Neuropathogenesis of Lentiviral Infection in Macaques
2002; Elsevier BV; Volume: 161; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)64241-1
ISSN1525-2191
AutoresAndrey Hicks, Raghava Potula, Yongjun Sui, François Villinger, David M. Pinson, Istvan Adany, Zhuang Li, Chloe Long, Paul D. Cheney, Joanne Marcario, Francis J. Novembre, Niklaus H. Mueller, Anil Kumar, Eugene O. Major, Opendra Narayan, Shilpa Buch,
Tópico(s)Herpesvirus Infections and Treatments
ResumoNeurological disease associated with lentiviral infection occurs mainly as a consequence of primary replication of the virus or a combination of the virus infection and replication of opportunistic pathogens in the central nervous system. Recent studies have shown that whereas the disease can be caused by CCR5 tropic viruses alone, its induction by CXCR4 (X4) tropic viruses occurred usually in association with infections caused by opportunistic pathogens and in the presence of a Th2 cytokine, interleukin (IL)-4.1Buch S Pinson D King CL Raghavan R Hou Y Li Z Adany I Hicks A Villinger F Kumar A Narayan O Inhibitory and enhancing effects of IFN-gamma and IL-4 on SHIV(KU) replication in rhesus macaque macrophages: correlation between Th(2)cytokines and productive infection in tissue macrophages during late-stage infection.Cytokine. 2001; 13: 295-304Crossref PubMed Scopus (23) Google Scholar, 2Buch S Villinger F Pinson D Hou Y Adany I Li Z Dalal R Raghavan R Kumar A Narayan O Innate differences between simian human immunodeficiency virus (SHIV)KU2-infected rhesus and pig-tailed macaques to development of neurological disease.Virology. 2002; 295: 54-62Crossref PubMed Scopus (17) Google Scholar Further, X4-mediated neurological disease developed preferentially in rhesus compared to pig-tailed macaques. Because macrophages are the target cells for lentiviral infection in the brain and because macrophage chemoattractant protein (MCP)-1 is one of the major chemokines that is closely associated with acquired immune deficiency syndrome (AIDS) dementia, we tested for correlations between MCP-1 production and virus tropism in macrophages from the two species of macaques. The studies showed that the higher susceptibility of rhesus macaques to X4 virus-mediated encephalitis correlated with heightened production of virus and MCP-1 in cultured macrophages from this species and that these effects were further enhanced with treatment with IL-4. However, the latter effect was restricted to macrophages infected with X4 viruses. IL-4 may therefore be a basic requirement for X4 viruses to cause central nervous system disease. Neurological disease associated with lentiviral infection occurs mainly as a consequence of primary replication of the virus or a combination of the virus infection and replication of opportunistic pathogens in the central nervous system. Recent studies have shown that whereas the disease can be caused by CCR5 tropic viruses alone, its induction by CXCR4 (X4) tropic viruses occurred usually in association with infections caused by opportunistic pathogens and in the presence of a Th2 cytokine, interleukin (IL)-4.1Buch S Pinson D King CL Raghavan R Hou Y Li Z Adany I Hicks A Villinger F Kumar A Narayan O Inhibitory and enhancing effects of IFN-gamma and IL-4 on SHIV(KU) replication in rhesus macaque macrophages: correlation between Th(2)cytokines and productive infection in tissue macrophages during late-stage infection.Cytokine. 2001; 13: 295-304Crossref PubMed Scopus (23) Google Scholar, 2Buch S Villinger F Pinson D Hou Y Adany I Li Z Dalal R Raghavan R Kumar A Narayan O Innate differences between simian human immunodeficiency virus (SHIV)KU2-infected rhesus and pig-tailed macaques to development of neurological disease.Virology. 2002; 295: 54-62Crossref PubMed Scopus (17) Google Scholar Further, X4-mediated neurological disease developed preferentially in rhesus compared to pig-tailed macaques. Because macrophages are the target cells for lentiviral infection in the brain and because macrophage chemoattractant protein (MCP)-1 is one of the major chemokines that is closely associated with acquired immune deficiency syndrome (AIDS) dementia, we tested for correlations between MCP-1 production and virus tropism in macrophages from the two species of macaques. The studies showed that the higher susceptibility of rhesus macaques to X4 virus-mediated encephalitis correlated with heightened production of virus and MCP-1 in cultured macrophages from this species and that these effects were further enhanced with treatment with IL-4. However, the latter effect was restricted to macrophages infected with X4 viruses. IL-4 may therefore be a basic requirement for X4 viruses to cause central nervous system disease. One of the major biological properties of all members of the lentivirus taxonomic group is that irrespective of virus-host pair interactions, the ability of the virus to replicate productively in macrophages of the host is fundamental to the susceptibility of the individual to neurological disease.3Gendelman HE Lipton SA Tardieu M Bukrinsky MI Nottet HS The neuropathogenesis of HIV-1 infection.J Leukoc Biol. 1994; 56: 389-398PubMed Google Scholar, 4Koenig S Gendelman HE Orenstein JM Dal Canto MC Pezeshkpour GH Yungbluth M Janotta F Aksamit A Martin MA Fauci AS Detection of AIDS virus in macrophages in brain tissue from AIDS patients with encephalopathy.Science. 1986; 233: 1089-1093Crossref PubMed Scopus (1348) Google Scholar, 5Gendelman HE Narayan O Molineaux S Clements JE Ghotbi Z Slow, persistent replication of lentiviruses: role of tissue macrophages and macrophage precursors in bone marrow.Proc Natl Acad Sci USA. 1985; 82: 7086-7090Crossref PubMed Scopus (226) Google Scholar, 6Sharma DP Zink MC Anderson M Adams R Clements JE Joag SV Narayan O Derivation of neurotropic simian immunodeficiency virus from exclusively lymphocytetropic parental virus: pathogenesis of infection in macaques.J Virol. 1992; 66: 3550-3556PubMed Google Scholar, 7Sharma DP Anderson M Zink MC Adams RJ Donnenberg AD Clements JE Narayan O Pathogenesis of acute infection in rhesus macaques with a lymphocyte-tropic strain of simian immunodeficiency virus.J Infect Dis. 1992; 166: 738-746Crossref PubMed Scopus (29) Google Scholar In human beings infected with human immunodeficiency virus (HIV), the human lentivirus, neurological disease has been associated primarily with infection of brain macrophages with viruses that use the CCR5 (R5) rather than those that use the CXCR4 (X4) co-receptor for entry into cells.8Albright AV Shieh JT Itoh T Lee B Pleasure D O'Connor MJ Doms RW Gonzalez-Scarano F Microglia express CCR5, CXCR4, and CCR3, but of these, CCR5 is the principal coreceptor for human immunodeficiency virus type 1 dementia isolates.J Virol. 1999; 73: 205-213Crossref PubMed Google Scholar, 9Choe H Farzan M Sun Y Sullivan N Rollins B Ponath PD Wu L Mackay CR LaRosa G Newman W Gerard N Gerard C Sodroski J The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates.Cell. 1996; 85: 1135-1148Abstract Full Text Full Text PDF PubMed Scopus (2078) Google Scholar Studies on replication of the two types of viruses in cultured macrophages have shown that the R5 viruses replicate much more efficiently in these cells than the X4 agents, and thus provided a rationale for the presence of R5 viruses in affected brains. This basic observation has been confirmed and extended in the macaque model of the neurological syndrome usingSIVmac,6Sharma DP Zink MC Anderson M Adams R Clements JE Joag SV Narayan O Derivation of neurotropic simian immunodeficiency virus from exclusively lymphocytetropic parental virus: pathogenesis of infection in macaques.J Virol. 1992; 66: 3550-3556PubMed Google Scholar, 7Sharma DP Anderson M Zink MC Adams RJ Donnenberg AD Clements JE Narayan O Pathogenesis of acute infection in rhesus macaques with a lymphocyte-tropic strain of simian immunodeficiency virus.J Infect Dis. 1992; 166: 738-746Crossref PubMed Scopus (29) Google Scholar and SIVsmmPGM10Novembre FJ De Rosayro J O'Neil SP Anderson DC Klumpp SA McClure HM Isolation and characterization of a neuropathogenic simian immunodeficiency virus derived from a sooty mangabey.J Virol. 1998; 72: 8841-8851PubMed Google Scholar (R5 viruses). However, neuropathology has also been observed in rhesus (Rh) macaques infected with the X4 tropic SHIVKU11Raghavan R Stephens EB Joag SV Adany I Pinson DM Li Z Jia F Sahni M Wang C Leung K Foresman L Narayan O Neuropathogenesis of chimeric simian/human immunodeficiency virus infection in pig-tailed and rhesus macaques.Brain Pathol. 1997; 7: 851-861Crossref PubMed Scopus (47) Google Scholar, 12Liu ZQ Muhkerjee S Sahni M McCormick-Davis C Leung K Li Z Gattone VH Tian C Doms RW Hoffman TL Raghavan R Narayan O Stephens EB Derivation and biological characterization of a molecular clone of SHIV(KU-2) that causes AIDS, neurological disease, and renal disease in rhesus macaques.Virology. 1999; 260: 295-307Crossref PubMed Scopus (70) Google Scholar and the X4/R5 dual-tropic SHIV89.6P.1Buch S Pinson D King CL Raghavan R Hou Y Li Z Adany I Hicks A Villinger F Kumar A Narayan O Inhibitory and enhancing effects of IFN-gamma and IL-4 on SHIV(KU) replication in rhesus macaque macrophages: correlation between Th(2)cytokines and productive infection in tissue macrophages during late-stage infection.Cytokine. 2001; 13: 295-304Crossref PubMed Scopus (23) Google Scholar, 13Hoffman TL Stephens EB Narayan O Doms RW HIV type I envelope determinants for use of the CCR2b, CCR3, STRL33, and APJ coreceptors.Proc Natl Acad Sci USA. 1998; 95: 11360-11365Crossref PubMed Scopus (100) Google Scholar The two R5 viruses,SIVmac251 and SIVsmmPGM, are highly macrophage-tropic in cultured macaque macrophages10Novembre FJ De Rosayro J O'Neil SP Anderson DC Klumpp SA McClure HM Isolation and characterization of a neuropathogenic simian immunodeficiency virus derived from a sooty mangabey.J Virol. 1998; 72: 8841-8851PubMed Google Scholar, 14Stephens EB Galbreath D Liu ZQ Sahni M Li Z Lamb-Wharton R Foresman L Joag SV Narayan O Significance of macrophage tropism of SIV in the macaque model of HIV disease.J Leukoc Biol. 1997; 62: 12-19PubMed Google Scholar and are also associated with primary lentiviral encephalopathy6Sharma DP Zink MC Anderson M Adams R Clements JE Joag SV Narayan O Derivation of neurotropic simian immunodeficiency virus from exclusively lymphocytetropic parental virus: pathogenesis of infection in macaques.J Virol. 1992; 66: 3550-3556PubMed Google Scholar, 10Novembre FJ De Rosayro J O'Neil SP Anderson DC Klumpp SA McClure HM Isolation and characterization of a neuropathogenic simian immunodeficiency virus derived from a sooty mangabey.J Virol. 1998; 72: 8841-8851PubMed Google Scholar, 11Raghavan R Stephens EB Joag SV Adany I Pinson DM Li Z Jia F Sahni M Wang C Leung K Foresman L Narayan O Neuropathogenesis of chimeric simian/human immunodeficiency virus infection in pig-tailed and rhesus macaques.Brain Pathol. 1997; 7: 851-861Crossref PubMed Scopus (47) Google Scholar, 15Westmoreland SV Halpern E Lackner AA Simian immunodeficiency virus encephalitis in rhesus macaques is associated with rapid disease progression.J Neurovirol. 1998; 4: 260-268Crossref PubMed Scopus (97) Google Scholar unlike the X4 simian human immunodeficiency viruses (SHIVs) that cause encephalopathy, usually in association with other opportunistic pathogens.2Buch S Villinger F Pinson D Hou Y Adany I Li Z Dalal R Raghavan R Kumar A Narayan O Innate differences between simian human immunodeficiency virus (SHIV)KU2-infected rhesus and pig-tailed macaques to development of neurological disease.Virology. 2002; 295: 54-62Crossref PubMed Scopus (17) Google Scholar Further studies aimed at exploring whether X4 virus-mediated neurological disease required synergy between the lentivirus and the opportunistic pathogen demonstrated that the infection of macaques with SHIVs, supplemented with injection of Schistosoma mansoni eggs (an experimental surrogate of opportunistic pathogens), into the animals resulted in development of lentiviral lesions in various tissues, including the central nervous system (CNS). These lesions were associated with heightened virus replication in macrophages in S. mansoni-induced granulomas that were rich in cells secreting Th2 cytokines, interleukin (IL)-4 and IL-10.1Buch S Pinson D King CL Raghavan R Hou Y Li Z Adany I Hicks A Villinger F Kumar A Narayan O Inhibitory and enhancing effects of IFN-gamma and IL-4 on SHIV(KU) replication in rhesus macaque macrophages: correlation between Th(2)cytokines and productive infection in tissue macrophages during late-stage infection.Cytokine. 2001; 13: 295-304Crossref PubMed Scopus (23) Google Scholar This suggested that the neuropathogenesis of X4 virus infections may be contingent on the presence of Th2 cytokines in the brain. Histologically, characteristic pathological findings of lentiviral encephalitis consist of microglial foci, dense infiltration of mononuclear cells (including monocyte/macrophages) in the perivascular sites and in the brain parenchyma, and formation of syncytia comprised mainly of infected, fused cells bearing the macrophage marker.16Brew BJ Rosenblum M Cronin K Price RW AIDS dementia complex and HIV-1 brain infection: clinical-virological correlations.Ann Neurol. 1995; 38: 563-570Crossref PubMed Scopus (237) Google Scholar, 17Kure K Weidenheim KM Lyman WD Dickson DW Morphology and distribution of HIV-1 gp41-positive microglia in subacute AIDS encephalitis. Pattern of involvement resembling a multisystem degeneration.Acta Neuropathol (Berl). 1990; 80: 393-400Crossref PubMed Scopus (193) Google Scholar, 18Masliah E Achim CL Ge N De Teresa R Wiley CA Cellular neuropathology in HIV encephalitis.Res Publ Assoc Res Nerv Ment Dis. 1994; 72: 119-131PubMed Google Scholar, 19Petito CK Cho ES Lemann W Navia BA Price RW Neuropathology of acquired immunodeficiency syndrome (AIDS): an autopsy review.J Neuropathol Exp Neurol. 1986; 45: 635-646Crossref PubMed Scopus (389) Google Scholar Although the mechanisms leading to monocytic infiltration in the CNS still remain unclear, it is speculated that secretory products, including CC chemokines, released by HIV-1-infected cells could be potentially important for recruitment of monocytes from blood to the brain. One such CC chemokine, monocyte chemoattractant protein-1 (MCP-1), has been shown to be the most potent of a variety of monocyte chemoattractants, including RANTES, macrophage inflammatory protein-1α (MIP-1α), MIP-1β, MCP-2, and MCP-3, at inducing the transmigration of monocytes across a model of the blood-brain barrier.20Uguccioni M D'Apuzzo M Loetscher M Dewald B Baggiolini M Actions of the chemotactic cytokines MCP-1, MCP-2, MCP-3, RANTES, MIP-1 alpha and MIP-1 beta on human monocytes.Eur J Immunol. 1995; 25: 64-68Crossref PubMed Scopus (324) Google Scholar The role of MCP-1 in monocyte recruitment in vivo in many organs, including the brain, has been supported by studies in transgenic mice overexpressing MCP-121Fuentes ME Durham SK Swerdel MR Lewin AC Barton DS Megill JR Bravo R Lira SA Controlled recruitment of monocytes and macrophages to specific organs through transgenic expression of monocyte chemoattractant protein-1.J Immunol. 1995; 155: 5769-5776PubMed Google Scholar, 22Gunn MD Nelken NA Liao X Williams LT Monocyte chemoattractant protein-1 is sufficient for the chemotaxis of monocytes and lymphocytes in transgenic mice but requires an additional stimulus for inflammatory activation.J Immunol. 1997; 158: 376-383PubMed Google Scholar, 23Grewal IS Rutledge BJ Fiorillo JA Gu L Gladue RP Flavell RA Rollins BJ Transgenic monocyte chemoattractant protein-1 (MCP-1) in pancreatic islets produces monocyte-rich insulitis without diabetes: abrogation by a second transgene expressing systemic MCP-1.J Immunol. 1997; 159: 401-408PubMed Google Scholar and in CCR2-deficient mice.24Kurihara T Warr G Loy J Bravo R Defects in macrophage recruitment and host defense in mice lacking the CCR2 chemokine receptor.J Exp Med. 1997; 186: 1757-1762Crossref PubMed Scopus (545) Google Scholar, 25Boring L Gosling J Chensue SW Kunkel SL Farese RVJ Broxmeyer HE Charo IF Impaired monocyte migration and reduced type 1 (Th1) cytokine responses in C-C chemokine receptor 2 knockout mice.J Clin Invest. 1997; 100: 2552-2561Crossref PubMed Scopus (859) Google Scholar, 26Kuziel WA Morgan SJ Dawson TC Griffin S Smithies O Ley K Maeda N Severe reduction in leukocyte adhesion and monocyte extravasation in mice deficient in CC chemokine receptor 2.Proc Natl Acad Sci USA. 1997; 94: 12053-12058Crossref PubMed Scopus (554) Google Scholar The selective accumulation of MCP-1, but not other chemokines, in the brains of patients with AIDS dementia,27Conant K Garzino-Demo A Nath A McArthur JC Halliday W Power C Gallo RC Major EO Induction of monocyte chemoattractant protein-1 in HIV-1 Tat-stimulated astrocytes and elevation in AIDS dementia.Proc Natl Acad Sci USA. 1998; 95: 3117-3121Crossref PubMed Scopus (500) Google Scholar in the cerebrospinal fluid (CSF) of AIDS patients with cytomegalovirus (CMV) infection,28Bernasconi S Cinque P Peri G Sozzani S Crociati A Torri W Vicenzi E Vago L Lazzarin A Poli G Mantovani A Selective elevation of monocyte chemotactic protein-1 in the cerebrospinal fluid of AIDS patients with cytomegalovirus encephalitis.J Infect Dis. 1996; 174: 1098-1101Crossref PubMed Scopus (83) Google Scholar and HIV-encephalitis29Cinque P Vago L Mengozzi M Torri V Ceresa D Vicenzi E Transidico P Vagani A Sozzani S Mantovani A Lazzarin A Poli G Elevated cerebrospinal fluid levels of monocyte chemotactic protein-1 correlate with HIV-1 encephalitis and local viral replication.AIDS. 1998; 12: 1327-1332Crossref PubMed Scopus (211) Google Scholar and in CSF of macaques with SIV encephalitis30Zink MC Coleman GD Mankowski JL Adams RJ Tarwater PM Fox K Clements JE Increased macrophage chemoattractant protein-1 in cerebrospinal fluid precedes and predicts simian immunodeficiency virus encephalitis.J Infect Dis. 2001; 184: 1015-1021Crossref PubMed Scopus (144) Google Scholar further supports the role of this chemokine in the pathogenesis of lentiviral neurological disease. In addition, the role of soluble factors such as HIV Tat that modulate the expression of MCP-1, and other β chemokines in microglia and astrocytes, lends further support to the complex interplay of viral infection, chemokines, and chemokine receptors in the inflammatory processes of HIV encephalitis.31McManus CM Weidenheim K Woodman SE Nunez J Hesselgesser J Nath A Berman JW Chemokine and chemokine-receptor expression in human glial elements: induction by the HIV protein, Tat, and chemokine autoregulation.Am J Pathol. 2000; 156: 1441-1453Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar In the present study, we examined whether there was a connection between the tropism of the virus for macrophages and the ability of the cells to produce MCP-1. The experiments confirmed that the higher susceptibility of Rh compared to pig-tailed (Pt) macaques for development of encephalitis correlated with the ability of macrophages from the Rh species to produce more virus and higher concentrations of MCP-1 than Pt macaques. The Th2 cytokine, IL-4 caused further enhancement of production of MCP-1 in macrophages from both species, but its enhancing effect on virus replication was confined to Rh macrophages infected with X4 viruses. We obtained SHIV-4 DNA encoding the env, tat, rev, and vpu genes of HIV-1 HXBc2 on a background of SIVmac23932Li J Lord CI Haseltine W Letvin NL Sodroski J Infection of cynomolgus monkeys with a chimeric HIV-1/SIVmac virus that expresses the HIV-1 envelope glycoproteins.J Acquir Immune Defic Syndr. 1992; 5: 639-646PubMed Google Scholar from Dr. Joseph Sodroski, Harvard University, Boston, MA. Viral DNA was transfected into CEMx174 cells to produce a virus that was used to initiate sequential passages in macaques. Virus isolated from cerebrospinal fluid obtained 8 weeks after inoculation from Pt macaque PNb in passage four was amplified in a culture of peripheral blood mononuclear cells from a normal macaque and subsequently designated as SHIVkU-1.33Joag SV Li Z Foresman L Stephens EB Zhao LJ Adany I Pinson DM McClure HM Narayan O Chimeric simian/human immunodeficiency virus that causes progressive loss of CD4+ T cells and AIDS in pig-tailed macaques.J Virol. 1996; 70: 3189-3197PubMed Google Scholar A further passage of this virus in Rh macaques gave rise to SHIVkU-2.34Joag SV Li Z Wang C Jia F Foresman L Adany I Pinson DM Stephens EB Narayan O Chimeric SHIV that causes CD4+ T cell loss and AIDS in rhesus macaques.J Med Primatol. 1998; 27: 59-64Crossref PubMed Scopus (40) Google Scholar SHIV89.6P was kindly provided by Dr. N. Letvin (Harvard University), SIVmac251 was a gift from Dr. R. Desrosiers (Harvard Medical School) and SIVsmmPGM from Dr. F. Novembre (Emory University, Atlanta, GA). Peripheral blood mononuclear cells from macaques were obtained by Ficoll-Hypaque (Sigma, St. Louis, MO) gradient centrifugation and suspended at a concentration of 2 × 106 cells/ml in macrophage differentiation medium consisting of RPMI medium supplemented with 20% heat-inactivated human serum, 5 U/ml of M-CSF (Genetics Institute), 100 U/ml of GM-CSF (Genetics Institute, Cambridge, MA) and 5% heat-inactivated Rh monkey serum. Six-well dishes (Costar, Cambridge, MA) were seeded with 3 ml of medium containing 6 to 10 × 106 cells/per well and incubated overnight at 37°C. Cultures were then rinsed to remove nonadherent cells, refed with macrophage differentiation medium, and maintained for 7 days to allow adherent monocytes to differentiate into mature macrophages. Cells in our cultures were exclusively macrophages because >98% of them expressed the CD14 monocyte/macrophage-specific cell surface marker (data not shown). These cells were then inoculated with cell-free SHIVkU-2, SHIV89.6P, or SIVmac251 at an multiplicity of infection (MOI) of 0.1 for 24 hours at 37°C. Cultures were then rinsed three times with R10 and replenished with macrophage medium. Studies were performed on macrophage cultures from at least three animals of either species and each experiment was performed in triplicate. Inoculated cultures were maintained in the presence or absence of recombinant macaque (rm) IL-4 at a concentration of 1.5 U/ml.35Valentin A Lu W Rosati M Schneider R Albert J Karlsson A Pavlakis GN Dual effect of interleukin 4 on HIV-1 expression: implications for viral phenotypic switch and disease progression.Proc Natl Acad Sci USA. 1998; 95: 8886-8891Crossref PubMed Scopus (126) Google Scholar The medium was replenished every third day with fresh rm IL-4. Supernatant fluids were collected at regular intervals for determination of MCP-1 content by enzyme-linked immunosorbent assay (ELISA) and virus concentrations using reverse transcriptase (RT) assays. Viral replication was monitored by assaying the RT activity in the supernatants as described previously.36Kumar A Stipp HL Sheffer D Narayan O Use of herpesvirus saimiri-immortalized macaque CD4(+) T cell clones as stimulators and targets for assessment of CTL responses in macaque/AIDS models.J Immunol Methods. 1999; 230: 47-58Crossref PubMed Scopus (16) Google Scholar Macrophages from normal animals were infected with the X4 and R5 viruses as described above followed by treatment with or without rm IL-4 for 7 days. The medium was replenished every third day with fresh rmIL-4 (1.5 U/ml). Studies were performed on macrophage cultures from at least three animals of either species and each experiment was performed in triplicate. MCP-1 was detected by ELISA (R & D systems, Minneapolis, MN). The ELISA could detect concentrations as low as 10 pg/ml. Protein measurements were determined by comparison to a standard curve, run in duplicate with each assay. For semiquantitative RT-PCR, RNA from Rh and Pt macaques, with and without IL-4 treatment, was extracted with Trizol reagent (Life Technologies, Inc., Grand Island, NY), and co-receptor mRNA levels were expressed as a function of the amount of β-actin mRNA for each RNA sample.36Kumar A Stipp HL Sheffer D Narayan O Use of herpesvirus saimiri-immortalized macaque CD4(+) T cell clones as stimulators and targets for assessment of CTL responses in macaque/AIDS models.J Immunol Methods. 1999; 230: 47-58Crossref PubMed Scopus (16) Google Scholar The sequences of primers used for RT-PCR reactions are shown in Table 1. For RT reactions, 1 μg of total RNA from each sample was used in the Titan One-Tube RT-PCR system (Boehringer-Mannheim, Indianapolis, IN). Presence of any DNA contamination in the RNA preparations was tested by performing duplicate reactions, and subjecting one of them to 99°C for 2 minutes followed by 3 minutes at 95°C, to inactivate the RT activity. When present, DNA was removed with DNase I (Life Technologies, Inc., Gaithersburg, MD), followed by extraction. The reactions were performed in a Perkin-Elmer (Emeryville, CA) DNA Thermal Cycler 480 with a temperature profile of 42°C for 30 minutes, one cycle; 94°C for 5 minutes, one cycle; 94°C for 30 seconds, 55°C for 30 seconds, 68°C for 45 seconds, 10 cycles; 94°C for 30 seconds, 55°C for 30 seconds, 68°C for 45 seconds, 5-second extension/cycle, 30 cycles; 68°C for 6 minutes. PCR products were resolved by electrophoresis in 1% agarose gels (SeaKem MR; FMC BioProducts) in 0.04 mol/L of Tris-acetate (pH 8.5) and 0.001 mol/L ethylenediaminetetraacetic acid containing 0.05 μg of ethidium bromide per ml. The UV fluorescence of cDNA bands was measured with a ChemiImagerv5.5 Digital Imaging System. Data are presented as measured fluorescence of co-receptor cDNA/measured fluorescence of β-actin cDNA. Studies were performed on macrophage cultures from at least three animals of either species and each experiment was performed in triplicate.Table 1Primers for Semiquantitative RT-PCR Analyses of Chemokine Receptor mRNAReceptorPrimerPositionSize of PCR product (bp)CCR5Forward: 5′-ATG ACT GAC ATC TAC CTG CTC-3′194–214Reverse: 5′-CAT TAA AGA TGG TCA TCT TGG GG-3′588–610416CXCR4Forward: 5′-CTC ATT TCA ATA GGA TCT TCC TGC C-3′101–125Reverse: 5′-CTT CTA CCC CAA TGA CTT GTG-3′564–584483β-ActinForward: 5′-AAC ACC CCA GCC ATG TAT GT-3′105–124Reverse: 5′-AGC ACT GTG TTG GCG TAC AG-3′600–619514CCR5 gene bank access number: gi 2305193 gb AF011538.1; CXCR4 gene bank access number: gi 1771982 gb U73740.1 MMU73740; β-actin gene bank access number: gi 6636343 gb AF209434.1 AF209434. Open table in a new tab CCR5 gene bank access number: gi 2305193 gb AF011538.1; CXCR4 gene bank access number: gi 1771982 gb U73740.1 MMU73740; β-actin gene bank access number: gi 6636343 gb AF209434.1 AF209434. Statistical analysis was performed using one-way analysis of variance. Archival brain tissue from uninfected Rh macaques and macaques that developed encephalitis after infection with SHIV89.6P and treatment with S. mansoni eggs were used in this study. RNA was isolated by homogenizing weighed portions of brain tissues in Trizol (Life Technologies) followed by precipitation with isopropanol. Air-dried RNA pellets were then resuspended in distilled water and their concentrations determined spectrophotometrically. RNA integrity was confirmed by fractionation on 1.2% (w:v) agarose-formaldehyde gels and staining the ribosomal bands with ethidium bromide. For slot blot analysis, 0.6- to 5.0-μg aliquots of RNA were applied in a final 200 μl solution of 6.15 mol/L formaldehyde and 10× standard saline citrate (1.5 mol/L sodium chloride and 0.15 mol/L sodium citrate, pH 7.0) onto a Hybond-N+ nylon membrane (Amersham, Arlington Heights, IL). An equal amount of ribosomal RNA was loaded onto the slot blot and used as a negative control. The specificity of the slot blot was confirmed by performing Northern analysis. Total RNA (20 μg) from deep white matter regions of the brains from normal and SHIV89.6P-infected Rh macaques with encephalitis was denatured in a loading buffer containing 2.2 mol/L of formaldehyde for 5 minutes at 65°C and electrophoresed in 1% (w:v) agarose gels containing 0.66 mol/L of formaldehyde. The RNAs were transferred to nylon membrane using the capillary transfer technique. After transfer the blots were air-dried and fixed in a UV crosslinker (Stratagene, La Jolla, CA). The probe was labeled with deoxycytidine 5′-[α-32P]triphosphate by a random-primed labeling system (Amersham), with a specific activity of 3.0 × 109 counts/min/μg DNA. Prehybridization (2 hours) and hybridization (overnight) conditions were performed as described.37Covert J Splitter G Detection of cytokine transcriptional profiles from bovine peripheral blood mononuclear cells and CD4+ lymphocytes by reverse transcriptase polymerase chain reaction.Vet Immunol Immunopathol. 1995; 49: 39-50Crossref PubMed Scopus (45) Google Scholar Animals were deeply anesthetized and CSF was withdrawn from the cisterna magnum. The animal was then exsanguinated by aortic cannulation and the vasculature perfused with 1 L of normal saline. The brain and spinal cord were removed and the brain bisected sagitally. The right half was transferred into 10% buffered formalin and the left half was dissected into nine anatomically distinct regions. All samples were snap-frozen in methylbutane chilled in liquid nitrogen and stored at −70°C. Portions of the left half of the brain and the entire right half were kept in buffered formalin for 3 to 4 days. Paraffin sections were stained with hematoxylin and eosin as a preliminary screen for morphological abnormalities, and serial sections were stained with Luxol Fast Blue and Sevier Munger stains for assessment of suspected myelin and axonal abnormalities, respectively.11Raghavan R Stephens EB Joag SV Adany I Pinson DM Li Z Jia F Sahni M Wang C Leung K Foresman L Narayan O Neuropathogenesis of chimeric simian/human immunodeficiency virus infection in pig-tailed and rhesus macaques.Brain Pathol. 1997; 7: 851-861Crossref PubMed Scopus (47) Google Scholar The criterion for choosing SHIV89.6P-infected macaque brains for this study stems from our earlier findings1Buch S Pinson D King CL Raghavan R Hou Y Li Z Adany I Hicks A Villinger F Kumar A Narayan O Inhibitory and enhanci
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