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1
Möller, Carolina, and Frank Marí. "A vasopressin/oxytocin-related conopeptide with γ-carboxyglutamate at position 8." Biochemical Journal 404, no. 3 (May 29, 2007): 413–19. http://dx.doi.org/10.1042/bj20061480.
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Vasopressins and oxytocins are homologous, ubiquitous and multifunctional peptides present in animals. Conopressins are vasopressin/oxytocin-related peptides that have been found in the venom of cone snails, a genus of marine predatory molluscs that envenom their prey with a complex mixture of neuroactive peptides. In the present paper, we report the purification and characterization of a unique conopressin isolated from the venom of Conus villepinii, a vermivorous cone snail species from the western Atlantic Ocean. This novel peptide, designated γ-conopressin-vil, has the sequence CLIQDCPγG* (γ is γ-carboxyglutamate and * is C-terminal amidation). The unique feature of this vasopressin/oxytocin-like peptide is that the eighth residue is γ-carboxyglutamate instead of a neutral or basic residue; therefore it could not be directly classified into either the vasopressin or the oxytocin peptide families. Nano-NMR spectroscopy of the peptide isolated directly from the cone snails revealed that the native γ-conopressin-vil undergoes structural changes in the presence of calcium. This suggests that the peptide binds calcium, and the calcium-binding process is mediated by the γ-carboxyglutamate residue. However, the negatively charged residues in the sequence of γ-conopressin-vil may mediate calcium binding by a novel mechanism not observed in other peptides of this family.
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Mason, R. D., M. I. Bowmer, C. M. Howley, and M. D. Grant. "Cross-Reactive Cytotoxic T Lymphocytes against Human Immunodeficiency Virus Type 1 Protease and Gamma Interferon-Inducible Protein 30." Journal of Virology 79, no. 9 (May 1, 2005): 5529–36. http://dx.doi.org/10.1128/jvi.79.9.5529-5536.2005.
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ABSTRACT The gamma interferon (IFN-γ)-inducible protein 30 (IP-30) signal peptide −11 to −3 (LLDVPTAAV) is a prominent self peptide expressed with the class I human histocompatibility leukocyte antigen A2 (HLA-A2). Stimulation of peripheral blood mononuclear cells (PBMC) from HLA-A2 human immunodeficiency virus type 1 (HIV-1)-infected individuals with an HLA-A2-restricted HIV protease (PR) peptide 76-84 (LVGPTPVNI) activated cytotoxic T lymphocytes (CTL) against the IP-30 signal peptide. Since HIV-1 PR 76-84 stimulated CD8+ T cells from these individuals to secrete IFN-γ, we tested whether the activation of IP-30-specific CTL in vitro resulted from T-cell cross-reactivity or from up-regulation of IP-30 by IFN-γ. Neither high levels of exogenous IFN-γ nor incubation of PBMC with other HIV peptides triggering substantial IFN-γ release activated IP-30-specific CTL. Although the IP-30 signal peptide did not stimulate IFN-γ release from freshly isolated PBMC, it activated CTL in vitro against itself and HIV PR 76-84. Peptide-stimulated IFN-γ release, cold target inhibition, and HLA-A2/immunoglobulin dimer-mediated binding and depletion of effector cells all indicated that in vitro stimulation with HIV PR 76-84 or the IP-30 signal peptide activated a comparable population of cross-reactive effector cells. Neither IP-30 nor HIV PR 76-84 activated CTL against themselves following in vitro stimulation of PBMC from non-HIV-infected HLA-A2 individuals. Peptide titrations indicated higher-avidity T-cell interactions with HIV PR 76-84 than with the IP-30 signal peptide. These data indicate that HIV PR 76-84 is a heteroclitic variant of the IP-30 signal peptide −11 to −3, which has implications for immune memory and autoimmunity.
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Huang, Xiao-Li, Zheng Fan, LuAnn Borowski, and Charles R. Rinaldo. "Multiple T-Cell Responses to Human Immunodeficiency Virus Type 1 Are Enhanced by Dendritic Cells." Clinical and Vaccine Immunology 16, no. 10 (August 19, 2009): 1504–16. http://dx.doi.org/10.1128/cvi.00104-09.
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ABSTRACT Human immunodeficiency virus type 1 (HIV-1)-specific T-cell reactivity has been related to protection from disease progression. Optimal T-cell reactivity to HIV-1 presumably requires antigen processing and presentation by professional antigen-presenting cells, particularly dendritic cells (DC). Here we examined whether multiple HIV-1-specific T-cell functions are enhanced by stimulation with HIV-1 peptide-loaded DC derived from HIV-1-infected subjects on antiretroviral therapy. We first found that mature DC increased the number of gamma interferon (IFN-γ)-producing T cells detected by enzyme-linked immunospot assay to overlapping 15-mer peptides of HIV-1 Gag and Nef, compared to stimulation with peptide-loaded, immature DC or to peptides without DC. IFN-γ production was lower in response to large pools of the Gag and Nef peptides, regardless of presentation by DC. We further observed that HIV-1 peptide-loaded, mature DC stimulated greater CD8+ and CD4+ T-cell proliferation than did the peptides without DC and that T-cell proliferation was lower in response to larger pools of the peptides. The lower T-cell IFN-γ and proliferation responses to the larger peptide pools were related to lower T-cell viability. Finally, the number of polyfunctional CD8+ and CD4+ T cells stimulated by HIV-1 peptide-loaded, mature DC, defined as positive by intracellular staining for more than one immune mediator (IFN-γ, interleukin 2, tumor necrosis factor alpha, macrophage inhibitory protein 1β, or CD107a), was greater than that stimulated by the peptides alone. These results indicate that DC can enhance multiple types of HIV-1-specific T-cell functions.
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Flood, Veronica H., Hamid A. Al-Mondhiry, Antony C. Bakke, and David H. Farrell. "Fibrinogen Hershey IV: A Novel Dysfibrinogen with a γ V411I Mutation in the Integrin αIibβ3 Binding Site." Blood 106, no. 11 (November 16, 2005): 2133. http://dx.doi.org/10.1182/blood.v106.11.2133.2133.
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Abstract The C-terminal segment of the fibrinogen γ chain plays a crucial role in platelet aggregation via its interaction with the platelet receptor αIIbβ3. It is well established that the last four amino acids of the γ chain (408 to 411) are critical for this function, as mutations or deletions of this region abrogate fibrinogen’s ability to bind αIIbβ3. We describe here the first naturally occurring fibrinogen mutation affecting the C-terminal region of the γ chain and investigate its effects on platelet interactions. The proband, a 49-year-old woman, was diagnosed with dysfibrinogenemia based on a prolonged thrombin time and low fibrinogen activity (55 mg/dL). Her bleeding history was significant for menorrhagia and one episode of post-operative hemorrhage. DNA sequencing of the fibrinogen genes demonstrated heterozygosity for two mutations, γ R275C and γ V411I. The latter γ V411I mutation represents a novel mutation affecting the C-terminal amino acid of the γ chain. We hypothesized that this mutation would decrease fibrinogen’s affinity for the platelet receptor αIIbβ3. In order to isolate the effects of this mutation on fibrinogen-platelet binding, γ 400-411 dodecapeptides were synthesized to mimic the C-terminal γ chain sequence. One peptide contained the wild-type sequence ending in valine (γ 400-V411), and the second peptide incorporated the isoleucine mutation (γ 400-I411). Previous studies have demonstrated that the wild type γ 400-V411 dodecapeptide inhibits platelet aggregation by competing for fibrinogen binding to αIIbβ3. We performed platelet aggregation studies comparing inhibition of aggregation with the wild-type γ 400-V411 and the mutant γ 400-I411 peptides. Washed platelets were obtained from a normal donor, and platelet aggregation monitored using the agonist ADP. The IC50 for the initial rate of aggregation with the γ 400-I411 peptide was 214 μM, compared to 133 μM with the wild-type peptide. We then examined the extent of aggregation in the presence of either wild-type or mutant peptide. Consistent with the previous results, total aggregation was lower with the wild-type peptide compared to the mutant peptide. The IC50 for the γ 400-I411 peptide was 450 μM compared to 250 μM with the γ 400-V411 peptide. Overall, these findings suggest that the γ I411 mutation results in a decreased ability to bind platelets. In the heterozygous state, however, the available amount of wild-type fibrinogen may be sufficient to support platelet aggregation. The bleeding diathesis observed for the proband could therefore reflect other factors, especially the γ R275C mutation.
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Farrell, David H., Rehana S. Lovely, and Lynn K. Boshkov. "Inhibition of Thrombin Cleavage of Factor VIII by Fibrinogen γ’ Chain Carboxyl Terminus." Blood 106, no. 11 (November 16, 2005): 1957. http://dx.doi.org/10.1182/blood.v106.11.1957.1957.
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Fibrinogen can contain two types of γ chains, γA and γ’, that have identical sequences for the first 407 amino acids, but differ at their carboxyl termini. The γA chain contains four amino acids from γA 408–411, whereas the γ’ chain contains a different, highly anionic sequence of twenty amino acids from γ’ 408–427. This small change results in profound differences in the fibrinogen isoforms containing these distinct chains. The γ’ chain contains high affinity binding sites for zymogen factor XIII and for thrombin, and fibrinogen containing γ’ chains forms fibrin clots that are resistant to fibrinolysis. We have shown previously that the fibrinogen γ’ chain carboxyl terminus binds to thrombin exosite II, an anion-binding exosite known for its heparin binding activity. Recent reports show that the presence of the γ’ chain in fibrinogen reduces prothrombin activation in whole plasma assays. This inhibition may correspond to an activity originally described by Seegers in 1945 as “antithrombin I”, the adsorption of thrombin to fibrin. We have therefore investigated the enzymatic effects of γ’ peptide binding to thrombin in order to determine the role of the γ’ chain in the inhibition of prothrombin activation. In whole plasma, the γ’410–427 peptide prolonged the activated partial thromboplastin time (aPTT) in a dose-dependent manner, causing a delay in clotting from 29 seconds to 47 seconds at a concentration of 500 μM. A scrambled control peptide at the same concentration had little effect, increasing the clotting time to only 31.4 seconds. A series of γ’ deletion peptides were tested for their ability to prolong the aPTT. The inhibitory effect of the peptides corresponded in rank order to their thrombin binding affinity. In contrast to the aPTT, the prothrombin time increased only slightly from 11.7 seconds to 12.8 seconds (INR from 1.04 to 1.14) with the γ’410–427 peptide. This suggested that the γ’ peptide exerted its inhibitory effect on (a) component(s) of the intrinsic pathway, rather than the extrinsic pathway. We therefore investigated the effect of the γ’ peptide on thrombin activation of factor VIII, a thrombin substrate that, unlike factor V, is unique to the intrinsic pathway. Using purified factor VIII and thrombin in vitro, we showed that the γ’ peptide inhibited thrombin cleavage of factor VIII. However, this inhibitory effect was not due to direct inhibition of the thrombin active site, since the free γ’ peptide had little effect on thrombin cleavage of a small peptidyl substrate, tosyl-glycyl-prolyl-arginine-4-nitranilide acetate. These results suggest that factor VIII interactions with thrombin exosite II are essential for efficient cleavage of factor VIII, and support recent findings by others that interactions between thrombin exosite II and the A2 domain of factor VIII facilitate thrombin-catalyzed cleavage. These findings may explain, at least in part, the inhibitory effect of the γ’ chain on prothrombin activation in plasma.
6
Hirano, Naoto, Marcus O. Butler, Zhinan Xia, Seiji Kojima, and Lee M. Nadler. "γ-Globin, a Tumor-Associated Antigen for Juvenile Myelomonocytic Leukemia (JMML): A Cell-Based Approach To Identify Tumor Antigenic Epitopes That Are Naturally Processed and Presented." Blood 104, no. 11 (November 16, 2004): 3418. http://dx.doi.org/10.1182/blood.v104.11.3418.3418.
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Abstract Juvenile myelomonocytic leukemia (JMML) is a rare clonal myeloproliferative disorder of early childhood. Although allogeneic stem cell transplantation can induce long-term remissions, relapse rates remain high, and innovative approaches are needed. Since donor lymphocyte infusion in JMML is efficacious, T cell mediated immunotherapy may be effective, and appropriate antigenic targets must be identified. One candidate tumor-associated antigen for the immunotherapy of JMML is γ-globin, which is expressed at high levels in most JMML patients. Most clonogenic JMML cells constitutively express this onco-fetal protein, which is not necessary for the normal erythropoesis of children and adults. To determine whether γ-globin can serve as a target for immunotherapy in JMML, we sought to determine whether γ-globin is naturally processed and presented by the HLA complex. Using conventional bioinformatic techniques and the T2 binding assay to predict candidate epitopes, we identified 4 γ-globin derived peptides (g031, g071, g105, and g106) that were predicted to bind to the HLA-A2 molecule in vitro. Since this strategy provides no evidence for which predicted epitopes are processed and presented by tumor cells in vivo, we employed a biochemical strategy to determine which peptides are naturally processed and presented. This step is critical in certifying that a candidate peptide epitope is an appropriate target for immunotherapy treatments. Using our K562-derived artificial APC (aAPC), an APC that expresses A2 and no other HLA allele, we introduced the EGFP-γ-globin fusion gene. We then acid stripped peptides directly from the surface of one billion aAPC/EGFP-γ-globin cells without subjecting the cells to detergent mediated lysis. Peptides less than 5 kDa in size were fractionated by reverse phased HPLC analysis and analyzed by mass spectrometry. We identified two mass spectrometry peaks which corresponded to γ-globin derived peptides, g031 and g105. Of these, the identity of one peak, g105, was successfully confirmed by peptide sequencing, providing strong evidence that g105 is naturally processed and presented by aAPC/EGFP-γ-globin cells. Next, to confirm that g105 is processed and presented by primary JMML cells, we generated γ-globin specific CD8+ cytotoxic T cells (CTL) from A2 positive healthy donors using synthetic g105 peptide. γ-Globin specific CTL were able to specifically cytolyze A2+ γ-globin+ JMML cells but not A2+ γ-globin- JMML cells. Specific cytotoxicity was blocked by anti-A2 mAb but not isotype control. These results show for the first time that the γ-globin derived peptide, g105, can serve as a target epitope for the CTL directed immunotherapy of JMML. Furthermore, these results illustrate an innovative aAPC based strategy that can identify the antigenic peptide epitopes of putative tumor associated antigens that are naturally processed by tumor cells, presented via HLA class I, and can serve as targets for effective anti-cancer immunotherapy.
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Hirao, Takashi, Masahide Sato, Akira Shirahata, and Yoshiyuki Kamio. "Covalent Linkage of Polyamines to Peptidoglycan inAnaerovibrio lipolytica." Journal of Bacteriology 182, no. 4 (February 15, 2000): 1154–57. http://dx.doi.org/10.1128/jb.182.4.1154-1157.2000.
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ABSTRACT Spermidine and cadaverine were found to be constituents of the cell wall peptidoglycan of Anaerovibrio lipolytica, a strictly anaerobic bacterium. The peptidoglycan was degraded with theN-acetylmuramyl-l-alanine amidase and endopeptidase into two peptide fragments, peptide I and peptide II, at a molar ratio of 4:1. Peptides I and II were identified asl-alanine–d-glutamic acid(αcadaverine)γ meso-diaminopimelic acid (DAP)–d-alanine andl-alanine–d-glutamic acid(αspermidine)γ meso-DAP–d-alanine, respectively. The N1-amino group of spermidine was linked to the α-carboxyl group of the d-glutamic acid residue of peptide II.
8
Mujtaba, Mustafa. "Antiviral inducing properties of staphylococcal enterotoxin mimetic peptides (VAC9P.1065)." Journal of Immunology 194, no. 1_Supplement (May 1, 2015): 145.5. http://dx.doi.org/10.4049/jimmunol.194.supp.145.5.
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Abstract Superantigens, like the staphylococcal enterotoxins, activate vast numbers of T-cells to produce large amounts of cytokines, including interferon-gamma (IFN-γ), and eventually cause these cells to undergo apoptosis. The objective of this research was to design mimetic peptides of staphylococcal enterotoxins that are not toxic to cells, but can produce antiviral activity in cells via production of IFN-γ. Based on the amino acid sequences of staphylococcal enterotoxins (SE) A, B, C, and toxic shock syndrome toxin, peptides were designed to mimic the antigenic sites of these superantigens. Whole protein superantigens (SEA) at concentrations ranging from 1.0 to 10.0ng/mL stimulated T-cell proliferation and induced IFN-γ production. Superantigen concentrations at or above 100ng/mL showed cellular toxicity. Of the eight mimetic peptides tested, SEA 3 was the only peptide that induced IFN-γ production, as determined by the IFN-γ ELISA kit, in HPBMC but did not induce cellular proliferation. SEA1, SEA2, and TSST peptides induced cellular proliferation but no IFN-γ stimulation. The peptides showed no toxicity directly on HeLa cells or HPBMC at 100 ug/mL or lower. Cell supernatant from the SEA3 peptide treated HPBMC also had antiviral activity against vesicular stomatitis virus. Thus, this study showed that mimetic peptides of superantigens could be developed that can induce T-cells to produce IFN-γ without the cellular toxicity associated with superantigens.
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Passero, Christopher J., Marcelo D. Carattino, Ossama B. Kashlan, Mike M. Myerburg, Rebecca P. Hughey, and Thomas R. Kleyman. "Defining an inhibitory domain in the gamma subunit of the epithelial sodium channel." American Journal of Physiology-Renal Physiology 299, no. 4 (October 2010): F854—F861. http://dx.doi.org/10.1152/ajprenal.00316.2010.
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Proteases activate the epithelial sodium channel (ENaC) by cleaving the large extracellular domains of the α- and γ-subunits and releasing peptides with inhibitory properties. Furin and prostasin activate mouse ENaC by cleaving the γ-subunit at sites flanking a 43 residue inhibitory tract (γE144-K186). To determine whether there is a minimal inhibitory region within this 43 residue tract, we generated serial deletions in the inhibitory tract of the γ-subunit in channels resistant to cleavage by furin and prostasin. We found that partial or complete deletion of a short segment in the γ-subunit, R158-N171, enhanced channel activity. Synthetic peptides overlapping this segment in the γ-subunit further identified a key 11-mer tract, R158-F168 (RFLNLIPLLVF), which inhibited wild-type ENaC expressed in Xenopus laevis oocytes, and endogenous channels in mpkCCD cells and human airway epithelia. Further studies with amino acid-substituted peptides defined residues that are required for inhibition in this key 11-mer tract. The presence of the native γ inhibitory tract in ENaC weakened the intrinsic binding constant of the 11-mer peptide inhibitor, suggesting that the γ inhibitory tract and the 11-mer peptide interact at overlapping sites within the channel.
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Zeng, Yi, Michael W. Graner, Sylvia Thompson, Marilyn Marron, and Emmanuel Katsanis. "Induction of BCR-ABL–specific immunity following vaccination with chaperone-rich cell lysates derived from BCR-ABL+ tumor cells." Blood 105, no. 5 (March 1, 2005): 2016–22. http://dx.doi.org/10.1182/blood-2004-05-1915.
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AbstractWe have previously reported that chaperonerich cell lysates (CRCL) derived from the BCR-ABL+ 12B1 leukemia activate dendritic cells (DCs) and stimulate leukemia-specific immune responses. Because CRCL contain a variety of heat shock/chaperone proteins, we theorized that CRCL obtained from BCR-ABL+ leukemias are likely to chaperone BCR-ABL–derived fusion peptides and that DCs pulsed with 12B1 CRCL could cross-present BCR-ABL fusion peptides to T cells. We found that splenocytes from mice vaccinated with BCR-ABL+ leukemia-derived CRCL secreted interferon-γ (IFN-γ) when restimulated with a BCR-ABL peptide, GFKQSSKAL, indicating that BCR-ABL peptides are chaperoned by leukemia-derived CRCL. We next eluted peptides from 12B1 leukemia-derived CRCL and used high-pressure liquid chromatography (HPLC) fractions to restimulate splenocytes harvested from mice vaccinated with DC/GFKQSSKAL or DC/12B1 CRCL. We found that the same peptide fractions derived from 12B1 CRCL and from “refractionated” GFKQSSKAL stimulated IFN-γ production, suggesting the presence of BCR-ABL peptides in the peptide repertoire of 12B1 CRCL. We also demonstrated that immunization with DCs loaded with leukemia-derived CRCL induced BCR-ABL–specific cytotoxic T lymphocytes (CTLs) in vivo. Moreover, mice immunized with DCs pulsed with 12B1-derived CRCL had superior survival (60%) when compared with those immunized with DCs pulsed with BCR-ABL peptide (20%), indicating that CRCL vaccines provide additional immune stimulus over and above individual peptide vaccination.
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Saini, Chaman, H. K. Prasad, Rajni Rani, A. Murtaza, Namita Misra, N. P. Shanker Narayan, and Indira Nath. "Lsr2 of Mycobacterium leprae and Its Synthetic Peptides Elicit Restitution of T Cell Responses in Erythema Nodosum Leprosum and Reversal Reactions in Patients with Lepromatous Leprosy." Clinical and Vaccine Immunology 20, no. 5 (February 27, 2013): 673–82. http://dx.doi.org/10.1128/cvi.00762-12.
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ABSTRACTThe Lsr2 protein ofMycobacterium lepraeand its synthetic peptides have been shown to elicit lymphoproliferation and gamma interferon (IFN-γ) release by peripheral blood mononuclear cells (PBMCs) of patients with lepromatous leprosy (M. Chaduvula, A. Murtaza, N. Misra, N. P. Narayan, V. Ramesh, H. K. Prasad, R. Rani, R. K. Chinnadurai, I. Nath, Infect. Immun. 80:742–752, 2012). PBMCs from 16 patients with lepromatous leprosy who were undergoing erythema nodosum leprosum (ENL) (type 2) and 5 patients with reversal reactions (RR) (type 1) were stimulated withM. leprae, recombinant Lsr2, and six end-to-end synthetic peptides (A through F) spanning the Lsr2 sequence. During the reaction all patients with ENL showed lymphoproliferation (stimulation index, >2) in response to peptides A and F, with other peptides eliciting responses in 75 to 88% of the subjects. In PBMC cultures, both lymphoproliferation and IFN-γ release for peptide E were significantly higher than for peptides B and C and recombinant Lsr2 (P< 0.05, Wilcoxon signed-rank test). Five patients with RR also showed enhanced lymphoproliferative responses and IFN-γ release in response to Lsr2,M. leprae, and peptide E. Six months postreaction, 14 patients with ENL continued to exhibit responses to Lsr2 and its peptides, with the highest responses being elicited by peptide E. However, 5 subjects showed no lymphoproliferation and had reduced IFN-γ release in response to Lsr2 peptides (P< 0.001, Kruskal-Wallis test) but responded to recombinant Lsr2. Six patients with ENL had HLA-A*68.01, which the STFPEITHI program showed to have high peptide-binding scores of 20 to 21 for peptides E, B, and C. Eleven patients had HLA-DRB1*1501 and HLA-DRB1*1502, which had high binding scores for peptides C and E. Thus, Lsr2 and its peptides are recognized in leprosy reactions during and well after the subsidence of clinical signs.
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Fuji, Shigeo, Julia Fischer, Markus Kapp, Thomas G. Bumm, Hermann Einsele, Stefan Stevanovic, Hans-Georg Rammensee, Thomas Hunig, and Götz Ulrich Grigoleit. "Wilms Tumor Protein-1-Derived 9-Mer Peptide Induces CD4 T-Cell Responses in an HLA-DR Restricted Manner." Blood 120, no. 21 (November 16, 2012): 4351. http://dx.doi.org/10.1182/blood.v120.21.4351.4351.
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Abstract Abstract 4351 Wilms‘ tumor protein-1 (WT1) is one of the most investigated tumor-associated antigens (TAA) in hematological malignancies. CD8 T-cell responses against several WT1-derived peptides have been characterized and are known to contribute to disease control after allogeneic hematopoietic stem cell transplantation (HSCT). Also the identification of human leukocyte antigen (HLA) class II-restricted CD4 T-cell epitopes from WT1 is a challenging task of T-cell-based cancer immunotherapy to improve the effectiveness of WT1 peptide vaccination. We found a highly immunogenic WT1 peptide composed of only 9 amino acids having the ability to induce IFN-γ secretion in CD4 T-cells in an HLA DR-restricted manner. This finding is of great interest as it was generally accepted that HLA class II binding peptides are composed of at least 12 amino acids being recognized by CD4 T-cells, whereas HLA class I binding peptides are composed of 8–11 amino acids being recognized by CD8 T-cells (Wang et al Mol. Immunol. 2002). However, both HLA class I and class II molecules bind to primary and secondary peptide anchor motifs covering the central 9–10 amino acids. Thus, considering this common structural basis for peptide binding there is a possibility that the WT1 9-mer peptide binds to HLA class II molecules, and induces CD4 T-cell responses. IFN-γ induction in response to several WT1 9-mer peptides was screened in 24 HLA-A*02:01 positive patients with acute myeloid leukemia or myelodysplastic syndrome after allogeneic HSCT. Responses to one WT1 9-mer peptide were exclusively detected in CD3+CD4+ T-cells of 2 patients after allogeneic HSCT, but not in CD3+CD4+ T-cells of their corresponding HSC donors. CD4+ T-cell responses to this WT1 9-mer peptide exhibited high levels of functional avidity, as IFN-γ induction was detected after stimulation with 100 ng peptide per mL. Peptide-induced IFN-γ production was confirmed with IFN-γ ELISPOT assays and the HLA restriction of the T-cell response was determined by HLA blocking antibodies. The reaction was significantly blocked by anti-pan HLA class II antibody (85 % reduction), but neither by pan-HLA class I nor by anti-HLA A2 antibody. To identify the subtype of HLA class II molecule, blocking assays with antibodies against HLA-DP, HLA-DR and HLA-DQ were performed. IFN-γ induction was completely abrogated by anti-HLA-DR antibody (99 % reduction) (fig 1, p value of unpaired student‘s t-test <0.0001 for the medium control vs anti-pan HLA class II antibody or anti-HLA-DR antibody, respectively). To test whether IFN-γ was exclusively induced in CD4 T cells, CD4 or CD8 T-cells were depleted from PBMC. Whereas CD8 T-cell depletion did not affect IFN-γ induction, CD4 T-cell depletion completely abrogated the WT1 9-mer peptide induced response (fig 2). CD4 T-cells responding to the WT1 9-mer peptide were indicated to be functional cytotoxic T-cells with an effector CD4 T-cell phenotype. Longitudinal analyses demonstrated the persistence and functionality of WT1 9-mer specific CD4 T-cells in PBMC of patients even at day 1368 after allogeneic HSCT. These data indicate for the first time that a TAA-derived 9-mer peptide can induce HLA class II-restricted CD4 T-cell responses. Vaccination with the characterized WT1 9-mer peptide can enhance the induction and maintenance of not only CD4 but also indirect CD8 T-cell responses. Considering that CD4 T-cells play an important role in tumor rejection, the possibility that other TAA-derived 9-mer peptides having the potential to induce CD4 T-cell responses should be explored in other settings of tumor immunology as well to improve vaccination strategies. Disclosures: No relevant conflicts of interest to declare.
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Bracci, Laura, David F. Stroncek, Stefanie Slezak, Giulio C. Spagnoli, and Maurizio Provenzano. "Comprehensive Analysis of CD8 T Cell Immune Response Specific for Two Novel HLA-A*0201 Restriced CMV pp65 Peptides." Blood 106, no. 11 (November 16, 2005): 3928. http://dx.doi.org/10.1182/blood.v106.11.3928.3928.
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Abstract In immune compromised subjects such as patients undergoing bone marrow, organ transplantation or immunosuppressive therapies, Cytomegalovirus (CMV) infection is associated with significant morbidity until the individual’s immune system is completely reconstituted. One method of preventing CMV infection during immune suppression in transplant patients is represented by adoptive administration of CMV peptide-specific cytotoxic T lymphocytes (CTLs) from HLA-matched donors. Despite the strong immunogenicity demonstrated by specific HLA class I peptides, the peptide’s responsiveness varies among individuals. Therefore, it is important to identify additional epitopes for each HLA determinant of interest. In this study we report a comprehensive analysis of CD8 T cell-specific activity against two novel CMV pp65 HLA-A*0201 associated peptides in CMV-experienced HLA-A*0201 subjects. PBMCs from CMV seropositive HLA-A*0201 restricted healthy subjects were peptide-stimulated ex vivo and IFN-γ gene expression was analyzed by qrt-PCR. An IFN-γ ELISPOT assay was carried out on peptide-specific elicited CD8 T lymphocytes to confirm the qrt-PCR results. Tetrameric HLA/epitope complexes (tHLA) were used to track the levels of peptide-specific CD8 T cells responsiveness to cognate epitopes. In addition, in vitro peptide-specific expanded populations of CTLs were used in 51Cr release assay. Based on qrt-PCR results, four out of eight HLA-A*0201 peptides identified by computer algorithms were selected. Two are preaviously published peptides: pp65495–503 (NLVPMVATV) and pp65347–355 (ALFFFDIDL), while two are novel: pp65340–348 (RQYDPVAAL) and pp65310–318 (LMNGQQIFL). In spite the four peptides induced comparable mRNA IFN-γ transcript production, peptides pp65495–503, pp65340–348 and pp65310–318 induced a consistent and sustained IFN-γ protein release and specific killing, while the pp65347–355 failed to induce IFN-γ protein secretion and killing activity (if we exclude a positive IFN-γ protein release after 2-week in vitro induction in one of the donors tested). Comparative assays carried on the functional activity of the three peptides pp65495–503, pp65340–348 and pp65310–318 revealed no intrinsic differences in term of IFN-γ protein release and cytotoxic activity save for the CTL affinity to the HLA-A*0201/epitope complexes (pp65495–503 ~2.6% in nearly 100% of donors vs pp65340–348 ~0.67% or pp65310–318 ~0.77% in nearly one third of the donors after 2-week in vitro induction). The tHLA binding results could be possibly ascribed to differencies in the peptide avidity and stability for the HLA class I. Taken together, these results lead to the conclusion that different peptides can induce a variable levels of immune responses ranging between mere cytokine gene expression and effective cytotoxic activity. In addition, the two novel peptides selected here broaden the panel of potential reagents useful for adoptive immune therapy. Thus, in anticipation of a specific epitope-targeted immune intervention, the three HLA-A*0201 peptides described could be used in combination for adoptive transfer of epitope-specific T cells or epitope-specific vaccination.
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Geluk, Annemieke, Jolien J. van der Ploeg-van Schip, Krista E. van Meijgaarden, Susanna Commandeur, Jan W. Drijfhout, Willemien E. Benckhuijsen, Kees L. M. C. Franken, Bernard Naafs, and Tom H. M. Ottenhoff. "Enhancing Sensitivity of Detection of Immune Responses to Mycobacterium leprae Peptides in Whole-Blood Assays." Clinical and Vaccine Immunology 17, no. 6 (April 28, 2010): 993–1004. http://dx.doi.org/10.1128/cvi.00046-10.
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ABSTRACT Although worldwide leprosy prevalence has been reduced considerably following multidrug therapy, new case detection rates remain relatively stable, suggesting that transmission of infection still continues. This calls for new efforts, among which is development of assays that can identify subclinical/early-stage Mycobacterium leprae-infected subjects, a likely source of transmission. Areas in which leprosy is endemic often lack sophisticated laboratories, necessitating development of field-friendly immunodiagnostic tests for leprosy, like short-term whole-blood assays (WBA). In classical, peripheral blood mononuclear cell (PBMC)-based gamma interferon (IFN-γ) release assays, M. leprae peptides have been shown to discriminate in a more specific fashion than M. leprae proteins between M. leprae-exposed contacts and patients as opposed to healthy controls from the same area of endemicity. However, peptides induced significantly lower levels of IFN-γ than did proteins, particularly when whole blood was used. Therefore, possibilities of specifically enhancing IFN-γ production in response to M. leprae peptides in 24-h WBA were sought by addition of various cytokines and antibodies or by mannosylation of peptides. In addition, other cytokines and chemokines were analyzed as potential biomarkers in WBA. We found that only interleukin 12 (IL-12), not other costimulants, increased IFN-γ production in WBA while maintaining M. leprae peptide specificity, as evidenced by lack of increase of IFN-γ in control samples stimulated with IL-12 alone. The IL-12-induced increase in IFN-γ was mainly mediated by CD4+ T cells that did not produce IL-2 or tumor necrosis factor (TNF). Mannosylation further allowed the use of 100-fold-less peptide. Although not statistically significantly, macrophage inflammatory protein 1β (MIP-1β) and macrophage c protein 1 (MCP-1) levels specific for M. leprae peptide tended to be increased by IL-12. IP-10 production was also found to be a useful marker of M. leprae peptide responses, but its production was enhanced by IL-12 nonspecifically. We conclude that IFN-γ-based WBA combined with IL-12 represents a more sensitive and robust assay for measuring reactivity to M. leprae peptides.
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DeLucia, Diana, Tiffany Pariva, Roland Strong, Owen Witte, and John Lee. "148 Identification of prostate-restricted epithelial antigens for transgenic T cell adoptive therapy against prostate cancer." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (November 2020): A161. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0148.
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BackgroundIn advanced prostate cancer (PCa), progression to castration-resistant PCa (CRPC) is inevitable and novel therapies for CRPC are needed. Adoptive transfer of T cells targeting tumor antigens is a promising approach in the cancer field. Unfortunately, identifying antigens expressed exclusively in prostate tumor cells has been challenging. Since the prostate is not an essential organ, we alternatively selected prostate-restricted epithelial antigens (PREAs) expressed in both malignant and normal prostate tissue for transgenic T cell studies.MethodsRNA-seq data sets identifying genes enriched in PCa were cross-referenced with the NIH Genotype-Expression database to identify PREAs. Using a novel molecular immunology approach, select PREAs and major histocompatibility complex class I (MHC-I) molecules were co-expressed in HEK293F cells, from which MHC–peptide complexes were efficiently isolated. Peptides were eluted and sequenced by mass spectrometry. Peptide–MHC binding was validated with a T2 stabilization assay and peptide immunodominance was determined using an interferon-γ (IFN-γ) ELISpot assay following stimulation of healthy HLA-A2+ peripheral blood mononuclear cells (PBMC) with peptide pools. Following peptide stimulation, CD8+ T cells with peptide-specific T cell receptors (TCR) were enriched by peptide–MHC-I dextramer labeling and fluorescence activated cell sorting for single cell TCR α/β chain sequencing.ResultsWe identified 11 A2+ peptides (8 previously unpublished) from prostatic acid phosphatase (ACPP), solute carrier family 45 member 3 (SLC45A3), and NK3 homeobox 1 (NKX3.1) that bound to HLA-A2 with varying affinities. Extended culture stimulation of PBMC with peptide pools from each PREA, compared to the standard overnight culture, revealed a greater number of IFN-γ producing cells overall and a greater breadth of response across all the peptides. Antigen specific CD8+ T cells were detectable at low frequencies in both male and female healthy PBMC for 7 of the 11 peptides. Dextramer-sorted antigen-specific cells were used for single-cell paired TCR αβ sequencing and transgenic T cell development.ConclusionsThrough this work we identified HLA-A2-presented antigenic peptides from the PREAs ACPP, SLC45A3, and NKX3.1 that can induce the expansion of IFN-γ producing CD8+ T cells. Through peptide–MHC-I dextramer labeling, we isolated PREA-specific CD8+ T cells and characterized TCR αβ sequences with potential anti-tumor functionality. Our results highlight a rapid and directed platform for the development of MHC-I-restricted transgenic CD8+ T cells targeting lineage-specific proteins expressed in prostate epithelia for adoptive therapy of advanced PCa.
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Carbone, David P., I. Frank Ciernik, Michael J. Kelley, M. Charles Smith, Sorena Nadaf, Denise Kavanaugh, V. Ellen Maher, et al. "Immunization With Mutant p53- and K-ras–Derived Peptides in Cancer Patients: Immune Response and Clinical Outcome." Journal of Clinical Oncology 23, no. 22 (August 1, 2005): 5099–107. http://dx.doi.org/10.1200/jco.2005.03.158.
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Purpose To determine the ability to induce tumor-specific immunity with individual mutant K-ras–or p53-derived peptides and to monitor clinical outcome. Patients and Methods Patients in varying stages of disease underwent genetic analysis for mutations in K-ras and p53. Thirty-nine patients were enrolled. Seventeen-mer peptides were custom synthesized to the corresponding mutation. Baseline immunity was assessed for cytotoxic T-lymphocyte (CTL) response and interferon gamma (IFN-γ) release from mutant peptide-primed lymphocytes. Patients' peripheral-blood mononuclear cells were pulsed with the corresponding peptide, irradiated, and applied intravenously. Patients were observed for CTL, IFN-γ, interleukin (IL) -2, IL-5, and granulocyte-macrophage colony-stimulating factor responses, for treatment-related toxicity, and for tumor response. Results No toxicity was observed. Ten (26%) of 38 patients had detectable CTL against mutant p53 or K-ras, and two patients were positive for CTL at baseline. Positive IFN-γ responses occurred in 16 patients (42%) after vaccination, whereas four patients had positive IFN-γ reaction before vaccination. Of 29 patients with evident disease, five experienced a period of stable disease. Favorable prognostic markers were detectable CTL activity and a positive IFN-γ reaction but not IL-5 release. Median survival times of 393 v 98 days for a positive versus negative CTL response (P = .04), respectively, and of 470 v 88 days for a positive versus negative IFN-γ response (P = .02), respectively, were detected. Conclusion Custom-made peptide vaccination is feasible without any toxicity. CTL and cytokine responses specific to a given mutation can be induced or enhanced with peptide vaccines. Cellular immunity to mutant p53 and K-ras oncopeptides is associated with longer survival.
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Sabatino, Denise E., Federico Mingozzi, Haifeng Chen, Peter Colosi, Hildegund C. J. Ertl, and Katherine A. High. "Identification of the AAV2 Capsid CD8+ T Cell Epitope in C57BL/6 Mice." Blood 104, no. 11 (November 16, 2004): 3188. http://dx.doi.org/10.1182/blood.v104.11.3188.3188.
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Abstract Recently, a clinical trial for adeno-associated virus serotype 2 (AAV2) mediated liver directed gene transfer of human Factor IX to subjects with severe hemophilia B revealed that two patients developed transient asymptomatic transaminitis following vector administration. Immunology studies in the second patient demonstrated a transient T cell response to AAV2 capsid peptides suggesting that the immune response to the AAV capsid may be related to the transient transaminitis. We hypothesized that the observations made in the human subjects were due to a CD8 T cell response to AAV2 capsid protein. Preclinical studies in mice and dogs, which are not naturally infected by wild type AAV2 viruses, did not predict these findings in the clinical study. Thus, we developed a mouse model in which we were able to mimic this phenomenon (Blood 102:493a). In an effort to further characterize the immune responses to AAV2 capsid proteins in this mouse model, we identified the T cell epitope in the AAV capsid protein recognized by murine C57Bl/6 CD8 T cells. A peptide library of AAV2 VP1 capsid peptides (n=145) that were synthesized as 15mers overlapping by 10 amino acids were divided into 6 pools each containing 24–25 peptides. C57Bl/6 mice were immunized intramuscularly with an adenovirus expressing AAV2 capsid protein. Nine days later the spleen was harvested and intracellular cytokine staining (ICS) was used to assess release of IFN-γ from CD8 T cells in response to 6 AAV2 capsid peptide pools. ICS demonstrated CD8 cells from mice immunized with Ad-AAV2 produced IFN-γ (3.5% of the CD8 cells) in response to Pool F (amino acid 119–145) while no IFN-γ release in CD8 cells was detected with Pool A to E (mean 0.28%±0.25%) compared to the media control (0.16%). This detection of IFN-γ release from CD8 T cells indicates a specific proliferation to a peptide(s) within this peptide pool (Pool F). A matrix approach was used to further define which peptide(s) contained the immunodominant epitope. Eleven small peptide pools of Pool F were created in which each peptide was represented in 2 pools. ICS of splenocytes from immunized (Ad-AAV2 capsid) C57Bl/6 mice demonstrated IFN-γ response from CD8 cells to 3 of the matrix pools corresponding to peptide 140 (PEIQYTSNYNKSVNV) and 141 (TSNYNKSVNVDFTVD) compared with media controls. To determine the exact peptide sequence that binds to the MHC Class I molecule, 9 amino acid peptides (n=7) were created that overlap peptide 140 and 141. Peptide SNYNKSVNV showed positive staining for both CD8 and IFN- γ(3.2%) compared with the six other peptides (0.14%±0.08%), media control (0.08%) and mice that were not immunized (0.11%). This epitope lies in the C terminus of the AAV2 VP1 capsid protein. Current studies using strains of mice with different MHC H2 haplotypes will allow us to determine which of the C57Bl/6 MHC alleles the epitope binds. These findings will provide us with a powerful tool for assessing immune responses to AAV capsid in the context of gene therapy. Specifically, they will allow us to determine how long immunologically detectable capsid sequences persist in an animal injected with AAV vectors. This in turn will provide a basis for a clinical study in which subjects are transiently immunosuppressed, from the time of vector injection until capsid epitopes are no longer detectable by the immune system.
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Amorín, M., V. Villaverde, L. Castedo, and J. R. Granja. "New α,γ-peptide tubulets." Journal of Drug Delivery Science and Technology 15, no. 1 (2005): 87–92. http://dx.doi.org/10.1016/s1773-2247(05)50011-x.
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Yang, Fang-Fang, Zhi-Quan Tu, Yi-Min Fang, Yan Li, Yi Peng, Tao Dong, Cong Wang, et al. "Monitoring of Peptide-Specific and Gamma Interferon-Productive T Cells in Patients with Active and Convalescent Tuberculosis Using an Enzyme-Linked Immunosorbent Spot Assay." Clinical and Vaccine Immunology 19, no. 3 (January 11, 2012): 401–10. http://dx.doi.org/10.1128/cvi.05544-11.
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ABSTRACTTo establish a high-efficiency gamma interferon-specific enzyme-linked immunosorbent spot assay (IFN-γ ELISPOT assay) for detection of tuberculosis (TB), peptides (E6, E7, and C14) and peptide mixtures (E6 plus E7 and E6 plus E7 plus C14) were used to monitor peripheral blood (PBL) samples from patients with pulmonary TB (PTB), as well as control samples. The positive detection rates of the five IFN-γ ELISPOT assays were 78.38%, 74.86%, 55.83%, 90.43%, and 91.51%, respectively, and there were similar detection rates between the two combined peptide mixture IFN-γ ELISPOT assays and the tuberculin skin test (TST) (90.62% versus 95.59%). No significant difference was found between the detection rates of the two combined peptide mixture IFN-γ ELISPOT assays and the T-SPOT.TB assay for 86 patients with PTB (P> 0.05), but the median number of spot-forming cells/106cells (SFP value) for positive results was higher by the former than by the latter assay (P< 0.05). In contrast, the 29.93% positive detection rate and median SFP value of 482 by the two combined peptide mixture IFN-γ ELISPOT assays were significantly higher than the corresponding values of 14.29% and 152 by T-SPOT.TB assay for the same 147 community donors (P< 0.05). For nine PTB patients tracked, the SFP value of 7 for the two peptide mixture IFN-γ ELISPOT assays began to decrease from the second month after regular treatment. A relatively low, almost normal, SFP level was reached and maintained after the third or fourth month. Two in-house IFN-γ ELISPOT assays and the T-SPOT.TB assay could reduce the false-positive and false-negative detection rates of TST and sputum acid-fast staining. Therefore, these two combined peptide mixture IFN-γ ELISPOT assays have a potential advantage, beyond their greater specificity and sensitivity, for use in screening and detection of active TB infection (TBI) and latent TB infection (LTBI) in China.
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Ahmed, Chulbul M. I., Marjorie A. Burkhart, Prem S. Subramaniam, Mustafa G. Mujtaba, and Howard M. Johnson. "Peptide Mimetics of Gamma Interferon Possess Antiviral Properties against Vaccinia Virus and Other Viruses in the Presence of Poxvirus B8R Protein." Journal of Virology 79, no. 9 (May 1, 2005): 5632–39. http://dx.doi.org/10.1128/jvi.79.9.5632-5639.2005.
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ABSTRACT We have developed peptide mimetics of gamma interferon (IFN-γ) that play a direct role in the activation and nuclear translocation of STAT1α transcription factor. These mimetics do not act through recognition by the extracellular domain of IFN-γ receptor but rather bind to the cytoplasmic domain of the receptor chain 1, IFNGR-1, and thereby initiate the cellular signaling. Thus, we hypothesized that these mimetics would bypass the poxvirus virulence factor B8R protein that binds to intact IFN-γ and prevents its interaction with the receptor. Human and murine IFN-γ mimetic peptides were introduced into an adenoviral vector for intracellular expression. Murine IFN-γ mimetic peptide was also expressed via chemical synthesis with an attached lipophilic group for penetration of cell plasma membrane. In contrast to intact human IFN-γ, the mimetics did not bind poxvirus B8R protein, a homolog of the IFN-γ receptor extracellular domain. Expression of B8R protein in WISH cells did not block the antiviral effect of the mimetics against encephalomyocarditis or vesicular stomatitis virus, while the antiviral activity of human IFN-γ was neutralized. Consistent with the antiviral activity, the upregulation of MHC class I molecules on WISH cells by the IFN-γ mimetics was not affected by B8R protein, while IFN-γ-induced upregulation was blocked. Finally, the mimetics, but not IFN-γ, inhibited vaccinia virus replication in African green monkey kidney BSC-40 cells. The data presented demonstrate that small peptide mimetics of IFN-γ can avoid the B8R virulence factor for poxviruses and, thus, are potential candidates for antivirals against smallpox virus.
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Illa, Ona, José-Antonio Olivares, Nerea Gaztelumendi, Laura Martínez-Castro, Jimena Ospina, María-Ángeles Abengozar, Giuseppe Sciortino, et al. "Chiral Cyclobutane-Containing Cell-Penetrating Peptides as Selective Vectors for Anti-Leishmania Drug Delivery Systems." International Journal of Molecular Sciences 21, no. 20 (October 12, 2020): 7502. http://dx.doi.org/10.3390/ijms21207502.
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Two series of new hybrid γ/γ-peptides, γ-CC and γ-CT, formed by (1S,2R)-3-amino-2,2,dimethylcyclobutane-1-carboxylic acid joined in alternation to a Nα-functionalized cis- or trans-γ-amino-l-proline derivative, respectively, have been synthesized and evaluated as cell penetrating peptides (CPP) and as selective vectors for anti-Leishmania drug delivery systems (DDS). They lacked cytotoxicity on the tumoral human cell line HeLa with a moderate cell-uptake on these cells. In contrast, both γ-CC and γ-CT tetradecamers were microbicidal on the protozoan parasite Leishmania beyond 25 μM, with significant intracellular accumulation. They were conjugated to fluorescent doxorubicin (Dox) as a standard drug showing toxicity beyond 1 μM, while free Dox was not toxic. Intracellular accumulation was 2.5 higher than with Dox-TAT conjugate (TAT = transactivator of transcription, taken as a standard CPP). The conformational structure of the conjugates was approached both by circular dichroism spectroscopy and molecular dynamics simulations. Altogether, computational calculations predict that the drug-γ-peptide conjugates adopt conformations that bury the Dox moiety into a cavity of the folded peptide, while the positively charged guanidinium groups face the solvent. The favorable charge/hydrophobicity balance in these CPP improves the solubility of Dox in aqueous media, as well as translocation across cell membranes, making them promising candidates for DDS.
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Chaitra, M. G., M. S. Shaila, and R. Nayak. "Evaluation of T-cell responses to peptides with MHC class I-binding motifs derived from PE_PGRS 33 protein of Mycobacterium tuberculosis." Journal of Medical Microbiology 56, no. 4 (April 1, 2007): 466–74. http://dx.doi.org/10.1099/jmm.0.46928-0.
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The PE and PPE proteins of Mycobacterium tuberculosis form a source of antigenic variation among different strains of M. tuberculosis. One of the PE_PGRS proteins, Rv1818c, plays a role in the pathogenesis of mycobacterial infection and specifically influences host-cell responses to tuberculosis infection. Although little is known about these two classes of protein, an immunoinformatics approach has indicated the possibility of their participation in eliciting a major histocompatibility complex (MHC) class I-mediated immune response against tuberculosis, as peptides derived from Rv1818c are predicted to bind to MHC class I molecules with high affinity. In the present work, a DNA vaccine was constructed encoding the full-length Rv1818c protein of M. tuberculosis and its immunogenicity was analysed in BALB/c mice. Immunization with Rv1818c DNA induced a strong CD8+ cytotoxic lymphocyte and Th1-type response, with high levels of gamma interferon (IFN-γ) and low levels of interleukin-4. Two nonameric peptides (Peptide6–14 and Peptide385–393) from Rv1818c were identified by their ability to induce the production of IFN-γ by CD8+ T cells in mice immunized with Rv1818c DNA. An epitope-specific response was demonstrated by the lysis of peptide-pulsed antigen-presenting cells, release of cytotoxic granules and IFN-γ production. These peptides bound with high affinity to MHC H-2Kd and showed low dissociation rates of peptide–MHC complexes. These results could form the basis for testing the identified T-cell epitopes of PE_PGRS proteins in the induction of protective immunity against M. tuberculosis challenge in the mouse model.
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Mustafa, Ghulam, Hafiza Salaha Mahrosh, Mahwish Salman, Sumaira Sharif, Raheela Jabeen, Tanveer Majeed, and Hafsah Tahir. "Identification of Peptides as Novel Inhibitors to Target IFN-γ, IL-3, and TNF-α in Systemic Lupus Erythematosus." BioMed Research International 2021 (November 13, 2021): 1–11. http://dx.doi.org/10.1155/2021/1124055.
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Autoimmune disorder is a chronic immune imbalance which is developed through a series of pathways. The defect in B cells, T cells, and lack of self-tolerance has been greatly associated with the onset of many types of autoimmune complications including rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis, and chronic inflammatory demyelinating polyneuropathy. The SLE is an autoimmune disease with a common type of lupus that causes tissue and organ damage due to the wide spread of inflammation. In the current study, twenty anti-inflammatory peptides derived from plant and animal sources were docked as ligands or peptides counter to proinflammatory cytokines. Interferon gamma (IFN-γ), interleukin 3 (IL-3), and tumor necrosis factor alpha (TNF-α) were targeted in this study as these are involved in the pathogenesis of SLE in many clinical studies. Two docking approaches (i.e., protein-ligand docking and peptide-protein docking) were employed in this study using Molecular Operating Environment (MOE) software and HADDOCK web server, respectively. Amongst docked twenty peptides, the peptide DEDTQAMMPFR with S -score of -11.3018 and HADDOCK score of − 10.3 ± 2.5 kcal/mol showed the best binding interactions and energy validation with active amino acids of IFN-γ protein in both docking approaches. Depending upon these results, this peptide could be used as a potential drug candidate to target IFN-γ, IL-3, and TNF-α proteins to control inflammatory events. Other peptides (i.e., QEPQESQQ and FRDEHKK) also revealed good binding affinity with IFN-γ with S -scores of -10.98 and -10.55, respectively. Similarly, the peptides KHDRGDEF, FRDEHKK, and QEPQESQQ showed best binding interactions with IL-3 with S -scores of -8.81, -8.64, and -8.17, respectively.
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Mittendorf, E. A., S. Khoo, C. E. Storrer, K. A. Harris, Y. H. Jama, Z. A. Dehqanzada, J. L. Murray, et al. "Early results of a phase I clinical trial of an Ii-Key/Her2/neu MHC class II peptide-based vaccine in breast cancer patients." Journal of Clinical Oncology 24, no. 18_suppl (June 20, 2006): 2532. http://dx.doi.org/10.1200/jco.2006.24.18_suppl.2532.
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2532 Background: HER2/neu is over-expressed in breast cancer (BCa) and is the source of immunogenic peptides currently being evaluated in clinical trials as cancer vaccines. Ii-Key is a 4-amino-acid modification of peptides that has been reported to increase their occupancy of MHC class II molecules and enhance CD4 T cell responses. We are currently conducting a phase I clinical trial with an Ii-Key/HER2/neu MHC class II peptide (AE37) in BCa patients. Methods: The dose escalation safety trial design is for 3 groups of 3 patients to receive either 100, 500, or 1,000 μg of AE37 with 250 μg of GM-CSF in 6 monthly inoculations. Six additional patients will then be vaccinated at the optimal dose. Local and systemic toxicity is monitored and graded by the Common Toxicity Criteria. Immunologic response is monitored by peptide-stimulated proliferation (3H-thymidine incorporation) and ELISPOT (IFN-γ) assays for both AE37 and AE36 [the unmodified peptide HER2 776–790 (GVGSPYVSRLLGICL)]. Thus far 6 BCa patients have been vaccinated. Results: The 100μg dose group has completed all 6 vaccinations with maximum local and systemic toxicities of grade 2 and 1, respectively. All 3 patients developed increasing peptide-specific proliferation post-vaccination to both AE37 and AE36. ELISPOT responses were more variable with 1 minimal responder (30 IFN-γ spot-forming cells (SFC)/106 cells), 1 moderate responder (600 IFN-γ SFC/106 cells), and 1 good responder (1500 IFN-γ SFC/106 cells) post-vaccination. The 500μg dose group has thus far experienced earlier and larger local reactions but limited systemic toxicity. All 3 patients have demonstrated >1500 IFN-γ SFC /106 cells after just 2 inoculations. Peptide-specific proliferation after 2 inoculations has ranged between 3200–11500 cpm for the 500μg patients compared with 1200–2000 cpm after 6 inoculations for the 100μg dose group. Conclusions: AE37 appears to be safe and well-tolerated. A dose-dependent immunologic response has been demonstrated to the Ii-Key modified peptide as well as the naturally occurring sequence. These encouraging early results suggest that AE37 may prove to be useful as a CD4-specific vaccine either alone or in combination with CD8-specific HER2/neu peptides for improved cancer immunotherapy. [Table: see text]
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Staska, Lauren M., Christopher J. Davies, Wendy C. Brown, Travis C. McGuire, Carlos E. Suarez, Joo Youn Park, Bruce A. Mathison, Jeffrey R. Abbott, and Timothy V. Baszler. "Identification of Vaccine Candidate Peptides in the NcSRS2 Surface Protein of Neospora caninum by Using CD4+ Cytotoxic T Lymphocytes and Gamma Interferon-Secreting T Lymphocytes of Infected Holstein Cattle." Infection and Immunity 73, no. 3 (March 2005): 1321–29. http://dx.doi.org/10.1128/iai.73.3.1321-1329.2005.
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ABSTRACT Previously, our laboratory showed that Holstein cattle experimentally infected with Neospora caninum develop parasite-specific CD4+ cytotoxic T lymphocytes (CTL) that lyse infected, autologous target cells through a perforin-granzyme pathway. To identify specific parasite antigens inducing bovine CTL and helper T-lymphocyte responses for vaccine development against bovine neosporosis, the tachyzoite major surface proteins NcSAG1 and NcSRS2 were targeted. In whole tachyzoite antigen-expanded bovine T-lymphocyte lines, recombinant NcSRS2 induced potent memory CD4+- and CD8+-T-lymphocyte activation, as indicated by proliferation and gamma interferon (IFN-γ) secretion, while recombinant NcSAG1 induced a minimal memory response. Subsequently, T-lymphocyte epitope-bearing peptides of NcSRS2 were mapped by using overlapping peptides covering the entire NcSRS2 sequence. Four experimentally infected cattle with six different major histocompatibility complex (MHC) class II haplotypes were the source of immune cells used to identify NcSRS2 peptides presented by Holstein MHC haplotypes. NcSRS2 peptides were mapped by using IFN-γ secretion by rNcSRS2-stimulated, short-term T-lymphocyte cell lines, IFN-γ enzyme-linked immunospot (ELISPOT) assay with peripheral blood mononuclear cells, and 51Cr release cytotoxicity assay of rNcSRS2-stimulated effector cells. Four N. caninum-infected Holstein cattle developed NcSRS2 peptide-specific T lymphocytes detected ex vivo in peripheral blood by IFN-γ ELISPOT and in vitro by measuring T-lymphocyte IFN-γ production and cytotoxicity. An immunodominant region of NcSRS2 spanning amino acids 133 to 155 was recognized by CD4+ T lymphocytes from the four cattle. These findings support investigation of subunit N. caninum vaccines incorporating NcSRS2 gene sequences or peptides for induction of NcSRS2 peptide-specific CTL and IFN-γ-secreting T lymphocytes in cattle with varied MHC genotypes.
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Liu, Jie, Tracy J. Ruckwardt, Man Chen, Teresa R. Johnson, and Barney S. Graham. "Characterization of Respiratory Syncytial Virus M- and M2-Specific CD4 T Cells in a Murine Model." Journal of Virology 83, no. 10 (March 4, 2009): 4934–41. http://dx.doi.org/10.1128/jvi.02140-08.
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ABSTRACT CD4 T cells have been shown to play an important role in the immunity and immunopathogenesis of respiratory syncytial virus (RSV) infection. We identified two novel CD4 T-cell epitopes in the RSV M and M2 proteins with core sequences M213-223 (FKYIKPQSQFI) and M227-37 (YFEWPPHALLV). Peptides containing the epitopes stimulated RSV-specific CD4 T cells to produce gamma interferon (IFN-γ), interleukin 2 (IL-2), and other Th1- and Th2-type cytokines in an I-Ab-restricted pattern. Construction of fluorochrome-conjugated peptide-I-Ab class II tetramers revealed RSV M- and M2-specific CD4 T-cell responses in RSV-infected mice in a hierarchical pattern. Peptide-activated CD4 T cells from lungs were more activated and differentiated, and had greater IFN-γ expression, than CD4 T cells from the spleen, which, in contrast, produced greater levels of IL-2. In addition, M209-223 peptide-activated CD4 T cells reduced IFN-γ and IL-2 production in M- and M2-specific CD8 T-cell responses to Db-M187-195 and Kd-M282-90 peptides more than M225-39 peptide-stimulated CD4 T cells. This correlated with the fact that I-Ab-M209-223 tetramer-positive cells responding to primary RSV infection had a much higher frequency of FoxP3 expression than I-Ab-M226-39 tetramer-positive CD4 T cells, suggesting that the M-specific CD4 T-cell response has greater regulatory function. Characterization of epitope-specific CD4 T cells by novel fluorochrome-conjugated peptide-I-Ab tetramers allows detailed analysis of their roles in RSV pathogenesis and immunity.
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Roussel-Jazédé, Virginie, Jesús Arenas, Jeroen D. Langereis, Jan Tommassen, and Peter van Ulsen. "Variable processing of the IgA protease autotransporter at the cell surface of Neisseria meningitidis." Microbiology 160, no. 11 (November 1, 2014): 2421–31. http://dx.doi.org/10.1099/mic.0.082511-0.
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As with all classical monomeric autotransporters, IgA protease of Neisseria meningitidis is a modular protein consisting of an N-terminal signal sequence, a passenger domain and a C-terminal translocator domain (TD) that assists in the secretion of the passenger domain across the outer membrane. The passenger of IgA protease consists of three separate domains: the protease domain, the γ-peptide and the α-peptide that contains nuclear localization signals (NLSs). The protease domain is released into the extracellular milieu either via autocatalytic processing or via cleavage by another autotransporter, NalP, expression of which is phase-variable. NalP-mediated cleavage results in the release of a passenger that includes the α- and γ-peptides. Here, we studied the fate of the α-peptide when NalP was not expressed and observed strain-dependent differences. In meningococcal strains where the α-peptide contained a single NLS, the α-peptide remained covalently attached to the TD and was detected at the cell surface. In other strains, the α-peptide contained four NLSs and was separated from the TD by an IgA protease autoproteolytic cleavage site. In many of those cases, the α-peptide was found non-covalently associated with the cells as a separate polypeptide. The cell surface association of the α-peptides may be relevant physiologically. We report a novel function for the α-peptide, i.e. the binding of heparin – an immune-modulatory molecule that in the host is found in the extracellular matrix and connected to cell surfaces.
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Kalantar, K., Z. Farzaneh, M. Eshkevar Vakili, M. H. Karimi, M. Asadi, S. Khosropanah, and M. Doroudchi. "T cell responses to an HLA-A2-restricted adipophilin peptide correlate with BMI in patients with atherosclerosis." Physiology International 107, no. 2 (June 2020): 280–93. http://dx.doi.org/10.1556/2060.2020.00023.
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AbstractIntroductionAtherosclerosis is an inflammatory disease causing a vast array of cardiovascular diseases. Adipophilin has been reported to be highly expressed in atherosclerotic lesions. This study investigated the possible existence of auto-reactive T cells against an HLA-A02-restricted adipophilin-derived peptide as well as peptides from Epstein-barr virus (EBV), Cytomegalovirus (CMV) and influenza (Flu) virus in patients with atherosclerosis.MethodsHLA-A02 expression on peripheral blood mononuclear cells (PBMCs) was examined by flow cytometry. PBMCs from HLA-A02 individuals were stimulated with adipophilin, CMV, EBV, and Flu peptides at a concentration of 10 µM. Interferon (IFN)-γ production was evaluated in the culture supernatant using a commercial ELISA test.ResultsThe levels of IFN-γ production against an HLA-A02-restricted adipophilin peptide and peptides from CMV, EBV, and Flu revealed no statistically significant differences between patients and healthy controls. However, we found a positive correlation between IFN-γ production against adipophilin and Body mass index (BMI) of patients (R = 0.8, P = 0.003), whereas no significant correlation was found in healthy controls (R = −0.267, P = 0.378). No correlation between BMI and IFN-γ production against CMV, EBV, or Flu peptides was found.DiscussionAtherosclerotic patients with higher BMIs might have greater numbers of T cells against adipophilin that is highly expressed in atherosclerotic plaques. Therefore, autoimmune reactions may have a greater role in the development of atherosclerosis in individuals with higher BMI.
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Humphreys, Michael H. "γ-MSH, sodium metabolism, and salt-sensitive hypertension." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 286, no. 3 (March 2004): R417—R430. http://dx.doi.org/10.1152/ajpregu.00365.2003.
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α-, β-, and γ-melanocyte stimulating hormones (MSHs) are melanotropin peptides that are derived from the ACTH/β-endorphin prohormone proopiomelanocortin (POMC). They have been highly conserved through evolutionary development, although their functions in mammals have remained obscure. The identification in the last decade of a family of five membrane-spanning melanocortin receptors (MC-Rs), for which the melanotropins are the natural ligands, has permitted the characterization of a number of important actions of these peptides, although the physiological function(s) of γ-MSH have remained elusive. Much evidence indicates that γ-MSH stimulates sympathetic outflow and raises blood pressure through a central mechanism. However, this review focuses on newer cardiovascular and renal actions of the peptide, acting in most cases through the MC3-R. In rodents, a high-sodium diet (HSD) increases the pituitary abundance of POMC mRNA and of γ-MSH content and results in a doubling of plasma γ-MSH concentration. The peptide is natriuretic and acts through renal MC3-Rs, which are also upregulated by the HSD. Thus the system appears designed to participate in the integrated response to dietary sodium excess. Genetic or pharmacologic induction of γ-MSH deficiency results in marked salt-sensitive hypertension that is corrected by the administration of the peptide, probably through a central site of action. Deletion of the MC3-R also produces salt-sensitive hypertension, which, however, is not corrected by infusion of the hormone. These observations in aggregate suggest the operation of a hormonal system important in blood pressure control and in the regulation of sodium excretion. The relationship of these two actions to each other and the significance of this system in humans are important questions for future research.
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Mohri, Hiroshi, and Takao Ohkubo. "Effects of Hybrid Peptide Analogs to Receptor Recognition Domains on α- and γ-Chains of Human Fibrinogen on Fibrinogen Binding to Platelets." Thrombosis and Haemostasis 69, no. 05 (1993): 490–95. http://dx.doi.org/10.1055/s-0038-1651639.
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SummaryWe synthesized a series of hybrid peptides that correspond to the γ-chain dodecapeptide (400-411), variable numbers of glycine residues, and the RGDS peptide [Y-HHLGGAK-QAGDV(G) n RGDS] to investigate the relationship of these receptor recognition domains of fibrinogen to platelet membrane glycoprotein IIb/IIIa. The tetrapeptide RGDS, the GRGDSPA peptide and the dodecapeptide inhibited binding of fibrinogen to GPIIb/IIIa by 50% (IC50) at concentrations of 17 ± 1.6 μM, 15 ± 2.1 μM, and 87 ± 6.8 μM, respectively. The inhibitory effect of hybrid peptides increased as the number of glycine residues increased, plateauing with 9-11 glycine residues in hybrid peptide analogs, which had an IC50 of 0.68 ± 0.14 μM. These hybrid peptides completely inhibited the binding of fibrinogen to activated platelets when used in sufficient concentrations. The peptide Y-HHLGGAKQAGDV(G)9RGDS blocked ADP-induced aggregation in citrated platelet-rich plasma with IC50 of 3.5 ± 0.6 μM. When the peptide Y-HHLGGAK-QAGDV(G)9RGDS was labeled with 125I to quantify its binding to platelets, maximal binding was observed within 30 min. The binding sites of the hybrid peptide were 43,600 molecules/platelet (K d = 3.1 ± 0.5 × 10-7 M) to stimulated platelets and 12,500 molecules/platelet (K d = 1.4 ± 0.2 × 10-7 M) to nonstimulated platelets. The hybrid peptides had the same binding affinity to platelets as fibrinogen and inhibited platelet function. Moreover, anti-GPIIb/IIIa antibody inhibited the binding of the labeled hybrid peptide to stimulated platelets. These results indicate that in the native fibrinogen molecule the presence of both RGD sequence or γ-chain domain at optimal distances increased the binding affinity to GPIIb/IIIa. These domains may be the source of hybrid peptide, expanding a new class of platelet inhibitors that act at membrane receptors for adhesive proteins.
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Shankar, Sudha, Junaid Ur Rahim, and Rajkishor Rai. "Self-Assembly in Peptides Containing β-and γ-amino Acids." Current Protein & Peptide Science 21, no. 6 (August 21, 2020): 584–97. http://dx.doi.org/10.2174/1389203721666200127112244.
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The peptides containing β-and γ-amino acids as building blocks display well-defined secondary structures with unique morphologies. The ability of such peptides to self-assemble into complex structures of controlled geometries has been exploited in biomedical applications. Herein, we have provided an updated overview about the peptides containing β-and γ-amino acids considering the significance and advancement in the area of development of peptide-based biomaterials having diverse applications.
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John, Chandy C., Ann M. Moormann, Peter O. Sumba, Ayub V. Ofulla, Daniel C. Pregibon, and James W. Kazura. "Gamma Interferon Responses to Plasmodium falciparum Liver-Stage Antigen 1 and Thrombospondin-Related Adhesive Protein and Their Relationship to Age, Transmission Intensity, and Protection against Malaria." Infection and Immunity 72, no. 9 (September 2004): 5135–42. http://dx.doi.org/10.1128/iai.72.9.5135-5142.2004.
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ABSTRACT Gamma interferon (IFN-γ) responses to the Plasmodium falciparum antigens liver-stage antigen 1 (LSA-1) and thrombospondin-related adhesive protein (TRAP) are thought to be important in protection against malaria. Optimal methods of testing and the effects of age and transmission intensity on these responses are unknown. IFN-γ responses to LSA-1 and TRAP peptides were assessed by the enzyme-linked immunospot assay (ELISPOT) and enzyme-linked immunosorbent assay (ELISA) in children and adults from areas of stable and unstable malaria transmission in Kenya. Adults in the areas of stable and unstable transmission had similar frequencies and levels of IFN-γ responses to LSA-1 and TRAP as determined by ELISPOT and ELISA. In contrast, IFN-γ responses to the LSA-1 T3 peptide (assessed by ELISPOT) and to any LSA-1 peptide (assessed by ELISA) were less frequent in children in the area of unstable transmission than in children in the area of stable transmission. IFN-γ responses to LSA-1 were more frequently detected by ELISA than by ELISPOT in the stable-transmission area. IFN-γ responses detected by ELISA and ELISPOT did not correlate with each other. In children in the stable-transmission area, IFN-γ responses to LSA-1 peptides assessed by ELISA, but not by ELISPOT, were associated with protection against clinical malaria and anemia. IFN-γ responses to LSA-1 appear to require repeated P. falciparum exposure and/or increased age and, as measured by ELISA, are associated with protection against clinical malaria and anemia.
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Yang, Juan, Weidong Bai, Xiaofang Zeng, and Chun Cui. "γ-[Glu](n=1,2)-Phe/-Met/-Val stimulates gastrointestinal hormone (CCK and GLP-1) secretion by activating the calcium-sensing receptor." Food & Function 10, no. 7 (2019): 4071–80. http://dx.doi.org/10.1039/c9fo00313d.
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This study was conducted to discover the effectiveness of dietary peptides (γ-[Glu](n=1,2)-Phe/-Met/-Val) as stimulators of cholecystokinin (CCK) and glucagon-like peptide 1 (GLP-1) secretion.
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Lovely, Rehana S., Chantelle M. Rein, Tara C. White, Sari A. Jouihan, Lynn K. Boshkov, Antony C. Bakke, Owen J. McCarty, and David H. Farrell. "γA/γ’ fibrinogen inhibits thrombin-induced platelet aggregation." Thrombosis and Haemostasis 100, no. 05 (2008): 837–46. http://dx.doi.org/10.1160/th08-03-0145.
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SummaryThe minor γA/γ’ fibrinogen isoform contains a high affinity binding site for thrombin exosite II that is lacking in the major γA/γA fibrinogen isoform. We therefore investigated the biological consequences of the γ’ chain binding to thrombin. Thrombin-induced platelet aggregation was inhibited by γA/γ’ fibrinogen.Carboxyl terminal peptide fragment γ’410–427 from the γ’ chain was also inhibitory, with an IC50 of ∼200 µM in whole plasma. Deletion of the peptide from either the amino or carboxyl end significantly decreased inhibition. In contrast to thrombin-induced platelet aggregation, aggregation induced by epinephrine, ADP, arachidonic acid, or SFLLRN peptide showed little inhibition by the γ’ peptide. The inhibition of thrombin-induced platelet aggregation was not due to direct inhibition of the thrombin active site, since cleavage of a small peptidyl substrate was 91% of normal even in the presence of 1 mM γ’410–427.The γ’410–427 peptide blocked platelet adhesion to immobilized thrombin under both static and flow conditions,blocked soluble thrombin binding to platelet GPIbα, and inhibited PAR1 cleavage by thrombin. These results suggest that the γ’ chain of fibrinogen inhibits thrombin-induced platelet aggregation by binding to thrombin exosite II. Thrombin that is bound to the γ’ chain is thereby prevented from activating platelets, while retaining its amidolytic activity.
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Ovsyannikova, Inna G., Kenneth L. Johnson, David C. Muddiman, Robert A. Vierkant, and Gregory A. Poland. "Identification and Characterization of Novel, Naturally Processed Measles Virus Class II HLA-DRB1 Peptides." Journal of Virology 78, no. 1 (January 1, 2004): 42–51. http://dx.doi.org/10.1128/jvi.78.1.42-51.2004.
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ABSTRACT Previously, we identified a naturally processed and presented measles virus (MV) 19-amino-acid peptide, ASDVETAEGGEIHELLRLQ (MV-P), derived from the phosphoprotein and eluted from the human leukocyte antigen (HLA) class II molecule by using mass spectrometry. We report here the identification of a 14-amino-acid peptide, SAGKVSSTLASELG, derived from the MV nucleoprotein (MV-N) bound to HLA-DRB1*0301. Peripheral blood mononuclear cells (PBMC) from 281 previously vaccinated measles-mumps-rubella II (MMR-II) subjects (HLA discordant) were studied for peptide recognition by T cells. Significant gamma interferon (IFN-γ) responses to MV-P and MV-N peptides were observed in 55.9 and 15.3% of subjects, respectively. MV-P- and MV-N-specific interleukin-4 (IL-4) responses were detected in 19.2 and 23.1%, respectively, of PBMC samples. Peptide-specific cytokine responses and HLA-DRB1 allele associations revealed that, for the MV-P peptide, the allele with the strongest association with both IFN-γ (P = 0.02) and IL-4 (P = 0.03) secretion was DRB1*0301. For MV-N, the allele with the strongest association with IFN-γ secretion was DRB1*1501 (P = 0.04), and the alleles with the strongest associations with IL-4 secretion were DRB1*1103 and DRB1*1303 (P = 0.01). These results indicate that HLA class II MV proteins can be processed, presented, and identified, and the ability to generate cell-mediated immune responses can be demonstrated. This information is promising for new vaccine design strategies with peptide-based vaccines.
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Legrand, Baptiste, and Ludovic T. Maillard. "α,β‐Unsaturated γ‐Peptide Foldamers." ChemPlusChem 86, no. 4 (April 2021): 629–45. http://dx.doi.org/10.1002/cplu.202100045.
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Misra, Rajkumar, Gijo George, Abhijith Saseendran, Srinivasarao Raghothama, and Hosahudya N. Gopi. "Ambidextrous α,γ‐Hybrid Peptide Foldamers." Chemistry – An Asian Journal 14, no. 23 (November 28, 2019): 4408–14. http://dx.doi.org/10.1002/asia.201901411.
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Domitrovic, Tatiana, Tsutomu Matsui, and John E. Johnson. "Dissecting Quasi-Equivalence in Nonenveloped Viruses: Membrane Disruption Is Promoted by Lytic Peptides Released from Subunit Pentamers, Not Hexamers." Journal of Virology 86, no. 18 (July 3, 2012): 9976–82. http://dx.doi.org/10.1128/jvi.01089-12.
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Nonenveloped viruses often invade membranes by exposing hydrophobic or amphipathic peptides generated by a proteolytic maturation step that leaves a lytic peptide noncovalently associated with the viral capsid. Since multiple copies of the same protein form many nonenveloped virus capsids, it is unclear if lytic peptides derived from subunits occupying different positions in a quasi-equivalent icosahedral capsid play different roles in host infection. We addressed this question withNudaurelia capensis omega virus(NωV), an insect RNA virus with an icosahedral capsid formed by protein α, which undergoes autocleavage during maturation, producing the lytic γ peptide. NωV is a unique model because autocatalysis can be precisely initiatedin vitroand is sufficiently slow to correlate lytic activity with γ peptide production. Using liposome-based assays, we observed that autocatalysis is essential for the potent membrane disruption caused by NωV. We observed that lytic activity is acquired rapidly during the maturation program, reaching 100% activity with less than 50% of the subunits cleaved. Previous time-resolved structural studies of partially mature NωV particles showed that, during this time frame, γ peptides derived from the pentamer subunits are produced and are organized in a vertical helical bundle that is projected toward the particle surface, while identical polypeptides in quasi-equivalent subunits are produced later or are in positions inappropriate for release. Our functional data provide experimental support for the hypothesis that pentamers containing a central helical bundle, observed in different nonenveloped virus families, are a specialized lytic motif.
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Baker, John R., Chengbao Liu, Shengli Dong, and David G. Pritchard. "Endopeptidase and Glycosidase Activities of the Bacteriophage B30 Lysin." Applied and Environmental Microbiology 72, no. 10 (October 2006): 6825–28. http://dx.doi.org/10.1128/aem.00829-06.
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ABSTRACT Synthetic peptides corresponding to portions of group B streptococcal peptidoglycan were used to show that the endopeptidase activity of bacteriophage B30 lysin cleaves between d-Ala in the stem peptide and l-Ala in the cross bridge and that the minimal peptide sequence cleaved is dl-γ-Glu-Lys-d-Ala-Ala-Ala. The only glycosidase activity present is that of N-acetyl-β-d-muramidase.
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Kariyone, Ai, Masashi Ikutani, Yoshinori Nagai, and Kiyoshi Takatsu. "Molecular mechanisms of Th1-mediated antigen cross-presentation: roles of Iigp1 and ingenol. (130.34)." Journal of Immunology 184, no. 1_Supplement (April 1, 2010): 130.34. http://dx.doi.org/10.4049/jimmunol.184.supp.130.34.
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Abstract Antitumor immunity requires activation of CD8+ cytotoxic T cells (CTL) by antigen-presenting cells (APCs) through interaction with tumor antigen peptides on MHC class I molecules. Intracellular mechanisms on MHC class I-restricted soluble antigen presentation (cross-presentation) remain elusive. Peptide-25 of Ag85B induces Th1 cell response in I-Ab mice that facilitate CTL generation against unrelated Ovalbumin (OVA) peptide when co-immunized with OVA, indicating that Peptide-25 augments cross-presentation by APCs. In this presentation we will discuss about roles of Iigp1 and effects of various natural products on Th1-mediated enhancement of the cross-presentation. We evaluated the cross-presentation by 2-step culture system. At first, APCs were cultured with P25 TCR-Tg CD4+ T cells, Peptide-25, OVA or natural products for overnight. After the culture the OVA-pulsed APCs were harvested and cultured with OVA specific OT-I Tg CD8+ T cells for 3 days. The cross-presentation activity was evaluated by OT-1 proliferation and IFN-γ production. Results revealed an indispensable roles of IFN-γ and IFN-γ-inducible genes such as Iigp1 in the Th1-mediated cross-presentation. We also found that some of natural products such as ingenol derivatives augment the cross-presentation in an Th1-independent manner.
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Grison, Claude, Stéphane Genève, Stéphanie Claudel, Philippe Coutrot, and Michel Marraud. "Catalytic hydrogenation of vinylogous peptides: a route towards γ-peptide foldamers." Tetrahedron Letters 44, no. 11 (March 2003): 2297–300. http://dx.doi.org/10.1016/s0040-4039(03)00272-7.
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Liu, Lei, Li Ding, Matteo Rovere, Michael S. Wolfe, and Dennis J. Selkoe. "A cellular complex of BACE1 and γ-secretase sequentially generates Aβ from its full-length precursor." Journal of Cell Biology 218, no. 2 (January 9, 2019): 644–63. http://dx.doi.org/10.1083/jcb.201806205.
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Intramembrane proteolysis of transmembrane substrates by the presenilin–γ-secretase complex is preceded and regulated by shedding of the substrate’s ectodomain by α- or β-secretase. We asked whether β- and γ-secretases interact to mediate efficient sequential processing of APP, generating the amyloid β (Aβ) peptides that initiate Alzheimer’s disease. We describe a hitherto unrecognized multiprotease complex containing active β- and γ-secretases. BACE1 coimmunoprecipitated and cofractionated with γ-secretase in cultured cells and in mouse and human brain. An endogenous high molecular weight (HMW) complex (∼5 MD) containing β- and γ-secretases and holo-APP was catalytically active in vitro and generated a full array of Aβ peptides, with physiological Aβ42/40 ratios. The isolated complex responded properly to γ-secretase modulators. Alzheimer’s-causing mutations in presenilin altered the Aβ42/40 peptide ratio generated by the HMW β/γ-secretase complex indistinguishably from that observed in whole cells. Thus, Aβ is generated from holo-APP by a BACE1–γ-secretase complex that provides sequential, efficient RIP processing of full-length substrates to final products.
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Bandyopadhyay, Anupam, and Hosahudya N. Gopi. "Hybrid Peptides: Direct Transformation of α/α, β-Unsaturated γ-Hybrid Peptides to α/γ-Hybrid Peptide 12-Helices." Organic Letters 14, no. 11 (May 18, 2012): 2770–73. http://dx.doi.org/10.1021/ol300987d.
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Shim, Wan Sub, Soo Kyung Kim, Hae Jin Kim, Eun Seok Kang, Chul Woo Ahn, Sung Kil Lim, Hyun Chul Lee, and Bong Soo Cha. "Decrement of postprandial insulin secretion determines the progressive nature of type-2 diabetes." European Journal of Endocrinology 155, no. 4 (October 2006): 615–22. http://dx.doi.org/10.1530/eje.1.02249.
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Objective: Type-2 diabetes is a progressive disease. However, little is known about whether decreased fasting or postprandial pancreatic β-cell responsiveness is more prominent with increased duration of diabetes. The aim of this study was to evaluate the relationship between insulin secretion both during fasting and 2 h postprandial, and the duration of diabetes in type-2 diabetic patients. Design: Cross-sectional clinical investigation. Methods: We conducted a meal tolerance test in 1466 type-2 diabetic patients and calculated fasting (M0) and postprandial (M1) β-cell responsiveness. Results: The fasting C-peptide, postprandial C-peptide, M0, and M1 values were lower, but HbA1c values were higher, in patients with diabetes duration > 10 years than those in other groups. There was no difference in the HbA1c levels according to the tertiles of their fasting C-peptide level. However, in a group of patients with highest postprandial C-peptide tertile, the HbA1c values were significantly lower than those in other groups. After adjustment of age, sex, and body mass index (BMI), the duration of diabetes was found to be negatively correlated with fasting C-peptide (γ = −0.102), postprandial C-peptide (γ = −0.356), M0 (γ = −0.263), and M1 (γ = −0.315; P < 0.01 respectively). After adjustment of age, sex, and BMI, HbA1c was found to be negatively correlated with postprandial C-peptide (γ = −0.264), M0 (γ = −0.379), and M1 (γ = −0.522), however, positively correlated with fasting C-peptide (γ = 0.105; P < 0.01 respectively). In stepwise multiple regression analysis, M0, M1, and homeostasis model assessment for insulin resistance (HOMA-IR) emerged as predictors of HbAlc after adjustment for age, sex, and BMI (R2 = 0.272, 0.080, and 0.056 respectively). Conclusions: With increasing duration of diabetes, the decrease of postprandial insulin secretion is becoming more prominent, and postprandial β-cell responsiveness may be a more important determinant for glycemic control than fasting β-cell responsiveness.
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Tebbey, Paul W., Michael Hagen, and Gerald E. Hancock. "Atypical Pulmonary Eosinophilia Is Mediated by a Specific Amino Acid Sequence of the Attachment (G) Protein of Respiratory Syncytial Virus." Journal of Experimental Medicine 188, no. 10 (November 16, 1998): 1967–72. http://dx.doi.org/10.1084/jem.188.10.1967.
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We analyzed the immune responses evoked by a series of overlapping peptides to better understand the molecular basis for respiratory syncytial virus (RSV) G protein–induced eosinophilia in BALB/c mice. In vitro stimulation of spleen cells from natural G protein–primed mice showed dominant proliferative and cytokine (interferon [IFN]-γ and interleukin [IL]-5) responses to a peptide encompassing amino acids 184–198. Mice vaccinated with peptide 184– 198 conjugated to keyhole limpet hemocyanin showed significant pulmonary eosinophilia (39.5%) after challenge with live RSV. In contrast, mice immunized with a peptide (208–222) conjugate associated with induction of IFN-γ secreting spleen cells did not exhibit pulmonary eosinophilia after challenge. The in vivo depletion of CD4+ cells abrogated pulmonary eosinophilia in mice vaccinated with the peptide 184–198 conjugate, whereas the depletion of CD8+ cells had a negligible effect. Therefore, we have identified an association between peptide 184– 198 of natural G protein and the CD4+ T cell–mediated induction of pulmonary eosinophilia after live RSV challenge. Out of 43 human donors, 6 provided peripheral blood mononuclear cells that showed reactivity to G protein from RSV A2, 3 of which responded to peptide 184– 198. The results have important implications for the development of a vaccine against RSV.
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Adibzadeh Sereshgi, Mohammad Mehdi, Sahar Salimi, and Hassan Noorbazargan. "AK2 Antibacterial Synthetic Peptide Can Potentiate Macrophage Responses." ImmunoRegulation 4, no. 2 (January 1, 2022): 73–82. http://dx.doi.org/10.32598/immunoregulation.4.2.1.
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Background: Emphasis on substitutional medications with the elevated bacterial resistance to current antibiotics is pivotal. We evaluated the antibacterial effect of AK2 by Minimum Bactericidal Concentration (MBC) and Minimum inhibitory Concentration (MIC) and its impact on macrophage responses in 17 strains of pathogenic bacteria. The gene expression of macrophage’s cytokines was evaluated. Accordingly, the bioinformatic assessment predicted this peptide’s physicochemical characteristics, behavior, and structures. The present study aimed to assess the antibacterial effect of AK2 peptides on Macrophage responses. Materials and Methods: Cytotoxicity level was assessed by MTT assay on the HeLa cell line. The hemolytic activity of peptides on red blood cells was evaluated. The Griess assay was performed to assess the amount of macrophage nitric oxide production. The real-time PCR method measured the iNOS, IFN-γ, and TNF-α gene expression in isolated macrophages. Results: Peptide concentrations (13-60 µg/mL) were observed as the MBC and MIC value results for various bacteria. No remarkable cytotoxicity was observed at 30 and 60 µg/ml concentrations after 24h. iNOS, IFN-γ, and TNF-α gene expression were upregulated. There was also a higher secretion of nitric oxide in 48 hour-culture of the cell line with peptide. Great antibacterial activity was observed in some bacterial strains, particularly B. melitensis. Conclusion: AK2 peptides display suitable antibacterial activity with negligible toxicity for host cells. This peptide could also stimulate macrophage responses through nitric oxide production and gene expression in proinflammatory cytokines.
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Haghighi, Farid Hajareh, Roya Binaymotlagh, Laura Chronopoulou, Sara Cerra, Andrea Giacomo Marrani, Francesco Amato, Cleofe Palocci, and Ilaria Fratoddi. "Self-Assembling Peptide-Based Magnetogels for the Removal of Heavy Metals from Water." Gels 9, no. 8 (August 1, 2023): 621. http://dx.doi.org/10.3390/gels9080621.
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In this study, we present the synthesis of a novel peptide-based magnetogel obtained through the encapsulation of γ-Fe2O3-polyacrylic acid (PAA) nanoparticles (γ-Fe2O3NPs) into a hydrogel matrix, used for enhancing the ability of the hydrogel to remove Cr(III), Co(II), and Ni(II) pollutants from water. Fmoc-Phe (Fluorenylmethoxycarbonyl-Phenylalanine) and diphenylalanine (Phe2) were used as starting reagents for the hydrogelator (Fmoc-Phe3) synthesis via an enzymatic method. The PAA-coated magnetic nanoparticles were synthesized in a separate step, using the co-precipitation method, and encapsulated into the peptide-based hydrogel. The resulting organic/inorganic hybrid system (γ-Fe2O3NPs-peptide) was characterized with different techniques, including FT-IR, Raman, UV-Vis, DLS, ζ-potential, XPS, FESEM-EDS, swelling ability tests, and rheology. Regarding the application in heavy metals removal from aqueous solutions, the behavior of the obtained magnetogel was compared to its precursors and the effect of the magnetic field was assessed. Four different systems were studied for the separation of heavy metal ions from aqueous solutions, including (1) γ-Fe2O3NPs stabilized with PAA, (γ-Fe2O3NPs); (2) Fmoc-Phe3 hydrogel (HG); (3) γ-Fe2O3NPs embedded in peptide magnetogel (γ-Fe2O3NPs@HG); and (4) γ-Fe2O3NPs@HG in the presence of an external magnetic field. To quantify the removal efficiency of these four model systems, the UV-Vis technique was employed as a fast, cheap, and versatile method. The results demonstrate that both Fmoc-Phe3 hydrogel and γ-Fe2O3NPs peptide magnetogel can efficiently remove all the tested pollutants from water. Interestingly, due to the presence of magnetic γ-Fe2O3NPs inside the hydrogel, the removal efficiency can be enhanced by applying an external magnetic field. The proposed magnetogel represents a smart multifunctional nanosystem with improved absorption efficiency and synergic effect upon applying an external magnetic field. These results are promising for potential environmental applications of γ-Fe2O3NPs-peptide magnetogels to the removal of pollutants from aqueous media.
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Al-Attiyah, R., and A. S. Mustafa. "Characterization of Human Cellular Immune Responses to Novel Mycobacterium tuberculosis Antigens Encoded by Genomic Regions Absent in Mycobacterium bovis BCG." Infection and Immunity 76, no. 9 (June 23, 2008): 4190–98. http://dx.doi.org/10.1128/iai.00199-08.
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ABSTRACT Comparative genomics has identified several regions of differences (RDs) between the infectious Mycobacterium tuberculosis and the vaccine strains of Mycobacterium bovis BCG. We aimed to evaluate the cellular immune responses induced by antigens encoded by genes predicted in 11 RDs. Synthetic peptides covering the sequences of RD1, RD4 to RD7, RD9 to RD13, and RD15 were tested for antigen-induced proliferation and secretion of Th1 cytokine, gamma interferon (IFN-γ), by peripheral blood mononuclear cells (PBMC) obtained from culture-proven pulmonary tuberculosis (TB) patients and M. bovis BCG-vaccinated healthy subjects. Among the peptide pools, RD1 induced the best responses in both donor groups and in both assays. In addition, testing of TB patients' PBMC for secretion of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin 6 [IL-6], IL-8, and IL-1β), Th1 cytokines (IFN-γ, IL-2, and TNF-β), and Th2 cytokines (IL-4, IL-5, and IL-10) showed differential effects of RD peptides in the secretion of IFN-γ and IL-10, with high IFN-γ/IL-10 ratios (32 to 5.0) in response to RD1, RD5, RD7, RD9, and RD10 and low IFN-γ/IL-10 ratios (<1.0) in response to RD12, RD13, and RD15. Peptide-mixing experiments with PBMC from healthy subjects showed that secretion of large quantities of IL-10 in response to RD12 and RD13 correlated with inhibition of Th1 responses induced by RD1 peptides. In conclusion, our results suggest that M. tuberculosis RDs can be divided into two major groups—one group that activates PBMC to preferentially secrete IFN-γ and another group that activates preferential secretion of IL-10—and that these two groups of RDs may have roles in protection against and pathogenesis of TB, respectively.
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Arsenault, Ryan J., Yue Li, Kelli Bell, Kimberley Doig, Andrew Potter, Philip J. Griebel, Anthony Kusalik, and Scott Napper. "Mycobacterium avium subsp. paratuberculosis Inhibits Gamma Interferon-Induced Signaling in Bovine Monocytes: Insights into the Cellular Mechanisms of Johne's Disease." Infection and Immunity 80, no. 9 (June 11, 2012): 3039–48. http://dx.doi.org/10.1128/iai.00406-12.
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ABSTRACTMycobacterium aviumsubsp.paratuberculosisis the causative agent of Johne's disease in cattle and may have implications for human health. Establishment of chronic infection byM. aviumsubsp.paratuberculosisdepends on its subversion of host immune responses. This includes blocking the ability of infected macrophages to be activated by gamma interferon (IFN-γ) for clearance of this intracellular pathogen. To define the mechanism by whichM. aviumsubsp.paratuberculosissubverts this critical host cell function, patterns of signal transduction to IFN-γ stimulation of uninfected andM. aviumsubsp.paratuberculosis-infected bovine monocytes were determined through bovine-specific peptide arrays for kinome analysis. Pathway analysis of the kinome data indicated activation of the JAK-STAT pathway, a hallmark of IFN-γ signaling, in uninfected monocytes. In contrast, IFN-γ stimulation ofM. aviumsubsp.paratuberculosis-infected monocytes failed to induce patterns of peptide phosphorylation consistent with JAK-STAT activation. The inability of IFN-γ to induce differential phosphorylation of peptides corresponding to early JAK-STAT intermediates in infected monocytes indicates thatM. aviumsubsp.paratuberculosisblocks responsiveness at, or near, the IFN-γ receptor. Consistent with this hypothesis, increased expression of negative regulators of the IFN-γ receptors SOCS1 and SOCS3 as well as decreased expression of IFN-γ receptor chains 1 and 2 is observed inM. aviumsubsp.paratuberculosis-infected monocytes. These patterns of expression are functionally consistent with the kinome data and offer a mechanistic explanation for this criticalM. aviumsubsp.paratuberculosisbehavior. Understanding this mechanism may contribute to the rational design of more effective vaccines and/or therapeutics for Johne's disease.
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Faith, Alexander, Cezmi A. Akdis, Mübeccel Akdis, Andrea Joss, Daniel Wymann, and Kurt Blaser. "An Altered Peptide Ligand Specifically Inhibits Th2 Cytokine Synthesis by Abrogating TCR Signaling." Journal of Immunology 162, no. 3 (February 1, 1999): 1836–42. http://dx.doi.org/10.4049/jimmunol.162.3.1836.
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Abstract Altered peptide ligands (APL) can modify T cell effector function by their diversity in binding to the TCR or MHC class II-presenting molecules. The capacity to inhibit Th2 cytokine production by allergen-specific T cells would contribute to combating allergic inflammation. The presence of APL generated by Ala-substitutions in a synthetic dodeca-peptide spanning an immunodominant epitope of bee venom phospholipase A2 (PLA) was investigated in human T cells. Four of five substituted peptides reduced proliferation, IL-4, and IFN-γ production by cloned PLA-specific Th0 cells proportionately. However, one APL, PLA-F82A, inhibited IL-4 but had no effect on IFN-γ production. This uncoupling of IL-4 from IFN-γ production was also observed on immunogenic restimulation of the cloned T cells pre-exposed to the APL/APCs. It appeared to result from lower affinity of binding to MHC class II by the APL compared with the native peptide. The APL also inhibited IL-4 production by polyclonal T cells. In consequence of the change in cytokine secretion, the production of IgG4 in vitro increased by PLA-F82A stimulation, compared with the native peptide. Exposure of the cloned T cells to either the APL or the native peptide, in the absence of professional APC, induced anergy such that proliferation and production of IL-4, IL-5, and IL-13 was abrogated on immunogenic rechallenge. Defective T cell activation appeared to result from alterations in transmembrane signaling through the TCR, specifically to lack of tyrosine phosphorylation of the tyrosine kinase, ZAP-70.