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• W1985918910 abstract "Few rodent models of human immunodeficiency virus type one (HIV-1) infection can reflect the course of viral infection in humans. To this end, we investigated the relationships between progressive HIV-1 infection, immune compromise, and neuroinflammatory responses in NOD/scid-IL-2Rγcnull mice reconstituted with human hematopoietic CD34+ stem cells. Human blood-borne macrophages repopulated the meninges and perivascular spaces of chimeric animals. Viral infection in lymphoid tissue led to the accelerated entry of human cells into the brain, marked neuroinflammation, and HIV-1 replication in human mononuclear phagocytes. A meningitis and less commonly an encephalitis followed cM-T807 antibody-mediated CD8+ cell depletion. We conclude that HIV-1–infected NOD/scid-IL-2Rγcnull humanized mice can, at least in part, recapitulate lentiviral neuropathobiology. This model of neuroAIDS reflects the virological, immunological, and early disease-associated neuropathological components of human disease. Few rodent models of human immunodeficiency virus type one (HIV-1) infection can reflect the course of viral infection in humans. To this end, we investigated the relationships between progressive HIV-1 infection, immune compromise, and neuroinflammatory responses in NOD/scid-IL-2Rγcnull mice reconstituted with human hematopoietic CD34+ stem cells. Human blood-borne macrophages repopulated the meninges and perivascular spaces of chimeric animals. Viral infection in lymphoid tissue led to the accelerated entry of human cells into the brain, marked neuroinflammation, and HIV-1 replication in human mononuclear phagocytes. A meningitis and less commonly an encephalitis followed cM-T807 antibody-mediated CD8+ cell depletion. We conclude that HIV-1–infected NOD/scid-IL-2Rγcnull humanized mice can, at least in part, recapitulate lentiviral neuropathobiology. This model of neuroAIDS reflects the virological, immunological, and early disease-associated neuropathological components of human disease. Reconstitution of mice with human cells susceptible to HIV-1 is an attractive approach to address the basic pathobiological mechanisms of viral infection and for the screening of therapeutic modalities. Application of mice injected intracranially with HIV-1 infected human monocyte-derived macrophages (MDM) alone or in combination with peripheral blood lymphocytes (PBL) reconstitution for the study of neuroAIDS is well established.1Persidsky Y Limoges J McComb R Bock P Baldwin T Tyor W Patil A Nottet HSLM Epstein L Gelbard H Flanagan E Reinhard J Pirruccello SJ Gendelman HE Human immunodeficiency virus encephalitis in SCID mice.Am J Pathol. 1996; 149: 1027-1053PubMed Google Scholar, 2Persidsky Y Buttini M Limoges J Bock P Gendelman HE An analysis of HIV-1-associated inflammatory products in brain tissue of humans and SCID mice with HIV-1 encephalitis.J Neurovirol. 1997; 3: 401-416Crossref PubMed Scopus (108) Google Scholar, 3Poluektova LY Munn DH Persidsky Y Gendelman HE Generation of cytotoxic T cells against virus-infected human brain macrophages in a murine model of HIV-1 encephalitis.J Immunol. 2002; 168: 3941-3949Crossref PubMed Scopus (60) Google Scholar, 4Poluektova L Gorantla S Faraci J Birusingh K Dou H Gendelman HE Neuroregulatory events follow adaptive immune-mediated elimination of HIV-1-infected macrophages: studies in a murine model of viral encephalitis.J Immunol. 2004; 172: 7610-7617Crossref PubMed Scopus (45) Google Scholar, 5Tyor WR Power C Gendelman HE Markham RB A model of human immunodeficiency virus encephalitis in scid mice.Proc Natl Acad Sci USA. 1993; 90: 8658-8662Crossref PubMed Scopus (107) Google Scholar, 6Poluektova L Meyer V Walters L Paez X Gendelman HE Macrophage-induced inflammation affects hippocampal plasticity and neuronal development in a murine model of HIV-1 encephalitis.Glia. 2005; 52: 344-353Crossref PubMed Scopus (50) Google Scholar However, traumatic injury to the brain from injection, PBL activation by the mouse environment and developed graft-versus host reactivity are significant disadvantages of these models. Long-term functional engraftment of a human immune system was achieved in immune deficient mice reconstituted with human hematopoietic stem cells (HSC) in divergent genetic backgrounds: NOD/scid-IL-2Rγcnull (NSG),7Gorantla S Makarov E Finke-Dwyer J Gebhart CL Domm W Dewhurst S Gendelman HE Poluektova LY CD8+ cell depletion accelerates HIV-1 immunopathology in humanized mice.J Immunol. 2010; 184: 7082-7091Crossref PubMed Scopus (65) Google Scholar, 8Watanabe S Terashima K Ohta S Horibata S Yajima M Shiozawa Y Dewan MZ Yu Z Ito M Morio T Shimizu N Honda M Yamamoto N Hematopoietic stem cell-engrafted NOD/SCID/IL2Rgamma null mice develop human lymphoid systems and induce long-lasting HIV-1 infection with specific humoral immune responses.Blood. 2007; 109: 212-218Crossref PubMed Scopus (164) Google Scholar, 9Watanabe S Ohta S Yajima M Terashima K Ito M Mugishima H Fujiwara S Shimizu K Honda M Shimizu N Yamamoto N Humanized NOD/SCID/IL2R{gamma}null mice transplanted with hematopoietic stem cells under nonmyeloablative conditions show prolonged life spans and allow detailed analysis of human immunodeficiency virus type 1 pathogenesis.J Virol. 2007; 81: 13259-13264Crossref PubMed Scopus (70) Google Scholar, 10Ito M Hiramatsu H Kobayashi K Suzue K Kawahata M Hioki K Ueyama Y Koyanagi Y Sugamura K Tsuji K Heike T Nakahata T NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse model for engraftment of human cells.Blood. 2002; 100: 3175-3182Crossref PubMed Scopus (1117) Google Scholar BALB/c-Rag2−/−γc−/− (BRG),11Baenziger S Tussiwand R Schlaepfer E Mazzucchelli L Heikenwalder M Kurrer MO Behnke S Frey J Oxenius A Joller H Aguzzi A Manz MG Speck RF Disseminated and sustained HIV infection in CD34+ cord blood cell-transplanted Rag2-/-gamma c-/- mice.Proc Natl Acad Sci USA. 2006; 103: 15951-15956Crossref PubMed Scopus (197) Google Scholar, 12Gorantla S Sneller H Walters L Sharp JG Pirruccello SJ West JT Wood C Dewhurst S Gendelman HE Poluektova L Human immunodeficiency virus type 1 pathobiology studied in humanized BALB/c-Rag2-/-gammac-/- mice.J Virol. 2007; 81: 2700-2712Crossref PubMed Scopus (107) Google Scholar, 13Berges B Akkina S Folkvord J Connick E Akkina R Mucosal transmission of R5 and X4 tropic HIV-1 via vaginal and rectal routes in humanized Rag2-/- gammac -/- (RAG-hu) mice.Virology. 2008; 373: 342-351Crossref PubMed Scopus (110) Google Scholar, 14An DS Poon B Fang RH Weijer K Blom B Spits H Chen IS Uittenbogaart CH Use of a novel chimeric mouse model with a functionally active human immune system to study human immunodeficiency virus type 1 infection.Clin Vaccine Immunol. 2007; 14: 391-396Crossref PubMed Scopus (57) Google Scholar, 15Zhang L Kovalev GI Su L HIV-1 infection and pathogenesis in a novel humanized mouse model.Blood. 2007; 109: 2978-2981PubMed Google Scholar and NOD/scid mice reconstituted with fetal thymus/liver and HSC (BLT).16Sun Z Denton P Estes J Othieno F Wei B Wege A Melkus M Padgett-Thomas A Zupancic M Haase A Garcia J Intrarectal transmission, systemic infection, and CD4+ T cell depletion in humanized mice infected with HIV-1.J Exp Med. 2007; 204: 705-714Crossref PubMed Scopus (212) Google Scholar, 17Kumar P Ban HS Kim SS Wu H Pearson T Greiner DL Laouar A Yao J Haridas V Habiro K Yang YG Jeong JH Lee KY Kim YH Kim SW Peipp M Fey GH Manjunath N Shultz LD Lee SK Shankar P T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice.Cell. 2008; 134: 577-586Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar Despite rapid research advances made in these rodent animal models on HIV-1 disease pathobiology, their importance for studies of HIV-1–related complications, including neuroAIDS, have not been explored. Central nervous system (CNS) inflammatory responses are driven by chronic low-level infection affecting activation of brain mononuclear phagocytes (MP; blood borne macrophage and microglia).18Eden A Price RW Spudich S Fuchs D Hagberg L Gisslen M Immune activation of the central nervous system is still present after >4 years of effective highly active antiretroviral therapy.J Infect Dis. 2007; 196: 1779-1783Crossref PubMed Scopus (145) Google Scholar, 19Clifford DB HIV-associated neurocognitive disease continues in the antiretroviral era.Top HIV Med. 2008; 16: 94-98PubMed Google Scholar, 20Ances BM Ellis RJ Dementia and neurocognitive disorders due to HIV-1 infection.Semin Neurol. 2007; 27: 86-92Crossref PubMed Scopus (240) Google Scholar, 21Letendre S Ances B Gibson S Ellis RJ Neurologic complications of HIV disease and their treatment.Top HIV Med. 2007; 15: 32-39PubMed Google Scholar, 22Jones G Power C Regulation of neural cell survival by HIV-1 infection.Neurobiol Dis. 2006; 21: 1-17Crossref PubMed Scopus (82) Google Scholar HIV-1–associated neurocognitive disorders (HAND), in particular, are a common cause of morbidity for virus-infected people.23Antinori A Arendt G Becker JT Brew BJ Byrd DA Cherner M Clifford DB Cinque P Epstein LG Goodkin K Gisslen M Grant I Heaton RK Joseph J Marder K Marra CM McArthur JC Nunn M Price RW Pulliam L Robertson KR Sacktor N Valcour V Wojna VE Updated research nosology for HIV-associated neurocognitive disorders.Neurology. 2007; 69: 1789-1799Crossref PubMed Scopus (1931) Google Scholar However, human disease can be paralleled only in studies of simian immunodeficiency virus (SIV)-infected rhesus macaques.24Narayan O Joag SV Stephens EB Selected models of HIV-induced neurological disease.Curr Top Microbiol Immunol. 1995; 202: 151-166PubMed Google Scholar, 25Lackner AA Smith MO Munn RJ Martfeld DJ Gardner MB Marx PA Dandekar S Localization of simian immunodeficiency virus in the central nervous system of rhesus monkeys.Am J Pathol. 1991; 139: 609-621PubMed Google Scholar Moreover, only the later stages of disease have been studied in detail where encephalitis is seen as a result of virus-infected MP and MP-derived multinucleated giant cells (MGC), astrogliosis, myelin pallor, and neuronal dropout predominate.26Budka H Wiley CA Kleihues P Artigas J Asbury AK Cho ES Cornblath DR Dal Canto MC DeGirolami U Dickson D et al.HIV-associated disease of the nervous system: review of nomenclature and proposal for neuropathology-based terminology.Brain Pathol. 1991; 1: 143-152Crossref PubMed Scopus (321) Google Scholar, 27Kraft-Terry SD Buch SJ Fox HS Gendelman HE A coat of many colors: neuroimmune crosstalk in human immunodeficiency virus infection.Neuron. 2009; 64: 133-145Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar Little is known about the early stages of disease after acute infection. Here the pathobiological findings include aseptic meningitis. This can consist of inflammatory T-cell reaction with overt vasculitis and leptomeningitis. Immune activation of brain parenchyma with increased number of microglial cells, up-regulation of major histocompatibility complex class II antigens, and local production of cytokines was described.28Gray F Scaravilli F Everall I Chretien F An S Boche D Adle-Biassette H Wingertsmann L Durigon M Hurtrel B Chiodi F Bell J Lantos P Neuropathology of early HIV-1 infection.Brain Pathol. 1996; 6: 1-15Crossref PubMed Scopus (238) Google Scholar, 29An SF Giometto B Scaravilli F HIV-1 DNA in brains in AIDS and pre-AIDS: correlation with the stage of disease.Ann Neurol. 1996; 40: 611-617Crossref PubMed Scopus (47) Google Scholar In both acute and chronic infection phases, CD8+ cytotoxic T lymphocytes (CTL) play a dual role: control viral replication and modulate neurological disease.30Marcondes MC Burudi EM Huitron-Resendiz S Sanchez-Alavez M Watry D Zandonatti M Henriksen SJ Fox HS Highly activated CD8(+) T cells in the brain correlate with early central nervous system dysfunction in simian immunodeficiency virus infection.J Immunol. 2001; 167: 5429-5438Crossref PubMed Scopus (96) Google Scholar, 31McCrossan M Marsden M Carnie FW Minnis S Hansoti B Anthony IC Brettle RP Bell JE Simmonds P An immune control model for viral replication in the CNS during presymptomatic HIV infection.Brain. 2006; 129: 503-516Crossref PubMed Scopus (46) Google Scholar, 32Sopper S Sauer U Hemm S Demuth M Muller J Stahl-Hennig C Hunsmann G ter Meulen V Dorries R Protective role of the virus-specific immune response for development of severe neurologic signs in simian immunodeficiency virus-infected macaques.J Virol. 1998; 72: 9940-9947PubMed Google Scholar, 33Williams K Westmoreland S Greco J Ratai E Lentz M Kim WK Fuller RA Kim JP Autissier P Sehgal PK Schinazi RF Bischofberger N Piatak M Lifson JD Masliah E Gonzalez RG Magnetic resonance spectroscopy reveals that activated monocytes contribute to neuronal injury in SIV neuroAIDS.J Clin Invest. 2005; 115: 2534-2545Crossref PubMed Scopus (107) Google Scholar, 34Bissel SJ Wang G Trichel AM Murphey-Corb M Wiley CA Longitudinal analysis of monocyte/macrophage infection in simian immunodeficiency virus-infected. CD8+ T-cell-depleted macaques that develop lentiviral encephalitis.Am J Pathol. 2006; 168: 1553-1569Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 35Schmitz JE Kuroda MJ Santra S Sasseville VG Simon MA Lifton MA Racz P Tenner-Racz K Dalesandro M Scallon BJ Ghrayeb J Forman MA Montefiori DC Rieber EP Letvin NL Reimann KA Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes.Science. 1999; 283: 857-860Crossref PubMed Scopus (1942) Google Scholar, 36Schmitz JE Simon MA Kuroda MJ Lifton MA Ollert MW Vogel CW Racz P Tenner-Racz K Scallon BJ Dalesandro M Ghrayeb J Rieber EP Sasseville VG Reimann KA A nonhuman primate model for the selective elimination of CD8+ lymphocytes using a mouse-human chimeric monoclonal antibody.Am J Pathol. 1999; 154: 1923-1932Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 37Matano T Shibata R Siemon C Connors M Lane HC Martin MA Administration of an anti-CD8 monoclonal antibody interferes with the clearance of chimeric simian/human immunodeficiency virus during primary infections of rhesus macaques.J Virol. 1998; 72: 164-169PubMed Google Scholar, 38Sadagopal S Lorey SL Barnett L Basham R Lebo L Erdem H Haman K Avison M Waddell K Haas DW Kalams SA Enhancement of human immunodeficiency virus (HIV)-specific CD8+ T cells in cerebrospinal fluid compared to those in blood among antiretroviral therapy-naive HIV-positive subjects.J Virol. 2008; 82: 10418-10428Crossref PubMed Scopus (26) Google Scholar In HIV-1–infected patients and SIV-infected monkeys, virus-induced innate and adaptive immune responses trigger activation of oxidative stress and lead to neuronal injury. These include secretion of viral proteins, proinflammatory cytokines, platelet-activating factor, arachidonic acid metabolites, quinolinic acid, and activation of inducible nitric oxide synthase (iNOS).2 iNOS produced in an oxidative environment with excessive production of NO leads to peroxynitrite formation and cell toxicity.39Koprowski H Zheng YM Heber-Katz E Fraser N Rorke L Fu ZF Hanlon C Dietzschold B In vivo expression of inducible nitric oxide synthase in experimentally induced neurologic diseases [published erratum appears in Proc Natl Acad Sci USA: 1993 Jun 1;90(11):5378].Proc Natl Acad Sci USA. 1993; 90: 3024-3027Crossref PubMed Scopus (472) Google Scholar, 40Adamson DC McArthur JC Dawson TM Dawson VL Rate and severity of HIV-associated dementia (HAD): correlations with Gp41 and iNOS.Mol Med. 1999; 5: 98-109Crossref PubMed Google Scholar, 41Lane TE Buchmeier MJ Watry DD Fox HS Expression of inflammatory cytokines and inducible nitric oxide synthase in brains of SIV-infected rhesus monkeys: applications to HIV-induced central nervous system disease.Mol Med. 1996; 2: 27-37Crossref PubMed Google Scholar, 42Nuovo G Alfieri M AIDS dementia is associated with massive, activated HIV-1 infection and concomitant expression of several cytokines.Mol Med. 1996; 2: 358-366Crossref PubMed Google Scholar, 43Hori K Burd PR Furuke K Kutza J Weih KA Clouse KA Human immunodeficiency virus-1-infected macrophages induce inducible nitric oxide synthase and nitric oxide (NO) production in astrocytes: astrocytic NO as a possible mediator of neural damage in acquired immunodeficiency syndrome.Blood. 1999; 93: 1843-1850Crossref PubMed Google Scholar However, the pathobiology of the early stage HAND remains unclear because investigations are not readily done in infected patients. Here, we investigated whether CNS pathologies could be seen in HIV-1–infected humanized mice. Several notable observations were made to human cells repopulation, progressive HIV-1 infection, and CD8+ cell depletion. First, brains were repopulated with human CD163+, CD14+ macrophages, predominantly located in meninges and perivascular spaces. Second, productive infection accelerated the entry of human cells into the brain. This was seen by immunostaining for human CD163 and HLA-DR+ cells and the expression of human HLA-DQ by real-time PCR. HIV-1 p24+ cells with macrophage and lymphocyte morphology were seen in the meninges and perivascular spaces. Third, CD8+ T cell depletion initiated by cM-T807 antibodies resulted in increased HIV-1gag RNA and iNOS expression in the brain. Fourth, the development of a meningitis and rarely a meningoencephalitis were observed. These findings, taken together, demonstrate a natural progression of HIV-1 CNS disease in rodents in a fashion qualitatively similar to SIV-infected macaques. This is the first study, to our knowledge, showing relationships between ongoing viral replication and CNS HIV-1 pathobiology in mice permanently reconstituted with a human immune system. NOD/scid-IL-2Rγcnull mice were obtained from the Jackson Laboratories (Bar Harbor, ME) and were bred under specific pathogen-free conditions in accordance with ethical guidelines for care of laboratory animals at the University of Nebraska Medical Center (UNMC) as set forth by the National Institutes of Health. Human umbilical cord blood was obtained with parental written informed consent from healthy full-term newborns (Department of Gynecology and Obstetrics, UNMC). After density gradient centrifugation, CD34+ cells were enriched using immunomagnetic beads according to the manufacturer's instructions (CD34+ selection kit; Miltenyi Biotec Inc., Auburn, CA). Purity of CD34+ cells was evaluated by flowcytometry and was >90%. Cells were either frozen or immediately transplanted into newborn mice irradiated at 1 Gy using a C9 cobalt 60 source (Picker Corporation, Cleveland, OH). The number of animal deaths was as follows: i) acute death during first 7 days after sublethal irradiation of newborn pups by 1 Gy <1%; ii) death during 1–2 months postirradiation <1%. CD34+ cells were injected intrahepatically (i.h.) at 105 cells per mouse in 20 μl phosphate-buffered saline (PBS) using a 30-gauge needle. Newborns received cells from single donors. Two to seven littermates were reconstituted with cells isolated from cord blood sample derived from one donor. The number of animals reconstituted was dependent on the number of CD34+ cells isolated from cord blood. Mice were weaned at 3 weeks of age. Mice were then evenly distributed between different experimental groups (Table 1). Animals that developed sign of chronic graft-versus host disease from 5 to 6 months of age (such as hair loss, loss of weight was observed in ∼5%) were sacrificed and were not included in the analysis.Table 1Profiles of HIV-1–Infected and CD8+ Cell–Depleted Animals Used in This Study*All rodent brains were evaluated by immunohistochemistry.Experimental GroupsNumber of AnimalsAge, WeeksHuman CD45+ Cells, %Viral Load, log10 RNA Copies/mlControl†Control, uninfected reconstituted mice.1825 (21–32)25.8 (3.8–50.5)NAHIV-1–infected‡Productive viral infection was assessed by the presence of viral RNA in peripheral blood, HIV-1p24+ cells in lymphoid tissues or HIV-1gag mRNA in brain tissue.2229 (26–36)28.7 (6.0–50.2)5.06 (4.12–6.18)HIV-1–infected/CD8+ cell depleted§Animals were CD8+ cell depleted at 2 weeks (n = 5) or 5–7 weeks after infection. Median and the range are shown in parentheses.1229 (26–32)20.2 (3.1–52.6)5.58 (4.39–7.54)CD8+ cell–depleted731 (26–35)16.5 (3.1–80.6)NA* All rodent brains were evaluated by immunohistochemistry.† Control, uninfected reconstituted mice.‡ Productive viral infection was assessed by the presence of viral RNA in peripheral blood, HIV-1p24+ cells in lymphoid tissues or HIV-1gag mRNA in brain tissue.§ Animals were CD8+ cell depleted at 2 weeks (n = 5) or 5–7 weeks after infection. Median and the range are shown in parentheses. Open table in a new tab The CCR5 coreceptor-using HIV-1ADA strain was propagated in human MDM. Monocytes were isolated from leukopaks and generating MDM.44Gendelman HE Orenstein JM Martin MA Ferrua C Mitra R Phipps T Wahl LA Lane HC Fauci AS Burke DS et al.Efficient isolation and propagation of human immunodeficiency virus on recombinant colony-stimulating factor 1-treated monocytes.J Exp Med. 1988; 167: 1428-1441Crossref PubMed Scopus (625) Google Scholar Viral preparations were screened and found to be negative for endotoxin (<10 pg/ml) (Associates of Cape Cod, Woods Hole, MA) and mycoplasma (Gen-Probe II; Gen-Probe, San Diego, CA). The viral titers were assayed on MDM and 105 tissue culture infectious dose50 (TCID50)/ml. HIV-1ADA was injected intraperitoneally (i.p.) at 104 TCID50. The levels of viral RNA copies/ml were analyzed by automated COBAS Amplicor System (Roche Molecular Diagnostics, Basel, Switzerland). For assay use, mouse plasma samples (20 μl each) were diluted to 700 μl with normal human serum which increased the detection limit to 1750 viral RNA copies/ml. HIV-1 infection was confirmed by virologic and histological examination in 26 animals (Table 1). Eighteen reconstituted animals not exposed to HIV-1 served as controls. No mortalities were induced by HIV-1 infection. The cM-T807 mAb was obtained from the National Institutes of Health/National Center for Research Resources. Each mouse received 10 mg/kg of cM-T807 subcutaneously (s.c.) and 5 mg/kg i.p. at 3 day intervals. Peripheral blood samples were collected from the submandibular vein in EDTA-coated tubes by using lancets (MEDIpoint, Inc., Mineola, NY) or by cardiocentesis at the end of observation. Blood leukocytes and cells suspensions from half of spleen were tested for human pan-CD45, CD3, CD4, CD8, CD11c, CD14, CD19, and HLA-DR markers as seven-color combinations. Antibodies and isotype controls were obtained from BD PharMingen (San Diego, CA), and staining was analyzed with a FACSDiva (BD Immunocytometry Systems, Mountain View, CA). Presence of CD8+ cells before and after depletion was assessed by using anti–CD8-PE clone DK25 (Dako, Carpinteria, CA). Results were expressed as percentages of total number of gated lymphocytes. The gating strategy was human CD45→CD3→CD4/CD8, CD45→CD19, CD45→CD14. Brains were removed immediately after euthanasia and processed. Tissue was divided by hemispheres (for sagittal section histology and real time PCR) or left as a whole for horizontal sectioning. Tissue samples (brain and spleen half) were fixed with 4% paraformaldehyde overnight and embedded in paraffin. Five-micron-thick sections were stained with mouse monoclonal antibodies for CD163 (clone 10D6, 1:50, Vector Laboratories, Burlingame, CA), HLA-DQ/DP/DR (clone CR3/43, 1:100), CD8 (clone 144, 1:50), CD68 (clone KP-1, 1:50), HIV-1 p24 (clone Kal-1, 1:10), CD3 (1:100, rabbit polyclonal), and glial fibrillary acidic protein (GFAP; 1:1000 rabbit polyclonal). Except for CD163 antibody, all other antibodies were obtained from Dako. Mouse monoclonal antibodies to human CD14 (clone 7, 1:50) and CD4 (clone 1F6, 1:40) were purchased from Biocare Medical, LLC (Concord, CA) and Novocastra (Norwell, MA), respectively. Ionized calcium-binding adaptor molecule 1 for mouse/human microglial/macrophages cells (Iba-1, 1:500; rabbit polyclonal) were purchased from Wako Chemicals USA, Inc. (Richmond, VA). The polymer-based HRP- and AP-conjugated anti-mouse and anti-rabbit Dako EnVision systems were used as secondary detection reagents, and 3,3′-diaminobenzidine (DAB) and Permanent Red (Dako) were used as chromogens. All paraffin-embedded sections were counterstained with Mayer's hematoxylin. Deletion of primary Ab or mouse IgG served as controls. Images were obtained by Optronics digital camera fixed to a Nikon Eclipse E800 (Nikon Instruments, Melville, NY) using MagnaFire 2.0 software. Tissue sections (2–4 sagittal, average total area of 78.8 mm2) were examined for human HLA-DR, CD163 and CD8 immunopositive cells using a ×40 objective. Number of cells in meninges, perivascular spaces, and parenchyma/section were calculated for each mouse. The assessments of cellular infiltrates in meninges, perivascular spaces, and parenchyma were done in blinded manner by four independent investigators. Intensity of astrocytes staining (GFAP), microglia and blood-borne macrophages (Iba-1) were scored by two investigators using ×10 and ×20 objectives. Findings were compared to animals that were not manipulated (score of 0). A score of 1 represented few activated astrocytes or activated microglial cells. A score of 2 is consistent with moderate activation of astrocytes and microglia. A score of 3 showed hypertrophic astrocytes with concomitant processes and microglial nodules. Total RNA from cortex, midbrain and cerebellum/brain stem sections were extracted with TRIzol (Invitrogen, Carlsbad, CA) and reverse transcribed to cDNA with random hexamers and Moloney murine leukemia virus reverse transcriptase (Invitrogen). Real-time quantitative PCR was performed with cDNA using an ABI PRISM 7000 sequence detector (Applied Biosystems, Foster City, CA). Human HLA-DQ,45Locardi C Puddu P Ferrantini M Parlanti E Sestili P Varano F Belardelli F Persistent infection of normal mice with human immunodeficiency virus.J Virol. 1992; 66: 1649-1654PubMed Google Scholar mouse TNF-α, Mac-1, GAPDH expression were analyzed using TaqMan gene expression assays, and for HIV-1gag the primers and probe used were: forward, 5′-ACATCA AGCCATGCAAAT-3′; reverse, 5′-ATCTGGCCTGGT GCAATAGG-3′; and probe (FAM), 5′-CATCAATGAGGA AGCTGCAGAATGGGATAG A-3′ (TAMRA).46Cota M Kleinschmidt A Ceccherini-Silberstein F Aloisi F Mengozzi M Mantovani A Brack-Werner R Poli G Upregulated expression of interleukin-8. RANTES and chemokine receptors in human astrocytic cells infected with HIV-1.J Neurovirol. 2000; 6: 75-83Crossref PubMed Scopus (58) Google Scholar Expression of mouse GFAP and iNOS were determined using SYBR-green method and the primers were: GFAP, forward 5′-ACTGGGTACCATGCCACGTT-3′; reverse 5′-GGGAGTGGAGGAGTCATTCG-3′, and iNOS forward 5′-GGCAGCCTGTGAGACCTTTG-3′; reverse 5′-GAAGCGTTTCGGGATCTGAA-3′. Quantification was done using standard curve method, as described in User Bulletin 2 obtained with ABI PRISM 7000 sequence detector. Gene expression including HIV-1 gag was expressed relative to GAPDH and used as an endogenous control. All PCR reagents were obtained from Applied Biosystems. Data were analyzed using GraphPad Prizm and Excel softwares; statistical tests used were nonparametric t-test (Mann–Whitney U-test), non-parametric Spearman correlation test, chi test (χ2), and one-way analysis of variance for comparisons of multiple groups. A P value of <0.05 was considered statistically significant. We reconstituted newborn NSG mice with CD34+ human HSC isolated from umbilical cord blood. Details of different experimental groups of animals are summarized in Table 1. Human lymphoid tissue development was evaluated by flow cytometric analysis of human cells in peripheral blood (CD45, CD3, CD4, CD8, CD19, and CD14) and in spleens at the end of observation. This was performed to determine the relative abundance of immune cell groups. We observed that HIV-1–infected animals showed peak viremia 5–6 weeks after viral challenge with HIV-1ADA at 104 TCID50. All infected animals by this time had detectable viral load at the range 3.76–6.50 Log10 copies/ml. CD8+ cell depletion was used to accelerate viral dynamics with two sequential injections of cM-T807 antibodies (s.c. and i.p. within 3 days interval), either at 2 weeks or 5–7 weeks after infection. After treatment, human CD8+ cells were absent in blood but reappeared in circulation 2–3 weeks postdepletion. CD8+ cell depletion at earlier stage of infection (2w postinfection) resulted in accelerated increase in viral load and Δ log10 was 2.02 (P = 0.04). In mice with established infection for 5–7 weeks, CD8+ cell depletion resulted in a Δ log10 of 0.87 (P = 0.013). In the same stage of disease, non-depleted animals did show decline of viral load over the same time interval. Transient antibody-mediated depletion of CD8+ cells increased the numbers and level of CD3+CD4+ cell proliferation. CD8+ cell depletion affected total number of human cells in lymphoid tissues. The effect of CD8+ cell depletion on viral load dynamics, total CD4+ T cells, other immune parameters, and pathologies were previously discussed.7Gorantla S Makarov E Finke-Dwyer J Gebhart CL Domm W Dewhurst S Gendelman HE Poluektova LY CD8+ cell depletion accelerates HIV-1 immunopathology in humanized mice.J Immunol. 2010; 184: 7082-7091Crossref PubMed Scopus (65) Google Scholar Repopulation of mouse brain with human cells was assessed by immunohistochemistry. Figure 1 represents brain sections from an uninfected mouse stained for different human cell markers. Human specific antibodies to CD163, CD14, HLA-DR, and CD68 detected MP in meninges. Twenty to 50% of CD163+ staining colocalized with human HLA-DR. Few human CD163+/HLA-DR+ cells had dendritic cell morphology. Among the reconstituted mice, a marked difference in the number of human cells infiltrated" @default.
• W1985918910 created "2016-06-24" @default.
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• W1985918910 date "2010-12-01" @default.
• W1985918910 modified "2023-10-17" @default.
• W1985918910 title "Links between Progressive HIV-1 Infection of Humanized Mice and Viral Neuropathogenesis" @default.
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• W1985918910 doi "https://doi.org/10.2353/ajpath.2010.100536" @default.
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• W1985918910 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/21088215" @default.
• W1985918910 hasPublicationYear "2010" @default.

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