model, which is immunologically similar to humans, we present a novel example of how viral proteins counteract host antiviral pathways to determine the infection outcome and pathogenesis.

  6. Wolbachia surface protein induces innate immune responses in mosquito cells

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    Pinto Sofia B

    2012-01-01

    Full Text Available Abstract Background Wolbachia endosymbiotic bacteria are capable of inducing chronic upregulation of insect immune genes in some situations and this phenotype may influence the transmission of important insect-borne pathogens. However the molecules involved in these interactions have not been characterized. Results Here we show that recombinant Wolbachia Surface Protein (WSP stimulates increased transcription of immune genes in mosquito cells derived from the mosquito Anopheles gambiae, which is naturally uninfected with Wolbachia; at least two of the upregulated genes, TEP1 and APL1, are known to be important in Plasmodium killing in this species. When cells from Aedes albopictus, which is naturally Wolbachia-infected, were challenged with WSP lower levels of upregulation were observed than for the An. gambiae cells. Conclusions We have found that WSP is a strong immune elicitor in a naturally Wolbachia-uninfected mosquito species (Anopheles gambiae while a milder elicitor in a naturally-infected species (Aedes albopictus. Since the WSP of a mosquito non-native (nematode Wolbachia strain was used, these data suggest that there is a generalized tolerance to WSP in Ae. albopictus.

  7. Screening and Identification of Bacteriocin-like Substance Producing Bacillus%一株产细菌素物质芽孢杆菌的筛选和鉴定

    Institute of Scientific and Technical Information of China (English)

    李俊峰; 李红芳; 段孝辉; 宿烽; 田智刚

    2011-01-01

    One Bacillus strain named SLY—3 producing bacteriocin-like substance ( BLS) was screened from the soil polluted by oil. The G +bacteria and molds were effectively inhibited by BLS produced by strain SLY—3. With the analysis of colony morphology, physiological and biochemistry experiments and 16S rDNA gene sequence, the strain SLY—3 was identified as Bacillus subtilis. The BLS secreted by Bacillus subtilis SLY—3 has the highest inhibitory activity when it is cultured for 24 h at initial pH 7.0 and 28 ℃ .%从石油污染的土壤中筛选出一株能产生细菌素类物质的芽孢杆菌,命名为SLY-3.该菌株分泌的活性物质抑菌活性好,对细菌主要是革兰氏阳性菌、霉菌都有抑制作用.从表型、生理生化反应及16S rDNA序列比对方面进行分析,最终确定菌株SLY-3 为枯草芽孢杆菌(Bacillus subtilis).培养基初始pH为7.0,28℃振荡培养24 h后,发酵产物抑菌活性最高.

  8. Immunity to distinct sand fly salivary proteins primes the anti-Leishmania immune response towards protection or exacerbation of disease.

    Directory of Open Access Journals (Sweden)

    Fabiano Oliveira

    Full Text Available BACKGROUND: Leishmania parasites are transmitted in the presence of sand fly saliva. Together with the parasite, the sand fly injects biologically active salivary components that favorably change the environment at the feeding site. Exposure to bites or to salivary proteins results in immunity specific to these components. Mice immunized with Phlebotomus papatasi salivary gland homogenate (SGH or pre-exposed to uninfected bites were protected against Leishmania major infection delivered by needle inoculation with SGH or by infected sand fly bites. Immunization with individual salivary proteins of two sand fly species protected mice from L. major infection. Here, we analyze the immune response to distinct salivary proteins from P. papatasi that produced contrasting outcomes of L. major infection. METHODOLOGY/PRINCIPAL FINDINGS: DNA immunization with distinct DTH-inducing salivary proteins from P. papatasi modulates L. major infection. PpSP15-immunized mice (PpSP15-mice show lasting protection while PpSP44-immunized mice (PpSP44-mice aggravate the infection, suggesting that immunization with these distinct molecules alters the course of anti-Leishmania immunity. Two weeks post-infection, 31.5% of CD4(+ T cells produced IFN-gamma in PpSP15-mice compared to 7.1% in PpSP44-mice. Moreover, IL-4-producing cells were 3-fold higher in PpSP44-mice. At an earlier time point of two hours after challenge with SGH and L. major, the expression profile of PpSP15-mice showed over 3-fold higher IFN-gamma and IL-12-Rbeta2 and 20-fold lower IL-4 expression relative to PpSP44-mice, suggesting that salivary proteins differentially prime anti-Leishmania immunity. This immune response is inducible by sand fly bites where PpSP15-mice showed a 3-fold higher IFN-gamma and a 5-fold lower IL-4 expression compared with PpSP44-mice. CONCLUSIONS/SIGNIFICANCE: Immunization with two salivary proteins from P. papatasi, PpSP15 and PpSP44, produced distinct immune profiles that

  9. R4 Regulator of G Protein Signaling (RGS) Proteins in Inflammation and Immunity.

    Science.gov (United States)

    Xie, Zhihui; Chan, Eunice C; Druey, Kirk M

    2016-03-01

    G protein-coupled receptors (GPCRs) have important functions in both innate and adaptive immunity, with the capacity to bridge interactions between the two arms of the host responses to pathogens through direct recognition of secreted microbial products or the by-products of host cells damaged by pathogen exposure. In the mid-1990s, a large group of intracellular proteins was discovered, the regulator of G protein signaling (RGS) family, whose main, but not exclusive, function appears to be to constrain the intensity and duration of GPCR signaling. The R4/B subfamily--the focus of this review--includes RGS1-5, 8, 13, 16, 18, and 21, which are the smallest RGS proteins in size, with the exception of RGS3. Prominent roles in the trafficking of B and T lymphocytes and macrophages have been described for RGS1, RGS13, and RGS16, while RGS18 appears to control platelet and osteoclast functions. Additional G protein independent functions of RGS13 have been uncovered in gene expression in B lymphocytes and mast cell-mediated allergic reactions. In this review, we discuss potential physiological roles of this RGS protein subfamily, primarily in leukocytes having central roles in immune and inflammatory responses. We also discuss approaches to target RGS proteins therapeutically, which represents a virtually untapped strategy to combat exaggerated immune responses leading to inflammation.

  10. Induction of mucosal immunity by intranasal immunization with recombinant adenovirus expressing major epitopes of Porcine circovirus-2 capsid protein.

    Science.gov (United States)

    Liu, Yu-feng; Guo, Quan-hai; Chen, Lu; Zhao, Jun; Chang, Hong-tao; Wang, Xin-wei; Yang, Xia; Wang, Chuan-qing

    2013-07-15

    Porcine circovirus-2 (PCV-2) is primarily transmitted through mucosa, thus the mucosal immunity may constitute an essential feature of vaccination strategies against PCV-2 infection. Mucosal immunity elicited by recombinant replication-deficient adenovirus expressing the major epitopes of PCV-2 capsid protein (rAd/Cap/518) via intranasal (i.n.), intramuscular (i.m.) or oral routes in mice were evaluated. Immunization with rAd/Cap/518 via i.n. route induced higher titers of IgA in saliva, bronchoalveolar and intestinal lavage fluid compared with those immunized via i.m. route. The proportions of CD3+, CD3+CD4+ and CD3+CD8+ T cells were significantly increased in mice immunized with rAd/Cap/518 via i.n. route compared with the control group. Higher levels of IFN-γ were detected in the spleen and mesenteric lymph nodes of mice immunized with rAd/Cap/518 via i.n. route compared with other groups, yet IL-4 was not detected in any group. Real-time PCR analysis confirmed viral DNA loads in the i.m. or i.n. immunization group was lower than that seen in the rAd immunization. These results indicate that i.n. administration of rAd/Cap/518 can elicit humoral and Th1-type cellular protective immunity in both systemic and mucosal immune compartments in mice, representing a promising mucosal vaccine candidate against PCV-2.

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

    Science.gov (United States)

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

    2014-01-01

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

  12. Novel Immunity Proteins Associated with Colicin M-like Bacteriocins Exhibit Promiscuous Protection in Pseudomonas

    Science.gov (United States)

    Ghequire, Maarten G. K.; Kemland, Lieselore; De Mot, René

    2017-01-01

    Bacteriocins related to colicin M, acting via cleavage of the cell wall precursor lipid II, have been characterized in γ- and β-proteobacteria. Depending on the species, immunity is provided by either an inner membrane-anchored periplasmic protein or by an integral membrane protein. In Pseudomonas however, the immunity partner of colicin M-like bacteriocins remains unknown. Based on an in silico analysis in pseudomonad genomes, we here identify a gene encoding a putative immunity partner that represents a novel type of integral membrane protein (PmiA, Pseudomonas colicin M-like immunity type A). By heterologous expression of pmiA genes in susceptible strains, we show that immunity to colicin M-like bacteriocins is indeed provided by the cognate PmiA. Sequence homology among PmiA proteins is essentially absent, except for a short motif with a conserved periplasm-exposed aspartate residue. However, PmiA's protective function is not abolished by changing this acidic residue to the uncharged alanine. Immunity by PmiAs appears promiscuous to the extent that PmiA homologs from a clade sharing bacteriocin linked to the original PmiA. This study shows that multiple immunity factors have evolved independently to silence lipid II-targeting enzymatic bacteriocins. Their relaxed bacteriocin immunization capacity contrasts to the strict specificity of immunity proteins shielding the enzymatic domain of nuclease bacteriocins. The nature of associated immune functions needs consideration when using such natural protein antibiotics or designing novel variants. PMID:28194143

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

    Science.gov (United States)

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

    2015-03-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2015-04-01

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

  16. Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium tuberculosis.

    OpenAIRE

    1995-01-01

    Tuberculosis, caused by the intracellular pathogen Mycobacterium tuberculosis, is the world's leading cause of death in humans from a single infectious agent. A safe and effective vaccine against this scourge is urgently needed. This study demonstrates that immunization with the 30-kDa major secretory protein, alone or in combination with other abundant extracellular proteins of M. tuberculosis, induces strong cell-mediated immune responses and substantial protective immunity against aerosol ...

  17. F-actin-binding protein drebrin regulates CXCR4 recruitment to the immune synapse.

    Science.gov (United States)

    Pérez-Martínez, Manuel; Gordón-Alonso, Mónica; Cabrero, José Román; Barrero-Villar, Marta; Rey, Mercedes; Mittelbrunn, María; Lamana, Amalia; Morlino, Giulia; Calabia, Carmen; Yamazaki, Hiroyuki; Shirao, Tomoaki; Vázquez, Jesús; González-Amaro, Roberto; Veiga, Esteban; Sánchez-Madrid, Francisco

    2010-04-01

    The adaptive immune response depends on the interaction of T cells and antigen-presenting cells at the immune synapse. Formation of the immune synapse and the subsequent T-cell activation are highly dependent on the actin cytoskeleton. In this work, we describe that T cells express drebrin, a neuronal actin-binding protein. Drebrin colocalizes with the chemokine receptor CXCR4 and F-actin at the peripheral supramolecular activation cluster in the immune synapse. Drebrin interacts with the cytoplasmic tail of CXCR4 and both proteins redistribute to the immune synapse with similar kinetics. Drebrin knockdown in T cells impairs the redistribution of CXCR4 and inhibits actin polymerization at the immune synapse as well as IL-2 production. Our data indicate that drebrin exerts an unexpected and relevant functional role in T cells during the generation of the immune response.

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

    Directory of Open Access Journals (Sweden)

    Edgar Rascón-Castelo

    2015-11-01

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

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

    Science.gov (United States)

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

    2015-06-01

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

  20. A protein in pig spleen similar to immune suppressive protein of stress

    Institute of Scientific and Technical Information of China (English)

    FUHong-Yan; ZHANGJie; DINGGui-Feng; FANShao-Guang

    2003-01-01

    AIM:To purify a protein in pig spleens, which was similar to immune suppressive protein of stress (ISPS), and characterize its properties and functions. METHODS: 1)Pig spleen was extracted in dilute hydrochloric acid. 2) The extract was ultra-filtrated for having high molecular weight proteins (Mr>30000). 3) The filtrates were purified with FPLC affinity chromatography. 4)The elute from FPLC was used for T-lymphocyte proliferation and ELISA test. 5) Lastly, SDS-PAGE was used to determine the molecular weight and purity of the final product. RESULTS: A protein purified from pig spleen (the pig ISPS homologue) inhibited concanavalin A(Con A)-induced mouse lymphocyte proliferation. The molecular weight of this protein was about Mr 190000. It has a stronger selectivity against T-lymphocyte line such as Jurkat cell line and mastocyte line (P815) and has a weaker inhibitory activity on macrophage line (U937). CONCLUSION: A protein similar to rat/mouse ISPS was found in pig spleen. This may provide an opportunity to study its roles in tumors and autoimmune diseases.

  1. Immunization with chlamydial plasmid protein pORF5 DNA vaccine induces protective immunity against genital chlamydial infection in mice

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    To validate the immune protective efficacy of pORF5 DNA vaccine and to analyze potential mechanisms related to this protection. In this study, pORF5 DNA vaccine was constructed and evaluated for its protective immunity in a mouse model of genital chlamydial infection. Groups of BALB/c mice were immunized intranasally with pORF5 DNA vaccine. Humoral and cell mediated immune responses were evaluated. The clearance ability of chlamydial challenge from the genital tract and the chlamy- dia-induced upper genital tract gross pathology and histopathological characterization were also de- tected. The results showed that the total and the IgG2a anti-pORF5 antibody levels in serum were sig- nificantly elevated after pcDNA3.1-pORF5 vaccination, as were the total antibody and IgA levels in vaginal fluids. pcDNA3.1-pORF5 induced a significantly high level of Th1 response as measured by robust gamma interferon (IFN-γ). Minimal IL-4 was produced by immune T cells in response to the re-stimulation with pORF5 protein or the inactive elementary body in vitro. pcDNA3.1-pORF5-vacci- nated mice displayed significantly reduced bacterial shedding upon a chlamydial challenge and an accelerated resolution of infection. 100% of pcDNA3.1-pORF5 vaccinated mice successfully resolved the infection by day 24. pcDNA3.1-pORF5-immunized mice also exhibited protection against patho- logical consequences of chlamydial infection. The stimulated index was significantly higher than that of mice immunized with pcDNA3.1 and PBS (P<0.05). Together, these results demonstrated that immu- nization with pORF5 DNA vaccine is a promising approach for eliciting a protective immunity against a genital chlamydial challenge.

  2. Cross-Protection against Challenge with Puumala Virus after Immunization with Nucleocapsid Proteins from Different Hantaviruses

    Science.gov (United States)

    de Carvalho Nicacio, Cristina; Gonzalez Della Valle, Marcelo; Padula, Paula; Björling, Ewa; Plyusnin, Alexander; Lundkvist, Åke

    2002-01-01

    Hantaviruses are rodent-borne agents that cause hemorrhagic fever with renal syndrome or hantavirus pulmonary syndrome in humans. The nucleocapsid protein (N) is relatively conserved among hantaviruses and highly immunogenic in both laboratory animals and humans, and it has been shown to induce efficient protective immunity in animal models. To investigate the ability of recombinant N (rN) from different hantaviruses to elicit cross-protection, we immunized bank voles with rN from Puumala (PUUV), Topografov (TOPV), Andes (ANDV), and Dobrava (DOBV) viruses and subsequently challenged them with PUUV. All animals immunized with PUUV and TOPV rN were completely protected. In the group immunized with DOBV rN, 7 of 10 animals were protected, while only 3 of 8 animals were protected in the group immunized with ANDV rN, which is more closely related to PUUV rN than DOBV rN. Humoral and cellular immune responses after rN immunization were also investigated. The highest cross-reactive humoral responses against PUUV antigen were detected in sera from ANDV rN-immunized animals, followed by those from TOPV rN-immunized animals, and only very low antibody cross-reactivity was observed in sera from DOBV rN-immunized animals. In proliferation assays, T lymphocytes from animals immunized with all heterologous rNs were as efficiently recalled in vitro by PUUV rN as were T lymphocytes from animals immunized with homologous protein. In summary, this study has shown that hantavirus N can elicit cross-protective immune responses against PUUV, and the results suggest a more important role for the cellular arm of the immune response than for the humoral arm in cross-protection elicited by rN. PMID:12050380

  3. Protein-Carbohydrate Interactions as Part of Plant Defense and Animal Immunity

    Directory of Open Access Journals (Sweden)

    Kristof De Schutter

    2015-05-01

    Full Text Available The immune system consists of a complex network of cells and molecules that interact with each other to initiate the host defense system. Many of these interactions involve specific carbohydrate structures and proteins that specifically recognize and bind them, in particular lectins. It is well established that lectin-carbohydrate interactions play a major role in the immune system, in that they mediate and regulate several interactions that are part of the immune response. Despite obvious differences between the immune system in animals and plants, there are also striking similarities. In both cases, lectins can play a role as pattern recognition receptors, recognizing the pathogens and initiating the stress response. Although plants do not possess an adaptive immune system, they are able to imprint a stress memory, a mechanism in which lectins can be involved. This review will focus on the role of lectins in the immune system of animals and plants.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Vladimir López

    2016-03-01

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

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

    Science.gov (United States)

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

    2016-03-01

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

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

    Science.gov (United States)

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

    2016-07-01

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

  8. The adaptive immune response to cow's milk proteins in allergy and tolerance

    NARCIS (Netherlands)

    Ruiter, B.

    2007-01-01

    Cow's milk (CM) and related products are an important source of protein in the diet. Unfortunately, cow's milk proteins (CMPs) can also be allergenic. IgE-mediated cow's milk allergy (CMA) occurs in 1.5% of infants, as well as in 0.3% of older children and adults. Insight into the immune response to

  9. Analysis of the humoral immune response to Chlamydia outer membrane protein 2

    DEFF Research Database (Denmark)

    Mygind, P; Christiansen, Gunna; Persson, K;

    1998-01-01

    The humoral immune response to Chlamydia outer membrane protein 2 (Omp2) was studied. Omp2 is a highly genus-conserved structural protein of all Chlamydia species, containing a variable N-terminal fragment. To analyze where the immunogenic parts were localized, seven highly purified truncated...

  10. Enhanced in vivo protein synthesis in circulating immune cells of ICU patients.

    Science.gov (United States)

    Januszkiewicz, Anna; Klaude, Maria; Loré, Karin; Andersson, Jan; Ringdén, Olle; Rooyackers, Olav; Wernerman, Jan

    2007-11-01

    Insufficient function of the immune system contributes to a poor prognosis in intensive care unit (ICU) patients. However, the immune system function is not easily monitored and evaluated. In vivo protein synthesis determination in immune competent cells offers a possibility to quantify immunological activation. The aim of this descriptive study was to determine the in vivo fractional protein synthesis rate (FSR) in immune cells of ICU patients during the initial phase of the critical illness. Patients (n = 20) on ventilator treatment in the general ICU were studied during their first week of ICU stay. FSR was determined in circulating T lymphocytes, mononuclear cells, the whole population of blood leukocytes, and in stationary immune cells of palatine tonsils during a 90-min period by a flooding technique. Healthy, adult subjects (n = 11), scheduled for elective ear, nose, and throat surgery served as a control group. The FSR in leukocytes and mononuclear cells of ICU patients was higher compared with the control group. In contrast, the FSR of circulating T lymphocytes and of tonsillar cells was not different from that in the healthy subjects. In summary, the ICU patients showed a distinct polarization of metabolic responses during the initial phase of the critical illness. The in vivo rate of protein synthesis was high in the circulating mononuclear cells and leukocytes, reflecting enhanced metabolic activity in these cell populations. Determination of the in vivo protein synthesis rate may be used as a tool to obtain additional information on activation of the immune system.

  11. Protein synthesis regulation, a pillar of strength for innate immunity?

    Science.gov (United States)

    Argüello, Rafael J; Rodriguez Rodrigues, Christian; Gatti, Evelina; Pierre, Philippe

    2015-02-01

    Recognition of pathogen derived molecules by Pattern Recognition Receptors (PRR) induces the production of cytokines (i.e. type I interferons) that stimulate the surrounding cells to transcribe and translate hundreds of genes, in order to prevent further infection and organize the immune response. Here, we report on the rising matter that metabolism sensing and gene expression control at the level of mRNA translation, allow swift responses that mobilize host defenses and coordinate innate responses to infection.

  12. Hot water extracts of Chlorella vulgaris improve immune function in protein-deficient weanling mice and immune cells.

    Science.gov (United States)

    An, Hyo-Jin; Rim, Hong-Kun; Jeong, Hyun-Ja; Hong, Seung-Heon; Um, Jae-Young; Kim, Hyung-Min

    2010-12-01

    The objective of this study was to investigate the effects of hot water extracts of Chlorella vulgaris (CVE) on a deteriorated immune function through utilization of a protein-energy malnutrition (PEM) diet. Unicellular algae, C. vulgaris, were used as biological response modifier. PEM is associated with decreased host immune defense. Male C57BL/6J mice, initially four weeks old, were fed for 8 days with standard diet or a PEM diet. Mice in the PEM diet group were orally administered 0.1 g/kg and 0.15 g/kg of CVE for the following week. Nutritional parameters such as the total protein, albumin, glucose, and interferon γ (IFN-γ) were increased in blood serum of the CVE-treated group compared with the non-treated group. The mononuclear cell numbers from spleen, superficial, and mesenteric lymph node were reduced in mice fed with PEM diet, but numbers from the spleen and superficial lymph node were increased by the CVE (0.1 and 0.15 g/kg) treatment. We also investigated the effect of CVE on the production of cytokines in human T-cell line, MOLT-4 cells, and primary cultured splenocytes. The CVE treatment significantly increased the production of both interleukin (IL)-2 and IL-4 compared with the media control, but did not affect the production of IFN-γ. These results suggest that CVE may be useful in improving the immune function.

  13. Salivary Defense Proteins: Their Network and Role in Innate and Acquired Oral Immunity

    Directory of Open Access Journals (Sweden)

    Gábor Fábián

    2012-04-01

    Full Text Available There are numerous defense proteins present in the saliva. Although some of these molecules are present in rather low concentrations, their effects are additive and/or synergistic, resulting in an efficient molecular defense network of the oral cavity. Moreover, local concentrations of these proteins near the mucosal surfaces (mucosal transudate, periodontal sulcus (gingival crevicular fluid and oral wounds and ulcers (transudate may be much greater, and in many cases reinforced by immune and/or inflammatory reactions of the oral mucosa. Some defense proteins, like salivary immunoglobulins and salivary chaperokine HSP70/HSPAs (70 kDa heat shock proteins, are involved in both innate and acquired immunity. Cationic peptides and other defense proteins like lysozyme, bactericidal/permeability increasing protein (BPI, BPI-like proteins, PLUNC (palate lung and nasal epithelial clone proteins, salivary amylase, cystatins, prolin-rich proteins, mucins, peroxidases, statherin and others are primarily responsible for innate immunity. In this paper, this complex system and function of the salivary defense proteins will be reviewed.

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

    Directory of Open Access Journals (Sweden)

    Aurélien Lathuilière

    2015-05-01

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

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

    Science.gov (United States)

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

    2016-02-17

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

  16. Immunity

    Institute of Scientific and Technical Information of China (English)

    1992-01-01

    920630 Effects of the spleen on immunestate of patients with gastric cancer.QIUDengbo (仇登波), et al. Dept General Surg,Union Hosp, Tongji Med Univ, Wuhan, 430022.Natl Med J China 1992; 72(6): 334-337. For analysing the effects of the spleen on im-mune state of gastric cancer patients.T-lym-

  17. Characteristics and optimization producing conditions of bacteriocin-like from Lactobacillus plantrum%植物乳酸菌类细菌素特性研究及其产生条件的优化

    Institute of Scientific and Technical Information of China (English)

    解俊梅; 文汉

    2011-01-01

    Lactobacillus production of lactic acid bacteria bacteriocin culture conditions was optimized by single factor experiment and orthogonal and its characteristics was analyzed.The results showed that:the best vegetative growth of lactic acid bacteria was a combination of culture conditions at 30℃,initial pH6.0 with inoculum amount of 4%,seed age 12h,anaerobic conditions for 40h.Lactobacillus streptozotocin on Gram-negative bacteria and yeast inhibition optimum culture conditions for the largest combination of the temperature 28℃,initial pH7.0 with inoculum amount of 2%,seed age 10h,anaerobic conditions for 24h.Gram-positive lactic acid bacteria Streptomyces antibacterial effects had the best combination of culture conditions of temperature 28℃,initial pH 6.5 medium,inoculum 1%,seed age 6h,anaerobic conditions for 40h.Without effect of organic acids and hydrogen peroxide,the cell-free liquid culture of Lactobacillus showed high inhibitory activity against gram-positive bacterias,including Bacillus subtilis and Staphyloccocus aureus Rosenbach and gram-negative bacterias,such as Escherichia coli and Salmonella typhimunius,but also inhibited some fungi.Therefore,the inhibitory substance produced by the lactic acid bacteria was assumed bacteriocin-like substance.Lactic acid bacteria showed it was stable to heat,active at low pH,not sensitive to proteinasea and broad antibacterial spectrum.%通过单因素实验和正交实验优化了乳酸菌产乳酸菌素的培养条件并分析了其特性。结果表明:植物性乳酸杆菌生长的最佳培养条件组合是温度30℃,培养基初始pH为6.0,接种量4%,种龄12h,厌氧条件下,培养时间40h;乳酸菌菌素对革兰氏阴性菌和酵母菌的抑菌作用最大的最佳培养条件组合是温度28℃,培养基初始pH为7.0,接种量2%,种龄10h,厌氧条件下,培养时间24h。乳酸菌菌素革兰氏阳性菌的抑菌作用最大的最佳培养条件组合是温度28℃,培养基初始pH为6.5,

  18. Protein-linked glycans in periodontal bacteria: prevalence and role at the immune interface

    Directory of Open Access Journals (Sweden)

    Rajendra P Settem

    2013-10-01

    Full Text Available Protein modification with complex glycans is increasingly being recognized in many pathogenic and non-pathogenic bacteria, and is now thought to be central to the successful life-style of those species in their respective hosts. This review aims to convey current knowledge on the extent of protein glycosylation in periodontal pathogenic bacteria and its role in the modulation of the host immune responses. The available data show that surface glycans of periodontal bacteria orchestrate dendritic cell cytokine responses to drive T cell immunity in ways that facilitate bacterial persistence in the host and induce periodontal inflammation. In addition, surface glycans may help certain periodontal bacteria protect against serum complement attack or help them escape immune detection through glycomimicry. In this review we will focus mainly on the generalized S-layer protein glycosylation system of the periodontal pathogen Tannerella forsythia in shaping innate and adaptive host immunity in the context of periodontal disease. In addition, we will also review the current state of knowledge of surface protein glycosylation and its potential for immune modulation in other periodontal pathogens.

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

    DEFF Research Database (Denmark)

    Christensen, Hanne Risager; Pedersen, Susanne Brix; Frøkiær, Hanne

    2004-01-01

    While allergic reactions to soya are increasingly investigated, the normal immune response to ingested soya is scarcely described. In the present study, we wanted to characterise the soya-specific immune response in healthy mice ingesting soya protein. Mice fed a soya-containing diet (F0) and mice...... of the first (F1) and second (F2) offspring generation bred on a soya protein-free diet were used either directly or were transferred between the soya-containing and soya protein-free diet during pregnancy or neonatal life. The mice were compared as to levels of naturally occurring specific antibodies analysed...... by ELISA, and to the presence of oral tolerance detected as a suppressed antibody and cell-proliferation response upon immunisation with soya protein. F0 mice generated soya-specific antibodies, while oral tolerance to the same soya proteins was also clearly induced. When F0 dams were transferred to soya...

  20. Production of bacteriocin-like inhibitory substances (BLIS by Streptococcus salivarius strains isolated from the tongue and throat of children with and without sore throat Produção de substâncias inibidoras semelhantes à bacteriocina por cepas de Streptococcus salivarius, isoladas da língua e garganta de crianças com e sem dor de garganta

    Directory of Open Access Journals (Sweden)

    Vera Fantinato

    1999-12-01

    Full Text Available Streptococcus salivarius strains, isolated from children with and without sore throat, were tested for bacteriocin production against Streptococcus pyogenes. S. salivarius strains producing bacteriocin-like inhibitory substances (BLIS against S. pyogenes were more frequently found in children without sore throat. These results suggest that these children may be protected against sore throat by the presence of BLIS-positive S. salivarius strains.Cepas de Streptococcus salivarius, isoladas de crianças com e sem dor de garganta, foram testadas quanto à produção de bacteriocina contra Streptococcus pyogenes. Os resultados mostraram que as crianças que não tinham dor de garganta possuiam, na boca, cepas de bactérias produtoras de substâncias inibidoras semelhantes à bacteriocina contra S. pyogenes.

  1. Vaccine platforms combining circumsporozoite protein and potent immune modulators, rEA or EAT-2, paradoxically result in opposing immune responses.

    Directory of Open Access Journals (Sweden)

    Nathaniel J Schuldt

    Full Text Available BACKGROUND: Malaria greatly impacts the health and wellbeing of over half of the world's population. Promising malaria vaccine candidates have attempted to induce adaptive immune responses to Circumsporozoite (CS protein. Despite the inclusion of potent adjuvants, these vaccines have limited protective efficacy. Conventional recombinant adenovirus (rAd based vaccines expressing CS protein can induce CS protein specific immune responses, but these are essentially equivalent to those generated after use of the CS protein subunit based vaccines. In this study we combined the use of rAds expressing CS protein along with rAds expressing novel innate immune response modulating proteins in an attempt to significantly improve the induction of CS protein specific cell mediated immune (CMI responses. METHODS AND FINDINGS: BALB/cJ mice were co-vaccinated with a rAd vectors expressing CS protein simultaneous with a rAd expressing either TLR agonist (rEA or SLAM receptors adaptor protein (EAT-2. Paradoxically, expression of the TLR agonist uncovered a potent immunosuppressive activity inherent to the combined expression of the CS protein and rEA. Fortunately, use of the rAd vaccine expressing EAT-2 circumvented CS protein's suppressive activity, and generated a fivefold increase in the number of CS protein responsive, IFNγ secreting splenocytes, as well as increased the breadth of T cells responsive to peptides present in the CS protein. These improvements were positively correlated with the induction of a fourfold improvement in CS protein specific CTL functional activity in vivo. CONCLUSION: Our results emphasize the need for caution when incorporating CS protein into malaria vaccine platforms expressing or containing other immunostimulatory compounds, as the immunological outcomes may be unanticipated and/or counter-productive. However, expressing the SLAM receptors derived signaling adaptor EAT-2 at the same time of vaccination with CS protein can

  2. Insight into bacterial virulence mechanisms against host immune response via the Yersinia pestis-human protein-protein interaction network.

    Science.gov (United States)

    Yang, Huiying; Ke, Yuehua; Wang, Jian; Tan, Yafang; Myeni, Sebenzile K; Li, Dong; Shi, Qinghai; Yan, Yanfeng; Chen, Hui; Guo, Zhaobiao; Yuan, Yanzhi; Yang, Xiaoming; Yang, Ruifu; Du, Zongmin

    2011-11-01

    A Yersinia pestis-human protein interaction network is reported here to improve our understanding of its pathogenesis. Up to 204 interactions between 66 Y. pestis bait proteins and 109 human proteins were identified by yeast two-hybrid assay and then combined with 23 previously published interactions to construct a protein-protein interaction network. Topological analysis of the interaction network revealed that human proteins targeted by Y. pestis were significantly enriched in the proteins that are central in the human protein-protein interaction network. Analysis of this network showed that signaling pathways important for host immune responses were preferentially targeted by Y. pestis, including the pathways involved in focal adhesion, regulation of cytoskeleton, leukocyte transendoepithelial migration, and Toll-like receptor (TLR) and mitogen-activated protein kinase (MAPK) signaling. Cellular pathways targeted by Y. pestis are highly relevant to its pathogenesis. Interactions with host proteins involved in focal adhesion and cytoskeketon regulation pathways could account for resistance of Y. pestis to phagocytosis. Interference with TLR and MAPK signaling pathways by Y. pestis reflects common characteristics of pathogen-host interaction that bacterial pathogens have evolved to evade host innate immune response by interacting with proteins in those signaling pathways. Interestingly, a large portion of human proteins interacting with Y. pestis (16/109) also interacted with viral proteins (Epstein-Barr virus [EBV] and hepatitis C virus [HCV]), suggesting that viral and bacterial pathogens attack common cellular functions to facilitate infections. In addition, we identified vasodilator-stimulated phosphoprotein (VASP) as a novel interaction partner of YpkA and showed that YpkA could inhibit in vitro actin assembly mediated by VASP.

  3. Innate immune suppression enables frequent transfection with RNA encoding reprogramming proteins.

    Directory of Open Access Journals (Sweden)

    Matthew Angel

    Full Text Available BACKGROUND: Generating autologous pluripotent stem cells for therapeutic applications will require the development of efficient DNA-free reprogramming techniques. Transfecting cells with in vitro-transcribed, protein-encoding RNA is a straightforward method of directly expressing high levels of reprogramming proteins without genetic modification. However, long-RNA transfection triggers a potent innate immune response characterized by growth inhibition and the production of inflammatory cytokines. As a result, repeated transfection with protein-encoding RNA causes cell death. METHODOLOGY/PRINCIPAL FINDINGS: RNA viruses have evolved methods of disrupting innate immune signaling by destroying or inhibiting specific proteins to enable persistent infection. Starting from a list of known viral targets, we performed a combinatorial screen to identify siRNA cocktails that could desensitize cells to exogenous RNA. We show that combined knockdown of interferon-beta (Ifnb1, Eif2ak2, and Stat2 rescues cells from the innate immune response triggered by frequent long-RNA transfection. Using this technique, we were able to transfect primary human fibroblasts every 24 hours with RNA encoding the reprogramming proteins Oct4, Sox2, Klf4, and Utf1. We provide evidence that the encoded protein is active, and we show that expression can be maintained for many days, through multiple rounds of cell division. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that suppressing innate immunity enables frequent transfection with protein-encoding RNA. This technique represents a versatile tool for investigating expression dynamics and protein interactions by enabling precise control over levels and timing of protein expression. Our finding also opens the door for the development of reprogramming and directed-differentiation methods based on long-RNA transfection.

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

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    Heli Salmela

    2015-07-01

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

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

    Directory of Open Access Journals (Sweden)

    Chang-Jin Park

    2015-12-01

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

  6. Pooled protein immunization for identification of cell surface antigens in Streptococcus sanguinis.

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    Xiuchun Ge

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

  7. Shigella manipulates host immune responses by delivering effector proteins with specific roles

    Directory of Open Access Journals (Sweden)

    Hiroshi eAshida

    2015-05-01

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

  8. Protein defence systems against the lantibiotic nisin: Function of the immunity protein NisI and the resistance protein NSR

    Directory of Open Access Journals (Sweden)

    Sakshi eKhosa

    2016-04-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2010-11-01

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

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

    OpenAIRE

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

    2016-01-01

    ABSTRACT Rho-associated kinase 1 (ROCK1) and ROCK2 are activated by Rho GTPase and control cytoskeleton rearrangement through modulating the phosphorylation of their down-stream effector molecules. Although these 2 isoforms share more than 90% homology within their kinase domain the question of whether ROCK proteins function identically in different cell types is not clear. By using both pharmacological inhibition and genetic knockdown approaches recent studies suggest that the ROCK2 isoform ...

  12. The Nanoscience of Polyvalent Binding by Proteins in the Immune Response

    DEFF Research Database (Denmark)

    Vorup-Jensen, Thomas

    2016-01-01

    Recent research has demonstrated that the successful use of nanometer-scaled material, such as nanoparticles, as medicines is often challenged by the host immune system. Mechanisms of the innate immunity seem to provide a swift response to administration of particulate nanomedicines, which may...... clear or in other way incapacitate the function of these drugs. To rationalize, why and how, the innate immune system especially interacts with nanomedicines, this chapter points to the prominent role of polyvalent interactions by large, immunoactive proteins with the surfaces of nanoparticles. From...... addressing the thermodynamics and ultrastructural properties of these interactions, it is proposed that the nm-scaled ligand presentation and symmetry on such surfaces is a determinant in the binding of these proteins. Better control over nanomedicine ultrastructure is consequently likely to provide...

  13. A force-activated trip switch triggers rapid dissociation of a colicin from its immunity protein.

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    Oliver E Farrance

    Full Text Available Colicins are protein antibiotics synthesised by Escherichia coli strains to target and kill related bacteria. To prevent host suicide, colicins are inactivated by binding to immunity proteins. Despite their high avidity (K(d ≈ fM, lifetime ≈ 4 days, immunity protein release is a pre-requisite of colicin intoxication, which occurs on a timescale of minutes. Here, by measuring the dynamic force spectrum of the dissociation of the DNase domain of colicin E9 (E9 and immunity protein 9 (Im9 complex using an atomic force microscope we show that application of low forces (<20 pN increases the rate of complex dissociation 10(6-fold, to a timescale (lifetime ≈ 10 ms compatible with intoxication. We term this catastrophic force-triggered increase in off-rate a trip bond. Using mutational analysis, we elucidate the mechanism of this switch in affinity. We show that the N-terminal region of E9, which has sparse contacts with the hydrophobic core, is linked to an allosteric activator region in E9 (residues 21-30 whose remodelling triggers immunity protein release. Diversion of the force transduction pathway by the introduction of appropriately positioned disulfide bridges yields a force resistant complex with a lifetime identical to that measured by ensemble techniques. A trip switch within E9 is ideal for its function as it allows bipartite complex affinity, whereby the stable colicin:immunity protein complex required for host protection can be readily converted to a kinetically unstable complex whose dissociation is necessary for cellular invasion and competitor death. More generally, the observation of two force phenotypes for the E9:Im9 complex demonstrates that force can re-sculpt the underlying energy landscape, providing new opportunities to modulate biological reactions in vivo; this rationalises the commonly observed discrepancy between off-rates measured by dynamic force spectroscopy and ensemble methods.

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

    OpenAIRE

    2015-01-01

    New minimal invasive diagnostic methods for early detection of lung cancer are urgently needed. It is known that the immune system responds to tumors with production of tumor-autoantibodies. Protein microarrays are a suitable highly multiplexed platform for identification of autoantibody signatures against tumor-associated antigens (TAA). These microarrays can be probed using 0.1 mg immunoglobulin G (IgG), purified from 10 µL of plasma. We used a microarray comprising recombinant proteins der...

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

    Science.gov (United States)

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

    2015-11-27

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

  16. Role of macrophage inflammatory protein-1alpha in T-cell-mediated immunity to viral infection

    DEFF Research Database (Denmark)

    Madsen, Andreas N; Nansen, Anneline; Christensen, Jan P

    2003-01-01

    The immune response to lymphocytic choriomeningitis virus in mice lacking macrophage inflammatory protein-1alpha (MIP-1alpha) was evaluated. Generation of virus-specific effector T cells is unimpaired in MIP-1alpha-deficient mice. Furthermore, MIP-1alpha is not required for T-cell-mediated virus...

  17. Listeria monocytogenes protein fraction induces dendritic cells maturation and T helper 1 immune responses.

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    Azad Saei

    2014-02-01

    Full Text Available Fully mature dendritic cells (DCs play pivotal role in inducing immune responses and converting naïve T lymphocytes into functional Th1 cells. We aimed to evaluate Listeria Monocytogenes-derived protein fractions to induce DC maturation and stimulating T helper (Th1 immune responses.In the present study, we fractionated Listeria Monocytogenes-derived proteins by adding of ammonium sulfate in a stepwise manner. DCs were also generated from C57BL/6 mice bone marrow precursor cells. Then, the effects of protein fractions on bone marrow derived DC (BMDC maturation were evaluated. In addition, we assessed the capacity of activated DCs to induce cytokine production and proliferation of lymphocytes.Listeria-derived protein fractions induced fully mature DCs expressing high costimulatory molecules such as CD80, CD86 and CD40. DCs that were activated by selected F3 fraction had low capacity to uptake exogenous antigens while secreted high levels of Interleukine (IL-12. Moreover, lymphocytes cultured with activated BMDCs produced high amounts of IFN-γ and showed higher proliferation than control. Listeria derived protein fractions differently influenced DC maturation.In conclusion, Listeria protein activated-BMDCs can be used as a cell based vaccine to induce anti-tumor immune responses.

  18. Sequence-structure-function relations of the mosquito leucine-rich repeat immune proteins

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    Povelones Michael

    2010-09-01

    Full Text Available Abstract Background The discovery and characterisation of factors governing innate immune responses in insects has driven the elucidation of many immune system components in mammals and other organisms. Focusing on the immune system responses of the malaria mosquito, Anopheles gambiae, has uncovered an array of components and mechanisms involved in defence against pathogen infections. Two of these immune factors are LRIM1 and APL1C, which are leucine-rich repeat (LRR containing proteins that activate complement-like defence responses against malaria parasites. In addition to their LRR domains, these leucine-rich repeat immune (LRIM proteins share several structural features including signal peptides, patterns of cysteine residues, and coiled-coil domains. Results The identification and characterisation of genes related to LRIM1 and APL1C revealed putatively novel innate immune factors and furthered the understanding of their likely molecular functions. Genomic scans using the shared features of LRIM1 and APL1C identified more than 20 LRIM-like genes exhibiting all or most of their sequence features in each of three disease-vector mosquitoes with sequenced genomes: An. gambiae, Aedes aegypti, and Culex quinquefasciatus. Comparative sequence analyses revealed that this family of mosquito LRIM-like genes is characterised by a variable number of 6 to 14 LRRs of different lengths. The "Long" LRIM subfamily, with 10 or more LRRs, and the "Short" LRIMs, with 6 or 7 LRRs, also share the signal peptide, cysteine residue patterning, and coiled-coil sequence features of LRIM1 and APL1C. The "TM" LRIMs have a predicted C-terminal transmembrane region, and the "Coil-less" LRIMs exhibit the characteristic LRIM sequence signatures but lack the C-terminal coiled-coil domains. Conclusions The evolutionary plasticity of the LRIM LRR domains may provide templates for diverse recognition properties, while their coiled-coil domains could be involved in the formation

  19. Conserved immune recognition hierarchy of mycobacterial PE/PPE proteins during infection in natural hosts.

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    H Martin Vordermeier

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

  20. Immune responsiveness of Japanese quail selected for egg yolk testosterone content under severe protein restriction.

    Science.gov (United States)

    Kankova, Zuzana; Okuliarova, Monika; Zeman, Michal

    2014-11-01

    Yolk testosterone concentrations vary in response to environmental conditions and different testosterone contents can subsequently modify the phenotypic traits of offspring. Apart from effects on growth, proactive behaviour and secondary sexual characteristics, the possible negative impacts of maternal testosterone on the immune system are often considered a limitation for its deposition. The effects of maternal testosterone can be modulated by postnatal environmental conditions, such as the availability of food resources. However, the majority of studies considering the effects of maternal testosterone on the immune system have been conducted under optimum conditions. We evaluated the influence of genetic selection for high (HET) and low (LET) egg testosterone content in Japanese quail on immune responsiveness of offspring to phytohaemagglutinin (PHA) and lipopolysaccharide (LPS) stimulation under severe protein restriction. Protein restriction negatively influenced body weight and performance in the PHA-test. We observed an increase in Cort (corticosterone) and He/Ly (heterophil/lymphocyte ratio) after LPS, while no changes occurred in total IgY levels in the protein-restricted group. HET quails showed higher body mass and total IgY levels and lower He/Ly ratio than LET quails, while the PHA index and Cort concentration did not differ between lines. No interactions were found between protein restriction and genetic line. In conclusion, the immune response was not compromised under conditions of severe protein restriction in the faster growing HET line compared with the LET line. We hypothesise that the immune responsiveness of birds with higher yolk testosterone may be linked with other maternally-derived substances in a context-dependent manner.

  1. Tumor necrosis factor-α-induced protein 1 and immunity to hepatitis B virus

    Institute of Scientific and Technical Information of China (English)

    Marie C Lin; Nikki P Lee; Ning Zheng; Pai-Hao Yang; Oscar G Wong; Hsiang-Fu Kung; Chee-Kin Hui; John M Luk; George Ka-Kit Lau

    2005-01-01

    AIM: To compare the gene expression profile in a pair of HBV-infected twins.METHODS: The gene expression profile was compared in a pair of HBV-infected twins.RESULTS: The twins displayed different disease outcomes. One acquired natural immunity against HBV,whereas the other became a chronic HBV carrier. Eightyeight and forty-six genes were found to be up- or downregulated in their PBMCs, respectively. Tumor necrosis factor-alpha-induced protein 1 (TNF-αIP1) that expressed at a higher level in the HBV-immune twins was identified and four pairs of siblings with HBV immunity by RTPCR. However, upon HBV core antigen stimulation,TNF-αIP1 was downregulated in PBMCs from subjects with immunity, whereas it was slightly upregulated in HBV carriers. Bioinformatics analysis revealed a K+channel tetramerization domain in TNF-αIP1 that shares a significant homology with some human, mouse, and C elegan proteins.CONCLUSION: TNF-αIP1 may play a role in the innate immunity against HBV.

  2. An insight into the immune roles of surfactant proteins, SP-A and SP-D

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    Annapurna eNayak

    2012-06-01

    Full Text Available Pulmonary surfactant proteins, SP-A and SP-D, are hydrophilic collagen-containing calcium-dependent lectins, which appear to have a range of innate immune functions at pulmonary as well as extrapulmonary sites. These proteins bind to target ligands on pathogens, allergens and apoptotic cells via C-terminal carbohydrate recognition domains while the collagen region brings about the effector functions. SP-A and SP-D deals with various pathogens triggering a range of innate immune mechanisms such as agglutination/aggregation, enhancement of phagocytosis and killing reactions, and growth inhibition. SP-A and SP-D have also been involved in the control of pulmonary inflammation including allergy and asthma, and hence, they are likely to central issues in a range of pathological situations. There is also emerging evidence to suggest that SP-A and SP-D are capable of linking innate immunity with adaptive immunity that includes modulation of dendritic cell function and helper T cell polarisation. It is clear now that SP-A and SP-D are potent innate immune molecules in the lungs. This review is a summary of their immunological properties in general and their importance in human health and disease.

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

    Science.gov (United States)

    Armstrong, Peter B

    2015-01-01

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

  4. Eimeria maxima microneme protein 2 delivered as DNA vaccine and recombinant protein induces immunity against experimental homogenous challenge.

    Science.gov (United States)

    Huang, Jingwei; Zhang, Zhenchao; Li, Menghui; Song, Xiaokai; Yan, Ruofeng; Xu, Lixin; Li, Xiangrui

    2015-10-01

    E. maxima is one of the seven species of Eimeria that infects chicken. Until now, only a few antigenic genes of E. maxima have been reported. In the present study, the immune protective effects against E. maxima challenge of recombinant protein and DNA vaccine encoding EmMIC2 were evaluated. Two-week-old chickens were randomly divided into five groups. The experimental group of chickens was immunized with 100 μg DNA vaccine pVAX1-MIC2 or 200 μg rEmMIC2 protein while the control group of chickens was injected with pVAX1 plasmid or sterile PBS. The results showed that the anti-EmMIC2 antibody titers of both rEmMIC2 protein and pVAX1-MIC2 groups were significantly higher as compared to PBS and pVAX1 control (Precombinant protein and the DNA vaccine could obviously alleviate jejunum lesions, body weight loss, increase oocyst, decrease ratio and provide ACIs of more than 165. All the above results suggested that immunization with EmMIC2 was effective in imparting partial protection against E. maxima challenge and it could be an effective antigen candidate for the development of new vaccines against E. maxima.

  5. Study on DNA Immunization by Recombinants Encoding Japanese Encephalitis Virus prME and E Proteins

    Institute of Scientific and Technical Information of China (English)

    冯国和; 赵桂珍; 窦晓光; 乔光彦; 周子文

    2003-01-01

    To study the expression characteristic of Japanese encephalitis virus (JEV) prME and E proteins and the efficacy of DNA immunization by different recombinant plasmids containing JEV prME (2001 bp) and E (1500 bp) genes, tworecombinants ( pJME and pJE) containing JEV prME and E genes fused with FLAG were constructed and then transfected into HepG2 and COS-1 cells by lipnsome fusion. The expression feature of FLAG-prME (about 72 kDa) and FLAG-E (about 54 kDa) proteins in transfected cells were analyzed by Western blot and two antibody systems (anti-FLAG and anti-E).BALB/c mice were immunized with 100 μg of two kinds of recombinants by intramuscular injection, and JEV JaGAr-01strains ( 105 PFU/100 μl)were given to BALB/c mice by intraperioneal injection 3 wk after twice DNA immunization by a lethal virus challenge. BALB/c mice were observed for 21 days after challenge. 80% plaque reduction neutralization test was performed to titrate neutralization antibody before and after viral challenge. It was found that the expression of proteins associated with pJME and pJE was determined in transfected cells with anti-FLAG and a new protein of 11 kDa was detected inHepG2 and COS-1 cells transfected with pJME. Only E (53 kDa) protein was identified as transfected with pJME using antiE. Higher level of neutralization antibodies and the efficacy of protective immunity were induced with pJME immunization,and were similar to those induced by inactivated Japanese encephalitis vaccine, but were better than those induced with pJE.It concludes that the expression level from prM to E proteins of JEV is different in vitro, and the in vitro expression efficiency of pJME was better than that of piE. FLAG-prME protein expressed by pJME could be cleaved by peptidase from host.The efficacy of DNA immunization is correlated to the expression characterization of related proteins expressed in vitro.

  6. Stealth Proteins: In Silico Identification of a Novel Protein Family Rendering Bacterial Pathogens Invisible to Host Immune Defense.

    Directory of Open Access Journals (Sweden)

    2005-11-01

    Full Text Available There are a variety of bacterial defense strategies to survive in a hostile environment. Generation of extracellular polysaccharides has proved to be a simple but effective strategy against the host's innate immune system. A comparative genomics approach led us to identify a new protein family termed Stealth, most likely involved in the synthesis of extracellular polysaccharides. This protein family is characterized by a series of domains conserved across phylogeny from bacteria to eukaryotes. In bacteria, Stealth (previously characterized as SacB, XcbA, or WefC is encoded by subsets of strains mainly colonizing multicellular organisms, with evidence for a protective effect against the host innate immune defense. More specifically, integrating all the available information about Stealth proteins in bacteria, we propose that Stealth is a D-hexose-1-phosphoryl transferase involved in the synthesis of polysaccharides. In the animal kingdom, Stealth is strongly conserved across evolution from social amoebas to simple and complex multicellular organisms, such as Dictyostelium discoideum, hydra, and human. Based on the occurrence of Stealth in most Eukaryotes and a subset of Prokaryotes together with its potential role in extracellular polysaccharide synthesis, we propose that metazoan Stealth functions to regulate the innate immune system. Moreover, there is good reason to speculate that the acquisition and spread of Stealth could be responsible for future epidemic outbreaks of infectious diseases caused by a large variety of eubacterial pathogens. Our in silico identification of a homologous protein in the human host will help to elucidate the causes of Stealth-dependent virulence. At a more basic level, the characterization of the molecular and cellular function of Stealth proteins may shed light on fundamental mechanisms of innate immune defense against microbial invasion.

  7. Stealth proteins: in silico identification of a novel protein family rendering bacterial pathogens invisible to host immune defense.

    Directory of Open Access Journals (Sweden)

    Peter Sperisen

    2005-11-01

    Full Text Available There are a variety of bacterial defense strategies to survive in a hostile environment. Generation of extracellular polysaccharides has proved to be a simple but effective strategy against the host's innate immune system. A comparative genomics approach led us to identify a new protein family termed Stealth, most likely involved in the synthesis of extracellular polysaccharides. This protein family is characterized by a series of domains conserved across phylogeny from bacteria to eukaryotes. In bacteria, Stealth (previously characterized as SacB, XcbA, or WefC is encoded by subsets of strains mainly colonizing multicellular organisms, with evidence for a protective effect against the host innate immune defense. More specifically, integrating all the available information about Stealth proteins in bacteria, we propose that Stealth is a D-hexose-1-phosphoryl transferase involved in the synthesis of polysaccharides. In the animal kingdom, Stealth is strongly conserved across evolution from social amoebas to simple and complex multicellular organisms, such as Dictyostelium discoideum, hydra, and human. Based on the occurrence of Stealth in most Eukaryotes and a subset of Prokaryotes together with its potential role in extracellular polysaccharide synthesis, we propose that metazoan Stealth functions to regulate the innate immune system. Moreover, there is good reason to speculate that the acquisition and spread of Stealth could be responsible for future epidemic outbreaks of infectious diseases caused by a large variety of eubacterial pathogens. Our in silico identification of a homologous protein in the human host will help to elucidate the causes of Stealth-dependent virulence. At a more basic level, the characterization of the molecular and cellular function of Stealth proteins may shed light on fundamental mechanisms of innate immune defense against microbial invasion.

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

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    Tsiona Eliyahu

    2013-07-01

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

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

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    Tuchscherer Margret

    2012-11-01

    Full Text Available Abstract Background Inadequate nutrition in utero may retard foetal growth and alter physiological development of offspring. This study investigated the effects of low and high protein diets fed to primiparous German Landrace sows throughout pregnancy on the immune function of their offspring at different ages. Sows were fed diets with adequate (AP, 12.1%; n = 13, low (LP, 6.5%; n = 15, or high (HP, 30%; n = 14 protein content, made isoenergetic by varying carbohydrate levels. Cortisol, total protein and immunoglobulin (IgG, IgM, IgA concentrations were measured in the blood of sows over the course of pregnancy. Cortisol, total protein, immunoglobulins, lymphocyte proliferation, immune cell counts, and cytokines were assessed in the blood of offspring at baseline and under challenging conditions (weaning; lipopolysaccharide (LPS administration. Results In sows, the LP diet increased cortisol (P P P P + cell percentage and the CD4+/CD8+ ratio increased after weaning (P P = 0.09 and HP (P P  Conclusions Our results indicate that both low and high protein:carbohydrate ratios in the diet of pregnant sows can induce short-term as well as long-lasting effects on immune competence in piglets that may have serious consequences for host defence against bacterial pathogens.

  10. Immune Reactivity of Brucella Melitensis–Vaccinated Rabbit Serum with Recombinant Omp31 and Dnak Proteins

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    Mahmood Jeddi-Tehrani

    2013-03-01

    Full Text Available Background and objectives: Brucella melitensis infection is still a major health problem for human and cattle in developing countries and the Middle East.Materials and Methods: In this study, in order to screen immunogenic candidate antigens for the development of a Brucella subunit vaccine, a cytoplasmic protein (DnaK and an outer membrane protein (Omp31 of B. melitensis were cloned, expressed in E.coli BL21 and then purified using Ni-NTA agarose. Immunized serum was prepared from a rabbit inoculated with attenuated B. melitensis.Results and Conclusion: It was proved that immunized serum contains antibodies against recombinant Omp31 (rOmp31 and DnaK (rDnaK by Western blot and ELISA assays. The results may suggest the importance of these proteins as subunit vaccines against B. melitensis as well as targets for immunotherapy.

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

    Science.gov (United States)

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

    2015-07-01

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

  12. Sublingual immunization with recombinant adenovirus encoding SARS-CoV spike protein induces systemic and mucosal immunity without redirection of the virus to the brain

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    Shim Byoung-Shik

    2012-09-01

    Full Text Available Abstract Background Sublingual (s.l. administration of soluble protein antigens, inactivated viruses, or virus-like particles has been shown to induce broad immune responses in mucosal and extra-mucosal tissues. Recombinant replication-defective adenovirus vectors (rADVs infect mucosa surface and therefore can serve as a mucosal antigen delivery vehicle. In this study we examined whether s.l. immunization with rADV encoding spike protein (S (rADV-S of severe acute respiratory syndrome-associated coronavirus (SARS-CoV induces protective immunity against SARS-CoV and could serve as a safe mucosal route for delivery of rADV. Results Here, we show that s.l. administration of rADV-S induced serum SARS-CoV neutralizing and airway IgA antibodies in mice. These antibody responses are comparable to those induced by intranasal (i.n. administration. In addition, s.l. immunization induced antigen-specific CD8+ T cell responses in the lungs that are superior to those induced by intramuscular immunization. Importantly, unlike i.n. administration, s.l. immunization with rADV did not redirect the rADV vector to the olfactory bulb. Conclusion Our study indicates that s.l. immunization with rADV-S is safe and effective in induction of a broad spectrum of immune responses and presumably protection against infection with SARS-CoV.

  13. Passive Immunization by Recombinant Ferric Enterobactin Protein (FepA from Escherichia Coli O157

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    Seyed Mehdi Larrie-Baghal

    2013-06-01

    Full Text Available Background: Enterohemorrhagic Escherichia coli (EHEC O157:H7 has been recognized as a major food borne pathogen responsible for frequent hemorrhagic colitis and hemolytic uremic syndrome in humans. Cattle are important reservoirs of E. coli O157:H7, in which the organism colonizes the intestinal tract and is shed in the feces.Objective: Vaccination of cattle has significant potential as a pre-harvest intervention strategy for E. coli O157:H7. The aim of this study was to evaluate active and passive immunization against E. coli O157:H7 using a recombinant protein.Materials and Methods: The recombinant FepA protein induced by IPTG was purified by nickel affinity chromatography. Antibody titre was determined by ELISA in FepA immunized rabbits sera. Sera collected from vaccinated animals were used for bacterial challenge in passive immunization studies.Results: The results demonstrate that passive immunization with serum raised against FepA protects rabbits from subsequent infection.Conclusion: Significant recognition by the antibody of ferric enterobactin binding protein may lead to its application in the restriction of Enterobacteriaceae propagation.

  14. Role of Streptococcus pneumoniae Proteins in Evasion of Complement-Mediated Immunity

    Science.gov (United States)

    Andre, Greiciely O.; Converso, Thiago R.; Politano, Walter R.; Ferraz, Lucio F. C.; Ribeiro, Marcelo L.; Leite, Luciana C. C.; Darrieux, Michelle

    2017-01-01

    The complement system plays a central role in immune defense against Streptococcus pneumoniae. In order to evade complement attack, pneumococci have evolved a number of mechanisms that limit complement mediated opsonization and subsequent phagocytosis. This review focuses on the strategies employed by pneumococci to circumvent complement mediated immunity, both in vitro and in vivo. At last, since many of the proteins involved in interactions with complement components are vaccine candidates in different stages of validation, we explore the use of these antigens alone or in combination, as potential vaccine approaches that aim at elimination or drastic reduction in the ability of this bacterium to evade complement. PMID:28265264

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

    Science.gov (United States)

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

    2002-03-15

    The immunological activities of five synthetic peptides of the prM protein of dengue-2 (DEN-2) virus containing B cell epitopes were evaluated in BALB/c mice. Two peptides elicited neutralizing antibodies against all four DEN serotypes. Virus-specific proliferative responses were demonstrated in mice immunized with four of the five peptides, demonstrating the presence of T cell epitopes. Mice immunized with three of the five peptides conjugated with bovine albumin showed statistically significant levels (Pdevelopment of anti-flaviviral vaccines.

  16. The innate immune protein Nod2 binds directly to MDP, a bacterial cell wall fragment.

    Science.gov (United States)

    Grimes, Catherine Leimkuhler; Ariyananda, Lushanti De Zoysa; Melnyk, James E; O'Shea, Erin K

    2012-08-22

    Mammalian Nod2 is an intracellular protein that is implicated in the innate immune response to the bacterial cell wall and is associated with the development of Crohn's disease, Blau syndrome, and gastrointestinal cancers. Nod2 is required for an immune response to muramyl dipeptide (MDP), an immunostimulatory fragment of bacterial cell wall, but it is not known whether MDP binds directly to Nod2. We report the expression and purification of human Nod2 from insect cells. Using novel MDP self-assembled monolayers (SAMs), we provide the first biochemical evidence for a direct, high-affinity interaction between Nod2 and MDP.

  17. Circumsporozoite Protein-Specific Kd-Restricted CD8+ T Cells Mediate Protective Antimalaria Immunity in Sporozoite-Immunized MHC-I-Kd Transgenic Mice

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    Jing Huang

    2014-01-01

    Full Text Available Although the roles of CD8+ T cells and a major preerythrocytic antigen, the circumsporozoite (CS protein, in contributing protective antimalaria immunity induced by radiation-attenuated sporozoites, have been shown by a number of studies, the extent to which these players contribute to antimalaria immunity is still unknown. To address this question, we have generated C57BL/6 (B6 transgenic (Tg mice, expressing Kd molecules under the MHC-I promoter, called MHC-I-Kd-Tg mice. In this study, we first determined that a single immunizing dose of IrPySpz induced a significant level of antimalaria protective immunity in MHC-I-Kd-Tg mice but not in B6 mice. Then, by depleting various T-cell subsets in vivo, we determined that CD8+ T cells are the main mediator of the protective immunity induced by IrPySpz. Furthermore, when we immunized (MHC-I-Kd-Tg × CS-Tg F1 mice with IrPySpz after crossing MHC-I-Kd-Tg mice with PyCS-transgenic mice (CS-Tg, which are unable to mount PyCS-specific immunity, we found that IrPySpz immunization failed to induce protective antimalaria immunity in (MHC-I-Kd-Tg × CS-Tg F1 mice, thus indicating the absence of PyCS antigen-dependent immunity in these mice. These results indicate that protective antimalaria immunity induced by IrPySpz in MHC-I-Kd-Tg mice is mediated by CS protein-specific, Kd-restricted CD8+ T cells.

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

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    Kuo Bi

    Full Text Available 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.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.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.

  19. Cross-species Virus-host Protein-Protein Interactions Inhibiting Innate Immunity

    Science.gov (United States)

    2016-07-01

    SUBJECTTERMS viral pathogen, zoonotic, arenavirus, host tropism, protein - protein interactions, RIG-I, Z protein , CARD domain, MAVS 16. SECURITY...individually subcloned into Checkmate M2H System (Promega) bait and prey reporter plasmids. The genes encoding the viral Z proteins were synthesized... viral proteins were calculated with PhyML. While several residue positions are highly conserved across Z proteins (Figure 8), significant sequence

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

    Science.gov (United States)

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

    2015-08-28

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

  1. Purification and Immunity Analysis of Recombinant 6His- HPT Protein Expressed in E.coli

    Institute of Scientific and Technical Information of China (English)

    LI-CHEN YANG; ZHEN ZHU; XIAO-GUANG YANG

    2003-01-01

    Objective To obtain HPT protein (Hygromycin B Phosphotransferase), a kind of plantselective maker gene product expressed from E. coli and to prepare the polyclonal antibody (pAbs)against it. Methods HPT cDNA fragment was obtained by PCR and was inserted into theprokaryotic expressing vector pBV222. Then the constructed recombinant plasmid pBV222-HPT wastransfered into E. coli DH5α for HPT expression. The recombinant expressing system was confirmedby restriction endonuclease digestion, DNA sequencing and protein expression. E. coli cells were lysedby sonication and detergent dissolution. After cell membrane was extracted, the inclusion bodies weredenatured by 8 mol/L Urea and purified with metal chelate affinity chromatography on Ni-NTAagarose under denaturing condition. The purified 6His-HPT was characterized by SDS-PAGE, andused to immunize rabbit. The titer and specificity of antisera were detected by ELISA and Westernblot respecitively. Results Analysis of DNA sequence and restricted enzymes showed that thesequence of PBV222-HPT plasmid was correct. The amount of recombinant HPT expressed in E. coliaccounted for 30% of total cellular proteins. From 1 liter of fermentative bacteria about 22 milligramsof pure recombinant HPT was isolated with purity above 95%. The recombinant HPT protein couldproduce high titer antiserum in rabbits and show good immunity activity. Western blot showedspecific binding reaction between the antiserum to the purified 6His-HPT protein and their expressedproducts (plants protein and bacterial protein). Conclusion HPT protein can be expressed andpurified from E. coli by a relatively simple method, which has high immunity activity.

  2. The Sbi protein is a multifunctional immune evasion factor of Staphylococcus aureus.

    Science.gov (United States)

    Smith, Emma Jane; Visai, Livia; Kerrigan, Steven W; Speziale, Pietro; Foster, Timothy J

    2011-09-01

    The second immunoglobulin-binding protein (Sbi) of Staphylococcus aureus has two N-terminal domains that bind the Fc region of IgG in a fashion similar to that of protein A and two domains that can bind to the complement protein C3 and promote its futile consumption in the fluid phase. It has been proposed that Sbi helps bacteria to avoid innate immune defenses. By comparing a mutant defective in Sbi with mutants defective in protein A, clumping factor A, iron-regulated surface determinant H, and capsular polysaccharide, it was shown that Sbi is indeed an immune evasion factor that promotes bacterial survival in whole human blood and the avoidance of neutrophil-mediated opsonophagocytosis. Sbi is present in the culture supernatant and is also associated with the cell envelope. S. aureus strains that expressed truncates of Sbi lacking N-terminal domains D1 and D2 (D1D2) or D3 and D4 (D3D4) or a C-terminal truncate that was no longer retained in the cell envelope were analyzed. Both the secreted and envelope-associated forms of Sbi contributed to immune evasion. The IgG-binding domains contributed only when Sbi was attached to the cell, while only the secreted C3-binding domains were biologically active.

  3. Cellular immune response of humans to the circumsporozoite protein of Plasmodium vivax

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    Mauricio M. Rodrigues

    1991-06-01

    Full Text Available The cellular immune response to the circumsporozoite (CS protein of plasmodium vivax of individuals from malaria-endemic areas of Brazil was studied. We examined the in vitro proliferative response of the peripheral blood mononuclear cells (PBMC of 22 individuals when stimulated with a CS recombinant protein (rPvCS-2 and two other synthetic peptides based on the sequenceof the P. vivax CS protein. Seven of the individuals from malaria-endemic area displayed an antigen specific in vitro proliferative responseto the recombinant protein PvCS-2 and one out of 6, proliferative response to the peptide 308-320. In contrast, none of the individuals displayed a proliferative reponse when stimulated with the D/A peptide which represent some of the repeated units present in this CS protein. Our study, therefore, provides evidence for the presence, withinthe major surface antigen of P. vivax sporozoites, of epitopes capble to induce proliferation of human PBMC.

  4. Special issue of clinical pharmacology: advances and applications in new protein therapeutics modulating tumor immunity

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    Frankel AE

    2013-11-01

    Full Text Available Arthur E Frankel Department of Internal Medicine, Simmons Comprehensive Cancer Center, The University of Texas Southwestern Medical Center, Dallas, TX, USA Until recent decades, the role of the immune system in harnessing tumor growth was based on anecdotal observations of increased cancers in immune-compromised patients, the benefits of graft-versus-leukemia in allogeneic stem cell transplants, and the limited but reproducible anticancer activity of several lymphokines, including interferon and interleukin (IL-2. Vaccine studies and infusions of "activated" lymphocytes yielded variable clinical responses and disease control. An improved understanding of the molecular and cell mechanisms of the innate and adaptive immune system in cancer-bearing animals and the discovery of an immune-suppressive tumor microenvironment then led to development and testing of a battery of new drug and cell-based approaches to trigger antitumor immunity. This issue of Clinical Pharmacology: Advances and Applications highlights some of the new protein-based compounds that are radically changing the cancer therapeutic landscape. The purpose of this collection of reviews is to inform the readership regarding the importance of the seismic change in cancer therapeutics and stimulate efforts to find novel niches and combinations of agents similar to recent advances in the application of cancer pathway inhibitors.

  5. Protein poly(ADP-ribosyl)ation regulates arabidopsis immune gene expression and defense responses.

    Science.gov (United States)

    Feng, Baomin; Liu, Chenglong; de Oliveira, Marcos V V; Intorne, Aline C; Li, Bo; Babilonia, Kevin; de Souza Filho, Gonçalo A; Shan, Libo; He, Ping

    2015-01-01

    Perception of microbe-associated molecular patterns (MAMPs) elicits transcriptional reprogramming in hosts and activates defense to pathogen attacks. The molecular mechanisms underlying plant pattern-triggered immunity remain elusive. A genetic screen identified Arabidopsis poly(ADP-ribose) glycohydrolase 1 (atparg1) mutant with elevated immune gene expression upon multiple MAMP and pathogen treatments. Poly(ADP-ribose) glycohydrolase (PARG) is predicted to remove poly(ADP-ribose) polymers on acceptor proteins modified by poly(ADP-ribose) polymerases (PARPs) with three PARPs and two PARGs in Arabidopsis genome. AtPARP1 and AtPARP2 possess poly(ADP-ribose) polymerase activity, and the activity of AtPARP2 was enhanced by MAMP treatment. AtPARG1, but not AtPARG2, carries glycohydrolase activity in vivo and in vitro. Importantly, mutation (G450R) in atparg1 blocks its activity and the corresponding residue is highly conserved and essential for human HsPARG activity. Consistently, mutant atparp1atparp2 plants exhibited compromised immune gene activation and enhanced susceptibility to pathogen infections. Our study indicates that protein poly(ADP-ribosyl)ation plays critical roles in plant immune gene expression and defense to pathogen attacks.

  6. Protein poly(ADP-ribosylation regulates arabidopsis immune gene expression and defense responses.

    Directory of Open Access Journals (Sweden)

    Baomin Feng

    2015-01-01

    Full Text Available Perception of microbe-associated molecular patterns (MAMPs elicits transcriptional reprogramming in hosts and activates defense to pathogen attacks. The molecular mechanisms underlying plant pattern-triggered immunity remain elusive. A genetic screen identified Arabidopsis poly(ADP-ribose glycohydrolase 1 (atparg1 mutant with elevated immune gene expression upon multiple MAMP and pathogen treatments. Poly(ADP-ribose glycohydrolase (PARG is predicted to remove poly(ADP-ribose polymers on acceptor proteins modified by poly(ADP-ribose polymerases (PARPs with three PARPs and two PARGs in Arabidopsis genome. AtPARP1 and AtPARP2 possess poly(ADP-ribose polymerase activity, and the activity of AtPARP2 was enhanced by MAMP treatment. AtPARG1, but not AtPARG2, carries glycohydrolase activity in vivo and in vitro. Importantly, mutation (G450R in atparg1 blocks its activity and the corresponding residue is highly conserved and essential for human HsPARG activity. Consistently, mutant atparp1atparp2 plants exhibited compromised immune gene activation and enhanced susceptibility to pathogen infections. Our study indicates that protein poly(ADP-ribosylation plays critical roles in plant immune gene expression and defense to pathogen attacks.

  7. Virus-Heat Shock Protein Interaction and a Novel Axis for Innate Antiviral Immunity

    Directory of Open Access Journals (Sweden)

    Michael Oglesbee

    2012-09-01

    Full Text Available Virus infections induce heat shock proteins that in turn enhance virus gene expression, a phenomenon that is particularly well characterized for the major inducible 70 kDa heat shock protein (hsp70. However, hsp70 is also readily induced by fever, a phylogenetically conserved response to microbial infections, and when released from cells, hsp70 can stimulate innate immune responses through toll like receptors 2 and 4 (TLR2 and 4. This review examines how the virus-hsp70 relationship can lead to host protective innate antiviral immunity, and the importance of hsp70 dependent stimulation of virus gene expression in this host response. Beginning with the well-characterized measles virus-hsp70 relationship and the mouse model of neuronal infection in brain, we examine data indicating that the innate immune response is not driven by intracellular sensors of pathogen associated molecular patterns, but rather by extracellular ligands signaling through TLR2 and 4. Specifically, we address the relationship between virus gene expression, extracellular release of hsp70 (as a damage associated molecular pattern, and hsp70-mediated induction of antigen presentation and type 1 interferons in uninfected macrophages as a novel axis of antiviral immunity. New data are discussed that examines the more broad relevance of this protective mechanism using vesicular stomatitis virus, and a review of the literature is presented that supports the probable relevance to both RNA and DNA viruses and for infections both within and outside of the central nervous system.

  8. EFFECTS OF HBV preS AS A HUMORAL ENHANCER ON THE ABILITIES OF HCV E2 PROTEIN TO INDUCE IMMUNE RESPONSES IN THE DNA-IMMUNIZED MICE

    Institute of Scientific and Technical Information of China (English)

    谢尧; 陶其敏; 高建恩

    2003-01-01

    Objective.To study whether the abilities of hepatitis C virus(HCV)E2 gene immunization to induce humoral and cellular immune responses to E2 protein were affected by hepatitis B virus(HBV)preS gene when they were fused in DNA-immunized mice.Methods.Mice were immunized with E2,preS-E2(preS gene was upstream of E2 gene),and E2-preS(preS gene was downstream of E2 gene)gene by their eukaryotic expression vectors,respectively.The anti-E2 or anti-preS antibodies were detected using the E2 and preS antigens.The cellular immune response to E2 pro-tein in immunized mice was presented by its survival time after injecting SP2/O myeloma cells expressing HCV E2 protein into the abdominal cavity.Results. Chimeric E2 and preS gene immunization can induce mice to develop anti-preS and anti-E2 antibodies.The number of the mice developing anti-E2 antibody and the antibody titers in preS-E2 gene-injected group were higher than those in E2-preS gene-immunized group.However,the mice injected with E2 gene did not develop the detectable anti-E2 antibodies until 12 weeks after DNA immunization.After the mice was injected with target cells,the average survival time of the mice in the group immunized with E2 gene alone was longer than that of the group injected with E2 gene fused with HBV preS and was significantly longer than that of the control(P< 0.05).Conclusion.HBV preS might be a humoral enhancer that can affect the abilities of HCV E2 protein to in-duce immune responses in DNA-immunized mice.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Litai Zhang

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Li Cheng

    2011-06-01

    Full Text Available Abstract Background Target specific antibodies are pivotal for the design of vaccines, immunodiagnostic tests, studies on proteomics for cancer biomarker discovery, identification of protein-DNA and other interactions, and small and large biochemical assays. Therefore, it is important to understand the properties of protein sequences that are important for antigenicity and to identify small peptide epitopes and large regions in the linear sequence of the proteins whose utilization result in specific antibodies. Results Our analysis using protein properties suggested that sequence composition combined with evolutionary information and predicted secondary structure, as well as solvent accessibility is sufficient to predict successful peptide epitopes. The antigenicity and the specificity in immune response were also found to depend on the epitope length. We trained the B-Cell Epitope Oracle (BEOracle, a support vector machine (SVM classifier, for the identification of continuous B-Cell epitopes with these protein properties as learning features. The BEOracle achieved an F1-measure of 81.37% on a large validation set. The BEOracle classifier outperformed the classical methods based on propensity and sophisticated methods like BCPred and Bepipred for B-Cell epitope prediction. The BEOracle classifier also identified peptides for the ChIP-grade antibodies from the modENCODE/ENCODE projects with 96.88% accuracy. High BEOracle score for peptides showed some correlation with the antibody intensity on Immunofluorescence studies done on fly embryos. Finally, a second SVM classifier, the B-Cell Region Oracle (BROracle was trained with the BEOracle scores as features to predict the performance of antibodies generated with large protein regions with high accuracy. The BROracle classifier achieved accuracies of 75.26-63.88% on a validation set with immunofluorescence, immunohistochemistry, protein arrays and western blot results from Protein Atlas database

  13. Immunization of chickens with quail and turkey perivitelline membrane proteins: production of antibodies and their effects on fertility.

    Science.gov (United States)

    Kapoor, P; Compton, M M; Howarth, B

    2000-02-01

    The cross-reactivity of antibodies developed against zona pellucida proteins and their subsequent deleterious effect on fertility in heterologous species is well documented. However, similar investigations have not been undertaken in avian species. In Experiment 1, White Leghorn hens were immunized with intact germinal discs (GD) of quail and chickens. Chicken GD proteins did not elicit an immune response in chicken hens, whereas quail GD proteins were highly immunogenic. Anti-quail GD antibodies did not bind with chicken inner perivitelline membrane (IPM) proteins as determined by immunoblot analysis. To examine the fertility of immunized hens, artificial insemination was performed at weekly intervals for 4 wk following the booster immunization. No significant differences were detected in fertility or hatchability of immunized hens when compared with unimmunized control hens. In Experiment 2, White Leghorn hens were immunized with intact turkey GD, solubilized turkey perivitelline membrane (PM) modified with dinitrophenol (DNP), and solubilized chicken IPM modified with DNP. High titers of antibodies were detected against the turkey GD and the DNP-modified turkey PM proteins. A weak immune response was observed in hens immunized with modified chicken IPM proteins. The fertility and hatchability of eggs laid by immunized hens, however, were not significantly different from those of unimmunized hens. Antibodies from immunized hens were further analyzed using an in vitro assay that assesses sperm penetration of intact IPM. Sperm penetration of intact IPM was inhibited to the same extent when IPM was preincubated with preimmune as well as anti-PM immunoglobulins. Collectively, these results suggested that the antibodies developed in these hens did not cross-react with the chicken IPM proteins involved in sperm-egg interaction and thus did not influence the fertility.

  14. High resolution crystal structure of PedB: a structural basis for the classification of pediocin-like immunity proteins

    Directory of Open Access Journals (Sweden)

    Cha Sun-Shin

    2007-05-01

    Full Text Available Abstract Background Pediocin-like bacteriocins, ribosomally-synthesized antimicrobial peptides, are generally coexpressed with cognate immunity proteins in order to protect the bacteriocin-producer from its own bacteriocin. As a step for understanding the mode of action of immunity proteins, we determined the crystal structure of PedB, a pediocin-like immunity protein conferring immunity to pediocin PP-1. Results The 1.6 Å crystal structure of PedB reveals that PedB consists of an antiparallel four-helix bundle with a flexible C-terminal end. PedB shows structural similarity to an immunity protein against enterocin A (EntA-im but some disparity to an immunity protein against carnobacteriocin B2 (ImB2 in both the C-terminal conformation and the local structure constructed by α3, α4, and their connecting loop. Structure-inspired mutational studies reveal that deletion of the last seven residues of the C-terminus of PedB almost abolished its immunity activity. Conclusion The fact that PedB, EntA-im, and ImB2 share a four-helix bundle structure strongly suggests the structural conservation of this motif in the pediocin-like immunity proteins. The significant difference in the core structure and the C-terminal conformation provides a structural basis for the classification of pediocin-like immunity proteins. Our mutational study using C-terminal-shortened PedBs and the investigation of primary sequence of the C-terminal region, propose that several polar or charged residues in the extreme C-terminus of PedB which is crucial for the immunity are involved in the specific recognition of pediocin PP-1.

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

    Science.gov (United States)

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

    2016-01-01

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

  16. Serotonergic chemosensory neurons modify the C. elegans immune response by regulating G-protein signaling in epithelial cells.

    Directory of Open Access Journals (Sweden)

    Alexandra Anderson

    Full Text Available The nervous and immune systems influence each other, allowing animals to rapidly protect themselves from changes in their internal and external environment. However, the complex nature of these systems in mammals makes it difficult to determine how neuronal signaling influences the immune response. Here we show that serotonin, synthesized in Caenorhabditis elegans chemosensory neurons, modulates the immune response. Serotonin released from these cells acts, directly or indirectly, to regulate G-protein signaling in epithelial cells. Signaling in these cells is required for the immune response to infection by the natural pathogen Microbacterium nematophilum. Here we show that serotonin signaling suppresses the innate immune response and limits the rate of pathogen clearance. We show that C. elegans uses classical neurotransmitters to alter the immune response. Serotonin released from sensory neurons may function to modify the immune system in response to changes in the animal's external environment such as the availability, or quality, of food.

  17. Identification of the major proteins of an immune modulating fraction from adult Fasciola hepatica released by Nonidet P40.

    Science.gov (United States)

    Morphew, Russell M; Hamilton, Clare M; Wright, Hazel A; Dowling, David J; O'Neill, Sandra M; Brophy, Peter M

    2013-01-31

    Fasciola hepatica NP-40 released protein extract (FhNPE) exhibits potent Th1 immunosuppressive properties in vitro and in vivo. However, the protein composition of this active fraction, responsible for Th1 immune modulatory activity, has yet to be resolved. Therefore, FhNPE, a Nonidet P-40 extract, was subjected to a proteomic analysis in order to identify individual protein components. This was performed using an in house F. hepatica EST database following 2D electrophoresis combined with de novo sequencing based mass spectrometry. The identified proteins, a mixture of excretory/secretory and membrane-associated proteins, are associated with stress response and chaperoning, energy metabolism and cytoskeletal components. The immune modulatory properties of these identified protein(s) are discussed and HSP70 from F. hepatica is highlighted as a potential host immune modulator for future study.

  18. Proteins of the Rpf Family: Immune Cell Reactivity and Vaccination Efficacy against Tuberculosis in Mice

    Science.gov (United States)

    Yeremeev, Vladimir V.; Kondratieva, Tatiana K.; Rubakova, Elvira I.; Petrovskaya, Svetlana N.; Kazarian, Konstantin A.; Telkov, Miroslav V.; Biketov, Sergej F.; Kaprelyants, Arseny S.; Apt, Alexander S.

    2003-01-01

    It was shown recently that Mycobacterium tuberculosis expresses five proteins that are homologous to Rpf (resuscitation promoting factor), which is secreted by growing cells of Micrococcus luteus. Rpf is required to resuscitate the growth of dormant Micrococcus luteus organisms, and its homologues may be involved in mycobacterial reactivation. Mycobacterial Rpf-like products are secreted proteins, which makes them candidates for recognition by the host immune system and anti-Rpf immune responses potentially protective against reactivated tuberculosis. Here we report that the Rpf protein itself and four out of five of its mycobacterial homologues, which were administered as subunit vaccines to C57BL/6 mice, are highly immunogenic. Rpf-like proteins elicit immunoglobulin G1 (IgG1) and IgG2a responses and T-cell proliferation and stimulate production of gamma interferon, interleukin-10 (IL-10), and IL-12 but not IL-4 or IL-5. Both humoral and T-cell responses against these antigens show a high degree of cross-reactivity. Vaccination of mice with Rpf-like proteins results in a significant level of protection against a subsequent high-dose challenge with virulent M. tuberculosis H37Rv, both in terms of survival times and mycobacterial multiplication in lungs and spleens. PMID:12874362

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

    Science.gov (United States)

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

    2016-02-01

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

  20. Pili-like proteins of Akkermansia muciniphila modulate host immune responses and gut barrier function

    Science.gov (United States)

    Reunanen, Justus; Meijerink, Marjolein; Pietilä, Taija E.; Kainulainen, Veera; Klievink, Judith; Huuskonen, Laura; Aalvink, Steven; Skurnik, Mikael; Boeren, Sjef; Satokari, Reetta; Mercenier, Annick; Palva, Airi; Smidt, Hauke; de Vos, Willem M.; Belzer, Clara

    2017-01-01

    Gut barrier function is key in maintaining a balanced response between the host and its microbiome. The microbiota can modulate changes in gut barrier as well as metabolic and inflammatory responses. This highly complex system involves numerous microbiota-derived factors. The gut symbiont Akkermansia muciniphila is positively correlated with a lean phenotype, reduced body weight gain, amelioration of metabolic responses and restoration of gut barrier function by modulation of mucus layer thickness. However, the molecular mechanisms behind its metabolic and immunological regulatory properties are unexplored. Herein, we identify a highly abundant outer membrane pili-like protein of A. muciniphila MucT that is directly involved in immune regulation and enhancement of trans-epithelial resistance. The purified Amuc_1100 protein and enrichments containing all its associated proteins induced production of specific cytokines through activation of Toll-like receptor (TLR) 2 and TLR4. This mainly leads to high levels of IL-10 similar to those induced by the other beneficial immune suppressive microorganisms such as Faecalibacterium prausnitzii A2-165 and Lactobacillus plantarum WCFS1. Together these results indicate that outer membrane protein composition and particularly the newly identified highly abundant pili-like protein Amuc_1100 of A. muciniphila are involved in host immunological homeostasis at the gut mucosa, and improvement of gut barrier function. PMID:28249045

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

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    Natalia V. Permyakova

    2015-01-01

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

  2. Impact of Dietary Protein Concentration and Quality on Immune Function of Cats

    Science.gov (United States)

    Paßlack, Nadine; Kohn, Barbara; Doherr, Marcus G.; Zentek, Jürgen

    2017-01-01

    Protein levels and quality in cat food can vary significantly and might affect immune function in various ways. In the present study, 3 diets with a low protein quality (LQ) and 3 diets with a high protein quality (HQ) were offered to 10 healthy adult cats for 6 weeks each, using a randomized cross-over design. The LQ and HQ diets differed in the collagen content and had low (36.7% and 36.2%), medium (45.0% and 43.3%) and high (56.1% and 54.9%) protein levels. At the end of each feeding period, blood was collected for phenotyping of leukocyte subsets, lymphocyte proliferation assay and cytokine measurements, phagocytosis assay and differential blood count. The results demonstrated no group differences for numbers of CD4+CD8-, CD4+CD8+, CD4-CD8+, MHCII+, CD21+, SWC3+ and CD14+ cells in the blood of the cats. Proliferative activity of lymphocytes when stimulated with pokeweed mitogen, Concanavalin A and Phytohemagglutinin, M form did not differ depending on the dietary protein concentration and quality. Concentrations of tumor necrosis factor alpha and interferon gamma in the supernatant of the proliferation assay were also not affected by the dietary treatment. Blood monocyte phagocytic activity was higher (P = 0.048) and cell numbers of eosinophilic granulocytes in the blood were lower (P = 0.047) when cats were fed the low protein diets. In conclusion, only a few differences in feline immune cell populations and activity depending on dietary protein supply could be detected. However, the observed increase of eosinophilic granulocytes by a higher protein intake indicates an activation of immunological mechanisms and requires further investigation. PMID:28072882

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

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    Brian A Chow

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

  4. HLA-associated immune escape pathways in HIV-1 subtype B Gag, Pol and Nef proteins.

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    Zabrina L Brumme

    Full Text Available BACKGROUND: Despite the extensive genetic diversity of HIV-1, viral evolution in response to immune selective pressures follows broadly predictable mutational patterns. Sites and pathways of Human Leukocyte-Antigen (HLA-associated polymorphisms in HIV-1 have been identified through the analysis of population-level data, but the full extent of immune escape pathways remains incompletely characterized. Here, in the largest analysis of HIV-1 subtype B sequences undertaken to date, we identify HLA-associated polymorphisms in the three HIV-1 proteins most commonly considered in cellular-based vaccine strategies. Results are organized into protein-wide escape maps illustrating the sites and pathways of HLA-driven viral evolution. METHODOLOGY/PRINCIPAL FINDINGS: HLA-associated polymorphisms were identified in HIV-1 Gag, Pol and Nef in a multicenter cohort of >1500 chronically subtype-B infected, treatment-naïve individuals from established cohorts in Canada, the USA and Western Australia. At q< or =0.05, 282 codons commonly mutating under HLA-associated immune pressures were identified in these three proteins. The greatest density of associations was observed in Nef (where close to 40% of codons exhibited a significant HLA association, followed by Gag then Pol (where approximately 15-20% of codons exhibited HLA associations, confirming the extensive impact of immune selection on HIV evolution and diversity. Analysis of HIV codon covariation patterns identified over 2000 codon-codon interactions at q< or =0.05, illustrating the dense and complex networks of linked escape and secondary/compensatory mutations. CONCLUSIONS/SIGNIFICANCE: The immune escape maps and associated data are intended to serve as a user-friendly guide to the locations of common escape mutations and covarying codons in HIV-1 subtype B, and as a resource facilitating the systematic identification and classification of immune escape mutations. These resources should facilitate

  5. Immunization of stromal cell targeting fibroblast activation protein providing immunotherapy to breast cancer mouse model.

    Science.gov (United States)

    Meng, Mingyao; Wang, Wenju; Yan, Jun; Tan, Jing; Liao, Liwei; Shi, Jianlin; Wei, Chuanyu; Xie, Yanhua; Jin, Xingfang; Yang, Li; Jin, Qing; Zhu, Huirong; Tan, Weiwei; Yang, Fang; Hou, Zongliu

    2016-08-01

    Unlike heterogeneous tumor cells, cancer-associated fibroblasts (CAF) are genetically more stable which serve as a reliable target for tumor immunotherapy. Fibroblast activation protein (FAP) which is restrictively expressed in tumor cells and CAF in vivo and plays a prominent role in tumor initiation, progression, and metastasis can function as a tumor rejection antigen. In the current study, we have constructed artificial FAP(+) stromal cells which mimicked the FAP(+) CAF in vivo. We immunized a breast cancer mouse model with FAP(+) stromal cells to perform immunotherapy against FAP(+) cells in the tumor microenvironment. By forced expression of FAP, we have obtained FAP(+) stromal cells whose phenotype was CD11b(+)/CD34(+)/Sca-1(+)/FSP-1(+)/MHC class I(+). Interestingly, proliferation capacity of the fibroblasts was significantly enhanced by FAP. In the breast cancer-bearing mouse model, vaccination with FAP(+) stromal cells has significantly inhibited the growth of allograft tumor and reduced lung metastasis indeed. Depletion of T cell assays has suggested that both CD4(+) and CD8(+) T cells were involved in the tumor cytotoxic immune response. Furthermore, tumor tissue from FAP-immunized mice revealed that targeting FAP(+) CAF has induced apoptosis and decreased collagen type I and CD31 expression in the tumor microenvironment. These results implicated that immunization with FAP(+) stromal cells led to the disruption of the tumor microenvironment. Our study may provide a novel strategy for immunotherapy of a broad range of cancer.

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

    Science.gov (United States)

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

    2011-10-01

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

  7. Immune response to dna vaccine expressing transferrin binding protein a gene of Pasteurella multocida

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    Satparkash Singh

    2011-06-01

    Full Text Available Haemorrhagic Septicaemia (HS, an acute and fatal disease of cattle and buffalo is primarily caused by serotype B:2 or E:2 of Pasteurella multocida. The transferrin binding protein A (TbpA has been found to act as immunogen and potent vaccine candidate in various Gram negative bacteria including P. multocida. The present study was carried out to evaluate the potential of this antigen as a DNA vaccine against HS in mice model. The tbpA gene of P. multocida serotype B:2 was cloned in a mammalian expression vector alone and along with murine IL2 gene as immunological adjuvant to produce monocistronic and bicistronic DNA vaccine constructs, respectively. The immune response to DNA vaccines was evaluated based on serum antibody titres and lymphocyte proliferation assay. A significant increase in humoral and cell mediated immune responses was observed in mice vaccinated with DNA vaccines as compared to non immunized group. Additionally, the bicistronic DNA vaccine provided superior immune response and protection level following challenge as compared to monocistronic construct. The study revealed that DNA vaccine presents a promising approach for the prevention of HS.

  8. Immune response to dna vaccine expressing transferrin binding protein a gene of Pasteurella multocida.

    Science.gov (United States)

    Singh, Satparkash; Singh, Vijendra Pal; Cheema, Pawanjit Singh; Sandey, Maninder; Ranjan, Rajeev; Gupta, Santosh Kumar; Sharma, Bhaskar

    2011-04-01

    Haemorrhagic Septicaemia (HS), an acute and fatal disease of cattle and buffalo is primarily caused by serotype B:2 or E:2 of Pasteurella multocida. The transferrin binding protein A (TbpA) has been found to act as immunogen and potent vaccine candidate in various Gram negative bacteria including P. multocida. The present study was carried out to evaluate the potential of this antigen as a DNA vaccine against HS in mice model. The tbpA gene of P. multocida serotype B:2 was cloned in a mammalian expression vector alone and along with murine IL2 gene as immunological adjuvant to produce monocistronic and bicistronic DNA vaccine constructs, respectively. The immune response to DNA vaccines was evaluated based on serum antibody titres and lymphocyte proliferation assay. A significant increase in humoral and cell mediated immune responses was observed in mice vaccinated with DNA vaccines as compared to non immunized group. Additionally, the bicistronic DNA vaccine provided superior immune response and protection level following challenge as compared to monocistronic construct. The study revealed that DNA vaccine presents a promising approach for the prevention of HS.

  9. Protection of chickens against avian hepatitis E virus (avian HEV) infection by immunization with recombinant avian HEV capsid protein.

    Science.gov (United States)

    Guo, H; Zhou, E M; Sun, Z F; Meng, X J

    2007-04-12

    Avian hepatitis E virus (avian HEV) is an emerging virus associated with hepatitis-splenomegaly syndrome in chickens in North America. Avian HEV is genetically and antigenically related to human HEV, the causative agent of hepatitis E in humans. In the lack of a practical animal model, avian HEV infection in chickens has been used as a model to study human HEV replication and pathogenesis. A 32 kDa recombinant ORF2 capsid protein of avian HEV expressed in Escherichia coli was found having similar antigenic structure as that of human HEV containing major neutralizing epitopes. To determine if the capsid protein of avian HEV can be used as a vaccine, 20 chickens were immunized with purified avian HEV recombinant protein with aluminum as adjuvant and another 20 chickens were mock immunized with KLH precipitated in aluminum as controls. Both groups of chickens were subsequently challenged with avian HEV. All the tested mock-immunized control chickens developed typical avian HEV infection characterized by viremia, fecal virus shedding and seroconversion to avian HEV antibodies. Gross hepatic lesions were also found in portion of these chickens. In contrast, none of the tested chickens immunized with avian HEV capsid protein had detectable viremia, fecal virus shedding or observable gross hepatitis lesions. The results from this study suggested that immunization of chickens with avian HEV recombinant ORF2 capsid protein with aluminum as adjuvant can induce protective immunity against avian HEV infection. Chickens are a useful small animal model to study anti-HEV immunity and pathogenesis.

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

    Science.gov (United States)

    Verthelyi, Daniela; Wang, Vivian

    2010-12-22

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

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

    Directory of Open Access Journals (Sweden)

    Daniela Verthelyi

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

  12. Similar Ability of FbaA with M Protein to Elicit Protective Immunity Against Group A Streptococcus Challenge in Mice

    Institute of Scientific and Technical Information of China (English)

    Cuiqing Ma; Caihong Li; Xiurong Wang; Ruihong Zeng; Xiaolin Yin; Huidong Feng; Lin Wei

    2009-01-01

    Group A streptococcus (GAS), an important human pathogen, can cause various kinds of infections including superficial infections and potentially lethal infections, and the search for an effective vaccine to prevent GAS infections has been ongoing for many years. This paper compares the immunogenicity and immunoprotection of FbaA (an Fn-binding protein expressed on the surface of GAS) with that of M protein, the best immunogen of GAS. Assay for immune response showed that FbaA, similar to M protein, could induce protein-specific high IgG titer in BALB/c mice. Furthermore, following GAS challenge, the mice immunized with FbaA showed the same protective rate as those with M protein. These results indicate that FbaA is similar in ability to M protein in inducing protective immunity against GAS challenge in mice.

  13. Isolation and Purification of an Antibacterial Protein from Immune Induced Haemolymph of American Cockroach, Periplaneta americana

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    Hamid Reza Basseri

    2016-10-01

    Full Text Available Background: Antimicrobial peptides play a role as effectors substances in the immunity of vertebrate and inverte­brate hosts. In the current study, antimicrobial peptide was isolated from the haemolymph of the American cock­roach, Periplaneta americana.Methods: Micrococcus luteus as Gram-positive bacteria and Escherichia coli as Gram-negative bacteria were candi­date for injection. Induction was done by injecting both bacteria into the abdominal cavity of two groups of cock­roaches separately. The haemolymphs were collected 24 hours after post injection and initially tested against both bacteria. Subsequently, the immune induced haemolymph was purified by high performance liquid chromatography (HPLC to separate the proteins responsible for the antibacterial activity.Results: The non-induced haemolymph did not show any activity against both bacteria whereas induced haemo­lymph exhibited high activity against M. luteus but did less against E. coli. Two fractions showed antibacterial activ­ity against M. luteus. Finally the molecular weight of the isolated antibacterial proteins were determined as 72 kDa and 62 kDa using SDS-PAGE.Conclusion: Induced haemolymph of American cockroaches has the ability to produce peptides to combat against Gram-positive bacteria when an immune challenge is mounted. Further work has to be done to sequence of the pro­tein, which it would be advantageous.

  14. Humoral Immune Response to Keyhole Limpet Haemocyanin, the Protein Carrier in Cancer Vaccines

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    A. Kantele

    2011-01-01

    Full Text Available Keyhole limpet haemocyanin (KLH appears to be a promising protein carrier for tumor antigens in numerous cancer vaccine candidates. The humoral immune response to KLH was characterized at the single-cell level with ELISPOT combined with separations of cell populations according to their expression of homing receptors (HRs. The analysis of HR expressions is expected to reveal the targeting of the immune response in the body. Eight orally primed and four nonprimed volunteers received KLH-vaccine subcutaneously. Circulating KLH-specific plasmablasts were found in all volunteers, 60 KLH-specific plasmablasts/106 PBMC in the nonprimed and 136/106 in the primed group. The proportion of L-selectin+ plasmablasts proved high and integrin α4β7+ low. KLH serving as protein carrier in several vaccines, the homing profile of KLH-specific response may be applicable to the cancer antigen parts in the same vaccines. The present data reflect a systemic homing profile, which appears advantageous for the targeting of immune response to cancer vaccines.

  15. Arabinogalactan Proteins from Baobab and Acacia Seeds Influence Innate Immunity of Human Keratinocytes in vitro.

    Science.gov (United States)

    Zahid, Abderrakib; Despres, Julie; Benard, Magalie; Nguema-Ona, Eric; Leprince, Jerome; Vaudry, David; Rihouey, Christophe; Vicré-Gibouin, Maité; Driouich, Azeddine; Follet-Gueye, Marie-Laure

    2016-10-13

    Plant derived arabinogalactan proteins (AGP) were repeatedly confirmed as immunologically as well as dermatologically active compounds. However little is currently known regarding their potential activity towards skin innate immunity. Here, we extracted and purified AGP from acacia (Acacia senegal) and baobab (Adansonia digitata) seeds to investigate their biological effects on the HaCaT keratinocyte cell line in an in vitro system. While AGP from both sources did not exhibit any cytotoxic effect, AGP from acacia seeds enhanced cell viability Moreover, real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that AGP extracted from both species induced a substantial overexpression of hBD-2, TLR-5, and IL1-α genes. These data suggest that plant AGP, already known to control plant defensive processes, could also modulate skin innate immune responses. This article is protected by copyright. All rights reserved.

  16. Is sporozoite refractile body protein expression different in Eimeria acervulina sporozoites isolated from non-immune versus immune chickens?

    Science.gov (United States)

    A hallmark of Eimeria infection in avians is the establishment of immunity against clinical signs of coccidiosis. Resistant birds experience improved weight gain and feed conversion efficiency and lack intestinal lesions. Oocysts excretion is reduced, but not eliminated, in such immune chickens. ...

  17. Innate immunity and protective neuroinflammation: new emphasis on the role of neuroimmune regulatory proteins.

    Science.gov (United States)

    Griffiths, M; Neal, J W; Gasque, P

    2007-01-01

    Brain inflammation due to infection, hemorrhage, and aging is associated with activation of the local innate immune system as expressed by infiltrating cells, resident glial cells, and neurons. The innate immune response relies on the detection of "nonself" and "danger-self" ligands behaving as "eat me signals" by a plethora of pattern recognition receptors (PRRs) expressed by professional and amateur phagocytes to promote the clearance of pathogens, toxic cell debris (amyloid fibrils, aggregated synucleins, prions), and apoptotic cells accumulating within the brain parenchyma and the cerebrospinal fluid (CSF). These PRRs (e.g., complement, TLR, CD14, scavenger receptors) are highly conserved between vertebrates and invertebrates and may represent the most ancestral innate scavenging system involved in tissue homeostasis. However, in some diseases, these protective mechanisms lead to neurodegeneration on the ground that several innate immune molecules have neurocytotoxic activities. The response is a "double-edged sword" representing a fine balance between protective and detrimental effects. Several key regulatory mechanisms have now been evidenced in the control of CNS innate immunity, and these could be harnessed to explore novel therapeutic avenues. We will herein provide new emphasis on the role of neuroimmune regulatory proteins (NIRegs), such as CD95L, TNF, CD200, CD47, sialic acids, CD55, CD46, fH, C3a, HMGB1, which are involved in silencing innate immunity at the cellular and molecular levels and suppression of inflammation. For instance, NIRegs may play an important role in controlling lymphocyte/macrophage/microglia hyperinflammatory responses, while sparing host defense and repair mechanisms. Moreover, NIRegs have direct beneficial effects on neurogenesis and contributing to brain tissue remodeling.

  18. Scanning the cell surface proteome of cancer cells and identification of metastasis-associated proteins using a subtractive immunization strategy

    DEFF Research Database (Denmark)

    Rasmussen, Nicolaj; Ditzel, Henrik J

    2009-01-01

    characterization of the identified proteins. The strategy is based on subtractive immunization of mice, and we used the two isogenic cell lines, NM-2C5 and M-4A4, derived from the MDA-MB-435 cancer cell line, as a model system. Although the two cell lines are equally tumorigenic, only M-4A4 has metastatic...... of the immune system itself to scan the cell surface proteome for differentially expressed proteins. The subtractive immunization strategy should be broadly applicable as a quantitative and comparative proteomic approach for screening the cell surface and also allow generation of mAbs to study these cell...

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

    Science.gov (United States)

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

    2016-01-01

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

  20. Increased titers of neutralizing antibodies after immunization with both envelope proteins of the porcine endogenous retroviruses (PERVs

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    Denner Joachim

    2012-11-01

    Full Text Available Abstract Despite enormous difficulties to induce antibodies neutralizing HIV-1, especially broadly neutralizing antibodies directed against the conserved membrane proximal external region (MPER of the transmembrane envelope protein, such antibodies can be easily induced in the case of gammaretroviruses, among them the porcine endogenous retroviruses (PERVs. In addition to neutralizing antibodies directed against the transmembrane envelope protein p15E, neutralizing antibodies were also induced by immunization with the surface envelope protein gp70. PERVs represent a special risk for xenotransplantation using pig tissues or organs since they are integrated in the genome of all pigs and infect human cells and a vaccine may protect from transmission to the recipient. To investigate the effect of simultaneous immunization with both proteins in detail, a study was performed in hamsters. Gp70 and p15E of PERV were produced in E. coli, purified and used for immunization. All animals developed binding antibodies against the antigens used for immunization. Sera from animals immunized with p15E recognized epitopes in the MPER and the fusion peptide proximal region (FPPR of p15E. One MPER epitope showed a sequence homology to an epitope in the MPER of gp41 of HIV-1 recognized by broadly neutralizing antibodies found in HIV infected individuals. Neutralizing antibodies were detected in all sera. Most importantly, sera from animals immunized with gp70 had a higher neutralizing activity when compared with the sera from animals immunized with p15E and sera from animals immunized with gp70 together with p15E had a higher neutralizing activity compared with sera from animals immunized with each antigen alone. These immunization studies are important for the development of vaccines against other retroviruses including the human immunodeficiency virus HIV-1.

  1. Response to Hepatocarcinoma Hca-F of Mice Immunized with Heat Shock Protein 70 from Elemene Combo Tumor Cell Vaccine

    Institute of Scientific and Technical Information of China (English)

    Lianying Guo; Guangxia Shi; Zhihong Gao; Jie Shen; Rong Xing; Zhenchao Qian

    2006-01-01

    To analyze immune response to murine hepatocarcinoma Hca-F of mice immunized with heat shock protein 70(HSP70) derived from elemene combo tumor cell vaccine (EC-TCV) of Hca-F, HSP70 was isolated from EC-TCV by ADP affinity chromatography. Mice were immunized with HSP70 intraperitoneally three times and spleen cells were sampled. For cells, their proliferation and cytotoxicity against Hca-F were measured with MTT assay and their phenotypes were analyzed with flow cytometry. Spleen cells of immunized mice with HSP70 exhibited more potent cytotoxicity against Hca-F and proliferation than that of normal control mice, but less potent than that of mice immunized with EC-TCV. Among three groups, the percent of γδ T lymphocytes in the mice immunized with HSP70 (35.5%) was the highest compared with 6.25% in normal mice, and 28.4% in the mice immunized with EC-TCV. Immunization of HSP70 derived from EC-TCV could elicit potent immune response to Hca-F. HSP70 is one of elements inducing anti-tumor immune responses against Hca-F. Cellular & Molecular Immunology. 2006;3(4):291-295.

  2. Rotavirus nonstructural protein 1 antagonizes innate immune response by interacting with retinoic acid inducible gene I

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    Qin Lan

    2011-12-01

    Full Text Available Abstract Background The nonstructural protein 1 (NSP1 of rotavirus has been reported to block interferon (IFN signaling by mediating proteasome-dependent degradation of IFN-regulatory factors (IRFs and (or the β-transducin repeat containing protein (β-TrCP. However, in addition to these targets, NSP1 may subvert innate immune responses via other mechanisms. Results The NSP1 of rotavirus OSU strain as well as the IRF3 binding domain truncated NSP1 of rotavirus SA11 strain are unable to degrade IRFs, but can still inhibit host IFN response, indicating that NSP1 may target alternative host factor(s other than IRFs. Overexpression of NSP1 can block IFN-β promoter activation induced by the retinoic acid inducible gene I (RIG-I, but does not inhibit IFN-β activation induced by the mitochondrial antiviral-signaling protein (MAVS, indicating that NSP1 may target RIG-I. Immunoprecipitation experiments show that NSP1 interacts with RIG-I independent of IRF3 binding domain. In addition, NSP1 induces down-regulation of RIG-I in a proteasome-independent way. Conclusions Our findings demonstrate that inhibition of RIG-I mediated type I IFN responses by NSP1 may contribute to the immune evasion of rotavirus.

  3. Immune response to Candida albicans is preserved despite defect in O-mannosylation of secretory proteins.

    Science.gov (United States)

    Corbucci, Cristina; Cenci, Elio; Skrzypek, Franck; Gabrielli, Elena; Mosci, Paolo; Ernst, Joachim F; Bistoni, Francesco; Vecchiarelli, Anna

    2007-12-01

    The PMT gene family in Candida albicans encodes five isoforms of the protein mannosyltransferases that initiate O-mannosylation of secretory proteins. Mutations at the Pmt level have been associated with differences in pathogenicity, e.g. in contrast to pmt5/pmt5, pmt2/PMT2 mutants showed poor virulence. Our objective was to determine whether these differences were related to the capacity of pmt2/PMT2 and pmt5/pmt5 to (i) express differences in selected virulence factors, and (ii) stimulate the natural immune system. The results show that pmt mutants (i) form hyphae in serum, (ii) show defective production of proteases but not of phospholipases with respect to the parental strain, (iii) undergo mycelial transition in the kidneys of hematogenously infected animals, (iv) are phagocytosed and killed by macrophages similar to the parental strain, although neutrophils are unable to destroy pmt5/pmt5, (v) engage TLR4 and stimulate MyD88 leading to NF-kappaB activation, and (vi) stimulate cytokine production by macrophages. Collectively our findings suggest that the defect in protein O-mannosylation in C. albicans cause attenuation of the virulence although the antigenic factors that retain the capacity to stimulate an efficient immune response are preserved.

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

    Science.gov (United States)

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

    2015-01-01

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

  5. The E3-ligase TRIM family of proteins regulates signaling pathways triggered by innate immune pattern-recognition receptors.

    Science.gov (United States)

    Versteeg, Gijs A; Rajsbaum, Ricardo; Sánchez-Aparicio, Maria Teresa; Maestre, Ana M; Valdiviezo, Julio; Shi, Mude; Inn, Kyung-Soo; Fernandez-Sesma, Ana; Jung, Jae; García-Sastre, Adolfo

    2013-02-21

    Innate immunity conferred by the type I interferon is critical for antiviral defense. To date only a limited number of tripartite motif (TRIM) proteins have been implicated in modulation of innate immunity and anti-microbial activity. Here we report the complementary DNA cloning and systematic analysis of all known 75 human TRIMs. We demonstrate that roughly half of the 75 TRIM-family members enhanced the innate immune response and that they do this at multiple levels in signaling pathways. Moreover, messenger RNA levels and localization of most of these TRIMs were found to be altered during viral infection, suggesting that their regulatory activities are highly controlled at both pre- and posttranscriptional levels. Taken together, our data demonstrate a very considerable dedication of this large protein family to the positive regulation of the antiviral response, which supports the notion that this family of proteins evolved as a component of innate immunity.

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

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, Ritesh K.; Li, Changzhao; Chaudhary, Sandeep C. [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL (United States); Ballestas, Mary E. [Department of Pediatrics Infectious Disease, Children' s of Alabama, School of Medicine, University of Alabama at Birmingham, AL (United States); Elmets, Craig A. [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL (United States); Robbins, David J. [Department of Surgery, Molecular Oncology Program, Miller School of Medicine, University of Miami, Miami (United States); Matalon, Sadis [Department of Anesthesiology, University of Alabama at Birmingham, Birmingham, AL (United States); Deshane, Jessy S. [Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, AL (United States); Afaq, Farrukh [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL (United States); Bickers, David R. [Department of Dermatology, Columbia University Medical Center, New York (United States); Athar, Mohammad, E-mail: mathar@uab.edu [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL (United States)

    2013-11-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Tianyu [Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Ding, Jinjing; Zhang, Ying; Wang, Da-Cheng, E-mail: dcwang@ibp.ac.cn [Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Liu, Wei, E-mail: dcwang@ibp.ac.cn [The Third Military Medical University, Chongqing 400038 (China); Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)

    2013-10-01

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

  8. STUDIES ON THE BLOOD PROTEINS : I. THE SERUM GLOBULINS IN BACTERIAL INFECTION AND IMMUNITY.

    Science.gov (United States)

    Hurwitz, S H; Meyer, K F

    1916-11-01

    The progress of an infection is usually associated with marked changes in the serum proteins. There may be an increase in the percentage of the total protein during some stage of the infection, and there is usually a change in the albumin-globulin ratio with an increase in the total globulins. This rise may antedate the development of any resistance by a considerable period of time. The non-protein constituents of the blood show fluctuations with a tendency to rise as the infection progresses. The process of immunization is in almost all instances associated with a definite increase in the globulins of the blood, and in some cases with a complete inversion of the normal albumin-globulin ratio. This may be produced both by living and dead organisms and by bacterial endotoxins. Massive doses usually result in an upset which shows no tendency to right itself during the period of observation. A rise in the globulins has been shown to occur long before the animal develops immune bodies in any appreciable concentration; and where the globulin curve and antibody curve appear to parallel one another, it can be shown by a careful analysis of both curves that there is a definite lack of correspondence at various periods of the experiment. Animals possessing a basic immunity show a more rapid rise in the globulin curve following inoculation. There is no parallelism between the leukocytic reaction and the globulin reaction. During periods of leukopenia the globulins may be as high as during the period of a leukocytosis. Bacterial endotoxins produce as striking an increase in the serum globulins as do living and killed bacteria. This would seem to indicate that a bacterial invasion of the organism is not absolutely essential for the globulin changes, and that the toxogenic factor in infection and immunity must play a part in the production of the changes noted. Inflammatory irritants injected intraperitoneally also result in a globulin increase. In this case the changes

  9. Cancer associated aberrant protein o-glycosylation can modify antigen processing and immune response

    DEFF Research Database (Denmark)

    Madsen, Caroline B; Petersen, Cecilie; Lavrsen, Kirstine

    2012-01-01

    Aberrant glycosylation of mucins and other extracellular proteins is an important event in carcinogenesis and the resulting cancer associated glycans have been suggested as targets in cancer immunotherapy. We assessed the role of O-linked GalNAc glycosylation on antigen uptake, processing...... response to a cancer related tumor antigen, Balb/c or B6.Cg(CB)-Tg(HLA-A/H2-D)2Enge/J (HLA-A2 transgenic) mice were immunized with a non-glycosylated or GalNAc-glycosylated MUC1 derived peptide followed by comparison of T cell proliferation, IFN-¿ release, and antibody induction. Gal...

  10. Inhibitor of apoptosis (IAP) proteins in regulation of inflammation and innate immunity

    DEFF Research Database (Denmark)

    Damgaard, Rune B; Gyrd-Hansen, Mads

    2011-01-01

    as important regulators of innate immune signaling downstream of pattern recognition receptors (PRRs) such as Toll-like receptor 4 (TLR4), the nucleotide-binding oligomerization domain 1 (NOD1) and NOD2 receptors, and the retinoic acid-inducible gene (RIG)-I receptor. Recent evidence suggests that cIAP1, cIAP2......, and XIAP facilitate ubiquitin-dependent signaling activated by these PRRs and mediate activation of nuclear factor-kappa B (NF-kappaB) transcription factors as well as the MAP kinases p38 and JNK. Here, we review the current understanding of IAP-mediated PRR signaling and how IAP proteins might present...

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

    Science.gov (United States)

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

    2016-02-01

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

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

    Science.gov (United States)

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

    2015-04-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  14. ISOLATION AND PARTIAL PURIFICATION OF ANTIMICROBIAL PEPTIDES/PROTEINS FROM DUNG BEETLE, ONTHOPHAGUS TAURUS IMMUNE HEMOLYMPH

    Directory of Open Access Journals (Sweden)

    Vasanth Patil H.B

    2013-06-01

    Full Text Available Antimicrobial peptides are important in the first line of the host defense system of all insect species. In the present study antimicrobial peptide(s were isolated from the hemolymph of the dung beetle Onthophagus taurus. Both non induced and immune induced hemolymphs were tested for their antimicrobial activity against different bacterial strains and C. albicans. Induction was done by injecting E. coli into the abdominal cavity of the O. taurus. The non induced hemolymph did not show activity against any of the tested fungal and bacterial strains where as induced hemolymph showed activity against all tested bacterial strains but no activity against C. albicans. The induced hemolymph was subjected to non reducing SDS-PAGE and UV wavelength scan was performed to detect the presence of peptides. The immune induced hemolymph was purified by gel filtration chromatography to separate the proteins responsible for the antibacterial activity. The fractions within the peak were tested against those bacteria which previously showed sensitivity to the crude immune induced hemolymph. All fractions were found to be active against all tested bacteria with difference in zone of inhibition. The peptides are active against prokaryotes & not against eukaryotes. These properties reveal its unique characteristics and therapeutic application.

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

    Science.gov (United States)

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

    2015-01-01

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

  16. Mycobacterium tuberculosis co-operonic PE32/PPE65 proteins alter host immune responses by hampering Th1 response

    Directory of Open Access Journals (Sweden)

    Mohd eKhubaib

    2016-05-01

    Full Text Available PE/PPE genes, present in cluster with ESAT-6 like genes, are suspected to have a role in antigenic variation and virulence of Mycobacterium tuberculosis. Their roles in immune evasion and immune modulation of host are also well documented. We present evidence that PE32/PPE65 present within the RD8 region are co-operonic, co-transcribed and co-translated, and play role in modulating host immune responses. Experiments with macrophage cell lines revealed that this protein complex suppresses pro-inflammatory cytokines such as TNF-α and IL-6 whereas also inducing high expression of anti-inflammatory IL-10. Immunization of mice with these recombinant proteins dampens an effective Th1 response as evident from reduced frequency of IFN-g and IL-2 producing CD4+ and CD8+ T cells. IgG sub-typing from serum of immunized mice revealed high levels of IgG1 when compared with IgG2a and IgG2b. Further IgG1/IgG2a ratio clearly demonstrated that the protein complex manipulates the host immune response favourable to the pathogen. Our results demonstrate that the co-transcribed and co-translated PE32 and PPE65 antigens are involved specifically in modulating anti-mycobacterial host immune response by hampering Th1 response.

  17. BtpB, a novel Brucella TIR-containing effector protein with immune modulatory functions.

    Science.gov (United States)

    Salcedo, Suzana P; Marchesini, María I; Degos, Clara; Terwagne, Matthieu; Von Bargen, Kristine; Lepidi, Hubert; Herrmann, Claudia K; Santos Lacerda, Thais L; Imbert, Paul R C; Pierre, Philippe; Alexopoulou, Lena; Letesson, Jean-Jacques; Comerci, Diego J; Gorvel, Jean-Pierre

    2013-01-01

    Several bacterial pathogens have TIR domain-containing proteins that contribute to their pathogenesis. We identified a second TIR-containing protein in Brucella spp. that we have designated BtpB. We show it is a potent inhibitor of TLR signaling, probably via MyD88. BtpB is a novel Brucella effector that is translocated into host cells and interferes with activation of dendritic cells. In vivo mouse studies revealed that BtpB is contributing to virulence and control of local inflammatory responses with relevance in the establishment of chronic brucellosis. Together, our results show that BtpB is a novel Brucella effector that plays a major role in the modulation of host innate immune response during infection.

  18. An improved and robust DNA immunization method to develop antibodies against extracellular loops of multi-transmembrane proteins.

    Science.gov (United States)

    Hazen, Meredith; Bhakta, Sunil; Vij, Rajesh; Randle, Steven; Kallop, Dara; Chiang, Vicki; Hötzel, Isidro; Jaiswal, Bijay S; Ervin, Karen E; Li, Bing; Weimer, Robby M; Polakis, Paul; Scheller, Richard H; Junutula, Jagath R; Hongo, Jo-Anne S

    2014-01-01

    Multi-transmembrane proteins are especially difficult targets for antibody generation largely due to the challenge of producing a protein that maintains its native conformation in the absence of a stabilizing membrane. Here, we describe an immunization strategy that successfully resulted in the identification of monoclonal antibodies that bind specifically to extracellular epitopes of a 12 transmembrane protein, multi-drug resistant protein 4 (MRP4). These monoclonal antibodies were developed following hydrodynamic tail vein immunization with a cytomegalovirus (CMV) promoter-based plasmid expressing MRP4 cDNA and were characterized by flow cytometry. As expected, the use of the immune modulators fetal liver tyrosine kinase 3 ligand (Flt3L) and granulocyte-macrophage colony-stimulating factor positively enhanced the immune response against MRP4. Imaging studies using CMV-based plasmids expressing luciferase showed that the in vivo half-life of the target antigen was less than 48 h using CMV-based plasmids, thus necessitating frequent boosting with DNA to achieve an adequate immune response. We also describe a comparison of plasmids, which contained MRP4 cDNA with either the CMV or CAG promoters, used for immunizations. The observed luciferase activity in this comparison demonstrated that the CAG promoter-containing plasmid pCAGGS induced prolonged constitutive expression of MRP4 and an increased anti-MRP4 specific immune response even when the plasmid was injected less frequently. The method described here is one that can be broadly applicable as a general immunization strategy to develop antibodies against multi-transmembrane proteins, as well as target antigens that are difficult to express or purify in native and functionally active conformation.

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

    Science.gov (United States)

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

    2015-03-01

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

  20. Evolutionary origin of peptidoglycan recognition proteins in vertebrate innate immune system

    Directory of Open Access Journals (Sweden)

    Tsujino Fumi

    2011-03-01

    Full Text Available Abstract Background Innate immunity is the ancient defense system of multicellular organisms against microbial infection. The basis of this first line of defense resides in the recognition of unique motifs conserved in microorganisms, and absent in the host. Peptidoglycans, structural components of bacterial cell walls, are recognized by Peptidoglycan Recognition Proteins (PGRPs. PGRPs are present in both vertebrates and invertebrates. Although some evidence for similarities and differences in function and structure between them has been found, their evolutionary history and phylogenetic relationship have remained unclear. Such studies have been severely hampered by the great extent of sequence divergence among vertebrate and invertebrate PGRPs. Here we investigate the birth and death processes of PGRPs to elucidate their origin and diversity. Results We found that (i four rounds of gene duplication and a single domain duplication have generated the major variety of present vertebrate PGRPs, while in invertebrates more than ten times the number of duplications are required to explain the repertoire of present PGRPs, and (ii the death of genes in vertebrates appears to be almost null whereas in invertebrates it is frequent. Conclusion These results suggest that the emergence of new PGRP genes may have an impact on the availability of the repertoire and its function against pathogens. These striking differences in PGRP evolution of vertebrates and invertebrates should reflect the differences in the role of their innate immunity. Insights on the origin of PGRP genes will pave the way to understand the evolution of the interaction between host and pathogens and to lead to the development of new treatments for immune diseases that involve proteins related to the recognition of self and non-self.

  1. The adaptor protein FHL2 enhances the cellular innate immune response to influenza A virus infection.

    Science.gov (United States)

    Nordhoff, Carolin; Hillesheim, Andrea; Walter, Beate M; Haasbach, Emanuel; Planz, Oliver; Ehrhardt, Christina; Ludwig, Stephan; Wixler, Viktor

    2012-07-01

    The innate immune response of influenza A virus-infected cells is predominantly mediated by type I interferon-induced proteins. Expression of the interferon β (IFNβ) itself is initiated by accumulating viral RNA and is transmitted by different signalling cascades that feed into activation of the three transcriptional elements located in the IFNβ promoter, AP-1, IRF-3 and NF-κB. FHL2 (four-and-a-half LIM domain protein 2) is an adaptor molecule that shuttles between membrane and nucleus regulating signalling cascades and gene transcription. Here we describe FHL2 as a novel regulator of influenza A virus propagation. Using mouse FHL2 wild-type, knockout and rescued cells and human epithelial cells with different expression levels of FHL2 we showed that FHL2 decreases influenza A virus propagation by regulating the intrinsic cellular antiviral immune response. On virus infection FHL2 translocates into the nucleus, potentiating the IRF-3-dependent transcription of the IFNβ gene.

  2. Ternary WD40 repeat-containing protein complexes: evolution, composition and roles in plant immunity

    Directory of Open Access Journals (Sweden)

    Jimi C. Miller

    2016-01-01

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

  3. Protection of mice against Trypanosoma cruzi by immunization with paraflagellar rod proteins requires T cell, but not B cell, function.

    Science.gov (United States)

    Miller, M J; Wrightsman, R A; Stryker, G A; Manning, J E

    1997-06-01

    Previous studies have shown that immunization of mice with the paraflagellar rod proteins (PAR) of Trypanosoma cruzi induces an immune response capable of protecting mice against an otherwise lethal challenge with this parasite. Herein, we define immunologic responses that do or do not play a critical role in PAR-mediated protection. Firstly, PAR-immunized Ab-deficient (muMT) strain mice survived an otherwise lethal T. cruzi challenge, indicating that a B cell response is not required for PAR-induced immunity. However, beta2m -/- mice, which are severely deficient in MHC class I and TCR alphabeta+ CD8+ CD4- T cells, did not survive challenge infection following PAR immunization, indicating that MHC class I/CD8+ T cell function is necessary for protection induced by PAR immunization. Surprisingly, PAR-immunized mice depleted of CD4+ T cells survived a T. cruzi challenge for >84 days postinfection while maintaining a parasitemia that is generally thought to be lethal (i.e., >10(6) trypomastigotes/ml), thus associating CD4+ T cell function with the process of parasite clearance. Consistent with this association, CD4+ T cells from PAR-immunized mice released INF-gamma and stimulated T. cruzi-infected macrophages to release nitric oxide. The importance of IFN-gamma in PAR-induced protective immunity is further indicated by the observation that PAR-immunized INF-gamma knockout mice developed an extremely high parasitemia and did not survive a challenge infection. Thus, while Ab-mediated immune mechanisms are not required for protection induced by PAR immunization, T cell responses are necessary for both elimination of bloodstream parasites and survival.

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

    Science.gov (United States)

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

    2011-07-01

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

  5. Antitumor immunity induced by DNA vaccine encoding alpha-fetoprotein/heat shock protein 70

    Institute of Scientific and Technical Information of China (English)

    Xiao-Ping Wang; Guo-Zhen Liu; Ai-Li Song; Hai-Yan Li; Yu Liu

    2004-01-01

    AIM: To construct a DNA vaccine encoding human alphafetoprotein (hAFP)/heat shock protein 70 (HSP70), and to study its ability to induce specific CTL response and its protective effect against AFP-expressing tumor.METHODS: A DNA vaccine was constructed by combining hAFP gene with HSP70 gene. SP2/0 cells were stably transfected with pBBS212-hAFP and pBBS212-hAFP/HSP70eukaryotic expression vectors. Mice were primed and boosted with DNA vaccine hAFP/HSP70 by intramuscular injection, whereas plasmid with hAFP or HSP70 was used as controls. ELISPOT and ELISA were used to detect IFN-γ-producing splenocytes and the level of serum anti-AFP antibody from immunized mice respectively. In vivo tumor challenge was measured to assess the immune effect of the DNA vaccine.RESULTS: By DNA vaccine immunization, the results of ELISPOT and ELISA showed that the number of IFN-γ-producing splenocytes and the level of serum anti-AFP antibody were significantly higher in rhAFP/HSP70 group than in hAFP and empty plasmid groups (95.50±10.90IFN-γ spots/106 cells vs 23.60±11.80 IFN-γ spots/106 cells,7.17±4.24 IFN-γ spots/106 cells, P<0.01; 126.50±8.22 μg/mL vs 51.72±3.40 μg/mL, 5.83±3.79 μg/mL, P<0.01). The tumor volume in rhAFP/HSP70 group was significantly smaller than that in pBBS212-hAFP and empty plasmid groups (37.41±7.34 mm3 vs381.13±15.48 mm3, 817.51±16.25 mm3,P<0.01).CONCLUSION: Sequential immunization with a recombinant DNA vaccine encoding AFP and heat shock protein70 could generate effective AFP-specific T cell responses and induce definite antitumor effects on AFP-producing tumors, which may be suitable for some clinical testing as a vaccine for HCC.

  6. The human metapneumovirus matrix protein stimulates the inflammatory immune response in vitro.

    Directory of Open Access Journals (Sweden)

    Audrey Bagnaud-Baule

    Full Text Available Each year, during winter months, human Metapneumovirus (hMPV is associated with epidemics of bronchiolitis resulting in the hospitalization of many infants. Bronchiolitis is an acute illness of the lower respiratory tract with a consequent inflammation of the bronchioles. The rapid onset of inflammation suggests the innate immune response may have a role to play in the pathogenesis of this hMPV infection. Since, the matrix protein is one of the most abundant proteins in the Paramyxoviridae family virion, we hypothesized that the inflammatory modulation observed in hMPV infected patients may be partly associated with the matrix protein (M-hMPV response. By western blot analysis, we detected a soluble form of M-hMPV released from hMPV infected cell as well as from M-hMPV transfected HEK 293T cells suggesting that M-hMPV may be directly in contact with antigen presenting cells (APCs during the course of infection. Moreover, flow cytometry and confocal microscopy allowed determining that M-hMPV was taken up by dendritic cells (moDCs and macrophages inducing their activation. Furthermore, these moDCs enter into a maturation process inducing the secretion of a broad range of inflammatory cytokines when exposed to M-hMPV. Additionally, M-hMPV activated DCs were shown to stimulate IL-2 and IFN-γ production by allogeneic T lymphocytes. This M-hMPV-mediated activation and antigen presentation of APCs may in part explain the marked inflammatory immune response observed in pathology induced by hMPV in patients.

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

    Science.gov (United States)

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

    2015-02-01

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

  8. Structures of the ultra-high-affinity protein–protein complexes of pyocins S2 and AP41 and their cognate immunity proteins from pseudomonas aeruginosa

    OpenAIRE

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

    2015-01-01

    How ultra-high-affinity protein–protein interactions retain high specificity is still poorly understood. The interaction between colicin DNase domains and their inhibitory immunity (Im) proteins is an ultra-high-affinity interaction that is essential for the neutralisation of endogenous DNase catalytic activity and for protection against exogenous DNase bacteriocins. The colicin DNase–Im interaction is a model system for the study of high-affinity protein–protein interactions. However, despit...

  9. Major basic protein from eosinophils and myeloperoxidase from neutrophils are required for protective immunity to Strongyloides stercoralis in mice.

    Science.gov (United States)

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

    2011-07-01

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

  10. Salicylic acid-dependent and -independent impact of an RNA-binding protein on plant immunity.

    Science.gov (United States)

    Hackmann, Christian; Korneli, Christin; Kutyniok, Magdalene; Köster, Tino; Wiedenlübbert, Matthias; Müller, Caroline; Staiger, Dorothee

    2014-03-01

    Plants overexpressing the RNA-binding protein AtGRP7 (AtGRP7-ox plants) constitutively express the PR-1 (PATHOGENESIS-RELATED-1), PR-2 and PR-5 transcripts associated with salicylic acid (SA)-mediated immunity and show enhanced resistance against Pseudomonas syringae pv. tomato (Pto) DC3000. Here, we investigated whether the function of AtGRP7 in plant immunity depends on SA. Endogenous SA was elevated fivefold in AtGRP7-ox plants. The elevated PR-1, PR-2 and PR-5 levels were eliminated upon expression of the salicylate hydroxylase nahG in AtGRP7-ox plants and elevated PR-1 levels were suppressed by sid (salicylic acid deficient) 2-1 that is impaired in SA biosynthesis. RNA immunoprecipitation showed that AtGRP7 does not bind the PR-1 transcript in vivo, whereas it binds PDF1.2. Constitutive or inducible AtGRP7 overexpression increases PR-1 promoter activity, indicating that AtGRP7 affects PR-1 transcription. In line with this, the effect of AtGRP7 on PR-1 is suppressed by npr (non-expressor of PR genes) 1. Whereas AtGRP7-ox plants restricted growth of Pto DC3000 compared with wild type (wt), sid2-1 AtGRP7-ox plants allowed more growth than AtGRP7-ox plants. Furthermore, we show an enhanced hypersensitive response triggered by avirulent Pto DC3000 (AvrRpt2) in AtGRP7-ox compared with wt. In sid2-1 AtGRP7-ox, an intermediate phenotype was observed. Thus, AtGRP7 has both SA-dependent and SA-independent effects on plant immunity.

  11. Immune response of broiler chickens immunized orally with the recombinant proteins flagellin and the subunit B of cholera toxin associated with Lactobacillus spp.

    Science.gov (United States)

    Baptista, A A S; Donato, T C; Garcia, K C O D; Gonçalves, G A M; Coppola, M P; Okamoto, A S; Sequeira, J L; Andreatti Filho, R L

    2014-01-01

    This study investigated the immune response of broiler chickens with oral treatment of a Lactobacillus spp. pool (PL) associated with microencapsulated recombinant proteins flagellin (FliC) and the subunit B of cholera toxin (CTB). Immune responses were evaluated by measuring IgA from intestinal fluid, serum IgY, and immunostaining of CD8(+) T lymphocytes present in the cecum. The evaluations were performed on d 0, 7, 14, 21, and 28 posttreatment. A significant increase (P < 0.05) was observed in IgA levels in all immunized groups, especially 3 wk after immunization. Treatments 2 (recombinant CTB) and 3 (recombinant FliC+CTB) showed the highest concentrations. Similarly, serum concentrations IgY (μg/mL) increased along the experiment, and the means for treatments 2 and 3 showed significant differences (P < 0.05) compared with controls, reaching concentrations of 533 and 540 μg/mL, respectively. The number of CD8(+) T lymphocytes in all treatments greatly differed (P < 0.05) compared with the negative control at 21 d posttreatment. However, only treatment 2 (recombinant CTB), 4 (PL), and 5 (recombinant FliC+ recombinant CTB + PL) remained significantly (P < 0.05) different from the control at 28 d posttreatment. Thus, it is concluded that the microencapsulated recombinant proteins administered orally to broiler chickens are capable of stimulating humoral and cellular immune response, and the combinations of these antigens with Lactobacillus spp. can influence the population of CD8(+) T cells residing in the cecum.

  12. Trypanosoma cruzi: protective immunity in mice immunized with paraflagellar rod proteins is associated with a T-helper type 1 response.

    Science.gov (United States)

    Miller, M J; Wrightsman, R A; Manning, J E

    1996-11-01

    We have examined the ability of mice to survive a lethal challenge with the parasitic hemoflagellate, Trypanosoma cruzi, following immunization with paraflagellar rod proteins (PAR) 1 and 2 either alone or in combination with the following adjuvants: Freund's, alum, QS-21, Ribi-700, or IL-12. PAR administered subcutaneously (sc) in combination with Freund's or alum provided significant protection, 100 and 83%, respectively, against a T. cruzi challenge. In contrast, PAR in combination with QS-21, Ribi-700, IL-12, or Freund's administered intraperitoneally (ip) or PAR alone provide no protection against a challenge. PAR-specific serum antibody titers and isotype profiles for several of the immunization regimens were determined, and no positive correlation could be seen between a protective immune response and either antibody titer or the subclass of antibody induced. We also examined the ability of PAR to stimulate T cells from the spleen and lymph nodes of mice immunized with PAR in combination with Freund's (sc), Freund's (ip), alum, or Ribi-700. Each of the adjuvants strongly enhanced the ability of enriched T cells to proliferate in a PAR-specific fashion, suggesting no obvious correlation between PAR-specific T cell activation and protection. However, examination of the cytokine profiles of the stimulated T cell groups showed that the protective groups differed from the nonprotective groups. While all four groups showed low levels of IL-10, the Freund's (sc) and alum groups had higher levels of IFN-gamma and IL-2 than Freund's (ip) and Ribi-700 groups, and most strikingly, no IL-4 could be detected in either the Freund's (sc) or the alum group, in contrast to significant levels of IL-4 in both the Freund's (ip) and the Ribi-700 group. These findings indicate that protective immunity in mice immunized with PAR is associated with a Th1-type response.

  13. Dengue virus targets the adaptor protein MITA to subvert host innate immunity.

    Directory of Open Access Journals (Sweden)

    Chia-Yi Yu

    Full Text Available Dengue is one of the most important arboviral diseases caused by infection of four serotypes of dengue virus (DEN. We found that activation of interferon regulatory factor 3 (IRF3 triggered by viral infection and by foreign DNA and RNA stimulation was blocked by DEN-encoded NS2B3 through a protease-dependent mechanism. The key adaptor protein in type I interferon pathway, human mediator of IRF3 activation (MITA but not the murine homologue MPYS, was cleaved in cells infected with DEN-1 or DEN-2 and with expression of the enzymatically active protease NS2B3. The cleavage site of MITA was mapped to LRR↓(96G and the function of MITA was suppressed by dengue protease. DEN replication was reduced with overexpression of MPYS but not with MITA, while DEN replication was enhanced by MPYS knockdown, indicating an antiviral role of MITA/MPYS against DEN infection. The involvement of MITA in DEN-triggered innate immune response was evidenced by reduction of IRF3 activation and IFN induction in cells with MITA knockdown upon DEN-2 infection. NS2B3 physically interacted with MITA, and the interaction and cleavage of MITA could be further enhanced by poly(dA:dT stimulation. Thus, we identified MITA as a novel host target of DEN protease and provide the molecular mechanism of how DEN subverts the host innate immunity.

  14. Activation of unfolded protein response and autophagy during HCV infection modulates innate immune response.

    Science.gov (United States)

    Estrabaud, Emilie; De Muynck, Simon; Asselah, Tarik

    2011-11-01

    Autophagy, a process for catabolizing cytoplasmic components, has been implicated in the modulation of interactions between RNA viruses and their host. However, the mechanism underlying the functional role of autophagy in the viral life cycle still remains unclear. Hepatitis C virus (HCV) is a single-stranded, positive-sense, membrane-enveloped RNA virus that can cause chronic liver disease. Here we report that HCV induces the unfolded protein response (UPR), which in turn activates the autophagic pathway to promote HCV RNA replication in human hepatoma cells. Further analysis revealed that the entire autophagic process through to complete autolysosome maturation was required to promote HCV RNA replication and that it did so by suppressing innate antiviral immunity. Gene silencing or activation of the UPR-autophagy pathway activated or repressed, respectively, IFN-β activation mediated by an HCV-derived pathogen-associated molecular pattern (PAMP). Similar results were achieved with a PAMP derived from Dengue virus (DEV), indicating that HCV and DEV may both exploit the UPR-autophagy pathway to escape the innate immune response. Taken together, these results not only define the physiological significance of HCV-induced autophagy, but also shed light on the knowledge of host cellular responses upon HCV infection as well as on exploration of therapeutic targets for controlling HCV infection.

  15. Dengue virus targets the adaptor protein MITA to subvert host innate immunity.

    Science.gov (United States)

    Yu, Chia-Yi; Chang, Tsung-Hsien; Liang, Jian-Jong; Chiang, Ruei-Lin; Lee, Yi-Ling; Liao, Ching-Len; Lin, Yi-Ling

    2012-01-01

    Dengue is one of the most important arboviral diseases caused by infection of four serotypes of dengue virus (DEN). We found that activation of interferon regulatory factor 3 (IRF3) triggered by viral infection and by foreign DNA and RNA stimulation was blocked by DEN-encoded NS2B3 through a protease-dependent mechanism. The key adaptor protein in type I interferon pathway, human mediator of IRF3 activation (MITA) but not the murine homologue MPYS, was cleaved in cells infected with DEN-1 or DEN-2 and with expression of the enzymatically active protease NS2B3. The cleavage site of MITA was mapped to LRR↓(96)G and the function of MITA was suppressed by dengue protease. DEN replication was reduced with overexpression of MPYS but not with MITA, while DEN replication was enhanced by MPYS knockdown, indicating an antiviral role of MITA/MPYS against DEN infection. The involvement of MITA in DEN-triggered innate immune response was evidenced by reduction of IRF3 activation and IFN induction in cells with MITA knockdown upon DEN-2 infection. NS2B3 physically interacted with MITA, and the interaction and cleavage of MITA could be further enhanced by poly(dA:dT) stimulation. Thus, we identified MITA as a novel host target of DEN protease and provide the molecular mechanism of how DEN subverts the host innate immunity.

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

    Science.gov (United States)

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

    2014-06-01

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

  17. Evasion of antiviral innate immunity by Theiler's virus L* protein through direct inhibition of RNase L.

    Directory of Open Access Journals (Sweden)

    Frédéric Sorgeloos

    Full Text Available Theiler's virus is a neurotropic picornavirus responsible for chronic infections of the central nervous system. The establishment of a persistent infection and the subsequent demyelinating disease triggered by the virus depend on the expression of L*, a viral accessory protein encoded by an alternative open reading frame of the virus. We discovered that L* potently inhibits the interferon-inducible OAS/RNase L pathway. The antagonism of RNase L by L* was particularly prominent in macrophages where baseline oligoadenylate synthetase (OAS and RNase L expression levels are elevated, but was detectable in fibroblasts after IFN pretreatment. L* mutations significantly affected Theiler's virus replication in primary macrophages derived from wild-type but not from RNase L-deficient mice. L* counteracted the OAS/RNase L pathway through direct interaction with the ankyrin domain of RNase L, resulting in the inhibition of this enzyme. Interestingly, RNase L inhibition was species-specific as Theiler's virus L* protein blocked murine RNase L but not human RNase L or RNase L of other mammals or birds. Direct RNase L inhibition by L* and species specificity were confirmed in an in vitro assay performed with purified proteins. These results demonstrate a novel viral mechanism to elude the antiviral OAS/RNase L pathway. By targeting the effector enzyme of this antiviral pathway, L* potently inhibits RNase L, underscoring the importance of this enzyme in innate immunity against Theiler's virus.

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

    Science.gov (United States)

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

    2009-08-01

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

  19. Microecology and local immune and nonspecific defensive proteins depending on different nutrition.

    Science.gov (United States)

    Kuvaeva, I B; Orlova, N G; Borovik, T E; Veselova, O L

    1987-01-01

    Breast-feeding is of high importance for the development of intestinal eubiosis. Before beginning with breast-feeding the coprofiltrates of newborns lack of IgA. Following the first feeding IgA concentration in the faeces sharply increases (up to 200 mg/100 g faeces). Comparable high values can be found in the coprofiltrates of breast-fed sick prematures. In the coprofiltrates of artificially fed healthy newborns and sick prematures no IgA is provable, within the first two weeks of age. Afterwards both the frequency of its evidence and its concentration gradually rise. This can be regarded as a sign of an increasing local production of immune proteins. Starting with the second year of life, only, the values of all the immunoglobulins fall again. It happens a microbial degradation. Increased concentrations of immunoglobulins in the coprofiltrates of children over 3 years must be evaluated as a sign of subclinical dysbacteriosis.

  20. Induced prion protein controls immune-activated retroviruses in the mouse spleen.

    Directory of Open Access Journals (Sweden)

    Marius Lötscher

    Full Text Available The prion protein (PrP is crucially involved in transmissible spongiform encephalopathies (TSE, but neither its exact role in disease nor its physiological function are known. Here we show for mice, using histological, immunochemical and PCR-based methods, that stimulation of innate resistance was followed by appearance of numerous endogenous retroviruses and ensuing PrP up-regulation in germinal centers of the spleen. Subsequently, the activated retroviruses disappeared in a PrP-dependent manner. Our results reveal the regular involvement of endogenous retroviruses in murine immune responses and provide evidence for an essential function of PrP in the control of the retroviral activity. The interaction between PrP and ubiquitous endogenous retroviruses may allow new interpretations of TSE pathophysiology and explain the evolutionary conservation of PrP.

  1. Vitamin A supplementation reduces the monocyte chemoattractant protein-1 intestinal immune response of Mexican children.

    Science.gov (United States)

    Long, Kurt Z; Santos, Jose Ignacio; Estrada Garcia, Teresa; Haas, Meredith; Firestone, Mathew; Bhagwat, Jui; Dupont, Herbert L; Hertzmark, Ellen; Rosado, Jorge L; Nanthakumar, Nanda N

    2006-10-01

    The impact of vitamin A supplementation on childhood diarrhea may be determined by the regulatory effect supplementation has on the mucosal immune response in the gut. Previous studies have not addressed the impact of vitamin A supplementation on the production of monocyte chemoattractant protein 1 (MCP-1), an essential chemokine involved in pathogen-specific mucosal immune response. Fecal MCP-1 concentrations, determined by an enzyme-linked immuno absorption assay, were compared among 127 Mexican children 5-15 mo of age randomized to receive a vitamin A supplement ( or =12 mo, 45,000 iu) every 2 mo or a placebo as part of a larger vitamin A supplementation trial. Stools collected during the summer months were screened for MCP-1 and gastrointestinal pathogens. Values of MCP-1 were categorized into 3 levels (nondetectable, or =median). Multinomial logistic regression models were used to determine whether vitamin A-supplemented children had different categorical values of MCP-1 compared with children in the placebo group. Differences in categorical values were also analyzed stratified by gastrointestinal pathogen infections and by diarrheal symptoms. Overall, children who received the vitamin A supplement had reduced fecal concentrations of MCP-1 compared with children in the placebo group (median pg/mg protein +/- interquartile range: 284.88 +/- 885.35 vs. 403.39 +/- 913.16; odds ratio 0.64, 95% CI 0.42-97, P = 0.03). Vitamin A supplemented children infected with enteropathogenic Escherichia coli (EPEC) had reduced MCP-1 levels (odds ratio = 0.38, 95% CI 0.18-0.80) compared with children in the placebo group. Among children not infected with Ascaris lumbricoides vitamin A supplemented children had reduced MCP-1 levels (OR = 0.62, 95% CI 0.41-0.94). These findings suggest that vitamin A has an anti-inflammatory effect in the gastrointestinal tract by reducing MCP-1 concentrations.

  2. Neuroblastoma after Childhood: Prognostic Relevance of Segmental Chromosome Aberrations, ATRX Protein Status, and Immune Cell Infiltration

    Directory of Open Access Journals (Sweden)

    Ana P. Berbegall

    2014-06-01

    Full Text Available Neuroblastoma (NB is a common malignancy in children but rarely occurs during adolescence or adulthood. This subgroup is characterized by an indolent disease course, almost uniformly fatal, yet little is known about the biologic characteristics. The aim of this study was to identify differential features regarding DNA copy number alterations, α-thalassemia/mental retardation syndrome X-linked (ATRX protein expression, and the presence of tumor-associated inflammatory cells. Thirty-one NB patients older than 10 years who were included in the Spanish NB Registry were considered for the current study; seven young and middle-aged adult patients (range 18-60 years formed part of the cohort. We performed single nucleotide polymorphism arrays, immunohistochemistry for immune markers (CD4, CD8, CD20, CD11b, CD11c, and CD68, and ATRX protein expression. Assorted genetic profiles were found with a predominant presence of a segmental chromosome aberration (SCA profile. Preadolescent and adolescent NB tumors showed a higher number of SCA, including 17q gain and 11q deletion. There was also a marked infiltration of immune cells, mainly high and heterogeneous, in young and middle-aged adult tumors. ATRX negative expression was present in the tumors. The characteristics of preadolescent, adolescent, young adult, and middle-aged adult NB tumors are different, not only from childhood NB tumors but also from each other. Similar examinations of a larger number of such tumor tissues from cooperative groups should lead to a better older age–dependent tumor pattern and to innovative, individual risk-adapted therapeutic approaches for these patients.

  3. Purification of heat shock protein 70-associated tumor peptides and their antitumor immunity to hepatoma in mice

    Institute of Scientific and Technical Information of China (English)

    Dai-Xiong Chen; Yan-Rong Su; Gen-Ze Shao; Zhen-Chao Qian

    2004-01-01

    AIM: To purify the heat shock protein (HSP) 70-associated tumor peptides and to observe its non-MHC-I molecule restrictive antitumor effect.METHODS: By ConA-sepharose affinity chromatography,ADP-agarose affinity chromatography, and DEAE anion exchange chromatography, we were able to purify HSP70-associated peptides from mouse hepatoma (HCaF) cells treated in heat shock at 42 ℃ . Specific active immunization and adoptive cellular immunization assay were adopted to observe the immunoprotective effect elicited by HSP70-associated peptide complexes isolated from HcaF.RESULTS: The finally purified HSP-associated peptides had a very high purity and specificity found by SDS-PAGE and Western blot. Mice immunized with HSP70-associated peptide complexes purified from HCaF cells were protected from HCaF living cell challenge. This effect was dose dependent.Adoptive immunization of immune spleen cells of mice immunized with HSP70-associated peptide complexes could elicit immunity against HCaF challenge, and the tumor-free mice could resist repeated challenges. This effect could be continuously enhanced by repeated challenge with HCaF living cells. The tumor-free mice could tolerate the challenge for as high as l×107 HCaF cells. The mice immunized once with spleen cells pulsed with HSP70-associated peptide complexes in vitro could also result in a certain adoptive immunity against HCaF.CONCLUSION: High purity and specificity of HSP70-associated peptides could be achieved from tumor cells by the low-pressure affinity chromatography method used in this study. HSP70-associated peptide complexes derived from the HCaF can elicit non-MHC-I molecule restrictive immunoprotective effect against HCaF. This effect can be transferred by adoptive immunization to mice and enhanced by repeated challenge with HCaF live cells.

  4. Induction of Boosted Immune Response in Mice by Leptospiral Surface Proteins Expressed in Fusion with DnaK

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    Marina V. Atzingen

    2014-01-01

    Full Text Available Leptospirosis is an important global disease of human and veterinary concern. Caused by pathogenic Leptospira, the illness was recently classified as an emerging infectious disease. Currently available veterinarian vaccines do not induce long-term protection against infection and do not provide cross-protective immunity. Several studies have suggested the use of DnaK as an antigen in vaccine formulation, due to an exceptional degree of immunogenicity. We focused on four surface proteins: rLIC10368 (Lsa21, rLIC10494, rLIC12690 (Lp95, and rLIC12730, previously shown to be involved in host-pathogen interactions. Our goal was to evaluate the immunogenicity of the proteins genetically fused with DnaK in animal model. The chosen genes were amplified by PCR methodology and cloned into pAE, an E. coli vector. The recombinant proteins were expressed alone or in fusion with DnaK at the N-terminus. Our results demonstrate that leptospiral proteins fused with DnaK have elicited an enhanced immune response in mice when compared to the effect promoted by the individual proteins. The boosted immune effect was demonstrated by the production of total IgG, lymphocyte proliferation, and significant amounts of IL-10 in supernatant of splenocyte cell cultures. We believe that this approach could be employed in vaccines to enhance presentation of antigens of Leptospira to professional immune cells.

  5. Paraflagellar rod proteins administered with alum and IL-12 or recombinant adenovirus expressing IL-12 generates antigen-specific responses and protective immunity in mice against Trypanosoma cruzi.

    Science.gov (United States)

    Wrightsman, R A; Manning, J E

    2000-01-31

    Successful vaccination of mice against an otherwise lethal challenge with the Peru strain of Trypanosoma cruzi necessitates the induction of a strong cell mediated immune response. Previously, immunization of mice with the paraflagellar rod proteins from Trypanosoma cruzi90% reduction in parasitemia in immunized mice challenged with the bloodstream stage of Trypanosoma cruzi.

  6. Reliability of the nanopheres-DNA immunization technology to produce polyclonal antibodies directed against human neogenic proteins.

    Science.gov (United States)

    Arnaoty, Ahmed; Gouilleux-Gruart, Valérie; Casteret, Sophie; Pitard, Bruno; Bigot, Yves; Lecomte, Thierry

    2013-08-01

    The molecular domestication of several DNA transposons that occurred during the evolution of the mammalian lineage, has led to the emergence of at least 43 genes, known as neogenes. To date, the limited availability of efficient commercial antibodies directed against most of their protein isoforms hampers investigation of their expression in vitro and in situ. Since immunization protocols using peptides or recombinant proteins have revealed that it is difficult to recover antibodies, we planned to produce antisera in mice using a new technique of nanopheres/DNA immunization, the ICANtibodies™ technology. Here, we investigate the possibilities of obtaining polyclonal antibodies for 24 proteins or protein domains using this immunization strategy. We successfully obtained 13 antisera that were able to detect neogenic proteins by Western blotting and ELISA in protein extracts of transiently-transfected cells and various cancer cell lines, plus another two that only detected the in ELISA and in in situ hybridizations. The features required for the production of these antibodies are analyzed and discussed, and examples are given of the advantages they offer for the study of neogenic proteins.

  7. Studies on Mucosal Immunity Induced by Transmissible Gastroenteritis Virus Nucleocapsid Protein Recombinant Lactobacillus casei in Mice and Sow

    Institute of Scientific and Technical Information of China (English)

    WANG Gui-hua; HOU Xi-lin; YU Li-yun; LIU Jian-kui; WEI Chun-hua

    2009-01-01

    Mucosal immunity plays an important role in protecting pigs against transmissible gastroenteritis virus (TGEV) infection. To elicit mucosal immune response against TGEV, we developed a surface antigen display system using the poly-γ-glutamate synthetase A (pgsA) protein of Bacillus subtilis as an anchoring matrix to express recombinant fusion proteins of pgsA and nucleocapsid protein of TGEV in Lactobacillus casei. Surface location of fusion protein was verified by ELISA and indirect immunofluorescence test. Oral and intranasal inoculations of pregnant sow and mice with recombinant L. casei resulted in high levels of serum immunoglobulin G (IgG) and secretory immunogiobulin A (sIgA) against recombinant N protein as demonstrated by ELISA. More importantly, the level of specific slgA in colostrum significantly increased compared with that of IgG. The serum lgG levels of the piglets increased after suckling coiostrum produced by sows was previously inoculated with recombinant L. casei. These results indicate that immunization with recombinant L. casei expressing TGEV N protein on its surface elicited high levels of specific slgA and circulating lgG against TGEV N protein.

  8. Proteomic identification of the related immune-enhancing proteins in shrimp Litopenaeus vannamei stimulated with vitamin C and Chinese herbs.

    Science.gov (United States)

    Qiao, Jie; Du, Zhiheng; Zhang, Yueling; Du, Hong; Guo, Lingling; Zhong, Mingqi; Cao, Jingsong; Wang, Xiuying

    2011-12-01

    Recently, strong interest has been focused on immunostimulants to reducing the diseases in shrimp aquaculture. However, information regarding to the related immune-enhancing proteins in shrimps is not available yet. In this study, vitamin C (Vc), Chinese herbs (CH), and the mixture of vitamin C and Chinese herbs (Mix) were tested for their enhancement on shrimp's immune activity. Compared with those in the control group, values of phenoloxidase (PO), superoxide dismutase (SOD) and antibacterial (Ua) activity in the Mix-treated group were improved significantly 12 or 24 days after the treatment. The cumulative mortality was also lower in the Mix-treated group after infection with Vibrio parahemolyticus. Furthermore, comparative proteomic approach was used to assess the protein expression profile in shrimps. Approximately 220-290 and 300-400 protein spots were observed in the 2-DE gels. Among them, 29 and 28 altered proteins from hemocytes and hepatopancreas, respectively, were subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) analysis. The results revealed that the main altered proteins showed high homologies with Litopenaeus vannamei hemocyanin, hemolymph clottable protein, hemoglobin beta, cytosolic MnSOD, trypsin, cathepsin I(L) and zinc proteinase Mpc1. Together, these studies found Vc and CH were suitable immunostimulants to shrimp L. vannamei, and 7 altered proteins could be involved in the enhanced immune activities.

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

    Science.gov (United States)

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

    2007-01-01

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

  10. The Study on The ImmuneResponse Induced by Expressing Recombinant Plasmid of Dengue Virus Type 2 NS3 Protein

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The PSV · NS3, an expressing recombinant plasmid of dengue virus type 2 NS3 protein, was in Jected directly into the quadriceps of Balb/C mice to explore whether it could inducing immune response. The splenic T cell subsets of two groups was analysed by flow cytometry. It was found that the percentage of CD4+ and CD8+ T cells of experimental group were significantly higher than those of the control group. The titer of IgG antibody was as high as 1:S 120 in experimental group, but it couldn't be detected in control group by ELISA. The western blot further proved that the IgG antibody was specific for NS3 protein. Those results Suggested that inoculation Balb/C mice with PSV · NS3 could inducing immune response, and the NS3 protein might be used as the candidate protein of DNA vaccine of dengue virus.

  11. Regulation of plant innate immunity by three proteins in a complex conserved across the plant and animal kingdoms.

    Science.gov (United States)

    Palma, Kristoffer; Zhao, Qingguo; Cheng, Yu Ti; Bi, Dongling; Monaghan, Jacqueline; Cheng, Wei; Zhang, Yuelin; Li, Xin

    2007-06-15

    Innate immunity against pathogen infection is an evolutionarily conserved process among multicellular organisms. Arabidopsis SNC1 encodes a Resistance protein that combines attributes of multiple mammalian pattern recognition receptors. Utilizing snc1 as an autoimmune model, we identified a discrete protein complex containing at least three members--MOS4 (Modifier Of snc1, 4), AtCDC5, and PRL1 (Pleiotropic Regulatory Locus 1)--that are all essential for plant innate immunity. AtCDC5 has DNA-binding activity, suggesting that this complex probably regulates defense responses through transcriptional control. Since the complex components along with their interactions are highly conserved from fission yeast to Arabidopsis and human, they may also have a yet-to-be-identified function in mammalian innate immunity.

  12. A recombinant DNA vaccine encoding C. andersoni oocyst wall protein induces immunity against experimental C. parvum infection.

    Science.gov (United States)

    Zheng, Jun; Ren, Wenzhi; Pan, Qingshan; Wang, Qiuyue; Elhag, I A Elfaki; Li, Jianhua; Li, Mingying; Gong, Pengtao; Liu, Yingli; Zhang, Xichen

    2011-06-30

    Cryptosporidium andersoni parasited in the abomasum has been demonstrated as a cause of reduction of milk production in dairy cow. In this study, a novel chimeric DNA vaccine pVAX1-AB was constructed and the efficacy against Cryptosporidium parvum was determined. BALB/c mice were divided into 3 groups and immunized with DNA vaccine expressing the oocyst wall protein, AB protein of C. andersoni, the recombinant plasmid containing the AB gene, respectively. After inoculation of 1 × 10(6) oocysts of C. parvum, the humoral and cellular immune responses were detected. Experimental results showed that the recombinant plasmid can induce corresponding specific antibody response, simultaneously influenced cellular immune responses, and provided greater protection rate (48.6%) than the other groups. These results indicated that chimeric DNA vaccine has a potential in Cryptosporidium vaccine development.

  13. Evaluation of immune effect of recombinant fusion protein targeting the prostate stem cell antigen based on PSCA and HSP70

    Directory of Open Access Journals (Sweden)

    Lei DONG

    2014-10-01

    Full Text Available Objective To explore the immune effect and antitumor activity of recombinant prostate stem cell protein (PSCA and heat shock protein 70 (HSP70 in a murine model of prostate cancer. Methods Twenty-five healthy male C57BL/6 mice were randomly divided into 5 groups (5 each: PSCA, HSP, PSCA+HSP, PSCA-HSP and control group. Mice in the first 4 groups were vaccinated with the corresponding proteins, and those in control group were faked with injection of phosphate buffer saline (PBS. After immunization with recombinant proteins, the PSCA-specific cellular immune responses were monitored with ELISPOT, intracellular cytokine staining assay, and flow cytometry, and ELISA assay was used to detect humoral immune responses. The tumor growth and survival of vaccined mice were observed. Results ELISPOT revealed that the mice in PSCA-HSP group generated much more IFN-γ spot-forming cells than those in other groups (P<0.05, and they could generate strong anti-PSCA antibody response. Results of flow cytometry showed that the number of CD8+/IFN-γ+ T cells was significantly higher in PSCAHSP group than that in other groups (P<0.05. ELISA results revealed that all the mice in PSCA, PSCA+HSP and PSCA-HSP group were induced to generate the PSCA-specific humoral immune response, and no statistical difference was found on the antibody levels among the three groups. Animal experiment showed that PSCA-HSP could inhibit the growth of PSCA-expressing tumors and prolong the survival time of vaccinated mice. Conclusion HSP70 is a chaperone with significant effect for protein vaccines, and the recombinant fusion protein PSCA-HSP70 could be of potential value for prostate cancer treatment. DOI: 10.11855/j.issn.0577-7402.2014.09.08

  14. Identification of the major proteins of an immune modulating fraction from adult Fasciola hepatica released by Nonidet P40

    OpenAIRE

    Morphew, R.M.; Hamilton, C M; Wright, H. A.; Dowling, D. J.; O’Neill, S. M.; Brophy, P.M.

    2012-01-01

    Fasciola hepatica NP-40 released antigens (FhTeg) exhibit potent Th1 immunosuppressive properties in vitro and in vivo. However, the protein composition of this active fraction, responsible for Th1 immune modulatory activity, has yet to be resolved. Therefore, FhTeg, a Nonidet P-40 extract, was subjected to a proteomic analysis in order to identify individual protein components. This was performed using an in house F. hepatica EST database following 2D electrophoresis combined with de novo se...

  15. Recombinant thioredoxin 1 protein: the immune-adjuvant effect of Streptococcus iniae and its safety in rock bream, Oplegnathus fasciatus.

    Science.gov (United States)

    Kim, Ju-Won; Shim, Sang Hee; Lee, Jung-Ho; Jeong, Ji-Min; Park, Chan-Il

    2014-08-01

    Adjuvant is an immune enhancer commonly used during vaccination to enhance the host immune response. In the present study, we analysed the recombinant protein produced from rock bream thioredoxin 1 cDNA (rRbTRx1). To verify the immune-stimulatory effect of this recombinant protein, changes in the expression level of several genes were investigated using the cDNA microarray chips in rock bream peripheral blood leukocytes stimulated with rRbTRx1. Furthermore, the immune responses of rock bream to Streptococcus iniae FKC (formalin-killed cell) vaccination alone or in combination with recombinant proteins were analysed after an experimental challenge with living S. iniae. Microarray analysis showed that 237 unique genes were upregulated more than two-fold after rRbTRx1 stimulation. Serum agglutination titres were relatively low; however, the FKC vaccine still conferred protection against S. iniae. Moreover, the adverse effects showed no statistically significant difference between fish injected with a concentration and non-injected fish. After experimental challenge to the rock bream by injection with living bacteria (S. iniae), the relative percent survival in the vaccinated groups with FKC + rRbTRx1 was significantly higher than that of the vaccinated groups with FKC alone, which were 85.9% and 68.2%, respectively. This indicated that the recombinant protein as an adjuvant showed synergism with the injected vaccine in rock bream.

  16. Nucleocapsid-like particles of dengue-2 virus enhance the immune response against a recombinant protein of dengue-4 virus.

    Science.gov (United States)

    Lazo, Laura; Gil, Lázaro; Lopez, Carlos; Valdes, Iris; Marcos, Ernesto; Alvarez, Mayling; Blanco, Aracelys; Romero, Yaremis; Falcon, Viviana; Guzmán, María G; Guillén, Gerardo; Hermida, Lisset

    2010-10-01

    In this study, we evaluate in mice a novel formulation containing nucleocapsid-like particles of dengue-2 virus (recNLP) co-immunized with a chimeric protein composed of the dengue-4 envelope domain III fused twice within the meningococcal P64k protein of Neisseria meningitidis (PD24). The animals receiving the PD24-recNLP mixture showed the highest levels of antiviral antibodies. Similar results were obtained for IFNγ secretion levels, indicating a functional Th1 cellular response. Consistently, the percentage of mice surviving after viral challenge was significantly higher for those immunized with the mixture than for those inoculated with PD24 protein alone. In addition, in vivo depletion experiments demonstrated the decisive role of CD4(+) and CD8(+) cells in the protection conferred by immunization with PD24-recNLP. In conclusion, this report demonstrates for the first time the adjuvant capacity of dengue-2 virus recNLP. Additionally, the evidence presented highlights the potential of these particles for enhancing the immune response against heterologous recombinant proteins.

  17. Combined immunity of DNA vector and recombinant vaccinia virus expressing Gag proteins of equine infectious anemia virus

    Institute of Scientific and Technical Information of China (English)

    DAI Chunming; ZHANG Xiaoyan; WANG Shuhui; LIU Ying; DUAN Danli; SHEN Rongxian; SHAO Yiming

    2004-01-01

    In order to develop a new vaccine candidate for equine infectious anemia virus (EIAV), gag gene of Chinese donkey leukocyte attenuated strain (EIAV DLV) and its parental virulent strain (EIAV LN) were inserted respectively into the TK region of the Tiantan strain (VV) of vaccinia virus by homologous recombination and the positive clone was confirmed by blue plaque assay. Protein expression was examined by Western blot. Prime and prime-boost procedures were used to immunize mice with two DNA vectors and two recombinant vaccinia viruses expressing EIAV Gag proteins. The results showed that the specific lysis of CTL responses in the DNA+rVV groups was stronger than those in the DNA groups, amounting to 31%. Although the levels of specific antibodies were not significantly different, we could conclude that the recombinant vaccinia virus could boost the cellular responses following DNA vector priming. There was no detectable difference between the immune responses induced by DLV and LN Gag proteins. This data demonstrates that the combined immunity of DNA vector and recombinant vaccinia virus expressing EIAV gag proteins, utilizing the prime-boost procedure, can drive immunized mice to produce powerful cellular responses. These results lay an important foundation for the development of a new EIAV genetic engineering vaccine.

  18. Receptor interacting protein kinase-2 inhibition by CYLD impairs anti-bacterial immune responses in macrophages

    Directory of Open Access Journals (Sweden)

    Katharina eWex

    2016-01-01

    Full Text Available Upon infection with intracellular bacteria, nucleotide oligomerization domain protein 2 (NOD2 recognizes bacterial muramyl dipeptide and binds, subsequently, to receptor-interacting serine/threonine kinase 2 (RIPK2. RIPK2 mediates the activation of immune responses via the nuclear factor-κB (NF-κB and extracellular-signal regulated kinase (ERK pathways. Previously, it has been shown that RIPK2 activation dependens on its K63-ubiquitination by the E3 ligases pellino-3 and ITCH, whereas the deubiquitinating enzyme A20 counter-regulates RIPK2 activity by cleaving K63-polyubiquitin chains from RIPK2. Here, we newly identify the deubiquitinating enzyme CYLD as a new interacting partner and inhibitor of RIPK2. We show that CYLD binds to and removes K63-polyubiquitin chains from RIPK2 in Listeria monocytogenes (Lm infected bone-marrow-derived macrophages (BMDM. CYLD-mediated K63-deubiquitination of RIPK2 resulted in an impaired activation of both NF-κB and ERK1/2 pathways, reduced production of proinflammatory cytokines (IL-6, IL-12, anti-listerial ROS and NO, and, finally, impaired pathogen control. In turn, RIPK2 inhibition by siRNA prevented activation of NF-κB and ERK1/2 and completely abolished the protective effect of CYLD-deficiency with respect to the production of IL-6, NO, ROS and pathogen control. Noteworthy, CYLD also inhibited autophagy of Listeria in a RIPK2-ERK1/2 dependent manner.The protective function of CYLD-deficiency was dependent on IFN-γ pre-stimulation of infected macrophages. Interestingly, the reduced NF-κB activation in CYLD-expressing macrophages limited the protective effect of IFN-γ by reducing NF-κB-dependent STAT1 activation. Taken together, our study identifies CYLD as an important inhibitor of RIPK2-dependent anti-bacterial immune responses in macrophages.

  19. Immunization strategy against cervical cancer involving an alphavirus vector expressing high levels of a stable fusion protein of human papillomavirus 16 E6 and E7

    NARCIS (Netherlands)

    Daemen, T; Regts, J; Holtrop, M; Wilschut, J

    2002-01-01

    We are developing immunization strategies against cervical carcinoma and premalignant disease, based on the use of recombinant Semliki Forest virus (SFV) encoding the onco-proteins E6 and E7 from high-risk human papilloma viruses (HPV). Thus far, protein-based, as well as genetic immunization studie

  20. Serological characterization of guinea pigs infected with H3N2 human influenza or immunized with hemagglutinin protein

    Directory of Open Access Journals (Sweden)

    Bushnell Ruth V

    2010-08-01

    Full Text Available Abstract Background Recent and previous studies have shown that guinea pigs can be infected with, and transmit, human influenza viruses. Therefore guinea pig may be a useful animal model for better understanding influenza infection and assessing vaccine strategies. To more fully characterize the model, antibody responses following either infection/re-infection with human influenza A/Wyoming/03/2003 H3N2 or immunization with its homologous recombinant hemagglutinin (HA protein were studied. Results Serological samples were collected and tested for anti-HA immunoglobulin by ELISA, antiviral antibodies by hemagglutination inhibition (HI, and recognition of linear epitopes by peptide scanning (PepScan. Animals inoculated with infectious virus demonstrated pronounced viral replication and subsequent serological conversion. Animals either immunized with the homologous HA antigen or infected, showed a relatively rapid rise in antibody titers to the HA glycoprotein in ELISA assays. Antiviral antibodies, measured by HI assay, were detectable after the second inoculation. PepScan data identified both previously recognized and newly defined linear epitopes. Conclusions Infection and/or recombinant HA immunization of guinea pigs with H3N2 Wyoming influenza virus resulted in a relatively rapid production of viral-specific antibody thus demonstrating the strong immunogenicity of the major viral structural proteins in this animal model for influenza infection. The sensitivity of the immune response supports the utility of the guinea pig as a useful animal model of influenza infection and immunization.

  1. Elongation Factor Tu and Heat Shock Protein 70 Are Membrane-Associated Proteins from Mycoplasma ovipneumoniae Capable of Inducing Strong Immune Response in Mice.

    Science.gov (United States)

    Jiang, Fei; He, Jinyan; Navarro-Alvarez, Nalu; Xu, Jian; Li, Xia; Li, Peng; Wu, Wenxue

    2016-01-01

    Chronic non-progressive pneumonia, a disease that has become a worldwide epidemic has caused considerable loss to sheep industry. Mycoplasma ovipneumoniae (M. ovipneumoniae) is the causative agent of interstitial pneumonia in sheep, goat and bighorn. We here have identified by immunogold and immunoblotting that elongation factor Tu (EF-Tu) and heat shock protein 70 (HSP 70) are membrane-associated proteins on M. ovipneumonaiea. We have evaluated the humoral and cellular immune responses in vivo by immunizing BALB/c mice with both purified recombinant proteins rEF-Tu and rHSP70. The sera of both rEF-Tu and rHSP70 treated BALB/c mice demonstrated increased levels of IgG, IFN-γ, TNF-α, IL-12(p70), IL-4, IL-5 and IL-6. In addition, ELISPOT assay showed significant increase in IFN-γ+ secreting lymphocytes in the rHSP70 group when compared to other groups. Collectively our study reveals that rHSP70 induces a significantly better cellular immune response in mice, and may act as a Th1 cytokine-like adjuvant in immune response induction. Finally, growth inhibition test (GIT) of M. ovipneumoniae strain Y98 showed that sera from rHSP70 or rEF-Tu-immunized mice inhibited in vitro growth of M. ovipneumoniae. Our data strongly suggest that EF-Tu and HSP70 of M. ovipneumoniae are membrane-associated proteins capable of inducing antibody production, and cytokine secretion. Therefore, these two proteins may be potential candidates for vaccine development against M. ovipneumoniae infection in sheep.

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

    Science.gov (United States)

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

    2015-01-01

    How ultra-high-affinity protein–protein interactions retain high specificity is still poorly understood. The interaction between colicin DNase domains and their inhibitory immunity (Im) proteins is an ultra-high-affinity interaction that is essential for the neutralisation of endogenous DNase catalytic activity and for protection against exogenous DNase bacteriocins. The colicin DNase–Im interaction is a model system for the study of high-affinity protein–protein interactions. However, despite the fact that closely related colicin-like bacteriocins are widely produced by Gram-negative bacteria, this interaction has only been studied using colicins from Escherichia coli. In this work, we present the first crystal structures of two pyocin DNase–Im complexes from Pseudomonas aeruginosa, pyocin S2 DNase–ImS2 and pyocin AP41 DNase–ImAP41. These structures represent divergent DNase–Im subfamilies and are important in extending our understanding of protein–protein interactions for this important class of high-affinity protein complex. A key finding of this work is that mutations within the immunity protein binding energy hotspot, helix III, are tolerated by complementary substitutions at the DNase–Immunity protein binding interface. Im helix III is strictly conserved in colicins where an Asp forms polar interactions with the DNase backbone. ImAP41 contains an Asp-to-Gly substitution in helix III and our structures show the role of a co-evolved substitution where Pro in DNase loop 4 occupies the volume vacated and removes the unfulfilled hydrogen bond. We observe the co-evolved mutations in other DNase–Immunity pairs that appear to underpin the split of this family into two distinct groups. PMID:26215615

  3. Expression profile and differential regulation of the Human I-mfa domain-Containing protein (HIC) gene in immune cells.

    Science.gov (United States)

    Gu, Lili; Dean, Jonathan; Oliveira, André L A; Sheehy, Noreen; Hall, William W; Gautier, Virginie W

    2009-04-27

    The Human I-mfa domain-Containing protein, HIC, is a 246 amino acid protein that functions as a transcriptional regulator. Although the precise function of HIC remains to be clarified, the association of the HIC gene locus with myeloid neoplasms, its interactions with lymphotropic viruses such as EBV, HIV-1 and HTLV-1 and its expression in immune tissues suggest that HIC might have a modulatory role in immune cells. To further characterise the HIC functional relationship with the immune system, we sought to analyse the HIC gene expression profile in immune cells and to determine if immunomodulatory cytokines, such as interleukin (IL)-2, could regulate the expression of HIC mRNA. Relative quantitative real-time RT-PCR revealed that HIC mRNA is highly expressed in PBMCs and in various hematopoietic cell lines. The immunomodulatory cytokine IL-2 up-regulated HIC gene expression in PBMCs, CEM, MT-2 and U937 but markedly reduced HIC gene expression in Raji. Addition of cycloheximide indicated that the IL-2 effects were independent of de novo protein synthesis and that the HIC gene is a direct target of IL-2. Two cell lines (Jurkat and BJAB) displayed a distinct loss in HIC gene expression. However, when these cell lines were subjected to a combination of DNA methyltransferase and histone-deacetylase inhibitors, (5-aza-2-deoxycytidine and trichostatin A, respectively), HIC expression was de-repressed, indicating possible epigenetic control of HIC expression. Overall, our study describes that the immune expression of HIC is cell-specific, dynamic, and identifies the HIC gene as an IL-2 responsive gene. Furthermore, our de-repression studies support the hypothesis that HIC might represent a candidate tumor suppressor gene. Overall, this report provides new insights for a putative role of HIC in the modulation of immune and inflammatory responses and/or hematological malignancies.

  4. Immune evasion activities of accessory proteins Vpu, Nef and Vif are conserved in acute and chronic HIV-1 infection.

    Science.gov (United States)

    Mlcochova, Petra; Apolonia, Luis; Kluge, Silvia F; Sridharan, Aishwarya; Kirchhoff, Frank; Malim, Michael H; Sauter, Daniel; Gupta, Ravindra K

    2015-08-01

    Heterosexual HIV-1 transmission has been identified as a genetic bottleneck and a single transmitted/founder (T/F) variant with reduced sensitivity to type I interferon initiates productive infection in most cases. We hypothesized that particularly active accessory protein(s) may confer T/F viruses with a selective advantage in establishing HIV infection. Thus, we tested vpu, vif and nef alleles from six T/F and six chronic (CC) viruses in assays for 9 immune evasion activities involving the counteraction of interferon-stimulated genes and modulation of ligands known to activate innate immune cells. All functions were highly conserved with no significant differences between T/F and CC viruses, suggesting that these accessory protein functions are important throughout the course of infection.

  5. Functions of innate and acquired immune system are reduced in domestic pigeons (Columba livia domestica) given a low protein diet.

    Science.gov (United States)

    Mabuchi, Yuko; Frankel, Theresa L

    2016-03-01

    Racing pigeons are exposed to and act as carriers of diseases. Dietary protein requirement for their maintenance has not been determined experimentally despite their being domesticated for over 7000 years. A maintenance nitrogen (protein) requirement (MNR) for pigeons was determined in a balance study using diets containing 6, 10 and 14% crude protein (CP). Then, the effects of feeding the diets were investigated to determine whether they were adequate to sustain innate and acquired immune functions. Nitrogen intake from the 6% CP diet was sufficient to maintain nitrogen balance and body weight in pigeons. However, the immune functions of phagocytosis, oxidative burst and lymphocyte proliferation in pigeons fed this diet were reduced compared with those fed 10 and 14% CP diets. Pigeons given the 6 and 10% CP diets had lower antibody titres following inoculation against Newcastle disease (ND) than those on the 14% CP diet. A confounding factor found on autopsy was the presence of intestinal parasites in some of the pigeons given the 6 and 10% CP diets; however, none of the pigeons used to measure MNR or acquired immunity to ND were infested with parasites. In conclusion, neither the 6 nor 10% CP diets adequately sustained acquired immune function of pigeons.

  6. Functional analysis of membrane-bound complement regulatory protein on T-cell immune response in ginbuna crucian carp.

    Science.gov (United States)

    Nur, Indriyani; Abdelkhalek, Nevien K; Motobe, Shiori; Nakamura, Ryota; Tsujikura, Masakazu; Somamoto, Tomonori; Nakao, Miki

    2016-02-01

    Complements have long been considered to be a pivotal component in innate immunity. Recent researches, however, highlight novel roles of complements in T-cell-mediated adaptive immunity. Membrane-bound complement regulatory protein CD46, a costimulatory protein for T cells, is a key molecule for T-cell immunomodulation. Teleost CD46-like molecule, termed Tecrem, has been newly identified in common carp and shown to function as a complement regulator. However, it remains unclear whether Tecrem is involved in T-cell immune response. We investigated Tecrem function related to T-cell responses in ginbuna crucian carp. Ginbuna Tecrem (gTecrem) proteins were detected by immunoprecipitation using anti-common carp Tecrem monoclonal antibody (mAb) and were ubiquitously expressed on blood cells including CD8α(+) and CD4(+) lymphocytes. gTecrem expression on leucocyte surface was enhanced after stimulation with the T-cell mitogen, phytohaemagglutinin (PHA). Coculture with the anti-Tecrem mAb significantly inhibited the proliferative activity of PHA-stimulated peripheral blood lymphocytes, suggesting that cross-linking of Tecrems on T-cells interferes with a signal transduction pathway for T-cell activation. These findings indicate that Tecrem may act as a T-cell moderator and imply that the complement system in teleost, as well as mammals, plays an important role for linking adaptive and innate immunity.

  7. Immunization with Protein D from Non-Typeable Haemophilus influenzae (NTHi) Induced Cytokine Responses and Bioactive Antibody Production

    Science.gov (United States)

    Davoudi Vijeh Motlagh, Atefeh; Siadat, Seyed Davar; Abedian Kenari, Saeid; Mahdavi, Mehdi; Behrouzi, Ava; Asgarian-Omran, Hossein

    2016-01-01

    Background Outer membrane protein D (PD) is a highly conserved and stable protein in the outer membrane of both encapsulated (typeable) and non-capsulated (non-typeable) strains of Haemophilus influenzae. As an immunogen, PD is a potential candidate vaccine against non-typeable H. influenzae (NTHi) strains. Objectives The aim of this study was to determine the cytokine pattern and the opsonic antibody response in a BALB/c mouse model versus PD from NTHi as a vaccine candidate. Methods Protein D was formulated with Freund’s and outer membrane vesicle (OMV) adjuvants and injected into experimental mice. Sera from all groups were collected. The bioactivity of the anti-PD antibody was determined by opsonophagocytic killing test. To evaluate the cytokine responses, the spleens were assembled, suspension of splenocytes was recalled with antigen, and culture supernatants were analyzed by ELISA for IL-4, IL-10, and IFN-γ cytokines. Results Anti-PD antibodies promoted phagocytosis of NTHi in both immunized mice groups (those administered PD + Freund’s and those administered PD + OMV adjuvants, 92.8% and 83.5%, respectively, compared to the control group). In addition, the concentrations of three cytokines were increased markedly in immunized mice. Conclusions We conclude that immunization with PD protects mice against NTHi. It is associated with improvements in both cellular and humoral immune responses and opsonic antibody activity. PMID:27942362

  8. Induction of a robust immunity response against novel duck reovirus in ducklings using a subunit vaccine of sigma C protein

    Science.gov (United States)

    Bi, Zhuangli; Zhu, Yingqi; Chen, Zongyan; Li, Chuanfeng; Wang, Yong; Wang, Guijun; Liu, Guangqing

    2016-01-01

    Novel duck reovirus (NDRV) disease emerged in China in 2011 and continues to cause high morbidity and about 5.0 to 50% mortality in ducklings. Currently there are no approved vaccines for the virus. This study aimed to assess the efficacy of a new vaccine created from the baculovirus and sigma C gene against NDRV. In this study, a recombinant baculovirus containing the sigma C gene was constructed, and the purified protein was used as a vaccine candidate in ducklings. The efficacy of sigma C vaccine was estimated according to humoral immune responses, cellular immune response and protection against NDRV challenge. The results showed that sigma C was highly expressed in Sf9 cells. Robust humoral and cellular immune responses were induced in all ducklings immunized with the recombinant sigma C protein. Moreover, 100% protection against lethal challenge with NDRV TH11 strain was observed. Summary, the recombinant sigma C protein could be utilized as a good candidate against NDRV infection. PMID:27974824

  9. Immunization and chemical conjugation of Bm95 obtained from Pichia pastoris enhances the immune response against vaccinal protein and Neisseria meningitidis capsular polysaccharide

    Directory of Open Access Journals (Sweden)

    Rodriguez-Valle M

    2014-03-01

    Full Text Available Manuel Rodriguez-Valle,1 Leonardo Canan-Hadden,2 Olivia Niebla2 1Animal Biotechnology Division, 2Analytical Division, Centre for Genetic Engineering and Biotechnology, Havana, Cuba Abstract: The ectoparasite Rhipicephalus (Boophilus microplus causes severe economic losses to the cattle industry in tropical and subtropical regions, and transmits endoparasites, such as Babesia bovis. The glycoprotein Bm95 is homologous to Bm86, a surface membrane protein of gut epithelial cells in R. microplus, and has been shown to efficiently control this ectoparasite in regions of the Americas. The immunostimulant properties of Bm86 have already been demonstrated after its coinjection with hepatitis B surface antigen (HBsAg and the infectious bovine rhinotracheitis virus. This study evaluated the carrier and immunostimulant properties of Bm95 using low immunogenic Neisseria meningitidis capsular C polysaccharide (Men CpS and HBsAg. We produced two polysaccharide-Bm95 conjugates by carbodiimide (MenCpSBm-c and reductive amination (MenCpSBm-ra methods. These conjugates were characterized and evaluated in mice. Antibody titers against Men CpS were significantly higher in mice immunized with MenCpSBm-ra (2,350±250, P<0.01 than in those immunized with MenCpSBm-c (250±75 or Men CpS (570±104. The study data indicate effective immunological memory after booster inoculation in mice immunized with MenCpSBm-ra. Additionally, significant humoral immunity against HBsAg was documented in mice coimmunized via the intranasal route with recombinant Bm95 (11,400±345 and HBsAg (128,000±250 compared with mice immunized only with HBsAg (400±40 or Bm95 (5,461±150, P<0.01. In conclusion, the immunostimulatory properties of recombinant Bm95 make it a useful element for developing safer conjugated vaccines against bacterial pathogens and for evaluation against ticks and tick-borne diseases in the context of a polyvalent veterinary vaccine. Keywords: glycoconjugate, Bm86

  10. Protein deprivation causes reversible impariment of mucosal immune response to cholera toxoid/toxin in rat gut.

    Science.gov (United States)

    Barry, W S; Pierce, N F

    1979-09-06

    Scretory antibodies may be the major defence against mucosal infections, especially those due to viruses and non-invasive pathogens such as Vibrio cholerae and toxinogenic Escherichia coli. The high incidence of mucosal infections in malnourished protein-deficient children may result from defective antibody production, but evidence for this is conflicting. We report here that protein deficiency markedly impairs the mucosal immune reponse to cholera toxiod/toxin (CT), a protein antigen, in rats and that this impairment is rapidly reversed by refeeding.

  11. Arabidopsis lysin-motif proteins LYM1 LYM3 CERK1 mediate bacterial peptidoglycan sensing and immunity to bacterial infection

    Science.gov (United States)

    Willmann, Roland; Lajunen, Heini M.; Erbs, Gitte; Newman, Mari-Anne; Kolb, Dagmar; Tsuda, Kenichi; Katagiri, Fumiaki; Fliegmann, Judith; Bono, Jean-Jacques; Cullimore, Julie V.; Jehle, Anna K.; Götz, Friedrich; Kulik, Andreas; Molinaro, Antonio; Lipka, Volker; Gust, Andrea A.; Nürnberger, Thorsten

    2011-01-01

    Recognition of microbial patterns by host pattern recognition receptors is a key step in immune activation in multicellular eukaryotes. Peptidoglycans (PGNs) are major components of bacterial cell walls that possess immunity-stimulating activities in metazoans and plants. Here we show that PGN sensing and immunity to bacterial infection in Arabidopsis thaliana requires three lysin-motif (LysM) domain proteins. LYM1 and LYM3 are plasma membrane proteins that physically interact with PGNs and mediate Arabidopsis sensitivity to structurally different PGNs from Gram-negative and Gram-positive bacteria. lym1 and lym3 mutants lack PGN-induced changes in transcriptome activity patterns, but respond to fungus-derived chitin, a pattern structurally related to PGNs, in a wild-type manner. Notably, lym1, lym3, and lym3 lym1 mutant genotypes exhibit supersusceptibility to infection with virulent Pseudomonas syringae pathovar tomato DC3000. Defects in basal immunity in lym3 lym1 double mutants resemble those observed in lym1 and lym3 single mutants, suggesting that both proteins are part of the same recognition system. We further show that deletion of CERK1, a LysM receptor kinase that had previously been implicated in chitin perception and immunity to fungal infection in Arabidopsis, phenocopies defects observed in lym1 and lym3 mutants, such as peptidoglycan insensitivity and enhanced susceptibility to bacterial infection. Altogether, our findings suggest that plants share with metazoans the ability to recognize bacterial PGNs. However, as Arabidopsis LysM domain proteins LYM1, LYM3, and CERK1 form a PGN recognition system that is unrelated to metazoan PGN receptors, we propose that lineage-specific PGN perception systems have arisen through convergent evolution. PMID:22106285

  12. Immunization with Brugia malayi Myosin as Heterologous DNA Prime Protein Boost Induces Protective Immunity against B. malayi Infection in Mastomys coucha

    Science.gov (United States)

    Gupta, Jyoti; Misra, Sweta; Misra-Bhattacharya, Shailja

    2016-01-01

    The current control strategies employing chemotherapy with diethylcarbamazine, ivermectin and albendazole have reduced transmission in some filaria-endemic areas, there is growing interest for complementary approaches, such as vaccines especially in light of threat of parasite developing resistance to mainstay drugs. We earlier demonstrated recombinant heavy chain myosin of B. malayi (Bm-Myo) as a potent vaccine candidate whose efficacy was enhanced by heterologous DNA prime/protein boost (Myo-pcD+Bm-Myo) vaccination in BALB/c mice. BALB/c mouse though does not support the full developmental cycle of B. malayi, however, the degree of protection may be studied in terms of transformation of challenged infective larvae (L3) to next stage (L4) with an ease of delineating the generated immunological response of host. In the current investigation, DNA vaccination with Bm-Myo was therefore undertaken in susceptible rodent host, Mastomys coucha (M. coucha) which sustains the challenged L3 and facilitates their further development to sexually mature adult parasites with patent microfilaraemia. Immunization schedule consisted of Myo-pcD and Myo-pcD+Bm-Myo followed by B. malayi L3 challenge and the degree of protection was evaluated by observing microfilaraemia as well as adult worm establishment. Myo-pcD+Bm-Myo immunized animals not only developed 78.5% reduced blood microfilarial density but also decreased adult worm establishment by 75.3%. In addition, 75.4% of the recovered live females revealed sterilization over those of respective control animals. Myo-pcD+Bm-Myo triggered higher production of specific IgG and its isotypes which induced marked cellular adhesion and cytotoxicity (ADCC) to microfilariae (mf) and L3 in vitro. Both Th1 and Th2 cytokines were significantly up-regulated displaying a mixed immune response conferring considerable protection against B. malayi establishment by engendering a long-lasting effective immune response and therefore emerges as a

  13. Fos/AP-1 proteins in bone and the immune system.

    Science.gov (United States)

    Wagner, Erwin F; Eferl, Robert

    2005-12-01

    The skeleton and the immune system share a variety of different cytokines and transcription factors, thereby mutually influencing each other. These interactions are not confined to the bone marrow cavity where bone cells and hematopoietic cells exist in proximity but also occur at locations that are target sites for inflammatory bone diseases. The newly established research area termed 'osteoimmunology' attempts to unravel these skeletal/immunological relationships. Studies towards a molecular understanding of inflammatory bone diseases from an immunological as well as a bone-centered perspective have been very successful and led to the identification of several signaling pathways that are causally involved in inflammatory bone loss. Induction of receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) signals by activated T cells and subsequent activation of the key transcription factors Fos/activator protein-1 (AP-1), NF-kappaB, and NF for activation of T cells c1 (NFATc1) are in the center of the signaling networks leading to osteoclast-mediated bone loss. Conversely, nature has employed the interferon system to antagonize excessive osteoclast differentiation, although this counteracting activity appears to be overruled under pathological conditions. Here, we focus on Fos/AP-1 functions in osteoimmunology, because this osteoclastogenic transcription factor plays a central role in inflammatory bone loss by regulating genes like NFATc1 as well as the interferon system. We also attempt to put potential therapeutic strategies for inflammatory bone diseases in perspective.

  14. Immunization of mice by Hollow Mesoporous Silica Nanoparticles as carriers of Porcine Circovirus Type 2 ORF2 Protein

    Directory of Open Access Journals (Sweden)

    Guo Hui-Chen

    2012-06-01

    Full Text Available Abstract Backgroud Porcine circovirus type 2 (PCV2 is a primary etiological agent of post-weaning multi-systemic wasting syndrome (PMWS, which is a disease of increasing importance to the pig industry worldwide. Hollow mesoporous silica nanoparticles (HMSNs have gained increasing interest for use in vaccines. Methods To study the potential of HMSNs for use as a protein delivery system or vaccine carriers. HMSNs were synthesized by a sol–gel/emulsion(oil-in-water/ethanol method, purified PCV2 GST-ORF2-E protein was loaded into HMSNs, and the resulting HMSN/protein mixture was injected into mice. The uptake and release profiles of protein by HMSNs in vitro were investigated. PCV2 GST-ORF2-E specific antibodies and secretion of IFN-γ were detected by enzyme-linked immunosorbent assays, spleen lymphocyte proliferation was measured by the MTS method, and the percentage of CD4+ and CD8+ were determined by flow cytometry. Results HMSNs were found to yield better binding capacities and delivery profiles of proteins; the specific immune response induced by PCV2 GST-ORF2-E was maintained for a relatively long period of time after immunization with the HMSN/protein complex. Conclusion The findings suggest that HMSNs are good protein carriers and have high potential for use in future applications in therapeutic drug delivery.

  15. The C-type lectin-like domain containing proteins Clec-39 and Clec-49 are crucial for Caenorhabditis elegans immunity against Serratia marcescens infection.

    Science.gov (United States)

    Miltsch, S M; Seeberger, P H; Lepenies, B

    2014-07-01

    Caenorhabditis elegans exhibits protective immunity against a variety of fungal and bacterial pathogens. Since C. elegans lacks an adaptive immune system, pathogen recognition is mediated entirely by innate immunity. To date, little is known about the involvement of pattern recognition receptors (PRRs) in pathogen sensing as part of the C. elegans immunity. C-type lectin-like domain (CTLD) containing proteins represent a superfamily of PRRs. A large number of genes encoding for CTLD proteins are present in the C. elegans genome, however the role of CTLD proteins in bacterial recognition and antibacterial immunity has not yet been determined. In this study, we investigated the function of selected C. elegans CTLD proteins during infection with the Gram-negative bacterium Serratia marcescens. Wild-type and CTLD gene-deficient C. elegans strains were compared in their susceptibility to S. marcescens infection. Interestingly, survival and egg laying were significantly reduced in strains deficient for clec-39 and clec-49 indicating a role for both CTLD proteins in C. elegans immune defense against bacteria as evidenced by using S. marcescens infection. Binding studies with recombinantly expressed Clec-39-Fc and Clec-49-Fc fusion proteins revealed that both CTLD proteins recognized live bacteria in a Ca(2+)-independent manner. This study provides insight into the role of CTLD proteins in C. elegans immunity and demonstrates their function during bacterial infection.

  16. T cell metabolism. The protein LEM promotes CD8⁺ T cell immunity through effects on mitochondrial respiration.

    Science.gov (United States)

    Okoye, Isobel; Wang, Lihui; Pallmer, Katharina; Richter, Kirsten; Ichimura, Takahuru; Haas, Robert; Crouse, Josh; Choi, Onjee; Heathcote, Dean; Lovo, Elena; Mauro, Claudio; Abdi, Reza; Oxenius, Annette; Rutschmann, Sophie; Ashton-Rickardt, Philip G

    2015-05-29

    Protective CD8(+) T cell-mediated immunity requires a massive expansion in cell number and the development of long-lived memory cells. Using forward genetics in mice, we identified an orphan protein named lymphocyte expansion molecule (LEM) that promoted antigen-dependent CD8(+) T cell proliferation, effector function, and memory cell generation in response to infection with lymphocytic choriomeningitis virus. Generation of LEM-deficient mice confirmed these results. Through interaction with CR6 interacting factor (CRIF1), LEM controlled the levels of oxidative phosphorylation (OXPHOS) complexes and respiration, resulting in the production of pro-proliferative mitochondrial reactive oxygen species (mROS). LEM provides a link between immune activation and the expansion of protective CD8(+) T cells driven by OXPHOS and represents a pathway for the restoration of long-term protective immunity based on metabolically modified cytotoxic CD8(+) T cells.

  17. The mitochondrial fission factor dynamin-related protein 1 modulates T-cell receptor signalling at the immune synapse.

    Science.gov (United States)

    Baixauli, Francesc; Martín-Cófreces, Noa B; Morlino, Giulia; Carrasco, Yolanda R; Calabia-Linares, Carmen; Veiga, Esteban; Serrador, Juan M; Sánchez-Madrid, Francisco

    2011-04-06

    During antigen-specific T-cell activation, mitochondria mobilize towards the vicinity of the immune synapse. We show here that the mitochondrial fission factor dynamin-related protein 1 (Drp1) docks at mitochondria, regulating their positioning and activity near the actin-rich ring of the peripheral supramolecular activation cluster (pSMAC) of the immune synapse. Mitochondrial redistribution in response to T-cell receptor engagement was abolished by Drp1 silencing, expression of the phosphomimetic mutant Drp1S637D and the Drp1-specific inhibitor mdivi-1. Moreover, Drp1 knockdown enhanced mitochondrial depolarization and T-cell receptor signal strength, but decreased myosin phosphorylation, ATP production and T-cell receptor assembly at the central supramolecular activation cluster (cSMAC). Our results indicate that Drp1-dependent mitochondrial positioning and activity controls T-cell activation by fuelling central supramolecular activation cluster assembly at the immune synapse.

  18. Induction of tetravalent protective immunity against four dengue serotypes by the tandem domain III of the envelope protein.

    Science.gov (United States)

    Chen, Shuiping; Yu, Man; Jiang, Tao; Deng, Yongqiang; Qin, Chengfeng; Qin, Ede

    2007-06-01

    In the present study, the domain IIIs of all four dengue virus (DENV) serotypes were connected sequentially to construct the tandem domain III. The resulting DNA fragment was then cloned into pBAD/Topo ThioFusion plasmid. After induction, the Escherichia coli expression protein was purified and used to immunize BALB/c mice by subcutaneous route. The sera from mice immunized with the purified protein were confirmed to contain specific high antibody titers against DEN1, DEN2, and DEN4, and moderate antibody titer against DEN3. In suckling mouse model, 70% of the mice challenged with DEN1, DEN2, and DEN4 in combination with sera from mice immunized with the purified protein were protected, and 18% of the mice challenged with DEN3 in combination with the same sera were protected. Our data suggest that the tandem domain III of the envelope protein can be used as a potential tetravalent dengue vaccine based on a single antigen.

  19. Mycoplasma hyopneumoniae and Mycoplasma flocculare differential domains from orthologous surface proteins induce distinct cellular immune responses in mice.

    Science.gov (United States)

    Leal, Fernanda Munhoz Dos Anjos; Virginio, Veridiana Gomes; Martello, Carolina Lumertz; Paes, Jéssica Andrade; Borges, Thiago J; Jaeger, Natália; Bonorino, Cristina; Ferreira, Henrique Bunselmeyer

    2016-07-15

    Mycoplasma hyopneumoniae and Mycoplasma flocculare are two genetically close species found in the swine respiratory tract. Despite their similarities, while M. hyopneumoniae is the causative agent of porcine enzootic pneumonia, M. flocculare is a commensal bacterium. Genomic and transcriptional comparative analyses so far failed to explain the difference in pathogenicity between these two species. We then hypothesized that such difference might be, at least in part, explained by amino acid sequence and immunological or functional differences between ortholog surface proteins. In line with that, it was verified that approximately 85% of the ortholog surface proteins from M. hyopneumoniae 7448 and M. flocculare present one or more differential domains. To experimentally assess possible immunological implications of this kind of difference, the extracellular differential domains from one pair of orthologous surface proteins (MHP7448_0612, from M. hyopneumoniae, and MF_00357, from M. flocculare) were expressed in E. coli and used to immunize mice. The recombinant polypeptides (rMHP61267-169 and rMF35767-196, respectively) induced distinct cellular immune responses. While, rMHP61267-169 induced both Th1 and Th2 responses, rMF35767-196 induced just an early pro-inflammatory response. These results indicate that immunological properties determined by differential domains in orthologous surface protein might play a role in pathogenicity, contributing to elicit specific and differential immune responses against each species.

  20. Protein malnutrition impairs the immune response and influences the severity of infection in a hamster model of chronic visceral leishmaniasis.

    Directory of Open Access Journals (Sweden)

    Eugenia Carrillo

    Full Text Available Leishmaniasis remains one of the world's most devastating neglected tropical diseases. It mainly affects developing countries, where it often co-exists with chronic malnutrition, one of the main risk factors for developing the disease. Few studies have been published, however, on the relationship between leishmaniasis progression and malnutrition. The present paper reports the influence of protein malnutrition on the immune response and visceral disease development in adult hamsters infected with Leishmania infantum fed either standard or low protein diets. The low protein diet induced severe malnutrition in these animals, and upon infection with L. infantum 33% had severe visceral leishmaniasis compared to only 8% of animals fed the standard diet. The infected, malnourished animals showed notable leukocyte depletion, mild specific antibody responses, impairment of lymphoproliferation, presence of parasites in blood (16.67% of the hamsters and significant increase of the splenic parasite burden. Animals fed standard diet suffered agranulocytosis and monocytopenia, but showed stronger specific immune responses and had lower parasite loads than their malnourished counterparts. The present results show that protein malnutrition promotes visceral leishmaniasis and provide clues regarding the mechanisms underlying the impairment of the immune system.

  1. Neural Network Enhanced Structure Determination of Osteoporosis, Immune System, and Radiation Repair Proteins Project

    Data.gov (United States)

    National Aeronautics and Space Administration — The proposed innovation will utilize self learning neural network technology to determine the structure of osteoporosis, immune system disease, and excess radiation...

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

    Science.gov (United States)

    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.

  3. IgG immune responses to different proteins of Helicobacter Pylori as defined by immunoblot assay

    Directory of Open Access Journals (Sweden)

    Raeiszadeh M

    2000-09-01

    Full Text Available Helicobacter pylori (H.pylori is an etiologic factor for chronic gastritis and peptic ulcers. Serological testing of H.pylori infection is common in Iran, as other parts of the world. There are geographical variations in the humoral immune response to various H. pylori strains in different parts of the worl. We studied the immunogenic proteins of H.pylori by means of an Immunoblot assay with antigens of H.pylori strains isolated in Iran. Sera of 64 patients suffering from dyspepsia were analyzed to determine antibodlies which were good marker of infection and the antibody patterns associated with peptic ulcer.54 out of 64 dyspeptic patients were infected by H. pylori based on positive culture or positive results of both rapid urease test and direct examination. 14 out of fity-four had peptic ulcers and the rest were catagoriied as patients with non-ulcer dyspepsia. Some of them had multiple erosions in the gut or deodenum. Tweny –two major bands were identified by immunoblot. Of these, IgG antibodies against 10 protients, and they produced immunoreative bands at 14, 16, 22, 26, 32 , 32, 44, 87, 92, 120 Kda. Antibody patterns were not identical in the patients. The presence of at least one band at 14, 16, 22, 26, 32, 35Kda was the best marker of infection(sensitivity, 90% and specificity, 80% Major serological cross reactions were found at moderate molecular weight bands (50, 52, 54, 60, 66 KDa. The presence of at least one band at 14, 16, 22, 26, 32, 35Kda was the best marker of infection (sensitivity, 90% and specificity, 80%. Major serological crossreactions were found at moderate molerate molecular weight bands (50, 52, 54, 60, 66 KDa. The presence of antibodies to 120 Kda protein (Cag A and 87 Kda Protein (Vac A were not associated with the presence of peptic ulcers. These were in contradiction to results obtained across Europe and U.S but in agreement with Asian studies. However the presence of at least one band at either 32 or 35 Kda was

  4. Targeting tumor-associated immune suppression with selective protein kinase A type I (PKAI) inhibitors may enhance cancer immunotherapy.

    Science.gov (United States)

    Hussain, Muzammal; Shah, Zahir; Abbas, Nasir; Javeed, Aqeel; Mukhtar, Muhammad Mahmood; Zhang, Jiancun

    2016-01-01

    Despite the tremendous progress in last few years, the cancer immunotherapy has not yet improved disease-free because of the tumor-associated immune suppression being a major barrier. Novel trends to enhance cancer immunotherapy aims at harnessing the therapeutic manipulation of signaling pathways mediating the tumor-associated immune suppression, with the general aims of: (a) reversing the tumor immune suppression; (b) enhancing the innate and adaptive components of anti-tumor immunosurveillance, and (c) protecting immune cells from the suppressive effects of T regulatory cells (Tregs) and the tumor-derived immunoinhibitory mediators. A particular striking example in this context is the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A type I (PKAI) pathway. Oncogenic cAMP/PKAI signaling has long been implicated in the initiation and progression of several human cancers. Emerging data indicate that cAMP/PKAI signaling also contributes to tumor- and Tregs-derived suppression of innate and adaptive arms of anti-tumor immunosurveillance. Therapeutically, selective PKAI inhibitors have been developed which have shown promising anti-cancer activity in pre-clinical and clinical settings. Rp-8-Br-cAMPS is a selective PKAI antagonist that is widely used as a biochemical tool in signal transduction research. Collateral data indicate that Rp-8-Br-cAMPS has shown immune-rescuing potential in terms of enhancing the innate and adaptive anti-tumor immunity, as well as protecting adaptive T cells from the suppressive effects of Tregs. Therefore, this proposal specifically implicates that combining selective PKAI antagonists/inhibitors with cancer immunotherapy may have multifaceted benefits, such as rescuing the endogenous anti-tumor immunity, enhancing the efficacy of cancer immunotherapy, and direct anti-cancer effects.

  5. PGRP-LB is a maternally transmitted immune milk protein that influences symbiosis and parasitism in tsetse’s offspring

    Science.gov (United States)

    Wang, Jingwen; Aksoy, Serap

    2012-01-01

    Beneficial microbe functions range from host dietary supplementation to development and maintenance of host immune system. In mammals, newborn progeny are quickly colonized with a symbiotic fauna that is provisioned in mother’s milk and that closely resembles that of the parent. Tsetse fly (Diptera: Glossinidae) also depends on the obligate symbiont Wigglesworthia for nutritional supplementation, optimal fecundity, and immune system development. Tsetse progeny develop one at a time in an intrauterine environment and receive nourishment and symbionts in mother’s milk. We show that the host Peptidoglycan Recognition Protein (PGRP-LB) is expressed only in adults and is a major component of the milk that nourishes the developing progeny. The amidase activity associated with PGRP-LB may scavenge the symbiotic peptidoglycan and prevent the induction of tsetse's Immune Deficiency pathway that otherwise can damage the symbionts. Reduction of PGRP-LB experimentally diminishes female fecundity and damages Wigglesworthia in the milk through induction of antimicrobial peptides, including Attacin. Larvae that receive less maternal PGRP-LB give rise to adults with fewer Wigglesworthia and hyperimmune responses. Such adults also suffer dysregulated immunity, as indicated by the presence of higher trypanosome densities in parasitized adults. We show that recPGRP-LB has antimicrobial and antitrypanosomal activities that may regulate symbiosis and impact immunity. Thus, PGRP-LB plays a pivotal role in tsetse’s fitness by protecting symbiosis against host-inflicted damage during development and by controlling parasite infections in adults that can otherwise reduce host fecundity. PMID:22689989

  6. Oral immune regulation using colitis extracted proteins for treatment of Crohn's disease: Results of a phase Ⅰ clinical trial

    Institute of Scientific and Technical Information of China (English)

    Eran Israeli; Eran Goldin; Oren Shibolet; Athalia Klein; Nilla Hemed; Dean Engelhardt; Elazar Rabbani; Yaron Ilan

    2005-01-01

    AIM: To evaluate safety and possible efficacy of induction of oral immune regulation using colitis extracted proteins (CEP) in Crohn's disease (CD) subjects. METHODS: Ten CDs were treated orally with autologous CEP thrice weekly for 16 wk. Subjects were monitored for CDAI and IBDQ. Immune modulatory effect was assessed by T-lymphocyte FACS analysis, CEP-specific IFNγ ELISPOT assay and cytokine levels. RESULTS: Induction of oral immune regulation significantly ameliorated disease activity. All (10/10) subjects had clinical response (CDAI ≤ 70) and 7/10 achieved clinical remission (CDAI ≤ 150). Significant increase in mean IBDQ score was noted (134±9 vs 164±12). No treatment-related adverse events were noted. High levels of CEP-specific IFNγ spot forming colonies were detected in five subjects prior to treatment and in all five, a marked decrease was observed. The CD4+/CD8+ lymphocyte ratio and peripheral NKT cell numbers increased significantly, in 7/10 and in 5/10 subjects, respectively. Significant increase in serum IL-10 and IL-4 levels was observed in 7/10 subjects during treatment period.CONCLUSION: Immune regulation via oral administration of CEP is a safe and possibly effective treatment for subjects with moderate CD and may provide means of antigen-specific immune modulation.

  7. Porcine reproductive and respiratory syndrome virus nonstructural protein 1beta modulates host innate immune response by antagonizing IRF3 activation.

    Science.gov (United States)

    Beura, Lalit K; Sarkar, Saumendra N; Kwon, Byungjoon; Subramaniam, Sakthivel; Jones, Clinton; Pattnaik, Asit K; Osorio, Fernando A

    2010-02-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) infection of swine leads to a serious disease characterized by a delayed and defective adaptive immune response. It is hypothesized that a suboptimal innate immune response is responsible for the disease pathogenesis. In the study presented here we tested this hypothesis and identified several nonstructural proteins (NSPs) with innate immune evasion properties encoded by the PRRS viral genome. Four of the total ten PRRSV NSPs tested were found to have strong to moderate inhibitory effects on beta interferon (IFN-beta) promoter activation. The strongest inhibitory effect was exhibited by NSP1 followed by, NSP2, NSP11, and NSP4. We focused on NSP1alpha and NSP1beta (self-cleavage products of NSP1 during virus infection) and NSP11, three NSPs with strong inhibitory activity. All of three proteins, when expressed stably in cell lines, strongly inhibited double-stranded RNA (dsRNA) signaling pathways. NSP1beta was found to inhibit both IFN regulatory factor 3 (IRF3)- and NF-kappaB-dependent gene induction by dsRNA and Sendai virus. Mechanistically, the dsRNA-induced phosphorylation and nuclear translocation of IRF3 were strongly inhibited by NSP1beta. Moreover, when tested in a porcine myelomonocytic cell line, NSP1beta inhibited Sendai virus-mediated activation of porcine IFN-beta promoter activity. We propose that this NSP1beta-mediated subversion of the host innate immune response plays an important role in PRRSV pathogenesis.

  8. Involvement of platelet-tumor cell interaction in immune evasion. Potential role of podocalyxin-like protein 1

    Directory of Open Access Journals (Sweden)

    Laura eAmo

    2014-09-01

    Full Text Available Besides their essential role in hemostasis and thrombosis, platelets are involved in the onset of cancer metastasis by interacting with tumor cells. Platelets release secretory factors that promote tumor growth, angiogenesis, and metastasis. Furthermore, the formation of platelet-tumor cell aggregates in the bloodstream provides cancer cells with an immune escape mechanism by protecting circulating malignant cells from immune-mediated lysis by natural killer (NK cells. Platelet-tumor cell interaction is accomplished by specific adhesion molecules, including integrins, selectins, and their ligands. Podocalyxin-like protein 1 (PCLP1 is a selectin ligand protein which overexpression has been associated with several aggressive cancers. PCLP1 expression enhances cell adherence to platelets in an integrin-dependent process and through the interaction with P-selectin expressed on activated platelets. However, the involvement of PCLP1-induced tumor-platelet interaction in tumor immune evasion still remains unexplored. The identification of selectin ligands involved in the interaction of platelets with tumor cells may provide help for the development of effective therapies to restrain cancer cell dissemination. This article summarizes the current knowledge on molecules that participate in platelet-tumor cell interaction as well as discusses the potential role of PCLP1 as a molecule implicated in tumor immune evasion.

  9. In silico interaction of methyl isocyanate with immune protein responsible for Mycobacterium tuberculosis infection using molecular docking.

    Science.gov (United States)

    Shrivastava, Rahul; Yasir, Mohammad; Tripathi, Manish; Singh, Pushpendra

    2016-01-01

    This article reports in silico analysis of methyl isocyanate (MIC) on different key immune proteins against Mycobacterium tuberculosis. The analysis shows that MIC is released in the Bhopal gas tragedy in 1984, which is highly toxic and extremely hazardous to human health. In this study, we have selected immune proteins to perform molecular docking with the help of Autodock 4.0. Results show that the CD40 ligand and alpha5beta1 integrin have higher inhibition compared to plasminogen activator urokinase, human glutathione synthetase, mitogen-activated protein kinase (P38 MAPK 14), surfactant protein-B, -D (SP-D), and pulmonary SP-D. MIC interacted with His-125, Try-146 residue of CD40 ligand and Ala-149, and Arg-152 residue of alpha5beta1 integrin and affects the proteins functioning by binding on their active sites. These inhibitory conformations were energetically and statistically favored and supported the evidence from wet laboratory experiments reported in the literature. We can conclude that MIC directly or indirectly affects these proteins, which shows that survivals of the disaster suffer from the diseases like tuberculosis infection and lung cancer.

  10. Ultrastructural localization of highly variable 185/333 immune response proteins in the coelomocytes of the sea urchin, Heliocidaris erythrogramma.

    Science.gov (United States)

    Dheilly, Nolwenn M; Birch, Debra; Nair, Sham V; Raftos, David A

    2011-11-01

    The 185/333 proteins of sea urchins represent a family of highly variable immune response molecules with unknown functions. In this study, we show that 185/333 proteins are expressed by three cell types: amoebocytes, colourless spherule cells and gut-associated amoebocytes. A sub-population of amoebocytes express 185/333 proteins on the membranes of vesicles emanating from the trans-Golgi and which later fuse with the plasma membranes of the cells. The previously uncharacterized gut-associated amoebocytes also show a high level of 185/333 protein expression on their internal vesicles and plasma membranes. Colourless spherule cells contain 185/333 proteins within large spherules (specialized intracellular vesicles). In the presence of bacteria and yeast, the ultrastucture of colourless spherule cells changes and 185/333 proteins disappear. In contrast, 185/333 proteins were not found in the phagosomes of coelomocytes. The 185/333-positive gut amoebocytes were often associated with anuclear bodies, which appeared to incorporate material of microbial origin that was surrounded by 185/333 proteins. The association between 185/333 proteins on gut amoebocytes and anuclear bodies suggests that these proteins may be involved in the phagocytosis of microbes in the gut epithelium.

  11. DMBT1 encodes a protein involved in the immune defense and in epithelial differentiation and is highly unstable in cancer

    DEFF Research Database (Denmark)

    Mollenhauer, J; Herbertz, S; Holmskov, U

    2000-01-01

    in the respiratory immune defense. Immunohistochemical analyses revealed that DMBT1 is produced by both tumor-associated macrophages and tumor cells and that it is deregulated in glioblastoma multiforme in comparison to normal brain tissue. Our data further suggest that the proteins CRP-ductin and hensin, both...... of which have been implicated in epithelial differentiation, are the DMBT1 orthologs in mice and rabbits, respectively. These findings and the spatial and temporal distribution of DMBT1 in fetal and adult epithelia suggest that DMBT1 further plays a role in epithelial development. Rearrangements of DMBT1......, DMBT1 is a gene that is highly unstable in cancer and encodes for a protein with at least two different functions, one in the immune defense and a second one in epithelial differentiation....

  12. 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.

    Science.gov (United States)

    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.

  13. Loss of Arabidopsis thaliana Dynamin-Related Protein 2B reveals separation of innate immune signaling pathways.

    Directory of Open Access Journals (Sweden)

    John M Smith

    2014-12-01

    Full Text Available Vesicular trafficking has emerged as an important means by which eukaryotes modulate responses to microbial pathogens, likely by contributing to the correct localization and levels of host components necessary for effective immunity. However, considering the complexity of membrane trafficking in plants, relatively few vesicular trafficking components with functions in plant immunity are known. Here we demonstrate that Arabidopsis thaliana Dynamin-Related Protein 2B (DRP2B, which has been previously implicated in constitutive clathrin-mediated endocytosis (CME, functions in responses to flg22 (the active peptide derivative of bacterial flagellin and immunity against flagellated bacteria Pseudomonas syringae pv. tomato (Pto DC3000. Consistent with a role of DRP2B in Pattern-Triggered Immunity (PTI, drp2b null mutant plants also showed increased susceptibility to Pto DC3000 hrcC-, which lacks a functional Type 3 Secretion System, thus is unable to deliver effectors into host cells to suppress PTI. Importantly, analysis of drp2b mutant plants revealed three distinct branches of the flg22-signaling network that differed in their requirement for RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD, the NADPH oxidase responsible for flg22-induced apoplastic reactive oxygen species production. Furthermore, in drp2b, normal MAPK signaling and increased immune responses via the RbohD/Ca2+-branch were not sufficient for promoting robust PR1 mRNA expression nor immunity against Pto DC3000 and Pto DC3000 hrcC-. Based on live-cell imaging studies, flg22-elicited internalization of the plant flagellin-receptor, FLAGELLIN SENSING 2 (FLS2, was found to be partially dependent on DRP2B, but not the closely related protein DRP2A, thus providing genetic evidence for a component, implicated in CME, in ligand-induced endocytosis of FLS2. Reduced trafficking of FLS2 in response to flg22 may contribute in part to the non-canonical combination of immune signaling defects

  14. Cancer associated aberrant protein O-glycosylation can modify antigen processing and immune response.

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    Caroline B Madsen

    Full Text Available Aberrant glycosylation of mucins and other extracellular proteins is an important event in carcinogenesis and the resulting cancer associated glycans have been suggested as targets in cancer immunotherapy. We assessed the role of O-linked GalNAc glycosylation on antigen uptake, processing, and presentation on MHC class I and II molecules. The effect of GalNAc O-glycosylation was monitored with a model system based on ovalbumin (OVA-MUC1 fusion peptides (+/- glycosylation loaded onto dendritic cells co-cultured with IL-2 secreting OVA peptide-specific T cell hybridomas. To evaluate the in vivo response to a cancer related tumor antigen, Balb/c or B6.Cg(CB-Tg(HLA-A/H2-D2Enge/J (HLA-A2 transgenic mice were immunized with a non-glycosylated or GalNAc-glycosylated MUC1 derived peptide followed by comparison of T cell proliferation, IFN-γ release, and antibody induction. GalNAc-glycosylation promoted presentation of OVA-MUC1 fusion peptides by MHC class II molecules and the MUC1 antigen elicited specific Ab production and T cell proliferation in both Balb/c and HLA-A2 transgenic mice. In contrast, GalNAc-glycosylation inhibited the presentation of OVA-MUC1 fusion peptides by MHC class I and abolished MUC1 specific CD8+ T cell responses in HLA-A2 transgenic mice. GalNAc glycosylation of MUC1 antigen therefore facilitates uptake, MHC class II presentation, and antibody response but might block the antigen presentation to CD8+ T cells.

  15. A role for plasma cell targeting agents in immune tolerance induction in autoimmune disease and antibody responses to therapeutic proteins.

    Science.gov (United States)

    Rosenberg, A S; Pariser, A R; Diamond, B; Yao, L; Turka, L A; Lacana, E; Kishnani, P S

    2016-04-01

    Antibody responses to life saving therapeutic protein products, such as enzyme replacement therapies (ERT) in the setting of lysosomal storage diseases, have nullified product efficacy and caused clinical deterioration and death despite treatment with immune-suppressive therapies. Moreover, in some autoimmune diseases, pathology is mediated by a robust antibody response to endogenous proteins such as is the case in pulmonary alveolar proteinosis, mediated by antibodies to Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF). In this work, we make the case that in such settings, when the antibody response is high titered, sustained, and refractory to immune suppressive treatments, the antibody response is mediated by long-lived plasma cells which are relatively unperturbed by immune suppressants including rituximab. However, long-lived plasma cells can be targeted by proteasome inhibitors such as bortezomib. Recent reports of successful reversal of antibody responses with bortezomib in the settings of ERT and Thrombotic Thrombocytopenic Purpura (TTP) argue that the safety and efficacy of such plasma cell targeting agents should be evaluated in larger scale clinical trials to delineate the risks and benefits of such therapies in the settings of antibody-mediated adverse effects to therapeutic proteins and autoantibody mediated pathology.

  16. High mobility group box 1 protein (HMGB1) as an immune-modulating factor for polarization of human T lymphocytes

    Institute of Scientific and Technical Information of China (English)

    Lifeng Huang; Yongming Yao; Haidong Meng; Xiaodong Zhao; Ning Dong; Yan Yu

    2008-01-01

    Objective This study was performed to investigate the effect of high mobility group box-1 protein (HMGB 1) on immune function of human T lymphocytes in vitro and explore its potential role in cell-mediated immune dysfunction.Methods Fresh blood was obtained from healthy adult volunteers and peripheral blood mononuclear cells (PBMCs) were isolated,then rhHMGB 1 was added to PBMCs.Four-color flow cytometric (FCM) analysis was used for the measurement of intracellular cytokine including interleukin Results (1) Different stimulating time and dosage of rhHMGB 1 did not alter the number of IFN-a positive cells (Th 1).rhHMGB 1 stimulation provoked a dose-dependent and time-dependent increase in Th2 subset and decrease in ratio of Th 1 to Th2.(2) Compared with the untreated cells,when the cells were coincubated with rhHMGB 1 (10-100ng/ml) for 12 hrs,protein release of IL-2 and sIL-2R were significantly up-regulated.At 48 hrs,in contrast,protein production was relatively lower in cells after exposure to 100-1000 ng/ml rhHMGBI.Conclusions These findings demonstrated that HMGB1 has a dual influence on immune functions of human T lymphocytes.

  17. Protein energy malnutrition during vaccination has limited influence on vaccine efficacy but abolishes immunity if administered during Mycobacterium tuberculosis infection

    DEFF Research Database (Denmark)

    Hoang, Truc; Agger, Else Marie; Cassidy, Joseph P;

    2015-01-01

    Protein energy malnutrition (PEM) increases susceptibility to infectious diseases, including tuberculosis (TB), but it is not clear how PEM influences vaccine-promoted immunity to TB. We demonstrate that PEM during low-level steady-state TB infection in a mouse model results in rapid relapse...... cytokine-producing CD4 T cell subsets, with the exception of CD4 T cells positive for TNF-α only. Importantly, this impairment was reversible and resupplementation of protein during infection rescued both the vaccine-promoted T cell response and the protective effect of the vaccine against M. tuberculosis...

  18. Tolerance and immune response to the porcine endogenous retrovirus in German landrace pigs immunised with viral proteins.

    Science.gov (United States)

    Denner, Joachim; Petersen, Björn; Niemann, Heiner

    2015-10-02

    Immunisation of goats, mice, rats, rabbits, guinea pigs, and hamsters with the recombinant ectodomain of the porcine endogenous retrovirus (PERV) transmembrane envelope (TM) protein (p15E) induced binding and neutralising immune antibodies in all animals. In contrast, no antibodies were induced when pigs were immunised with p15E, indicating that pigs are tolerant to their endogenous retroviruses, at least to the TM protein. To answer the question of whether pigs are tolerant to other structural proteins of PERV, we immunised German landrace pigs with p15E, this time in conjunction with the surface envelope proteins gp70 and the core capsid Gag protein p27CA. To ensure that the pigs were immunocompetent and that immunisation was successful, all animals also received an injection of an unrelated protein, keyhole limpet hemocyanin (KLH). Whereas all animals produced antibodies against KLH, no animals produced antibodies against the viral envelope proteins, thus confirming previous results for p15E and extending them to the other envelope protein, gp70. However, the pigs did produce antibodies against p27CA, indicating that there is no tolerance to the core capsid protein of PERV.

  19. Towards development of novel immunization strategies against leishmaniasis using PLGA nanoparticles loaded with kinetoplastid membrane protein-11

    Directory of Open Access Journals (Sweden)

    Santos DM

    2012-04-01

    Full Text Available Diego M Santos1, Marcia W Carneiro1, Tatiana R de Moura1, Kiyoshi Fukutani1, Jorge Clarencio1, Manuel Soto2, Socorro Espuelas3,4, Claudia Brodskyn1,5, Aldina Barral1,5, Manoel Barral-Netto1,5, Camila I de Oliveira1,51Centro de Pesquisas Gonçalo Moniz, FIOCRUZ, Salvador, BA, Brazil; 2Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientificas, Departamento de Biologia Molecular, Universidad Autonoma de Madrid, Madrid; 3Departamento de Farmacia y Tecnología Farmacéutica, 4Instituto de Salud Tropical, Facultad de Farmacia, Universidad de Navarra, Pamplona, Spain; 5Instituto de Investigação em Imunologia, Salvador, BA, BrazilBackground: Vaccine development has been a priority in the fight against leishmaniases, which are vector-borne diseases caused by Leishmania protozoa. Among the different immunization strategies employed to date is inoculation of plasmid DNA coding for parasite antigens, which has a demonstrated ability to induce humoral and cellular immune responses. In this sense, inoculation of plasmid DNA encoding Leishmania kinetoplasmid membrane protein-11 (KMP-11 was able to confer protection against visceral leishmaniasis. However, recently the use of antigen delivery systems such as poly(lactic-co-glycolic acid (PLGA nanoparticles has also proven effective for eliciting protective immune responses.Methods: In the present work, we tested two immunization strategies with the goal of obtaining protection, in terms of lesion development and parasite load, against cutaneous leishmaniasis caused by L. braziliensis. One strategy involved immunization with plasmid DNA encoding L. infantum chagasi KMP-11. Alternatively, mice were primed with PLGA nanoparticles loaded with the recombinant plasmid DNA and boosted using PLGA nanoparticles loaded with recombinant KMP-11.Results: Both immunization strategies elicited detectable cellular immune responses with the presence of both proinflammatory and anti

  20. Plant pathogenesis-related proteins PR-10 and PR-14 as components of innate immunity system and ubiquitous allergens.

    Science.gov (United States)

    Ovchinnikova, Tatiana V; Finkina, Ekaterina I; Melnikova, Daria N; Bogdanov, Ivan V

    2016-10-26

    Pathogenesis-related (PR) proteins are components of innate immunity system in plants. They play an important role in plant defense against pathogens. Lipid transfer proteins (LTPs) and Bet v 1 homologues comprise two separate families of PR-proteins. Both LTPs (PR-14) and Bet v 1 homologues (PR-10) are multifunctional small proteins involving in plant response to abiotic and biotic stress conditions. The representatives of these PR-protein families do not show any sequence similarity but have other common biochemical features such as low molecular masses, the presence of hydrophobic cavities, ligand binding properties, and antimicrobial activities. Besides, many members of PR-10 and PR-14 families are ubiquitous plant panallergens which are able to cause sensitization of human immune system and cross-reactive allergic reactions to plant food and pollen. This review is aimed at comparative analysis of structure-functional and allergenic properties of the PR-10 and PR-14 families, as well as prospects for their medicinal application.

  1. Receptor-like kinase SOBIR1/EVR interacts with receptor-like proteins in plant immunity against fungal infection.

    Science.gov (United States)

    Liebrand, Thomas W H; van den Berg, Grardy C M; Zhang, Zhao; Smit, Patrick; Cordewener, Jan H G; America, Antoine H P; America, Antione H P; Sklenar, Jan; Jones, Alexandra M E; Tameling, Wladimir I L; Robatzek, Silke; Thomma, Bart P H J; Joosten, Matthieu H A J

    2013-06-11

    The plant immune system is activated by microbial patterns that are detected as nonself molecules. Such patterns are recognized by immune receptors that are cytoplasmic or localized at the plasma membrane. Cell surface receptors are represented by receptor-like kinases (RLKs) that frequently contain extracellular leucine-rich repeats and an intracellular kinase domain for activation of downstream signaling, as well as receptor-like proteins (RLPs) that lack this signaling domain. It is therefore hypothesized that RLKs are required for RLPs to activate downstream signaling. The RLPs Cf-4 and Ve1 of tomato (Solanum lycopersicum) mediate resistance to the fungal pathogens Cladosporium fulvum and Verticillium dahliae, respectively. Despite their importance, the mechanism by which these immune receptors mediate downstream signaling upon recognition of their matching ligand, Avr4 and Ave1, remained enigmatic. Here we show that the tomato ortholog of the Arabidopsis thaliana RLK Suppressor Of BIR1-1/Evershed (SOBIR1/EVR) and its close homolog S. lycopersicum (Sl)SOBIR1-like interact in planta with both Cf-4 and Ve1 and are required for the Cf-4- and Ve1-mediated hypersensitive response and immunity. Tomato SOBIR1/EVR interacts with most of the tested RLPs, but not with the RLKs FLS2, SERK1, SERK3a, BAK1, and CLV1. SOBIR1/EVR is required for stability of the Cf-4 and Ve1 receptors, supporting our observation that these RLPs are present in a complex with SOBIR1/EVR in planta. We show that SOBIR1/EVR is essential for RLP-mediated immunity and propose that the protein functions as a regulatory RLK of this type of cell-surface receptors.

  2. Intestinal transport of sulfanilic acid in rats immunized with protein-sulfanilic acid conjugate.

    Science.gov (United States)

    Yamamoto, A; Kawaratani, T; Kawashima, K; Hashida, M; Sezaki, H

    1990-07-01

    Intestinal transport of sulfanilic acid was examined by means of an in vitro everted sac technique in rats immunized with a bovine gamma-globulin-sulfanilic acid conjugate. At a low concentration of sulfanilic acid, the intestinal transport of sulfanilic acid was decreased in rats immunized with bovine gamma-globulin-sulfanilic acid conjugate. This phenomenon was dose dependent and antigen specific, since there was no difference in the transport of sulfanilic acid at a high concentration and of an unrelated hapten. These results suggested that parenteral immunization impaired not only the intestinal transport of macromolecular antigens, as previously shown, but also the transport of the low molecular weight hapten, sulfanilic acid.

  3. Sulfated galactans from Gracilaria fisheri bind to shrimp haemocyte membrane proteins and stimulate the expression of immune genes.

    Science.gov (United States)

    Rudtanatip, Tawut; Withyachumnarnkul, Boonsirm; Wongprasert, Kanokpan

    2015-11-01

    Previous studies demonstrated that sulfated galactans (SG) from Gracilaria fisheri (G. fisheri) exhibit immunostimulant activity in shrimp. The present study was conducted to test the hypothesis that SG stimulates signaling molecules of the immune response of shrimp by binding to receptors on the host cell membrane. Accordingly, we evaluated the ability of SG to bind to shrimp haemocytes and showed that SG bound to the shrimp haemocyte membrane (SHM), potentially to specific receptors. Furthermore, this binding was associated with an activation of immune response genes of shrimp. Data from confocal laser scanning micrographs revealed that FITC-labeled SG bound to haemocytes. Far western blot analysis demonstrated that SHM peptides, with molecular sizes of 13, 14, 15, 17, and 25 kDa, were associated with SG. Peptide sequence analysis of the isolated bands using LC-MS/MS and NCBI blast search revealed the identity of the 13, 14, and 17 kDa peptides as lipopolysaccharide and β-1,3-glucan binding protein (LGBP). SG induced the expression of immune related genes and downstream signaling mediators of LGBP including IMD, IKKs, NF-κB, antimicrobial peptides (crustin and PEN-4), the antiviral immunity (dicer), and proPO system (proPO-I and proPO-II). A LGBP neutralizing assay with anti-LGBP antibody indicated a decrease in SG-induced expression of LGBP downstream signaling mediators and the immune related genes. In conclusion, this study demonstrated that the SG-stimulated immune activity in haemocytes is mediated, in part, through the LGBP, and IMD-NF-κB pathway.

  4. DNA vaccination with a gene encoding Toxoplasma gondii Rhoptry Protein 17 induces partial protective immunity against lethal challenge in mice

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    Wang Hai-Long

    2016-01-01

    Full Text Available Toxoplasma gondii is an obligate intracellular apicomplexan parasite that affects humans and various vertebrate livestock and causes serious economic losses. To develop an effective vaccine against T. gondii infection, we constructed a DNA vaccine encoding the T. gondii rhoptry protein 17 (TgROP17 and evaluated its immune protective efficacy against acute T. gondii infection in mice. The DNA vaccine (p3×Flag-CMV-14-ROP17 was intramuscularly injected to BALB/c mice and the immune responses of the vaccinated mice were determined. Compared to control mice treated with empty vector or PBS, mice immunized with the ROP17 vaccine showed a relatively high level of specific anti-T. gondii antibodies, and a mixed IgG1/IgG2a response with predominance of IgG2a production. The immunized mice also displayed a specific lymphocyte proliferative response, a Th1-type cellular immune response with production of IFN-γ and interleukin-2, and increased number of CD8+ T cells. Immunization with the ROP17 DNA significantly prolonged the survival time (15.6 ± 5.4 days, P < 0.05 of mice after challenge infection with the virulent T. gondii RH strain (Type I, compared with the control groups which died within 8 days. Therefore, our data suggest that DNA vaccination with TgROP17 triggers significant humoral and cellular responses and induces effective protection in mice against acute T. gondii infection, indicating that TgROP17 is a promising vaccine candidate against acute toxoplasmosis.

  5. Protein Malnutrition Modifies Innate Immunity and Gene Expression by Intestinal Epithelial Cells and Human Rotavirus Infection in Neonatal Gnotobiotic Pigs

    Science.gov (United States)

    Paim, Francine C.; Kandasamy, Sukumar; Alhamo, Moyasar A.; Fischer, David D.; Langel, Stephanie N.; Deblais, Loic; Kumar, Anand; Chepngeno, Juliet; Shao, Lulu; Huang, Huang-Chi; Candelero-Rueda, Rosario A.; Rajashekara, Gireesh

    2017-01-01

    ABSTRACT Malnutrition affects millions of children in developing countries, compromising immunity and contributing to increased rates of death from infectious diseases. Rotavirus is a major etiological agent of childhood diarrhea in developing countries, where malnutrition is prevalent. However, the interactions between the two and their combined effects on immune and intestinal functions are poorly understood. In this study, we used neonatal gnotobiotic (Gn) pigs transplanted with the fecal microbiota of a healthy 2-month-old infant (HIFM) and fed protein-deficient or -sufficient bovine milk diets. Protein deficiency induced hypoproteinemia, hypoalbuminemia, hypoglycemia, stunting, and generalized edema in Gn pigs, as observed in protein-malnourished children. Irrespective of the diet, human rotavirus (HRV) infection early, at HIFM posttransplantation day 3 (PTD3), resulted in adverse health effects and higher mortality rates (45 to 75%) than later HRV infection (PTD10). Protein malnutrition exacerbated HRV infection and affected the morphology and function of the small intestinal epithelial barrier. In pigs infected with HRV at PTD10, there was a uniform decrease in the function and/or frequencies of natural killer cells, plasmacytoid dendritic cells, and CD103+ and apoptotic mononuclear cells and altered gene expression profiles of intestinal epithelial cells (chromogranin A, mucin 2, proliferating cell nuclear antigen, SRY-Box 9, and villin). Thus, we have established the first HIFM-transplanted neonatal pig model that recapitulates major aspects of protein malnutrition in children and can be used to evaluate physiologically relevant interventions. Our findings provide an explanation of why nutrient-rich diets alone may lack efficacy in malnourished children. IMPORTANCE Malnutrition and rotavirus infection, prevalent in developing countries, individually and in combination, affect the health of millions of children, compromising their immunity and increasing

  6. Insect immune activation by apolipophorin III is correlated with the lipid-binding properties of this protein.

    Science.gov (United States)

    Niere, M; Dettloff, M; Maier, T; Ziegler, M; Wiesner, A

    2001-09-25

    Apolipophorin III (apoLp-III) is an exchangeable insect apolipoprotein consisting of five amphipathic alpha-helices. The protein is able to open reversibly on associating with hydrophobic surfaces and plays a role both in lipid transport and induction of immune responses. Point mutations were introduced at positions 66 (N-->D) and/or 68 (K-->E) between helices 2 and 3, a region possibly serving as a hinge for the opening of the molecule when associating with lipids. The lipid-binding properties of the mutant proteins were analyzed and compared with their immune inducing activities. Structural properties of the proteins were studied by far UV circular dichroism spectroscopy and their abilities to form discoidal complexes of dimyristoyl phosphatidylcholine (DMPC) vesicles were investigated. In comparison to wild-type apoLp-III, apoLp-III(N66D/K68E), and apoLp-III(K68E) displayed significantly decreased lipid-binding abilities and immune stimulating activities, while these effects were less noticeable with apoLp-III(N66D). The secondary structure of the double mutant apoLp-III(N66D/K68E) was similar to that of wild-type apoLp-III. A noticeable reduction of alpha-helical content could be observed for the single mutants apoLp-III(N66D) and apoLp-III(K68E), which was accompanied by an increase in percentage amount of beta-turns. The stability of the secondary structure determined by heat denaturation was not affected by mutagenesis. Furthermore, the ability of all proteins to form discoidal complexes of equal size and shape in the presence of dimyristoyl phosphatidylcholine indicated that the mutagenesis did not affect the molecular architecture in the lipid-associated conformation. The relationship between reduced lipid association and reduced immune stimulating activity supports the hypothesis that apoLp-III-induced immune activation is triggered by the conformational change of the protein.

  7. Modulation of immune response to Toxoplasma gondii in sheep by immunization with a DNA vaccine encoding ROP1 antigen as a fusion protein with ovine CD154.

    Science.gov (United States)

    Hiszczyńska-Sawicka, Elżbieta; Li, Hong; Xu, Janet Boyu; Holec-Gąsior, Lucyna; Kur, Józef; Sedcole, Richard; Bickerstaffe, Roy; Stankiewicz, Mirosław

    2011-12-29

    CD154 is a cell surface molecule expressed by activated T cells. CD40 and CD154 interaction is critically important in regulating humoral and cell-mediated immune responses. In this study we have investigated whether a DNA vaccine encoding rhoptry protein 1 (ROP1) of Toxoplasma gondii, and encoding ovine CD154 induces an enhanced ROP1-specific immune response in sheep. Two groups of twelve animals received two intramuscular injections, of a DNA plasmid encoding T. gondii ROP1 antigen (group 1) or an ROP1 antigen fused to ovine CD154 (group 2). There were two control groups of sheep. One was injected with an empty vector (group 3) and the other received no injections at all (group 4). The injection of the plasmid containing ROP1 (group 1) at weeks 0 and 4 induced a significant IgG2 response at week 2 which was amplified at week 4 after the booster injection and persisted to week 8 compared to the control animals in groups 3 and 4. For IgG1, significant differences from the control animals were only observed from week 5 onwards. The fusion of CD154 and ROP1 elicited significant IgG1 and IgG2 responses from week 1 which were amplified from weeks 5 to 8 compared to the control animals in groups 3 and 4. The IgG1 response was significantly higher in group 2 animals receiving pROP1-CD154 compared to group 1 receiving pROP1 only. There was no significant difference in IgG2 responses between groups 1 and 2. Significant differences in IFN-γ levels were only observed in treatment group 1 at week 2 and treatment group 2 at weeks 1 and 2 compared to the control animals. The results demonstrated that an intramuscular injection of pROP1-CD154 gene to sheep significantly enhanced their immune response and induced a mixed Th1/Th2 response while the intramuscular injection of pROP1 only induced a Th1-specific immune response.

  8. Chikungunya virus induces IPS-1-dependent innate immune activation and protein kinase R-independent translational shutoff.

    Science.gov (United States)

    White, Laura K; Sali, Tina; Alvarado, David; Gatti, Evelina; Pierre, Philippe; Streblow, Daniel; Defilippis, Victor R

    2011-01-01

    Chikungunya virus (CHIKV) is an arthritogenic mosquito-transmitted alphavirus that is undergoing reemergence in areas around the Indian Ocean. Despite the current and potential danger posed by this virus, we know surprisingly little about the induction and evasion of CHIKV-associated antiviral immune responses. With this in mind we investigated innate immune reactions to CHIKV in human fibroblasts, a demonstrable in vivo target of virus replication and spread. We show that CHIKV infection leads to activation of the transcription factor interferon regulatory factor 3 (IRF3) and subsequent transcription of IRF3-dependent antiviral genes, including beta interferon (IFN-β). IRF3 activation occurs by way of a virus-induced innate immune signaling pathway that includes the adaptor molecule interferon promoter stimulator 1 (IPS-1). Despite strong transcriptional upregulation of these genes, however, translation of the corresponding proteins is not observed. We further demonstrate that translation of cellular (but not viral) genes is blocked during infection and that although CHIKV is found to trigger inactivation of the translational molecule eukaryotic initiation factor subunit 2α by way of the double-stranded RNA sensor protein kinase R, this response is not required for the block to protein synthesis. Furthermore, overall diminution of cellular RNA synthesis is also observed in the presence of CHIKV and transcription of IRF3-dependent antiviral genes appears specifically blocked late in infection. We hypothesize that the observed absence of IFN-β and antiviral proteins during infection results from an evasion mechanism exhibited by CHIKV that is dependent on widespread shutoff of cellular protein synthesis and a targeted block to late synthesis of antiviral mRNA transcripts.

  9. Highly efficient expression, purification of recombinant LTB protein and its activity against mucosal immunoadjuvant by nasal immunization

    Institute of Scientific and Technical Information of China (English)

    王静; 李琳琳; 郑瑾; 于军; 杨筱凤; 耿宜平; 来宝长; 王一理; 司履生

    2003-01-01

    Objective To develop an efficient expression, purification system of recombinant Escherichia coli heat-labile enterotoxin B subunit (rLTB) and study its activity against mucosal immunoadjuvant by nasal immunization.Methods A recombinant, pMMB68-LTB was generated by cloning the LTB cDNA fragment into an expression vector (pMMB68) and transformed it into the host strain marine vibrio VSP60. The relevant target protein was identified using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Sephacryl S-100 gel filtration chromatography was carried out for purification of rLTB in engineering bacteria VSP60. BALB/c mice received hen egg lysozyme (HEL) alone or combined with rLTB by nasal administration. After three times immunization, IgG and IgA antibody levels in serum or small intestine wash samples were determined using ELISA.Results rLTB protein was highly expressed in VSP60. After gel filtration with Sephacryl S-100, the purity of rLTB reached 98.1%, the yield rate was about 52%. After immunization, IgG and IgA antibody responses specific to HEL in system and mucosa of HEL+rLTB groups were significantly increased, compared with the HEL alone group (P<0.001).Conclusions A set of protocols for large-scale rLTB preparation has been established, which is simple, efficient and applicable. The rLTB protein we prepared was proved to be a powerful mucocal adjuvant, which could greatly enhance systemic and mucosal immune responses to nasally co-administered antigen.

  10. Analysis of Globodera rostochiensis effectors reveals conserved functions of SPRYSEC proteins in suppressing and eliciting plant immune responses

    KAUST Repository

    Ali, Shawkat

    2015-08-11

    Potato cyst nematodes (PCNs), including Globodera rostochiensis (Woll.), are important pests of potato. Plant parasitic nematodes produce multiple effector proteins, secreted from their stylets, to successfully infect their hosts. These include proteins delivered to the apoplast and to the host cytoplasm. A number of effectors from G. rostochiensis predicted to be delivered to the host cytoplasm have been identified, including several belonging to the secreted SPRY domain (SPRYSEC) family. SPRYSEC proteins are unique to members of the genus Globodera and have been implicated in both the induction and the repression of host defense responses. We have tested the properties of six different G. rostochiensis SPRYSEC proteins by expressing them in Nicotiana benthamiana and N. tabacum. We have found that all SPRYSEC proteins tested are able to suppress defense responses induced by NB-LRR proteins as well as cell death induced by elicitors, suggesting that defense repression is a common characteristic of members of this effector protein family. At the same time, GrSPRYSEC-15 elicited a defense responses in N. tabacum, which was found to be resistant to a virus expressing GrSPRYSEC-15. These results suggest that SPRYSEC proteins may possess characteristics that allow them to be recognized by the plant immune system.

  11. Effects of fish protein hydrolysate on growth performance and humoral immune response in large yellow croaker (Pseudosciaena crocea R.)

    Institute of Scientific and Technical Information of China (English)

    Hong-gang TANG; Tian-xing WU; Zhan-yu ZHAO; Xiao-dong PAN

    2008-01-01

    We investigated the effects of fish protein hydrolysate (FPH) on growth performance and humoral immune response of the large yellow croaker (Pseudosciaena crocea R.). One thousand and two hundred large yellow croakers [initial average weight: (162.75±23.85) g] were divided into four groups and reared in floating sea cages (3 m×3 m×3 m). The animals were fed with 4 diets: basal diet only (control) or diets supplemented with 5%, 10% and 15% (w/w) FPH. The results show that dietary FPH levels significantly influenced the growth and immunity of the large yellow croaker. Compared with the control group, total weight gain (TWG) in all treatment groups, relative weight gain (RWG) and specific growth rate (SGR) in fish fed with diets supplemerited with 10% and 15% FPH were significantly increased (P<0.05). Similar results were observed in immune parameters [lysozyme activity, serum complements, immunoglobulin M (IgM)]. Lysozyme activity, complement C4 and IgM were also significantly increased (P<0.05) in fish fed with diets supplemented with 10% and 15% FPH, while complement C3 level was significantly increased (P<0.05) in all treatment groups. In general, with the supplementation of FPH, particularly at dose of 10%,the growth performance and immunity of the large yellow croaker can be improved effectively.

  12. The Salmonella Effector Protein SopA Modulates Innate Immune Responses by Targeting TRIM E3 Ligase Family Members.

    Directory of Open Access Journals (Sweden)

    Jana Kamanova

    2016-04-01

    Full Text Available Salmonella Typhimurium stimulates inflammatory responses in the intestinal epithelium, which are essential for its ability to replicate within the intestinal tract. Stimulation of these responses is strictly dependent on the activity of a type III secretion system encoded within its pathogenicity island 1, which through the delivery of effector proteins, triggers signaling pathways leading to inflammation. One of these effectors is SopA, a HECT-type E3 ligase, which is required for the efficient stimulation of inflammation in an animal model of Salmonella Typhimurium infection. We show here that SopA contributes to the stimulation of innate immune responses by targeting two host E3 ubiquitin ligases, TRIM56 and TRIM65. We also found that TRIM65 interacts with the innate immune receptor MDA5 enhancing its ability to stimulate interferon-β signaling. Therefore, by targeting TRIM56 and TRIM65, SopA can stimulate signaling through two innate immune receptors, RIG-I and MDA5. These findings describe a Salmonella mechanism to modulate inflammatory responses by directly targeting innate immune signaling mechanisms.

  13. Dengue-1 envelope protein domain III along with PELC and CpG oligodeoxynucleotides synergistically enhances immune responses.

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    Chen-Yi Chiang

    Full Text Available The major weaknesses of subunit vaccines are their low immunogenicity and poor efficacy. Adjuvants can help to overcome some of these inherent defects with subunit vaccines. Here, we evaluated the efficacy of the newly developed water-in-oil-in-water multiphase emulsion system, termed PELC, in potentiating the protective capacity of dengue-1 envelope protein domain III. Unlike aluminum phosphate, dengue-1 envelope protein domain III formulated with PELC plus CpG oligodeoxynucleotides induced neutralizing antibodies against dengue-1 virus and increased the splenocyte secretion of IFN-γ after in vitro re-stimulation. The induced antibodies contained both the IgG1 and IgG2a subclasses. A rapid anamnestic neutralizing antibody response against a live dengue virus challenge was elicited at week 26 after the first immunization. These results demonstrate that PELC plus CpG oligodeoxynucleotides broaden the dengue-1 envelope protein domain III-specific immune responses. PELC plus CpG oligodeoxynucleotides is a promising adjuvant for recombinant protein based vaccination against dengue virus.

  14. Dietary supplementation with Lactobacilli improves emergency granulopoiesis in protein-malnourished mice and enhances respiratory innate immune response.

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    Matias Herrera

    Full Text Available This work studied the effect of protein malnutrition on the hemato-immune response to the respiratory challenge with Streptococcus pneumoniae and evaluated whether the dietary recovery with a probiotic strain has a beneficial effect in that response. Three important conclusions can be inferred from the results presented in this work: a protein-malnutrition significantly impairs the emergency myelopoiesis induced by the generation of the innate immune response against pneumococcal infection; b repletion of malnourished mice with treatments including nasally or orally administered Lactobacillus rhamnosus CRL1505 are able to significantly accelerate the recovery of granulopoiesis and improve innate immunity and; c the immunological mechanisms involved in the protective effect of immunobiotics vary according to the route of administration. The study demonstrated that dietary recovery of malnourished mice with oral or nasal administration of L. rhamnosus CRL1505 improves emergency granulopoiesis and that CXCR4/CXCR12 signaling would be involved in this effect. Then, the results summarized here are a starting point for future research and open up broad prospects for future applications of probiotics in the recovery of immunocompromised malnourished hosts.

  15. A novel homolog of protein tyrosine kinase Fyn identified in Lampetra japonica with roles in the immune response.

    Science.gov (United States)

    Zhang, Qiong; Song, Xueying; Su, Peng; Li, Ranran; Liu, Chang; Gou, Meng; Wang, Hao; Liu, Xin; Li, Qingwei

    2016-04-01

    The non-receptor protein tyrosine kinase (nrPTK) Fyn, a member of the avian sarcoma virus transforming gene (Src) kinase family, plays a very significant role in cell growth, survival, apoptosis, tumor formation and immune response. In this study, a homolog of nrPTK Fyn was identified for the first time in the lamprey, Lampetra japonica and was named "Lja-Fyn". The cDNA fragment of lamprey lja-fyn contains a 1611-bp open reading frame, which encodes a protein of 537 amino acids. Multiple sequence alignment analysis showed that it shares four conserved domains (Src homology (SH) 4, SH3, SH2 and protein kinases catalytic domains) and a variable unique domain with vertebrates Fyn molecules. Though Lja-Fyn has high sequence similarity with typical Fyn and Yes molecules of jawed vertebrates, the identities among Lja-Fyn and typical Fyn molecules in unique domain are relatively higher than that among Lja-Fyn and typical Yes molecules. The result indicates that Lja-Fyn is a homolog of Fyn rather than Yes. The phylogenetic analysis showed that Fyn, Yes and Src molecules are grouped into three distinct phylogenetic clusters, and Lja-Fyn is grouped as a single branch in Fyn cluster. The real-time quantitative PCR assay revealed the wide distribution of the lja-fyn mRNA in lamprey immune related tissues. After stimulation with mixed antigens, the levels of lja-fyn mRNA were obviously up-regulated in the gill and lymphocyte-like cells, and the similar results were got by western blot analysis of Lja-Fyn protein expression. These results indicated that nrPTK Lja-Fyn was likely to be involved in immune response. Furthermore, our present findings also provide the necessary information for understanding the distinction between lamprey Lja-Fyn and other members of jawed vertebrates in Src family.

  16. Chapter 11 Unexpected Turns and Twists in Structure/Function of PR-Proteins that Connect Energy Metabolism and Immunity

    KAUST Repository

    Narasimhan, Meena L.

    2009-01-01

    Innate immunity in plants is manifested by a complex array of antimicrobial processes that includes induction of sets of pathogenesis-related (PR) proteins. The availability of genomic data has made clear that each PR-protein family in a species is represented by several genes. Microarray data in public databases show that in most families, including the PR-5 family surveyed here, the expression of only few family members is defense associated. Genetic studies show that depending on their nutrient acquisition strategy, pathogens induce distinct but overlapping sets of PR genes, suggesting a connection to energy or resource allocation. PR-5 proteins have a clearly recognizable structure that is referred to as the thaumatin (THN) domain, which can be overlapped with mammalian Complement 1q-tumor necrosis factor (C1q-TNF) domains such as that of the mammalian hormone adiponectin. The occurrence of THN domain proteins is widespread. Similarities between THN domain proteins and mammalian C1q-TNF family proteins include their ligands and their subcellular locations. Osmotin (tobacco PR-5c) regulates energy balance signaling in mammalian cells by interaction with adiponectin receptors by a pathway that shares components with plant energy and stress signaling pathways. These data suggest additional roles for PR-5 proteins, as scaffolds and/or in signaling, particularly in regulating energy balance. © 2009 Elsevier Ltd. All rights reserved.

  17. Heat-shock protein 70 from plant biofactories of recombinant antigens activate multiepitope-targeted immune responses.

    Science.gov (United States)

    Buriani, Giampaolo; Mancini, Camillo; Benvenuto, Eugenio; Baschieri, Selene

    2012-04-01

    Although a physiological role of heat-shock proteins (HSP) in antigen presentation and immune response activation has not been directly demonstrated, their use as vaccine components is under clinical trial. We have previously demonstrated that the structure of plant-derived HSP70 (pHSP70) can be superimposed to the mammalian homologue and similarly to the mammalian counterpart, pHSP70-polypeptide complexes can activate the immune system. It is here shown that pHSP70 purified from plant tissues transiently expressing the influenza virus nucleoprotein are able to induce both the activation of major histocompatibility complex class I-restricted polyclonal T-cell responses and antibody production in mice of different haplotypes without the need of adjuvant co-delivery. These results indicate that pHSP70 derived from plants producing recombinant antigens may be used to formulate multiepitope vaccines.

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

    Science.gov (United States)

    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.

  19. Proteins of Leishmania (Viannia shawi confer protection associated with Th1 immune response and memory generation

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    Passero Luiz Felipe D

    2012-03-01

    Full Text Available Abstract Background Leishmania (Viannia shawi parasite was first characterized in 1989. Recently the protective effects of soluble leishmanial antigen (SLA from L. (V. shawi promastigotes were demonstrated using BALB/c mice, the susceptibility model for this parasite. In order to identify protective fractions, SLA was fractionated by reverse phase HPLC and five antigenic fractions were obtained. Methods F1 fraction was purified from L. (V. shawi parasite extract by reverse phase HPLC. BALB/c mice were immunized once a week for two consecutive weeks by subcutaneous routes in the rump, using 25 μg of F1. After 1 and 16 weeks of last immunization, groups were challenged in the footpad with L. (V. shawi promastigotes. After 2 months, those same mice were sacrificed and parasite burden, cellular and humoral immune responses were evaluated. Results The F1 fraction induced a high degree of protection associated with an increase in IFN-γ, a decrease in IL-4, increased cell proliferation and activation of CD8+T lymphocytes. Long-term protection was acquired in F1-immunized mice, associated with increased CD4+ central memory T lymphocytes and activation of both CD4+ and CD8+ T cells. In addition, F1-immunized groups showed an increase in IgG2a levels. Conclusions The inductor capability of antigens to generate memory lymphocytes that can proliferate and secrete beneficial cytokines upon infection could be an important factor in the development of vaccine candidates against American Tegumentary Leishmaniasis.

  20. Culture and the Immune System: Cultural Consonance in Social Support and C-reactive Protein in Urban Brazil.

    Science.gov (United States)

    Dressler, William W; Balieiro, Mauro C; Ribeiro, Rosane P; Dos Santos, José Ernesto

    2016-06-01

    In this article, we examine the distribution of a marker of immune system stimulation-C-reactive protein-in urban Brazil. Social relationships are associated with immunostimulation, and we argue that cultural dimensions of social support, assessed by cultural consonance, are important in this process. Cultural consonance is the degree to which individuals, in their own beliefs and behaviors, approximate shared cultural models. A measure of cultural consonance in social support, based on a cultural consensus analysis regarding sources and patterns of social support in Brazil, was developed. In a survey of 258 persons, the association of cultural consonance in social support and C-reactive protein was examined, controlling for age, sex, body mass index, low-density lipoprotein cholesterol, depressive symptoms, and a social network index. Lower cultural consonance in social support was associated with higher C-reactive protein. Implications of these results for future research are discussed.

  1. Immunization with recombinant Pb27 protein reduces the levels of pulmonary fibrosis caused by the inflammatory response against Paracoccidioides brasiliensis.

    Science.gov (United States)

    Morais, Elis Araujo; Martins, Estefânia Mara do Nascimento; Boelone, Jankerle Neves; Gomes, Dawidson Assis; Goes, Alfredo Miranda

    2015-02-01

    Paracoccidioidomycosis (PCM) is a systemic mycosis in which the host response to the infectious agent typically consists of a chronic granulomatous inflammatory process. This condition causes lesions that impair lung function and lead to chronic pulmonary insufficiency resulting from fibrosis development, which is a sequel and disabling feature of the disease. The rPb27 protein has been studied for prophylactic and therapeutic treatment against PCM. Previous studies from our laboratory have shown a protective effect of rPb27 against PCM. However, these studies have not determined whether rPb27 immunization prevents lung fibrosis. We therefore conducted this study to investigate fibrosis resulting from infection by Paracoccidioides brasiliensis in the lungs of animals immunized with rPb27. Animals were immunized with rPb27 and subsequently infected with a virulent strain of P. brasiliensis. Fungal load was evaluated by counting colony-forming units, and Masson's trichrome staining was performed to evaluate fibrosis at 30 and 90 days post-infection. The levels of CCR7, active caspase 3, collagen and cytokines were analyzed. At the two time intervals mentioned, the rPb27 group showed lower levels of fibrosis on histology and reduced levels of collagen and the chemokine receptor CCR7 in the lungs. CCR7 was detected at higher levels in the control groups that developed very high levels of pulmonary fibrosis. Additionally, the immunized groups showed high levels of active caspase 3, IFN-γ, TGF-β and IL-10 in the early phase of P. brasiliensis infection. Immunization with Pb27, in addition to its protective effect, was shown to prevent pulmonary fibrosis.

  2. Rickettsia rickettsii outer membrane protein YbgF induces protective immunity in C3H/HeN mice.

    Science.gov (United States)

    Gong, Wenping; Qi, Yong; Xiong, Xiaolu; Jiao, Jun; Duan, Changsong; Wen, Bohai

    2015-01-01

    Rickettsia rickettsii is the etiological agent of Rocky Mountain spotted fever (RMSF). YbgF and TolC are outer membrane-associated proteins of R. rickettsii that play important roles in its interaction with host cells. We investigated the immunogenicity of YbgF and TolC for protection against RMSF. We immunized C3H/HeN mice with recombinant R. rickettsii YbgF (rYbgF) or TolC (rTolC). Rickettsial burden and impairment in the lungs, spleens, and livers of rYbgF-immunized mice were significantly lower than in rTolC-immunized mice. The ratio of IgG2a to IgG1 in rYbgF-immunized mice continued to increase over the course of our experiments, while that in rTolC-immunized mice was reduced. The proliferation and cytokine secretion of CD4(+) and CD8(+) T cells isolated from R. rickettsii-infected mice were analyzed following antigen stimulation. The results indicated that proliferation and interferon (IFN)-γ secretion of CD4(+) or CD8(+) T cells in R. rickettsii-infected mice were significantly greater than in uninfected mice after stimulation with rYbgF. YbgF is a novel protective antigen of R. rickettsii. Protection conferred by YbgF is dependent upon IFN-γ-producing CD4(+) and CD8(+) T cells and IgG2a, which act in synergy to control R. rickettsii infection.

  3. Immunization with FSHβ fusion protein antigen prevents bone loss in a rat ovariectomy-induced osteoporosis model

    Energy Technology Data Exchange (ETDEWEB)

    Geng, Wenxin; Yan, Xingrong; Du, Huicong; Cui, Jihong; Li, Liwen, E-mail: liven@nwu.edu.cn; Chen, Fulin, E-mail: chenfl@nwu.edu.cn

    2013-05-03

    Highlights: •A GST-FSH fusion protein was successfully expressed in E. coli. •Immunization with GST-FSH antigen can raise high-titer anti-FSH polyclonal sera. •Anti-FSH polyclonal sera can neutralize osteoclastogenic effect of FSH in vitro. •FSH immunization can prevent bone loss in a rat osteoporosis model. -- Abstract: Osteoporosis, a metabolic bone disease, threatens postmenopausal women globally. Hormone replacement therapy (HTR), especially estrogen replacement therapy (ERT), is used widely in the clinic because it has been generally accepted that postmenopausal osteoporosis is caused by estrogen deficiency. However, hypogonadal α and β estrogen receptor null mice were only mildly osteopenic, and mice with either receptor deleted had normal bone mass, indicating that estrogen may not be the only mediator that induces osteoporosis. Recently, follicle-stimulating hormone (FSH), the serum concentration of which increases from the very beginning of menopause, has been found to play a key role in postmenopausal osteoporosis by promoting osteoclastogenesis. In this article, we confirmed that exogenous FSH can enhance osteoclast differentiation in vitro and that this effect can be neutralized by either an anti-FSH monoclonal antibody or anti-FSH polyclonal sera raised by immunizing animals with a recombinant GST-FSHβ fusion protein antigen. Moreover, immunizing ovariectomized rats with the GST-FSHβ antigen does significantly prevent trabecular bone loss and thereby enhance the bone strength, indicating that a FSH-based vaccine may be a promising therapeutic strategy to slow down bone loss in postmenopausal women.

  4. Partial protective effect of intranasal immunization with recombinant Toxoplasma gondii rhoptry protein 17 against toxoplasmosis in mice.

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    Hai-Long Wang

    Full Text Available Toxoplasma gondii (T. gondii is an obligate intracellular protozoan parasite that infects a variety of mammals, including humans. An effective vaccine for this parasite is therefore needed. In this study, RH strain T. gondii rhoptry protein 17 was expressed in bacteria as a fusion with glutathione S-transferase (GST and the recombinant proteins (rTgROP17 were purified via GST-affinity chromatography. BALB/c mice were nasally immunised with rTgROP17, and induction of immune responses and protection against chronic and lethal T. gondii infections were investigated. The results revealed that mice immunised with rTgROP17 produced high levels of specific anti-rTgROP17 IgGs and a mixed IgG1/IgG2a response of IgG2a predominance. The systemic immune response was associated with increased production of Th1 (IFN-γand IL-2 and Th2 (IL-4 cytokines, and enhanced lymphoproliferation (stimulation index, SI in the mice immunised with rTgROP17. Strong mucosal immune responses with increased secretion of TgROP17-specific secretory IgA (SIgA in nasal, vaginal and intestinal washes were also observed in these mice. The vaccinated mice displayed apparent protection against chronic RH strain infection as evidenced by their lower liver and brain parasite burdens (59.17% and 49.08%, respectively than those of the controls. The vaccinated mice also exhibited significant protection against lethal infection of the virulent RH strain (survival increased by 50% compared to the controls. Our data demonstrate that rTgROP17 can trigger strong systemic and mucosal immune responses against T. gondii and that ROP17 is a promising candidate vaccine for toxoplasmosis.

  5. Vaccination with TAT-antigen fusion protein induces protective, CD8(+) T cell-mediated immunity against Leishmania major.

    Science.gov (United States)

    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.

  6. Inflammasome adaptor protein Apoptosis-associated speck-like protein containing CARD (ASC) is critical for the immune response and survival in west Nile virus encephalitis.

    Science.gov (United States)

    Kumar, Mukesh; Roe, Kelsey; Orillo, Beverly; Muruve, Daniel A; Nerurkar, Vivek R; Gale, Michael; Verma, Saguna

    2013-04-01

    West Nile virus (WNV) is a neurotropic flavivirus that has emerged globally as a significant cause of viral encephalitis in humans. The WNV-induced innate immune response, including production of antiviral cytokines, is critical for controlling virus infection. The adaptor protein ASC mediates a critical step in innate immune signaling by bridging the interaction between the pathogen recognition receptors and caspase 1 in inflammasome complexes, but its role in WNV immunopathogenesis is not defined. Here, we demonstrate that ASC is essential for interleukin-1β (IL-1β) production and development of effective host immunity against WNV. ASC-deficient mice exhibited increased susceptibility to WNV infection, and reduced survival was associated with enhanced virus replication in the peripheral tissues and central nervous system (CNS). Infection of cultured bone marrow-derived dendritic cells showed that ASC was essential for the activation of caspase 1, a key component of inflammasome assembly. ASC(-/-) mice exhibited attenuated levels of proinflammatory cytokines in the serum. Intriguingly, infected ASC(-/-) mice also displayed reduced levels of alpha interferon (IFN-α) and IgM in the serum, indicating the overall protective role of ASC in restricting WNV infection. However, brains from ASC(-/-) mice displayed unrestrained inflammation, including elevated levels of proinflammatory cytokines and chemokines, such as IFN-γ, CCL2, and CCL5, which correlated with more pronounced activation of the astrocytes, enhanced infiltration of peripheral immune cells in the CNS, and increased neuronal cell death. Collectively, our data provide new insights into the role of ASC as an essential modulator of inflammasome-dependent and -independent immune responses to effectively control WNV infection.

  7. HIV-1 structural proteins serve as PAMPs for TLR2 heterodimers significantly increasing infection and innate immune activation

    Directory of Open Access Journals (Sweden)

    Kenneth Lee Rosenthal

    2015-08-01

    Full Text Available Immune activation is critical to HIV infection and pathogenesis; however, our understanding of HIV innate immune activation remains incomplete. Recently we demonstrated that soluble TLR2 (sTLR2 physically inhibited HIV-induced, NFκB activation and inflammation, as well as HIV-1 infection. In light of these findings, we hypothesized that HIV-1 structural proteins may serve as pathogen-associated molecular patterns (PAMPs for cellular TLR2 heterodimers. These studies made use of primary human T cells and TZMbl cells stably transformed to express TLR2 (TZMbl-2. Our results demonstrated that cells expressing TLR2 showed significantly increased proviral DNA compared to cells lacking TLR2, and mechanistically this may be due to a TLR2-mediated increased CCR5 expression. Importantly, we show that HIV-1 structural proteins, p17, p24, and gp41 act as viral PAMPs signalling through TLR2 and its heterodimers leading to significantly increased immune activation via the NFκB signaling pathway. Using co-immunoprecipitation and a dot blot method, we demonstrated direct protein interactions between these viral PAMPs and TLR2, while only p17 and gp41 bound to TLR1. Specifically, TLR2/1 heterodimer recognized p17 and gp41, while p24 lead to immune activation through TLR2/6. These results were confirmed using TLR2/1 siRNA knock down assays which ablated p17 and gp41-induced cellular activation and through studies of HEK293 cells expressing selected TLRs. Interestingly, our results show in the absence of TLR6, p24 bound to TLR2 and blocked p17 and gp41-induced activation, thus providing a novel mechanism by which HIV-1 can manipulate innate sensing. Taken together, our results identified, for the first time, novel HIV-1 PAMPs that play a role in TLR2-mediated cellular activation and increased proviral DNA. These findings have important implications for our fundamental understanding of HIV-1 immune activation and pathogenesis, as well as HIV-1 vaccine development.

  8. Antimicrobial proteins from snake venoms: direct bacterial damage and activation of innate immunity against Staphylococcus aureus skin infection.

    Science.gov (United States)

    Samy, R P; Stiles, B G; Gopalakrishnakone, P; Chow, V T K

    2011-01-01

    The innate immune system is the first line of defense against microbial diseases. Antimicrobial proteins produced by snake venoms have recently attracted significant attention due to their relevance to bacterial infection and potential development into new therapeutic agents. Staphylococcus aureus is one of the major human pathogens causing a variety of infections involving pneumonia, toxic shock syndrome, and skin lesions. With the recent emergence of methicillin (MRSA) and vancomycin (VRSA) resistance, S. aureus infection is a serious clinical problem that will have a grave socio-economic impact in the near future. Although S. aureus susceptibility to innate antimicrobial peptides has been reported recently, the protective effect of snake venom phospholipase A₂ (svPLA₂) proteins on the skin from S. aureus infection has been understudied. This review details the protective function of svPLA₂s derived from venoms against skin infections caused by S. aureus. We have demonstrated in vivo that local application of svPLA₂ provides complete clearance of S. aureus within 2 weeks after treatment compared to fusidic acid ointment (FAO). In vitro experiments also demonstrate that svPLA₂ proteins have inhibitory (bacteriostatic) and killing (bactericidal) effects on S. aureus in a dose-dependant manner. The mechanism of bacterial membrane damage and perturbation was clearly evidenced by electron microscopic studies. In summary, svPLA₂s from Viperidae and Elapidae snakes are novel molecules that can activate important mechanisms of innate immunity in animals to endow them with protection against skin infection caused by S. aureus.

  9. Immunization with a streptococcal multiple-epitope recombinant protein protects mice against invasive group A streptococcal infection

    Science.gov (United States)

    Kuo, Chih-Feng; Tsao, Nina; Hsieh, I-Chen; Lin, Yee-Shin; Wu, Jiunn-Jong; Hung, Yu-Ting

    2017-01-01

    Streptococcus pyogenes (group A Streptococcus; GAS) causes clinical diseases, including pharyngitis, scarlet fever, impetigo, necrotizing fasciitis and streptococcal toxic shock syndrome. A number of group A streptococcus vaccine candidates have been developed, but only one 26-valent recombinant M protein vaccine has entered clinical trials. Differing from the design of a 26-valent recombinant M protein vaccine, we provide here a vaccination using the polyvalence epitope recombinant FSBM protein (rFSBM), which contains four different epitopes, including the fibronectin-binding repeats domain of streptococcal fibronectin binding protein Sfb1, the C-terminal immunogenic segment of streptolysin S, the C3-binding motif of streptococcal pyrogenic exotoxin B, and the C-terminal conserved segment of M protein. Vaccination with the rFSBM protein successfully prevented mortality and skin lesions caused by several emm strains of GAS infection. Anti-FSBM antibodies collected from the rFSBM-immunized mice were able to opsonize at least six emm strains and can neutralize the hemolytic activity of streptolysin S. Furthermore, the internalization of GAS into nonphagocytic cells is also reduced by anti-FSBM serum. These findings suggest that rFSBM can be applied as a vaccine candidate to prevent different emm strains of GAS infection. PMID:28355251

  10. Multidimensional protein fractionation using ProteomeLab PF 2D™ for profiling amyotrophic lateral sclerosis immunity: A preliminary report

    Directory of Open Access Journals (Sweden)

    Mosley R Lee

    2008-09-01

    Full Text Available Abstract Background The ProteomeLab™ PF 2D platform is a relatively new approach to global protein profiling. Herein, it was used for investigation of plasma proteome changes in amyotrophic lateral sclerosis (ALS patients before and during immunization with glatiramer acetate (GA in a clinical trial. Results The experimental design included immunoaffinity depletion of 12 most abundant proteins from plasma samples with the ProteomeLab™ IgY-12 LC10 column kit as first dimension separation, also referred to as immuno-partitioning. Second and third dimension separations of the enriched proteome were performed on the PF 2D platform utilizing 2D isoelectric focusing and RP-HPLC with the resulting fractions collected for analysis. 1D gel electrophoresis was added as a fourth dimension when sufficient protein was available. Protein identification from collected fractions was performed using nano-LC-MS/MS approach. Analysis of differences in the resulting two-dimensional maps of fractions obtained from the PF 2D and the ability to identify proteins from these fractions allowed sensitivity threshold measurements. Masked proteins in the PF 2D fractions are discussed. Conclusion We offer some insight into the strengths and limitations of this emerging proteomic platform.

  11. Regulation of in vitro and in vivo immune functions by the cytosolic adaptor protein SKAP-HOM.

    Science.gov (United States)

    Togni, M; Swanson, K D; Reimann, S; Kliche, S; Pearce, A C; Simeoni, L; Reinhold, D; Wienands, J; Neel, B G; Schraven, B; Gerber, A

    2005-09-01

    SKAP-HOM is a cytosolic adaptor protein representing a specific substrate for the Src family protein tyrosine kinase Fyn. Previously, several groups have provided experimental evidence that SKAP-HOM (most likely in cooperation with the cytosolic adaptor protein ADAP) is involved in regulating leukocyte adhesion. To further assess the physiological role of SKAP-HOM, we investigated the immune system of SKAP-HOM-deficient mice. Our data show that T-cell responses towards a variety of stimuli are unaffected in the absence of SKAP-HOM. Similarly, B-cell receptor (BCR)-mediated total tyrosine phosphorylation and phosphorylation of Erk, p38, and JNK, as well as immunoreceptor-mediated Ca(2+) responses, are normal in SKAP-HOM(-/-) animals. However, despite apparently normal membrane-proximal signaling events, BCR-mediated proliferation is strongly attenuated in the absence of SKAP-HOM(-/-). In addition, adhesion of activated B cells to fibronectin (a ligand for beta1 integrins) as well as to ICAM-1 (a ligand for beta2 integrins) is strongly reduced. In vivo, the loss of SKAP-HOM results in a less severe clinical course of experimental autoimmune encephalomyelitis following immunization of mice with the encephalitogenic peptide of MOG (myelin oligodendrocyte glycoprotein). This is accompanied by strongly reduced serum levels of MOG-specific antibodies and lower MOG-specific T-cell responses. In summary, our data suggest that SKAP-HOM is required for proper activation of the immune system, likely by regulating the cross-talk between immunoreceptors and integrins.

  12. Immune complex modulation by plasma proteins. With special reference to the complement system and autoimmune diseases

    DEFF Research Database (Denmark)

    Baatrup, G

    1989-01-01

    The complement (C) system consists of two activation pathways, the classical and the alternative, which may both be activated by immune complexes (IC). C activation products become attached to the IC during activation leading to profound changes in the properties of the complexes. The common term...

  13. Interleukin-17-induced protein lipocalin 2 is dispensable for immunity to oral candidiasis.

    Science.gov (United States)

    Ferreira, Maria Carolina; Whibley, Natasha; Mamo, Anna J; Siebenlist, Ulrich; Chan, Yvonne R; Gaffen, Sarah L

    2014-03-01

    Oropharyngeal candidiasis (OPC; thrush) is an opportunistic fungal infection caused by the commensal microbe Candida albicans. Immunity to OPC is strongly dependent on CD4+ T cells, particularly those of the Th17 subset. Interleukin-17 (IL-17) deficiency in mice or humans leads to chronic mucocutaneous candidiasis, but the specific downstream mechanisms of IL-17-mediated host defense remain unclear. Lipocalin 2 (Lcn2; 24p3; neutrophil gelatinase-associated lipocalin [NGAL]) is an antimicrobial host defense factor produced in response to inflammatory cytokines, particularly IL-17. Lcn2 plays a key role in preventing iron acquisition by bacteria that use catecholate-type siderophores, and lipocalin 2(-/-) mice are highly susceptible to infection by Escherichia coli and Klebsiella pneumoniae. The role of Lcn2 in mediating immunity to fungi is poorly defined. Accordingly, in this study, we evaluated the role of Lcn2 in immunity to oral infection with C. albicans. Lcn2 is strongly upregulated following oral infection with C. albicans, and its expression is almost entirely abrogated in mice with defective IL-17 signaling (IL-17RA(-/-) or Act1(-/-) mice). However, Lcn2(-/-) mice were completely resistant to OPC, comparably to wild-type (WT) mice. Moreover, Lcn2 deficiency mediated protection from OPC induced by steroid immunosuppression. Therefore, despite its potent regulation during C. albicans infection, Lcn2 is not required for immunity to mucosal candidiasis.

  14. Immune evasion by varicelloviruses : the identification of a new family of TAP-inhibiting proteins

    NARCIS (Netherlands)

    Koppers-Lalić, Danijela

    2007-01-01

    From the earliest times of their evolution, multi-cellular organisms have been defending themselves against infectious agents like nucleic acids, viruses, bacteria, fungi and parasites. Continuous selection pressure resulted in the development of sophisticated immune systems, which in their adaptive

  15. Apoplastic venom allergen-like proteins of cyst nematodes modulate the activation of basal plant innate immunity by cell surface receptors.

    Science.gov (United States)

    Lozano-Torres, Jose L; Wilbers, Ruud H P; Warmerdam, Sonja; Finkers-Tomczak, Anna; Diaz-Granados, Amalia; van Schaik, Casper C; Helder, Johannes; Bakker, Jaap; Goverse, Aska; Schots, Arjen; Smant, Geert

    2014-12-01

    Despite causing considerable damage to host tissue during the onset of parasitism, nematodes establish remarkably persistent infections in both animals and plants. It is thought that an elaborate repertoire of effector proteins in nematode secretions suppresses damage-triggered immune responses of the host. However, the nature and mode of action of most immunomodulatory compounds in nematode secretions are not well understood. Here, we show that venom allergen-like proteins of plant-parasitic nematodes selectively suppress host immunity mediated by surface-localized immune receptors. Venom allergen-like proteins are uniquely conserved in secretions of all animal- and plant-parasitic nematodes studied to date, but their role during the onset of parasitism has thus far remained elusive. Knocking-down the expression of the venom allergen-like protein Gr-VAP1 severely hampered the infectivity of the potato cyst nematode Globodera rostochiensis. By contrast, heterologous expression of Gr-VAP1 and two other venom allergen-like proteins from the beet cyst nematode Heterodera schachtii in plants resulted in the loss of basal immunity to multiple unrelated pathogens. The modulation of basal immunity by ectopic venom allergen-like proteins in Arabidopsis thaliana involved extracellular protease-based host defenses and non-photochemical quenching in chloroplasts. Non-photochemical quenching regulates the initiation of the defense-related programmed cell death, the onset of which was commonly suppressed by venom allergen-like proteins from G. rostochiensis, H. schachtii, and the root-knot nematode Meloidogyne incognita. Surprisingly, these venom allergen-like proteins only affected the programmed cell death mediated by surface-localized immune receptors. Furthermore, the delivery of venom allergen-like proteins into host tissue coincides with the enzymatic breakdown of plant cell walls by migratory nematodes. We, therefore, conclude that parasitic nematodes most likely utilize

  16. Reduction of Factor VIII Inhibitor Titers During Immune Tolerance Induction With Recombinant Factor VIII-Fc Fusion Protein.

    Science.gov (United States)

    Groomes, Charles L; Gianferante, David M; Crouch, Gary D; Parekh, Dina S; Scott, David W; Lieuw, Kenneth

    2016-05-01

    The development of inhibitors toward factor VIII (FVIII) is a common and serious complication of hemophilia A (HA) therapy. Patients with hemophilia who develop inhibitors often undergo time- and resource-intensive immune tolerance induction (ITI) protocols. We report a 15-month-old male with severe HA and a high-titer inhibitor that occurred while receiving prophylactic treatment with recombinant FVIII (rFVIII), in whom significant inhibitor titer reduction was achieved with thrice weekly infusions of a new, prolonged half-life rFVIII-Fc fusion protein product (trade name Eloctate). Further studies are warranted to explore the potential of Eloctate in ITI protocols.

  17. Structural Basis for the Development of Avian Virus Capsids That Display Influenza Virus Proteins and Induce Protective Immunity

    OpenAIRE

    Pascual, Elena; Mata, Carlos P.; Gómez-Blanco, Josué; Moreno, Noelia; Bárcena, Juan; Blanco, Esther; Rodríguez-Frandsen, Ariel; Nieto, Amelia; Carrascosa, José L.; Castón, José R.

    2014-01-01

    Bioengineering of viruses and virus-like particles (VLPs) is a well-established approach in the development of new and improved vaccines against viral and bacterial pathogens. We report here that the capsid of a major avian pathogen, infectious bursal disease virus (IBDV), can accommodate heterologous proteins to induce protective immunity. The structural units of the ∼70-nm-diameter T=13 IBDV capsid are trimers of VP2, which is made as a precursor (pVP2). The pVP2 C-terminal domain has an am...

  18. 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.

    Directory of Open Access Journals (Sweden)

    Jacqueline Araújo Fiuza

    2016-01-01

    Full Text Available 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.

  19. 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

    Science.gov (United States)

    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

  20. Intramammary Immunization of Pregnant Mice with Staphylococcal Protein A Reduces the Post-Challenge Mammary Gland Bacterial Load but Not Pathology.

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    Jully Gogoi-Tiwari

    Full Text Available Protein A, encoded by the spa gene, is one of the major immune evading MSCRAMM of S. aureus, demonstrated to be prevalent in a significant percentage of clinical bovine mastitis isolates in Australia. Given its' reported significance in biofilm formation and the superior performance of S. aureus biofilm versus planktonic vaccine in the mouse mastitis model, it was of interest to determine the immunogenicity and protective potential of Protein A as a potential vaccine candidate against bovine mastitis using the mouse mastitis model. Pregnant Balb/c mice were immunised with Protein A emulsified in an alum-based adjuvant by subcutaneous (s/c or intramammary (i/mam routes. While humoral immune response of mice post-immunization were determined using indirect ELISA, cell-mediated immune response was assessed by estimation of interferon-gamma (IFN-γ produced by protein A-stimulated splenocyte supernatants. Protective potential of Protein A against experimental mastitis was determined by challenge of immunized versus sham-vaccinated mice by i/mam route, based upon manifestation of clinical symptoms, total bacterial load and histopathological damage to mammary glands. Significantly (p<0.05 higher levels of IgG1 isotype were produced in mice immunized by the s/c route. In contrast, significantly higher levels of the antibody isotype IgG2a were produced in mice immunized by the i/mam route (p<0.05. There was significant reduction (p<0.05 in bacterial loads of the mammary glands of mice immunized by Protein A regardless of the route of immunization, with medium level of clinical symptoms observed up to day 3 post-challenge. However, Protein A vaccine failed to protect immunized mice post-challenge with biofilm producing encapsulated S. aureus via i/mam route, regardless of the route of immunization, as measured by the level of mammary tissue damage. It was concluded that, Protein A in its' native state was apparently not a suitable candidate for inclusion

  1. Immunization of Male Mice with a New Recombinant GnRH Fusion Protein Reduces the Testicular Function

    Institute of Scientific and Technical Information of China (English)

    FANG Fu-gui; YANG Ya-ping; LIU Ya; ZHANG Yun-hai; TAO Yong; WANG Suo-lu; PU Yong; ZHANG Xiao-rong

    2009-01-01

    The objective of the present study was to evaluate the effect of an immunogenie maltose-binding protein-gonadotropin releasing hormone (GnRH-6-MBP) using genetic engineering. The synthetic mammalian tandem repeated GnRH hexamer gene was inserted into the expression plasmid pMAL-c2x. Recombinant GnRH-6-MBP protein was over-expressed in E.coli strain BL21. Amylose resin with affinity chromatograph was used to purify target protein. The reaetiongenicity of fusion protein was identified by indirect enzyme linked immunosorbent assay (ELISA), and the antigenicity and biological effects of GnRH-6-MBP were tested in mice. In the experiment, 20 male Kunming white mice of 20 d old were randomly divided into treatment and control group. Ten mice were immunized with 100 μgGnRH-6-MBP administered subcutaneously (s.c.) thrice at 2-week intervals with GnRH-6-MBP. Mice were sacrificed after 3 weeks following the booster injection, the testis was removed, weighed and measured, and the histological structure was observed. The reactiongenieity of fusion protein to GnRH antibody was much higher than the control.Active immunization against GnRH-6-MBP reduced remarkably (P < 0.01) the length and weight of the testis, and shortened the girth and width of the testis (P < 0.05), and suppressed testieular spermatogenesis compared to the control mice. These results indicate that the recombinant GnRH-6-MBP acted as a strong immunogen and caused atrophy of the testis.

  2. Newcastle disease virus fusion protein is the major contributor to protective immunity of genotype-matched vaccine.

    Science.gov (United States)

    Kim, Shin-Hee; Wanasen, Nanchaya; Paldurai, Anandan; Xiao, Sa; Collins, Peter L; Samal, Siba K

    2013-01-01

    Virulent strains of Newcastle disease virus (NDV) can cause devastating disease in chickens worldwide. Although the current vaccines are substantially effective, they do not completely prevent infection, virus shedding and disease. To produce genotype-matched vaccines, a full-genome reverse genetics system has been used to generate a recombinant virus in which the F protein cleavage site has been changed to that of avirulent vaccine virus. In the other strategy, the vaccines have been generated by replacing the F and HN genes of a commercial vaccine strain with those from a genotype-matched virus. However, the protective efficacy of a chimeric virus vaccine has not been directly compared with that of a full-genome virus vaccine developed by reverse genetics. Therefore, in this study, we evaluated the protective efficacy of genotype VII matched chimeric vaccines by generating three recombinant viruses based on avirulent LaSota (genotype II) strain in which the open reading frames (ORFs) encoding the F and HN proteins were replaced, individually or together, with those of the circulating and highly virulent Indonesian NDV strain Ban/010. The cleavage site of the Ban/010 F protein was mutated to the avirulent motif found in strain LaSota. In vitro growth characteristics and a pathogenicity test indicated that all three chimeric viruses retained the highly attenuated phenotype of the parental viruses. Immunization of chickens with chimeric and full-length genome VII vaccines followed by challenge with virulent Ban/010 or Texas GB (genotype II) virus demonstrated protection against clinical disease and death. However, only those chickens immunized with chimeric rLaSota expressing the F or F plus HN proteins of the Indonesian strain were efficiently protected against shedding of Ban/010 virus. Our findings showed that genotype-matched vaccines can provide protection to chickens by efficiently preventing spread of virus, primarily due to the F protein.

  3. Humoral immune response against contractile proteins (actin and myosin) during cardiovascular disease.

    Science.gov (United States)

    De Scheerder, I K; De Buyzere, M; Delanghe, J; Maas, A; Clement, D L; Wieme, R

    1991-08-01

    Sensitive and highly specific ELISA assays were developed to determine humoral immune response against actin and myosin in 122 patients suffering from various cardiovascular diseases: acute viral myocarditis (n = 10, MYO), acute myocardial infarction (n = 28, AMI), valve surgery (n = 35, VALVE), coronary bypass surgery (n = 35, CABG), and peripheral vascular surgery (n = 14, VASC). Anti-actin and anti-myosin antibodies were determined on admission and serially during a period of 90 days. Anti-actin and anti-myosin immune response (IgG, IgM) was expressed comparing absorbance of the patients' serum with a reference serum. In the different patient groups significantly (P less than 0.01) higher anti-actin and anti-myosin antibody concentrations were found on admission compared with age-matched control groups. During follow-up, all patient groups except the vascular surgery group showed a significant immune response against actin and myosin, with an immune response ratio (peak/admission) for AMA IgG and IgM respectively of 2.12 and 2.40 in the VALVE group, 1.30 and 1.99 in the CABG group, 1.42 and 1.48 in the AMI group and 1.66 and 1.25 in the MYO group; and for AAA IgG and IgM respectively of 1.57 and 3.00 in the VALVE group, 1.54 and 1.64 in the CABG group, 1.25 and 1.07 in the AMI group, and 1.42 and 1.42 in the MYO group. A significant correlation between pre-cardiac injury and peak post-cardiac injury anti-myosin and anti-actin autoantibody levels could be demonstrated suggesting that pre-injury sensitization to these antigens plays an important role in evoking post-cardiac injury immune response.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Preparation and immune activity analysis of H5N1 subtype avian influenza virus recombinant protein-based vaccine.

    Science.gov (United States)

    Xie, Q M; Ji, J; Du, L Q; Cao, Y C; Wei, L; Xue, C Y; Qin, J P; Ma, J Y; Bi, Y Z

    2009-08-01

    Avian influenza is a severe disease among farmed poultry and free-living birds and a constant threat to the commercial chicken industry around the world. Hemagglutinin (HA) is the major immunogen on the envelope of influenza A virus and is the predominant inducer of neutralizing antibody. To obtain the bioactive antigen proteins in large quantities, a new protein expression vector pBCX was constructed, which is based on the pET32a vector. The HA gene of the H5N1 subtype of avian influenza virus (AIV) was inserted into the pBCX vector and expressed efficiently in Escherichia coli BL21 (DE3). Fused expression of the exogenous gene and msyB produced a 97-kDa msyB-HA fusion protein. Sodium dodecyl sulfate-PAGE combined with scanning analysis demonstrated that the msyB-HA fusion protein accounted for 29.5% of the total bacterial protein, 90.5% being soluble. The msyB-HA fusion protein was purified with nondenaturing 50% Ni-NTA column chromatography, and the result showed that 24 mg of purified msyB-HA fusion protein could be obtained from 1 L of induced expression bacterial culture medium. The comparative results in the present study showed that pBCX was superior to pET32a as a protein expression vector. Western blotting showed the recombinant msyB-HA (rHA) to have better antigenic activity, which may be the result from the better posttranslation protein modification and folding in the pBCX expression system. With the rHA fusion protein as antigen, we successfully prepared and screened specific monoclonal antibodys against the H5N1 subtype AIV, which indicated that the rHA had antigen epitopes and biofunctions. The immune test confirmed that the rHA protein vaccine could also induce high neutralizing antibodies, and the AIV challenge test proved that the rHA protein-based vaccine could prevent the corresponding infection. This study demonstrates that the recombinant HA protein produced by the pBCX expression system could be used as a recombinant protein-based vaccine

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

    NARCIS (Netherlands)

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

    2016-01-01

    Food texture can be improved by enzyme-mediated covalent cross-linking of different food components, such as proteins and carbohydrates. Cross-linking changes the biological and immunological properties of proteins and may change the sensitizing potential of food allergens. In this study we applied

  6. Circulating immune complexes, immunoglobulin G, salivary proteins and salivary immunoglobulin A in patients with Sjögren's syndrome

    Directory of Open Access Journals (Sweden)

    Hadži-Mihailović Miloš

    2009-01-01

    Full Text Available Introduction. Sjögren's syndrome (SS is a chronic autoimmune disorder, with its major clinical manifestations resulting from changes in exocrine glands. Objective The aim of this study was to evaluate serum concentrations of circulating immune complexes (CIC and immunoglobulin G (IgG, and salivary proteins (SP and salivary immunoglobulin A (sIgA in 40 patients with SS, and to correlate these values among themselves, as well as with the unstimulated salivary flow rate (USFR and the duration of disease. Methods. The total of 40 patients were included in this research. CIC was determined using the solution of polyethylene glycol and IgG with the standard procedure of radial immunodiffusion. SP was investigated by the method of Lowry and sIgA was separated from the whole saliva using the method of immune chromatography. Results. The values of most of the studied parameters exceeded the normal range in a high degree: CIC 72.5%, IgG 70%, SP 80%. The concentrations of CIC were significantly higher in the patients with the duration of disease less than 10 years. With the decrease of USFR, the concentration of sIgA and IgG were increased with statistical significance. Conclusion The increased prevalence of abnormal values of CIC, IgG and SP indicate that the patients with SS have developed a higher level of immune reactivity. These results could be useful in diagnosis and disease activity monitoring.

  7. Immunization with HBsAg-Fc fusion protein induces a predominant production of Th1 cytokines and reduces HBsAg level in transgenic mice

    Institute of Scientific and Technical Information of China (English)

    MENG Zhe-feng; WANG Hua-jing; YAO Xin; WANG Xuan-yi; WEN Yu-mei; DAI Jian-xin; XIE You-hua; XU Jian-qing

    2012-01-01

    Background The Fc receptor associated pathway might improve the immune responses against hepatitis B virus (HBV) as previously described by us.In addition,the Flt3 ligand (FL) has been reported to potentiate antigen presenting cells in vivo and may act as a potential adjuvant to boost antigen-specific immune responses.In this study,the immune efficacies of a set of fusion proteins of HBsAg and Fc and/or FL were evaluated in HBsAg transgenic mice.Methods The fusion proteins composed of HBsAg and the Fc domain of murine IgG1 (HBsAg-Fc) and/or the Flt3 ligand,and yeast-derived recombinant HBsAg were used as immunogen to immunize HBsAg transgenic mice,respectively.Serum and liver HBsAg levels,serum anti-HBsAg and cytokine profile,and the activities of alanine aminotransferase (ALT)/AST were investigated after immunization.Results After six injections,the most pronounced decrease in serum and liver HBsAg levels was observed in the HBsAg-Fc immunized group.In addition,serum Th1 cytokines and ALT/AST activities were highest in this group,indicating an effective induction of a favorable cellular immune response.Interestingly,the fusion protein containing HBsAg-Fc and the Flt3 ligand stimulated an alternative Th1-type immune response featured with high level productions of tumor necrosis factor α (TNF- α) and monocyte chemoabstractant protein 1 (MCP-1),causing a more severe cytotoxicity in hepatocytes while showed less effective in reducing serum HBsAg level.Conclusion HBsAg-Fc is effective in eliciting both the humoral and cellular immune responses against HBsAg in HBsAg transgenic mice,which makes it a potential immunogen for the immunotherapy of chronic hepatitis B.

  8. Elicitation of strong immune responses by a DNA vaccine expressing a secreted form of hepatitis C virus envelope protein E2 in murine and porcine animal models

    Institute of Scientific and Technical Information of China (English)

    Yi-Ping Li; Hye Na Kang; Lorne A Babiuk; Qiang Liu

    2006-01-01

    AIM: To characterize the immunogenicity of a hepatitis C virus (HCV) E2 DNA vaccine alone or with a protein vaccine boost in murine and porcine animal models.METHODS: A DNA vaccine expressing a secreted form of HCV E2 protein was constructed and used to vaccinate mice and piglets with or without boosting with a recombinant E2 protein vaccine formulated with CpG ODN and 10% Emulsigen. The immunogenicity of HCV E2 vaccines was analyzed by ELISA for antibody responses, MTT assay for lymphocyte proliferation,ELISPOT for the number of interferon-γ secreting cells,and cytotoxic T lymphocyte assays.RESULTS: Intradermal injection of E2 DNA vaccine induced strong Th1-like immune responses in mice. In piglets, E2 DNA vaccine elicited moderate and more balanced immune responses. A DNA vaccine prime and protein boost vaccination strategy induced significantly higher E2-specific antibody levels and shifted the immune response towards Th2-like ones in piglets.CONCLUSION: A DNA vaccine expressing a secreted form of HCV E2 protein elicited E2-specific immune responses in mice and piglets. Recombinant E2 protein vaccination following DNA immunization significantly increased the antibody response in piglets. These HCV E2 vaccines may represent promising hepatitis C vaccine candidates for further investigations.

  9. Induction of Wnt-inducible signaling protein-1 correlates with invasive breast cancer oncogenesis and reduced type 1 cell-mediated cytotoxic immunity: a retrospective study.

    Science.gov (United States)

    Klinke, David J

    2014-01-01

    Innate and type 1 cell-mediated cytotoxic immunity function as important extracellular control mechanisms that maintain cellular homeostasis. Interleukin-12 (IL12) is an important cytokine that links innate immunity with type 1 cell-mediated cytotoxic immunity. We recently observed in vitro that tumor-derived Wnt-inducible signaling protein-1 (WISP1) exerts paracrine action to suppress IL12 signaling. The objective of this retrospective study was three fold: 1) to determine whether a gene signature associated with type 1 cell-mediated cytotoxic immunity was correlated with overall survival, 2) to determine whether WISP1 expression is increased in invasive breast cancer, and 3) to determine whether a gene signature consistent with inhibition of IL12 signaling correlates with WISP1 expression. Clinical information and mRNA expression for genes associated with anti-tumor immunity were obtained from the invasive breast cancer arm of the Cancer Genome Atlas study. Patient cohorts were identified using hierarchical clustering. The immune signatures associated with the patient cohorts were interpreted using model-based inference of immune polarization. Reverse phase protein array, tissue microarray, and quantitative flow cytometry in breast cancer cell lines were used to validate observed differences in gene expression. We found that type 1 cell-mediated cytotoxic immunity was correlated with increased survival in patients with invasive breast cancer, especially in patients with invasive triple negative breast cancer. Oncogenic transformation in invasive breast cancer was associated with an increase in WISP1. The gene expression signature in invasive breast cancer was consistent with WISP1 as a paracrine inhibitor of type 1 cell-mediated immunity through inhibiting IL12 signaling and promoting type 2 immunity. Moreover, model-based inference helped identify appropriate immune signatures that can be used as design constraints in genetically engineering better pre

  10. Staphylococcus aureus surface protein SdrE binds complement regulator factor H as an immune evasion tactic.

    Directory of Open Access Journals (Sweden)

    Julia A Sharp

    Full Text Available Similar to other highly successful invasive bacterial pathogens, Staphylococcus aureus recruits the complement regulatory protein factor H (fH to its surface to inhibit the alternative pathway of complement. Here, we report the identification of the surface-associated protein SdrE as a fH-binding protein using purified fH overlay of S. aureus fractionated cell wall proteins and fH cross-linking to S. aureus followed by mass spectrometry. Studies using recombinant SdrE revealed that rSdrE bound significant fH whether from serum or as a purified form, in both a time- and dose-dependent manner. Furthermore, rSdrE-bound fH exhibited cofactor functionality for factor I (fI-mediated cleavage of C3b to iC3b which correlated positively with increasing amounts of fH. Expression of SdrE on the surface of the surrogate bacterium Lactococcus lactis enhanced recruitment of fH which resulted in increased iC3b generation. Moreover, surface expression of SdrE led to a reduction in C3-fragment deposition, less C5a generation, and reduced killing by polymorphonuclear cells. Thus, we report the first identification of a S. aureus protein associated with the staphylococcal surface that binds factor H as an immune evasion mechanism.

  11. A novel lumazine synthase molecule from Brucella significantly promotes the immune-stimulation effects of antigenic protein.

    Science.gov (United States)

    Du, Z Q; Wang, J Y

    2015-10-27

    Brucella, an intracellular parasite that infects some livestock and humans, can damage or destroy the reproductive system of livestock. The syndrome is referred to as brucellosis and often occurs in pastoral areas; it is contagious from livestock to humans. In this study, the intact Brucella suis outer membrane protein 31 (omp31) gene was cloned, recombinantly expressed, and examined as a subunit vaccine candidate. The intact Brucella lumazine synthase (bls) gene was cloned and recombinantly expressed to study polymerization function in vitro. Non-reducing gel electrophoresis showed that rBs-BLS existed in different forms in vitro, including as a dimer and a pentamer. An enzyme-linked immunosorbent assay result showed that rOmp31 protein could induce production of an antibody in rabbits. However, the rOmp31-BLS fusion protein could elicit a much higher antibody titer in rabbits; this construct involved fusion of the Omp31 molecule with the BLS molecule. Our results indicate that Omp31 is involved in immune stimulation, while BLS has a polymerizing function based on rOmp31-BLS fusion protein immunogenicity. These data suggest that Omp31 is an ideal subunit vaccine candidate and that the BLS molecule is a favorable transport vector for antigenic proteins.

  12. A Phytophthora sojae effector suppresses endoplasmic reticulum stress-mediated immunity by stabilizing plant Binding immunoglobulin Proteins

    Science.gov (United States)

    Jing, Maofeng; Guo, Baodian; Li, Haiyang; Yang, Bo; Wang, Haonan; Kong, Guanghui; Zhao, Yao; Xu, Huawei; Wang, Yan; Ye, Wenwu; Dong, Suomeng; Qiao, Yongli; Tyler, Brett M.; Ma, Wenbo; Wang, Yuanchao

    2016-01-01

    Phytophthora pathogens secrete an array of specific effector proteins to manipulate host innate immunity to promote pathogen colonization. However, little is known about the host targets of effectors and the specific mechanisms by which effectors increase susceptibility. Here we report that the soybean pathogen Phytophthora sojae uses an essential effector PsAvh262 to stabilize endoplasmic reticulum (ER)-luminal binding immunoglobulin proteins (BiPs), which act as negative regulators of plant resistance to Phytophthora. By stabilizing BiPs, PsAvh262 suppresses ER stress-triggered cell death and facilitates Phytophthora infection. The direct targeting of ER stress regulators may represent a common mechanism of host manipulation by microbes. PMID:27256489

  13. Immunization with Individual Proteins of the Lrp/AsnC Family Induces Protection Against Brucella melitensis 16M Challenges in Mice

    Directory of Open Access Journals (Sweden)

    Xinhui eWang

    2015-10-01

    Full Text Available 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 6 proteins were identified as members of the Lrp/AsnC family. Lrp/AsnC proteins have 2 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 6 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 6 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 6 proteins of the Lrp/AsnC family in Brucella could induce protective immune responses in mice.

  14. Regulation of Toll-like receptor 4-mediated immune responses through Pasteurella multocida toxin-induced G protein signalling

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    Hildebrand Dagmar

    2012-08-01

    Full Text Available Abstract Background Lipopolysaccharide (LPS-triggered Toll-like receptor (TLR 4-signalling belongs to the key innate defence mechanisms upon infection with Gram-negative bacteria and triggers the subsequent activation of adaptive immunity. There is an active crosstalk between TLR4-mediated and other signalling cascades to secure an effective immune response, but also to prevent excessive inflammation. Many pathogens induce signalling cascades via secreted factors that interfere with TLR signalling to modify and presumably escape the host response. In this context heterotrimeric G proteins and their coupled receptors have been recognized as major cellular targets. Toxigenic strains of Gram-negative Pasteurella multocida produce a toxin (PMT that constitutively activates the heterotrimeric G proteins Gαq, Gα13 and Gαi independently of G protein-coupled receptors through deamidation. PMT is known to induce signalling events involved in cell proliferation, cell survival and cytoskeleton rearrangement. Results Here we show that the activation of heterotrimeric G proteins through PMT suppresses LPS-stimulated IL-12p40 production and eventually impairs the T cell-activating ability of LPS-treated monocytes. This inhibition of TLR4-induced IL-12p40 expression is mediated by Gαi-triggered signalling as well as by Gβγ-dependent activation of PI3kinase and JNK. Taken together we propose the following model: LPS stimulates TLR4-mediated activation of the NFĸB-pathway and thereby the production of TNF-α, IL-6 and IL-12p40. PMT inhibits the production of IL-12p40 by Gαi-mediated inhibition of adenylate cyclase and cAMP accumulation and by Gβγ-mediated activation of PI3kinase and JNK activation. Conclusions On the basis of the experiments with PMT this study gives an example of a pathogen-induced interaction between G protein-mediated and TLR4-triggered signalling and illustrates how a bacterial toxin is able to interfere with the host’s immune

  15. Silencing the shutoff protein of Epstein-Barr virus in productively infected B cells points to (innate) targets for immune evasion

    NARCIS (Netherlands)

    van Gent, Michiel; Gram, Anna M; Boer, Ingrid G J; Geerdink, Ruben J; Lindenbergh, Marthe F S; Lebbink, Robert Jan; Wiertz, Emmanuel J H J; Ressing, Maaike E

    2015-01-01

    During productive infection with Epstein-Barr virus (EBV), a dramatic suppression of cellular protein expression is caused by the viral alkaline exonuclease BGLF5. Among the proteins downregulated by BGLF5 are multiple immune components. Here, we show that shutoff reduces expression of the innate EB

  16. Evolution of the Immune Response against Recombinant Proteins (TcpA, TcpB, and FlaA as a Candidate Subunit Cholera Vaccine

    Directory of Open Access Journals (Sweden)

    Neda Molaee

    2017-01-01

    Full Text Available Vibrio cholerae is the causative agent of cholera and annually leads to death of thousands of people around the globe. Two factors in the pathogenesis of this bacterium are its pili and flagella. The main subunits of pili TcpA, TcpB, and FlaA are the constituent subunit of flagella. In this study, we studied the ability of pili and flagella subunits to stimulate immune responses in mice. After amplification of TcpA, TcpB, and FlaA genes using PCR, they were cloned in expression plasmids. After production of the above-mentioned proteins by using IPTG, the proteins were purified and then approved using immunoblot method. After injection of the purified proteins to a mice model, immune response stimulation was evaluated by measuring the levels of IgG1 and IgG2a antibody titers, IL5 and IFN-γ. Immune response stimulation against pili and flagella antigens was adequate. Given the high levels of IL5 titer and IgG1 antibody, the stimulated immune response was toward Th1. Humoral immune response stimulation is of key importance in prevention of cholera. Our immunological analysis shows the appropriate immune response in mice model after vaccination with recombinant proteins. The high level of IL5 and low level of IFN-γ show the activation of Th2 cell response.

  17. Evolution of the Immune Response against Recombinant Proteins (TcpA, TcpB, and FlaA) as a Candidate Subunit Cholera Vaccine.

    Science.gov (United States)

    Molaee, Neda; Mosayebi, Ghasem; Amozande-Nobaveh, Alireza; Soleyman, Mohammad Reza; Abtahi, Hamid

    2017-01-01

    Vibrio cholerae is the causative agent of cholera and annually leads to death of thousands of people around the globe. Two factors in the pathogenesis of this bacterium are its pili and flagella. The main subunits of pili TcpA, TcpB, and FlaA are the constituent subunit of flagella. In this study, we studied the ability of pili and flagella subunits to stimulate immune responses in mice. After amplification of TcpA, TcpB, and FlaA genes using PCR, they were cloned in expression plasmids. After production of the above-mentioned proteins by using IPTG, the proteins were purified and then approved using immunoblot method. After injection of the purified proteins to a mice model, immune response stimulation was evaluated by measuring the levels of IgG1 and IgG2a antibody titers, IL5 and IFN-γ. Immune response stimulation against pili and flagella antigens was adequate. Given the high levels of IL5 titer and IgG1 antibody, the stimulated immune response was toward Th1. Humoral immune response stimulation is of key importance in prevention of cholera. Our immunological analysis shows the appropriate immune response in mice model after vaccination with recombinant proteins. The high level of IL5 and low level of IFN-γ show the activation of Th2 cell response.

  18. Evolution of the Immune Response against Recombinant Proteins (TcpA, TcpB, and FlaA) as a Candidate Subunit Cholera Vaccine

    Science.gov (United States)

    Molaee, Neda; Amozande-Nobaveh, Alireza; Soleyman, Mohammad Reza

    2017-01-01

    Vibrio cholerae is the causative agent of cholera and annually leads to death of thousands of people around the globe. Two factors in the pathogenesis of this bacterium are its pili and flagella. The main subunits of pili TcpA, TcpB, and FlaA are the constituent subunit of flagella. In this study, we studied the ability of pili and flagella subunits to stimulate immune responses in mice. After amplification of TcpA, TcpB, and FlaA genes using PCR, they were cloned in expression plasmids. After production of the above-mentioned proteins by using IPTG, the proteins were purified and then approved using immunoblot method. After injection of the purified proteins to a mice model, immune response stimulation was evaluated by measuring the levels of IgG1 and IgG2a antibody titers, IL5 and IFN-γ. Immune response stimulation against pili and flagella antigens was adequate. Given the high levels of IL5 titer and IgG1 antibody, the stimulated immune response was toward Th1. Humoral immune response stimulation is of key importance in prevention of cholera. Our immunological analysis shows the appropriate immune response in mice model after vaccination with recombinant proteins. The high level of IL5 and low level of IFN-γ show the activation of Th2 cell response. PMID:28191473

  19. Oral Immunization with Recombinant Vaccinia Virus Prime and Intramuscular Protein Boost Provides Protection against Intrarectal Simian-Human Immunodeficiency Virus Challenge in Macaques.

    Science.gov (United States)

    Thippeshappa, Rajesh; Tian, Baoping; Cleveland, Brad; Guo, Wenjin; Polacino, Patricia; Hu, Shiu-Lok

    2015-12-30

    Human immunodeficiency virus type 1 (HIV-1) acquisition occurs predominantly through mucosal transmission. We hypothesized that greater mucosal immune responses and protective efficacy against mucosal HIV-1 infection may be achieved by prime-boost immunization at mucosal sites. We used a macaque model to determine the safety, immunogenicity, and protective efficacy of orally delivered, replication-competent but attenuated recombinant vaccinia viruses expressing full-length HIV-1 SF162 envelope (Env) or simian immunodeficiency virus (SIV) Gag-Pol proteins. We examined the dose and route that are suitable for oral immunization with recombinant vaccinia viruses. We showed that sublingual inoculation of two vaccinia virus-naive pigtailed macaques with 5 × 10(8) PFU of recombinant vaccinia viruses was safe. However, sublingual inoculation with a higher dose or tonsillar inoculation resulted in secondary oral lesions, indicating the need to optimize the dose and route for oral immunization with replication-competent vaccinia virus vectors. Oral priming alone elicited antibody responses to vaccinia virus and to the SF162 Env protein. Intramuscular immunization with the SF162 gp120 protein at either 20 or 21 weeks postpriming resulted in a significant boost in antibody responses in both systemic and mucosal compartments. Furthermore, we showed that immune responses induced by recombinant vaccinia virus priming and intramuscular protein boosting provided protection against intrarectal challenge with the simian-human immunodeficiency virus SHIV-SF162-P4.

  20. Evidence of a humoral immune response against the prokaryotic expressed N-terminal autoprotease (Npro) protein of bovine viral diarrhoea virus

    Indian Academy of Sciences (India)

    Niranjan Mishra; Katherukamem Rajukumar; Shruti Shrikant Pitale; Anil Prakash; Ram Kumar Nema; Sthita Pragnya Behera; Shiv Chandra Dubey

    2010-03-01

    Bovine viral diarrhoea virus (BVDV) is an economically important pathogen of cattle and sheep belonging to the genus Pestivirus of the family Flaviviridae. Although the BVDV non-structural N-terminal protease (Npro) acts as an interferon antagonist and subverts the host innate immunity, little is known about its immunogenicity. Hence, we expressed a recombinant BVDV Npro–His fusion protein (28 kDa) in E. coli and determined the humoral immune response generated by it in rabbits. The antigenicity of the Npro protein was confirmed by western blot using anti-BVDV hyperimmune cattle, sheep and goat serum, and anti-Npro rabbit serum. When rabbits were immunized with the Npro protein, a humoral immune response was evident by 4 weeks and persisted till 10 weeks post immunization as detected by ELISA and western blot. Despite Npro-specific antibodies remaining undetectable in 80 serum samples from BVDV-infected sheep and goats, BVDV hyperimmune sera along with some of the field cattle, sheep and goat sera with high BVDV neutralizing antibody titres were found positive for Npro antibodies. Our results provide evidence that despite the low immunogenicity of the BVDV Npro protein, a humoral immune response is induced in cattle, sheep and goats only with repeated BVDV exposure.

  1. Mucosal immunization with PsaA protein, using chitosan as a delivery system, increases protection against acute otitis media and invasive infection by Streptococcus pneumoniae.

    Science.gov (United States)

    Xu, J-H; Dai, W-J; Chen, B; Fan, X-Y

    2015-03-01

    As infection with Streptococcus pneumoniae (mainly via the mucosal route) is a leading cause of acute otitis media, sinus and bacterial pneumonia, the mucosal immunity plays an important role in the prevention of pneumococcal diseases. Therefore, intranasal vaccination may be an effective immunization strategy, but requires appropriate mucosal vaccine delivery systems. In this work, chitosan was used as a mucosal delivery system to form chitosan-PsaA nanoparticles based on ionotropic gelation methods and used to immunize BALB/c mice intranasally. Compared to mice immunized with naked PsaA, levels of IFN-γ, IL-17A and IL-4 in spleen lymphocytes, the systemic (IgG in serum) and mucosal (IgA in mucosal lavage) specific antibodies were enhanced significantly in mice inoculated with chitosan-PsaA. Furthermore, increased protection against acute otitis media following middle ear challenge with pneumococcus serotype 14, and improved survival following intraperitoneal challenge with pneumococcus serotype 3 or serotype 14, was found in the mice immunized with chitosan-PsaA nanoparticles. Thus, intranasal immunization with chitosan-PsaA can successfully induce mucosal and systemic immune responses and increase protection against pneumococcal acute otitis media and invasive infections. Hence, intranasal immunization with PsaA protein, based on chitosan as a delivery system, is an efficient immunization strategy for preventing pneumococcal infections.

  2. Immunization with the Recombinant Cholera Toxin B Fused to Fimbria 2 Protein Protects against Bordetella pertussis Infection

    Directory of Open Access Journals (Sweden)

    Noelia Olivera

    2014-01-01

    Full Text Available This study examined the immunogenic properties of the fusion protein fimbria 2 of Bordetella pertussis (Fim2—cholera toxin B subunit (CTB in the intranasal murine model of infection. To this end B. pertussis Fim2 coding sequence was cloned downstream of the cholera toxin B subunit coding sequence. The expression and assembly of the fusion protein into pentameric structures (CTB-Fim2 were evaluated by SDS-PAGE and monosialotetrahexosylgaglioside (GM1-ganglioside enzyme-linked immunosorbent assay (ELISA. To evaluate the protective capacity of CTB-Fim2, an intraperitoneal or intranasal mouse immunization schedule was performed with 50 μg of CTB-Fim2. Recombinant (rFim2 or purified (BpFim2 Fim2, CTB, and phosphate-buffered saline (PBS were used as controls. The results showed that mice immunized with BpFim2 or CTB-Fim2 intraperitoneally or intranasally presented a significant reduction in bacterial lung counts compared to control groups (P<0.01 or P<0.001, resp.. Moreover, intranasal immunization with CTB-Fim2 induced significant levels of Fim2-specific IgG in serum and bronchoalveolar lavage (BAL and Fim2-specific IgA in BAL. Analysis of IgG isotypes and cytokines mRNA levels showed that CTB-Fim2 results in a mixed Th1/Th2 (T-helper response. The data presented here provide support for CTB-Fim2 as a promising recombinant antigen against Bordetella pertussis infection.

  3. Enhanced antitumor immunity of nanoliposome-encapsulated heat shock protein 70 peptide complex derived from dendritic tumor fusion cells.

    Science.gov (United States)

    Zhang, Yunfei; Luo, Wen; Wang, Yucai; Chen, Jun; Liu, Yunyan; Zhang, Yong

    2015-06-01

    Tumor-derived heat shock proteins peptide complex (HSP.PC-Tu) has been regarded as a promising antitumor agent. However, inadequate immunogenicity and low bioavailability limit the clinical uses of this agent. In a previous study, we first produced an improved HSP70.PC-based vaccine purified from dendritic cell (DC)-tumor fusion cells (HSP70.PC-Fc) which had increased immunogenicity due to enhanced antigenic tumor peptides compared to HSP70.PC-Tu. In order to increase the bioavailability of HSP70.PC-Fc, the peptide complex was encapsulated with nanoliposomes (NL-HSP70.PC-Fc) in this study. After encapsulation, the tumor immunogenicity was observed using various assays. It was demonstrated that the NL-HSP70.PC-Fc has acceptable stability. The in vivo antitumor immune response was increased with regard to T-cell activation, CTL response and tumor therapy efficiency compared to that of HSP70.PC-Fc. In addition, it was shown that DC maturation was improved by NL-HSP70.PC-Fc, which added to the antitumor immunity. The results obtained for NL-HSP70.PC-Fc, which improved immunogenicity and increases the bioavailability of HSP70.PC, may represent superior heat shock proteins (HSPs)-based tumor vaccines. Such vaccines deserve further investigation and may provide a preclinical rationale to translate findings into early phase trials for patients with breast tumors.

  4. Aptamer-conjugated live human immune cell based biosensors for the accurate detection of C-reactive protein

    Science.gov (United States)

    Hwang, Jangsun; Seo, Youngmin; Jo, Yeonho; Son, Jaewoo; Choi, Jonghoon

    2016-10-01

    C-reactive protein (CRP) is a pentameric protein that is present in the bloodstream during inflammatory events, e.g., liver failure, leukemia, and/or bacterial infection. The level of CRP indicates the progress and prognosis of certain diseases; it is therefore necessary to measure CRP levels in the blood accurately. The normal concentration of CRP is reported to be 1–3 mg/L. Inflammatory events increase the level of CRP by up to 500 times; accordingly, CRP is a biomarker of acute inflammatory disease. In this study, we demonstrated the preparation of DNA aptamer-conjugated peripheral blood mononuclear cells (Apt-PBMCs) that specifically capture human CRP. Live PBMCs functionalized with aptamers could detect different levels of human CRP by producing immune complexes with reporter antibody. The binding behavior of Apt-PBMCs toward highly concentrated CRP sites was also investigated. The immune responses of Apt-PBMCs were evaluated by measuring TNF-alpha secretion after stimulating the PBMCs with lipopolysaccharides. In summary, engineered Apt-PBMCs have potential applications as live cell based biosensors and for in vitro tracing of CRP secretion sites.

  5. The involvement of cysteine-rich intestinal protein in early development and innate immunity of Asiatic hard clam, Meretrix meretrix.

    Science.gov (United States)

    Chen, Hongjian; Yang, Xue; Tang, Ting; Li, Juan; Liu, Baozhong; Liu, Fengsong; Xie, Song

    2014-10-01

    Cysteine-rich intestinal protein (CRIP), a Zn(2+)-binding protein, contains a single copy of the highly conserved double-zinc-finger structure known as the LIM (lin-11-isl-1-mec-3) motif. In this paper, a cDNA encoding MmCRIP was isolated from the Asiatic hard clam Meretrix meretrix. The full-length cDNA of MmCRIP consists of a 237-bp open reading frame that encodes a polypeptide of 78 amino acids with a predicted molecular weight (MW) of 8635.8 Da and theoretical isoelectric point (pI) of 9.01. Bioinformatics analysis showed that it belonged to a new member of the CRIP subfamily. Relationship analysis revealed that MmCRIP has high-levels of sequence similarity to many CRIPs reported in other animals, particularly in invertebrates. Real-time PCR analysis showed that the highest level of MmCRIP expression was in hemocyte tissue and at pediveligers stage. To investigate immune function, mature clams were challenged with Aeromonas hydrophila. During A. hydrophila infection, up-regulation of MmCRIP transcript in clam's hemocyte, gill and hepatopancreas was detected. DsRNAi (double-strand RNA interference) approach was employed to study the function of MmCRIP and the data showed that inactivation of the MmCRIP gene blocked larvae development and caused mass mortalities. The probable roles of MmCRIP in clam early development and innate immunity are presented for the first time.

  6. Dominant-Negative Proteins in Herpesviruses – From Assigning Gene Function to Intracellular Immunization

    Directory of Open Access Journals (Sweden)

    Zsolt Ruzsics

    2009-10-01

    Full Text Available Investigating and assigning gene functions of herpesviruses is a process, which profits from consistent technical innovation. Cloning of bacterial artificial chromosomes encoding herpesvirus genomes permits nearly unlimited possibilities in the construction of genetically modified viruses. Targeted or randomized screening approaches allow rapid identification of essential viral proteins. Nevertheless, mapping of essential genes reveals only limited insight into function. The usage of dominant-negative (DN proteins has been the tool of choice to dissect functions of proteins during the viral life cycle. DN proteins also facilitate the analysis of host-virus interactions. Finally, DNs serve as starting-point for design of new antiviral strategies.

  7. Mycobacterium tuberculosis PE25/PPE41 protein complex induces activation and maturation of dendritic cells and drives Th2-biased immune responses.

    Science.gov (United States)

    Chen, Wei; Bao, Yige; Chen, Xuerong; Burton, Jeremy; Gong, Xueli; Gu, Dongqing; Mi, Youjun; Bao, Lang

    2016-04-01

    Mycobacterium tuberculosis evades innate host immune responses by parasitizing macrophages and causes significant morbidity and mortality around the world. A mycobacterial antigen that can activate dendritic cells (DCs) and elicit effective host innate immune responses will be vital to the development of an effective TB vaccine. The M. tuberculosis genes PE25/PPE41 encode proteins which have been associated with evasion of the host immune response. We constructed a PE25/PPE41 complex gene via splicing by overlapping extension and expressed it successfully in E. coli. We investigated whether this protein complex could interact with DCs to induce effective host immune responses. The PE25/PPE41 protein complex induced maturation of isolated mouse DCs in vitro, increasing expression of cell surface markers (CD80, CD86 and MHC-II), thereby promoting Th2 polarization via secretion of pro-inflammatory cytokines IL-4 and IL-10. In addition, PE25/PPE41 protein complex-activated DCs induced proliferation of mouse CD4(+) and CD8(+) T cells, and a strong humoral response in immunized mice. The sera of five TB patients were also highly reactive to this antigen. These findings suggest that interaction of the PE25/PPE41 protein complex with DCs may be of great immunological significance.

  8. Multiple myeloma cell lines and primary tumors proteoma: protein biosynthesis and immune system as potential therapeutic targets

    Science.gov (United States)

    Mazzotti, Diego Robles; Evangelista, Adriane Feijó; Braga, Walter Moisés Tobias; de Lourdes Chauffaille, Maria; Leme, Adriana Franco Paes; Colleoni, Gisele Wally Braga

    2015-01-01

    Despite great advance in multiple myeloma (MM) treatment since 2000s, it is still an incurable disease and novel therapies are welcome. Therefore, the purpose of this study was to explore MM plasma cells' (MM-PC) proteome, in comparison with their normal counterparts (derived from palatine tonsils of normal donors, ND-PC), in order to find potential therapeutic targets expressed on the surface of these cells. We also aimed to evaluate the proteome of MM cell lines with different genetic alterations, to confirm findings obtained with primary tumor cells. Bone marrow (BM) samples from eight new cases of MM and palatine tonsils from seven unmatched controls were submitted to PC separation and, in addition to two MM cell lines (U266, RPMI-8226), were submitted to protein extraction for mass spectrometry analyses. A total of 81 proteins were differentially expressed between MM-PC and ND-PC - 72 upregulated and nine downregulated; U266 vs. RPMI 8226 cell lines presented 61 differentially expressed proteins - 51 upregulated and 10 downregulated. On primary tumors, bioinformatics analyses highlighted upregulation of protein biosynthesis machinery, as well as downregulation of immune response components, such as MHC class I and II, and complement receptors. We also provided comprehensive information about U266 and RPMI-8226 cell lines' proteome and could confirm some patients' findings. PMID:26807199

  9. Expression of innate immune genes, proteins and microRNAs in lung tissue of pigs infected experimentally with influenza virus (H1N2)

    DEFF Research Database (Denmark)

    Skovgaard, Kerstin; Cirera, Susanna; Vasby, Ditte;

    2013-01-01

    This study aimed at providing a better understanding of the involvement of innate immune factors, including miRNA, in the local host response to influenza virus infection. Twenty pigs were challenged by influenza A virus subtype H1N2. Expression of microRNA (miRNA), mRNA and proteins were...... results suggest that, in addition to a wide range of innate immune factors, miRNAs may also be involved in controlling acute influenza infection in pigs....

  10. Super-resolution microscopy reveals protein spatial reorganization in early innate immune responses.

    Energy Technology Data Exchange (ETDEWEB)

    Carson, Bryan D.; Aaron, Jesse S.; Timlin, Jerilyn Ann

    2010-10-01

    Over the past decade optical approaches were introduced that effectively break the diffraction barrier. Of particular note were introductions of Stimulated Emission/Depletion (STED) microscopy, Photo-Activated Localization Microscopy (PALM), and the closely related Stochastic Optical Reconstruction Microscopy (STORM). STORM represents an attractive method for researchers, as it does not require highly specialized optical setups, can be implemented using commercially available dyes, and is more easily amenable to multicolor imaging. We implemented a simultaneous dual-color, direct-STORM imaging system through the use of an objective-based TIRF microscope and filter-based image splitter. This system allows for excitation and detection of two fluorophors simultaneously, via projection of each fluorophor's signal onto separate regions of a detector. We imaged the sub-resolution organization of the TLR4 receptor, a key mediator of innate immune response, after challenge with lipopolysaccharide (LPS), a bacteria-specific antigen. While distinct forms of LPS have evolved among various bacteria, only some LPS variations (such as that derived from E. coli) typically result in significant cellular immune response. Others (such as from the plague bacteria Y. pestis) do not, despite affinity to TLR4. We will show that challenge with LPS antigens produces a statistically significant increase in TLR4 receptor clusters on the cell membrane, presumably due to recruitment of receptors to lipid rafts. These changes, however, are only detectable below the diffraction limit and are not evident using conventional imaging methods. Furthermore, we will compare the spatiotemporal behavior of TLR4 receptors in response to different LPS chemotypes in order to elucidate possible routes by which pathogens such as Y. pestis are able to circumvent the innate immune system. Finally, we will exploit the dual-color STORM capabilities to simultaneously image LPS and TLR4 receptors in the

  11. Phosphorylation of mouse immunity-related GTPase (IRG resistance proteins is an evasion strategy for virulent Toxoplasma gondii.

    Directory of Open Access Journals (Sweden)

    Tobias Steinfeldt

    Full Text Available Virulence of complex pathogens in mammals is generally determined by multiple components of the pathogen interacting with the functional complexity and multiple layering of the mammalian immune system. It is most unusual for the resistance of a mammalian host to be overcome by the defeat of a single defence mechanism. In this study we uncover and analyse just such a case at the molecular level, involving the widespread intracellular protozoan pathogen Toxoplasma gondii and one of its most important natural hosts, the house mouse (Mus musculus. Natural polymorphism in virulence of Eurasian T. gondii strains for mice has been correlated in genetic screens with the expression of polymorphic rhoptry kinases (ROP kinases secreted into the host cell during infection. We show that the molecular targets of the virulent allelic form of ROP18 kinase are members of a family of cellular GTPases, the interferon-inducible IRG (immunity-related GTPase proteins, known from earlier work to be essential resistance factors in mice against avirulent strains of T. gondii. Virulent T. gondii strain ROP18 kinase phosphorylates several mouse IRG proteins. We show that the parasite kinase phosphorylates host Irga6 at two threonines in the nucleotide-binding domain, biochemically inactivating the GTPase and inhibiting its accumulation and action at the T. gondii parasitophorous vacuole membrane. Our analysis identifies the conformationally active switch I region of the GTP-binding site as an Achilles' heel of the IRG protein pathogen-resistance mechanism. The polymorphism of ROP18 in natural T. gondii populations indicates the existence of a dynamic, rapidly evolving ecological relationship between parasite virulence factors and host resistance factors. This system should be unusually fruitful for analysis at both ecological and molecular levels since both T. gondii and the mouse are widespread and abundant in the wild and are well-established model species with

  12. Recombinant paracoccin reproduces the biological properties of the native protein and induces protective Th1 immunity against Paracoccidioides brasiliensis infection.

    Directory of Open Access Journals (Sweden)

    Ana Claudia Paiva Alegre

    2014-04-01

    Full Text Available BACKGROUND: Paracoccin is a dual-function protein of the yeast Paracoccidioides brasiliensis that has lectin properties and N-acetylglucosaminidase activities. Proteomic analysis of a paracoccin preparation from P. brasiliensis revealed that the sequence matched that of the hypothetical protein encoded by PADG-3347 of isolate Pb-18, with a polypeptide sequence similar to the family 18 endochitinases. These endochitinases are multi-functional proteins, with distinct lectin and enzymatic domains. METHODOLOGY/PRINCIPAL FINDINGS: The multi-exon assembly and the largest exon of the predicted ORF (PADG-3347, was cloned and expressed in Escherichia coli cells, and the features of the recombinant proteins were compared to those of the native paracoccin. The multi-exon protein was also used for protection assays in a mouse model of paracoccidioidomycosis. CONCLUSIONS/SIGNIFICANCE: Our results showed that the recombinant protein reproduced the biological properties described for the native protein-including binding to laminin in a manner that is dependent on carbohydrate recognition-showed N-acetylglucosaminidase activity, and stimulated murine peritoneal macrophages to produce high levels of TNF-α and nitric oxide. Considering the immunomodulatory potential of glycan-binding proteins, we also investigated whether prophylactic administration of recombinant paracoccin affected the course of experimental paracoccidioidomycosis in mice. In comparison to animals injected with vehicle (controls, mice treated with recombinant paracoccin displayed lower pulmonary fungal burdens and reduced pulmonary granulomas. These protective effects were associated with augmented pulmonary levels of IL-12 and IFN-γ. We also observed that injection of paracoccin three days before challenge was the most efficient administration protocol, as the induced Th1 immunity was balanced by high levels of pulmonary IL-10, which may prevent the tissue damage caused by exacerbated

  13. Cyclic di-GMP sensing via the innate immune signaling protein STING.

    Science.gov (United States)

    Yin, Qian; Tian, Yuan; Kabaleeswaran, Venkataraman; Jiang, Xiaomo; Tu, Daqi; Eck, Michael J; Chen, Zhijian J; Wu, Hao

    2012-06-29

    Detection of foreign materials is the first step of successful immune responses. Stimulator of interferon genes (STING) was shown to directly bind cyclic diguanylate monophosphate (c-di-GMP), a bacterial second messenger, and to elicit strong interferon responses. Here we elucidate the structural features of the cytosolic c-di-GMP binding domain (CBD) of STING and its complex with c-di-GMP. The CBD exhibits an α + β fold and is a dimer in the crystal and in solution. Surprisingly, one c-di-GMP molecule binds to the central crevice of a STING dimer, using a series of stacking and hydrogen bonding interactions. We show that STING is autoinhibited by an intramolecular interaction between the CBD and the C-terminal tail (CTT) and that c-di-GMP releases STING from this autoinhibition by displacing the CTT. The structures provide a remarkable example of pathogen-host interactions in which a unique microbial molecule directly engages the innate immune system.

  14. Transcriptome analysis to identify genes for peptides and proteins involved in immunity and reproduction from male accessory glands and ejaculatory duct of Bactrocera dorsalis.

    Science.gov (United States)

    Wei, Dong; Tian, Chuan-Bei; Liu, Shi-Huo; Wang, Tao; Smagghe, Guy; Jia, Fu-Xian; Dou, Wei; Wang, Jin-Jun

    2016-06-01

    In the male reproductive system of insects, the male accessory glands and ejaculatory duct (MAG/ED) are important organs and their primary function is to enhance the fertility of spermatozoa. Proteins secreted by the MAG/ED are also known to induce post-mating changes and immunity responses in the female insect. To understand the gene expression profile in the MAG/ED of the oriental fruit fly Bactrocera dorsalis (Hendel), that is an important pest in fruits, we performed an Illumina-based deep sequencing of mRNA. This yielded 54,577,630 clean reads corresponding to 4.91Gb total nucleotides that were assembled and clustered to 30,669 unigenes (average 645bp). Among them, 20,419 unigenes were functionally annotated to known proteins/peptides in Gene Orthology, Clusters of Orthologous Groups, Kyoto Encyclopedia of Genes and Genomes pathway databases. Typically, many genes were involved in immunity and these included microbial recognition proteins and antimicrobial peptides. Subsequently, the inducible expression of these immunity-related genes was confirmed by qRT-PCR analysis when insects were challenged with immunity-inducible factors, suggesting their function in guaranteeing fertilization success. Besides, we identified some important reproductive genes such as juvenile hormone- and ecdysteroid-related genes in this de novo assembly. In conclusion, this transcriptomic sequencing of B. dorsalis MAG/ED provides insights to facilitate further functional research of reproduction, immunity and molecular evolution of reproductive proteins in this important agricultural pest.

  15. Macrophage Immune Response Suppression by Recombinant Mycobacterium tuberculosis Antigens, the ESAT-6, CFP-10, and ESAT-6/CFP-10 Fusion Proteins

    Science.gov (United States)

    Seghatoleslam, Atefeh; Hemmati, Mina; Ebadat, Saeedeh; Movahedi, Bahram; Mostafavi-Pour, Zohreh

    2016-01-01

    Background: Macrophage immune responses are affected by the secretory proteins of Mycobacterium tuberculosis (Mtb). This study aimed to examine the immune responses of macrophages to Mtb secretory antigens, namely ESAT-6, CFP-10, and ESAT-6/CFP-10. Methods: THP-1 cells (a human monocytic cell line) were cultured and differentiated to macrophages by phorbol 12-myristate 13-acetate. The cytotoxicity of the recombinant Mtb proteins was assessed using the MTT assay. Two important immune responses of macrophages, namely NO and ROS production, were measured in response to the ESAT-6, CFP-10, and ESAT-6/CFP-10 antigens. The data were analyzed using one-way ANOVA with SPSS, version 16, and considered significant at P<0.05. Results: The results showed that the ESAT-6, CFP-10, and ESAT-6/CFP-10 proteins markedly reduced macrophage immune response. The treatment of the THP-1-differentiated cells with ESAT-6, CFP-10, and ESAT-6/CFP-10 reduced NO and ROS production. The treated THP-1-differentiated cells exhibited less inducible NO synthase activity than did the untreated cells. No toxic effect on macrophage viability was observed for the applied proteins at the different concentrations. Conclusion: It seems that the decline in macrophage immune response is due to the suppression of NO and ROS production pathways without any effect on cell viability. PMID:27365551

  16. In vivo effects of Escherichia coli lipopolysaccharide on regulation of immune response and protein expression in striped catfish (Pangasianodon hypophthalmus).

    Science.gov (United States)

    Hang, Bui Thi Bich; Milla, Sylvain; Gillardin, Virginie; Phuong, Nguyen Thanh; Kestemont, Patrick

    2013-01-01

    Lipolysaccharide (LPS), a component of outer membrane protein of gram-negative bacteria, reportedly stimulates fish immune system. However, mechanisms driving this immunomodulatory effect are yet unknown. To determine effects of Escherichia coli lipopolysaccharide on regulation of immune response and protein expression of striped catfish (Pangasianodon hypophthalmus), juvenile fish (20-25 g) were injected with 3, 15 or 45 mg E.coli LPS/kg and challenged with Edwardsiella ictaluri. Plasma cortisol and glucose were rather low and did not differ (p<0.05) among treatments. All LPS treatments differed regarding blood cell count and immune variables such as plasma and spleen lysozyme, complement activity and antibody titer, 3mg LPS/kg yielding best results; red blood cell count was not affected by LPS treatment. Accumulated mortalities after bacterial challenge were 23.4, 32.8, 37.7 and 52.5% for treatment 3, 15, 45 mg LPS/kg fish and control respectively. Proteomic analysis of peripheral blood mononuclear cells (PBMC) confirmed that LPS induced differentially over-expressed immune proteins such as complement component C3 and lysozyme C2 precursor. Regulation of other proteins such as Wap65, alpha-2 macroglobulin-3 and transferrin precursor was also demonstrated. Striped catfish injected with E.coli LPS enhanced innate immune responses.

  17. Innate immune response, intestinal morphology and microbiota changes in Senegalese sole fed plant protein diets with probiotics or autolysed yeast.

    Science.gov (United States)

    Batista, S; Medina, A; Pires, M A; Moriñigo, M A; Sansuwan, K; Fernandes, J M O; Valente, L M P; Ozório, R O A

    2016-08-01

    The effects of using plant ingredients in Senegalese sole (Solea senegalensis) diet on immune competence and intestine morphology and microbial ecology are still controversial. Probiotics or immunostimulants can potentially alter the intestinal microbiota in a way that protects fish against pathogens. The current study aimed to examine the intestine histology and microbiota and humoral innate immune response in juvenile sole fed diets with low (35 %) or high (72 %) content of plant protein (PP) ingredients supplemented with a multispecies probiotic bacteria or autolysed yeast. Fish fed the probiotic diet had lower growth performance. Lysozyme and complement activities were significantly higher in fish fed PP72 diets than in their counterparts fed PP35 diets after 17 and 38 days of feeding. At 2 days of feeding, fish fed unsupplemented PP72 showed larger intestine section area and longer villus than fish fed unsupplemented PP35. At 17 days of feeding, fish fed unsupplemented PP72 showed more goblet cells than the other dietary groups, except the group fed yeast supplemented PP35 diet. High dietary PP level, acutely stimulate fish innate immune defence of the fish after 2 and 17 days of feeding. However, this effect does not occur after 73 days of feeding, suggesting a habituation to dietary treatments and/or immunosuppression, with a reduction in the number of the goblet cells. Fish fed for 38 days with diets supplemented with autolysed yeast showed longer intestinal villus. The predominant bacteria found in sole intestine were Vibrio sp. and dietary probiotic supplementation caused a reduction in Vibrio content, regardless of the PP level.

  18. Construction of prokaryotic expression system of TGF-β1 epitope gene and identification of recombinant fusion protein immunity

    Institute of Scientific and Technical Information of China (English)

    Yong-Hong Guo; Zhi-Ming Hao; Jin-Yan Luo; Jun-Hong Wang

    2005-01-01

    AIM: To insert the constructed TGF-β1the el loop of C-terminus of truncated hepatitis B core antigen to increase TGF-β1expression system and to identify immunity of the expressed recombinant protein in order to exploit the possibility for obtaining anti- TGF-β1METHODS: The TGF-β1mature TGF-β1TGF-32) was amplified by polymerase chain reaction from the recombinant pGEM-7z/TGF-β1HBcAg gene fragments (encoding HBcAg from 1-71 and 89-144 amino acid residues) were amplified from PYTA1-HBcAg vector. The recombinant vector pGEMEX-1 was used to insert HBcAg1-71, TGF-β1into restrictive endonuclease enzyme and ligated with T4ligase. The fusion gene fragments HBcAg1-71-TGF-β1HBcAg89-144 were recloned to pET28a(+) and the DNA sequence was confirmed by the dideoxy chain termination method. The recombinant vector pET28a (+)/CTC was transformed and expressed in E.. Coli BL21 (DE3)under induction of IPTG. After purification with Ni+2-NTA agarose resins, the antigenicity of purified protein was detected by ELISA and Western blot and visualized under electron microscope.RESULTS: Enzyme digestion analysis and sequencing showed that TGF-β1loop of C-terminus of truncated hepatitis B core antigen.SDS-PAGE analysis showed that relative molecular mass(Mr) of the expressed product by pET28a (+)/CTC was Mr 24 600.The output of the target recombinant protein was approximately 34.8% of the total bacterial protein,mainly presented in the form of inclusion body. Western blotting and ELISA demonstrated that the fusion protein could combine with anti-TGF-β1not with anti-HBcAg. The purity of protein was about 90% and the protein was in the form of self-assembling particles visualized under electron microscope. This fusion protein had good anti-TGF-β1could be used as anti-TGF-β1CONCLUSION: A recombinant prokaryotic expression system with high expression efficiency of the target TGF- epitope gene was successfully established.The fusion protein is in the form of self-assembling particles

  19. Strain-transcending immune response generated by chimeras of the malaria vaccine candidate merozoite surface protein 2

    Science.gov (United States)

    Krishnarjuna, Bankala; Andrew, Dean; MacRaild, Christopher A.; Morales, Rodrigo A. V.; Beeson, James G.; Anders, Robin F.; Richards, Jack S.; Norton, Raymond S.

    2016-01-01

    MSP2 is an intrinsically disordered protein that is abundant on the merozoite surface and essential to the parasite Plasmodium falciparum. Naturally-acquired antibody responses to MSP2 are biased towards dimorphic sequences within the central variable region of MSP2 and have been linked to naturally-acquired protection from malaria. In a phase IIb study, an MSP2-containing vaccine induced an immune response that reduced parasitemias in a strain-specific manner. A subsequent phase I study of a vaccine that contained both dimorphic forms of MSP2 induced antibodies that exhibited functional activity in vitro. We have assessed the contribution of the conserved and variable regions of MSP2 to the generation of a strain-transcending antibody response by generating MSP2 chimeras that included conserved and variable regions of the 3D7 and FC27 alleles. Robust anti-MSP2 antibody responses targeting both conserved and variable regions were generated in mice, although the fine specificity and the balance of responses to these regions differed amongst the constructs tested. We observed significant differences in antibody subclass distribution in the responses to these chimeras. Our results suggest that chimeric MSP2 antigens can elicit a broad immune response suitable for protection against different strains of P. falciparum. PMID:26865062

  20. Enhancement of immunohistochemical staining of scrapie proteins and immune cells within lymph nodes of early scrapie-infected sheep.

    Science.gov (United States)

    Furr, Annissa; Knudsen, David; Hildreth, Michael B; Young, Alan J

    2011-08-31

    Transmissible spongiform encephalopathies (TSE) are a group of fatal neurodegenerative diseases that affect animals as well as humans. The oldest of these diseases is Scrapie seen in sheep. Scrapie is caused by an altered form (PrP(sc)), capable of inducing "self-replication" of the normal host prion protein (PrP(c)). There is currently no universal standard for antigen retrieval when using immunohistochemistry to simultaneously stain the PrP(c) protein and other cellular markers. The use of formalin-fixed tissue creates a challenge by concealing the antigenic sites where an antibody would bind, and lengthy antigen retrieval methods must be applied in order to facilitate staining. Further complicating sheep tissue immunohistochemistry is a significant lack of commercial antibodies to sheep cell markers available in research. Here we developed a novel immunohistochemical technique using trypsin, formic acid, and hydrated autoclaving using citraconic anhydride buffer to increase sensitivity of staining for scrapie proteins and immune cell subsets. This allowed us to stain and identify cells within lymphoid tissue associated with early lymphoid pathogenesis in scrapie.

  1. The Serine Protease Pic From Enteroaggregative Escherichia coli Mediates Immune Evasion by the Direct Cleavage of Complement Proteins.

    Science.gov (United States)

    Abreu, Afonso G; Fraga, Tatiana R; Granados Martínez, Adriana P; Kondo, Marcia Y; Juliano, Maria A; Juliano, Luiz; Navarro-Garcia, Fernando; Isaac, Lourdes; Barbosa, Angela S; Elias, Waldir P

    2015-07-01

    Enteroaggregative and uropathogenic Escherichia coli, Shigella flexneri 2a, and the hybrid enteroaggregative/Shiga toxin-producing E. coli strain (O104:H4) are important pathogens responsible for intestinal and urinary tract infections, as well as sepsis and hemolytic uremic syndrome. They have in common the production of a serine protease called Pic. Several biological roles for Pic have been described, including protection of E. coli DH5α from complement-mediated killing. Hereby we showed that Pic significantly reduces complement activation by all 3 pathways. Pic cleaves purified C3/C3b and other proteins from the classic and lectin pathways, such as C4 and C2. Cleavage fragments of C3, C4, and C2 were also observed with HB101(pPic1) culture supernatants, and C3 cleavage sites were mapped by fluorescence resonance energy transfer peptides. Experiments using human serum as a source of complement proteins confirmed Pic proteolytic activity on these proteins. Furthermore, Pic works synergistically with the human complement regulators factor I and factor H, promoting inactivation of C3b. In the presence of both regulators, further degradation of C3 α' chain was observed. Therefore, Pic may contribute to immune evasion of E. coli and S. flexneri, favoring invasiveness and increasing the severity of the disorders caused by these pathogens.

  2. Disruption of Protein Mannosylation Affects Candida guilliermondii Cell Wall, Immune Sensing, and Virulence

    Directory of Open Access Journals (Sweden)

    María J. Navarro-Arias

    2016-12-01

    Full Text Available The fungal cell wall contains glycoproteins that interact with the host immune system. In the prominent pathogenic yeast Candida albicans, Pmr1 acts as a Golgi-resident ion pump that provides cofactors to mannosyltransferases, regulating the synthesis of mannans attached to glycoproteins. To gain insight into a putative conservation of such a crucial process within opportunistic yeasts, we were particularly interested in studying the role of the PMR1 homolog in a low-virulent species that rarely causes candidiasis, Candida guilliermondii. We disrupted C. guilliermondii PMR1 and found that loss of Pmr1 affected cell growth and morphology, biofilm formation, susceptibility to cell wall perturbing agents, mannan levels, and the wall composition and organization. Despite there was a significant increment in the amount of β1,3-glucan exposed at the wall surface, this positively influenced only the ability of the mutant to stimulate IL-10 production by human monocytes, suggesting that recognition of both mannan and β1,3-glucan, is required to stimulate strong levels of pro-inflammatory cytokines. Accordingly, our results indicate C. guilliermondii sensing by monocytes was critically dependent on the recognition of N-linked mannans and β1,3-glucan, as reported in other Candida species. In addition, chemical remotion of cell wall O-linked mannans was found to positively influence the recognition of C. guilliermondii by human monocytes, suggesting that O-linked mannans mask other cell wall components from immune cells. This observation contrasts with that reported in C. albicans. Finally, mice infected with C. guilliermondii pmr1 null mutant cells had significantly lower fungal burdens compared to animals challenged with the parental strain. Accordingly, the null mutant showed inability to kill larvae in the Galleria mellonella infection model. This study thus demonstrates that mannans are relevant for the C. guilliermondii-host interaction, with

  3. Disruption of Protein Mannosylation Affects Candida guilliermondii Cell Wall, Immune Sensing, and Virulence

    Science.gov (United States)

    Navarro-Arias, María J.; Defosse, Tatiana A.; Dementhon, Karine; Csonka, Katalin; Mellado-Mojica, Erika; Dias Valério, Aline; González-Hernández, Roberto J.; Courdavault, Vincent; Clastre, Marc; Hernández, Nahúm V.; Pérez-García, Luis A.; Singh, Dhirendra K.; Vizler, Csaba; Gácser, Attila; Almeida, Ricardo S.; Noël, Thierry; López, Mercedes G.; Papon, Nicolas; Mora-Montes, Héctor M.

    2016-01-01

    The fungal cell wall contains glycoproteins that interact with the host immune system. In the prominent pathogenic yeast Candida albicans, Pmr1 acts as a Golgi-resident ion pump that provides cofactors to mannosyltransferases, regulating the synthesis of mannans attached to glycoproteins. To gain insight into a putative conservation of such a crucial process within opportunistic yeasts, we were particularly interested in studying the role of the PMR1 homolog in a low-virulent species that rarely causes candidiasis, Candida guilliermondii. We disrupted C. guilliermondii PMR1 and found that loss of Pmr1 affected cell growth and morphology, biofilm formation, susceptibility to cell wall perturbing agents, mannan levels, and the wall composition and organization. Despite the significant increment in the amount of β1,3-glucan exposed at the wall surface, this positively influenced only the ability of the mutant to stimulate IL-10 production by human monocytes, suggesting that recognition of both mannan and β1,3-glucan, is required to stimulate strong levels of pro-inflammatory cytokines. Accordingly, our results indicate C. guilliermondii sensing by monocytes was critically dependent on the recognition of N-linked mannans and β1,3-glucan, as reported in other Candida species. In addition, chemical remotion of cell wall O-linked mannans was found to positively influence the recognition of C. guilliermondii by human monocytes, suggesting that O-linked mannans mask other cell wall components from immune cells. This observation contrasts with that reported in C. albicans. Finally, mice infected with C. guilliermondii pmr1Δ null mutant cells had significantly lower fungal burdens compared to animals challenged with the parental strain. Accordingly, the null mutant showed inability to kill larvae in the Galleria mellonella infection model. This study thus demonstrates that mannans are relevant for the C. guilliermondii-host interaction, with an atypical role for O

  4. The hnRNP-Q protein LIF2 participates in the plant immune response.

    Directory of Open Access Journals (Sweden)

    Clémentine Le Roux

    Full Text Available Eukaryotes have evolved complex defense pathways to combat invading pathogens. Here, we investigated the role of the Arabidopsis thaliana heterogeneous nuclear ribonucleoprotein (hnRNP-Q LIF2 in the plant innate immune response. We show that LIF2 loss-of-function in A. thaliana leads to changes in the basal expression of the salicylic acid (SA- and jasmonic acid (JA- dependent defense marker genes PR1 and PDF1.2, respectively. Whereas the expression of genes involved in SA and JA biosynthesis and signaling was also affected in the lif2-1 mutant, no change in SA and JA hormonal contents was detected. In addition, the composition of glucosinolates, a class of defense-related secondary metabolites, was altered in the lif2-1 mutant in the absence of pathogen challenge. The lif2-1 mutant exhibited reduced susceptibility to the hemi-biotrophic pathogen Pseudomonas syringae and the necrotrophic ascomycete Botrytis cinerea. Furthermore, the lif2-1 sid2-2 double mutant was less susceptible than the wild type to P. syringae infection, suggesting that the lif2 response to pathogens was independent of SA accumulation. Together, our data suggest that lif2-1 exhibits a basal primed defense state, resulting from complex deregulation of gene expression, which leads to increased resistance to pathogens with various infection strategies. Therefore, LIF2 may function as a suppressor of cell-autonomous immunity. Similar to its human homolog, NSAP1/SYNCRIP, a trans-acting factor involved in both cellular processes and the viral life cycle, LIF2 may regulate the conflicting aspects of development and defense programs, suggesting that a conserved evolutionary trade-off between growth and defense pathways exists in eukaryotes.

  5. The Structure of the Toxin and Type Six Secretion System Substrate Tse2 in Complex with Its Immunity Protein.

    Science.gov (United States)

    Robb, Craig S; Robb, Melissa; Nano, Francis E; Boraston, Alisdair B

    2016-02-01

    Tse2 is a cytoactive toxin secreted by a type six secretion apparatus of Pseudomonas aeruginosa. The Tse2 toxin naturally attacks a target in the cytoplasm of bacterial cells, and can cause toxicity if artificially introduced into eukaryotic cells. The X-ray crystal structure of the complex of Tse2 and its cognate immunity protein Tsi2 revealed a heterotetrameric structure with an extensive binding interface. Structural identity was found between Tse2 and NAD-dependent enzymes, especially ADP-ribosylating toxins, which facilitated the identification of the Tse2 active site and revealed it to be occluded upon binding the inhibitor Tsi2. The structural identity shared with NAD-dependent enzymes, including conserved catalytic residues, suggests that the mechanism of Tse2 toxicity may be NAD dependent.

  6. Heat-shock protein 70 expression in shrimp Fenneropenaeus chinensis during thermal and immune-challenged stress

    Institute of Scientific and Technical Information of China (English)

    GUO Zhenyu; JIAO Chuanzhen; XIANG Jianhai

    2004-01-01

    Using western immunoblotting, we obtained heat-shock protein 70 (HSP70) induction data and distribution in different tissues from shrimp Fenneropenaeus chinensis during thermal and immune-challenged stresses. This is probably the first report of the effects of various stressors on the expression of HSP70 in shrimp. HSP70 was prominently induced in hepatopancreas and gills, but not in muscle, eyestalk and hemolymph, when the shrimp were exposed to heat shock and Vibrio anguillavium-challenged stresses. Cold shock and WSSV treatment had no significant effects on the levels of HSP70 expression in all tissues examined. HSP70 induction was greatest after 2 h exposure to heat shock stress, which was elevated after acute heat shock exposure of 10℃ above ambient temperature.

  7. A monoclonal antibody against the human SUMO-1 protein obtained by immunization with recombinant protein and CpG-DNA-liposome complex.

    Science.gov (United States)

    Kim, Dongbum; Lee, Joo Young; Song, Dae-Geun; Kwon, Sanghoon; Lee, Younghee; Pan, Cheol-Ho; Kwon, Hyung-Joo

    2013-10-01

    Post-translational modification regulated by conjugation of a small ubiquitin-like modifier (SUMO) is involved in various cellular processes. In this study, we expressed and purified recombinant human SUMO-1 (hSUMO-1). BALB/c mice were immunized with a complex of hSUMO-1 protein and Lipoplex(O) to produce hSUMO-1-specific antibodies. Using conventional hybridoma technology, we obtained four hybridoma clones derived from the mouse with the highest antibody titer against hSUMO-1. Based on Western blot analysis, our hSUMO-1 monoclonal antibody specifically recognizes hSUMO-1, but not other SUMO proteins. These results support that the anti-hSUMO-1 monoclonal antibody produced with the aid of Lipoplex(O) adjuvant is specific and that Lipoplex(O) is useful for development of monoclonal antibodies against recombinant protein. In addition, we analyzed human tissues to examine the distribution of hSUMO-1. Higher expression of hSUMO-1 was detected in normal adrenal gland, esophagus, pancreas, liver, stomach, kidney, and uterus than in corresponding cancer tissues, suggesting a tumor suppressive function of hSUMO-1.

  8. Enhancing mucosal immunity in mice by recombinant adenovirus expressing major epitopes of porcine circovirus-2 capsid protein delivered with cytosine-phosphate-guanosine oligodeoxynucleotides.

    Science.gov (United States)

    Chang, Hong-Tao; He, Xiu-Yuan; Liu, Yu-Feng; Chen, Lu; Guo, Quan-Hai; Yu, Qiu-Ying; Zhao, Jun; Wang, Xin-Wei; Yang, Xia; Wang, Chuan-Qing

    2014-01-01

    A recombinant replication-defective adenovirus expressing the major epitopes of porcine circovirus-2 (PCV-2) capsid protein (rAd/Cap/518) was previously constructed and shown to induce mucosal immunity in mice following intranasal delivery. In the present study, immune responses induced by intranasal immunization with a combination of rAd/Cap/518 and cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) were evaluated in mice. The levels of PCV-2-specific IgG in serum and IgA in saliva, lung, and intestinal fluids were significantly higher in the group immunized with rAd/Cap/518 and CpG ODN than animals immunized with rAd/Cap/518 alone. The frequencies of IL-2-secreting CD4⁺ T cells and IFN-γ-producing CD8⁺ T cells were significantly higher in the combined immunization group than mice immunized with rAd/Cap/518 alone. The frequencies of CD3⁺, CD3⁺CD4⁺CD8⁻, and CD3⁺CD4⁻CD8⁺ T cells in the combined immunization group were similar to that treated with CpG ODN alone, but significantly higher than mice that did not receive CpG ODN. PCV-2 load after challenge in the combined immunization group was significantly lower than that in the phosphate-buffered saline placebo group and approximately 7-fold lower in the group treated with CpG ODN alone. These results indicate that rAd/Cap/518 combined with CpG ODN can enhance systemic and local mucosal immunity in mice, and represent a promising synergetic mucosal vaccine against PCV-2.

  9. Immunization of mice with recombinant mosquito salivary protein D7 enhances mortality from subsequent West Nile virus infection via mosquito bite.

    Directory of Open Access Journals (Sweden)

    Krystle L Reagan

    Full Text Available BACKGROUND: Mosquito salivary proteins (MSPs modulate the host immune response, leading to enhancement of arboviral infections. Identification of proteins in saliva responsible for immunomodulation and counteracting their effects on host immune response is a potential strategy to protect against arboviral disease. We selected a member of the D7 protein family, which are among the most abundant and immunogenic in mosquito saliva, as a vaccine candidate with the aim of neutralizing effects on the mammalian immune response normally elicited by mosquito saliva components during arbovirus transmission. METHODOLOGY/PRINCIPAL FINDINGS: We identified D7 salivary proteins of Culex tarsalis, a West Nile virus (WNV vector in North America, and expressed 36 kDa recombinant D7 (rD7 protein for use as a vaccine. Vaccinated mice exhibited enhanced interferon-γ and decreased interleukin-10 expression after uninfected mosquito bite; however, we found unexpectedly that rD7 vaccination resulted in enhanced pathogenesis from mosquito-transmitted WNV infection. Passive transfer of vaccinated mice sera to naïve mice also resulted in increased mortality rates from subsequent mosquito-transmitted WNV infection, implicating the humoral immune response to the vaccine in enhancement of viral pathogenesis. Vaccinated mice showed decreases in interferon-γ and increases in splenocytes producing the regulatory cytokine IL-10 after WNV infection by mosquito bite. CONCLUSIONS/SIGNIFICANCE: Vector saliva vaccines have successfully protected against other blood-feeding arthropod-transmitted diseases. Nevertheless, the rD7 salivary protein vaccine was not a good candidate for protection against WNV disease since immunized mice infected via an infected mosquito bite exhibited enhanced mortality. Selection of salivary protein vaccines on the bases of abundance and immunogenicity does not predict efficacy.

  10. An Eimeria vaccine candidate based on Eimeria tenella immune mapped protein 1 and the TLR-5 agonist Salmonella typhimurium FliC flagellin

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Guangwen; Qin, Mei [National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Liu, Xianyong [National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Key Laboratory of Zoonosis, China Ministry of Agriculture and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Suo, Jingxia; Tang, Xinming; Tao, Geru [National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Han, Qian [Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061 (United States); Suo, Xun [National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Key Laboratory of Zoonosis, China Ministry of Agriculture and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Wu, Wenxue, E-mail: labboard@126.com [National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Key Laboratory of Zoonosis, China Ministry of Agriculture and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China)

    2013-10-25

    Highlights: •We found a new protective protein – (IMPI) in Eimeria tenella. •EtIMP1-flagellin fusion protein is an effective immunogen against Eimeria infection. •Flagellin can be as an apicomplexan parasite vaccine adjuvant in chickens. -- Abstract: Immune mapped protein-1 (IMP1) is a new protective protein in apicomplexan parasites, and exits in Eimeria tenella. But its structure and immunogenicity in E. tenella are still unknown. In this study, IMPI in E. tenella was predicted to be a membrane protein. To evaluate immunogenicity of IMPI in E. tenella, a chimeric subunit vaccine consisting of E. tenella IMP1 (EtIMP1) and a molecular adjuvant (a truncated flagellin, FliC) was constructed and over-expressed in Escherichia coli and its efficacy against E. tenella infection was evaluated. Three-week-old AA broiler chickens were vaccinated with the recombinant EtIMP1-truncated FliC without adjuvant or EtIMP1 with Freund’s Complete Adjuvant. Immunization of chickens with the recombinant EtIMP1-truncated FliC fusion protein resulted in stronger cellular immune responses than immunization with only recombinant EtIMP1 with adjuvant. The clinical effect of the EtIMP1-truncated FliC without adjuvant was also greater than that of the EtIMP1 with adjuvant, which was evidenced by the differences between the two groups in body weight gain, oocyst output and caecal lesions of E. tenella-challenged chickens. The results suggested that the EtIMP1-flagellin fusion protein can be used as an effective immunogen in the development of subunit vaccines against Eimeria infection. This is the first demonstration of antigen-specific protective immunity against avian coccidiosis using a recombinant flagellin as an apicomplexan parasite vaccine adjuvant in chickens.

  11. Cloning and Expression of Plantaricin W Produced by Lactobacillus plantarum U10 Isolate from "Tempoyak" Indonesian Fermented Food as Immunity Protein in Lactococcus lactis.

    Science.gov (United States)

    Lages, Aksar Chair; Mustopa, Apon Zaenal; Sukmarini, Linda; Suharsono

    2015-10-01

    Plantaricins, one of bacteriocin produced by Lactobacillus plantarum, are already known to have activities against several pathogenic bacterium. L. plantarum U10 isolated from "tempoyak," an Indonesian fermented food, produced one kind of plantaricin designated as plantaricin W (plnW). The plnW is suggested as a putative membrane location of protein and has similar conserved motif which is important as immunity to bacteriocin itself. Thus, due to study about this plantaricin, several constructs have been cloned and protein was analyzed in Lactococcus lactis. In this study, plnW gene was successfully cloned into vector NICE system pNZ8148 and created the transformant named L. lactis NZ3900 pNZ8148-WU10. PlnW protein was 25.3 kDa in size. The concentration of expressed protein was significantly increased by 10 ng/mL nisin induction. Furthermore, PlnW exhibited protease activity with value of 2.22 ± 0.05 U/mL and specific activity about 1.65 ± 0.03 U/mg protein with 50 ng/mL nisin induction. Immunity study showed that the PlnW had immunity activity especially against plantaricin and rendered L. lactis recombinant an immunity broadly to other bacteriocins such as pediocin, fermentcin, and acidocin.

  12. Analysis of skin and secretions of Dybowski's frogs (Rana dybowskii) exposed to Staphylococcus aureus or Escherichia coli identifies immune response proteins.

    Science.gov (United States)

    Xiao, Xiang-Hong; Miao, Hui-Min; Xu, Yi-Gang; Zhang, Jing-Yu; Chai, Long-Hui; Xu, Jia-Jia

    2014-04-01

    The aim of the present study was to investigate responses in Dybowski's frogs (Rana dybowskii) exposed to bacteria, using proteomic and transcriptomic approaches. Staphylococcus aureus and Escherichia coli were used as representative Gram-positive and Gram-negative bacteria, respectively, in an infectious challenge model. Frog skin and skin secretions were collected and protein expression in infected frogs compared to control frogs by two-dimensional gel electrophoresis, silver staining, and image analysis. Proteins that demonstrated differential expression were analysed by mass spectrometry and identified by searching protein databases. More than 180 protein spots demonstrated differential expression in E. coli- or S. aureus-challenged groups and, of these, more than 55 spots were up- or down-regulated at least sixfold, post-infection. Proteins with a potential function in the immune response were identified, such as stathmin 1a, annexin A1, superoxide dismutase A, C-type lectin, lysozyme, antimicrobial peptides, cofilin-1-B, mannose receptor, histone H4, prohormone convertase 1, carbonyl reductase 1 and some components of the Toll-like receptor (TLR) signalling pathway. These molecules are potential candidates for further investigation of immune mechanisms in R. dybowskii; in particular, TLR-mediated responses, which might be activated in frogs exposed to pathogenic bacteria as part of innate immune defence, but which might also impact on adaptive immunity to infection.

  13. Erwinia amylovora effector protein Eop1 suppresses PAMP-triggered immunity in Malus

    Science.gov (United States)

    Erwinia amylovora (Ea) utilizes a type three secretion system (T3SS) to deliver effector proteins into plant host cells. Several Ea effectors have been identified based on their sequence similarity to plant and animal bacterial pathogen effectors; however, the function of the majority of Ea effecto...

  14. Inflammation-associated activation of coagulation and immune regulation by the protein C pathway.

    Science.gov (United States)

    Weiler, Hartmut

    2014-05-01

    The inflammation-induced activation of the protein C pathway provides negative feedback inhibition of coagulation and exerts coagulation-independent anti-inflammatory and cytoprotective effects. The balance between these activities of aPC modulates the outcome of diverse inflammatory diseases such as encephalitis, diabetes, and sepsis; and is affected by naturally occurring aPC-resistance of coagulation factor V Leiden.

  15. Respiratory innate immune proteins differentially modulate the neutrophil respiratory burst response to influenza A virus

    DEFF Research Database (Denmark)

    White, Mitchell R; Crouch, Erika; Vesona, Jenny;

    2005-01-01

    Oxidants and neutrophils contribute to lung injury during influenza A virus (IAV) infection. Surfactant protein (SP)-D plays a pivotal role in restricting IAV replication and inflammation in the first several days after infection. Despite its potent anti-inflammatory effects in vivo, preincubation...

  16. Novel mechanism of bacteriocin secretion and immunity carried out by lactococcal multidrug resistance proteins

    NARCIS (Netherlands)

    Gajic, O; Buist, G; Kojic, M; Topisirovic, L; Kuipers, OP; Kok, J

    2003-01-01

    A natural isolate of Lactococcus lactis was shown to produce two narrow spectrum class II bacteriocins, designated LsbA and LsbB. The cognate genes are located on a 5.6-kb plasmid within a gene cluster specifying LmrB, an ATP-binding cassette-type multidrug resistance transporter protein. LsbA is a

  17. Induction of immune response in mice with a DNA vaccine encoding outer membrane protein (omp31) of Brucella melitensis 16M.

    Science.gov (United States)

    Gupta, V K; Rout, P K; Vihan, V S

    2007-06-01

    Brucellosis causes serious economic losses to goat farmers by way of reproductive losses in the form of abortions and stillbirths. Nucleic acid vaccines provide an exciting approach for antigen presentation to the immune system. In this study, we evaluated the ability of DNA vaccine encoding the omp31 protein of Brucella melitensis 16M to induce cellular and humoral immune responses in mice. We constructed eukaryotic expression vectors called pTargeTomp31, encoding outer membrane protein (omp31) of B. melitensis 16M. pTargeTomp31 was injected intramuscularly three times, at 3-week intervals in groups of mice 6 weeks of age. pTargeTomp31 induced good antibody response in ELISA . pTargeTomp31 elicited a T-cell-proliferative response and also induced a strong gamma interferon production upon restimulation with either the omp31 antigen or B. melitensis 16M extract. We also demonstrate that animals immunized with this plasmid elicited a strong and long-lived memory immune response. Furthermore, pTargeTomp31 elicited a typical T-helper 1-dominated immune response in mice, as determined by immunoglobulin G isotype analysis. This vaccine also provided the moderate degree of protection to the mice. This study for the first time focuses on DNA immunization of a gene from B. melitensis. These results may lead to the development of a DNA-based vaccine for the control of brucellosis in goats.

  18. CRACC-targeting Fc-fusion protein induces activation of NK cells and DCs and improves T cell immune responses to antigenic targets.

    Science.gov (United States)

    Aldhamen, Yasser A; Rastall, David P W; Chen, Weimin; Seregin, Sergey S; Pereira-Hicks, Cristiane; Godbehere, Sarah; Kaminski, Norbert E; Amalfitano, Andrea

    2016-06-08

    The CD2-like receptor activating cytotoxic cell (CRACC) receptor is a member of the SLAM family of receptors that are found on several types of immune cells. We previously demonstrated that increasing the abundance of the adaptor protein EAT-2 during vaccination enhanced innate and adaptive immune responses to vaccine antigens. Engagement of the CRACC receptor in the presence of the EAT-2 adaptor generally results in immune cell activation, while activating CRACC signaling in cells that lack EAT-2 adaptor inhibits their effector and regulatory functions. As EAT-2 is the only SAP adaptor that interacts with the CRACC receptor, we hypothesized that technologies that specifically modulate CRACC signaling during vaccination may also improve antigen specific adaptive immune responses. To test this hypothesis, we constructed a CRACC-targeting Fc fusion protein and included it in vaccination attempts. Indeed, mice co-vaccinated with the CRACC-Fc fusion protein and an adenovirus vaccine expressing the HIV-Gag protein had improved Gag-specific T cell responses, as compared to control mice. These responses are characterized by increased numbers of Gag-specific tetramer+ CD8+ T cells and increases in production of IFNγ, TNFα, and IL2, by Gag-specific CD8+ T cells. Moreover, our results revealed that use of the CRACC-Fc fusion protein enhances vaccine-elicited innate immune responses, as characterized by increased dendritic cells (DCs) maturation and IFNγ production from NK cells. This study highlights the importance of CRACC signaling during the induction of an immune response generally, and during vaccinations specifically, and also lends insight into the mechanisms underlying our prior results noting EAT-2-dependent improvements in vaccine efficacy.

  19. The Non-structural Protein 5 and Matrix Protein Are Antigenic Targets of T Cell Immunity to Genotype 1 Porcine Reproductive and Respiratory Syndrome Viruses

    Science.gov (United States)

    Mokhtar, Helen; Pedrera, Miriam; Frossard, Jean-Pierre; Biffar, Lucia; Hammer, Sabine E.; Kvisgaard, Lise K.; Larsen, Lars E.; Stewart, Graham R.; Somavarapu, Satyanarayana; Steinbach, Falko; Graham, Simon P.

    2016-01-01

    The porcine reproductive and respiratory syndrome virus (PRRSV) is the cause of one of the most economically important diseases affecting swine worldwide. Efforts to develop a next-generation vaccine have largely focused on envelope glycoproteins to target virus-neutralizing antibody responses. However, these approaches have failed to demonstrate the necessary efficacy to progress toward market. T cells are crucial to the control of many viruses through cytolysis and cytokine secretion. Since control of PRRSV infection is not dependent on the development of neutralizing antibodies, it has been proposed that T cell-mediated immunity plays a key role. Therefore, we hypothesized that conserved T cell antigens represent prime candidates for the development a novel PRRS vaccine. Antigens were identified by screening a proteome-wide synthetic peptide library with T cells from cohorts of pigs rendered immune by experimental infections with a closely related (subtype 1) or divergent (subtype 3) PRRSV-1 strain. Dominant T cell IFN-γ responses were directed against the non-structural protein 5 (NSP5), and to a lesser extent, the matrix (M) protein. The majority of NSP5-specific CD8 T cells and M-specific CD4 T cells expressed a putative effector memory phenotype and were polyfunctional as assessed by coexpression of TNF-α and mobilization of the cytotoxic degranulation marker CD107a. Both antigens were generally well conserved among strains of both PRRSV genotypes. Thus, M and NSP5 represent attractive vaccine candidate T cell antigens, which should be evaluated further in the context of PRRSV vaccine development. PMID:26909080

  20. The non-structural protein 5 and matrix protein are antigenic targets of T cell immunity to genotype 1 porcine reproductive and respiratory syndrome viruses

    Directory of Open Access Journals (Sweden)

    Helen eMokhtar

    2016-02-01

    Full Text Available The porcine reproductive and respiratory syndrome virus (PRRSV is the cause of one of the most economically important diseases affecting swine worldwide. Efforts to develop a next-generation vaccine have largely focussed on envelope glycoproteins to target virus-neutralising antibody responses. However, these approaches have failed to demonstrate the necessary efficacy to progress towards market. T cells are crucial to the control of many viruses through cytolysis and cytokine secretion. Since control of PRRSV infection is not dependent on the development of neutralising antibodies, it has been proposed that T cell mediated immunity plays a key role. We therefore hypothesised that conserved T cell antigens represent prime candidates for the development a novel PRRS vaccine. Antigens were identified by screening a proteome-wide synthetic peptide library with T cells from cohorts of pigs rendered immune by experimental infections with a closely-related (subtype 1 or divergent (subtype 3 PRRSV-1 strain. Dominant T cell IFN-γ responses were directed against the non-structural protein 5 (NSP5, and to a lesser extent, the matrix (M protein. The majority of NSP5-specific CD8 T cells and M-specific CD4 T cells expressed a putative effector memory phenotype and were polyfunctional as assessed by co-expression of TNF-α and mobilisation of the cytotoxic degranulation marker CD107a. Both antigens were generally well conserved amongst strains of both PRRSV genotypes. Thus M and NSP5 represent attractive vaccine candidate T cell antigens which should be evaluated further in the context of PRRSV vaccine development.

  1. Protective anti-Pseudomonas aeruginosa humoral and cellular mucosal immunity by AdC7-mediated expression of the P. aeruginosa protein OprF.

    Science.gov (United States)

    Krause, Anja; Whu, Wen Zhu; Xu, Yaqin; Joh, Ju; Crystal, Ronald G; Worgall, Stefan

    2011-03-03

    Replication-deficient adenoviral (Ad) vectors are an attractive platform for a vaccine against lung infections caused by Pseudomonas aeruginosa. Ad vectors based on non-human serotypes have been developed to circumvent the problem of pre-existing anti-Ad immunity in humans. The present study analyzes the anti-P. aeruginosa systemic and lung mucosal immunity elicited by a non-human primate-based AdC7 vector expressing the outer membrane protein F (AdC7OprF) of P. aeruginosa. Intramuscular immunization of mice with AdC7OprF induced similar levels of serum and mucosal anti-OprF IgG and increased levels of anti-OprF IgA in lung epithelial lining fluid (ELF) compared to immunization with a human serotype Ad5OprF vector (p>0.05). OprF-specific INF-γ in splenic T cells stimulated with OprF-pulsed syngeneic splenic dendritic cells (DC) was similar following immunization with AdC7OprF compared to Ad5OprF (p>0.05). In contrast, OprF-specific INF-γ responses in lung T cells stimulated with either spleen or lung DC were increased following immunization with AdC7OprF compared to Ad5OprF (pmucosal immunity.

  2. Structurally well-defined macrophage activating factor derived from vitamin D3-binding protein has a potent adjuvant activity for immunization.

    Science.gov (United States)

    Yamamoto, N; Naraparaju, V R

    1998-06-01

    Freund's adjuvant produced severe inflammation that augments development of antibodies. Thus, mixed administration of antigens with adjuvant was not required as long as inflammation was induced in the hosts. Since macrophage activation for phagocytosis and antigen processing is the first step of antibody development, inflammation-primed macrophage activation plays a major role in immune development. Therefore, macrophage activating factor should act as an adjuvant for immunization. The inflammation-primed macrophage activation process is the major macrophage activating cascade that requires participation of serum vitamin D3-binding protein (DBP; human DBP is known as Gc protein) and glycosidases of B and T lymphocytes. Stepwise incubation of Gc protein with immobilized beta-galactosidase and sialidase efficiently generated the most potent macrophage activating factor (designated GcMAF) we have ever encountered. Administration of GcMAF (20 or 100 pg/mouse) resulted in stimulation of the progenitor cells for extensive mitogenesis and activation of macrophages. Administration of GcMAF (100 pg/mouse) along with immunization of mice with sheep red blood cells (SRBC) produced a large number of anti-SRBC antibody secreting splenic cells in 2-4 days. Thus, GcMAF has a potent adjuvant activity for immunization. Although malignant tumours are poorly immunogenic, 4 days after GcMAF-primed immunization of mice with heat-killed Ehrlich ascites tumour cells, the ascites tumour was no longer transplantable in these mice.

  3. Memory T-Cell-Mediated Immune Responses Specific to an Alternative Core Protein in Hepatitis C Virus Infection

    Science.gov (United States)

    Bain, Christine; Parroche, Peggy; Lavergne, Jean Pierre; Duverger, Blandine; Vieux, Claude; Dubois, Valérie; Komurian-Pradel, Florence; Trépo, Christian; Gebuhrer, Lucette; Paranhos-Baccala, Glaucia; Penin, François; Inchauspé, Geneviève

    2004-01-01

    In vitro studies have described the synthesis of an alternative reading frame form of the hepatitis C virus (HCV) core protein that was named F protein or ARFP (alternative reading frame protein) and includes a domain coded by the +1 open reading frame of the RNA core coding region. The expression of this protein in HCV-infected patients remains controversial. We have analyzed peripheral blood from 47 chronically or previously HCV-infected patients for the presence of T lymphocytes and antibodies specific to the ARFP. Anti-ARFP antibodies were detected in 41.6% of the patients infected with various HCV genotypes. Using a specific ARFP 99-amino-acid polypeptide as well as four ARFP predicted class I-restricted 9-mer peptides, we show that 20% of the patients display specific lymphocytes capable of producing gamma interferon, interleukin-10, or both cytokines. Patients harboring three different viral genotypes (1a, 1b, and 3) carried T lymphocytes reactive to genotype 1b-derived peptides. In longitudinal analysis of patients receiving therapy, both core and ARFP-specific T-cell- and B-cell-mediated responses were documented. The magnitude and kinetics of the HCV antigen-specific responses differed and were not linked with viremia or therapy outcome. These observations provide strong and new arguments in favor of the synthesis, during natural HCV infection, of an ARFP derived from the core sequence. Moreover, the present data provide the first demonstration of the presence of T-cell-mediated immune responses directed to this novel HCV antigen. PMID:15367612

  4. DNA immunization with fusion of CTLA-4 to hepatitis B virus (HBV core protein enhanced Th2 type responses and cleared HBV with an accelerated kinetic.

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    Ying Yin

    Full Text Available BACKGROUND: Typically, DNA immunization via the intramuscular route induces specific, Th1-dominant immune responses. However, plasmids expressing viral proteins fused to cytotoxic T lymphocyte antigen 4 (CTLA-4 primed Th2-biased responses and were able to induced effective protection against viral challenge in the woodchuck model. Thus, we addressed the question in the mouse model how the Th1/Th2 bias of primed immune responses by a DNA vaccine influences hepatitis B virus (HBV clearance. PRINCIPAL FINDINGS: Plasmids expressing HBV core protein (HBcAg or HBV e antigen and HBcAg fused to the extracellular domain of CTLA-4 (pCTLA-4-HBc, CD27, and full length CD40L were constructed. Immunizations of these DNA plasmids induced HBcAg-specific antibody and cytotoxic T-cell responses in mice, but with different characteristics regarding the titers and subtypes of specific antibodies and intensity of T-cell responses. The plasmid pHBc expressing HBcAg induced an IgG2a-dominant response while immunizations of pCTLA-4-HBc induced a balanced IgG1/IgG2a response. To assess the protective values of the immune responses of different characteristics, mice were pre-immunized with pCTLA-4-HBc and pHBc, and challenged by hydrodynamic injection (HI of pAAV/HBV1.2. HBV surface antigen (HBsAg and DNA in peripheral blood and HBcAg in liver tissue were cleared with significantly accelerated kinetics in both groups. The clearance of HBsAg was completed within 16 days in immunized mice while more than 50% of the control mice are still positive for HBsAg on day 22. Stronger HBcAg-specific T-cell responses were primed by pHBc correlating with a more rapid decline of HBcAg expression in liver tissue, while anti-HBs antibody response developed rapidly in the mice immunized with pCTLA-4-HBc, indicating that the Th1/Th2 bias of vaccine-primed immune responses influences the mode of viral clearance. CONCLUSION: Viral clearance could be efficiently achieved by Th1/Th2-balanced

  5. Engineering the vaccinia virus L1 protein for increased neutralizing antibody response after DNA immunization

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    Moss Bernard

    2009-03-01

    Full Text Available Abstract Background The licensed smallpox vaccine, comprised of infectious vaccinia virus, has associated adverse effects, particularly for immunocompromised individuals. Therefore, safer DNA and protein vaccines are being investigated. The L1 protein, a component of the mature virion membrane that is conserved in all sequenced poxviruses, is required for vaccinia virus entry into host cells and is a target for neutralizing antibody. When expressed by vaccinia virus, the unglycosylated, myristoylated L1 protein attaches to the viral membrane via a C-terminal transmembrane anchor without traversing the secretory pathway. The purpose of the present study was to investigate modifications of the gene expressing the L1 protein that would increase immunogenicity in mice when delivered by a gene gun. Results The L1 gene was codon modified for optimal expression in mammalian cells and potential N-glycosylation sites removed. Addition of a signal sequence to the N-terminus of L1 increased cell surface expression as shown by confocal microscopy and flow cytometry of transfected cells. Removal of the transmembrane domain led to secretion of L1 into the medium. Induction of binding and neutralizing antibodies in mice was enhanced by gene gun delivery of L1 containing the signal sequence with or without the transmembrane domain. Each L1 construct partially protected mice against weight loss caused by intranasal administration of vaccinia virus. Conclusion Modifications of the vaccinia virus L1 gene including codon optimization and addition of a signal sequence with or without deletion of the transmembrane domain can enhance the neutralizing antibody response of a DNA vaccine.

  6. Extensive changes in innate immune gene expression in obese Göttingen minipigs do not lead to changes in concentrations of circulating cytokines and acute phase proteins

    DEFF Research Database (Denmark)

    Højbøge, Tina Rødgaard; Skovgaard, Kerstin; Moesgaard, S. G.;

    2014-01-01

    The usefulness of Göttingen minipigs as models for obesity and obesity-related pathologies is well established. The low-grade inflammation associated with obesity involves a range of innate immune factors; however, to our knowledge, the impact of obesity on innate immune factor expression has...... between adipose tissues and a decreased tissue-specific expression of cytokines and chemokines. In contrast to obese humans, no changes in serum concentrations of haptoglobin, C-reactive protein, serum amyloid A, tumor necrosis factor-α and interleukin 6 were found in obese Göttingen minipigs....... not been studied in Göttingen minipigs. Therefore, we studied the expression of innate immune genes in liver and adipose tissues as well as serum concentrations of cytokines and acute phase proteins in obese vs. lean Göttingen minipigs. In the liver, of 35 investigated genes, the expression of nine...

  7. Expression profile of key immune-related genes in Penaeus monodon juveniles after oral administration of recombinant envelope protein VP28 of white spot syndrome virus.

    Science.gov (United States)

    Thomas, Ancy; Sudheer, Naduvilamuriparampu Saidumuhammed; Kiron, Viswanath; Bright Singh, Issac S; Narayanan, Rangarajan Badri

    2016-07-01

    White spot syndrome virus (WSSV) is the most catastrophic pathogen the shrimp industry has ever encountered. VP28, the abundant envelope protein of WSSV was expressed in bacteria, the purified protein administered orally to Penaeus monodon juveniles and its immune modulatory effects examined. The results indicated significant up-regulation of caspase, penaeidin, crustin, astakine, syntenin, PmRACK, Rab7, STAT and C-type lectin in animals orally administered with this antigen. This revealed the immune modulations in shrimps followed by oral administration of rVP28P which resulted in the reduced transcription of viral gene vp28 and delay in mortality after WSSV challenge. The study suggests the potential of rVP28P to elicit a non-specific immune stimulation in shrimps.

  8. Targeting the non-structural protein 1 from dengue virus to a dendritic cell population confers protective immunity to lethal virus challenge.

    Science.gov (United States)

    Henriques, Hugo R; Rampazo, Eline V; Gonçalves, Antonio J S; Vicentin, Elaine C M; Amorim, Jaime H; Panatieri, Raquel H; Amorim, Kelly N S; Yamamoto, Marcio M; Ferreira, Luís C S; Alves, Ada M B; Boscardin, Silvia B

    2013-01-01

    Dengue is the most prevalent arboviral infection, affecting millions of people every year. Attempts to control such infection are being made, and the development of a vaccine is a World Health Organization priority. Among the proteins being tested as vaccine candidates in preclinical settings is the non-structural protein 1 (NS1). In the present study, we tested the immune responses generated by targeting the NS1 protein to two different dendritic cell populations. Dendritic cells (DCs) are important antigen presenting cells, and targeting proteins to maturing DCs has proved to be an efficient means of immunization. Antigen targeting is accomplished by the use of a monoclonal antibody (mAb) directed against a DC cell surface receptor fused to the protein of interest. We used two mAbs (αDEC205 and αDCIR2) to target two distinct DC populations, expressing either DEC205 or DCIR2 endocytic receptors, respectively, in mice. The fusion mAbs were successfully produced, bound to their respective receptors, and were used to immunize BALB/c mice in the presence of polyriboinosinic: polyribocytidylic acid (poly (I:C)), as a DC maturation stimulus. We observed induction of strong anti-NS1 antibody responses and similar antigen binding affinity irrespectively of the DC population targeted. Nevertheless, the IgG1/IgG2a ratios were different between mouse groups immunized with αDEC-NS1 and αDCIR2-NS1 mAbs. When we tested the induction of cellular immune responses, the number of IFN-γ producing cells was higher in αDEC-NS1 immunized animals. In addition, mice immunized with the αDEC-NS1 mAb were significantly protected from a lethal intracranial challenge with the DENV2 NGC strain when compared to mice immunized with αDCIR2-NS1 mAb. Protection was partially mediated by CD4(+) and CD8(+) T cells as depletion of these populations reduced both survival and morbidity signs. We conclude that targeting the NS1 protein to the DEC205(+) DC population with poly (I:C) opens

  9. Targeting the non-structural protein 1 from dengue virus to a dendritic cell population confers protective immunity to lethal virus challenge.

    Directory of Open Access Journals (Sweden)

    Hugo R Henriques

    Full Text Available Dengue is the most prevalent arboviral infection, affecting millions of people every year. Attempts to control such infection are being made, and the development of a vaccine is a World Health Organization priority. Among the proteins being tested as vaccine candidates in preclinical settings is the non-structural protein 1 (NS1. In the present study, we tested the immune responses generated by targeting the NS1 protein to two different dendritic cell populations. Dendritic cells (DCs are important antigen presenting cells, and targeting proteins to maturing DCs has proved to be an efficient means of immunization. Antigen targeting is accomplished by the use of a monoclonal antibody (mAb directed against a DC cell surface receptor fused to the protein of interest. We used two mAbs (αDEC205 and αDCIR2 to target two distinct DC populations, expressing either DEC205 or DCIR2 endocytic receptors, respectively, in mice. The fusion mAbs were successfully produced, bound to their respective receptors, and were used to immunize BALB/c mice in the presence of polyriboinosinic: polyribocytidylic acid (poly (I:C, as a DC maturation stimulus. We observed induction of strong anti-NS1 antibody responses and similar antigen binding affinity irrespectively of the DC population targeted. Nevertheless, the IgG1/IgG2a ratios were different between mouse groups immunized with αDEC-NS1 and αDCIR2-NS1 mAbs. When we tested the induction of cellular immune responses, the number of IFN-γ producing cells was higher in αDEC-NS1 immunized animals. In addition, mice immunized with the αDEC-NS1 mAb were significantly protected from a lethal intracranial challenge with the DENV2 NGC strain when compared to mice immunized with αDCIR2-NS1 mAb. Protection was partially mediated by CD4(+ and CD8(+ T cells as depletion of these populations reduced both survival and morbidity signs. We conclude that targeting the NS1 protein to the DEC205(+ DC population with poly (I

  10. Gp130-dependent release of acute phase proteins is linked to the activation of innate immune signaling pathways.

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    Maren Luchtefeld

    Full Text Available BACKGROUND: Elevated levels of acute phase proteins (APP are often found in patients with cardiovascular diseases. In a previous study, we demonstrated the importance of the IL-6-gp130 axis -as a key regulator of inflammatory acute phase signaling in hepatocytes-for the development of atherosclerosis. BACKGROUND/PRINCIPAL FINDINGS: Gp130-dependent gene expression was analyzed in a previously established hepatocyte-specific gp130 knockout mouse model. We performed whole transcriptome analysis in isolated hepatocytes to measure tissue specific responses after proinflammatory stimulus with IL-6 across different time points. Our analyses revealed an unexpected small gene cluster that requires IL-6 stimulus for early activation. Several of the genes in this cluster are involved in different cell defense mechanisms. Thus, stressors that trigger both general stress and inflammatory responses lead to activation of a stereotypic innate cellular defense response. Furthermore, we identified a potential biomarker Lipocalin (LCN 2 for the gp130 dependent early inflammatory response. CONCLUSIONS/SIGNIFICANCE: Our findings suggest a complex network of tightly linked genes involved in the early activation of different parts of the innate immune response including acute phase proteins, complement and coagulation cascade.

  11. The tomato NBARC-LRR protein Prf interacts with Pto kinase in vivo to regulate specific plant immunity.

    Science.gov (United States)

    Mucyn, Tatiana S; Clemente, Alfonso; Andriotis, Vasilios M E; Balmuth, Alexi L; Oldroyd, Giles E D; Staskawicz, Brian J; Rathjen, John P

    2006-10-01

    Immunity in tomato (Solanum lycopersicum) to Pseudomonas syringae bacteria expressing the effector proteins AvrPto and AvrPtoB requires both Pto kinase and the NBARC-LRR (for nucleotide binding domain shared by Apaf-1, certain R gene products, and CED-4 fused to C-terminal leucine-rich repeats) protein Prf. Pto plays a direct role in effector recognition within the host cytoplasm, but the role of Prf is unknown. We show that Pto and Prf are coincident in the signal transduction pathway that controls ligand-independent signaling. Pto and Prf associate in a coregulatory interaction that requires Pto kinase activity and N-myristoylation for signaling. Pto interacts with a unique Prf N-terminal domain outside of the NBARC-LRR domain and resides in a high molecular weight recognition complex dependent on the presence of Prf. In this complex, both Pto and Prf contribute to specific recognition of AvrPtoB. The data suggest that the role of Pto is confined to the regulation of Prf and that the bacterial effectors have evolved to target this coregulatory molecular switch.

  12. Protein energy malnutrition during vaccination has limited influence on vaccine efficacy but abolishes immunity if administered during Mycobacterium tuberculosis infection.

    Science.gov (United States)

    Hoang, Truc; Agger, Else Marie; Cassidy, Joseph P; Christensen, Jan P; Andersen, Peter

    2015-05-01

    Protein energy malnutrition (PEM) increases susceptibility to infectious diseases, including tuberculosis (TB), but it is not clear how PEM influences vaccine-promoted immunity to TB. We demonstrate that PEM during low-level steady-state TB infection in a mouse model results in rapid relapse of Mycobacterium tuberculosis, as well as increased pathology, in both Mycobacterium bovis BCG-vaccinated and unvaccinated animals. PEM did not change the overall numbers of CD4 T cells in BCG-vaccinated animals but resulted in an almost complete loss of antigen-specific cytokine production. Furthermore, there was a change in cytokine expression characterized by a gradual loss of multifunctional antigen-specific CD4 T cells and an increased proportion of effector cells expressing gamma interferon and tumor necrosis factor alpha (IFN-γ(+) TNF-α(+) and IFN-γ(+) cells). PEM during M. tuberculosis infection completely blocked the protection afforded by the H56-CAF01 subunit vaccine, and this was associated with a very substantial loss of the interleukin-2-positive memory CD4 T cells promoted by this vaccine. Similarly, PEM during the vaccination phase markedly reduced the H56-CAF01 vaccine response, influencing all cytokine-producing CD4 T cell subsets, with the exception of CD4 T cells positive for TNF-α only. Importantly, this impairment was reversible and resupplementation of protein during infection rescued both the vaccine-promoted T cell response and the protective effect of the vaccine against M. tuberculosis infection.

  13. Induction of HCA587-specific antitumor immunity with HCA587 protein formulated with CpG and ISCOM in mice.

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    Juanjuan Chen

    Full Text Available HCA587 (also known as MAGE-C2 is a "cancer-testis" antigen highly expressed in a number of malignancies with unique immunological properties, making it a promising target for tumor immunotherapy. In this report, we demonstrated that HCA587 protein, when formulated with adjuvants CpG-containing oligodeoxynucleotides (CpG ODN and ISCOM, was capable of inducing a potent cellular and humoral immune response as indicated by the presence of a large number of HCA587-specific, IFN-γ-producing CD4(+ T cells and high levels of HCA587-specific antibodies. More importantly, vaccination with HCA587 conferred protection against challenge with HCA587-expressing B16 melanoma in prophylactic and therapeutic settings. In analysis of the mechanisms underlying the protective effect, we showed that the vaccination was followed by enhanced accumulation of tumor-infiltrating lymphocytes (TILs with enrichment of conventional CD4(+ T cells but reduced representation of Treg cells. Further, the antitumor effect was largely abrogated in mice either depleted of CD4(+ T cells or deficient for IFN-γ. These results indicate that HCA587 protein vaccine possesses evident antitumor activity in a mouse model and holds promise for treatment of human cancers.

  14. Evolutionary Analyses Suggest a Function of MxB Immunity Proteins Beyond Lentivirus Restriction.

    Science.gov (United States)

    Mitchell, Patrick S; Young, Janet M; Emerman, Michael; Malik, Harmit S

    2015-12-01

    Viruses impose diverse and dynamic challenges on host defenses. Diversifying selection of codons and gene copy number variation are two hallmarks of genetic innovation in antiviral genes engaged in host-virus genetic conflicts. The myxovirus resistance (Mx) genes encode interferon-inducible GTPases that constitute a major arm of the cell-autonomous defense against viral infection. Unlike the broad antiviral activity of MxA, primate MxB was recently shown to specifically inhibit lentiviruses including HIV-1. We carried out detailed evolutionary analyses to investigate whether genetic conflict with lentiviruses has shaped MxB evolution in primates. We found strong evidence for diversifying selection in the MxB N-terminal tail, which contains molecular determinants of MxB anti-lentivirus specificity. However, we found no overlap between previously-mapped residues that dictate lentiviral restriction and those that have evolved under diversifying selection. Instead, our findings are consistent with MxB having a long-standing and important role in the interferon response to viral infection against a broader range of pathogens than is currently appreciated. Despite its critical role in host innate immunity, we also uncovered multiple functional losses of MxB during mammalian evolution, either by pseudogenization or by gene conversion from MxA genes. Thus, although the majority of mammalian genomes encode two Mx genes, this apparent stasis masks the dramatic effects that recombination and diversifying selection have played in shaping the evolutionary history of Mx genes. Discrepancies between our study and previous publications highlight the need to account for recombination in analyses of positive selection, as well as the importance of using sequence datasets with appropriate depth of divergence. Our study also illustrates that evolutionary analyses of antiviral gene families are critical towards understanding molecular principles that govern host-virus interactions and

  15. Evolutionary Analyses Suggest a Function of MxB Immunity Proteins Beyond Lentivirus Restriction.

    Directory of Open Access Journals (Sweden)

    Patrick S Mitchell

    2015-12-01

    Full Text Available Viruses impose diverse and dynamic challenges on host defenses. Diversifying selection of codons and gene copy number variation are two hallmarks of genetic innovation in antiviral genes engaged in host-virus genetic conflicts. The myxovirus resistance (Mx genes encode interferon-inducible GTPases that constitute a major arm of the cell-autonomous defense against viral infection. Unlike the broad antiviral activity of MxA, primate MxB was recently shown to specifically inhibit lentiviruses including HIV-1. We carried out detailed evolutionary analyses to investigate whether genetic conflict with lentiviruses has shaped MxB evolution in primates. We found strong evidence for diversifying selection in the MxB N-terminal tail, which contains molecular determinants of MxB anti-lentivirus specificity. However, we found no overlap between previously-mapped residues that dictate lentiviral restriction and those that have evolved under diversifying selection. Instead, our findings are consistent with MxB having a long-standing and important role in the interferon response to viral infection against a broader range of pathogens than is currently appreciated. Despite its critical role in host innate immunity, we also uncovered multiple functional losses of MxB during mammalian evolution, either by pseudogenization or by gene conversion from MxA genes. Thus, although the majority of mammalian genomes encode two Mx genes, this apparent stasis masks the dramatic effects that recombination and diversifying selection have played in shaping the evolutionary history of Mx genes. Discrepancies between our study and previous publications highlight the need to account for recombination in analyses of positive selection, as well as the importance of using sequence datasets with appropriate depth of divergence. Our study also illustrates that evolutionary analyses of antiviral gene families are critical towards understanding molecular principles that govern host

  16. Humoral and cell-mediated immunity following vaccination with synthetic Candida cell wall mannan derived heptamannoside-protein conjugate: immunomodulatory properties of heptamannoside-BSA conjugate.

    Science.gov (United States)

    Paulovičová, Lucia; Paulovičová, Ema; Karelin, Alexander A; Tsvetkov, Yury E; Nifantiev, Nikolay E; Bystrický, Slavomír

    2012-10-01

    Chemically defined glycoprotein conjugate composed of synthetically prepared mannan-derived heptamannoside with terminal β-1,2-linked mannose residue attached to the α-1,3-linked mannose residues and BSA as carrier protein (M7-BSA conjugate) was analysed for the capacity to induce protective humoral immunity and appropriate alteration cellular immunity. To identify protective antigenic structure of Candida cell wall mannan M7-BSA conjugate was used for BALB/c mice immunization. The obtained results were compared with placebo group and with heat-inactivated C. albicans whole cells immunization. The administration route of M7-BSA conjugate secondary booster injection significantly affected the intensity of humoral immune response and the specificity of produced antibodies. All prepared sera were able to elevate candidacidal activity of polymorphonuclear leukocytes (PMN) in cooperation with complement. Moreover, polyclonal sera obtained after secondary subcutaneous (s.c.) booster injection of M7-BSA conjugate were able to induce candidacidal activity of PMN also in complement independent manner. M7-BSA conjugate immunization induced increases of phagocytic activity and respiratory burst of granulocytes, caused a raise of the proportion of CD3(+) T lymphocytes and increased the CD4(+)/CD8(+) T lymphocyte ratio. We observed also an increasing proportion of CD4(+)CD25(+) T cells compared to immunization with heat inactivated whole C. albicans cells, which in turn promoted an increase of the CD8(+)CD25(+) cell proportion. Immunization with M7-BSA conjugate induced Th1, Th2 and Th17 immune responses as indicated by the elevation of relevant cytokines levels. These data provide some insights on the immunomodulatory properties of oligomannosides and contribute to the development of synthetic oligosaccharide vaccines against fungal diseases.

  17. Roles of receptor for activated protein kinase C1 for modulating immune responses in white shrimp Litopenaeus vannamei.

    Science.gov (United States)

    Chang, Zhong-Wen; Chang, Chin-Chyuan

    2015-10-01

    Complementary (c)DNA encoding a receptor for activated protein kinase C1 (RACK1) messenger (m)RNA of the white shrimp Litopenaeus vannamei, designated LvRACK1, consisted a 1136-bp cDNA containing an open reading frame (ORF) of 954 bp, a 111-bp 5'-untranslated region (UTR), and a 71-bp 3'-UTR, which is a 36 kDa cytosolic protein, belonging to the Trp-Asp40 (WD40) family of proteins, characterized by containing seven highly conserved Trp-Asp40 (WD40) internal repeats, and a poly A tail. The WD repeat of LvRACK1 can be predicted to form a seven-bladed propeller structure with each WD repeat composed of four antiparallel β-sheets. The WD40 domains have been implicated in protein-protein interactions. A comparison of amino acid sequences showed that LvRACK1 was closely related to arthropods RACK1. LvRACK1 cDNA was synthesized in all tested tissues detected with real-time PCR including haemocytes, hepatopancreas, gills, muscles, subcuticular epithelium, intestines, abdominal nervous ganglia, thoracic nervous ganglia, lymphoid organ, stomach, heart, and antennal gland, especially in subcuticular epithelium and gill. LvRACK1 mRNA transcription in haemocytes of L. vannamei injected with Vibrio alginolyticus decreased. The depletion of LvRACK1 of haemocytes in L. vannamei received its dsRNA revealed the increased respiratory bursts per haemocyte, superoxide dismutase (SOD), activity, glutathione peroxidase (GPx) activity, and clotting time, but showed the decreased total haemocyte count (THC), hyaline cells (HCs), phagocytic activity, and transglutaminase (TG) activity. LvRACK1 silenced shrimp showed the upregulated gene expressions of cyMnSOD, mtMnSOD, peroxinectin (PE), and TGI, and showed the downregulated α2-macroglobulin (α2-M), clottable protein (CP), lysozyme, and crustin gene expressions. It is therefore concluded that LvRACK1 is involved in immune defense and signaling transduction in haemocytes of L. vannamei infected with V. alginolyticus.

  18. Avr4 promotes Cf-4 receptor-like protein association with the BAK1/SERK3 receptor-like kinase to initiate receptor endocytosis and plant immunity

    NARCIS (Netherlands)

    Postma, Jelle; Liebrand, Thomas; Bi, Guozhi; Evrard, Alexandre; Bye, Ruby R.; Mbengue, Malick; Kuhn, Hannah; Joosten, Matthieu H.A.J.; Robatzek, Silke

    2016-01-01

    The first layer of plant immunity is activated by cell surface receptor-like kinases (RLKs) and proteins (RLPs) that detect infectious pathogens. Constitutive interaction with the SUPPRESSOR OF BIR1 (SOBIR1) RLK contributes to RLP stability and kinase activity. As RLK activation requires transpho

  19. Molecular Characterization and Immune Protection of a New Conserved Hypothetical Protein of Eimeria tenella.

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    Qi Zhai

    Full Text Available The genome sequences of Eimeria tenella have been sequenced, but >70% of these genes are currently categorized as having an unknown function or annotated as conserved hypothetical proteins, and few of them have been studied. In the present study, a conserved hypothetical protein gene of E. tenella, designated EtCHP559, was cloned using rapid amplification of cDNA 5'-ends (5'RACE based on the expressed sequence tag (EST. The 1746-bp full-length cDNA of EtCHP559 contained a 1224-bp open reading frame (ORF that encoded a 407-amino acid polypeptide with the predicted molecular weight of 46.04 kDa. Real-time quantitative PCR analysis revealed that EtCHP559 was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts and second generation merozoites. The ORF was inserted into pCold-TF to produce recombinant EtCHP559. Using western blotting, the recombinant protein was successfully recognized by rabbit serum against E. tenella sporozoites. Immunolocalization by using EtCHP559 antibody showed that EtCHP559 was mainly distributed on the parasite surface in free sporozoites and became concentrated in the anterior region after sporozoites were incubated in complete medium. The EtCHP559 became uniformly dispersed in immature and mature schizonts. Inhibition of EtCHP559 function using anti-rEtCHP559 polyclonal antibody reduced the ability of E. tenella sporozoites to invade host cells by >70%. Animal challenge experiments demonstrated that the recombinant EtCHP559 significantly increased the average body weight gain, reduced the oocyst outputs, alleviated cecal lesions of the infected chickens, and resulted in anticoccidial index >160 against E. tenella. These results suggest that EtCHP559 plays an important role in sporozoite invasion and could be an effective candidate for the development of a new vaccine against E. tenella.

  20. An unconventional antimicrobial protein histone from freshwater prawn Macrobrachium rosenbergii: analysis of immune properties.

    Science.gov (United States)

    Arockiaraj, Jesu; Gnanam, Annie J; Kumaresan, Venkatesh; Palanisamy, Rajesh; Bhatt, Prasanth; Thirumalai, Muthukumaresan Kuppusamy; Roy, Arpita; Pasupuleti, Mukesh; Kasi, Marimuthu

    2013-11-01

    In this study, we have reported the first histone characterized at molecular level from freshwater prawn Macrobrachium rosenbergii (MrHis). A full length cDNA of MrHis (751 base pairs) was identified from an established M. rosenbergii cDNA library using GS-FLX technique. It encodes 137 amino acid residues with a calculated molecular mass of 15 kDa and an isoelectric point of 10.5. MrHis peptide contains a histone H2A signature between 21 and 27 amino acids. Homologous analysis showed that MrHis had a significant sequence identity (99%) with other known histone H2A groups especially from Penaeus monodon. Phylogenetic analysis of MrHis showed a strong relationship with other amino acid sequences from histone H2A arthropod groups. Further phylogenetic analysis showed that the MrHis belongs to histone H2A superfamily and H2A1A sub-family. Secondary structure of MrHis showed that the protein contains 50.36% α-helical region and 49.64% coils. The 3D model of MrHis was predicted by I-Tasser program and the model was evaluated for quality analysis including C-score analysis, Ramachandran plot analysis and RMSD analysis. The surface view analysis of MrHis showed the active domain at the N terminal. The antimicrobial property of MrHis protein was confirmed by the helical structure and the total hydrophobic surface along with its net charge. The MFE of the predicted RNA structure of MrHis is -128.62 kcal/mol, shows its mRNA stability. Schiffer-Edmundson helical wheel analysis of the N-terminal of MrHis showed a perfect amphipathic nature of the peptide. Significantly (P < 0.05) highest gene expression was noticed in the hemocyte and is induced with viral (WSBV and MrNV) and bacteria (A eromonas hydrophila and Vibrio harveyi) infections. The coding sequence of recombinant MrHis protein was expressed in a pMAL vector and purified to study the antimicrobial properties. The recombinant product showed antimicrobial activity against both Gram negative and Gram positive bacteria

  1. Divergence in the plasminogen-binding group a streptococcal M protein family: functional conservation of binding site and potential role for immune selection of variants.

    Science.gov (United States)

    Sanderson-Smith, Martina; Batzloff, Michael; Sriprakash, Kabada S; Dowton, Mark; Ranson, Marie; Walker, Mark J

    2006-02-10

    Group A streptococci (GAS) display receptors for the human zymogen plasminogen on the cell surface, one of which is the plasminogen-binding group A streptococcal M protein (PAM). Characterization of PAM genes from 12 GAS isolates showed significant variation within the plasminogen-binding repeat motifs (a1/a2) of this protein. To determine the impact of sequence variation on protein function, recombinant proteins representing five naturally occurring variants of PAM, together with a recombinant M1 protein, were expressed and purified. Equilibrium dissociation constants for the interaction of PAM variants with biotinylated Glu-plasminogen ranged from 1.58 to 4.99 nm. Effective concentrations of prototype PAM required for 50% inhibition of plasminogen binding to immobilized PAM variants ranged from 0.68 to 22.06 nm. These results suggest that although variation in the a1/a2 region of the PAM protein does affect the comparative affinity of PAM variants, the functional capacity to bind plasminogen is conserved. Additionally, a potential role for the a1 region of PAM in eliciting a protective immune response was investigated by using a mouse model for GAS infection. The a1 region of PAM was found to protect immunized mice challenged with a PAM-positive GAS strain. These data suggest a link between selective immune pressure against the plasminogen-binding repeats and the functional conservation of the binding domain in PAM variants.

  2. MONOCLONAL ANTIBODIES AND RECOMBINANT PROTEINS OF FILOVIRUSES: IMMUNOCHEMICAL PROPERTIES AND EVALUATION OF THEIR EFFICIENCY FOR IMMUNE DIAGNOSTICS OF MARBURG AND EBOLA VIRUSES

    Directory of Open Access Journals (Sweden)

    E. I. Kazachinskaia

    2010-01-01

    Full Text Available A panel of monoclonal antibodies (MAbs against VP35, VP40 and NP viral proteins of Marburg and Ebola viruses, as well as recombinant VP35, VP40 and NP proteins were generated and tested for their capacity to specific immune reactions. Monoclonal antibodies to appropriate viral proteins effectively recognized the VP35, VP40 and NP recombinant proteins, thus allowing to develop a variant of a MAb-based ELISA analysis with different types of biotin-labeled MAbs, using these antibodies for capturing viral and recombinant antigens of Marburg and Ebola viruses. These techniques were able to detect viral and recombinant proteins in a concentration range between 1 and 150 ng/ml. We conclude that the recombinant VP35, VP40 and NP proteins of filoviruses, as well as MAbs against these viral proteins represent a promising tool for a new generation of immunodiagnostic kits and studying immunological features of filovirus infection.

  3. The role of G protein gene GNB3 C825T Polymorphism in HIV-1 acquisition, progression and immune activation

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    Juno Jennifer

    2012-01-01

    Full Text Available Abstract Background The GNB3 C825T polymorphism is associated with increased G protein-mediated signal transduction, SDF-1α-mediated lymphocyte chemotaxis, accelerated HIV-1 progression, and altered responses to antiretroviral therapy among Caucasian subjects. The GNB3 825T allele is highly prevalent in African populations, and as such any impact on HIV-1 acquisition or progression rates could have a dramatic impact. This study examines the association of the 825T polymorphism with HIV-1 acquisition, disease progression and immune activation in two African cohorts. GNB3 825 genotyping was performed for enrolees in both a commercial sex worker cohort and a perinatal HIV transmission (PHT cohort in Nairobi, Kenya. Ex vivo immune activation was quantified by flow cytometry, and plasma chemokine levels were assessed by cytokine bead array. Results GNB3 genotype was not associated with sexual or vertical HIV-1 acquisition within these cohorts. Within the Pumwani cohort, GNB3 genotype did not affect HIV-1 disease progression among seroconverters or among HIV-1-positive individuals after adjustment for baseline CD4 count. Maternal CD4 decline and viral load increase in the PHT cohort did not differ between genotypes. Multi-parametric flow cytometry assessment of T cell activation (CD69, HLA-DR, CD38 and Treg frequency (CD25+FOXP3+ found no differences between genotype groups. Plasma SDF-1α, MIP-1β and TRAIL levels quantified by cytokine bead array were also similar between groups. Conclusions In contrast to previous reports, we were unable to provide evidence to suggest that the GNB3 C825T polymorphism affects HIV-1 acquisition or disease progression within African populations. Ex vivo immune activation and plasma chemokine levels were similarly unaffected by GNB3 genotype in both HIV-1-negative and HIV-1-positive individuals. The paucity of studies investigating the impact of GNB3 polymorphism among African populations and the lack of mechanistic

  4. [Genetics and Genomics of Wheat: Storage Proteins, Ecological Plasticity, and Immunity].

    Science.gov (United States)

    Novoselskaya-Dragovich, A Yu

    2015-05-01

    Recent advances in genetics and genetic research methods made it possible to explain the large polymorphism observed among storage proteins of wheat weevil (gliadins and glutenins), to determine their genetic control mechanism, and to develop a system for the identification of wheat genotypes on the basis of multiple allelism. This system has extremely high sensitivity and efficiency, which makes it possible to conduct studies to determine the purity and authenticity of wheat varieties, the dynamics of alleles diversity in time and space, the phylogenetics, etc., through the use of an extensive database on the allelic composition of gliadin loci. An investigation of the molecular structure of genes controlling the synthesis of storage proteins and their organization on chromosomes, as well as an analysis of wheat genome structure, revealed the molecular mechanisms of variability in the wheat genome and its reorganization in response to changes in environmental conditions and cultivation technologies. The multilevel genetic system of protection against pathogens and adverse environmental factors that developed in the course of wheat evolution continues to astound researchers' imagination with new resistance genes and novel types of antimicrobial peptides having been discovered and sequenced in recent years and the diversity of their structures and mechanisms of action in response to pathogens. An analysis of gene sequences involved in wheat domestication, namely, those that define ecological plasticity, i. e. the type of plant development (Vrn genes), and those responsible for spikelet traits (Q genes), which ensured the successful cultivation of wheat by humans, revealed that the basis for these features are specific mutations.

  5. Copy number of FCGR3B, which is associated with systemic lupus erythematosus, correlates with protein expression and immune complex uptake.

    Science.gov (United States)

    Willcocks, Lisa C; Lyons, Paul A; Clatworthy, Menna R; Robinson, James I; Yang, Wanling; Newland, Stephen A; Plagnol, Vincent; McGovern, Naomi N; Condliffe, Alison M; Chilvers, Edwin R; Adu, Dwomoa; Jolly, Elaine C; Watts, Richard; Lau, Yu Lung; Morgan, Ann W; Nash, Gerard; Smith, Kenneth G C

    2008-07-07

    Copy number (CN) variation (CNV) has been shown to be common in regions of the genome coding for immune-related genes, and thus impacts upon polygenic autoimmunity. Low CN of FCGR3B has recently been associated with systemic lupus erythematosus (SLE). FcgammaRIIIb is a glycosylphosphatidylinositol-linked, low affinity receptor for IgG found predominantly on human neutrophils. We present novel data demonstrating that both in a family with FcgammaRIIIb-deficiency and in the normal population, FCGR3B CNV correlates with protein expression, with neutrophil uptake of and adherence to immune complexes, and with soluble serum FcgammaRIIIb. Reduced FcgammaRIIIb expression is thus likely to contribute to the impaired clearance of immune complexes, which is a feature of SLE, explaining the association between low FCGR3B CNV and SLE that we have confirmed in a Caucasian population. In contrast, antineutrophil cytoplasmic antibody-associated systemic vasculitis (AASV), a disease not associated with immune complex deposition, is associated with high FCGR3B CN. Thus, we define a role for FCGR3B CNV in immune complex clearance, a function that may explain why low FCGR3B CNV is associated with SLE, but not AASV. This is the first report of an association between disease-related gene CNV and variation in protein expression and function that may contribute to autoimmune disease susceptibility.

  6. Intranasal immunization with fusion protein MrpH·FimH and MPL adjuvant confers protection against urinary tract infections caused by uropathogenic Escherichia coli and Proteus mirabilis.

    Science.gov (United States)

    Habibi, Mehri; Asadi Karam, Mohammad Reza; Shokrgozar, Mohammad Ali; Oloomi, Mana; Jafari, Anis; Bouzari, Saeid

    2015-04-01

    Urinary tract infections (UTIs) caused by Uropathogenic Escherichia coli (UPEC) and Proteus mirabilis are among the most common infections in the world. Currently there are no vaccines available to confer protection against UTI in humans. In this study, the immune responses and protection of FimH of UPEC with MrpH antigen of P. mirabilis in different vaccine formulations with and without MPL adjuvant were assessed. Mice intranasally immunized with the novel fusion protein MrpH·FimH induced a significant increase in IgG and IgA in serum, nasal wash, vaginal wash, and urine samples. Mice immunized with fusion MrpH·FimH also showed a significant boost in cellular immunity. Addition of MPL as the adjuvant enhanced FimH and MrpH specific humoral and cellular responses in both systemic and mucosal samples. Vaccination with MrpH·FimH alone or in combination with MPL showed the highest efficiency in clearing bladder and kidney infections in mice challenged with UPEC and P. mirabilis. These findings may indicate that the protection observed correlates with the systemic, mucosal and cellular immune responses induced by vaccination with these preparations. Our data suggest MrpH·FimH fusion protein with or without MPL as adjuvant could be potential vaccine candidates for elimination of UPEC and P. mirabilis. These data altogether are promising and these formulations are good candidates for elimination of UPEC and P. mirabilis.

  7. The Ag85B protein of Mycobacterium tuberculosis may turn a protective immune response induced by Ag85B-DNA vaccine into a potent but non-protective Th1 immune response in mice.

    Science.gov (United States)

    Palma, Carla; Iona, Elisabetta; Giannoni, Federico; Pardini, Manuela; Brunori, Lara; Orefici, Graziella; Fattorini, Lanfranco; Cassone, Antonio

    2007-06-01

    Clarifying how an initial protective immune response to tuberculosis may later loose its efficacy is essential to understand tuberculosis pathology and to develop novel vaccines. In mice, a primary vaccination with Ag85B-encoding plasmid DNA (DNA-85B) was protective against Mycobacterium tuberculosis (MTB) infection and associated with Ag85B-specific CD4+ T cells producing IFN-gamma and controlling intramacrophagic MTB growth. Surprisingly, this protection was eliminated by Ag85B protein boosting. Loss of protection was associated with a overwhelming CD4+ T cell proliferation and IFN-gamma production in response to Ag85B protein, despite restraint of Th1 response by CD8+ T cell-dependent mechanisms and activation of CD4+ T cell-dependent IL-10 secretion. Importantly, these Ag85B-responding CD4+ T cells lost the ability to produce IFN-gamma and control MTB intramacrophagic growth in coculture with MTB-infected macrophages, suggesting that the protein-dependent expansion of non-protective CD4+ T cells determined dilution or loss of the protective Ag85B-specific CD4+ induced by DNA-85B vaccination. These data emphasize the need of exerting some caution in adopting aggressive DNA-priming, protein-booster schedules for MTB vaccines. They also suggest that Ag85B protein secreted during MTB infection could be involved in the instability of protective anti-tuberculosis immune response, and actually concur to disease progression.

  8. The antitumor immune responses induced by nanoemulsion-encapsulated MAGE1-HSP70/SEA complex protein vaccine following different administration routes.

    Science.gov (United States)

    Ge, Wei; Hu, Pei-Zhen; Huang, Yang; Wang, Xiao-Ming; Zhang, Xiu-Min; Sun, Yu-Jing; Li, Zeng-Shan; Si, Shao-Yan; Sui, Yan-Fang

    2009-10-01

    Our previous study showed that nanoemulsion-encapsulated MAGE1-HSP70/SEA (MHS) complex protein vaccine elicited MAGE-1 specific immune response and antitumor effects against MAGE-1-expressing tumor and nanoemulsion is a useful vehicle with possible important implications for cancer biotherapy. The purpose of this study was to compare the immune responses induced by nanoemulsion-encapsulated MAGE1-HSP70 and SEA as NE(MHS) vaccine following different administration routes and to find out the new and effective immune routes. Nanoemulsion vaccine was prepared using magnetic ultrasound methods. C57BL/6 mice were immunized with NE(MHS) via po., i.v., s.c. or i.p., besides mice s.c. injected with PBS or NE(-) as control. The cellular immunocompetence was detected by ELISpot assay and LDH release assay. The therapeutic and tumor challenge assay were also examined. The results showed that the immune responses against MAGE-1 expressing murine tumors elicited by NE(MHS) via 4 different routes were approximately similar and were all stronger than that elicited by PBS or NE(-), suggesting that this novel nanoemulsion carrier can exert potent antitumor immunity against antigens encapsulated in it. Especially, the present results indicated that nanoemulsion vaccine adapted to administration via different routes including peroral, and may have broader applications in the future.

  9. Immunization of chickens with a recombinant Ascaridia galli protein results in parasite-specific IgG with no protective effect against infection

    DEFF Research Database (Denmark)

    Fink, Dorte Rosenbek; Schou, T. W.; Norup, L. R.

    in the number of chicken flocks held in floor pens and free-range systems, which are associated with higher parasite burdens. In order to prevent infections with the nematode Ascaridia galli, development of a vaccine is desirable. In this study, three groups of 10 chickens were immunized with three different...... adjuvants together with a recombinant A. galli antigen. The adjuvants were CAF01, Emulsigen, and STV, and the antigen was Ag-NPA-1, a lipid-binding protein from the nematode polyprotein allergen/antigen family. Three immunizations were given i.m. with three-week intervals. A fourth group of 10 chickens...... was immunized with CAF01 and Ag-NPA-1, but only the first immunization was i.m., the next two immunizations were oral. A fifth group of 10 birds was injected i.m. with PBS as a control. The three groups that only received i.m. immunizations developed significantly higher Ag-NPA-1-specific serum IgG levels than...

  10. Non structural protein of avian influenza A (H11N1 virus is a weaker suppressor of immune responses but capable of inducing apoptosis in host cells

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    Mukherjee Sanjay

    2012-08-01

    Full Text Available Abstract Background The Non-Structural (NS1 protein of Influenza A viruses is an extensively studied multifunctional protein which is commonly considered as key viral component to fight against host immune responses. Even though there has been a lot of studies on the involvement of NS1 protein in host immune responses there are still ambiguities regarding its role in apoptosis in infected cells. Interactions of NS1 protein with host factors, role of NS1 protein in regulating cellular responses and apoptosis are quite complicated and further studies are still needed to understand it completely. Results NS1 genes of influenza A/Chicken/India/WBNIV2653/2008 (H5N1 and A/Aquatic bird/India/NIV-17095/2007(H11N1 were cloned and expressed in human embryonic kidney (293T cells. Microarray based approach to study the host cellular responses to NS1 protein of the two influenza A viruses of different pathogenicity showed significant differences in the host gene expression profile. NS1 protein of H5N1 resulted in suppression of IFN-β mediated innate immune responses, leading to down-regulation of the components of JAK-STAT pathway like STAT1 which further suppressed the expression of pro-inflammatory cytokines like CXCL10 and CCL5. The degree of suppression of host immune genes was found considerable with NS1 protein of H11N1 but was not as prominent as with H5N1-NS1. TUNEL assay analyses were found to be positive in both the NS1 transfected cells indicating both H5N1 as well as H11N1 NS1 proteins were able to induce apoptosis in transfected cells. Conclusions We propose that NS1 protein of both H5N1 and H11N1 subtypes of influenza viruses are capable of influencing host immune responses and possess necessary functionality to support apoptosis in host cells. H11N1, a low pathogenic virus without any proven evidence to infect mammals, contains a highly potential NS1 gene which might contribute to greater virus virulence in different gene combinations.

  11. Candida Immunity

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    Julian R. Naglik

    2014-01-01

    Full Text Available The human pathogenic fungus Candida albicans is the predominant cause of both superficial and invasive forms of candidiasis. C. albicans primarily infects immunocompromised individuals as a result of either immunodeficiency or intervention therapy, which highlights the importance of host immune defences in preventing fungal infections. The host defence system utilises a vast communication network of cells, proteins, and chemical signals distributed in blood and tissues, which constitute innate and adaptive immunity. Over the last decade the identity of many key molecules mediating host defence against C. albicans has been identified. This review will discuss how the host recognises this fungus, the events induced by fungal cells, and the host innate and adaptive immune defences that ultimately resolve C. albicans infections during health.

  12. Bovine milk proteome in the first 9 days: protein interactions in maturation of the immune and digestive system of the newborn.

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    Lina Zhang

    Full Text Available In order to better understand the milk proteome and its changes from colostrum to mature milk, samples taken at seven time points in the first 9 days from 4 individual cows were analyzed using proteomic techniques. Both the similarity in changes from day 0 to day 9 in the quantitative milk proteome, and the differences in specific protein abundance, were observed among four cows. One third of the quantified proteins showed a significant decrease in concentration over the first 9 days after calving, especially in the immune proteins (as much as 40 fold. Three relative high abundant enzymes (XDH, LPL, and RNASE1 and cell division and proliferation protein (CREG1 may be involved in the maturation of the gastro-intestinal tract. In addition, high correlations between proteins involved in complement and blood coagulation cascades illustrates the complex nature of biological interrelationships between milk proteins. The linear decrease of protease inhibitors and proteins involved in innate and adaptive immune system implies a protective role for protease inhibitor against degradation. In conclusion, the results found in this study not only improve our understanding of the role of colostrum in both host defense and development of the newborn calf but also provides guidance for the improvement of infant formula through better understanding of the complex interactions between milk proteins.

  13. Cauliflower mosaic virus protein P6 inhibits signaling responses to salicylic acid and regulates innate immunity.

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    Andrew J Love

    Full Text Available Cauliflower mosaic virus (CaMV encodes a multifunctional protein P6 that is required for translation of the 35S RNA and also acts as a suppressor of RNA silencing. Here we demonstrate that P6 additionally acts as a pathogenicity effector of an unique and novel type, modifying NPR1 (a key regulator of salicylic acid (SA- and jasmonic acid (JA-dependent signaling and inhibiting SA-dependent defence responses We find that that transgene-mediated expression of P6 in Arabidopsis and transient expression in Nicotiana benthamiana has profound effects on defence signaling, suppressing expression of representative SA-responsive genes and increasing expression of representative JA-responsive genes. Relative to wild-type Arabidopsis P6-expressing transgenics had greatly reduced expression of PR-1 following SA-treatment, infection by CaMV or inoculation with an avirulent bacterial pathogen Pseudomonas syringae pv tomato (Pst. Similarly transient expression in Nicotiana benthamiana of P6 (including a mutant form defective in translational transactivation activity suppressed PR-1a transcript accumulation in response to Agrobacterium infiltration and following SA-treatment. As well as suppressing the expression of representative SA-regulated genes, P6-transgenic Arabidopsis showed greatly enhanced susceptibility to both virulent and avirulent Pst (titres elevated 10 to 30-fold compared to non-transgenic controls but reduced susceptibility to the necrotrophic fungus Botrytis cinerea. Necrosis following SA-treatment or inoculation with avirulent Pst was reduced and delayed in P6-transgenics. NPR1 an important regulator of SA/JA crosstalk, was more highly expressed in the presence of P6 and introduction of the P6 transgene into a transgenic line expressing an NPR1:GFP fusion resulted in greatly increased fluorescence in nuclei even in the absence of SA. Thus in the presence of P6 an inactive form of NPR1 is mislocalized in the nucleus even in uninduced plants

  14. Branched-chain Amino Acids are Beneficial to Maintain Growth Performance and Intestinal Immune-related Function in Weaned Piglets Fed Protein Restricted Diet.

    Science.gov (United States)

    Ren, M; Zhang, S H; Zeng, X F; Liu, H; Qiao, S Y

    2015-12-01

    As a novel approach for disease control and prevention, nutritional modulation of the intestinal health has been proved. However, It is still unknown whether branched-chain amino acid (BCAA) is needed to maintain intestinal immune-related function. The objective of this study was to determine whether BCAA supplementation in protein restricted diet affects growth performance, intestinal barrier function and modulates post-weaning gut disorders. One hundred and eight weaned piglets (7.96±0.26 kg) were randomly fed one of the three diets including a control diet (21% crude protein [CP], CON), a protein restricted diet (17% CP, PR) and a BCAA diet (BCAA supplementation in the PR diet) for 14 d. The growth performance, plasma amino acid concentrations, small intestinal morphology and intestinal immunoglobulins were tested. First, average daily gain (ADG) (pBCAA group improved ADG (pBCAA groups was not different (p>0.05). The PR and BCAA treatments had a higher (pBCAA supplementation significantly increased BCAA concentrations (pBCAA supplementation increased villous height in the duodenum (pBCAA supplementation increased levels of jejunal and ileal immunoglobulin mentioned above. In conclusion, BCAA supplementation to protein restricted diet improved intestinal immune defense function by protecting villous morphology and by increasing levels of intestinal immunoglobulins in weaned piglets. Our finding has the important implication that BCAA may be used to reduce the negative effects of a protein restricted diet on growth performance and intestinal immunity in weaned piglets.

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

    Science.gov (United States)

    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.

  16. Different immunity elicited by recombinant H5N1 hemagglutinin proteins containing pauci-mannose, high-mannose, or complex type N-glycans.

    Directory of Open Access Journals (Sweden)

    Shih-Chang Lin

    Full Text Available Highly pathogenic avian influenza H5N1 viruses can result in poultry and occasionally in human mortality. A safe and effective H5N1 vaccine is urgently needed to reduce the pandemic potential. Hemagglutinin (HA, a major envelope protein accounting for approximately 80% of spikes in influenza virus, is often used as a major antigen for subunit vaccine development. In this study, we conducted a systematic study of the immune response against influenza virus infection following immunization with recombinant HA proteins expressed in insect (Sf9 cells, insect cells that contain exogenous genes for elaborating N-linked glycans (Mimic and mammalian cells (CHO. While the antibody titers are higher with the insect cell derived HA proteins, the neutralization and HA inhibition titers are much higher with the mammalian cell produced HA proteins. Recombinant HA proteins containing tri- or tetra-antennary complex, terminally sialylated and asialyated-galactose type N-glycans induced better protective immunity in mice to lethal challenge. The results are highly relevant to issues that should be considered in the production of fragment vaccines.

  17. Expression of innate immune genes, proteins and microRNAs in lung tissue of pigs infected experimentally with influenza virus (H1N2)

    DEFF Research Database (Denmark)

    Skovgaard, Kerstin; Cirera, Susanna; Vasby, Ditte

    2013-01-01

    This study aimed at providing a better understanding of the involvement of innate immune factors, including miRNA, in the local host response to influenza virus infection. Twenty pigs were challenged by influenza A virus subtype H1N2. Expression of microRNA (miRNA), mRNA and proteins were...... results suggest that, in addition to a wide range of innate immune factors, miRNAs may also be involved in controlling acute influenza infection in pigs....... differentiation associated protein-5), IFN and IFN-induced genes (IFN-β, IFN-γ, IRF7, STAT1, ISG15 and OASL), cytokines (IL-1 β, IL-1RN, IL-6, IL-7, IL-10, IL-12A, TNF-α, CCL2, CCL3 and CXCL10) and several acute phase proteins. Likewise, the following miRNAs were differentially expressed in one or more time...

  18. A suicidal DNA vaccine expressing the fusion protein of peste des petits ruminants virus induces both humoral and cell-mediated immune responses in mice.

    Science.gov (United States)

    Wang, Yong; Yue, Xiaolin; Jin, Hongyan; Liu, Guangqing; Pan, Ling; Wang, Guijun; Guo, Hao; Li, Gang; Li, Yongdong

    2015-12-01

    Peste des petits ruminants (PPR), a highly contagious disease induced by PPR virus (PPRV), affects sheep and goats. PPRV fusion (F) protein is important for the induction of immune responses against PPRV. We constructed a Semliki Forest virus (SFV) replicon-vectored DNA vaccine ("suicidal DNA vaccine") and evaluated its immunogenicity in BALB/c mice. The F gene of PPRV was cloned and inserted into the SFV replicon-based vector pSCA1. The antigenicity of the resultant plasmid pSCA1/F was identified by indirect immunofluorescence and western blotting. BALB/c mice were then intramuscularly injected with pSCA1/F three times at 14-d intervals. Specific antibodies and virus-neutralizing antibodies against PPRV were quantified by indirect ELISA and microneutralization tests, respectively. Cell-mediated immune responses were examined by cytokine and lymphocyte proliferation assays. The pSCA1/F expressed F protein in vitro and induced specific and neutralizing antibody production, and lymphocyte proliferation in mice. Mice vaccinated with pSCA1/F had increased IL-2 and IL-10 levels after 24-h post first immunization. IFN-γ and TNF-α levels increased from that time point and gradually decreased thereafter. Thus, the Semliki Forest virus replicon-vectored DNA vaccine expressing the F protein of PPRV induced both humoral and cell-mediated immune responses in mice. This could be considered as a novel strategy for vaccine development against PPR.

  19. Broad T Cell Immunity to the LcrV Virulence Protein is Induced by Targeted Delivery to DEC-205/CD205-Positive Mouse Dendritic Cells

    Science.gov (United States)

    2007-08-13

    Frontline: Broad T cell immunity to the LcrV virulence protein is induced by targeted delivery to DEC-205/CD205-positive mouse dendritic cells Yoonkyung...Received 30/8/07 Accepted 8/11/07 [DOI 10.1002/eji.200737799] Key words: CD205/DEC-205 Cellular immunity Dendritic cells LcrV Yersinia pestis...positive mouse dendritic cells . European Journal of Immunology 38:20 -29 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S

  20. Interferon-inducible p200-family protein IFI16, an innate immune sensor for cytosolic and nuclear double-stranded DNA: regulation of subcellular localization.

    Science.gov (United States)

    Veeranki, Sudhakar; Choubey, Divaker

    2012-01-01

    The interferon (IFN)-inducible p200-protein family includes structurally related murine (for example, p202a, p202b, p204, and Aim2) and human (for example, AIM2 and IFI16) proteins. All proteins in the family share a partially conserved repeat of 200-amino acid residues (also called HIN-200 domain) in the C-terminus. Additionally, most proteins (except the p202a and p202b proteins) also share a protein-protein interaction pyrin domain (PYD) in the N-terminus. The HIN-200 domain contains two consecutive oligosaccharide/oligonucleotide binding folds (OB-folds) to bind double stranded DNA (dsDNA). The PYD domain in proteins allows interactions with the family members and an adaptor protein ASC. Upon sensing cytosolic dsDNA, Aim2, p204, and AIM2 proteins recruit ASC protein to form an inflammasome, resulting in increased production of proinflammatory cytokines. However, IFI16 protein can sense cytosolic as well as nuclear dsDNA. Interestingly, the IFI16 protein contains a nuclear localization signal (NLS). Accordingly, the initial studies had indicated that the endogenous IFI16 protein is detected in the nucleus and within the nucleus in the nucleolus. However, several recent reports suggest that subcellular localization of IFI16 protein in nuclear versus cytoplasmic (or both) compartment depends on cell type. Given that the IFI16 protein can sense cytosolic as well as nuclear dsDNA and can initiate different innate immune responses (production of IFN-β versus proinflammatory cytokines), here we evaluate the experimental evidence for the regulation of subcellular localization of IFI16 protein in various cell types. We conclude that further studies are needed to understand the molecular mechanisms that regulate the subcellular localization of IFI16 protein.

  1. BTB-BACK Domain Protein POB1 Suppresses Immune Cell Death by Targeting Ubiquitin E3 ligase PUB17 for Degradation

    Science.gov (United States)

    Mesmar, Joelle; McLellan, Hazel; Yang, Chengwei; Craig, Adam; Zhang, Cunjin; Moore, Jonathan David; Tian, Zhendong; Birch, Paul R. J.; Sadanandom, Ari

    2017-01-01

    Hypersensitive response programmed cell death (HR-PCD) is a critical feature in plant immunity required for pathogen restriction and prevention of disease development. The precise control of this process is paramount to cell survival and an effective immune response. The discovery of new components that function to suppress HR-PCD will be instrumental in understanding the regulation of this fundamental mechanism. Here we report the identification and characterisation of a BTB domain E3 ligase protein, POB1, that functions to suppress HR-PCD triggered by evolutionarily diverse pathogens. Nicotiana benthamiana and tobacco plants with reduced POB1 activity show accelerated HR-PCD whilst those with increased POB1 levels show attenuated HR-PCD. We demonstrate that POB1 dimerization and nuclear localization are vital for its function in HR-PCD suppression. Using protein-protein interaction assays, we identify the Plant U-Box E3 ligase PUB17, a well established positive regulator of plant innate immunity, as a target for POB1-mediated proteasomal degradation. Using confocal imaging and in planta immunoprecipitation assays we show that POB1 interacts with PUB17 in the nucleus and stimulates its degradation. Mutated versions of POB1 that show reduced interaction with PUB17 fail to suppress HR-PCD, indicating that POB1-mediated degradation of PUB17 U-box E3 ligase is an important step for negative regulation of specific immune pathways in plants. Our data reveals a new mechanism for BTB domain proteins in suppressing HR-PCD in plant innate immune responses. PMID:28056034

  2. The hypersensitive induced reaction and leucine-rich repeat proteins regulate plant cell death associated with disease and plant immunity.

    Science.gov (United States)

    Choi, Hyong Woo; Kim, Young Jin; Hwang, Byung Kook

    2011-01-01

    Pathogen-induced programmed cell death (PCD) is intimately linked with disease resistance and susceptibility. However, the molecular components regulating PCD, including hypersensitive and susceptible cell death, are largely unknown in plants. In this study, we show that pathogen-induced Capsicum annuum hypersensitive induced reaction 1 (CaHIR1) and leucine-rich repeat 1 (CaLRR1) function as distinct plant PCD regulators in pepper plants during Xanthomonas campestris pv. vesicatoria infection. Confocal microscopy and protein gel blot analyses revealed that CaLRR1 and CaHIR1 localize to the extracellular matrix and plasma membrane (PM), respectively. Bimolecular fluorescent complementation and coimmunoprecipitation assays showed that the extracellular CaLRR1 specifically binds to the PM-located CaHIR1 in pepper leaves. Overexpression of CaHIR1 triggered pathogen-independent cell death in pepper and Nicotiana benthamiana plants but not in yeast cells. Virus-induced gene silencing (VIGS) of CaLRR1 and CaHIR1 distinctly strengthened and compromised hypersensitive and susceptible cell death in pepper plants, respectively. Endogenous salicylic acid levels and pathogenesis-related gene transcripts were elevated in CaHIR1-silenced plants. VIGS of NbLRR1 and NbHIR1, the N. benthamiana orthologs of CaLRR1 and CaHIR1, regulated Bax- and avrPto-/Pto-induced PCD. Taken together, these results suggest that leucine-rich repeat and hypersensitive induced reaction proteins may act as cell-death regulators associated with plant immunity and disease.

  3. Lysophospholipid Growth Factors and Their G Protein-Coupled Receptors in Immunity, Coronary Artery Disease, and Cancer

    Directory of Open Access Journals (Sweden)

    Edward J. Goetzl

    2002-01-01

    Full Text Available The physiological lysophospholipids (LPLs, exemplified by lysophosphatidic acid (LPA and sphingosine 1-phosphate (S1P, are omnific mediators of normal cellular proliferation, survival, and functions. Although both LPA and S1P attain micromolar concentrations in many biological fluids, numerous aspects of their biosynthesis, transport, and metabolic degradation are unknown. Eight members of a new subfamily of G protein-coupled LPA/S1P receptors, originally termed Edg Rs, bind either LPA or S1P with high affinity and transduce a series of growth-related and/or cytoskeleton-based functional responses. The most critical areas of LPL biology and pathobiology are neural development and neurodegeneration, immunity, atherosclerosis and myocardial injury, and cancer. Data from analyses of T cells established two basic points: (1 the plasticity and adaptability of expression of LPA/S1P Rs by some cells as a function of activation, and (2 the role of opposing signals from two different receptors for the same ligand as a mechanism for fine control of effects of LPLs. In the heart, LPLs may promote coronary atherosclerosis, but are effectively cytoprotective for hypoxic cardiac myocytes and those exposed to oxygen free radicals. The findings of production of LPA by some types of tumor cells, overexpression of selected sets of LPA receptors by the same tumor cells, and augmentation of the effects of protein growth factors by LPA have suggested pathogenetic roles for the LPLs in cancer. The breadth of physiologic and pathologic activities of LPLs emphasizes the importance of developing bioavailable nonlipid agonists and antagonists of the LPA/S1P receptors for diverse therapeutic applications.

  4. GTPase of the Immune-Associated Nucleotide Protein 5 Regulates the Lysosomal Calcium Compartment in T Lymphocytes

    Science.gov (United States)

    Serrano, Daniel; Ghobadi, Farnaz; Boulay, Guylain; Ilangumaran, Subburaj; Lavoie, Christine; Ramanathan, Sheela

    2017-01-01

    T lymphocytes from Gimap5lyp/lyp rats carrying a recessive mutation in the GTPase of immune-associated protein 5 (Gimap5) gene undergo spontaneous apoptosis. Molecular mechanisms underlying this survival defect are not yet clear. We have shown that Gimap5lyp/lyp T lymphocytes display reduced calcium influx following T cell antigen receptor (TCR) stimulation that was associated with impaired buffering of calcium by mitochondria. Here, we investigated the subcellular localization of GIMAP5 and its influence on Ca2+ response in HEK293T cells and T lymphocytes. The more abundantly expressed GIMAP5v2 localizes to the lysosome and certain endosomal vesicles. Gimap5lyp/lyp T lymphocytes showed increased accumulation of calcium in the lysosomes as evidenced by Gly-Phe β-naphthylamide (GPN) triggered Ca2+ release. As a corollary, GPN-induced Ca2+ flux was decreased in HEK293T cells expressing GIMAP5v2. Strikingly, TCR stimulation of rat, mouse, and human T lymphocytes increased lysosomal calcium content. Overall, our findings show that lysosomes modulate cellular Ca2+ response during T cell activation and that GIMAP5 regulates the lysosomal Ca2+ compartment in T lymphocytes. PMID:28223986

  5. The role of endosomal escape and mitogen-activated protein kinases in adenoviral activation of the innate immune response.

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    Jeffrey S Smith

    Full Text Available Adenoviral vectors (AdV activate multiple signaling pathways associated with innate immune responses, including mitogen-activated protein kinases (MAPKs. In this study, we investigated how systemically-injected AdVs activate two MAPK pathways (p38 and ERK and the contribution of these kinases to AdV-induced cytokine and chemokine responses in mice. Mice were injected intravenously either with a helper-dependent Ad2 vector that does not express viral genes or transgenes, or with the Ad2 mutant ts1, which is defective in endosomal escape. We found that AdV induced rapid phosphorylation of p38 and ERK as well as a significant cytokine response, but ts1 failed to activate p38 or ERK and induced only a limited cytokine response. These results demonstrate that endosomal escape of virions is a critical step in the induction of these innate pathways and responses. We then examined the roles of p38 and ERK pathways in the innate cytokine response by administering specific kinase inhibitors to mice prior to AdV. The cytokine and chemokine response to AdV was only modestly suppressed by a p38 inhibitor, while an ERK inhibitor has mixed effects, lowering some cytokines and elevating others. Thus, even though p38 and ERK are rapidly activated after i.v. injection of AdV, cytokine and chemokine responses are mostly independent of these kinases.

  6. Respiratory syncytial virus fusion glycoprotein expressed in insect cells form protein nanoparticles that induce protective immunity in cotton rats.

    Directory of Open Access Journals (Sweden)

    Gale Smith

    Full Text Available Respiratory Syncytial Virus (RSV is an important viral agent causing severe respiratory tract disease in infants and children as well as in the elderly and immunocompromised individuals. The lack of a safe and effective RSV vaccine represents a major unmet medical need. RSV fusion (F surface glycoprotein was modified and cloned into a baculovirus vector for efficient expression in Sf9 insect cells. Recombinant RSV F was glycosylated and cleaved into covalently linked F2 and F1 polypeptides that formed homotrimers. RSV F extracted and purified from insect cell membranes assembled into 40 nm protein nanoparticles composed of multiple RSV F oligomers arranged in the form of rosettes. The immunogenicity and protective efficacy of purified RSV F nanoparticles was compared to live and formalin inactivated RSV in cotton rats. Immunized animals induced neutralizing serum antibodies, inhibited virus replication in the lungs, and had no signs of disease enhancement in the respiratory track of challenged animals. RSV F nanoparticles also induced IgG competitive for binding of palivizumab neutralizing monoclonal antibody to RSV F antigenic site II. Antibodies to this epitope are known to protect against RSV when passively administered in high risk infants. Together these data provide a rational for continued development a recombinant RSV F nanoparticle vaccine candidate.

  7. Immunization against Clostridium perfringens cells elicits protection against Clostridium tetani in mouse model: identification of cross-reactive proteins using proteomic methodologies

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    Singh Lokendra

    2008-11-01

    Full Text Available Abstract Background Clostridium tetani and Clostridium perfringens are among the medically important clostridial pathogens causing diseases in man and animals. Several homologous open reading frames (ORFs have been identified in the genomes of the two pathogens by comparative genomic analysis. We tested a likelihood of extensive sharing of common epitopes between homologous proteins of these two medically important pathogens and the possibility of cross-protection using active immunization. Results Eight predominant cross-reactive spots were identified by mass spectrometry and had hits in the C. tetani E88 proteome with significant MOWSE scores. Most of the cross-reactive proteins of C. tetani shared 65–78% sequence similarity with their closest homologues in C. perfringens ATCC13124. Electron transfer flavoprotein beta-subunit (CT3 was the most abundant protein (43.3%, followed by methylaspartate ammonia-lyase (36.8% and 2-phosphoglycerate dehydratase (35.6%. All the proteins were predicted to be cytoplasmic by PSORT protein localization algorithm. Active immunization with C. perfringens whole cells elicited cross-protective immunity against C. tetani infection in a mouse model. Conclusion Most of the dominant cross-reactive proteins of C. tetani belonged to the cluster of orthologous group (COG functional category, either of posttranslational modification, protein turnover, and chaperones (O or energy production and conversion (C. The homologs of the identified proteins have been shown to play role in pathogenesis in other Gram-positive pathogenic bacteria. Our findings provide basis for the search of potential vaccine candidates with broader coverage, encompassing more than one pathogenic clostridial species.

  8. 14-3-3 gamma and zeta protein expression in active microglia Immune response mechanisms of Parkinson's disease

    Institute of Scientific and Technical Information of China (English)

    Jing He; Shenggang Sun; Xiaowu Chen

    2008-01-01

    BACKGROUND: The progressive degeneration of dopaminergic neurons in Parkinson's disease is associated with an activated glial reaction, combined with an inflammatory process. These responses lead to the production of cytokines, such as interferon-γ, tumor necrosis factor-α(TNF-α), and interleukin-1β. In addition, 14-3-3 protein is a component of Lewy bodies in Parkinson's disease.OBJECTIVE: To observe the expression of 14-3-3 γ and ζ protein, as well as TNF-α, in mouse microglia, as well as changes after lipopolysaccharide (LPS) activation. To investigate possible mechanisms of dopaminergic neuronal injury due to activated microglia. To and clarify the immune response mechanisms of Parkinson's disease.DESIGN: Randomized controlled observation, cell study.SETTING: Laboratory of Department of Neurology, the Affiliated Union Hospital of Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: The BV-2 immortalized murine microglia cell line was purchased from China Unit cell center. LPS was provided by Sigma Company. Cell cultures were purchased from Gibco. Phospho-(Ser) 14-3-3 binding motif antibody was purchased from Santa Cruz Biotechnologies. FITC was provided by Linfei Biotechnology, Wuhan, China. TNF-α ELISA was provided by Jingmei Biotech Co, Wuhan, China. The flow cytometer was provided by Becton Dickinson, Canada.METHODS: The present experiment was performed at the Laboratory of Department of Neurology, the Affiliated Union Hospital of Tongji Medical College, Huazhong University of Science and Technology from April to December 2006. The microglial cell line, BV-2, was cultured in vitro and stimulated with LPS for 2, 6, 12, and 24 hours. BV-2 cultures without LPS were used as controls.MAIN OUTCOME MEASURES: Expression of 14-3-3 γ protein was detected by flow cytometry. 14-3-3 ζ percentage expression and the mean fluorescence intensity was detected by immunofluorescence. TNF-αexpression was detected by ELISA.RESULTS: 14-3-3

  9. The impaired intestinal mucosal immune system by valine deficiency for young grass carp (Ctenopharyngodon idella) is associated with decreasing immune status and regulating tight junction proteins transcript abundance in the intestine.

    Science.gov (United States)

    Luo, Jian-Bo; Feng, Lin; Jiang, Wei-Dan; Liu, Yang; Wu, Pei; Jiang, Jun; Kuang, Sheng-Yao; Tang, Ling; Zhang, Yong-An; Zhou, Xiao-Qiu

    2014-09-01

    This study investigated the effects of dietary valine on the growth, intestinal immune response, tight junction proteins transcript abundance and gene expression of immune-related signaling molecules in the intestine of young grass carp (Ctenopharyngodon idella). Six iso-nitrogenous diets containing graded levels of valine (4.3-19.1 g kg(-)(1) diet) were fed to the fish for 8 weeks. The results showed that percentage weight gain (PWG), feed intake and feed efficiency of fish were the lowest in fish fed the valine-deficient diet (P valine deficiency decreased lysozyme, acid phosphatase activities and complement 3 content in the intestine (P valine deficiency significantly decreased transcript of Occludin, Claudin b, Claudin c, Claudin 3, and ZO-1 (P valine did not have a significant effect on expression of Claudin 12 in the intestine of grass carp (P > 0.05). In conclusion, valine deficiency decreased fish growth and intestinal immune status, as well as regulated gene expression of tight junction proteins, NF-κB P65, IκBα and TOR in the fish intestine. Based on the quadratic regression analysis of lysozyme activity or PWG, the dietary valine requirement of young grass carp (268-679 g) were established to be 14.47 g kg(-1) diet (4.82 g 100 g(-1) CP) or 14.00 g kg(-1) diet (4.77 g 100 g(-1) CP), respectively.

  10. Non-lethal heat shock increased Hsp70 and immune protein transcripts but not Vibrio tolerance in the white-leg shrimp.

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    Nguyen Hong Loc

    Full Text Available Non-lethal heat shock boosts bacterial and viral disease tolerance in shrimp, possibly due to increases in endogenous heat shock protein 70 (Hsp70 and/or immune proteins. To further understand the mechanisms protecting shrimp against infection, Hsp70 and the mRNAs encoding the immune-related proteins prophenoloxidase (proPO, peroxinectin, penaeidin, crustin and hemocyanin were studied in post-larvae of the white-leg shrimp Litopenaeus vannamei, following a non-lethal heat shock. As indicated by RT-qPCR, a 30 min abrupt heat shock increased Hsp70 mRNA in comparison to non-heated animals. Immunoprobing of western blots and quantification by ELISA revealed that Hsp70 production after heat shock was correlated with enhanced Hsp70 mRNA. proPO and hemocyanin mRNA levels were augmented, whereas peroxinectin and crustin mRNA levels were unchanged following non-lethal heat shock. Penaeidin mRNA was decreased by all heat shock treatments. Thirty min abrupt heat shock failed to improve survival of post-larvae in a standardized challenge test with Vibrio harveyi, indicating that under the conditions of this study, L. vannamei tolerance to Vibrio infection was influenced neither by Hsp70 accumulation nor the changes in the immune-related proteins, observations dissimilar to other shrimp species examined.

  11. Inhibition of Translation Initiation by Protein 169: A Vaccinia Virus Strategy to Suppress Innate and Adaptive Immunity and Alter Virus Virulence.

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    Pavla Strnadova

    2015-09-01

    Full Text Available Vaccinia virus (VACV is the prototypic orthopoxvirus and the vaccine used to eradicate smallpox. Here we show that VACV strain Western Reserve protein 169 is a cytoplasmic polypeptide expressed early during infection that is excluded from virus factories and inhibits the initiation of cap-dependent and cap-independent translation. Ectopic expression of protein 169 causes the accumulation of 80S ribosomes, a reduction of polysomes, and inhibition of protein expression deriving from activation of multiple innate immune signaling pathways. A virus lacking 169 (vΔ169 replicates and spreads normally in cell culture but is more virulent than parental and revertant control viruses in intranasal and intradermal murine models of infection. Intranasal infection by vΔ169 caused increased pro-inflammatory cytokines and chemokines, infiltration of pulmonary leukocytes, and lung weight. These alterations in innate immunity resulted in a stronger CD8+ T-cell memory response and better protection against virus challenge. This work illustrates how inhibition of host protein synthesis can be a strategy for virus suppression of innate and adaptive immunity.

  12. Impacts of different expressions of PA-X protein on 2009 pandemic H1N1 virus replication, pathogenicity and host immune responses.

    Science.gov (United States)

    Lee, Jinhwa; Yu, Hai; Li, Yonghai; Ma, Jingjiao; Lang, Yuekun; Duff, Michael; Henningson, Jamie; Liu, Qinfang; Li, Yuhao; Nagy, Abdou; Bawa, Bhupinder; Li, Zejun; Tong, Guangzhi; Richt, Juergen A; Ma, Wenjun

    2017-04-01

    Although several studies have investigated the functions of influenza PA-X, the impact of different expressions of PA-X protein including full-length, truncated or PA-X deficient forms on virus replication, pathogenicity and host response remains unclear. Herein, we generated two mutated viruses expressing a full-length or deficient PA-X protein based on the A/California/04/2009 (H1N1) virus that expresses a truncated PA-X to understand three different expressions of PA-X protein on virus replication, pathogenicity and host immune responses. The results showed that expression of either full-length or truncated PA-X protein enhanced viral replication and pathogenicity as well as reduced host innate immune response in mice by host shutoff activity when compared to the virus expressing the deficient PA-X form. Furthermore, the full-length PA-X expression exhibited a greater effect on virus pathogenicity than the truncated PA-X form. Our results provide novel insights of PA-X on viral replication, pathogenicity and host immune responses.

  13. Virtual Screening of M3 Protein Antagonists for Finding a Model to Study the Gammaherpesvirus Damaged Immune System and Chemokine Related Diseases

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    Ibrahim Torktaz

    2013-12-01

    Full Text Available Introduction: M3 protein is a chemokine decoy receptor involved in pathogenesis of persistent infection with gammaherpesvirus and complications related to the latency of this pathogen. We proposed that antagonists of the M3 would provide a unique opportunity for studying new therapeutic strategies in disordered immune system, immune-deficient states and role of chemokines in pathogenesis development. Methods: Comparative modeling and fold recognition algorithms have been used for prediction of M3 protein 3-D model. Evaluation of the models using Q-mean and ProSA-web score, has led to choosing predicted model by fold recognition algorithm as the best model which was minimized regarding energy level using Molegro Virtual Docker 2011.4.3.0 (MVD software. Pockets and active sites of model were recognized using MVD cavity detection, and MetaPocket algorithms. Ten thousand compounds accessible on KEGG database were screened; MVD was used for computer simulated docking study; MolDock SE was selected as docking scoring function and final results were evaluated based on MolDock and Re-rank score. Results: Docking data suggested that prilocaine, which is generally applied as a topical anesthetic, binds strongly to 3-D model of M3 protein. Conclusion: This study proposes that prilocaine is a potential inhibitor of M3 protein and possibly has immune enhancing properties.

  14. Identification by Mass Spectrometry and Immune Response Analysis of Guinea Pig Cytomegalovirus (GPCMV Pentameric Complex Proteins GP129, 131 and 133

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    Josephine S. Gnanandarajah

    2014-02-01

    Full Text Available Development of a vaccine against congenital infection with human cytomegalovirus (HCMV is a major public health priority. A potential vaccine target receiving considerable recent attention is the pentameric complex (PC of HCMV proteins consisting of gL, gH, UL128, UL130, and UL131, since some antibodies against these target proteins are capable of potently neutralizing virus at epithelial and endothelial cell surfaces. Recently, homologous proteins have been described for guinea pig cytomegalovirus (GPCMV, consisting of gH, gL, and the GPCMV proteins GP129, GP131, and GP133. To investigate these proteins as potential vaccine targets, expression of GP129-GP133 transcripts was confirmed by reverse-transcriptase PCR. Mass spectrometry combined with western blot assays demonstrated the presence of GP129, GP131, and GP133 proteins in virus particles. Recombinant proteins corresponding to these PC proteins were generated in baculovirus, and as GST fusion proteins. Recombinant proteins were noted to be immunoreactive with convalescent sera from infected animals, suggesting that these proteins are recognized in the humoral immune response to GPCMV infection. These analyses support the study of PC-based recombinant vaccines in the GPCMV congenital infection model.

  15. Humoral immune responses induced by anti-idiotypic antibody fusion protein of 6B11scFv/hGM-CSF in BALB/c mice

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Background We have previously developed and characterized a monoclonal anti-idiotype antibody, designated 6B11, which mimics an ovarian carcinoma associated antigen OC166-9 and whose corresponding monoclonal antibody is COC166-9 (Ab1). In this study, we evaluate the humoral immune responses induced by the fusion protein 6B11 single-chain variable fragment (scFv)/human granulocyte macrophage colony-stimulating factor (hGM-CSF) and 6B11scFv in BALB/c mice. Methods The fusion protein 6B11scFv/hGM-CSF was constructed by fusing a recombinant single-chain variable fragment of 6B11scFv to GM-CSF. BALB/c mice were administrated by 6B11scFv/hGM-CSF and 6B11scFv, respectively. Results The fusion protein 6B11scFv/hGM-CSF retained binding to the anti-mouse F(ab)2' and was also biologically active as measured by proliferation of human GM-CSF dependent cell TF1 in vitro. After immunization with the 6B11scFv/hGM-CSF and 6B11ScFv, BALB/c mice showed significantly enhanced Ab3 antibody responses to 6B11scFv/hGM-CSF compared with the 6B11scFv alone. The level of Ab3 was the highest after the first week and maintained for five weeks after the last immunization. Another booster was given when the Ab3 titer descended, and it would reach to the high level in a week. Conclusion The fusion protein 6B11scFv/hGM-CSF can induce humoral immunity against ovarian carcinoma in vivo. We also provide the theoretical foundation for the application of the fusion protein 6B11scFv/hGM-CSF for active immunotherapy of ovarian cancer.

  16. [A novel immunization strategy to induce strong humoral responses against HIV-1 using combined DNA, recombinant vaccinia virus and protein vaccines].

    Science.gov (United States)

    Liu, Chang; Wang, Shu-hui; Ren, Li; Hao, Yan-ling; Zhang, Qi-cheng; Liu, Ying

    2014-11-01

    To optimize the immunization strategy against HIV-1, a DNA vaccine was combined with a recombinant vaccinia virus (rTV) vaccine and a protein vaccine. Immune responses against HIV-1 were detected in 30 female guinea pigs divided into six groups. Three groups of guinea pigs were primed with HIV-1 DNA vaccine three times, boosted with rTV at week 14, and then boosted with gp140 protein at intervals of 4, 8 or 12 weeks. Simultaneously, the other three groups of animals were primed with rTV vaccine once, and then boosted with gp140 after 4, 8 or 12 weeks. The HIV-1 specific binding antibody and neutralizing antibody, in addition to the relative affinity of these antibodies, were detected at different time points after the final administration of vaccine in each group. The DNA-rTV-gp140 immune regimen induced higher titers and affinity levels of HIV-1 gp120/gp140 antibodies and stronger V1V2-gp70 antibodies than the rTV-gp140 regimen. In the guinea pigs that underwent the DNA-rTV-gp140 regimen, the highest V1V2-gp70 antibody was induced in the 12-week-interval group. However, the avidity of antibodies was improved in the 4-week-interval group. Using the rTV-gp140 immunization strategy, guinea pigs boosted at 8 or 12 weeks after rTV priming elicited stronger humoral responses than those boosted at 4 weeks after priming. In conclusion, this study shows that the immunization strategy of HIV-1 DNA vaccine priming, followed by rTV and protein vaccine boosting, could strengthen the humoral response against HIV-1. Longer intervals were better to induce V1V2-gp70-specific antibodies, while shorter intervals were more beneficial to enhance the avidity of antibodies.

  17. Characterization of the mucosal and systemic immune response induced by Cry1Ac protein from Bacillus thuringiensis HD 73 in mice

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    Vázquez-Padrón R.I.

    2000-01-01

    Full Text Available The present paper describes important features of the immune response induced by the Cry1Ac protein from Bacillus thuringiensis in mice. The kinetics of induction of serum and mucosal antibodies showed an immediate production of anti-Cry1Ac IgM and IgG antibodies in serum after the first immunization with the protoxin by either the intraperitoneal or intragastric route. The antibody fraction in serum and intestinal fluids consisted mainly of IgG1. In addition, plasma cells producing anti-Cry1Ac IgG antibodies in Peyer's patches were observed using the solid-phase enzyme-linked immunospot (ELISPOT. Cry1Ac toxin administration induced a strong immune response in serum but in the small intestinal fluids only anti-Cry1Ac IgA antibodies were detected. The data obtained in the present study confirm that the Cry1Ac protoxin is a potent immunogen able to induce a specific immune response in the mucosal tissue, which has not been observed in response to most other proteins.

  18. Immunization with LytB protein of Streptococcus pneumoniae activates complement-mediated phagocytosis and induces protection against pneumonia and sepsis.

    Science.gov (United States)

    Corsini, Bruno; Aguinagalde, Leire; Ruiz, Susana; Domenech, Mirian; Antequera, María Luisa; Fenoll, Asunción; García, Pedro; García, Ernesto; Yuste, Jose

    2016-12-07

    The cell wall glucosaminidase LytB of Streptococcus pneumoniae is a surface exposed protein involved in daughter cell separation, biofilm formation and contributes to different aspects of the pathogenesis process. In this study we have characterized the antibody responses after immunization of mice with LytB in the presence of alhydrogel as an adjuvant. Enzyme-linked immunosorbent assays measuring different subclasses of immunoglobulin G, demonstrated that the antibody responses to LytB were predominantly IgG1 and IgG2b, followed by IgG3 and IgG2a subclasses. Complement-mediated immunity against two different pneumococcal serotypes was investigated using sera from immunized mice. Immunization with LytB increased the recognition of S. pneumoniae by complement components C1q and C3b demonstrating that anti-LytB antibodies trigger activation of the classical pathway. Phagocytosis assays showed that serum containing antibodies to LytB stimulates neutrophil-mediated phagocytosis against S. pneumoniae. Animal models of infection including invasive pneumonia and sepsis were performed with two different clinical isolates. Vaccination with LytB increased bacterial clearance and induced protection demonstrating that LytB might be a good candidate to be considered in a future protein-based vaccine against S. pneumoniae.

  19. Proteomics and insect immunity

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    L Shi

    2006-01-01

    Full Text Available Insect innate immunity is both a model for vertebrate immunity as well as a key system that impactsmedically important pathogens that are transmitted by insects. Recent developments in proteomics andprotein identification techniques combined with the completion of genome sequences for Anophelesgambiae and Drosophila melanogaster provided the tools for examining insect immunity at a new level ofmolecular detail. Application of proteomics to insect immunity resulted in predictions of new roles inimmunity for proteins already known in other contexts (e.g. ferritin, transferrin, Chi-lectins and helped totarget specific members of multi-gene families that respond to different pathogens (e.g. serine proteases,thioester proteins. In addition, proteomics studies verify that post-translational modifications play a keyrole in insect immunity since many of the identified proteins are modified in some way. These studiescomplement recent work on insect transcriptomes and provide new directions for further investigation ofinnate immunity.

  20. Coupling Peptide Antigens to Virus-Like Particles or to Protein Carriers Influences the Th1/Th2 Polarity of the Resulting Immune Response

    Directory of Open Access Journals (Sweden)

    Rattanaruji Pomwised

    2016-05-01

    Full Text Available We have conjugated the S9 peptide, a mimic of the group B streptococcal type III capsular polysaccharide, to different carriers in an effort to elicit an optimal immune response. As carriers, we utilized the soluble protein keyhole limpet hemocyanin and virus-like particles (VLPs from two plant viruses, Cowpea Chlorotic Mottle Virus and Cowpea Mosaic Virus. We have found that coupling the peptide to the soluble protein elicits a Th2 immune response, as evidenced by the production of the peptide-specific IgG1 antibody and IL-4/IL-10 production in response to antigen stimulation, whereas the peptide conjugated to VLPs elicited a Th1 response (IgG2a, IFN-γ. Because the VLPs used as carriers package RNA during the assembly process, we hypothesize that this effect may result from the presence of nucleic acid in the immunogen, which affects the Th1/Th2 polarity of the response.

  1. Requirement for C-X-C chemokines (macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant) in IgG immune complex-induced lung injury

    DEFF Research Database (Denmark)

    Shanley, T P; Schmal, H; Warner, R L

    1997-01-01

    The C-X-C chemokines of the IL-8 family possess potent chemotactic activity for neutrophils, but their in vivo role in inflammatory responses is not well understood. In the IgG immune complex-induced model of acute lung inflammatory injury in the rat we have evaluated the roles of two rat...... chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC). Both mRNA and protein for MIP-2 and CINC appeared in a time-dependent manner after initiation of IgG immune complex deposition in lung. There exists a 69% homology between the amino acid sequences...... by 125I-labeled albumin leakage from the pulmonary vasculature) and reduced neutrophil accumulation in the lung (as determined by myeloperoxidase (MPO content) and neutrophil counts in bronchoalveolar lavage (BAL) fluids); however, no change in TNF-alpha levels in BAL fluids was found. Chemotactic...

  2. 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.

    Science.gov (United States)

    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.

  3. Influence of Foot-and-Mouth Disease Virus O/CHN/Mya98/33-P Strain Leader Protein on Viral Replication and Host Innate Immunity.

    Science.gov (United States)

    Jiang, Shaodong; Bai, Xingwen; Li, Pinghua; Zhang, Meng; Bao, Huifang; Sun, Pu; Lu, Zengjun; Cao, Yimei; Chen, Yingli; Li, Dong; Fu, Yuanfang; Liu, Zaixin

    2015-09-01

    Foot-and-mouth disease virus (FMDV) O/CHN/Mya98/33-P strain was isolated from the esophageal-pharyngeal fluid sample of cattle, and was shown to cause persistent infection. Its leader protein contains 200 amino acids with one amino acid deletion, which is upstream and next to the second initiation codon compared with the majority of FMDV Mya98 strains. The FMDV genome includes two initiation codons that can produce two different leader proteins, Lab (from the first AUG) and Lb (from the second AUG). For convenience, the inter-AUG region was named as La. Previously, it was found that a recombinant virus with Lab of FMDV O/CHN/Mya98/33-P strain had higher proliferation efficiency, and better ability to inhibit the host innate immune response. Three full-length infectious cDNA clones-rHN33-Lb, rHN33-La, and rHNGSLX-Lb-containing the FMDV O/CHN/Mya98/33-P strain leader proteins Lb, La, or the FMDV O/GSLX/2010 strain leader protein Lb, respectively, were constructed based on an established infectious clone r-HN rescued from FMDV O/HN/CHN/93 strain. After infecting pig kidney primary cells, rHN33-La showed higher replication efficiency than r-HN, and rHN33-Lb displayed better ability to resist host innate immunity than rHNGSLX-Lb. These results demonstrated that the inter-AUG region of FMDV strain O/CHN/Mya98/33-P leader protein must be involved in increasing viral replication efficiency. Additionally, the Lb of FMDV O/CHN/Mya98/33-P must be involve in increasing its ability to inhibit host innate immune response, and the distinctive amino acids G56 and/or R118 of FMDV leader protein may play essential roles in it.

  4. Sub-inhibitory tigecycline concentrations induce extracellular matrix binding protein Embp dependent Staphylococcus epidermidis biofilm formation and immune evasion.

    Science.gov (United States)

    Weiser, Julian; Henke, Hanae A; Hector, Nina; Both, Anna; Christner, Martin; Büttner, Henning; Kaplan, Jeffery B; Rohde, Holger

    2016-09-01

    Biofilm-associated Staphylococcus epidermidis implant infections are notoriously reluctant to antibiotic treatment. Here we studied the effect of sub-inhibitory concentrations of penicillin, oxacillin, vancomycin, daptomycin, linezolid and tigecycline on S. epidermidis 1585 biofilm formation, expression of extracellular matrix binding protein (Embp) and potential implications for S. epidermidis - macrophage interactions. Penicillin, vancomycin, daptomycin, and linezolid had no biofilm augmenting effect at any of the concentrations tested. In contrast, at sub-inhibitory concentrations tigecycline and oxacillin exhibited significant biofilm inducing activity. In S. epidermidis 1585, SarA is a negative regulator of giant 1 MDa Embp, and down regulation of sarA induces Embp-dependent assembly of a multi-layered biofilm architecture. Dot blot immune assays, confocal laser scanning microscopy, and qPCR showed that under biofilm inducing conditions, tigecycline augmented embp expression compared to the control grown without antibiotics. Conversely, expression of regulator sarA was suppressed, suggesting that tigecycline exerts its effects on embp expression through SarA. Tigecycline failed to induce biofilm formation in embp transposon mutant 1585-M135, proving that under these conditions Embp up-regulation is necessary for biofilm accumulation. As a functional consequence, tigecycline induced biofilm formation significantly impaired the up-take of S. epidermidis by mouse macrophage-like cell line J774A.1. Our data provide novel evidence for the molecular basis of antibiotic induced biofilm formation, a phenotype associated with inherently increased antimicrobial tolerance. While this could explain failure of antimicrobial therapies, persistence of S. epidermidis infections in the presence of sub-inhibitory antimicrobials is additionally propelled by biofilm-related impairment of macrophage-mediated pathogen eradication.

  5. The role of mammalian antimicrobial peptides and proteins in awakening of innate host defenses and adaptive immunity.

    Science.gov (United States)

    Yang, D; Chertov, O; Oppenheim, J J

    2001-06-01

    Since we live in a dirty environment, we have developed many host defenses to contend with microorganisms. The epithelial lining of our skin, gastrointestinal tract and bronchial tree produces a number of antibacterial peptides, and our phagocytic neutrophils rapidly ingest and enzymatically degrade invading organisms, as well as produce peptides and enzymes with antimicrobial activities. Some of these antimicrobial moieties also appear to alert host cells involved in both innate host defense and adaptive immune responses. The epithelial cells are a source of constitutively produced beta defensin (HBD1) and proinflammatory cytokine-inducible beta defensins (HBD2 and -3) and cathelicidin (LL37). The neutrophils-derived antimicrobial peptides are released on demand from their cytoplasmic granules. They include the enzymes cathepsin G and chymase, azurocidin, a defensins and cathelicidin. In contrast, C5a and C3b are produced by activation of the serum complement cascade. The antimicrobial moieties direct the migration and activate target cells by interacting with selected G-protein-coupled seven-transmembrane receptors (GPCRs) on cell surfaces. The beta defensins interact with the CCR6 chemokine GPCRs, whereas cathelicidins interact with the low-affinity FPRL-1 receptors. The neutrophil-derived cathepsin G acts on the high-affinity FMLP receptor (GPCR) known as FPR, while the receptors for chymase and azurocidin have not been identified as yet. The serum-derived C5a uses a GPCR known as C5aR to mediate its chemotactic and cell-activating effects. Consequently, all these ligand-receptor interactions in addition to mediating chemotaxis also activate receptor-expressing cells to produce other mediators of inflammation.

  6. Bacteriocin like substance production by Carnobacterium piscicola in a continuous system with three culture broths. Study of antagonism against Listeria monocytogenes on vacuum packaged salmon Produção de substâncias semelhantes à bacteriocinas por C. piscicola em um sistema contínuo com três meios de cultura e seu antagonismo contra L. monocytogenes em salmão embalado a vácuo

    Directory of Open Access Journals (Sweden)

    Renate P. Schöbitz

    2006-03-01

    Full Text Available Three culture media were studied for the bacteriocin like substance (BLS production from Carnobacterium piscicola L 103 in a batch and continuous culture system. The efficacy of the antagonistic substance against Listeria monocytogenes was tested on vacuum packaged salmon. BLS was produced in a 1.0 L bioreactor by batch and continuous culture using D-MRS, mod. D-MRS and APT as nutrient broths. Salmon fillets were inoculated with BLS (200 AU mL-1 and 800 AU mL-1 and 8.0 x 10¹ cfu cm-2 of L. monocytogenes and stored at 4ºC. The growth of L. monocytogenes was determined every 5 days during 15 days. After 12 h of batch culture the stationary growth phase of C. piscicola L 103 started, with a BLS activity of 800 AU mL-1 in D-MRS and mod. D-MRS broth, and of 400 AU mL-1 in APT broth. During continuous culture BLS activity increased to 6400 AU mL-1 in both types of MRS broths, while in APT the activity decreased to 50 AU mL-1, indicating a clear advantage of the first two culture media and also of the continuous culture system. BLS had a bacteriostatic effect on L. monocytogenes when inoculated on salmon, with counts of 6.0 x 10³ cfu cm-2 after 15 days. No significant differences were found between the two BLS activities used. In the control without BLS, L. monocytogenes counts increased to 1.0 x 10(6 cfu cm-2 after 15 days of storage.Três meios de cultura foram avaliados quanto a produção de substâncias semelhantes a bacteriocinas (SSB por Carnobacterium piscicola L 103, utilizando um sistema contínuo de cultura. A eficácia da substância antagonista contra Listeria monocytogenes foi testada em salmão embalado a vácuo. As SSB foram produzidas em bioreator de 1.0 L, em cultura contínua e em cultura estacionária, utilizando D-MRS, mod. D-MRS e APT como meios de cultivo. Filés de salmão foram inoculados com SSB (200 AU mL-1 e 800 AU mL-1 e L. monocytogenes (8.0 x 10¹ ufc cm-2 e mantidos a 4ºC. O crescimento de L. monocytogenes foi

  7. Toxicological effects of nickel chloride on the cytokine mRNA expression and protein levels in intestinal mucosal immunity of broilers.

    Science.gov (United States)

    Wu, Bangyuan; Cui, Hengmin; Peng, Xi; Fang, Jing; Zuo, Zhicai; Deng, Junliang; Wang, Xun; Huang, Jianying

    2015-11-01

    The purpose of this study was to examine the toxicological effects of nickel chloride (NiCl2 ; 300, 600, and 900 mg kg(-1) diet) on the cytokine mRNA expression and protein levels in the intestinal mucosa and cecal tonsil, and on the ileac and cecal tonsil T cells in broilers by the methods of qRT-PCR, flow cytometry and ELISA for 42 days. Results showed that the IL-2, IL-6, IL-10, IL-17, IFN-γ, and TNF-α (LITAF) cytokine mRNA expression and protein levels were lower (P mucosal immunity.

  8. Detection of the host immune response to Burkholderia mallei heat-shock proteins GroEL and DnaK in a glanders patient and infected mice.

    Science.gov (United States)

    Amemiya, Kei; Meyers, Jennifer L; Deshazer, David; Riggins, Renaldo N; Halasohoris, Stephanie; England, Marilyn; Ribot, Wilson; Norris, Sarah L; Waag, David M

    2007-10-01

    We examined, by enzyme-linked immunosorbent assay and Western blot analysis, the host immune response to 2 heat-shock proteins (hsps) in a patient and mice previously infected with Burkholderia mallei. The patient was the first reported human glanders case in 50 years in the United States. The expression of the groEL and dnaK operons appeared to be dependent upon a sigma(32) RNA polymerase as suggested by conserved heat-shock promoter sequences, and the groESL operon may be negatively regulated by a controlling invert repeat of chaperone expression (CIRCE) site. In the antisera, the GroEL protein was found to be more immunoreactive than the DnaK protein in both a human patient and mice previously infected with B. mallei. Examination of the supernatant of a growing culture of B. mallei showed that more GroEL protein than DnaK protein was released from the cell. This may occur similarly within an infected host causing an elevated host immune response to the B. mallei hsps.

  9. Systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type 5 or 41 vector-based vaccines carrying the spike protein of Middle East respiratory syndrome coronavirus.

    Science.gov (United States)

    Guo, Xiaojuan; Deng, Yao; Chen, Hong; Lan, Jiaming; Wang, Wen; Zou, Xiaohui; Hung, Tao; Lu, Zhuozhuang; Tan, Wenjie

    2015-08-01

    An ideal vaccine against mucosal pathogens such as Middle East respiratory syndrome coronavirus (MERS-CoV) should confer sustained, protective immunity at both systemic and mucosal levels. Here, we evaluated the in vivo systemic and mucosal antigen-specific immune responses induced by a single intramuscular or intragastric administration of recombinant adenoviral type 5 (Ad5) or type 41 (Ad41) -based vaccines expressing the MERS-CoV spike (S) protein. Intragastric administration of either Ad5-S or Ad41-S induced antigen-specific IgG and neutralizing antibody in serum; however, antigen-specific T-cell responses were not detected. In contrast, after a single intramuscular dose of Ad5-S or Ad41-S, functional antigen-specific T-cell responses were elicited in the spleen and pulmonary lymphocytes of the mice, which persisted for several months. Both rAd-based vaccines administered intramuscularly induced systemic humoral immune responses (neutralizing IgG antibodies). Our results show that a single dose of Ad5-S- or Ad41-S-based vaccines represents an appealing strategy for the control of MERS-CoV infection and transmission.

  10. Comparative Immunization in BALB/c Mice with Recombinant Replication-Defective Adenovirus Vector and DNA Plasmid Expressing a SARS-CoV Nucleocapsid Protein Gene

    Institute of Scientific and Technical Information of China (English)

    Chunling Ma; Kun Yao; Feng Zhou; Minsheng Zhu

    2006-01-01

    In order to investigate immunogenicity in the induction of humoral and cellular immune responses, severe acute respiratory syndrome associated coronavirus (SARS-CoV)-N gene recombinant replication-defective adenoviral vector, rAd-N, was generated and immunized BALB/c mice in a pcDNA3.1-N prime-rAd-N boost regimen. After humoral and cellular immune response detection, different levels of SARS-CoV N protein specific antibodies and interferon-γ (IFN-γ) secretion are shown compared to controls. The humoral immune response was induced more effectively by the DNA priming and recombinant adenovirus boosting regimen. There is a significant difference between heterogeneous and homologous vaccinations. The heterogeneous combinations were all higher than those of the homologous combinations in the induction of anti-N antibody response. Among the three heterogeneous combinations, pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/rAd-N induced the strongest antibody response. In the induction of IFN-γ production, the homologous combination of rAd-N/rAd-N/rAd-N/rAd- N was significantly stronger than that of pcDNA3.1-N/pcDNA3. 1-N/pcDNA3.1-N/pcDNA3.1-N, but was relatively weaker than the heterogeneous combination of pcDAN3.1-N/pcDAN3.1-N/pcDAN3.1-N/rAd-N. This combination was a most efficient immunization regimen in induction of SARS-CoV-N-specific (IFN-γ) secretion just as the antibody response. These results suggest that DNA immunization followed by recombinant adenovirus boosting could be used as a potential SARS-CoV vaccine.

  11. Cell type and gender-dependent differential regulation of the p202 and Aim2 proteins: implications for the regulation of innate immune responses in SLE.

    Science.gov (United States)

    Panchanathan, Ravichandran; Duan, Xin; Arumugam, Muthuvel; Shen, Hui; Liu, Hongzhu; Choubey, Divaker

    2011-10-01

    Upon sensing cytosolic double-stranded DNA (dsDNA), the murine Aim2 (encoded by the Aim2 gene) protein forms an inflammasome and promotes the secretion of proinflammatory cytokines, such as IL-1β and IL-18. In contrast, the p202 protein (encoded by the Ifi202 gene) does not form an inflammasome. Previously, we have reported that the interferon (IFN) and female sex hormone-induced increased nuclear levels of p202 protein in immune cells are associated with increased susceptibility to develop a lupus-like disease. However, signaling pathways that regulate the expression of Aim2 protein remain unknown. Here we report that the expression of Aim2 gene is induced in bone marrow-derived macrophages (BMDMs) by IFN-α treatment and the expression is, in part, STAT1-dependent. However, treatment of splenic T or B cells with IFN-α or their stimulation, which induced the expression of Ifi202 gene, did not induce the expression of Aim2 gene. Furthermore, treatment of cells with the male hormone androgen increased levels of Aim2 mRNA and protein. Moreover, treatment of murine macrophage cell lines (RAW264.7 and J774A.1) with IFN-α differentially induced the expression of Aim2 and p202 proteins and regulated their sub-cellular localization. Additionally, activation of Toll-like receptors (TLR3, 4, and 9) in BMDMs and cell lines also differentially regulated the expression of Aim2 and Ifi202 genes. Our observations demonstrate that cell type and gender-dependent factors differentially regulate the expression of the Aim2 and p202 proteins, thus, suggesting opposing roles for these two proteins in innate immune responses in lupus disease.

  12. Vaccination with Altered Peptide Ligands of a Plasmodium berghei Circumsporozoite Protein CD8 T-Cell Epitope: A Model to Generate T Cells Resistant to Immune Interference by Polymorphic Epitopes

    Science.gov (United States)

    Minigo, Gabriela; Flanagan, Katie L.; Slattery, Robyn M.; Plebanski, Magdalena

    2017-01-01

    Many pathogens, including the malaria parasite Plasmodium falciparum, display high levels of polymorphism within T-cell epitope regions of proteins associated with protective immunity. The T-cell epitope variants are often non-cross-reactive. Herein, we show in a murine model, which modifies a protective CD8 T-cell epitope from the circumsporozoite protein (CS) of Plasmodium berghei (SYIPSAEKI), that simultaneous or sequential co-stimulation with two of its putative similarly non-cross-reactive altered peptide ligand (APL) epitopes (SYIPSAEDI or SYIPSAEAI) has radically different effects on immunity. Hence, co-immunization or sequential stimulation in vivo of SYIPSAEKI with its APL antagonist SYIPSAEDI decreases immunity to both epitopes. By contrast, co-immunization with SYIPSAEAI has no apparent initial effect, but it renders the immune response to SYIPSAEKI resistant to being turned off by subsequent immunization with SYIPSAEDI. These results suggest a novel strategy for vaccines that target polymorphic epitopes potentially capable of mutual immune interference in the field, by initiating an immune response by co-immunization with the desired index epitope, together with a carefully selected “potentiator” APL peptide.

  13. Enhanced inhibition of tumour growth and metastasis, and induction of antitumour immunity by IL-2-IgG2b fusion protein.

    Science.gov (United States)

    Budagian, V; Nanni, P; Lollini, P L; Musiani, P; Di Carlo, E; Bulanova, E; Paus, R; Bulfone-Paus, S

    2002-05-01

    Cytokine-immunoglobulin (Ig)-fusion proteins have attracted increasing interest as antitumour agents. Here, we have investigated the antimetastatic and antitumour responses elicited in vivo by mammary adenocarcinoma cells (TS/A) engineered to secrete interleukin (IL)-2-IgG fusion proteins. TS/A cells were transfected with DNA coding for IL-2-IgG2b, IgG2b or IL-2, and injected subcutaneously into syngeneic mice. Animals injected with TS/A-IL-2 or TS/A-IL-2-IgG2b both efficiently rejected tumours, whereas treatment with parental cells or TS/A-IgG2b was lethal. Interestingly, only mice vaccinated with IL-2-IgG2b fusion protein-secreting cells showed a long-lasting protective immunity against a later challenge with parental tumour cells. Moreover, the metastatic potential of TS/A-IL-2-IgG2b-transfected cells was dramatically decreased compared with TS/A-IL-2-cells, with a virtual absence of lung metastases after intravenous injection. Adenocarcinomas secreting IL-2-IgG2b exhibited a more prominent, early and persistent infiltration of CD4+, CD8+ and natural killer (NK) cells than TS/A-IL-2 cells. Therefore, upon transfection into adenocarcinoma cells, the IgG2b part of IL-2 fusion protein exerts intriguing added antitumour properties over IL-2 alone, thus contributing to a long-lasting tumour immunity, probably by the recruitment of specific immune effector cells. These findings suggest a promising new oncotherapeutic strategy for poorly immunogenic tumours: vaccination with tumour cells engineered to secrete IL-2-IgG2b fusion protein.

  14. Protein Nutrition of Southern Plains Small Mammals: Immune Response to Variation in Maternal and Offspring Dietary Nitrogen

    Science.gov (United States)

    Maternal nutrition during pregnancy and postnatal offspring nutrition may influence offspring traits. We investigated the effects of maternal and postweaning offspring dietary nitrogen on immune function and hematology in two species of rodent: the hispid cotton rat (Sigmodon his...

  15. Despite sequence homologies to gluten, salivary proline-rich proteins do not elicit immune responses central to the pathogenesis of celiac disease.

    Science.gov (United States)

    Tian, Na; Leffler, Daniel A; Kelly, Ciaran P; Hansen, Joshua; Marietta, Eric V; Murray, Joseph A; Schuppan, Detlef; Helmerhorst, Eva J

    2015-12-01

    Celiac disease (CD) is an inflammatory disorder triggered by ingested gluten, causing immune-mediated damage to the small-intestinal mucosa. Gluten proteins are strikingly similar in amino acid composition and sequence to proline-rich proteins (PRPs) in human saliva. On the basis of this feature and their shared destination in the gastrointestinal tract, we hypothesized that salivary PRPs may modulate gluten-mediated immune responses in CD. Parotid salivary secretions were collected from CD patients, refractory CD patients, non-CD patients with functional gastrointestinal complaints, and healthy controls. Structural similarities of PRPs with gluten were probed with anti-gliadin antibodies. Immune responses to PRPs were investigated toward CD patient-derived peripheral blood mononuclear cells and in a humanized transgenic HLA-DQ2/DQ8 mouse model for CD. Anti-gliadin antibodies weakly cross-reacted with the abundant salivary amylase but not with PRPs. Likewise, the R5 antibody, recognizing potential antigenic gluten epitopes, showed negligible reactivity to salivary proteins from all groups. Inflammatory responses in peripheral blood mononuclear cells were provoked by gliadins whereas responses to PRPs were similar to control levels, and PRPs did not compete with gliadins in immune stimulation. In vivo, PRP peptides were well tolerated and nonimmunogenic in the transgenic HLA-DQ2/DQ8 mouse model. Collectively, although structurally similar to dietary gluten, salivary PRPs were nonimmunogenic in CD patients and in a transgenic HLA-DQ2/DQ8 mouse model for CD. It is possible that salivary PRPs play a role in tolerance induction to gluten early in life. Deciphering the structural basis for the lack of immunogenicity of salivary PRPs may further our understanding of the toxicity of gluten.

  16. Sporozoite neutralizing antibodies elicited in mice and rhesus macaques immunized with a Plasmodium falciparum repeat peptide conjugated to meningococcal outer membrane protein complex

    Directory of Open Access Journals (Sweden)

    Craig ePrzysiecki

    2012-11-01

    Full Text Available Antibodies that neutralize infectivity of malaria sporozoites target the central repeat region of the circumsporozoite (CS protein, which in Plasmodium falciparum is comprised primarily of 30-40 tandem NANP tetramer repeats. We evaluated immunogenicity of an alum-adsorbed (NANP6 peptide conjugated to an outer membrane protein complex (OMPC derived from Neisseria meningitidis, a carrier protein used in a licensed H. influenzae pediatric vaccine. Mice immunized with alum-adsorbed (NANP6-OMPC, with or without Iscomatrix© as co-adjuvant, developed high levels of anti-repeat peptide antibody that inhibited in vitro invasion of human hepatoma cells by transgenic P. berghei sporozoites that express P. falciparum CS repeats (PfPb. Inhibition of sporozoite invasion in vitro correlated with in vivo resistance to challenge by the bites of PfPb infected mosquitoes. Challenged mice had > 90% reduction of hepatic stage parasites as measured by real-time PCR, and either sterile immunity, i.e. no detectable blood stage parasites, or delayed prepatent periods which indicate neutralization of a majority, but not all, sporozoites. Rhesus macaques immunized with two doses of (NANP6-OMPC/MAA formulated with Iscomatrix© developed anti-repeat antibodies that persisted for ~2 years. A third dose of (NANP6-OMPC/MAA+ Iscomatrix© at that time elicited strong anamnestic antibody responses. Rhesus macaque immune sera obtained post second and third dose of vaccine displayed high levels of sporozoite neutralizing activity in vitro that correlated with presence of high anti-repeat antibody titers. These preclinical studies in mice of different MHC haplotypes and a non-human primate support use of CS peptide-OMPC conjugates as a highly immunogenic platform to evaluate CS protective epitopes. Potential pre-erythrocytic vaccines can be combined with sexual blood stage vaccines as a multi-antigen malaria vaccine to block invasion and transmission of Plasmodium parasites

  17. Wolbachia endosymbiont of Brugia malayi elicits a T helper type 17-mediated pro-inflammatory immune response through Wolbachia surface protein.

    Science.gov (United States)

    Pathak, Manisha; Verma, Meenakshi; Srivastava, Mrigank; Misra-Bhattacharya, Shailja

    2015-02-01

    Wolbachia is an endosymbiotic bacterium of the filarial nematode Brugia malayi. The symbiotic relationship between Wolbachia and its filarial host is dependent on interactions between the proteins of both organisms. However, little is known about Wolbachia proteins that are involved in the inflammatory pathology of the host during lymphatic filariasis. In the present study, we cloned, expressed and purified Wolbachia surface protein (r-wsp) from Wolbachia and administered it to mice, either alone or in combination with infective larvae of B. malayi (Bm-L3) and monitored the developing immune response in infected animals. Our results show that spleens and mesenteric lymph nodes of mice immunized with either r-wsp or infected with Bm-L3 show increased percentages of CD4(+) T helper type 17 (Th17) cells and Th1 cytokines like interferon-γ and interleukin-2 (IL-2) along with decreased percentages of regulatory T cells, Th2 cytokines like IL-4 and IL-10 and transforming growth factor β (TGF-β) levels in culture supernatants of splenocytes. These observations were stronger in mice immunized with r-wsp alone. Interestingly, when mice were first immunized with r-wsp and subsequently infected with Bm-L3, percentages of CD4(+) Th17 cells and Th1 cytokines increased even further while that of regulatory T cells, Th2 cytokines and TGF-β levels decreased. These results for the first time show that r-wsp acts synergistically with Bm-L3 in promoting a pro-inflammatory response by increasing Th17 cells and at the same time diminishes host immunological tolerance by decreasing regulatory T cells and TGF-β secretion.

  18. Comparison of the local immune response against Giardia lamblia cyst wall protein 2 induced by recombinant Lactococcus lactis and Streptococcus gordonii.

    Science.gov (United States)

    Lee, Peter; Abdul-Wahid, Aws; Faubert, Gaétan M

    2009-01-01

    Lactococcus lactis and Streptococcus gordonii are lactic acid bacteria (LAB) currently being advocated for use as live antigen delivery vehicles to mucosal sites. Since both vehicles differ in their capability to persist within the small intestine and in their mode of antigen delivery, we sought to compare them to determine which one was superior. In this study, we compared the efficacy of recombinant L. lactis and S. gordonii to stimulate intestinal immune responses against Giardia lamblia cyst wall protein-2 in BALB/c mice. Oral administration of either vector significantly increased the number of CD4(+) T helper and B-cells in the mesenteric lymph nodes (MLN) and Peyer's patches (PP) of immunized animals. Delivery of recombinant CWP2 (rCWP2) by L. lactis stimulated a balanced IFN-gamma/IL-4 response (MLN and PP cells) and a CWP2-specific intestinal IgA antibody response. Alternatively, delivery of rCWP2 by S. gordonii stimulated a higher frequency of IFN-gamma secreting MLN and PP cells, as well as doubling the amount of CWP2-specific intestinal IgA. In challenge studies, L. lactis and S. gordonii reduced cyst output by 71 and 90%, respectively. When compared to each other, S. gordonii-immunized animals shed 65% fewer cysts than their L. lactis-immunized counterparts. Based on these findings, we concluded that S. gordonii was superior to L. lactis as an intestinal vaccine delivery vehicle.

  19. Protection against Salmonella typhi infection in mice after immunization with outer membrane proteins isolated from Salmonella typhi 9,12,d, Vi.

    Science.gov (United States)

    Isibasi, A; Ortiz, V; Vargas, M; Paniagua, J; González, C; Moreno, J; Kumate, J

    1988-01-01

    The current studies were undertaken to assess the ability of the outer membrane proteins (OMPs) of Salmonella typhi to induce protection against challenge with the bacteria in mucin. OMPs were isolated as described by Schnaitman (J. Bacteriol. 108:553-556, 1971) and were found to be contaminated with approximately 4% lipopolysaccharide (LPS). Immunization with as little as 30 micrograms of OMPs conferred 100% protection to mice challenged with up to 1,000 50% lethal doses (LD50) of two strains of S. typhi (9,12,d, Vi and Ty2). In addition, 30% protection against challenge with up to 500 LD50 of Salmonella typhimurium was achieved. Immunization with LPS at doses equivalent to those found in the OMPs was considerably inferior to the OMPs in the induction of an immune status. Moreover, LPS was effective only when the challenge was performed with S. typhi 9,12,d, Vi (40% protection to 100 LD50). An antiserum raised in rabbits reacted mainly against the bands of the molecular weights corresponding to the so-called porins contained in the OMP preparation as shown by Western blotting (immunoblotting). This rabbit antiserum protected 100% of mice against challenge with 100 LD50 of either strain of S. typhi and 80% of mice against challenge with the same LD50 of S. typhimurium. These results indicate the usefulness of OMPs in the induction of active immunity against S. typhi in mice. Images PMID:2844676

  20. Protein-based profiling of the immune response to uropathogenic Escherichia coli in adult patients immediately following hospital admission for acute cystitis.

    Science.gov (United States)

    Sundac, Lana; Dando, Samantha J; Sullivan, Matthew J; Derrington, Petra; Gerrard, John; Ulett, Glen C

    2016-08-01

    Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) are common infections in humans. Despite the substantial healthcare cost represented by these infections, the human immune response associated with the infection immediately following the onset of symptoms in patients remains largely undefined. We performed a prospective study aimed at defining the milieu of urinary cytokines in adult inpatients in the 24-48 h period immediately following hospital admission for acute cystitis due to UPEC. Urine samples, analyzed using 27-target multiplex protein assays, were used to generate immune profiles for patients and compared to age- and gender-matched healthy controls. The levels of multiple pro-inflammatory cytokines were significantly elevated in urine as a result of infection, an observation consistent with prior findings in murine models and clinical literature. We also identified significant responses for several novel factors not previously associated with the human response to UTI, including Interleukin (IL)-4, IL-7, IL-9, IL-17A, eotaxin, Granulocyte-macrophage colony-stimulating factor (GM-CSF) and several growth factors. These data establish crucial parallels between the human immune response to UPEC and murine model UTI studies, and emphasize the complex but poorly defined nature of the human immune response to UPEC, particularly in the immediate period following the onset of symptoms for acute cystitis.