FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2106364045> ?p ?o ?g. }

• W2106364045 endingPage "1574" @default.
• W2106364045 startingPage "1569" @default.
• W2106364045 abstract "CD8+ T-cells are a major source for the production of non-cytolytic factors that inhibit HIV-1 replication. In order to characterize further these factors, we analyzed gene expression profiles of activated CD8+ T-cells using a human cDNA expression array containing 588 human cDNAs. mRNA for the chemokine I-309 (CCL1), the cytokines granulocyte-macrophage colony-stimulating factor and interleukin-13, and natural killer cell enhancing factors (NKEF) -A and -B were up-regulated in bulk CD8+ T-cells from HIV-1 seropositive individuals compared with seronegative individuals. Recombinant NKEF-A and NKEF-B inhibited HIV-1 replication when exogenously added to acutely infected T-cells at an ID50 (dose inhibiting HIV-1 replication by 50%) of ∼130 nm (3 μg/ml). Additionally, inhibition against dual-tropic simian immunodeficiency virus and dual-tropic simian-human immunodeficiency virus was found. T-cells transfected with NKEF-A or NKEF-B cDNA were able to inhibit 80–98% HIV-1 replication in vitro. Elevated plasma levels of both NKEF-A and NKEF-B proteins were detected in 23% of HIV-infected non-treated individuals but not in persons treated with highly active antiviral therapy or uninfected persons. These results indicate that the peroxiredoxin family members NKEF-A and NKEF-B are up-regulated in activated CD8+ T-cells in HIV infection, and suggest that these antioxidant proteins contribute to the antiviral activity of CD8+ T-cells. CD8+ T-cells are a major source for the production of non-cytolytic factors that inhibit HIV-1 replication. In order to characterize further these factors, we analyzed gene expression profiles of activated CD8+ T-cells using a human cDNA expression array containing 588 human cDNAs. mRNA for the chemokine I-309 (CCL1), the cytokines granulocyte-macrophage colony-stimulating factor and interleukin-13, and natural killer cell enhancing factors (NKEF) -A and -B were up-regulated in bulk CD8+ T-cells from HIV-1 seropositive individuals compared with seronegative individuals. Recombinant NKEF-A and NKEF-B inhibited HIV-1 replication when exogenously added to acutely infected T-cells at an ID50 (dose inhibiting HIV-1 replication by 50%) of ∼130 nm (3 μg/ml). Additionally, inhibition against dual-tropic simian immunodeficiency virus and dual-tropic simian-human immunodeficiency virus was found. T-cells transfected with NKEF-A or NKEF-B cDNA were able to inhibit 80–98% HIV-1 replication in vitro. Elevated plasma levels of both NKEF-A and NKEF-B proteins were detected in 23% of HIV-infected non-treated individuals but not in persons treated with highly active antiviral therapy or uninfected persons. These results indicate that the peroxiredoxin family members NKEF-A and NKEF-B are up-regulated in activated CD8+ T-cells in HIV infection, and suggest that these antioxidant proteins contribute to the antiviral activity of CD8+ T-cells. human immunodeficiency virus, type 1 natural killer cell enhancing factor interleukin-309 granulocyte-macrophage colony-stimulating factor interleukin-13 glyceraldehyde-3-phosphate dehydrogenase phosphate-buffered saline long terminal repeat CD8+ T-lymphocyte antiviral factors enzyme-linked immunosorbent assay simian immunodeficiency virus simian-human immunodeficiency virus tumor necrosis factor CD8+ T-cells inhibit HIV-11 replication by both cytolytic and non-cytolytic mechanisms (1Yang O.O. Walker B.D. Adv. Immunol. 1997; 66: 273-311Crossref PubMed Google Scholar). The importance of cell-mediated cytotoxic immunity for the partial control of human immunodeficiency virus type 1 (HIV-1) replication in infected individuals is now widely recognized (2Harrer T. Harrer E. Kalams S.A. Elbeik T. Staprans S.I. Feinberg M.B. Cao Y., Ho, D.D. Yilma T. Caliendo A.M. Johnson R.P. Buchbinder S.P. Walker B.D. AIDS Res. Hum. Retroviruses. 1996; 12: 585-592Crossref PubMed Scopus (292) Google Scholar, 3Fowke K.R. Kaul R. Rosenthal K.L. Oyugi J. Kimani J. Rutherford W.J. Nagelkerke N.J. Ball T.B. Bwayo J.J. Simonsen J.N. Shearer G.M. Plummer F.A. Immunol. Cell Biol. 2000; 78: 586-595Crossref PubMed Scopus (93) Google Scholar, 4Kaul R. Plummer F.A. Kimani J. Dong T. Kiama P. Rostron T. Njagi E. MacDonald K.S. Bwayo J.J. McMichael A.J. Rowland-Jones S.L. J. Immunol. 2000; 164: 1602-1611Crossref PubMed Scopus (342) Google Scholar, 5Letvin N.L. J. Clin. Invest. 1998; 102: 1643-1644Crossref PubMed Scopus (13) Google Scholar, 6McMichael A. Cell. 1998; 93: 673-676Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 7McMichael A.J. Ogg G. Wilson J. Callan M. Hambleton S. Appay V. Kelleher T. Rowland-Jones S. Philos. Trans. R. Soc. Lond-Biol. Sci. 2000; 355: 363-367Crossref PubMed Scopus (40) Google Scholar, 8Ogg G.S. Kostense S. Klein M.R. Jurriaans S. Hamann D. McMichael A.J. Miedema F. J. Virol. 1999; 73: 9153-9160Crossref PubMed Google Scholar, 9Rosenberg E.S. Altfeld M. Poon S.H. Phillips M.N. Wilkes B.M. Eldridge R.L. Robbins G.K. D'Aquila R.T. Goulder P.J. Walker B.D. Nature. 2000; 407: 523-526Crossref PubMed Scopus (910) Google Scholar, 10Walker B.D. Rosenberg E.S. Hay C.M. Basgoz N. Yang O.O. Adv. Exp. Med. Biol. 1998; 452: 159-167Crossref PubMed Scopus (17) Google Scholar). The direct killing of virus-infected cells by antigen-specific cytotoxic T-lymphocytes is considered to be the dominant mechanism of virus suppression. Nevertheless, chemokines (MIP-1α, MIP-1β, and regulated on activation normal T-cellexpressed and secreted (RANTES)) produced by CD8+ T-cells have been shown to inhibit HIV-1 replication in vitro (11Cocchi F. DeVico A.L. Garzino-Demo A. Arya S.K. Gallo R.C. Lusso P. Science. 1995; 270: 1811-1815Crossref PubMed Scopus (2626) Google Scholar, 12Pal R. Garzino-Demo A. Markham P.D. Burns J. Brown M. Gallo R.C. DeVico A.L. Science. 1997; 278: 695-698Crossref PubMed Scopus (191) Google Scholar) at the level of viral entry (13Rana S. Besson G. Cook D.G. Rucker J. Smyth R.J., Yi, Y. Turner J.D. Guo H.H., Du, J.G. Peiper S.C. Lavi E. Samson M. Libert F. Liesnard C. Vassart G. Doms R.W. Parmentier M. Collman R.G. J. Virol. 1997; 71: 3219-3227Crossref PubMed Google Scholar, 14Rucker J. Edinger A.L. Sharron M. Samson M. Lee B. Berson J.F., Yi, Y. Margulies B. Collman R.G. Doranz B.J. Parmentier M. Doms R.W. J. Virol. 1997; 71: 8999-9007Crossref PubMed Google Scholar) and may play a critical role in vivo as an antiviral host defense (15Samson M. Libert F. Doranz B.J. Rucker J. Liesnard C. Farber C.M. Saragosti S. Lapoumeroulie C. Cognaux J. Forceille C. Muyldermans G. Verhofstede C. Burtonboy G. Georges M. Imai T. Rana S., Yi, Y. Smyth R.J. Collman R.G. Doms R.W. Vassart G. Parmentier M. Nature. 1996; 382: 722-725Crossref PubMed Scopus (2447) Google Scholar, 16Furci L. Scarlatti G. Burastero S. Tambussi G. Colognesi C. Quillent C. Longhi R. Loverro P. Borgonovo B. Gaffi D. Carrow E. Malnati M. Lusso P. Siccardi A.G. Lazzarin A. Beretta A. J. Exp. Med. 1997; 186: 455-460Crossref PubMed Scopus (115) Google Scholar). CD8+ T-lymphocytes can suppress human immunodeficiency virus type I (HIV-1) replicationin vitro by secreting a soluble factor(s) that differs from the chemokines in the mechanism of inhibition. These factors remain undefined (17Walker C.M. Moody D.J. Stites D.P. Levy J.A. Science. 1986; 234: 1563-1566Crossref PubMed Scopus (846) Google Scholar, 18Chen C.H. Weinhold K.J. Bartlett J.A. Bolognesi D.P. Greenberg M.L. AIDS Res. Hum. Retroviruses. 1993; 9: 1079-1086Crossref PubMed Scopus (95) Google Scholar, 19Le Borgne S. Fevrier M. Callebaut C. Lee S.P. Riviere Y. J. Virol. 2000; 74: 4456-4464Crossref PubMed Scopus (42) Google Scholar, 20Locher C.P. Blackbourn D.J. Levy J.A. Immunol. Lett. 1999; 66: 151-157Crossref PubMed Scopus (19) Google Scholar, 21Mackewicz C.E. Blackbourn D.J. Levy J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2308-2312Crossref PubMed Scopus (226) Google Scholar, 22Mackewicz C.E. Patterson B.K. Lee S.A. Levy J.A. J. Gen. Virol. 2000; 81: 1261-1264Crossref PubMed Scopus (42) Google Scholar, 23Tomaras G.D. Lacey S.F. McDanal C.B. Ferrari G. Weinhold K.J. Greenberg M.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3503-3508Crossref PubMed Scopus (72) Google Scholar, 24Yang O.O. Kalams S.A. Trocha A. Cao H. Luster A. Johnson R.P. Walker B.D. J. Virol. 1997; 71: 3120-3128Crossref PubMed Google Scholar, 25Walker C.M. Erickson A.L. Hsueh F.C. Levy J.A. J. Virol. 1991; 65: 5921-5927Crossref PubMed Google Scholar, 26Geiben-Lynn R. Kursar M. Brown N. Kerr E. Luster A. Walker B. J. Virol. 2001; 75: 8306-8316Crossref PubMed Scopus (35) Google Scholar) and have been termed CD8+ T-lymphocyte antiviral factors (CAF). Although the cytotoxic T-lymphocytes response is major histocompatibility complex class I-restricted, this restriction does not apply to inhibition of HIV-1 replication by CAF (17Walker C.M. Moody D.J. Stites D.P. Levy J.A. Science. 1986; 234: 1563-1566Crossref PubMed Scopus (846) Google Scholar, 25Walker C.M. Erickson A.L. Hsueh F.C. Levy J.A. J. Virol. 1991; 65: 5921-5927Crossref PubMed Google Scholar). Moreover, the production of CAF appears to be the property of stimulated CD8+ T-cells and does not require HIV infection (19Le Borgne S. Fevrier M. Callebaut C. Lee S.P. Riviere Y. J. Virol. 2000; 74: 4456-4464Crossref PubMed Scopus (42) Google Scholar, 20Locher C.P. Blackbourn D.J. Levy J.A. Immunol. Lett. 1999; 66: 151-157Crossref PubMed Scopus (19) Google Scholar, 26Geiben-Lynn R. Kursar M. Brown N. Kerr E. Luster A. Walker B. J. Virol. 2001; 75: 8306-8316Crossref PubMed Scopus (35) Google Scholar, 27Castro B.A. Walker C.M. Eichberg J.W. Levy J.A. Cell. Immunol. 1991; 132: 246-255Crossref PubMed Scopus (78) Google Scholar). One site of CAF action is the inhibition of HIV-1 RNA transcription, particularly at the long terminal repeat (LTR) that is assumed to function through down-regulation of the NF-κB pathway (19Le Borgne S. Fevrier M. Callebaut C. Lee S.P. Riviere Y. J. Virol. 2000; 74: 4456-4464Crossref PubMed Scopus (42) Google Scholar,21Mackewicz C.E. Blackbourn D.J. Levy J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2308-2312Crossref PubMed Scopus (226) Google Scholar, 22Mackewicz C.E. Patterson B.K. Lee S.A. Levy J.A. J. Gen. Virol. 2000; 81: 1261-1264Crossref PubMed Scopus (42) Google Scholar, 23Tomaras G.D. Lacey S.F. McDanal C.B. Ferrari G. Weinhold K.J. Greenberg M.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3503-3508Crossref PubMed Scopus (72) Google Scholar). It seems probable that the antiviral action of CAF is achieved by more than one cytokine or chemokine secreted by CD8+T-cells, perhaps acting in concert (28Moriuchi H. Moriuchi M. Combadiere C. Murphy P.M. Fauci A.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15341-15345Crossref PubMed Scopus (147) Google Scholar, 29Bailer R.T. Lee B. Montaner L.J. Eur. J. Immunol. 2000; 30: 1340-1349Crossref PubMed Scopus (51) Google Scholar). Recently we showed that CAF consists at least two components (26Geiben-Lynn R. Kursar M. Brown N. Kerr E. Luster A. Walker B. J. Virol. 2001; 75: 8306-8316Crossref PubMed Scopus (35) Google Scholar), a heparin-binding >50-kDa molecule, which we identified as a modified form of antithrombin (30Geiben-Lynn R. Brown N. Walker B.D. Luster A.D. J. Biol. Chem. 2002; 277: 42352-42357Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), and a smaller molecule, possibly the α-defensins (31Zhang L., Yu, W., He, T., Yu, J. Caffrey R.E. Dalmasso E.A., Fu, S. Pham T. Mei J., Ho, J.J. Zhang W. Lopez P. Ho D.D. Science. 2002; 298: 995-1000Crossref PubMed Scopus (445) Google Scholar). In this study, we used a gene array to perform a more comprehensive analysis of gene expression by CD8+T-cells from HIV-infected persons. We demonstrate here that the peroxiredoxin family proteins NKEF-A and NKEF-B are up-regulated by CD8+ T-cells following activation. These proteins exogenously added or intracellularly expressed have anti-HIV activity and have been described to increase natural killer cell activity (32Shau H. Gupta R.K. Golub S.H. Cell. Immunol. 1993; 147: 1-11Crossref PubMed Scopus (134) Google Scholar, 33Shau H. Kim A. Biochem. Biophys. Res. Commun. 1994; 199: 83-88Crossref PubMed Scopus (63) Google Scholar, 34Shau H. Butterfield L.H. Chiu R. Kim A. Immunogenetics. 1994; 40: 129-134Crossref PubMed Scopus (118) Google Scholar, 35Sauri H. Ashjian P.H. Kim A.T. Shau H. J. Leukocyte Biol. 1996; 59: 925-931Crossref PubMed Scopus (50) Google Scholar), increase cell resistance to oxidative stress (36Sarafian T.A. Rajper N. Grigorian B. Kim A. Shau H. Free Radic. Res. 1997; 26: 281-289Crossref PubMed Scopus (22) Google Scholar, 37Shau H. Kim A.T. Hedrick C.C. Lusis A.J. Tompkins C. Finney R. Leung D.W. Paglia D.E. Free Radic. Biol. Med. 1997; 22: 497-507Crossref PubMed Scopus (60) Google Scholar), and regulate transcription activator protein (AP-1) (see Ref. 38Shau H. Huang A.C. Faris M. Nazarian R. de Vellis J. Chen W. Biochem. Biophys. Res. Commun. 1998; 249: 683-686Crossref PubMed Scopus (43) Google Scholar and reviewed in Ref. 39Butterfield L.H. Merino A. Golub S.H. Shau H. Antioxid. Redox Signal. 1999; 1: 385-402Crossref PubMed Scopus (121) Google Scholar). We show that recombinant NKEF-A and NKEF-B protein exogenously added to HIV-1 cultures can inhibit HIV-1 replication and that T-cells transfected with NKEF-A or NKEF-B cDNA were resistant to HIV-1 infection. Plasma was obtained from 13 long term nonprogressors. Control plasma samples were obtained from 13 HIV-1 seronegative, healthy donors. Additional plasma samples from 6 asymptomatic, 5 symptomatic, and 2 AIDS patients, all under highly active antiviral therapy treatment for progressive disease, were investigated. Bulk CD8+ T-cells were purified, expanded, and stimulated as described previously (26Geiben-Lynn R. Kursar M. Brown N. Kerr E. Luster A. Walker B. J. Virol. 2001; 75: 8306-8316Crossref PubMed Scopus (35) Google Scholar) by anti-CD3 cross-linking from peripheral blood mononuclear cells, which were obtained from six HIV-1-infected long term non-progessors (17393, 15188, CTS-02, NEW, RK2000, and CX741) (26Geiben-Lynn R. Kursar M. Brown N. Kerr E. Luster A. Walker B. J. Virol. 2001; 75: 8306-8316Crossref PubMed Scopus (35) Google Scholar) and from seven HIV-1 seronegative individuals. Bulk CD8+ T-cells for each individual were treated separately. Long term non-progression was defined as being infected for more than 10 years, having plasma HIV-1 loads <400 RNA copies per ml, and CD4+ T-cell counts >500 per μl in the absence of therapy. For the inhibition tests, HIV-1IIIB, a T-cell tropic strain of HIV-1 (ATCC CRL-8543) was used. H9 cells (HLA Al, B6, Bw62, and Cw3) were acutely infected with HIV-1IIIB at a multiplicity of infection of 10−2 TCID50/ml and resuspended in RPMI 1640 (Sigma) supplemented with 20% (v/v) heat-inactivated fetal calf serum (Sigma; R20 medium) (26Geiben-Lynn R. Kursar M. Brown N. Kerr E. Luster A. Walker B. J. Virol. 2001; 75: 8306-8316Crossref PubMed Scopus (35) Google Scholar). The cells were then plated in 2 ml of R20 medium at 5 × 105 cells/ml in a 24-well plate. H9 cell supernatant (1 ml) was removed every 3 days and replaced with 1 ml of fresh R20 medium supplemented with recombinant NKEF-A or NKEF-B protein. After 9 days the concentration of p24-antigen was measured using an HIV-1 p24 ELISA kit (PerkinElmer Life Sciences). For the dual-tropic SIV-239 or dual-tropic SHIVKU-1 studies, the human T-cell line 174xCEM was infected at a multiplicity of infection of 10−2 TCID50/ml and resuspended in R20. The cells were then plated as described above. After 9 days the concentration of SIV p27-antigen was measured by ELISA (Coulter, Miami, FL). The medium controls demonstrated p24 or p27 antigen levels in excess of 100 ng/ml at day 9 and were used to calculate percent virus inhibition. Jurkat cells were used for inhibition experiments following transfection with NKEF-A and NKEF-B. NKEF-A- and NKEF-B-pBacPAK9 vectors (38Shau H. Huang A.C. Faris M. Nazarian R. de Vellis J. Chen W. Biochem. Biophys. Res. Commun. 1998; 249: 683-686Crossref PubMed Scopus (43) Google Scholar) were digested withBamHI and XhoI (New England Biolabs, Beverly, MA). The digest was treated with T4 polymerase (Invitrogen) for blunt-end ligation according to the manufacturer's instructions. The NKEF-A and NKEF-B fragments were then inserted into the SmaI cloning site of the pIRES2-EGFP expression vector (Clontech, Palo Alto, CA) and cultured inEscherichia coli. Plasmid DNAs were purified, and the direction of the inserts was confirmed by DNA sequencing. Jurkat cells were then transfected with a DNA/liposome mixture (FuGENE, Roche Diagnostics) and selected under G418 (Sigma) pressure (1.5 mg/ml). Stable transfected cells were then used in the above described inhibition test using 1.5 mg/ml G418. For days 1–9 the concentration of p24 antigen was measured using an HIV-1 p24 ELISA kit (PerkinElmer Life Sciences). The percentage of inhibition was calculated against a control with the empty pIRES2-EGFP vector. For total RNA extraction cell pellets of 0 or 4 h, anti-CD3-activated bulk CD8+ T-cells (107) were lysed with 1 ml of RNA STAT60 (Tel-Test B, Friendswood, TX), and the cellular RNA was purified using the RNA STAT60 protocol. To eliminate the DNA contaminant of the RNA the CleanMessageTM Kit (Genhunter, Nashville, TN) was used. 10 μg of total cellular RNA was fractionated on a 1.2% agarose, 0.7% formaldehyde gel and transferred to a GeneScreen membrane (DuPont). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific DNA probes (Clontech) and NKEF-A-specific DNA probes (33Shau H. Kim A. Biochem. Biophys. Res. Commun. 1994; 199: 83-88Crossref PubMed Scopus (63) Google Scholar) were 32P-radiolabeled using the DECAprimeTM Kit (Ambion, Austin, TX). Membranes were sequentially hybridized with the 32P-radiolabeled cDNA probes. Blots were washed at high stringency (0.2× SSC, 55 °C) and were measured with Molecular PhosphorImager System GS-363 (Bio-Rad) for an equal amount of time. Signal intensity calculations were performed using the supporting software program Molecular AnalystTMand calculated against the GAPDH signal. The ATLAS ArrayTM(Clontech) is a nitrocellulose membrane with 588 spotted cDNAs. For the hybridization polyadenylated (poly(A)+) mRNA was prepared from 100 μg of total RNA of 4 h CD3-cross-linked or untreated bulk CD8+T-cells. Bulk CD8+ T-cells for each individual were examined separately. The mRNA was suspended in diethyl pyrocarbonate-treated water and separated on poly(A) Quik® mRNA Columns (Stratagene, La Jolla, CA) according to the manufacturer's protocol. 1 μg (in 2 μl) of each poly(A)+ mRNA sample was transcribed to radiolabeled cDNA using 1 μl of Moloney murine leukemia virus-reverse transcriptase (50 units/ul; Stratagene) and 3.5 μl [α-32P]dATP (3000 Ci/mmol, 10 mCi/ml) according to the ATLAS assay protocol and used for hybridization. The binding of radioactivity to membrane was measured with the Molecular PhosphorImager System GS-363 (Bio-Rad) for an equal amount of time. Signal intensity calculations were performed using the supporting software program Molecular AnalystTM and calculated against the GAPDH signal. To produce the NKEF-A and NKEF-B proteins for the above described inhibition test, the NKEF-A and NKEF-B genes were cloned into the baculovirus expression vector pBacPAK9 (Clontech) and overexpressed in Spodoptera frugiperda (Sf21 cells; Clontech) as described (33Shau H. Kim A. Biochem. Biophys. Res. Commun. 1994; 199: 83-88Crossref PubMed Scopus (63) Google Scholar). After transfections Sf21 cells were harvested and lysed with insect lysis buffer (Pharmingen) at days 2–4. Afterward recombinant protein was purified as described earlier (35Sauri H. Ashjian P.H. Kim A.T. Shau H. J. Leukocyte Biol. 1996; 59: 925-931Crossref PubMed Scopus (50) Google Scholar). For the NKEF-A and NKEF-B ELISAs 2 μg/ml of monoclonal mouse anti-NKEF-A or anti-NKEF-B antibody (Pharmingen) was incubated overnight at 4 °C on protein high-binding EIA/RIA plates (Costar, Cambridge, MA) in coating buffer (0.05 mCO32−/HCO3−buffer, pH 9.6). Plates were washed with PBST buffer (phosphate-buffered saline (PBS), 0.05% (v/v) Triton X-100 (Sigma), pH 7.4) and blocked for 2 h at 37 °C with blocking buffer (PBS, 3% (v/v) goat serum, 3% (w/v) bovine serum albumin). Plates were washed with PBST buffer. Standard protein or samples were incubated for 2 h at 37 °C. Plates were washed with PBST buffer. A rabbit polyclonal NKEF-A/NKEF-B detection antibody (32Shau H. Gupta R.K. Golub S.H. Cell. Immunol. 1993; 147: 1-11Crossref PubMed Scopus (134) Google Scholar), which recognizes both forms of the NKEFs, was diluted 1:1000 in PBSBT buffer (PBS, 0.1% (w/v) bovine serum albumin, 0.05% (v/v) Triton X-100) and was added at 37 °C for 30 min. After washing with PBST buffer a horseradish peroxidase-coupled anti-rabbit antibody (Vector, Burlingame, CA) was used at 1:50,000 dilution at room temperature for 20 min. After washing with PBST buffer the ELISA was developed for 30 min at room temperature with a 1:1 dilution of peroxidase solution B and TMB peroxidase substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) and stopped with 4 n H2SO4. SDS-PAGE and Western blot was carried out as described previously (26Geiben-Lynn R. Kursar M. Brown N. Kerr E. Luster A. Walker B. J. Virol. 2001; 75: 8306-8316Crossref PubMed Scopus (35) Google Scholar) using polyclonal NKEF-A- and NKEF-B-specific antibodies (32Shau H. Gupta R.K. Golub S.H. Cell. Immunol. 1993; 147: 1-11Crossref PubMed Scopus (134) Google Scholar) at a dilution of 1:10,000. Protein concentration was determined by the BCA method (Pierce). Fisher's exact test was used to determine significance. Standard error is shown as error bars in the figures. CD8+T-cells are a major source for inhibitory non-cytolytic factors in HIV-1-infected persons. We have shown previously (26Geiben-Lynn R. Kursar M. Brown N. Kerr E. Luster A. Walker B. J. Virol. 2001; 75: 8306-8316Crossref PubMed Scopus (35) Google Scholar) that secretion of soluble antiviral factors is elevated in expanded CD8+T-cells from HIV-infected persons. In order to assess potential differences in gene expression, which might also be responsible for the antiviral activity, we evaluated mRNA derived from CD8+T-cells using the ATLAS array, which contained 588 unique genes. Expanded CD8+ T-cell populations of HIV-1 seronegative and the HIV-1 seropositive untreated individuals were evaluated prior to stimulation and 4 h following stimulation with anti-CD3. Although the ATLAS array includes a wide spectrum of genes, significant differences were limited to the expression of only four genes. These included significant differences (p < 0.001) in mRNA levels for the chemokine I-309, the cytokines GM-CSF and IL-13, and the peroxiredoxin gene NKEF-B (Fig. 1 A). The peroxiredoxin data were confirmed by Northern blot analysis using NKEF-A cDNA, a homologue of NKEF-B, which showed differences for this peroxiredoxin gene as well (Fig. 1 B). Both proteins have antioxidant function, and a natural killer cell activity was found for the NKEF-A and NKEF-B complex or for recombinant NKEF-A (reviewed in Ref. 39Butterfield L.H. Merino A. Golub S.H. Shau H. Antioxid. Redox Signal. 1999; 1: 385-402Crossref PubMed Scopus (121) Google Scholar). These mRNA results are consistent with previous studies showing higher secretion of I-309, GM-CSF, and IL-13 in stimulated CD8+ T-cells from seropositive persons (26Geiben-Lynn R. Kursar M. Brown N. Kerr E. Luster A. Walker B. J. Virol. 2001; 75: 8306-8316Crossref PubMed Scopus (35) Google Scholar), and they extend these prior studies by demonstrating the elevation in these peroxiredoxin family mRNAs in CD8+ T-cells from HIV-1 seropositive persons. By having demonstrated that GM-CSF, I-309, IL-13, and the NKEFs were preferentially expressed in CD3-activated, HIV-1-infected but untreated individuals, we next evaluated whether some of these gene products might contribute antiviral activity. Although GM-CSF, I-309, and IL-13 have been shown to influence HIV-1 replication in somein vitro systems (40Crowe S.M. Lopez A. J. Leukocyte Biol. 1997; 62: 41-48Crossref PubMed Scopus (26) Google Scholar), we found that these proteins did not inhibit HIV-1IIIB replication in acutely infected T-cells when added up to 1 μg/ml, demonstrating that they do not contribute to the observed inhibition (see Ref. 26Geiben-Lynn R. Kursar M. Brown N. Kerr E. Luster A. Walker B. J. Virol. 2001; 75: 8306-8316Crossref PubMed Scopus (35) Google Scholar and data not shown). In contrast, we found that recombinant NKEF-A and NKEF-B proteins that we expressed in Sf21 cells using a baculovirus expression system and purified to homogeneity (Fig. 2 A) inhibited HIV-1IIIB replication at an ID50 of 130 nm (3 μg/ml), respectively (Fig. 2 B). Additionally, using the NKEF-B protein for the inhibition assay with dual-tropic SHIV and dual-tropic SIV strains, we found nearly complete suppression of these viruses at 3 μg/ml (Fig. 2, C andD). Total protein lysate of untransfected Sf21 cells was used as a control and demonstrated no inhibition, excluding contaminating Sf21 protein as being responsible for inhibition (data not shown). The observed inhibition did not correlate with a decrease in cell count as measured at log phase of cell growth from day 2 to 6 by trypan blue staining (data not shown). Our data indicate that recombinant NKEFs can inhibit replication of X4 HIV-1, dual-tropic SIV, and dual-tropic SHIV. By having demonstrated that the NKEFs can inhibit HIV-1 replication, we next tested whether the NKEFs are secreted or released from stimulated CD8+ T-cells, and whether these proteins contribute to the antiviral activity of these cells (26Geiben-Lynn R. Kursar M. Brown N. Kerr E. Luster A. Walker B. J. Virol. 2001; 75: 8306-8316Crossref PubMed Scopus (35) Google Scholar). Although NKEF-A and NKEF-B proteins were originally described as endogenous proteins (33Shau H. Kim A. Biochem. Biophys. Res. Commun. 1994; 199: 83-88Crossref PubMed Scopus (63) Google Scholar, 37Shau H. Kim A.T. Hedrick C.C. Lusis A.J. Tompkins C. Finney R. Leung D.W. Paglia D.E. Free Radic. Biol. Med. 1997; 22: 497-507Crossref PubMed Scopus (60) Google Scholar, 41Sarafian T.A. Huang C. Kim A. de Vellis J. Shau H. Mol. Chem. Neuropathol. 1998; 34: 39-51Crossref PubMed Scopus (12) Google Scholar), thioredoxin (also termed adult T-cell leukemia-derived factor), another protein that serves as the electron donor for most peroxiredoxins (reviewed in Ref. 42Perl A. Banki K. Antioxid. Redox Signal. 2000; 2: 551-573Crossref PubMed Scopus (73) Google Scholar), has been shown to be secreted through a novel pathway despite having no signal sequence (43Rubartelli A. Bajetto A. Allavena G. Wollman E. Sitia R. J. Biol. Chem. 1992; 267: 24161-24164Abstract Full Text PDF PubMed Google Scholar). A similar observation has been confirmed for peroxiredoxin IV (44Okado-Matsumoto A. Matsumoto A. Fujii J. Taniguchi N. J. Biochem. (Tokyo). 2000; 127: 493-501Crossref PubMed Scopus (150) Google Scholar). We found that both NKEF-A and NKEF-B proteins were secreted after 4 h, regardless of whether the cells were stimulated. The concentrations produced averaged 15–40 ng/ml (Fig. 3) or were at least 10–20 times higher than seen for the chemokines and cytokines at this 4-h time point (26Geiben-Lynn R. Kursar M. Brown N. Kerr E. Luster A. Walker B. J. Virol. 2001; 75: 8306-8316Crossref PubMed Scopus (35) Google Scholar) and were up to ∼125 ng/ml at 16 h. The secretion was observed in stimulated CD8+ T-cells from both infected and uninfected individuals (Fig. 3). Thus, despite higher levels of NKEF mRNA in cells from seropositive persons, supernatants of activated CD8+ T-cells from both infected and uninfected individuals contained comparable amounts of these proteins, and this might be the result of the missing active protein secretion. This indicates that although NKEFs are able to exert antiviral activity, they are not the elusive CAF, which is produced in greater amounts from CD8+T-cells of infected persons (26Geiben-Lynn R. Kursar M. Brown N. Kerr E. Luster A. Walker B. J. Virol. 2001; 75: 8306-8316Crossref PubMed Scopus (35) Google Scholar). Additionally, the concentrations of secreted NKEFs at 4 h (15–40 ng/ml) are at levels below those causing significant inhibition (Fig. 2) at a time when significant inhibition by HIV-1-suppressive factor(s) was observed (26Geiben-Lynn R. Kursar M. Brown N. Kerr E. Luster A. Walker B. J. Virol. 2001; 75: 8306-8316Crossref PubMed Scopus (35) Google Scholar). By having demonstrated that the NKEFs are secreted, we next tested blood plasma levels by a specific ELISA to evaluate if plasma concentrations were sufficient to mediate inhibition of HIV-1 replication in vivo. Additionally, we tested if blood plasma levels are dependent on whether individuals are infected or not and treated or untreated. We could not detect a significant difference in NKEF-A or NKEF-B levels in plasma among the uninfected and the long term nonprogressor population. Nevertheless, plasma levels of the NKEFs were elevated (up to 500 ng/ml) in 3 of 13 (∼23%) HIV-1-infected but untreated persons tested, with levels 2.5–8 times higher than the uninfected or treated HIV patients (Fig. 4). For HIV-1-infected but untreated persons NKEF-A and NKEF-B were found at levels up to 1 μg/ml. At this concentration HIV-1 inhibition was detectable in vitro (Fig. 2 B), and an increase of natural killer cell activity in vitro has been noted (35Sauri H. Ashjian P.H. Kim A.T. Shau H. J. Leukocyte Biol. 1996; 59: 925-931Crossref PubMed Scopus (50) Google Scholar), demonstrating that NKEFs might have an antiviral influence in vivo. We demonstrated that blood plasma of long term non-progressors have significantly more NKEF-B compared with blood plasma of asymptomatic and symptomatic patients (Fig. 4). Our data indicate that el" @default.
• W2106364045 created "2016-06-24" @default.
• W2106364045 creator A5003060618 @default.
• W2106364045 creator A5011032016 @default.
• W2106364045 creator A5013787984 @default.
• W2106364045 creator A5016518899 @default.
• W2106364045 creator A5028769708 @default.
• W2106364045 creator A5044802324 @default.
• W2106364045 creator A5055995920 @default.
• W2106364045 creator A5060011924 @default.
• W2106364045 date "2003-01-01" @default.
• W2106364045 modified "2023-10-18" @default.
• W2106364045 title "HIV-1 Antiviral Activity of Recombinant Natural Killer Cell Enhancing Factors, NKEF-A and NKEF-B, Members of the Peroxiredoxin Family" @default.
• W2106364045 cites W1481615440 @default.
• W2106364045 cites W1567647424 @default.
• W2106364045 cites W1601810440 @default.
• W2106364045 cites W161561713 @default.
• W2106364045 cites W173718920 @default.
• W2106364045 cites W1815763197 @default.
• W2106364045 cites W1972139662 @default.
• W2106364045 cites W1977780484 @default.
• W2106364045 cites W1979063108 @default.
• W2106364045 cites W1980169905 @default.
• W2106364045 cites W1982145118 @default.
• W2106364045 cites W1984811742 @default.
• W2106364045 cites W1994050787 @default.
• W2106364045 cites W1995963105 @default.
• W2106364045 cites W2002123191 @default.
• W2106364045 cites W2003227439 @default.
• W2106364045 cites W2005045747 @default.
• W2106364045 cites W2005200639 @default.
• W2106364045 cites W2006235185 @default.
• W2106364045 cites W2010019194 @default.
• W2106364045 cites W2014271950 @default.
• W2106364045 cites W2019974254 @default.
• W2106364045 cites W2020847705 @default.
• W2106364045 cites W2023247989 @default.
• W2106364045 cites W2023482280 @default.
• W2106364045 cites W2026444780 @default.
• W2106364045 cites W2032915600 @default.
• W2106364045 cites W2035713858 @default.
• W2106364045 cites W2040495282 @default.
• W2106364045 cites W2043089116 @default.
• W2106364045 cites W2045911924 @default.
• W2106364045 cites W2046579202 @default.
• W2106364045 cites W2046594902 @default.
• W2106364045 cites W2049160074 @default.
• W2106364045 cites W2054049253 @default.
• W2106364045 cites W2055264167 @default.
• W2106364045 cites W2057481548 @default.
• W2106364045 cites W2059129711 @default.
• W2106364045 cites W2080970400 @default.
• W2106364045 cites W2082730339 @default.
• W2106364045 cites W2098822919 @default.
• W2106364045 cites W2103059036 @default.
• W2106364045 cites W2111732142 @default.
• W2106364045 cites W2116296362 @default.
• W2106364045 cites W2123931382 @default.
• W2106364045 cites W2130558808 @default.
• W2106364045 cites W2139113799 @default.
• W2106364045 cites W2142580865 @default.
• W2106364045 cites W2156694786 @default.
• W2106364045 cites W2161222674 @default.
• W2106364045 cites W2161268860 @default.
• W2106364045 cites W2162337628 @default.
• W2106364045 cites W2164206375 @default.
• W2106364045 cites W2164745336 @default.
• W2106364045 cites W2165347895 @default.
• W2106364045 cites W2165744111 @default.
• W2106364045 cites W2166730310 @default.
• W2106364045 cites W2358581388 @default.
• W2106364045 cites W2408230628 @default.
• W2106364045 cites W2472772724 @default.
• W2106364045 doi "https://doi.org/10.1074/jbc.m209964200" @default.
• W2106364045 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12421812" @default.
• W2106364045 hasPublicationYear "2003" @default.
• W2106364045 type Work @default.
• W2106364045 sameAs 2106364045 @default.
• W2106364045 citedByCount "87" @default.
• W2106364045 countsByYear W21063640452012 @default.
• W2106364045 countsByYear W21063640452013 @default.
• W2106364045 countsByYear W21063640452014 @default.
• W2106364045 countsByYear W21063640452015 @default.
• W2106364045 countsByYear W21063640452017 @default.
• W2106364045 countsByYear W21063640452018 @default.
• W2106364045 countsByYear W21063640452019 @default.
• W2106364045 countsByYear W21063640452020 @default.
• W2106364045 countsByYear W21063640452021 @default.
• W2106364045 countsByYear W21063640452022 @default.
• W2106364045 crossrefType "journal-article" @default.
• W2106364045 hasAuthorship W2106364045A5003060618 @default.
• W2106364045 hasAuthorship W2106364045A5011032016 @default.
• W2106364045 hasAuthorship W2106364045A5013787984 @default.
• W2106364045 hasAuthorship W2106364045A5016518899 @default.
• W2106364045 hasAuthorship W2106364045A5028769708 @default.
• W2106364045 hasAuthorship W2106364045A5044802324 @default.
• W2106364045 hasAuthorship W2106364045A5055995920 @default.
• W2106364045 hasAuthorship W2106364045A5060011924 @default.

• next