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Effects of low dose gamma radiation on the human immunodeficiency virus-1 Xu, Yingdong 2001

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EFFECTS OF LOW DOSE G A M M A RADIATION ON THE H U M A N IMMUNODEFICIENCY VIRUS-1 BY YINGDONG X U B.Sc, Suzhou Medical College, 1983 M.Sc, Suzhou Medical College, 1988 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE F A C U L T Y OF G R A D U A T E STUDIES (EXPERIMENTAL MEDICINE PROGRAM) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March 2001 © Yingdong Xu, 2001 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT Lymphocytes are highly radiosensitive and the major host cells of HIV-1. To evaluate the effects of low doses of gamma radiation on HIV-1 replication, models of acute HIV-1 infection, PBMCs of HIV-1-infected patients and HIV-1 positive cell lines were used. The results demonstrated that HTLV-IIIB virus (laboratory strain of HIV-1) replication significantly increased in cultures initiated from PBMCs exposed to gamma radiation at the dose of 50 cGy given prior to PHA stimulation and acute infection. Similar results were also obtained in purified CD4 cells. The mechanism underlying these observations may be related to oxidative stress since pre-infection treatment with 35 nM H2O2 increased the susceptibility of PBMCs to acute infection in a similar way. Exposure of the cells to gamma radiation after in vitro infection had no significant stimulatory effect. In HIV-1-infected PBMCs taken from a patient with high levels of HIV-1 replication in the plasma, viral replication was clearly stimulated by exposure to 50 cGy as compared to the non-irradiated control. The highest stimulatory effect was found when acute infection following host cell irradiation was delayed for 24 hours. It seems that this stimulatory effect is due to host cells becoming more susceptible to viral infection rather than latent viral DNA becoming transcriptionally active following low doses of ionizing radiation. Radiation effects on viral replication are closely related to its timing, the stimulatory effects being more obvious in the early stage of the viral life cycle (perhaps before the integration of viral DNA). The stimulatory response of low doses of gamma radiation on viral replication is a trigger-type rather than dose-dependent response. Results support a mechanism in which low doses of gamma exposure induce IL2a receptors on the surface of mitogen-stimulated PBMCs that makes cells more susceptible to virus infection. Viral replication is continuous throughout the course of HIV-1 infection and a delicate balance among a wide array of host factors likely determines the net rate of viral replication. In light of our results, we recommend that extra precautions be considered when treating HIV-1 infected patients with radiation for medical purposes. ii T A B L E OF CONTENTS Page Abstract i i List of Tables vii List of Figures x List of Abbreviations and Symbols xi Acknowledgements xv Dedication xvi Parti 1.0 General Discussion 1 1.1 HIV-1 1 1.2 Life Cycle of HIV-1 5 1.3 Immunopathogenesis of HIV-1 Infection 11 2.0 Review of the Effects of Radiation on Lymphocytes and Viruses 14 2.1 Radiation Biophysics - An Introduction 14 2.2 Radiation Effects on Viruses 17 2.3 Specific Effects of Low Doses of Radiation on the Immune System 19 3.0 Ionizing Radiation and HIV-1: Practical and Theoretical Considerations. 22 3.1 The Possible Role of Radiation to Prevent HIV-1 Infection 24 3.2 Radiosensitivity of HIV-1 27 3.2.1. Direct and Indirect Radiation Effects on HIV-1 27 3.2.2. Effect of Radiation Sterilization on Biological Function 28 3.2.3. What is the Proper Dose of Radiation to Inactivate HIV-1? 30 3.3 Radiation Therapy of HIV-1-associated Conditions 30 3.4 Experimental Applications of Radiation Therapy 37 iii 3.5 Radiation-Induced Enhancement of HIV-1 Replication: An Area of Concern? 38 4.0 Thesis Hypothesis, General Objective and Specific Objectives 44 Part II 5.0 Materials, Methods and Infection Models 45 5.1 Material and Methods 45 5.1.1. Isolation and stimulation of donor peripheral blood mononuclear cells (PBMCs) 45 5.1.2. HTLV-IIIB and Titration of viral stock 45 5.1.3. Separation of CD4 and CD8 cells 46 5.1.4. Radiation facilities 47 5.1.5. Radiation and interleukin-2ct receptor detection 47 5.1.6. HIV-1 infection 48 5.1.7. Detection of p24 in culture supernatants 49 5.1.8. Detection of IL-2R and IL-10 in culture supernatants 49 5.1.9. DNA polymerase chain reaction (PCR) 50 5.1.10. Detection of CD3, CD4 and IL-2a receptor on HIV-1 infected cells 51 5.1.11. Stable CD4+ cell lines 52 5.1.12. Statistical analysis 52 5.2 HIV-1 Infection Models 53 5.2.1. Acute infection models 53 5.2.1.1. Pre-infection radiation model with PBMCs 53 5.2.1.2. Pre-infection radiation model with CD4, CD8 55 5.2.1.3. Post-infection radiation model with PBMCs 56 5.2.2. Evaluation of PBMCs of HIV-1-infected patients 56 5.2.3. Evaluation of cell lines stably infected with HIV-1 57 iv Part III 6.0 Results 6.1. HIV-1 infection in PBMCs of different donors 59 6.2 Effect of acute HIV-1 infection on IL2Ra expression on PHA-stimulated PBMCs 6 1 6.3 Effect of gamma radiation on IL- 2Ra expression 65 6.4 IL- 2Ra expression and cell-cell communication between non-irradiated and irradiated PBMCs 68 6.5 Effect of radiation of PBMCs on H T L V IIIB replication following subsequent acute infection ("pre-infection" model) 72 6.6 Kinetics of viral replication in PBMCs exposed to radiation prior to acute HIV-1 infection 77 6.7 Effect of radiation and H 2 0 2 of CD4+ cells on H T L V IIIB replication following subsequent acute infection ("pre-infection" model) 79 6.8 Effect of radiation of PBMCs cells on H T L V IIIB replication administered after acute infection ("post-infection" model) 85 6.9 Radiation, cell-cell communication and HIV-1 replication in the acute infection model ("pre-infection" model) 88 6.10 Effect of radiation of PBMCs and cell-to-cell communication on H T L V IIIB replication administered after acute infection ("post-infection" model) 91 6.11 Effect of "post-infection" irradiated culture supernatants on HIV-1 replication 94 6.12 Effects of gamma radiation on PBMCs of HIV-1-infected patients 96 6.13 Effects of gamma radiation on viral replication in chronically infected cell lines 100 6.14 Effect of radiation on viral replication in the presence of zidovudine (ZDV) 105 6.15 Radiation effect on IL10 expression 107 v Part IV 7.0 General Discussion 110 7.1 UV, Ionizing Radiation, and HIV-1 110 7.2 Radiation, Free Radicals, Cell Activation, and IL-2Ra Expression 113 7.3 Acute Infection Models, PBMCs of HIV-1-infected individuals and Stable HIV-1-infected CD4+ cell lines 116 7.4 Differential Radiation Effects and Cell-Cell Communication 120 7.5 Suggestions for Future Experiments 122 References 124 vi List of Tables Table 1. Function of HIV-1 genes 4 Table 2. Comparison of the visible electromagnetic spectrum with that of ultraviolet, X-ray and gamma radiation 23 Table 3. Inactivation of HIV-1 by gamma radiation 26 Table 4. Radiation therapy of HIV-1- associated conditions 34 Table 5. Radiation-induced enhancement of HIV-1 replication 40 Table 6. Experimental evaluation of the effects of radiation on HIV-1 replication 43 Table 7. Percentage of PBMCs expressing IL-2Ra over time as a function of PHA stimulation, acute HIV-1 infection and/or exposure to gamma radiation in a single experiment 63 Table 8. Percentage of PBMCs showing both IL-2Ra and CD4 after radiation, PHA stimulation and acute HIV-1 infection in a single experiment 64 Table 9. Enhancement of IL-2a receptor expression by exposure of ConA-stimulated PBMCs 66 TablelO. Enhancement of IL-2a receptor expression by exposure of ConA-stimulated PBMCs 67 Tablel 1. The percentage of PBMCs expressing IL-2Ra after ConA stimulation 70 Table 12. Effect of irradiated cells or medium on the expression of IL-2Ra on non-irradiated control cells 71 Table 13 . IL-2Ra levels (pg/mL) in culture supernatants following infection of PHA-stimulated control PBMCs taken from donor A and D 74 Table 14. Comparison of the mean, median and range of p24 antigen levels in culture supernatants of control and irradiated PBMCs infected with HTLV-IIIB 78 Table 15. p24 antigen levels in culture supernatants following infection of PHA-stimulated control, irradiated and H202-treated purified CD4 cells taken from donor F 81 Table 16. p24 antigen levels in culture supernatants following infection of PHA-stimulated control, irradiated and H202-treated PBMCs taken from donor G 82 vii Table 17. p24 antigen levels in culture supernatants following infection of PHA-stimulated control, irradiated and H202-treated CD4 cells taken from donor G 83 Table 18. p24 antigen levels in culture supernatants following infection of PHA-stimulated control, irradiated and H2O2 treated recombined CD4 and CD8 cells taken from donor G 84 Table 19. p24 antigen levels in culture supernatants of HTLV-IIIB-infected cells taken form donor H exposed to radiation immediately after infection 86 Table 20. p24 antigen levels in culture supernatants of HTLV-IIIB-infected cells taken from donors I-N exposed to radiation 18 hours after infection 87 Table 21. p24 antigen levels in culture supernatants of mixed irradiated and non-radiation PBMCs taken from donor D 89 Table 22. p24 antigen levels in culture supernatants of mixed irradiated and non-radiation PBMCs taken from donor A 90 Table 23. p24 antigen levels in culture supernatants of mixed irradiated and non-radiation PBMCs taken from donor H 92 Table 24. p24 antigen levels in culture supernatants of HTLV-IIIB-infected cells taken from donors I-L exposed to radiation 18 hours after infection 93 Table 25. p24 levels of viral infected non-irradiated cells which were incubated in the post- viral irradiated culture supernatant 95 Table 26. p24 level in the culture supernatant of PBMCs from an HIV-1-positive patients with a positive baseline plasma p24 antigen level 97 Table 27. Proviral load in PBMCs from HIV-1- positive patients following 21 days in culture 98 Table 28. Proviral load in PBMCs from HIV-1- positive patients following 21 days in culture 99 Table 29. p24 antigen levels in culture supernatants of H9RF cells following radiation 101 Table 30. p24 antigen levels in culture supernatants of mixed irradiated and non-irradiated H9RF cells 102 Table 31. p24 antigen levels in culture supernatants of 8E5 cells 104 viii Table 32. p24 antigen levels in culture supernatants of mixed irradiated and non-irradiated 8E5 cells 105 Table 33. p24 antigen levels in the supernatants of cultures of acutely infected PBMCs in the presence of 0.1 uM ZDV 106 Table 34. IL-10 levels in the culture supernatants of PBMCs exposed to radiation followed by acute HIV-1 infection 108 Table 35. IL-10 levels in the culture supernatants of mixed irradiated and non-irradiated PBMCs acutely infected with HIV-1 109 ix List of Figures Figure 1. Virion structure of HIV -1 2 Figure 2. Genomic organization of HIV -1 3 Figure 3. The general overview of HIV-1 life cycle 6 Figure 4. Specific binding regions of interest in the viral LTR 9 Figure 5. Development of radiobiological effect 16 Figure 6. H T L V IIIB infection in PBMCs of different donors 60 Figure 7. The p24 antigen levels in the culture supernatants following HIV-1 infection of PHA-stimulated PBMCs exposed to 0-200 cGy gamma-radiation 75 Figure 8. PBMCs counts (106/mL) on day 11 following H T L V IIIB infection 76 r List of Abbreviations and Symbols A D C C antibody depend cytotoxicity A E C L Atomic Energy of Canada Limited AIDS acquired immunodeficiency syndrome A N O V A analysis of variance C A capsid CAF CD8 activating factor CCR3 beta-chemokine receptor C-C CKR-3 CCR5 beta-chemokine receptor C-C CKR-5 CD clusters of differentiation cDNA complementary DNA cGy 0.01 Gray oc-chemokine C-X-C chemokine, preferentially act on polymorphonuclear leukocytes P-chemokine C-C chemokine, preferentially acts on monocytes, including RANTES, MIP-1 alpha, MIP-1 beta cis-UCA cis isoform urocanic acid C M V cytomegalovirus ConA concanavalin A COUP chicken ovalbumin upstream promoter C T L Cytotoxic T lymphocytes CXCR4 (fusin) alpha-chemokine receptor CXC-CKR-4 D10 average dose of gamma radiation necessary to reduce the amount of viral infectivity by 90%. DNA deoxyribonucleic acid dsDNA double strand DNA dsRNA double strand RNA EBV Epstein-Barr virus ELISA enzyme-linked immunosorbent assay xi ELOSA enzyme-linked oligonucleotidesorbent assay ESR electron spin resonance gpl20 glycoprotein 120 found in HIV envelope Gy Gray HBV hepatitis B virus HHV6 human herpesvirus 6 HIV-1 human immunodeficiency virus type 1 HIV-2 human immunodeficiency virus type 2 HRS hyper radiosensitivity HSV herpes simplex virus HTLV-I human T-cell leukemia type I virus HTLV-II human T-cell leukemia type II virus H T L V IIIB human T-lymphotropic retrovirus 3B IFN interferon IL interleukin IL-2Ra interleukin-2 receptor a INT transcription initiation IRR increased radiosensitivity KS Kaposi's sarcoma LET linear energy transfer LPS lipopolysaccharide LTNP long term non progressor LTR long term repeat Mif macrophage MCAF(MCP-l) macrophage chemoattractant and activating factor M E D minimal erythema dose mGy O.OOlGy M H C major histocompatibility complex MIP l a macrophage inflammatory protein -1 alpha MIP 1(3 macrophage inflammatory protein -1 beta MOI multiplicity of infection xii mRNA MuLV NC N A NF-AT N F - K B NHL N K NSI p24 PBMCs PBS PCR PHA psi PUVA Rantes RNA ROS RT SD SE SI ssDNA (+) ssRNA (-) ssRNA messenger RNA murine leukemia virus nucleocapasid neutralizing antibody nuclear factor of activated T cells nuclear factor K B Non-Hodgkin's lymphoma Natural killer non syncytium inducing the core protein found in the capsid surrounding the HIV-1 peripheral blood mononuclear cells phosphate buffered saline polymerase chain reaction phytohemagglutinin Discrimination among multiple viral and nonviral mRNAs and the fell-length genomic RNA for Gag encapsidation lies in a cis-directed sequence designated psi. This site is located at the 5' region of HIV genome and is not present in spliced viral mRNA transcripts. psoralen ultraviolet A receptor-activated neutrophil T cell expression and secretion ribonucleic acid reactive oxygen species reverse transcriptase standard deviation standard error syncytium-inducing single strand DNA positive sense of single strand RNA negative sense of single strand RNA xiii Sv Sievert TAR transactive response element TBP-1 T A T A binding protein T C F - l a T cell factor 1 a TCID 5 0 50% of tissue culture infection dose TCR T cell receptor Thl T helper type 1 Th2 T helper type 2 tRNA transfer RNA USF upstream stimulatory factor U V R ultraviolet radiation V Z V varicella zoster virus XP xeroderma pigmentisum ZDV Zidovudine xiv Acknowledgements I would like to thank my many friends and colleagues for their wonderful love and encouragement that enriched my life greatly and sustained me throughout all the ups and downs of the last ten years in Canada. I would acknowledge that this work was supported by the Musculoskeletal Transplant Foundation, the Bickell Foundation, and the British Columbia Centre for Excellence in HIV/AIDS. The experiments were conducted in University of British Columbia, University of Ottawa and Chalk River Laboratories. I would express my sincere appreciation to Brian Conway, my thesis supervisor, for his keen and creative thinking, time and financial support. I wish to thank Clive Greenstock, Steve Pelech for their incredible support, encouragement, suggestions and time. I also would express my special appreciation to Ron Mitchel, who invited me to Canada and has helped me greatly in many ways. I would like to thank Grant Stiver, David Godin, David Burdge, Aslam Anis, Nasreen Khalil, Kirsten Skov, Stephen Sacks, Neil Reiner, Liaoyuan Su, Jonathan B. Angel, Ken Dimock, S. A. Sattar, Leon Fillion, for their valuable teaching and support that made this work possible. I am very grateful for Norman Wong, Anita Keister, Linda Lee whose hard work has made experimental graduate study program very successful. I would also thank my first English teacher in Canada, Ruth Harrison who helped me prepared TOEFL. I would give many thanks to Eva Gallerger, Arturo Sy, Ann and Gershon Davis for their generosity and friendship. I wish to acknowledge my friend and husband Paul Tien Hua for his kind support and love. I would also acknowledge my daughter for her love and music. xv Dedication This thesis is dedicated to my parents Yi Xing Huang and Wen Fang Xu. PARTI 1.0 GENERAL INTRODUCTION 1.1. HIV-1 HIV-1, the most common cause of AIDS throughout the world, is a lentivirus, within the family of retroviruses. Based on the degree of sequence diversity within certain genes, eight major HIV -1 genotypes (or clades) have been identified (A-H). These clades have geographically distinct distributions, linked to their initial point of origin. More recently, additional clades that are truly unique (such as clade O, for "outliers") have been identified. These could help us identify the origins of HIV-1, and how it first came to infect human beings (1). Mature virions are spherical, about 100 nm in diameter, and are characterized by an electron-dense cylindrical core structure surrounded by a lipid envelope. The core of the virus contains two copies of the single stranded RNA genome encoding a series of structural, regulatory and enzymatic proteins (Figure 1). The HIV-1 genome is about 10 kB in length, and contains two flanking long- terminal repeat (LTR) sequences. From the 5' to 3' direction, the genome consists of the gag gene (encoding structural viral proteins), the pol gene (encoding the viral protease, reverse transcriptase and RNase H activities), and the env gene (encoding viral envelope glycoproteins). The HIV-1 genome contains at least six additional regulatory genes (tat, rev, nef, vpr, vpu and vif) which encode proteins that regulate transcription and translation of viral proteins and enhance assembly and release of particles from infected cells (Figure 2, Table 1). For completeness, it should be stated that the viral core also contains pre-formed RNA-dependent DNA polymerase (reverse transcriptase, or RT), which catalyzes the initial transcription of genomic RNA to proviral DNA, an essential early step of the viral life cycle(2-5). 1 gp120 gp41 viral envelope p17 p24 protease reverse transcriptase RNA Figure 1 V i r i o n structure of H I V -1 As seen by electron microscopy, the HIV-1 virion is an enveloped icosahedral structure. It has a cone shape core capsid covered with a lipid bilayer membrane. The diameter of a HIV-1 virion is about 100 nm. The capsid contains 2 identical copies of positive sense RNA genome. The genomic size of HIV-1 is about 9.8 kb. p24 is the main structural protein forming the capsid. A number of viral enzymes are pre-formed and present inside this capsid. These include the HIV-1 reverse transcriptase, protease and integrase. The lipid bilayer is produced when the virion buds off from the cellular plasma membrane and is thus host cell-derived. On this membrane, the major components are the gpl20 external or surface protein and the gp41 transmembrane protein. These two proteins are critical for viral binding, fusion and entry into the host cells. 2 Figure 2 Genomic organization of H I V -1 5'LTR GAG POL ma ca nc pr rt in fVPR VPU ENV su(gpl20) U3 R U5 rviF tm(gp41) NEF 3'LTR TAT U3 R U5 REV The genome of HIV-1 consists of at least nine genes: G A G , POL, ENV, TAT, REV, NEF, VIF, VPU and VPR. LTRs are located at each end of the viral genome. 3 Table 1 Function of HIV-1 genes Gene Protein size(kd) Function Gag p24 capsid (CA) structural protein pi7 matrix (MA) protein p7 RNA binding protein p6 RNA binding protein Pol p66,p51 reverse transcriptase (RT); RNaseH plO protease (PR) p32 integrase (INT) Env gpl20 envelop surface (SU) protein gp41 envelope transmembrane (TM) protein Tat pi4 transcriptional activator Rev p 19 required for expression of HIV-1 unspliced and single spliced mRNA Vif p23 promotes viral infectivity and viral assembly Vpr pi5 reactivates virus from latency Vpu pi6 required for efficient virion maturation and budding Nef p27 downregulates CD4, induces viral replication from quiescent lymphocytes and necessary for in vivo pathogenesis 4 1.2 Life Cycle of HIV-1 (Figure 3) The life cycle of HIV-1 consists a series of steps: 1. Binding and entry 2. Reverse transcription, nuclear import and integration of proviral DNA 3. Cell activation and viral transcription 4. Translation of early and late HIV-1 proteins 5. Virion assembly, budding and release 5 Figure 3 The general overview of HIV-1 life cycle Mature Virus 6 1. Binding and entry The life cycle begins with the attachment of the HIV-1 surface glycoprotein (gpl20) to a cell surface receptor (usually the CD4 molecule) and subsequently to a co-receptor. CD4 is found on the surface of certain T lymphocytes, macrophages, dendritic cells of the skin, and microglial cells of brain, among other cells. Once binding to the CD4 molecule has occurred, binding to a secondary co-receptor of the chemokine family may proceed. These seven-transmembrane domain G proteins are the key determinants of cellular tropism of viral isolates. Viruses binding to the CCR5 (or, less commonly, the CCR3 receptor) infect macrophages, while those binding to CXCR4 (also called fusin) infect T lymphocytes. Unlike other retroviruses, which may enter cells by receptor-mediated endocytosis, HIV-1 enters cells by fusion of the envelope with cellular plasma membrane. After this fusion occurs, the nucleocapsid is released into the cytoplasm, the viral capsid is disrupted and the viral reverse transcriptase is fully activated (2-3). 2. Reverse transcription, nuclear import and integration of proviral DNA Replication proceeds through a DNA proviral intermediate. The viral reverse transcriptase uses virion tRNA as a primer for the initiation of RNA-based DNA synthesis. After tRNA binds to the primer-binding site, the RT enzyme initiates synthesis of the negative-strand proviral DNA. RNase H degrades the RNA except for the polypurine tract that is used as primer to initiate the synthesis of positive-strand DNA. These latter steps follow specific template jumps, allowing for the synthesis of both linear and circular double-stranded proviral cDNA. The circular intermediate (containing one or two LTRs) cannot be integrated into the host cell genome. The amount of this non-integrated DNA may play a key role in the pathogenesis of the infection as a whole. The linear proviral cDNA (with one complete LTR at each end) is the only useful replication intermediate. This is maintained in a nucleoprotein complex, transported to the nucleus of an infected cell. This process occurs in two phases: 7 a. Prenuclear pore transport (low energy process) b. Nuclear pore transport (high energy process) Phosphorylated M A protein and Vpr protein are important at this stage of the viral life cycle as the nuclear localization signals in Vpr are similar to those in the M A protein. The proviral genome is integrated at random sites throughout host cell genome by covalent linkage, mediated by the viral integrase (4-5). 3. Cell activation and viral transcription The integrated provirus remains quiescent until the infected cell is activated. Functionally, the HIV-1 provirus acts as a cellular gene, with viral replication depending on the capabilities of the host cell's enhancer proteins (such as NF-kB, for example) to bind to specific regions of the proviral 5' LTR. Other important factors include the cell's growth rate and the degree of viral DNA methylation. Specific binding regions of interest in the viral LTR are shown in Figure 4. HIV-1 RNA transcription initiates at the beginning of the R region in the 5' LTR and terminates at the end of R region in the 3' LTR. The 5' LTR contains a strong enhancer-promoter and 3' LTR contains a polyadenylated site. Transcription of provirus is mediated by cellular RNA polymerase II, which results in three types of viral RNA: genomic viral RNA, mRNA for translation of gag, gag-pol and polyproteins in the cytoplasm, and shorter multiply spliced RNA species (2-5). 8 Figure 4 Specific binding regions of interest in the viral LTR AP-1 C O U P N F - A T USF T C F - l a N F - K b SP1 TBP INT UBP-l/LBP-1 UBP-2 CTF/NF1 f i n A A o o r~i A U n r n n n o •453 +1 +60 I I I 1 M O D U L A T O R Y CORE T A R I 1 ENHANCER Cellular protein binding sites: Once HIV-I integrates into the host-cell genome, gene expression is regulated by cellular transcription factors in a manner similar to that of endogenous cellular genes. AP-1 site: binding site for transcription factor AP-1. C O U P (Chicken ovalbumin upstream promoter) site: C O U P proteins belong to members of steroid/thyroid hormone receptor superfamily. C O U P protein bind to HIV-1 LTR may have a negative regulating effect on HIV-1 gene expression. N F - A T (Nuclear factor of activated T cell) site: When T cell is activated, binding activity of N F - A T will increase dramatically. NF-AT exerts a positive effect on HIV-1 gene expression. USF (Upstream stimulatory factor) site: This element may have both positive and negative regulatory effect on HIV-1 gene expression. It also mediates synergistic activation of HIV-1 in the presence of cytomegalovirus (CMV) immediately-early protein. T C F - l a (T cell factor l a ) site: It is a T cell-specific cellular transcription factor that may be important to HIV-1 tropism and replication in T cells. N F - K b (Nuclear factor -kappa B) site: HIV -1 has two functional N F - K B sites. In uninfected cells, N F - K b controls transcription of several cellular genes such as Ig k light chain, class I and class II M H C , IL2, IL2Ra, (5 interferon and TNF-a . N F - K B also regulates transcription directed by the promoters of several virus. Such as EB virus, C M V , HHV-6, adenovirus and HTLV-I . SP1 site: Sp-1 protein acts as anchor to stabilize HIV-1 LTR directed R N A transcription. TBP (The T A T A binding proteins) site: They regulate basal HIV-1 expression. PNT (transcription initiation) site: Sequence around HIV-1 R N A initiation sites. Uperstream binding protein 1 (UBP-1) site Leader binding protein (LBP-1) site: LBP-1 binding to strong site activates transcription. LBP-1 binding to weak site inhibits transcription. Upstream binding protein 2 (UBP-2) site Trans-acting response (TAR): Tat interaction with the T A R through its element and upstream HIV-1 long terminal repeat promoter binding site. Cellular factors play an important role in facilitating transcription activation by Tat cis-acting elements in LTR that is important in regulation of viral replication. 9 4. Translation of early and late HIV-1 proteins Early on, multiply spliced viral mRNAs are produced (at very low levels) that encode the tat, rev and nef proteins. At the beginning, the level of viral-specific transcription is low in the absence of tat protein. When this protein is present in sufficient amounts, it binds to a three-nucleotide bulge region in a stem-loop secondary structure of HIV-1 TAR (trans-acting response element) RNA in concert with one or more cellular proteins. This leads to a significant up-regulation of viral RNA and protein synthesis, allowing the translation of structural proteins encoded in the gag- pol, env genes, accessory proteins encoded in vpu, vif and vpr genes. Either rev-independent or rev-dependent transport pathways transport the viral transcripts in the nucleus. The rev protein binds to the RRE (rev-responsive element) and shifts the balance from transport of multiply spliced transcripts to singly spliced or unspliced viral mRNA into cytoplasm, with the generation of full length viral genomes (4-5). 5. Virion assembly, budding and release The final stages leading to the production of infectious virus particles occur as a coordinated process. This begins with the generation of gag and gag/pol precursors, it is followed by polyprotein processing (mediated by the viral protease), and ends with final assembly and viral budding. Assembly begins with an interaction between the gag polyprotein (Pr55gag) in the cytosol or at the plasma membrane and a cis-acting packaging element located near the 5' end of full-length viral transcript. Each virion contains two single-stranded viral RNA genomes. The nucleocapsid domain of the gag polyprotein may facilitate formation of this dimeric RNA complex by interacting with psi. Pr55 g a g also interacts with the transmembrane subunit of the envelope glycoprotein in the process of assembly. The viral protease cleaves the gag-pol protein in an ordered fashion, leading to the production of pi 5 NC protein, and then p24 C A protein, and lastly p7 and p6. This 10 is the key step in the production of the viral core, and is essential to the generation of infectious progeny. In the meantime, a lipid bilayer from the plasma membrane develops around the viral core. Precursor gpl60 env undergoes glycosylation and oligomerization in the endoplasmic reticulum and the gp41 (transmembrane domain) and gpl20 (extracellular domain) of the surface glycoproteins are produced and inserted into the cell membrane. The infectious virus is then released from the plasma membrane (4-5). 1.3 Immunopathogenesis of HIV-1 infection When humans are exposed to HIV-1, the outcome could be: 1. Abortive infection without cell entry 2. Abortive infection with a specific cytotoxic response 3. Most commonly, productive infection with a variable rate of disease progression A whole host of factors help determine the rate of viral growth and dissemination. Among these are the route of infection, the infectious inoculum and its virulence, and the type and intensity of the host immune response(6-9). From a clinical perspective, HIV-1 infection can be divided into three stages: acute or primary infection, a prolonged phase of clinical latency and a final symptomatic phase. The development of symptoms is generally associated with activation of viral replication. Inducers of HIV-1 gene expression may include endogenous cellular transcription factors such as NF-kB or TNF-oc, viral regulatory proteins such as tat and rev, and specific or non-specific immune activation, such as can be induced by co-infection with C M V , HSV, HBV, HHV-6 or HTLV-I. This is accompanied by progressive immunologic derangement, with depletion and dysfunction of CD4 cells, decreased MHC-II expression, decreased monocyte chemotaxis, altered cytokine expression, decreased cytotoxic T lymphocyte (CTL) and natural killer (NK) cell function, lymphopenia, and polyclonal B lymphocyte activation(lO). The primary specific effector responses responsible for controlling HIV-1 11 infection are neutralizing antibodies (NAs) and cytotoxic T cells (CTLs). NAs recognize either free virus or virally-infected cells, and act by directly neutralizing free virus or killing infected cells through complement-mediated pathways or via antibody-dependent cellular cytotoxicity (ADCC). CTLs are highly effective in eliminating virally-infected cells and controlling viral replication by cytolysis and/or lymphokine release. CD8-mediated MHC-I-restricted killing (directed against a broad range of viral epitopes) may also play a significant role in the immune response to infection(l 1). HIV-1 uses several strategies to evade an otherwise potentially effective immune response, such as antigenic variation through point mutations. Furthermore, direct virally-induced CD4 cell killing contributes significantly to immune disease progression. This occurs through a number of different mechanisms (6,10,11-15): 1. Direct single cell killing 2. Syncytium formation between infected and non-infected cells 3. Immune-based cell lysis (CTL or ADCC) 4. Viral-induced T cell anergy 5. Apoptosis 6. Superantigen formation 7. Autoimmunity The main features of progression are depletion of CD4 cells and loss of CD4 T helper (Th) function. This is associated with switching of cytokine production from a type 1 (Thl) to a type 2 (Th2) pattern( 15-22). In general, the Thl cytokines (interferon y, IL-2, IL-12) enhance cell-mediated immunity, inhibit humoral immunity and provide protective immunity in the setting of HIV-1 infection. The Th2 cytokines (IL-4, IL-5, IL-10, IL-13) amplify humoral immunity and suppress cell-mediated immunity, and are associated with progression of HIV-1 disease. The use of cytokines (especially IL-2) for the treatment of 12 HIV-1 infection in clinical trials is based on this aspect of the pathogenesis of disease progression, with a view to restoring a more favorable pattern of cytokine production(15-22). 13 2.0 REVIEW OF THE EFFECTS OF RADIATION ON LYMPHOCYTES AND VIRUSES 2.1 Radiation biophysics - an introduction Our world is a radioactive one, as radionuclides can be found in air, water and soil. In general terms, radiation represents the emission of energy in the form of waves or particles. Ionizing radiation (natural or man-made), produces positively charged particles or ions, and represents only a small part of the electromagnetic spectrum (Table 1). Short wave length (<200 nM) ultraviolet photons and, of greater biological importance, X-rays and gamma rays, may cause ionization. X-rays produced by high speed electrons hitting a heavy metal target are identical to gamma rays in their physical properties, as both originate from energy given off by unstable atoms. Gamma rays are ionizing photons from a radioactive source. X-rays, produced in a specialized tube, are considered to be polyenergetic (emitted with many energies). In contrast, gamma rays are considered to be a monoenergetic form of radiation. It is important to know the biological effects of ionizing radiation not only for understanding radiation injury and radiation therapy, but also for designing appropriate strategies to prevent radiation-induced injuries. The potentially negative effects of radiation on living organisms are well described. These effects occur quite rapidly and are countered by slower biological repair mechanisms (Figure 5). Initially, the effects depend on the amount of radiation absorbed by individual cells. Other relevant variables include the type of radiation, dose rate, temporal period of exposure (single dose, fractionated dose or chronic exposure), energy of the radiation and mode of exposure (external or internal, homogenous or not). Specific biological effects depend on the way energy is transferred to the tissue, which can be measured as linear energy transfer (LET). Radiation sources are generally classified as high or low LET. Both X-rays and gamma rays are highly penetrating, low 14 LET forms of radiation. The effectiveness of radiation is due to localized deposition of energy (which may be cumulative over time), which may in turn affect critical cellular structures, either directly or indirectly. As such, energy is directly absorbed by, and acts on, the target macromolecules themselves. Indirectly, the radiation (ionization and/or excitation) involves the radiolysis of water and generates free radicals (chiefly OH', H', H02' and their reaction product H2O2), that enter into chemical reactions with the target macromolecules. Oxygen and water may act to sensitize a cell to these effects, by providing additional substrate molecules for the generation of free radicals. All of these events occur in a matter of seconds to minutes. Even before the radiation stimulus is removed, enzymatic repair of molecular damage is initiated. Over time (perhaps years) biologic re-population occurs from the pool of surviving cells. In the course of this process, long term deleterious effects, such as malignant transformation of cells may, unfortunately, occur. The ultimate radiation effects could be either genetic and/or somatic. Genetic effects usually are stochastic, while somatic effects may be stochastic or not. Understanding the biological effects of radiation is usually achieved by measuring biochemical and genetic changes, alterations of certain cell functional capabilities, cell survival/death and cell transformation (23-27). 15 Figure 5 Development of radiobiological effect Radiation effect Time after radiation Radiation u Energy absorption li excitation and ionization (interaction with biological molecules and water) Ji Fixation of damage (direct and indirect effects) at physics level at chemistry level up to 10"^ second up to 10 second Enzymatic repair of molecular damage u at biochemistry level minutes Biological re-population from surviving cells at biology level hours, years 16 2.2 Radiation effects on viruses The biological effects of radiation are quite complex and all three types of chemical bonds (covalent, hydrogen, Van der Waals) can be damaged by the action of radiation which releases a large amount of energy within a small volume of biological material (28). Understanding the action of ionizing radiation on viruses will help us to gain new insight into the viral life cycle, including the radiosensitivity of specific viral organisms. In turn, this will help to establish parameters for radiation inactivation of viruses, and the effects (if any) of radiotherapy on viral pathogens in infected patients. Viruses differ from other infectious organisms in their structure and biology, especially in the ways they reproduce. Compared to bacterial, plant and animal cells, more biological diversity exists within viruses, especially based on the viral genome which can be single-or double-stranded RNA or DNA (29). Furthermore, as with HIV-1, an RNA genome may have a DNA intermediate in its life cycle. The interaction of radiation, viruses and host cells is very complex due to the heterogeneity of radiochemical lesions and viruses, the varying repair potential of host cells and the immune state of the host. The radiosensitivity of viruses is lower than that of bacteria and bacterial spores. The fully dry virus is the least sensitive and the dose of radiation that is lethal to all but 10% of the cells of the (Dio) generally ranges from 3.9-5.3 kGy. Simpler single-stranded RNA or DNA viruses are more sensitive than those with more complex double-stranded RNA or DNA structures (30). When a host cell is subjected to radiation or other DNA-damaging agents, the replication of any viral pathogens they contain may actually be enhanced. This has been shown for cells exposed to UV radiation, then infected with herpes or adenoviruses. It was demonstrated that active protein synthesis was required for the effect to occur. The mechanism of this effect does not depend upon excision repair since this phenomenon was also observed in xeroderma pigmentosum (XP) cells and could be inhibited by 17 cycloheximide(31). Jeeves et al. (32) showed that exposure of fibroblast cells to U V or 10 Gy y-radiation enhanced the replication of adenovirus in these cells. In another model, mutagenesis of parvovirus was used to infect irradiated cells. This was not observed in a herpes virus model, likely due to the fact that parvoviruses make use of the host cell DNA polymerase in their life cycle (32). Radiation can have suppressive or stimulatory effects on the host's immune system in the setting of viral infection. This has been studied in many models: murine leukemia virus (MuLV) (33); human T cell leukemia type I /type II virus (HTLV-I/II) (34,35); varicella zoster virus (VSV) and herpes simplex virus (HSV) (36,37); rabies virus (38); theiler murine encephalomyelitis virus (39); murine hepatitis virus (40). It has long been known that exposure to U V and ionizing radiation can trigger the reactivation of herpes virus. In one experiment, C57BL/6 mice infected with LP-BM5 MuLV were exposed to low doses of UV radiation before and after viral inoculation. This in vivo UV exposure enhanced the immunosuppressive effects of the retroviral infection, as measured by mitogenic responses of lymphocytes to PHA, ConA and LPS. Matsuo et al. (34) observed that exposure to ionizing radiation induced changes in HTLV-I antibody levels. Studies are currently underway to investigate the influence of radiation in the Chernobyl accident on the possible incidence of HTLV-related adult T-cell leukemia/lymphoma (35). This is done in the context of previous work showing impairment of antibody production against certain type A influenza viruses and increased prevalence of the hepatitis B virus surface antigen in serum among atom bomb survivors. These individuals were also found to have more frequent reactivation of Epstein Barr virus (EBV), due to impairment of T cell immune function (41). Whole body irradiation and head-protected body irradiation (but not the cephalic irradiation) at a dose of 6 Gy increase the degree and duration of rabies virus infection in mice. Exposure of mice to 3 Gy gamma radiation induced susceptibility to theiler murine encephalitis virus-induced demyelination. Finally, Wang et al. (40) demonstrated that total body gamma radiation (8.5 Gy) might prevent demyelination caused by the neurotropic mouse hepatitis virus (JHM strain). 18 In summary, the interaction of radiation and viruses is a complex one, and merits further study. This is particularly true for HIV-1, which is associated with a number of medical conditions for which therapeutic radiation therapy is often administered. 2.3 Specific Effects of Low Doses of Radiation on the Immune System The initial response to a viral infection involves the synthesis of a number of inflammatory mediators such as interleukins (IL-1, IL-6, IL- 8, IL-12), chemokines (MIP-1P, RANTES, lymphoactin), interferon and TNF-a, among others. If the virus persists, both humoral and cell-mediated immune responses are important in controlling the subsequent course of infection. A proper response is based on a delicate balance between macrophages, N K cells, and both T and B lymphocytes. The secretion of cytokines (both Thl and Th2 types) by CD4 T helper lymphocytes is at the heart of the specific immune response. Significant amounts of data are now available on the differential radiosensitivity of lymphocyte subsets, including alteration of cytokine networks, radiation- induced apoptosis, and the potential for carcinogenesis. Lymphocytes are highly sensitive to radiation, as exposure to 0.02-0.04 Gy causes significant alterations in motility and morphology of human lymphocytes in vitro (42). The CD8 T lymphocyte is more radiosensitive than B and CD4 lymphocytes, although this can be reduced by prior PHA stimulation (43). A selective cell renewal characterized by an increasing absolute CD4 cell number was found in Austrian workers who received occupational external radiation (up to 9.8 mSv) and internal radiation (up to 2.8 mSv) exposure (44). In atomic bomb survivors exposed to doses > 1 Gy, there were significant alterations in the differentiation and development of T cells, and the number and function of T and B lymphocytes (45). On the one hand, low dose long-term exposure to uranium resulted in high rates of chromosomal aberrations and low neutrophil counts (46). On the other hand, for dependent temporal foreign antigen stimulation, low dose radiation might enhance the overall immune response and facilitate elimination of foreign antigens (47). 19 Radiation hormesis is a stimulatory effect where small doses of radiation physically activate living organisms, while larger doses of radiation are inhibitory (48). A stimulatory effect in the spleen and thymus was observed in mice receiving chronic gamma radiation at a very low dose rate (49). A reduction in the incidence of cancers was observed among the inhabitants of regions where the background level of radiation is high (50). Low dose radiation (below 250 mGy) stimulated DNA repair and enzyme activity (51). Total-body irradiation of mice with 0.01 Gy once a day for 20 days resulted in augmentation of the proliferative response of effector T cells (52). Finally, 50 cGy X-ray radiation enhanced expression of CD2 antigen on the surface of T lymphocytes cultured for 24 hours with or without PHA (53). This is a reliable marker of the effect of the intervention, as the CD2 molecule is involved in T lymphocyte differentiation and activation. Beneficial effects of low doses of radiation have also been recognised. Fractionated radiation of mice increased their life expectancy. When lymphocytes and other cells are pre-exposed to very low doses of ionizing radiation, fewer chromosome aberrations are found in lymphocytes subsequently exposed to a high dose, a presumably adaptive response (54). Significantly less chromosome damage was found in lymphocytes from children who were continuously exposed to low levels of 1 3 7 Cs (by internal contamination) as compared to other children following the Chernobyl nuclear accident (55). This is further suggestive of a priming or adaptive response induced by the long-term low-level prior radiation exposure. U V exposure has been shown to up-regulate production of a number of cytokines, perhaps in response to DNA damage: IL-1 (56, 57), IL-6 (58, 59), IL-8 (60), IL-10 (61). The alteration of cytokine pattern could modulate the immune response in favor of switching from a Thl to a Th2 type response (62). It was hypothesized that CD4 Thl and CD8 C T L could exhibit cytotoxicty against radiation-induced pre-leukemia cells, whereas oncofoetal antigen (OFA)-specific CD8 T cells secrete IL-10 and inhibit C T L 20 activity, causing mice to develop lymphomas (63). Administration of a Thl cytokine such as IL-12 might overcome the IL-10-induced immune system suppression that follows exposure to radiation (61, 64). Enhanced production of IL-1, IL-2 and TNF-oc has also been reported following exposure to ionizing radiation, and single whole body X-radiation doses (2.5 cGy) augmented IL-1 production and T lymphocyte proliferation induced by suboptimal doses of mitogens such as ConA, PHA or LPS (65, 66). The pattern of cytokine gene expression has been found to be dependent on rate of dosing as the levels of IL-1 p and IL-6 mRNA in human glioblastoma cell lines were increased by 10 Gy gamma radiation exposure at 200 cGy/min or 4.1 cGy/min, but were decreased at 1 cGy/min or 0.35 cGy/min (67). Physiologic cytokine networks regulate the degree of HIV-1 activation and expression both positively and/or negatively (68). IL-2 is one of the cytokines which acts as a growth factor for T, N K and B cells. The high affinity of IL- 2 receptors is due to three distinct membrane components, termed a, P and y chains. IL-2ct is undetectable in resting T cells but is induced upon T cell activation. A positive feedback loop exists, wherein an increase in IL-2 secretion induces an increase in IL-2 production and IL-2 receptor expression (69). Low doses of radiation cause changes or damage in the cell membrane, an effect that may serve to modulate gene expression (70). As such, ionizing radiation may change the balance between the appropriate immune effector systems and viral growth and dissemination, and may therefore alter the natural course of viral infection. It has been reported that pro-oxidant conditions exist in HIV-1- infected patients, and ionizing radiation-induced oxidative stress may be physiologically significant. In our work, experiments have been performed to test the hypothesis that effects such as radiation-induced enhancement of IL- 2 receptor expression may activate HIV-1 replication and/or enhance target cells to HIV-1 infection. 21 3.0 IONIZING RADIATION AND HIV-1: PRACTICAL AND THEORETICAL CONSIDERATIONS Ultraviolet, gamma and X-radiation have all been used to inactivate HIV-1 in a variety of different settings (Table 2). Radiation may act specifically and non-specifically to inactivate HIV-1, either directly or indirectly. Although high doses of radiation may reduce viral infectivity, they may also damage the host's biological system. Lower doses of radiation, useful in medical therapeutics for HIV-1-related conditions, may modulate or suppress the immune response, activate viral replication and promote viral oncogenesis. Thus, the interaction of HIV-1 and radiation is a complex one that must be carefully reviewed before any meaningful experiments can be designed (71-75). 22 Table 2 Comparison of the visible electromagnetic spectrum with that of ultraviolet, X-ray and gamma radiation Frequency (GHz) Wavelength (nm) Visible 0.42-0.75x106 400-700 Ultraviolet 0.75-1.5xl06 200-400 X-ray 3 x l 0 6 - 1 3 0.00001-100 Gamma 3xl0 9 " 1 0 0.01-0.1 23 3.1 The Possible Role of Radiation to Prevent HIV-1 Infection The first case of HIV-1 transmission by bone transplantation was reported in February 1988 (76). The female recipient who developed full-blown AIDS had no known risk factors for HIV-1 infection other than the bone transplantation she had received in November 1984. Prior to surgery, the donor's bone had been harvested under sterile conditions and stored at -80°C. No sterilizing procedures were performed on the sample. The donor was subsequently found to be infected with HIV-1 and was diagnosed with AIDS in July 1986. In 1991, a second patient was reported whose only risk factor for HIV-1 infection was also transplantation of a bone allograft in 1985. In this case, the donor had been extensively screened and tested. Unfortunately, multiple organs and tissues had been harvested from her. Of the 48 identified recipients, 41 tested positive for HIV-1 antibodies (77). Subsequent investigation revealed that the tissues had been procured between the time the donor became infected and the appearance of measureable anti-HIV-1 antibodies. According to the estimation of Buck et al. (78,79), the chance of acute infection following receipt of a bone allograft from an HIV-1-infected donor if adequate precautions are not taken (for example, by testing only for antibodies to HIV-1 without extensive donor screening) may be as high as 1/161 (0.6%). However, this is reduced to less than 1/106 if rigorous donor selection procedures are also implemented. This can possibly be reduced even further if appropriate sterilization procedures are implemented on a routine basis. Ionizing radiation is destructive and inactivates viruses quantitatively. As such, gamma radiation has been used to sterilize medical equipment and supplies, medications and pharmaceutical starting materials, cosmetics, biological tissues and other biological 24 preparations (80). The nucleic acid is generally considered to be the radiation-sensitive target of the virus. Virtually all viruses that have been subjected to radiation target analysis are inactivated by a first-order process (71,81,). The use of gamma radiation to inactivate HIV-1 has been studied sporadically. The effects of gamma radiation on HIV-1 alone, or within banked bone (82-92), fascia lata (93), plasma and coagulation factor concentrates (85), or other body fluids (86) have attracted particular attention in recent years (Table 3). 25 Table 3 Inactivation of HIV-1 by gamma radiation Dose(kGy)/Temp(°C) Viral Titer Effective Dose Toxicity < 1 OkGy/ 4°C 5000 units/mL 25 kGy 2.5-40 kGy/-40°C i 0 6 - 5 TCID50 0.1-1 kGy/24°C 5 x l 0 7 - l x l 0 n particles/mL 2.5 kGy 25 kGy 40 kGy 0.75 kGy 0.4 - 5.6 kGy/-70°C u p t Q 1 Q 7 T C I D 5 0 D 1 0 approx. 4 kGy 4 kGy/-70°C 102- 103TCID50 viral stock 1 . 5 - 2 0 k G y / - 7 0 ° C 1 0 2 - 103 TCID50 2.5-100 kGy -80°Cand 15°C viral stock 1 0 5 . 6 - 6 . 2 T C I D 5 ( ) D 1 0 > 4 k G D,0=5.6kGy 25kGy 14-38% loss of certain plasma clotting factors Hemolysis 10 kGy (yes) 0.25-3 kGy (no) Reference 83 84 85 86 87 88 89 90 26 3.2 Radiosensitivity of HIV-1 3.2.1 Direct and indirect radiation effects on HIV-1 The virucidal effectiveness of gamma radiation is directly related to damage to the viral genome. Cleavage of the peptide bond of proteins has been observed at higher doses. The indirect effect is due to diffusible free radicals produced by absorption of the radiation by the medium in which the viruses are suspended. Previous studies have shown that the purine and pyrimidine moieties of the nucleic acids are the major foci of chemical change when nucleic acids are irradiated in dilute solution, while the pentose phosphate backbone is damaged less frequently. In the frozen and lyophilized state, the direct action of radiation predominantly affects the viral genome. In the liquid state, indirect radiation affects on viral proteins and nucleic acid are more significant. Thus, if one radiates the virus in the dry state, thereby emphasizing the direct effect of ionization within the molecular structure itself, and in dilute solution, thereby emphasizing the action of radicals, peroxides and other reactive oxygen species, the sensitivities to inactivation may differ widely. Hiemstra et al. (90) reported that a reduction in the HIV-1 virus titer by 5 or 6 orders of magnitude was achieved with doses of 50-100 kGy at -80°C in the dry state. When the virus was placed in dilute solution at 15°C, similar effects only required exposure to 25 kGy. It is quite clear that our understanding of the effect of radiation on HIV-1 remains incomplete. If radiation causes HIV-1 nucleic acid breaks that interfere with some vital function of the virus (such as reverse transcription), then it would undoubtedly be lethal to the virus. If not, all that may occur is a delay in viral replication. The radiosensitivity of different microorganisms is expressed in terms of DlO values (the dose required to reduce the population by a factor of 10 or 90%), although a number of authors have chosen to report their results using less standard approaches. Kitchen et al. (85) indicated that 40 kGy would inactivate 1 0 ^ 50% tissue culture 27 infective closes of HIV-1 (TCID50)/mL. Salai et al. (84) found that 25 kGy of gamma radiation "completely" inactivated HIV-1 in banked bone. Bigbee et al. (86) reported that at highly concentrated levels of HIV-1 (10^ * viral particles/mL), doses between 50 and 75 kGy were required to completely inactivate the virus based on analysis of pi 7 and p24 viral core protein in supernatants of cultures established using irradiated viruses. At less concentrated serial dilutions of the virus (5x10 viral particles/mL), a 25 kGy dose of X-rays appeared to lead to complete inactivation. Spire et al. (83) failed to determine the threshold dose of radiation required to inactivate HIV-1, but doses well below 10 kGy were used. Previous data from Conway et al. (88, 89) showed that 4 kGy led to a reduction in 9 4 viral load of approximately 1.0 TCID50/mL. Beginning with treated inocula of 10 - 10 TCID50/mL, productive infection developed in all cultures set up with viral samples which received 4 kGy, as evaluated by the measurement of p24 antigen in culture supernatants. A delay in the kinetics of infections was observed in all treated samples. 7 3 Additional experiments using HTLV-IIIB at physiologic bioburdens (10 -103 TCID50/mL) exposed to 1.5-10 kGy indicated that the DlO was approximately 5.6 kGy. 3.2.2 Effect of radiation sterilization on biological function Hiemstra et al. (90) examined the effect of gamma radiation on the biologic activity of a number of coagulation factors in plasma and in lyophilized concentrates of factor VIII and prothrombin complex. They showed that the tolerable dose for preservation of coagulation factor activity in plasma should not exceed 14 kGy at -80°C, and 2 and 4 kGy for factor VIII concentrate irradiated at 15°C and -80°C, respectively. Kitchen et al. (85) found that 40 kGy of gamma radiation caused a 14-38% loss of the activity of certain plasma clotting factors. Thus, since large radiation doses cause profound changes in important haemostatic variables, gamma radiation should not be 28 considered for sterilization of plasma and plasma-derived products, as required doses would render these products clinically useless. In clinical practice, doses of 25 kGy are used to inactivate microbial pathogens in banked tissues, such as fascia lata (91). This dose may alter important tissue constituents, such as enzymes and proteins. However, according to Bedrossian (93), there is no significant difference in peak breaking tension between fascia lata treated with a 40 kGy dose of radiation and its matched control. Transmission electron microscopic examination shows that the large doses do not change the 67 nm periodicity of the collagen fibrils. The functions of a bone graft are to stimulate new bone formation and provide some internal structural support. Deleterious effects of radiation sterilization of banked bone have been observed, as radiation can alter these morphogenetic properties (94, 95). According to Urist and Hernandez (96), undemineralized bone has higher concentration than demineralized bone or bone matrix derivatives and thus could absorb a larger quantity of ionizing radiation. Their results showed that 35 kGy of gamma radiation could completely destroy a morphogenetic response of bone matrix from the mesenchymal cells of the recipient. Comparison of five methods of biological inactivation of demineralized rat bone 85 was performed by Munting et al. (97). Dry weight, calcium content, and Sr incorporation of the induced ossicles were used to examine new bone formation. They found that a glutaraldehyde solution, formaldehyde gas, and ethylene oxide destroyed almost all the bone-inductive capacity while bone exposed to a 25 kGy dose of gamma radiation retained approximately half of its inductive capacity. A dose of 60 kGy had deleterious effects on compression, torsion and bending. However, 10-40 kGy did not have significant deleterious effects on these biomechanical properties of bone, and may be sufficient to inactivate HIV-1 (98). Additional studies based on graft function post-radiation have yielded mixed results, casting doubt on whether this approach is appropriate for sterilization in this specific setting (99, 100). 29 Other methods of sterilizing biological materials include photochemical decontamination of red blood cell concentrates and platelet concentrates to eliminate pathogens that might be present in donated blood (101-104). Exposure to 30-40 J/cm U V A may possibly inactivate without a reduction in platelet haemostatic function (104). 3.2.3 What is the proper dose of radiation to inactivate HIV-1 ? In practice, doses up to 40 kGy are used for radiation sterilization. At these doses, the biologic properties of other tissues, such as bone, are relatively preserved. Variable effects in practice are related to the radiation dose, the particle size of the graft, and the extent of mineralization in the bone matrix. Given the measured Din for HIV-1, doses of 40 kGy cannot reduce the infectious burden of tissues to levels consistent with absolute sterility in a reliable manner. At the present time, no definitive recommendation for radiation sterilization can be issued (101). According Campbell et al's result, the radiation dose required to achieve a sterility assurance level of 10"6 was 89 kGy. Since 89 kGy exceeds current recommendations for sterilizing medical products and the current practice of many bone banks, gamma irradiation should not be considered as a significant virus inactivation method for bone allografts (102). The current standards of the American Association of Tissue Banks (103) regarding strict donor screening and testing must be observed to ensure the safety of banked tissues and organs for transplantation. 3.3 Radiation Therapy of HIV-1-associated Conditions The use of antiretroviral agents and improved therapy for opportunistic infections has significantly lengthened the life span of HIV-1-infected patients. However, HIV-1 infection and subsequent immuno-suppression result in an increased risk of malignancies and other conditions. Radiotherapy has been used extensively for the treatment of a number of malignant conditions in this setting, most notably Kaposi's sarcoma (KS) (106-108). 30 In the pre-AIDS era, endemic KS was highly radiosensitive, and could be controlled with a radiation dose of 8 Gy, given as a single or as a fractionated exposure (108). Stein et al. (109) reported that radiation with 8-10 Gy (single dose) or 14-24 Gy (fractionated exposure over 1-3 weeks) led to 32% and 54% complete and partial regression of cutaneous lesions of endemic KS, respectively. KS in AIDS patients is also highly radiosensitive, and radiotherapy can achieve effective palliation with minimal side effects (110). Plettenbergy et al. (Il l) reported that using fractionated radiotherapy to treat HIV-1-associated KS led to significant partial (76%) or complete (17%) responses. In a case report of a 39-year-old homosexual patient on zidovudine (as antiretroviral therapy), plaque-like KS lesions responded dramatically to low-dose X-radiation (111). Forty-three patients with AIDS-associated KS lesions of the skin or oral cavity were treated with radiotherapy. The fraction of patients with complete response was much higher if patients received doses above 20Gy (66-100%), although complete and partial response rates (34 and 55%, respectively) can also be attained with the same dose given in multiple fractions (113,114). HIV-1 infection is also associated with an increasing incidence of non-Hodgkin's lymphomas (NHL) (115-117). The most prevalent lymphomas exhibit a follicular or diffuse histological pattern. Follicular lymphomas are curable with radiation therapy when they are localized to lymph nodes. The use of additional therapy (e.g. interferon, high-dose chemotherapy, and immunologic therapy) to treat advanced follicular lymphomas after induction of an initial complete response has improved the cure rate significantly (124). Dewesse et al. (125) treated 16 patients with AIDS-associated NHL with megavoltage X-rays, using doses ranging from 10.5 Gy in 1.5 weeks to 50.4 Gy in 6 weeks. All 11 patients with CNS lymphoma died within a range of 0.2 to 5.3 months (median survival 2.2 months). However, 5 patients with non-CNS NHL responded better to radiotherapy, one of them achieving a 40-month disease-free survival. Smith et al. 31 (137) reported a patient with a soft tissue lymphoma who responded well to surgery and radiotherapy (44 Gy at 2 Gy/day for 22 days). The New York Hospital-Cornell Medical Centre has used brain radiation to treat 25 men with AIDS-related CNS NHL (138). The whole brain was treated with a total dose ranging from 30 to 40 Gy over 3-4 weeks. If a response was obtained, a "boost" to the tumor bed was delivered (in general, 14 Gy was given in about one week). Of 24 evaluable patients, 19 exhibited improvement in their neurological manifestations (79.2%). No subject who responded to treatment had any recurrence of neurological dysfunction prior to death. Side effects were minimal and temporary and were limited to skin erythema and alopecia. Blood cell counts were unaffected. None of the subjects required interruption of their antiretroviral therapy. The four-month survival rate was 68%>, with a mean survival time of 4.8 months. In comparison, the mean survival in patients with untreated AIDS-related CNS NHL was less than 2 months. Radiation therapy thus appears to play an important role in palliation of this condition, with a significant improvement in the quality of life. Kao et al. (139) retrospectively analyzed a cohort of 8 HIV-positive patients with Kaposi sarcoma (KS), lymphoma, or squamous carcinoma of the oral cavity oropharynx who were consecutively treated during a single year with radiation therapy at a tertiary care referral center. All patients had partial and complete responses to treatment. Although it was considerably better tolerated by patients with non-KS tumors, HIV infection is not a contraindication when aggressive radiation therapy is needed in select patients. Anal cancer, often seen in AIDS patients, is treated with a standardized protocol of chemotherapy and low-dose radiotherapy (143). Constantinou et al.(144) reviewed 50 patients who received this regimen for local/regional anal carcinoma from 1984-1994. Radiation doses of 54 Gy or more are associated with significantly improved survival and local control of disease. Patients with HIV-1 infection respond just as well, and exhibit disease progression without any evidence of recurrence of their anal carcinoma. Cleator et al's results also shows radical chemoradiation may be given safely at conventional doses in HIV-positive anal carcinoma, with a high complete response rate (143). 32 As shown in Table 4, non-malignant conditions associated with HIV-1 infection (such as multicystic lymphoepithelial lesions of the parotid gland and autoimmune thrombocytopenia) can also be successfully treated with radiotherapy. In one study conducted between 1994-96, 17 patients with thrombocytopenia were treated with low-dose spleenic radiation. A total dose of 9 Gy was delivered in nine one Gy fractions over 3 weeks. One month after radiotherapy, platelet counts of 6 patients increased significantly, and hemorrhagic symptoms were absent in all cases. However, the duration of the radiotherapy response was quite transitory (141). 33 Table 4 Radiation therapy of HIV-1- associated conditions Condition Kaposi's sarcoma Kaposi's sarcoma Kaposi's sarcoma Kaposi's sarcoma N Radiation Results 111 20Gy 34%complete, 55% partial response 14 8-40 Gy 83% complete response with 40Gy 187 8-40 Gy Median time to Progression 21 months 1 15 Gy Good response Reference 113 118 119 120 Kaposi's sarcoma Kaposi's sarcoma Kaposi's sarcoma (GI disease) 3 4-15 Gy 2/3 objective response 121 453 10-30 Gy 85% complete 122 response 1 20 Gy Control of 123 hemorrhage Non-Hodgkin's Lymphoma Non-Hodgkin's Lymphoma Primary CNS Lymphoma Primary CNS Lymphoma Primary CNS Lymphoma 16 25 10 46 Gy Response 10-50 Gy Response 30-40 Gy 4 complete and 5 partial 22-55 Gy 6 complete and 1 partial response 40 Gy Response 124 125 126 127 128 34 Primary CNS 17 Lymphoma Primary CNS 32 Lymphoma Lymphomatous 13 Meningitis Parotitis 4 Parotitis 12 Parotitis 20 Autoimmune 3 Thrombocytopenia Autoimmune 8 Thrombocytopenia Autoimmune 17 Thrombocytopenia 30-57 Gy 12 responses 129 30 Gy 50% responses 130 15-40 Gy 69%o responses 131 median survival 4.2 months 10 Gy 2 complete and 2 132 partial responses 8-10 Gy 5 complete and 7 133 partial responses 18-24 Gy 14 complete 134 responses 10 Gy Complete response 135 10 Gy Complete response 136 9Gy Complete response 141 35 Squamous cell 8 30Gy Complete response 142 carcinoma of the anal canal Squamous cell carcinoma 12 38-51 Gy 9/11 complete of the anal canal 10-18 Gy for response 143 boost Cervical carcinoma 1 45 Gy poor response 148 36 Despite its successful use even in HIV-1-infected patients, the adverse effects of radiotherapy must be carefully considered (145). It was reported that three HIV-1 -positive homosexual male patients with Kaposi's sarcoma treated with fractionated soft X-rays (total dose 30 Gy, single fraction dose 3 Gy) developed large bulbous cutaneous eruptions in the radiation field within 3 days of completion of treatment. These lesions resolved after 2-4 weeks, leaving post-inflammatory hyperpigmentation. This rare reaction to soft X-ray therapy suggests an altered radiosensitivity in HIV-1-infected patients with KS (146). Smith et al. (147) also reported that three HIV-1 infected patients developed cutaneous toxic reactions to radiation therapy, two with KS and one with NHL. Further study of the mechanisms underlying these severe adverse reactions associated with radiotherapy would be useful. A novel approach to improving the efficacy of radiation therapy that has produced impressive results is gamma knife radiosurgery. In two patients with deep-seated histologically verified malignant NHL, the minimal peripheral doses to the tumor were 21-25 Gy, with maximal doses of 55.5-70 Gy. Both showed a virtually complete response, with significant neurological improvement (149). Delivering therapy with higher dose-intensity could improve treatment results of NHL (115). If such higher doses were to be used on a more widespread basis, we would have to be sure that there are no deleterious effects on the replication of HIV-1 or any other virus that may be present in the host. 3.4 Experimental Applications of Radiation Therapy Shen et al.(150) reported that low dose total body radiation acted as a potent antiretroviral agent in vivo against Friend leukemia virus in mice. Del Regato et al. (151) hypothesized that simultaneous exposure of all lymphoid organs and tissues to ionizing radiation may destroy all virus-containing cells (including deep tissue stores) in HIV-1-infected humans. Any surviving viruses would be released into the circulation and be readily susceptible to anti-retroviral drugs. In addition, repeated exposure to ionizing radiation may be virucidal in itself. Thus, in the context of a study, they submitted their 37 patients to 10 consecutive daily exposures of approximately 0.1 Gy and to a weekly booster of approximately 0.05 Gy thereafter. A repeated series of 10 daily treatments was completed over a few months, to a total dose of approximately 20 Gy. A preliminary report published in 1989, was encouraging. However, to this date, the final analysis has yet to be published. Kliman et al. (152) tested the effect of cyclophosphamide, total body radiation and zidovudine on retroviral proliferation and disease progression in a murine AIDS model. Their results showed no benefit of radiation over zidovudine alone. 3.5 Radiation-Induced Enhancement of HIV-1 Replication: An Area of Concern? Preliminary data indicate that low dose radiation may increase HIV-1 replication (159). In addition, HIV-1-infected patients are frequently co-infected with other viruses (HSV-1, HSV-2, HBV and HCV, as examples). It is important to understand the radiation effects on viral replication in host cells, particularly if activation of viral replication (whether it be HIV-1 or other viruses) may be the result of radiation exposure (142). In other virologic models, radiation of target cells (before or after infection) or the viruses themselves may enhance the infectivity of these agents under certain circumstances. This has been shown for cells exposed to UV radiation, then infected with herpes or adenoviruses. It was demonstrated that active protein synthesis was required for the effect to occur. The mechanism of this effect does not depend upon excision repair since this phenomenon was also observed in XP cells and could be inhibited by cycloheximide (31). Jeeves and Rainbow (32) showed that exposure of fibroblast cells to 10 Gy U V or y radiation enhanced the replication of adenovirus in these cells. In another model, mutagenesis of parvovirus was used to infect irradiated cells. This was not observed in a herpesvirus model, likely due to the fact that parvoviruses make use of the host cell DNA polymerase in their life cycle. 38 Given that ionizing radiation is known to activate gene expression of certain molecules that enhance HIV-1 replication and that, as described above, gamma radiation is often used in vivo, the question of any relevant interaction must at least be considered. Much of the work in this field (summarized in Table 5) has been done using UV and X-radiation. HIV-1-infected patients frequently develop skin diseases including psoriasis, eosinophilic folliculitis and eczemas, for which UV therapy may be prescribed. We hypothesized that sunlight exposure or medical UV treatment may induce progression of AIDS based on the data that experimental exposure of HIV-1-infected cells to such radiation enhances HIV-1 replication in vitro, and UV itself inhibits cell- mediated immune responses. Intense sun exposure enhances the level of TNF-a and cis-UCA (urocanic acid), both of which may activate HIV-1 replication. Strong epidemiological evidence exists showing that exposure to solar UV radiation is an important cause of squamous cell carcinoma of the conjunctiva and that HIV-1 infection may be an additional predisposing factor (161-163). Thus, the safety of phototherapy in HIV-1-infected individuals remains to be definitively addressed. 39 Table 5 Radiation-induced enhancement of HIV-1 replication Type of radiation Max Dose U V A U V C uvc U V C uvc X-rays 600 KJ/m 2 30 J/m 2 28.8 K J / m 2 30 J/m 2 25-35.5 J/m 2 3 Gy Model HIV cat/HeLa X-rays 2.5 Gy HIV cat/HeLa HIV-LTR/CAT HIV-LTR/CAT HIV-LTR/CAT HTLV-IIIB/MT4 Clinical isolates/ PBMCs HTLV-IIIB/MT4 Enhancement of HIV-1 replication Yes, with U V A and photosensitizers Yes yes, up to 3 OX Yes yes, up to 29 fold yes up to 3 OX Ref 154 155 156 157 158 159 Yes, in 4 cell types 160 40 It was reported that these HIV-1-infected individuals have increased sensitivity to light, even in the early stages of disease. Although the pathogenesis of these light reactions is not known, they may be related to the depletion of endogenous scavengers, resulting from metabolic deregulation induced by HIV-1 disease (164,165). The Multicenter AIDS Cohort Study of 1155 white HIV-1-seronegative and 496 white HIV-1-seropositive homosexual men (of whom 142 became infected during the study) demonstrated that high U V sensitivity was not strongly correlated with the decline in the CD4 cell count or progression to AIDS (166). In one experiment, Saah et al. (166) administered psoralen plus U V A (total dose 91-1401 J/m ) to 8 patients with HIV-1-related skin disease that were unresponsive to other treatment modalities. No evidence of disease progression was observed, with respect to the HIV-1 infection, as a result of this therapy. Furthermore, the minimal U V B dose causing erythema was determined in 57 consecutive HIV-1-infected patients, as compared to a control group of 57 individuals with skin diseases. There was no difference between the groups (167), nor was the plasma viral load increased in four HIV-1-infected patients receiving U V A therapy for psoriasis (168). In the final analysis, some stimulatory effect of radiation on HIV-1 gene expression was observed, including at least one study evaluating clinical strains in human mononuclear cells. The mechanism of the observed effect is likely multifactorial and may relate to radiation-induced cell activation (as a result of DNA damage, for instance), specific or non-specific signal transduction (with increased production of intracellular TNF-a, for instance), or more generalized release of soluble mediators facilitating viral replication. U V radiation causes DNA damage including the formation of pyrimidine cyclobutane dimers (169). It has been suggested that induction of nucleotide excision repair by UV-induced DNA damage may lead to the activation of HIV-1 gene expression (170). Valerie et al. 41 (171) proposed that HIV-1 transcriptional responses to UV-C radiation are not the result of TNF-a stimulation and subsequent downstream cytoplasmic signaling events in HeLa cells. They suggest that U V induces bulky DNA damage that activates HIV-1 by chromatin decondensation associated with the repair process (171,172). Transcriptional regulation plays an important role in cellular responses to radiation. NF-kB, the intracellular concentration of which is increased in the setting of any form of cellular activation, can also activate certain viral promoters in the 5'LTR of the HIV-1 genome (173). In conclusion, there is little evidence of any deleterious effect of U V radiation in vivo with respect to HIV-1. However, the multiple in vitro findings remain (174-183). Once their mechanism has been explained, we may be in a better position to determine if there is a real possibility of any deleterious effects in vivo. We have gone on to evaluate the effect of gamma radiation in acute HIV-1 infection models (73). Mononuclear cells from uninfected healthy donors were exposed to 0.01-2 Gy gamma radiation. It appeared that the cells exposed to certain doses of radiation (especially 0.5 Gy) were more susceptible to infection with HIV-1. It has been shown that gamma radiation at doses of 2-50 Gy induced expression and binding activity of NF-kB in human KG-1 myeloid leukemia cell (174), which may be an explanation for our preliminary findings. It should be emphasized that our results and those of others remain laboratory observations, the in vivo correlates of which remain, at best, theoretical. An outline of proposed areas of investigation is shown in Table 6. These experiments, taken together, will allow us to address these issues. Practically, it is important to establish whether additional precautions (such as ensuring that suppressive anti-retroviral therapy is being taken) are indicated in patients treated with radiation for HIV-1-related conditions. Information generated in the course of these studies may also help us gain new insight into specific pathways involved in viral replication that could subsequently be used to design new strategies to inhibit viral replication. 42 Table 6 Experimental evaluation of the effects of radiation on HIV-1 replication A. In vitro Evaluation 1. Effect of gamma radiation (2-10 Gy) on replication of clinical HIV-1 isolates in mononuclear cells. a. Viral replication b. Intracellular and extracellular cytokine levels (IL-2, IL-4, IL-6/IL-10, IL-12) c. Intracellular redox potential d. Intracellular second messenger (TNF-a/NFkB) 2. Inhibition/enhancement of observed effect a. Addition of cytokines/ cytokine antibodies b. Addition of antioxidants c. Stimulation / inhibition of TNF-a/NFkB B. In vivo Evaluation 1. Serial viral load measurements in HIV-1-infected patients treated with radiotherapy a. Plasma viral load b. Cell-associated viral load c. Viral infectivity 2. Appropriate intervention studies, based on results of above evaluations. a. Recommendations for use of antiretroviral therapy with radiation b. Design of new radiation therapy protocols 43 4.0 THESIS HYPOTHESIS, GENERAL OBJECTIVES AND SPECIFIC OBJECTIVES 4.1 HYPOTHESIS Low-dose gamma radiation may activate HIV-1 replication and/or enhance HIV-1 infection in PBMCs. The extent of the effect will be dependent on the dose and the timing of radiation and may vary in different experimental settings. The communication between irradiated cells and non-irradiated cells may also influence the radiation effect on activation of PBMCs and HIV-1 replication. 4.2 GENERAL OBJECTIVE To increase our understanding of the effects of gamma radiation on the interaction of HIV-1 and host cells using appropriate in vitro model systems and to generate appropriate parameters to evaluate the potential effects of radiation therapy used in HIV-1-infected patients in clinical practice. 4.3 SPECIFIC OBJECTIVES 1. To study the immune stimulatory effects of low doses of gamma radiation on the activation of lymphocytes. 2. To determine whether low dose gamma radiation modulates HIV-1 replication in models of acutely infected PBMCs. 3. To determine whether low dose gamma radiation modulates HIV-1 replication in PBMCs of HIV-1-infected patients 4. To characterize the effect of timing of radiation on any observed effect. 5. To study the role of cell-cell communication in influencing the effects of radiation on lymphocyte activation and HIV-1 replication. 6. To characterize the dose-response curve for the interaction of radiation on host cell activation and HIV-1 replication, as well as the mechanism of any observed effect. 44 PART II 5.0 MATERIALS, METHODS AND INFECTION MODELS 5.1 Materials and Methods 5.1.1 Isolation and stimulation of donor peripheral blood mononuclear cells (PBMCs) Whole blood units taken from individuals known to be HIV-1-negative were obtained from the Canadian Red Cross. The blood was centrifuged to remove plasma, and then diluted with 0.01 M phosphate-buffered saline (PBS), pH 7.4. PBMCs were separated on a Ficoll-Hypaque density gradient. The isolated cells were washed twice with PBS, enumerated and cultured in R-P medium (RPMI 1640 with 2 mM L-glutamine, 10 mM HEPES, 10% heat-inactivated fetal bovine serum (Gibco BRL), 10 mg/mL PHA (Sigma Chemicals), 50 U/mL IL-2 (NIAID AIDS Research and Reference Reagent Program, Rockville, USA), 100 U/mL penicillin and 100 ug/mL streptomycin for 3 days. Concentration was adjusted to 2 x 106 PBMCs/mL. 5.1.2 HTLV-IIIB and titration of viral stocks 5.1.2.1 HTLV-IIIB H9 infected with HTLV-IIIB was obtained from Dr. Robert Gallo through the AIDS research and Reference Reagent Program, Division of AIDS, NIAID, NIH. H T L V -IIIB is dual tropic, that is, it is able to replicate both in T cells and macrophage (186). The virus containing culture supernatant of HTLV-IIIB H9 cells was collected in level-two laboratory at University of Ottawa. The virus containing fluid stored at -80°C as HTLV-IIIB stocks for further experiments. . The HTLV-IIIB containing culture supernatant was prepared by Ms. Dorren Ko, a technician in Dr. Conway's laboratory at the University of Ottawa. The HTLV-IIIB The titer of the HTLV-IIIB is 105 4 TCID 5 0 45 (titration of HTLV-IIIB was performed by myself). 5.1.2.2. Titration of viral stocks The stimulated PBMCs were then centrifuged at 400 g for 10 minutes at 22°C, the supernatant was removed and discarded and the cells were resuspended in fresh culture medium (as described above, but without the PHA). The cells were enumerated and adjusted to a concentration of 4 x 106 PBMCs/mL. For the viral titrations themselves, seven serial four-fold dilutions of virus-containing supernatant fluids (dilutions ranging from 1:16 through 1:64,536) were inoculated in triplicate in the middle wells of 96 well flat-bottom tissue culture plates, with a total volume of 200 uL being added to each well. Cells (50 uL stimulated PBMCs, or 2xl0 5 cells/well) were added to each well, with a separate culture established without HIV-1, as a negative control. The plate was incubated at 37°C and 5% CO2 in a humidified chamber. On day 4, cells were resuspended and 125 uL of the cell suspension were discarded, and replaced with 150 uL of fresh culture medium. On day 7, the assay was terminated and the appropriate supernatants were tested for HIV-1 p24 antigen levels, as a marker of viral replication. A well was scored "positive" if the p24 values exceeded 30 pg/mL. The viral titer was computed by the Spearman-Karber's method, and expressed as the TCIDso/mL (50% tissue culture infective doses/mL). 5.1.3 Separation of CD4 and CD8 cells Isolated PBMCs were washed twice with PBS and the cell pellet resuspended in 80uL of PBS supplemented with 5 mM EDTA and 0.5% bovine serum albumin per 107 cells. The CD4 and CD8 cells were then separated with the MiniMACS System using colloidal super-paramagnetic microbeads conjugated with monoclonal anti-human CD4 and CD8 antibodies (MACS, Mitenyi Biotec Inc, Sunnyvale, USA). The PBMCs were resuspended in 1 mL of PBS containing 0.5% BSA (Sigma Chemical Co). After 46 centrifugation, 20 uL of MACS CD4 or CD8 microbeads/107 cells were added to the pellet, which was then incubated at 4-6°C for 15 minutes. Five minutes before the end of the incubation, a magnet was mounted on the column, a plate flow adapter added to its end and the column was then washed with 0.5 mL of cold PBS-BSA. The cells suspended in 500 uL of buffer were applied to the top of a pre-filled and washed column. Effluent was collected as a negative cell fraction. After washing the column with 1 mL PBS-BSA, the column was removed from the magnet, placed in an appropriate tube (labeled "CD4" or "CD8") and filled with 2 mL of PBS-BSA. The labeled cells were pushed through with a plunger and eluted from the MACS as a positive fraction. 5.1.4 Radiation facilities During the entire course of these studies, there were three types of radiation facilities which were made available to us: 6 0 Co Gammabeam 150 (AECL, Radiochemical Company) located at Chalk River laboratories, dose rate ~ 3mGy/min. 137 Cs Gammacell 40 (AECL Radiochemical Company) located at the University of Ottawa), dose rate 52mGy/min and Model 81-14-R Beam Irradiator (J. L. Shepherd and Associates, San Fernando) located in the B. C. Cancer Agency, Vancouver, dose rate ~ 112 mGy/min. Both control cells as well as the cells to be irradiated were handled and processed in the same way except for the radiation exposure. During exposure of the test cells, the control cells were held under the same conditions of dilution and temperature in an area just outside the radiation facility. 5.1.5 Radiation and interleukin-2a receptor detection Separated PBMCs from each donor were divided into separate aliquots to serve as controls or samples to be irradiated, both types of samples being diluted to the same concentration (5-10 x 106 cells/mL). Following radiation, the cells were resuspended in viral culture medium containing 5 ug/mL of T-cell mitogen ConA ( Sigma Chemical 47 Co.), 2 mM glutamine and 10% (v/v) heat-inactivated fetal bovine serum, 100 unit/mL penicillin and 100 u,g/mL streptomycin, then incubated at 37°C in the presence of 5% CO2 for up to 72 h. When harvested, the cells were centrifuged and washed once in PBS containing 2% FCS at 0°C and once in PBS without FCS. Fluorescently labeled (FITC) monoclonal antibody to the IL-2Ra chain (Becton Dickinson, CD25) diluted in PBS was incubated with the cells for 1 h at 0°C. The cells were washed three times at 0°C in PBS and re-suspended in PBS at 0°C for flow cytometry analysis (Ortho model 50 H Cytofluorograf). Labeled cells were scored using a two-parameter analysis of axial light loss vs. FITC fluorescence that discriminates between live and dead cells. This analysis, after correcting for autofluorescence, non-specific fluorescence and signals from background debris and apoptotic or dead cells using pulse discrimination, provided data on the fraction of mononuclear cells expressing IL-2Roc. The above experiments were performed at Atomic Energy of Canada Limited, Chalk River Laboratories. 5.1.6 HIV-1 infection HIV-1 culture was performed in a level II laboratory at the University of Ottawa and in a level III laboratory at the BC Center for Disease Control. Strict sterile technique was required when HIV-1 culture was performed. All control and test cells were stimulated in complete RPMI- 1640 medium containing 10 ug/mL phytohemagglutinin-P (PHA) (Sigma Chemical Co.) and 50 units/mL interleukin- 2 (IL-2; NIAID AIDS Research and Reference Reagent Program, Rockville, MD) and seeded at a concentration of lx 106 cells/mL. All tissue culture flasks (VWR Canlab) were incubated overnight at 37 0 C with 5% C 0 2 . After 18-24 h of PHA stimulation, the cultures were harvested and all control and test PBMCs were centrifuged down, washed in phosphate-buffered saline (PBS), then resuspended at 107 cells/mL in complete RPMI 1640 medium containing 2 mM glutamine, 10% heat-inactivated fetal bovine serum, 100 unit/mL penicillin and 100 p.g/mL streptomycin without PHA and IL-2. The cells were infected with titrated HIV-1 virus stock at a multiplicity of infection (MOI) of 0.001 (104 TCID 5 0 / 107 PBMCs). A laboratory strain of HIV-1, HTLV-IIIB (NIAID AIDS Research and Reference Reagent 48 Program. Rockville, MD) was used. After gently mixing cells and virus stock, the suspensions were incubated at 37 °C with 5% CO2 for 1 hour, vortexed gently, and then incubated for an additional hour. To remove extracellular virus from the preparation, the infected PBMC suspension was brought up to 12 mL with viral culture medium. After brief centrifugation, the supernatant was then removed and the cells were resuspended in complete RPMI 1640 medium with 50 unit/mL IL-2, but without PHA. On days 4, 7, 11 and 14, the cultures were fed (replacement of half the medium with fresh supplemented RPMI 1640) and an aliquot of the harvested supernatant was frozen at -70 °C. At the time of each feeding, cells were also enumerated, with cell viability assessed by Trypan blue dye exclusion. 5.1.7 Detection of p24 in culture supernatants Viral replication was evaluated by the measurement of p24 antigen levels in these supernatants using a p24 Enzyme-Linked Immuno-Sorbent Assay (ELISA). This was done using commercial HIV-1 p24 antigen assay kits (Coulter Corp., Hialeah, FL and Organon Teknika Corp., Durham, N C ) , according to the manufacturer's instructions. The limit of the p24 detection is 3 pg/mL. Briefly, lysed culture supernatant samples were incubated in 96-well plate containing murine anti-p24 monoclonal antibody coated micro wells. Following plate washing, a biotinylated anti-HIV-1 antibody was added, which could subsequently react with a second antibody conjugated with streptavidin and horseradish peroxidase (HRP). After appropriate incubations, the plate was washed and a substrate to HRP was added, leading to the development of a blue color in positive wells. The reaction was terminated by adding a stopping reagent (H2SO4). The plate was then read on a microtiter plate reader set at 450 nM with a reference wavelength of 570 nM. The intensity color is directly proportional to the amount of HIV-1 antigen in the culture supernatant. 5.1.8 Detection of IL-2R and IL-10 in culture supernatants 49 BioSource International Cytoscreen human interleukin-2R ELISA kits and BioSource International Cytoscreen human interleukin -10 ELISA kits (Serotec Ltd, Toronto) were used for quantitation in cell culture supernatants. Briefly, standards, controls and specimens were added to the appropriate microtiter wells coated with an antibody specific for IL-2R or IL-10, followed by the addition of a second biotinylated antibody. During the first incubation, the IL-2R and IL-10 antigens bound simultaneously to the immobilized antibody on one site, and to the solution phase biotinylated antibody on a second site. Streptavidin/Peroxidase working conjugate solution was added to wells after washing to remove excess second antibody. After a second incubation and washing to remove all of the unbound enzyme, the TMB solution was added which acted upon by the bound enzyme to produce color. One hundred uL of stop solution was added to each well and the absorbance of each well was read at 450 nM with a reference wavelength of 570 nM. The yellow color developed is directly proportional to the amount of IL-2R and IL-10 in the culture supernatant. 5.1.9 DNA polymerase chain reaction (PCR) 5.1.9.1 Cell lysis Cell pellets which had been frozen at -70°C were re-suspended in lysis buffer #1 (lm M Tris, 50 mM KC1, 2.5 mM MgC^). After resuspension, an equal volume of lysis buffer #2 (lysis buffer #1 containing 1% Tween 20 and 1% NP40) was added and mixed. For every 50 uL of cell lysate which contained 1.5 x 106 PBMCs, 5 uL of proteinase K stock solution were then added to each tube. The tubes of lysate were then placed in a heating block at 56°C for 40 minutes, then 94°C for 20 minutes. The lysate was spun down at 4000 rpm for 10 minutes in a microcentrifuge, the supernatant was then removed and transferred to fresh 1.5 mL tubes and stored in the refrigerator at 4°C. 5.1.9.2 Polymerase chain reaction amplification of HIV-1 pol region 50 The sequences of PCR pol 1 and pol 2 primers as follows: pol 1 5'-CTA A A G G A A GCT C A T T T A GA-3' and pol 2 5'-GTC A A T GGC C A T TGT T T A AC-3'. These primers amplify a conserved 326 base pair segment in the pol region of the HIV-1 BRU isolate. The PCR mixture contained double-distilled H 2 0 (20.3 uL), 10 x PCR buffer II (5 uL), 25 mM MgCl 2 (4 uL), dNTP mix (1.25 mM each nucleotide, 8 uL), 20 uM pol 1 (1.235 uL), 20 uM pol 2 (1.235 uL), Taq (0.234 uL). The sample (10 uL) was then added to each tube (total volume, with sample, 50 uL). The positive control consisted of 8E5 cells (15, 150 and 1500 cells, each of which contains a single copy of integrated HIV-1 DNA). The amplification was performed in an automated thermal cycler, programmed for 30 cycles (denaturation at 95°C for 1 minute, extension at 72°C for 1 minute, annealing at 45°C for 1 min). 5.1.9.3 Enzyme-linkedoligonucleotidesorbent assay (ELOSA) Twenty uL of PCR product was added to each streptavidin-coated in a 96-well plate. First, 100 uL of hybridization buffer (10% formamide, 6xSSC at pH7.0,1.2% Triton X-100, 0.12% bovine serum albumin) containing 10 nM biotin -labeled capture probe (sequence 5'-CCC T A T C A T TTT T G G TTT C C A T-3') and 5 nM fluorescein-labelled reporter probe (sequence 5'-CTG TCT T A C TTT GAT A A A A C C TC-3') was then added to each well and the plate was incubated at 37°C for 60 minutes. After washing six times using a plate washer, 100 uL of 1:50 diluted anti-fluorescein BP-1 enzyme conjugate in anti-fluorescein dilution buffer (lx Dupont plate wash buffer, 5%> calf serum, 0.2%> casein) was then added to each well, and the plate was then incubated at room temperature for 60 minutes. Thereafter, 100 uL TMB substrate were added to each well and the plate was incubated at room temperature for another 60 minutes. A stop solution was added, and the plate was read in the plate reader at 450 nm with a reference wavelength of 620 nM. 5.1.10 Detection of CD3, CD4 and IL-2 receptor a on HIV-1-infected cells 51 To measure the percentages of PHA stimulated HIV-1-infected PBMCs that express CD3, CD4 and IL-2 receptor a expression, anti-CD3-ECD, anti-CD4-PE and anti-IL-2R a (anti-CD 25)-FITC monoclonal antibodies were used, as supplied by the manufacturer. Three color immunoflurence analysis was performed with an standard EPICS X L (Coulter Corp.). The method of detection IL-2 receptor a was different from the method described on section 5.15, because it measured the percentage of IL-2 receptor a expreesion on CD4+ subpopulation of T lymphocytes (CD3+). This part of experiments was conducted at the University of Ottawa. 5.1.11 Stable CD4+ cell lines Human CD4+ neoplastic cell lines (H9, H 9 R F , 8E5, and 8E5L, NIAID AIDS Research and Reference Reagent Program, Rockville, MD) were used in the studies. H9, a T-lymphoblastoid CD4 cell line, mycoplasma-free, is a clonal derivative of HUT78, a human T-cell line derived from the peripheral blood of a patient with cutaneous T-cell lymphoma. The HUT78 line and its clonal derivative H9, secrete IL-2 and express markers of mature helper T- cells (CD3+, CD4+, IL-2R+, MHC class II antigens). The presence of adventitious viruses, mycoplasma, fungi and protozoa has been excluded in all cases. The 8E5 cell line is a clonal derivative of CCRF-CEM, a T cell leukemia line derived from a patient with acute lymphoblastic leukemia. The C C R F - C E M and its clonal derivatives exhibit relatively immature T- lymphocyte markers (CD3-/CD4+/MHC class II antigen-). The 8E5L cell line was used in our experiments to study the induction of virus expression since it behaves as a latently- infected cell. 5.1.12 Statistical analysis 5.1.12.1 IL-2R data analysis For any individual person, the data points reported are the average of the indicated number (n) of samples, and the standard deviation (SD) is given numerically or as error 52 bars. Significant variation in response between individuals was noted where the data are averaged for a significant number of separate blood samples from different individuals, and the degree of variation is given as the standard error of the mean (SE). Except for the data presented in the Tables, results from different individuals were not averaged, and the number of separate blood samples from a single individual are presented. Where relatively precise results from repetitive samples from a single individual are shown, they are representative of the qualitative but statistically significant results obtained from any and all the individuals studied. When averaged data from irradiated cells were compared with control cells, differences were tested for statistical significance using the analysis of variance test, and P values are given. Since the percentages of IL-2 receptors obeyed a binomial distribution, arc-sine square root transformation was used to change the data into an approximately normal distribution. One-way and two-way analyses of variance (ANOVA) were employed (Epidemiology Statistic Software available at AECL). 5.1.12.2 p24 data analysis The analysis consisted of comparing the maximal levels of p24 antigen in the control and irradiated cultures. The statistical significance of any observed differences was assessed using the Wilcoxon signed-rank test ( Statistic Analysis Software at the Canadian HIV Trials Network). In addition, to ensure that any of these differences were not due to selective cell killing, cells were enumerated for the various experimental conditions. 5.1.12.3 DNA PCR data analysis The analysis consisted of comparing HIV-1 DNA copies in the control and irradiated cells using the Wilcoxon signed-rank test ( Statistic Analysis Software available at Canadian HIV Trials Network). 5.2 HIV-1 infection Models. 53 5.2.1. Acute infection models 5.2.1.1. Pre-infection radiation model with PBMCs Leukocyte-enriched whole blood from HIV-1 seronegative donors was obtained on five separate occasions and PBMCs were isolated by density gradient centrifugation. The cells were then incubated overnight at 37^C with 5% C02 at a concentration of 1x10^ cells/mL in tissue culture flasks in complete RPMI 1640 medium. Thereafter, the cells were enumerated and suspended in multiple aliquots of 5-10xl07 cells/mL (depending on cell yield) in 1.5 mL polypropylene tubes prior to radiation. Doses of 1, 5, 10, 50, 100 and 200 cGy were administered using a Cs gamma irradiator (Gammacell 40, A E C L Radiochemical Company). To reduce the dose-rate from 1.25 Gy/min to 5.2 cGy/min, the samples were placed in a double container, then shielded by lead blocks. The doses received were measured using ESR alanine dosimeters calibrated against an ionizing chamber (AECL, Chalk River Laboratories). In each of the five experiments, control cells were processed in parallel in an identical manner except for their exposure to radiation. After radiation, all control and irradiated cells were cultured separately in complete RPMI 1640 medium containing 10 ug/mL PHA and 50 unit/mL IL-2 (NIAID AIDS Research and Reference Reagent Program) at a concentration of lx l0 6 cell/mL with 12- well flat-bottom plates (VWR, Westwood, MA) .All tissue culture flasks were incubated at 37^C with 5% C02 overnight. After incubation for 24 hours, the cells were aspirated and pelleted and all control and irradiated PBMCs were infected with titrated HIV-1 virus stock at an MOI of 0.001 (104 TCID50 A O 7 PBMCs), using standard protocols for acute infection (186-187). A laboratory strain of HIV-1 (HTLV-IIIB, NIAID AIDS Research and Reference Reagent Program) was used. After infection, the cells were resuspended in complete RPMI1640 medium with 50 units/mL IL-2, without PHA. On days 4, 7, 11 and 14, the cultures were fed (replacing half the medium with fresh supplemented RPMI1640 medium containing 50 unit/mL IL-2, without PHA). On days 54 4, 7, 11 and 14, the cultures were fed (replacing half the medium with fresh supplemented RPMI1640) and the cells were enumerated, with cell viability assessed by Trypan blue dye exclusion. An aliquot of the supernatant was harvested and frozen at -70^C. Viral replication was evaluated by the measurement of p24 antigen levels in the supernatants using a commercial assay (Coulter Corp). The IL-2R levels, IL-10 levels in the culture supernatants and CD3, CD4 counts were measured at day 4, 7, 11 and 14. In selected experiments, PBMCs were treated with 35 nM H2O2 rather than gamma radiation, In selected experiments, PBMCs were treated with 35 nM H202 provides an concentration of H202 equivalent to that generated by 50 cGy radiation (188). 5.2.1.2. Pre-infection radiation model with CD4, CD8 For these experiments, volumes 35-40 mL of peripheral blood were obtained from HIV-1 seronegative donors and PBMCs were isolated by density gradient centrifugation. CD4 and CD8 cells were sorted from PBMCs using colloidal super-paramagnetic microbeads conjugated with monoclonal anti-human CD4 and CD8 antibodies (MACS, Mitenyi Biotec Inc). Isolated CD4 cells, recombined CD4 and CD8 cells and unseparated PBMCs were exposed to gamma radiation at doses of 25-200 cGy or treated with 35 nM H 2 0 2 . Control cells were processed in parallel, except for exposure to radiation or H2O2. Thereafter, all cells were cultured separately in complete RPMI 1640 medium containing 10 ug/mL PHA and 50 unit/mL IL-2 (NIAID AIDS Research and Reference Reagent Program) at a concentration of 0.5-1x10^ cell/mL. Following 24 hours of PHA stimulation, the cells were washed and pelleted and all the samples were infected with titrated HIV-1 stock virus stock at an MOI of 0.001 (104 TCID50/IO7 PBMCs), using standard protocols for acute infection (187). A laboratory strain of HIV-1 (HTLV-IIIB, NIAID AIDS Research and Reference Reagent Program) was used. The cells were then resuspended in complete RPMI 1640 medium with 50 unit/mL IL-2, without PHA. On days 4, 7,11 and 14, the cultures were fed (replacing half the medium with fresh supplemented RPMI 1640) and the cells were enumerated, with cell viability assessed by Trypan blue dye exclusion. An aliquot of the supernatant was harvested and frozen at -70 55 Viral replication was evaluated by the measurement of p24 antigen levels in these supernatants (Organon Teknika Corp). 5.2.1.3 Post-infection radiation model with PBMCs Whole blood from HIV-1 seronegative donors was obtained and mononuclear cells(PBMCs) were isolated by density gradient centrifugation. Cells were cultured in complete RPMI-1640 medium containing 10 u.g/mL PHA and 50 unit/mL IL-2 (NIAID AIDS Research and Reference Reagent Program) at a concentration of lx l0 6 cell/mL. All tissue culture flasks were incubated at 37^C with 5% C02 overnight. After PHA stimulation for 24 hours, the cells were harvested and washed, and all the samples were infected with titrated HIV-1 stock at an MOI of 0.001 (104 TCID50/IO7 PBMCs), using standard protocols for acute infection. A laboratory strain of HIV-1 (HTLV-IIIB, NIAID AIDS Research and Reference Reagent Program) was used. Doses of 1, 5, 10, 50, 100 and 200 cGy were administered using Model 81-14-R beam irradiator (J.L. Shepherd and Associates) immediately after viral infection or 18 hours after viral infection. In each of the experiments, control cells were processed in parallel in an identical manner except for their exposure to radiation. The cells were then re-suspended in complete RPMI 1640 medium with 50 unit/mL IL-2, without PHA. On days 4, 7, 11 and 14, the cultures were fed (replacing half the medium with fresh supplemented RPMI 1640 medium with 50 unit/mL IL-2, without PHA) and the cells were enumerated, with cell viability assessed by Trypan blue dye exclusion. An aliquot of the supernatant was harvested and frozen at -70^C. Viral replication was evaluated by the measurement of p24 antigen levels in these supernatant (Organon Teknika Corp). 5.2.2. Evaluation of PBMCs of HIV-1-infected patients Blood was obtained from nine HIV-1-infected individuals attending the Infectious Disease Clinic at St. Paul's Hospital (Vancouver, B.C.). These patients were selected as 56 having negative plasma p24 antigen levels. It is not known whether they were on active antiretroviral therapy or not. PBMCs were isolated by density gradient centrifugation. The cells were enumerated and suspended in multiple aliquots of 1x10^ cells/mL in 1.5 mL polypropylene tubes prior to radiation. Doses of 5, 25, and 50 cGy were administered using a Model 81-14-R beam irradiator (J.L. Shepherd and Associates). Following radiation, all control and irradiated cells were cultured separately in complete RPMI1640 medium containing 10 u.g/mL PHA-P and 50 unit/mL IL-2 at a concentration of 0.5 -l x l O 6 cell/mL in 24 well tissue culture plates at 37°C with 5% C02 overnight for 18-24 hours. The PBMCs were then resuspended in complete RPMI1640 medium with 50 unit/mL IL-2, without PHA-P. On days 4, 7, 11, 14, 17 and 21, the cultures were fed (replacement of half the medium with fresh supplemented RPMI-1640) and an aliquot of the harvested supernatant was frozen at -70^C. Viral replication was evaluated by the measurement of p24 antigen levels in these supernatants (Coulter Corp). At the time of each culture feeding, cells were also enumerated, with cell viability assessed by Trypan blue dye exclusion. On day 21, the cultures were harvested and the cells were enumerated, pelletted and kept frozen at -70^C. Cell-associated pro-viral load was quantitated using a DNA PCR assay designed in our facility (178,179). 5.2.3 Evaluation of cell lines stably infected with HIV-1 H9RF, 8E5 and 8E5L cells were thawed in a 37°C water bath and washed once with warm R-10 medium. The cell suspensions were transferred into T25 flasks containing 10 mL RPMI 1640 medium containing 2 mM L-glutamine, 10 mM HEPES, 10% fetal bovine serum (heat-inactivated), 100 units/mL penicillin and lOOug/mL streptomycin. The cells were enumerated and suspended in multiple aliquots of l x l 0 7 cell/mL in 1.5 mL polypropylene tubes prior to irradiation. Doses of 1,5, 10, 50,100, 200 and 400 cGy were administered using a Model 81-14-R beam irradiator (J.L. Shepherd and Associates) at the B.C. Cancer Agency. Immediately after irradiation, all control and tested cells were cultured in RPMI 1640 medium containing 2 mM L-glutamine; 10 mM 57 HEPES, 10% fetal bovine serum (heat-inactivated); 100 units/mL penicillin, 100 ug/mL streptomycin at a concentration of lx l0 6 cells/mL (H9RF) or 2xl0 6 cells/mL (8E5 and 8E5L) in 24 well tissue culture plates at 37°C with 5% CO2. On days 4, 7, 11, 14, the cells were enumerated and cultures were fed (replacement of half the medium with fresh supplemented RPMI-1640) and an aliquot of the harvested supernatant was frozen at -70^C. Viral replication was evaluated by the measurement of p24 antigen levels in these supernatants (Organon Teknika). At the time of each feeding, cells were also enumerated, with cell viability assessed by Trypan blue dye exclusion. 58 PART III 6.0 RESULTS 6.1. HIV-1 infection in PBMCs of different donors. Productive HIV-1 infection in normal PBMCs could not be achieved in the absence of PHA-P stimulation (data not shown). After PHA-P stimulation for 18-24 hours, PBMCs were infected with HTLV-IIIB (a laboratory strain of HIV-1) at a multiplicity of infection (MOI) of 0.001, p24 was measured by ELISA. p24 is HIV-1 core protein and the presence of HIV-1 p24 is evidence of viral replication. The concentration of p24 directly represents the amount of HIV-1 replication. Results from Figure 6 showed that a productive infection was established in three of the five experiments (donor A, B and C); however, the levels of HIV-1 replication were quite different among these three donors although the multiplicity of infection was the same (MOI=0.001). Figure 6 is a comparison of p24 levels in culture supernatants of three productive infections harvested at day 4, 7, 11 and 14, showing great inter-individual variability. Two of the five experiments (donor D and E) failed to establish a productive HIV-1 infection. In donor D, the p24 levels in the culture supernatant of PBMCs decreased with the time; on day 4, it was 8 pg/mL and then on day 7, only 3 pg/mL was detected, and by the day 11, p24 returned to undetectable level. In the experiment with donor E, there was no detectable p24 in the culture supernatant of PBMCs after they were infected with HTLV-IIIB. The results indicate that PBMCs from the same donor should be used to observe any low dose radiation effect on HIV-1 replication since great variation in HIV-1 infection is present among different individual donors. The data also suggested that it was necessary to culture PBMCs up to 11 or 14 days to verify HIV infection of PBMCs. 59 Rgtre 6 HTIV-111B infection in PBMCs of dfferent donors Days after HTLVIIIB infection 60 6.2. Effect of acute HIV-1 infection on IL2Rot expression in PHA-stimulated PBMCs Activation of CD4 cells is essential for effective HIV-1 replication. Lymphocytes are highly radiosensitive, while viruses are more radioresistant. To study low dose radiation effects on HIV replication, it is important to examine whether there is an immune stimulatory effect induced by low dose radiation on activation of lymphocytes. Only activated T cells express the high affinity IL2Rcc, since resting T cells only express IL-2R(3y. The high-affinity IL2 receptor allows growth stimulation at physiologic IL-2 concentrations. IL-2Roc expression on PHA -stimulated PBMCs subjected to acute HIV-1 infection was measured as a marker for IL-2 production since activated T cells produce IL-2 which further enhances IL2Rct expression and IL 2 synthesis, providing a positive amplification system. IL2 is an important autocrine and paracrine growth factor produced by activated CD4 (Thn and Thj) cells, and in lesser quantities by CD8 cells. The magnitude of IL-2 synthesis plays an important role in determining the magnitude of cellular and humoral immune response. Table 7 summarizes an experiment in which PBMCs were infected with H T L V -IIIB following PHA stimulation for 18 hours, and were subsequently maintained in culture medium containing IL-2. There were only 5% of PBMCs which expressed IL-2Rot when they were isolated and before PHA stimulation and HTLV-IIIB infection. IL-2Ra expression on PBMCs increased dramatically following infection as well as in culture medium containing IL-2. The highest percentage of IL-2Ra on PBMCs was observed on day 4, then decreased with the time in culture. However, on day 14, although 59% of IL2Rcc was detected on the surface of PBMCs infected with HTLV-IIB, only 33% of IL2Ra was expressed on the uninfected PBMCs. HIV-1-induced high affinity IL2cc receptor expression on PBMCs seems important to maintain active HIV-1 replication, as HIV-1 infection takes 14 days to reach the peak of acute HIV-1 infection (Figure 6). Overall, no significant changes of IL-2Ra expression were observed in the samples subjected to radiation. 61 Table 8 indicates that, as expected, the number of CD4 cells expressing IL-2Rcc decreased after HIV-1 infection. Interestingly, they also decreased with time in culture, even without infection, suggesting that CD4 molecules can be down-regulated by mitogen/IL-2 stimulation and HIV-1 infection. It is also possible that the number of cells expressing these molecules could be decreased over time. Although the peak of viral replication was on days 11 or 14 in acute HIV-1 infection, only 1-2 % CD4 cells bearing IL-2Ra were detected in the cultured cells. Since the major target cells of HIV-1 infection are CD4 cells, this could have indicated that CD4 cells were alive but CD4 molecules on the cell surface couldn't be detected due to CD4 molecule down-regulation. Even in the absence of HIV-1 infection, CD4 molecules still disappeared from the surface following PHA stimulation, this may represent a negative regulating mechanism to prevent further immune activation. 62 Table 7 Percentage of PBMCs expressing IL-2Ra over time as a function of PHA stimulation, acute HIV-1 infection and/or exposure to gamma radiation in a single experiment Days after HTLV IIIB infection' 4 7 11 14 Control/Uninfected 83 85 50 33 Control/Infected 90 92 78 59 Irradiated cells (Infected) 1 cGy 87 93 78 68 5 cGy 92 92 79 53 lOcGy 87 87 72 47 50cGy 91 90 81 52 100 cGy 84 93 74 52 200 cGy 94 89 70 42 (MOI=0.001) aWhen PBMCs were isolated and before PHA stimulation and HTLV-IIIB infection, the baseline of IL-2Ra was 5%. 63 Table 8 Percentage of PBMCs showing both IL-2Ra and CD4 after radiation, PHA stimulation and acute HIV-1 infection in a single experiment Days after HTLV-IIIB infection 4 7 11 14 Control 14 21 9 2 Control/Infected 7 6 1 1 Irradiated cells (Infected) 1 cGy 8 2 1 1 5 cGy 7 3 1 1 lOcGy 5 1 1 2 50cGy 8 3 1 2 100 cGy 6 2 1 <1 200 cGy 8 2 1 2 (MOI=0.001) 64 6.3. Effect of gamma radiation on IL- 2Ra expression The preliminary experiments had shown that when PBMCs were stimulated with PHA and maintained in a medium containing IL-2, IL-2Ra expression was detected on 90% of cells after four days. It was difficult to tell whether there was a low dose radiation effect on IL-2Roc expression since 90% of infected PBMCs were expressing IL-2Ra in culture conditions required for acute in vitro HIV-1 infection. Acute in vitro HIV-1 infection requires PHA stimulation and the presence of IL-2 in the viral culture medium. To examine whether low dose radiation affects lymphocyte activation, in follow-up studies, PBMCs taken from HIV-1-uninfected donors were first exposed to low doses of gamma radiation and then stimulated with ConA at sub-optimal concentration (5ug/mL) in the absence of IL-2. Table 9 shows results averaged from 11 different individuals, showing a statistically significant response to radiation. After ConA stimulation, control PBMCs expressed 14.3%, 22.4% and 23.3% IL2Ra on their surface after 24, 48 and 72 hours. There was increased IL-2Roc expression when PBMCs were exposed to low dose gamma radiation. This stimulatory effect could be detected as low as 0.25 mGy, as 19.1%, 27.9% and 29.4% of PBMCs expressed IL-2Rcc after ConA stimulation at 24, 48, and 72 hours respectively. Although low doses of radiation enhanced IL-2Ra on lymphocytes in a statistically significant manner, there was also a statistically significant variability among different donors (PO.05, two-way A N O V A tests). To evaluate this further, 10 multiple replicate samples of PBMCs from the same donor were tested. Table 10 shows typical results that 10 mGy radiation significantly enhanced IL-2Roc expression on the cellular surface by 48 hours. 65 Table 9 Enhancement of IL-2oc receptor expression by exposure of ConA-stimulated P B M C s No. of donors Hours after ConA stimulation' 24 48 72 Control 11 14.312.2 22.413.3 23.313.0 Irradiated (dose in mGy) 0.25 11 19.1+3.5* 27.914.9* 29.415.5* 1.0 11 16.0+2.5* 27.314.1* 31.515.2* 2.0 11 16.412.6* 29.514.6* 30.714.5* 5.0 11 20.313.4* 27.812.9* 31.313.6* 10 11 22.714.6* 28.214.0* 36.815.4* 20 11 21.713.5* 29.013.8* 33.114.3* 50 11 19.913.1* 29.714.6* 31.813.9* a Data represent that meanlSE of percentage of cell expressing IL-2Ra on PBMCs taken from 11 different donors. *P<0.05 (Control vs. Irradiated) 66 Table 10 Enhancement of IL-2a receptor expression by exposure of C o n A -stimulated P B M C s Dose No. of samples Hours after ConA stimulation3 0 24 48 72 Control 10 3.3+1.1 9.6±0.1 23.1+1.0 28.5+0.4 Irradiated (10 mGy) 10 2.2+0.8 11.1+0.4* 34.2+0.4* 36.5+0.2* a Data represents mean±SE for n=7. PBMCs were isolated from a single donor. * P<0.01 (Control vs. Irradiated ) 67 6.4. IL-2Ra expression and cell-cell communication between non-irradiated and irradiated PBMCs. To determine whether IL-2Ra expression was altered in cultures containing mixtures of non-irradiated and irradiated PBMCs, cells which had been exposed to 10 mGy 24 hours previously were mixed with control cells and cultured in the presence of ConA. The stimulating effect of mixing culture of irradiated cells and control cells (1:1 ratio) on IL-2Ra expression was similar to that observed in 100% irradiated cells. The results indicated that cell-cell communication played a role in this the stimulatory effect as radiation enhanced IL-2Ra could be extended to control cells (Table 11). To determine whether the stimulatory effect was due to a factor/factors released in the medium following radiation, the exposed cells and the culture supernatant were tested separately. PBMCs were subjected to 10 mGy radiation and stimulated with ConA for 24 hours, and then the culture supernatants were separated from the cells. Therefore, the culture supernatants were called "irradiated supernatants" and the cells were called "irradiated cells". Control cells were incubated for 24 hours in the presence of ConA and separated from their culture supernatants in the same way as 10 mGy irradiated cells. The culture supernatants were called "control supernatants" and the cells were called "control cells". The "irradiated cells" were washed once in PBS and incubated in the supernatants from control cells for 24 hours with ConA as above. These "control cells" (without their previous culture supernatant) were then suspendended and cultured in the previous supernatant removed from 10 mGy irradiated cells and incubated for a further 24 hours. There was about 32% IL2Ra expression on the surface of control cell in control supernatants, 41% of IL2Ra on control cells in irradiated supernatant, and only 30% on irradiated cells in control supernatant. IL-2Ra expression on control cells was stimulated by irradiated supernatants; this was not observed when irradiated cells were suspended in control supernatant, suggesting the observed effect was mediated by a soluble factor. The results showed that when irradiated cells were further incubated with control cells in 68 control supernatants, there was no elevated IL-2Ra expression on the cell surface. It seemed that control cells and their culture supernatant exerted a suppressive effect on irradiated cells and abolished the stimulatory effect of low dose radiation on IL-2Ra expression. 69 Table 11 The percentage of P B M C s expressing IL-2Rcc after Con A stimulation 3 Hours after C o n A n control cells Irradiated cells 50% control cells+ stimulation ( l O m G y ) 50% irradiated cells 24 7 7.7+4.1 17.8+3.3 22.6+4.8 p<0.001b p<0.0001b 48 7 32.4+3.2 42.5+4.1 41.5+2.2 p<0.01b p<0.001b 72 7 54.4+5.2 70.4+1.8 69.1+3.3 p<0.001b p<0.01b a The values given are the mean±S.D for n=7. These data represent results from lymphocyte taken from a single donor. b Compared to control cells 70 Table 12 Effect of irradiated cells or medium on the expression of IL-2Ra on non-irradiated control cells8 Control cellsb Irradiated cells0 Control supernatantsb 32.4 ± 3 . 2 30 ± 4.2 Irradiated supernatants0 40.9 ± 3 . 6 42.5 ± 4 . 1 a These data represented the percentage of IL-2Rcc expressing on PBMCs taken from a single donor. The values given are the mean±SD for n=7. b PBMCs were stimulated with ConA for 24 hours, then culture supernatant was removed from control cells. The culture supernatant was called "control medium" and the cells were called "control cells". Control cells were further cultured with irradiated medium or with irradiated cells in control medium for another 24 hours. c PBMCs were irradiated at 10 mGy and stimulated with ConA. Then, culture supernatant was removed from the cells. The culture supernatant was called "irradiated medium" and the cells were called "irradiated cells". 71 6.5. Effect of radiation of PBMCs on HTLV IIIB replication following subsequent acute infection ("pre-infection" model) PBMC samples were obtained from five individuals not infected with HIV-1. The cells were first exposed to low doses of gamma radiation and stimulated with PHA for 18- 24 hours, then were infected with HTLV-IIIB at 0.001 MOI and maintained in culture for up to 14 days. HIV-1 replication was assessed by measuring p24 antigen levels in culture supernatants that were harvested twice weekly. In the control samples, a productive infection was established in three of five experiments (Figure 7), with evidence of ongoing active viral replication on day 11. Figure 7 shows that in the irradiated samples, productive infection was established in all five cases. The results of the experiment with PBMCs taken from donor D indicates that the p24 level in the culture supernatant declines with time. At a dose of 50 cGy on day 4, there was a significant increase of HIV-1 replication. On day 4, the p24 value of the control sample was 8 pg/mL, vs 50 pg/mL in the 50 cGy sample. On day 7, these values were 3 pg/mL and 2160 pg/mL, respectively. On days 11 and 14, there was no evidence of a productive infection in the control culture, but the p24 level in the 50 cGy sample continued to increase up to 12,000 and 29,800 pg/mL. The PBMCs of donor A to HIV-1 infection was dramatically different from that of donor D, as the p24 value of the control sample of donor A increased with the time. Even given this, a positive effect of radiation on p24 production was still observed. On days 7 and 11, following low dose radiation at 1 to 50 cGy, p24 production in PBMCs was enhanced by 10 to 1000 fold. Although there were considerable variations regarding the magnitude and kinetics of the stimulatory effects of radiation on HIV-1 replication, an overall stimulatory effect was observed. No significant radiation-induced cell number reduction was observed following exposure to radiation at doses up to 50 cGy (Figure 8). However, a reduction in cell numbers was observed following exposure to higher doses of radiation. The cells isolated from donor A, B and C) that had high cell counts following 11 days of culture were those 72 donors that had established HIV-1 infection in the absence of radiation. The results indicated that the level of proliferation is positively correlated with the establishment of productive HIV-1 infection. It is interesting to note that in donors A, B and C the highest rates of infection were observed. It was further confirmed by measuring LL-2Roc level in the culture supernatant generated from donor A and donor D PBMCs. Overall, at days of 4, 7 and 11, the levels of IL-2Ra in the control culture supernatants of individual A were higher than these of individual D, which may explain why the cells were more easily infected with HIV-1 (Table 13). 73 Table 13 IL-2Ra levels (pg/mL) in culture supernatants following infection of PHA-stimulated control PBMCs taken from donor A and D Days after HTLV-IIIB infection1 Donor A Donor D 4 >30000 13700 7 >30000 17100 11 >30000 25300 aValues represent IL2Ra in culture supernatants at the concentration of pg/mL after cells were infected with HTLV-IIIB at MOI=0.001. 74 Donor B 100000 O) 0) O c o o CM CL 4 7 11 Days after HTLV IIIB infection Donor C Days after HTLV IIIB infection Donor E Figure 7 The p24 antigen levels in the culture supernatants following HIV-1 infection of PHA-stimulated PBMCs exposed to 0-200 cGy gamma radiation 75 Figure 8 Counts of P B M C s on day 11 following infection 76 6.6. Kinetics of viral replication in PBMCs exposed to radiation prior to acute HIV-1 infection Results from Figure 7 showed that p24 antigen in culture supernatant could be detected on day 4 after acute HIV-1 infection. Maximal p24 levels in the culture supernatant were reached at days 11 and 14. When PBMCs were exposed to low doses of gamma radiation over the range of 1 -200 cGy and then were stimulated with PHA and infected with HTLV-IIIB, up to 1000-fold increases in p24 levels was observed in some cases. The maximal radiation stimulatory effect varied widely and was not always observed at the same dose of radiation, which suggested that radiation-enhanced HIV-1 replication was a trigger-type response as compared to a dose-response. The statistical significance was achieved by comparing the maximal levels of p24 antigen in the sample control and 50 cGy cultures from five experiments(p<0.05, Wilcoxon signed-rank test, table 14). 77 Table 14 Comparison of the mean, median and range of p24 antigen levels in culture supernatants of control and irradiated PBMCs infected with HTLV-IIIB mean3 median range day 7 Control (n=5) Irradiated (50 cGy, n=5) p<0.05* day 11 Control (n=5) 20 4 0-63 Irradiated (50 cGy, n=5) 37 14 0-92 p<0.05* aThe value represents the concentration of p24 in the culture supernatants (in ng/mL). Cells were infected with HTLV-IIIB at MOI=0.001. ""Comparison of the maximal levels of p24 antigen in control and irradiated cultures using the Wilcoxon signed-rank test. 2 0 0-7 19 14 0-54 78 6.7. Effect of radiation and H2O2 of CD4+ cells on HTLV IIIB replication following subsequent acute infection ("pre-infection" model) To determine whether the mechanism underlying the observed stimulatory effects of radiation relates specifically to CD4 and/or CD8 cells, and whether they can be mimicked by other pro-oxidant conditions, isolated CD4 cells were evaluated. In the experiments, PBMCs and isolated and recombined CD4 and CD8 cells were also exposed to gamma radiation at doses of 25, 200 cGy or treated with 35 nM H2O2, providing an amount of H 2 0 2 equivalent to that generated by 50 cGy radiation exposure (187). Control cells were processed in parallel, except for exposure to radiation or H2O2. All cells were then stimulated with PHA for 24 hours, then infected with HTLV-IIIB and maintained in culture for 14 days. Table 15 shows our first experiment with PBMCs taken from donor F, in whom we were unable to establish a productive culture in control CD4 cells. In contrast, active viral replication was successfully established following exposure to 25 cGy gamma radiation, and to a greater extent following exposure to 35 nM H2O2. PBMCs , CD4 and CD8 cells were obtained from another subject (donor G). No productive viral replication was established in control PBMCs , while enhanced HIV-1 replication occurred following exposure to radiation (200 cGy) or H2O2 before acute viral infection (Table 16). In the same donor G, no productive viral replication was obtained in control CD4 cells or in the same cells exposed to 35 nM H2O2. However, maximal active viral replication was established following exposure to 200 cGy y radiation (Table 17). To test whether the presence of CD8 cells could influence the radiation effect, isolated CD4 and CD8 cells taken from donor G were recombined and cultured together. No productive viral replication was established in any irradiated cells. In contrast, active viral replication was established following exposure to 35 nM H202(Table 18). The dominant effect of low LET radiation is indirect through the generation of reactive oxygen species (ROS). The ROS generated by gamma radiation is not evenly distributed extracellularly and intracellularlly. The addition of H 2 0 2 into culture medium will stimulate cells in a 79 more uniform manner. It was interesting to notice that different cell subpopulations could change the effects of gamma radiation and H2O2 on HIV-1 replication. Both 200 cGy y-rays and 35 nM H2O2 could stimulate HTLV-IIIB replication in PBMCs of donor G (Table 16). 200 cGy y-rays only enhanced HTLV-IIIB replication in CD4 cells but not in recombined CD4 and CD8 cells isolated from donor G (Table 17). However, 35 nM H2O2 could stimulate HTLV-IIIB replication in recombined CD4 and CD8 cells, but not in purified CD4 cells. The results demonstrated that the subpopulations of lymphocytes was one of the factors in determining the outcome of the radiation effect on HIV-1 replication. 80 Table 15 p24 antigen levels in culture supernatants following infection of PHA-stimulated control, irradiated and H202-treated purified CD4 cells taken from donor F Days after HTLV-IIIB infection3 4 7 11 14 Control 0 0 0 0 25cGy 0 1 10 18 200 cGy 0 0 0 0 35nMH202 0 9 21 94 3The value represents the p24 in the culture supernatants at concentration of ng/mL after cell were infected with HTLV-IIIB at MOI = 0.001. bThe yield of radiolysis product = G G(H2C>2) = 0.7 molecules/100 eV of energy absorbed = 0.7x0.1036 umol/J = 0.0725 uM/Gy 1 Gy - 100 cGy Because radiation at 1 Gy produces 0.0725 uM H2O2, the yield of H2O2 is 36 nM when radiation is at 50 cGy. 81 Table 16 p24 antigen levels (ng/mL) in culture supernatants following infection of PHA-stimulated control, irradiated and H202-treated PBMCs taken from donor G Days after HTLV-IIIB infection1 7 11 14 Control 0 0 0 25cGy 0 0 0 200 cGy 122 210 465 35nMH202 1 145 130 aValues represent p24 in culture supernatants at the concentration of ng/mL after cells were infected with HTLV-IIIB at MOI=0.001. 82 Table 17 p24 antigen levels in culture supernatants following infection of PHA-stimulated control, irradiated and H202-treated CD4 cells taken from donor G Days after HTLV-IIIB infection1 7 11 14 Control 0 0 0 25cGy 0 0 0 200 cGy 0 1231 3294 35nMH202 0 0 0 8Values represent p24 in culture supernatants at the concentration of ng/mL after cells were infected with HTLV-IIIB at MOI=0.001. 83 Table 18 p24 antigen levels in culture supernatants following infection of PHA-stimulated control, irradiated and H 2 O 2 treated recombined CD4 and CD8 cells taken from donor G Days after HTLV-IIIB infection1 7 11 14 Control 0 0 0 25cGy 0 0 0 200 cGy 0 0 0 35nMH202 157 321 1600 "Values represent p24 in culture supernatants at the concentration of ng/mL after cells were infected with HTLV-IIIB at MOI=0.001. 84 6.8. Effect of radiation of PBMCs cells on H T L V IIIB replication administered after acute infection ("post-infection" model) In the "pre-infection" model, low dose radiation enhanced HIV-1 replication. To further characterize the effect of timing of radiation on HIV-1 replication, a "post-infection" model was designed. PBMCs were first stimulated with PHA for 18 hours and then infected with HTLV-IIIB. The infected PBMCs of donor H were then exposed to radiation immediately. In this experiment, productive viral replication was established in the control condition as the concentration of p24 in the clture supernatants was increased with time. No productive viral infection could be detected when cells were subjected to gamma radiation at doses of 1 to 100 cGy after infection. An inhibitory effect of radiation was observed, except following exposure to 200 cGy (Table 19). To confirm and expand this observation, the infected PBMCs of donors I, J, K, L, M , and N were then exposed to radiation after 18 hours acute HTLV-IIIB infection. Exposure to radiation 18 hours after infection also showed an inhibitory effect in the majority of cases (Table 20). The results indicated that timing of radiation was important in determining whether radiation will exert stimulatory or inhibitory effect on HIV-1 replication. 85 Table 19 p24 antigen levels in culture supernatants of HTLV-IIIB-infected PBMCs taken from donor H exposed to radiation immediately after infection Days after radiation/infection3 Dose (cGy) 7 11 14 0 0 5 55 1 0 0 0 5 0 0 0 10 0 0 0 50 0 0 0 100 0 0 0 200 0 180 32 "Values represent p24 in culture supernatants at the concentration of ng/mL after cells were infected with HTLV-IIIB at MOI=0.001. 86 Table 20 p24 antigen levels in culture supernatants of HTLV-IIIB-infected PBMCs taken form donors I-N exposed to radiation 18 hours after infection Donor Days Doses(cGy)3 0 1 5 10 50 100 200 400 I 7 0 0 0 11 1 0 0 14 150 0 0 J 7 0 0 0 11 105 1 24 14 427 720 113 K 7 68 28 28 11 143 42 30 14 263 81 195 L 7 38 56 0 11 588 66 0 14 262 103 0 M 7 2 2 0 11 18 61 0 14 74 185 0 N 7 3 0 0 11 647 0 0 14 563 0 0 0 0 0 0 0 0 1 0 0 0 1 112 0 0 0 1 0 0 0 0 857 0 20 5 0 117 1 73 364 2 42 64 34 63 66 75 93 64 113 144 78 109 116 305 289 88 0 0 0 0 76 0 0 0 0 583 0 0 0 0 0 1 0 0 0 9 72 0 603 0 12 148 0 270 0 2 0 0 0 0 18 2 0 2 23 40 6 1 7 343 "Values represent p24 in culture supernatants at the concentration of ng/mL after cells were infected with HTLV-IIIB at MOI=0.001. 87 6.9. Radiation, cell-cell communication and HIV-1 replication in the acute infection model ("pre-infection" model) PBMCs taken from Donor A and Donor D ( Figure 7) were chosen to test the role of cell-cell communication in the up-regulation of HIV-1 replication in the acute infection model with radiation administered prior to infection. After irradiated PBMCs of donor D were mixed and cultured with non-irradiated cells and stimulated with PHA for 18 hours, the mixed cells were infected with HTLV-IIIB. The p24 antigen levels in the culture supernatants were measured at days 7, 11 and 14. As shown in Table 21, the non-irradiated cells exert a suppressive effect on viral replication in cells exposed to 1-10 cGy, but not 50 cGy. Instead, the magnitude of viral replication in the sample of mixed cultures was the same as in the sample that contained 100% 50 cGy irradiated cells. The results were replicated in a differen donor (donor A) (Table 22). The viral inhibitory effect was observed in samples of donor A and donor D by mixing irradiated and non-irradiated cells; however, the inhibitory effect was more obvious with donor D than with donor A. Here again, the only dose at which inhibition of viral replication by non-irradiated cells was not observed was in the cells previously exposed to 50 cGy. Since the most effective dose in "pre-infection " model to stimulate HIV-1 replication was 50 cGy, it seemed that it was difficult for control cells to eliminate this strong viral stimulatory effect. The balance between host cells and HIV-1 determined the outcome of HIV-1 infection and low dose radiation obviously had an effect on shifting this balance. The mechanism of the effect of 50 cGy in achieving maximum stimulation of HIV replication in the studies is unknown. It may be related to changes in some gene expression (i.e. N F - K B ) or, alternatively, destruction of a very radiosensitive CD8 lymphocyte subpopulation. 88 Table 21 p24 antigen levels in culture supernatants of mixed irradiated and non-irradiated PBMCs taken from Donor D Days after infection Dose(cGy) 4 7 11 14 I a II b I II I II I II Control 8 9 3 5 0 1 0 0 1 cGy 8 8 2 3 0 0 0 0 5 cGy 13 13 63 6 336 2 339 0 lOcGy 10 11 28 4 680 3 1050 0 50cGy 58 10 2610 221 12000 5070 29800 27701 a p24 levels (pg/mL) in cultures established from irradiated cells infected with HTLV-IIIB atMOI=0.001. bp24 levels (pg/mL) in cultures established from irradiated and non-irradiated cells (1:1 ratio) infected with HTLV-IIIB at MOI=0.001. 89 Table 22 p24 antigen levels in culture supernatants of mixed irradiated and non-irradiated PBMCs taken from donor A Days after infection Dose (cGy) 4 7 11 14 r II b I II I II I II Control 16 20 54 97 3460 4020 41600 11600 1 cGy 23 92 234 263 11100 2100 12200 11900 5cGy 16 10 245 456 3910 0 22200 0 lOcGy 199 11 10100 435 20400 136 180000 291 50cGy 50 36 26100 2260 14300 27700 34300 32700 a p24 levels (pg/mL) in cultures established from irradiated cells infected with HTLV-IIIB atMOI=0.001. bp24 levels (pg/mL) in cultures established from irradiated and non-irradiated cells (1:1 ratio) infected with HTLV-IIIB at MOI=0.001. 90 6.10. Effect of radiation of PBMCs and cell-to-cell communication on HTLV IIIB replication administered after acute infection ("post-infection" model) To further study the role of cell-cell communication in influencing the effects of radiation on HIV-1 replication, we also evaluated the effects of adding non-irradiated cells to cells that had been exposed to radiation immediately after acute HIV-1 infection. Results in Table 23 indicate that the addition of the non-irradiated cells reversed the inhibitory effects of radiation. We extended our observations to four additional donors, from whom PBMCs were isolated. Half were infected then irradiated 18 hours later, and the other half were maintained in culture, then added back immediately after the infected cells were irradiated . As shown in Table 24, there was usually an inhibitory effect of the radiation that could not be completely overcome by the addition of non-irradiated cells. Actually, in most cases (donor I, J and L), the viral inhibitory effect of "post-infection" radiation could be extended to control cells from irradiated cells in the condition of mixing culture. However, this effect was quite variable and, in some experiments (such as donor K), the addition of the non-irradiated cells essentially restored the culture to control conditions. 91 Table 23 p24 antigen levels in culture supernatants of mixed irradiated and non-radiation PBMCs taken from donor H Days after radiation 11 14 Dose(cGy) I a II b I II control 5 3 55 78 1 0 0 0 0 5 0 0 0 32 10 0 0 0 3 50 0 0 0 0 a p24 levels (pg/mL) in cultures established from irradiated cells infected with H T L V -IIIB at MOI=0.001. b p24 levels (pg/mL) in cultures established from irradiated and non-irradiated cells (1:1 ratio) infected with HTLV-IIIB at MOI=0.001. 92 Table 24 p24 antigen levels in culture supernatants of HTLV-IIIB-infected cells taken form donors I - L exposed to radiation 18 hours after infection Donor Days Doses(cGy)3 0 1 5 10 50 100 200 400 _ J K L 7 0 0 0 0 0 0 0 0 11 1 0 0 0 0 0 0 0 14 97 0 0 0 0 0 0 0 7 0 1 1 0 0 0 0 0 11 44 58 69 50 0 122 0 0 14 957 976 287 82 96 254 629 735 7 908 126 102 114 80 35 30 200 11 96 199 291 127 86 82 55 778 14 264 339 370 336 198 152 110 785 7 49 0 0 18 29 80 0 0 11 564 0 0 136 62 72 0 0 14 837 0 0 580 808 482 0 0 a Data represent p24 in the culture supernatants at the concentration of ng/mL established from irradiated and non-irradiated cells (1:1 ratio) after cells were infected with H T L V -IIIB at MOI=0.001 for 18 hours when radiation was administed. 93 6.11. Effect of "post-infection" irradiated culture supernatants on HIV-1 replication In the "pre-infection" model, the dominant radiation effect on HIV-1 replication was stimulatory. However, in the "post infection" model, the dominant was inhibitory. To evaluate if the supernatant taken from "post-infection" irradiated cell cultures contained a factor which may inhibit viral replication, aliquots of such supernatants were added to established cultures taken from two donors (donor M and N). PBMCs were stimulated with PHA and infected with HTLV-IIIB. PBMCs were then exposed to radiation at doses of 5, 50, 200 and 400 cGy and cultured for 24 hours. Control cells were treated the same as irradiated cells except for radiation. The culture supernatants were removed from control cells and irradiated supernatants were added back to these same control cells, The same supernatant failed to inhibit HIV-1 replication (Table 25). In fact, if anything, a stimulatory effect was observed. In donor N, irradiated supernatants showed a stimulatory effect of irradiated culture supernatants at doses of 5 cGy to 400 cGy, which was especially noticeable at 50 cGy at day 14. The balance between radiation viral inhibitory and stimulatory effects could influence the levels of HIV-1 replication. The results indicated that the "post infection" radiation-induced inhibitory effect required the presence of irradiated cells. 94 Table 25 p24 levels of viral infected non-irradiated cells which were incubated in the post-viral irradiated culture supernatant Days in culture3 7 11 14 Donor M control supernatant 3 18 23 5 cGy supernatant 3 221 126 50 cGy supernatant 35 425 804 200 cGy supernatant 2 126 113 400 cGy supernatant 70 389 515 Donor N control supernatant 1 87 257 5 cGy supernatant 2 139 597 50 cGy supernatant 2 26 298 200 cGy supernatant 2 54 126 400 cGy supernatant 2 45 276 aThe data represents p24 levels in the culture supernatant at the concentration of ng/mL. 95 6.12. Effects of gamma radiation on PBMCs of HIV-1-infected patients To test whether there were any effects of radiation on HIV-1 replication in PBMCs of HIV-1-infected patients(preferably one which approximates in vivo conditions), PBMCs were taken from nine HIV-1-positive patients with negative and low levels of p24 (plasma p24 antigen levels < 5 pg/mL). An aliquot of these chronically infected PBMCs were exposed to 5-50 cGy gamma radiation, with a separate aliquot serving as a non-irradiated control. After radiation, all control and irradiated cells were stimulated with PHA for 24 hours and then incubated in culture medium containing only IL-2 without PHA for up to 21 days. No productive HIV-1 replication was established in cultures derived from the 8 patients with negative p24 antigen levels either under control conditions or following 5, 25, 50 cGy gamma radiation and PHA stimulation (data not shown). The culture initiated from the PBMCs of the patient with a positive plasma p24 antigen level (3.73 pg/mL) showed productive HIV-1 replication in the control condition and following exposure to 5, 25 and 50 cGy gamma radiation, with a stimulatory effect of radiation (vs. the control culture) at the 50 cGy dose (Table 26). Cell-associated proviral load was quantitated using a DNA PCR assay developed in our facility. Higher proviral loads are present in 4 samples exposed to radiation and maintained in culture for 21 days (Table 27). When considering the maximal proviral load in the irradiated cells (whether exposed to 5, 25 or 50 cGy), an increase was observed when compared to the control condition but this was not statistically significant (p>0.05). Taken together, these results demonstrated that exposure to gamma radiation at doses of 5-50 cGy could stimulate viral replication in chronically-infected PBMCs (Table 28), most significantly in a patient with a positive plasma p24 antigen level. Thus, the stimulatory effects of radiation on viral replication may be most significant in cells with higher viral replication rates at baseline. 96 Table 26 p24 level in the culture supernatant of PBMCs from an HIV-positive patients with a positive baseline plasma p24 antigen level Days control 50 cGy 14 10 47 17 228 704 21 606 7073 The values given represent p24 level(pg/mL) in culture supernatants. 97 Table 27 Proviral load in PBMCs from HIV-1- positive patients following 21 days in culture Individual Control 5 cGy 25 cGy 50cGy 1 0 39 N/A 199 2 0 0 16 32 3 35 0 0 0 4 0 0 0 0 5 15 15 17 0 6 27 0 28 0 7 0 51 0 0 8 0 18 0 143 9 634 1321 687 634 Data represent the HIV-1 DNA copies l\.5x10^ cells. 98 Table 28 Proviral load in PBMCs from HIV-1- positive patients following 21 days in culture Mean Range P value control (n=9) 79 0-634 >0.05 irradiated (n=9) 123 0-1321 Data represent the HIV-1 DNA copies /1.5xl0^ cells. 99 6.13. Effects of gamma radiation on viral replication in chronically infected cell lines Only 5-10% PBMC taken from infected individuals actually carry actively replicating HIV-1 at any one time (175). To evaluate the effects of gamma radiation on cultures established from cells with a high degree of infection (approaching 100%), appropriate cells lines were used: H9RF, 8E5, and 8E5L (the latter having only latent, integrated provirus in the resting state). Logarithmic growth-phase cells (>95% viability) were irradiated in the dose range of 1-400 cGy. Following radiation, the cells were maintained in culture up to 14 days, at 1-2 x 106 cells/mL. Table 29 shows that there was no significant change in the p24 antigen levels in the culture supernatants derived from H9RF cells exposed to radiation. Table 30 shows that p24 levels were not affected by the presence of non-irradiated cells in the culture. The p24 values in the supernatants of mixing culture of irradiated H9RF and non-irradiated H9RF cells were the same as these of cells with or without exposure to low dose radiation. Similarly, viral replications in 8E5 cells were not modulated by radiation (Table 31 and Table 32). Finally, radiation exposure could not activate viral replication in 8E5L cells, as all the value of p24 in the culture supernatants were negative (data not shown). Taken together, the results demonstrated that radiation does not accelerate HIV-1 replication in H9RF and 8E5 cell lines, nor does it seem to have an effect on latently infected cells (8E5L), with no active turnover at baseline. Low dose radiation could not activate latent provirus in 8E5L cells. The results further indicated that there was little viral stimulatory radiation effect on HIV-1 positive cells carrying a relatively high (MOI-l .0) viral burden at baseline. 100 Table 29 p24 antigen levels in culture supernatants of H9RF cells following radiation Days after radiation Dose(cGy) 7 11 14 Control 167 177 292 1 cGy 125 200 188 5 cGy 123 174 243 lOcGy 152 260 248 50cGy 137 70 235 100 cGy 92 177 235 200 cGy 136 197 226 400 cGy 129 194 233 Values given represent p24 level (ng/mL) in culture supernatants of H9RF cells. 101 Table 30 p24 antigen levels in culture supernatants of mixed irradiated and non-irradiated H9RF cells Days after radiation Dose(cGy) 7 11 14 Control 137 195 227 1 cGy 185 119 236 5cGy 175 126 248 lOcGy 146 271 210 50cGy 144 298 265 100 cGy 162 229 214 200 cGy 148 215 202 400 cGy 184 190 270 Values given represent p24 level (ng/mL) in culture supernatants of mixed irradiated and non-irradiated cells H9RF cells (1:1 ratio). 102 Table 31 p24 antigen levels in culture supernatants of 8E5 cells Days after radiation Dose(cGy) 7 11 14 Control 29 23 43 1 cGy 13 14 28 5 cGy 36 38 41 lOcGy 29 11 52 50 cGy 28 26 32 100 cGy 10 15 46 200 cGy 19 30 38 400 cGy 20 14 41 Values given represent p24 level (ng/mL) in culture supernatants of 8E5cells. 103 Table 32 p24 antigen levels in culture supernatants of mixed irradiated and non-irradiated 8E5 cells Dose(cGy) 7 Days after radiation3 11 14 Control 27 25 35 1 cGy 21 21 33 5 cGy 29 37 36 lOcGy 21 54 22 50cGy 13 35 45 100 cGy 16 42 38 200 cGy 6 32 27 400 cGy 25 32 29 aValues given represent p24 level (ng/mL) in culture supernatants of mixed irradiated and non-irradiated cells 8E5 cells (1:1 ratio). 104 6.14. Effect of radiation on viral replication in the presence of zidovudine (ZDV) PBMC taken from donors I, J, K and L were exposed to 1-400 cGy, stimulated with PHA for 24 hours, infected with HTLV-IIIB (MOI=0.001) and maintained in culture in the presence of the antiretroviral agent zidovudine (ZDV). ZDV is a nucleoside analog that is a potent inhibitor of the HIV-1 reverse transcriptase. Since low dose radiation enhanced host cells to HIV-1 infection in the acute "pre-infection" radiation model, two experiments were performed to test whether ZDV would abort this viral stimulatory effect. Cells of donors I and J were maintained with 0.5 uM ZDV, and cells of donors K and L were treated with 0.1 uM ZDV. Based on p24 antigen levels in culture supernatants to day 14, radiation was not able to overcome the antiviral effect of 0.5 uM as there were no detectable p24 antigen in the culture supernatants in the presence of 0.5 uM of ZDV. However, as shown in Table 33, 50 cGy radiation enhanced viral replication in the cells of donor K, despite the presence of 0.1 uM ZDV in the culture medium. No such stimulatory effect was observed for donor L, although in contrast to donor K, the control culture showed significant viral replication in the presence of ZDV. 105 Table 33 p24 antigen levels in the supernatants of cultures of acutely infected P B M C in the presence of 0.1 u M Z D V 4 Days after infection3 7 11 14 Donor K control 0 0 0 0 1 cGy 0 61 115 4682 5 cGy 0 0 0 42 lOcGy 0 0 0 0 50cGy 0 11 22 9260 100 cGy 0 0 0 0 200 cGy 0 22 32 10 400 cGy 0 0 0 0 Donor L control 58 187 368 350 1 cGy 39 133 257 356 5 cGy 48 87 95 89 10 61 185 44 755 50cGy 50 213 27 277 100 cGy 80 516 76 846 200 cGy 104 497 75 576 400 cGy 36 322 29 676 'Value given represents p24 level (ng/mL) in culture supernatants. 106 6.15. Radiation effect on IL10 expreesion In the acute viral infection model, we measured IL-10 levels in culture supernatants, to determine whether the radiation-associated enhancement of viral replication was the result of overexpresion of IL10 since IL10 is one of Th2-associated cytokines. There were significant differences in IL-10 levels between the two donors studied in this experiment. However, in both cases, there was no systematic change in IL-10 levels, whether these were measured in cultures established from irradiated (Table 34) or mixed irradiated /non-irradiated (Table 35) cells, although where radiation-induced viral stimulatory effect was evident. The results indicated that the viral stimulatory conditions developed in our experimental models were not associated with the levels of IL-10 expression of PBMC. 107 Table 34 IL-10 levels in the culture supernatants of PBMC exposed to radiation followed by acute HIV-1 infection Days after radiation3 11 14 Donor A control 0 7 6 lcGy 7 2 8 5cGy 19 7 11 lOcGy 0 14 10 50cGy 0 24 25 lOOcGy 0 10 15 200cGy 0 8 30 Donor B control 42 73 48 lcGy 107 30 43 5cGy 40 28 83 lOcGy 56 40 43 50cGy 51 134 184 lOOcGy 58 38 77 200cGy 80 38 36 aValues given represent IL10 level (ng/mL) in culture supernatants when PBMC infected with HTLV-IIIB at MOI=0.001. 108 Table 35 IL-10 levels in the culture supernatants of mixed irradiated and non-irradiated PBMC acutely infected with HIV-1 Days after radiation 7 11 14 Donor A control 0 7 6 1 0 5 12 5 1 2 6 10 3 3 8 50 0 14 7 100 0 12 21 200 0 6 20 Donor B control 42 73 48 1 64 59 46 5 63 185 48 10 34 35 123 50 36 218 94 100 87 36 56 200 36 47 52 aValues given represent IL-10 level (pg/mL) in culture supernatants of mixed irradiated and non-irradiated PBMC (mixed irradiated /non-irradiated cells, 1:1 ratio) w hen PBMC were infected with HTLV-IIIB at MOI=0.001. 109 PART IV 7.0 GENERAL DISCUSSION 7.1 UV, Ionizing Radiation and HIV-1 HIV-1 mainly infects and destroys CD4-positive T lymphocytes (6). After infection is established, the most efficient host defense mechanism is the cell-mediated immune response, in which the CD4 cells themselves play a central role. In the late stages of infection, patients frequently develop malignancies and other opportunistic conditions (189). As reviewed previously, radiotherapy is used extensively for the treatment of a number of these HIV-1-associated conditions such as Kaposi's sarcoma, non-Hodgkin's lymphoma and autoimmune thrombocytopenic purpura. It was also reported that low dose total body radiation could act as a potent anti-retroviral agent in vivo in mice, and this has been used under strict experimental conditions in humans. However, ionizing radiation is known to activate gene expression of certain molecules known to stimulate HIV-1 replication (NF-kB, TNFa and certain oncogenes) (182-183). In other virologic models, exposure of target cells or the viruses themselves to radiation may also enhance their infectivity (190-195). The question of the interaction of radiation and HIV-1 in vivo is complex, and can only be predicted by knowing the specific effect on HIV-1 itself, the host cell, and the immune system. More data are available on the effects of UVR, which can cause a different spectrum of DNA damage (196). U V C and U V B induce the formation of DNA pyrimidine dimers, while U V A produces DNA cross-links and strand breaks. In contrast, ionizing radiation is rather non-specific in its interaction with DNA, causing mostly DNA strand breaks (197). In addition to damaging DNA, ionizing radiation produces a variety of oxidative stresses via reactive oxygen species, including malondialdehyde and products of membrane and protein peroxidation. Some of these free radical intermediates 110 may also serve as second messengers in intracellular signaling or cell-to-cell communication. Like UVR, ionizing radiation exposure is transient, but the molecular damage to membranes, proteins and DNA, if unrepaired, will produce sustained biological effects analogous to those of oxidants and other toxic chemicals. It was reported that U V R and ionizing radiation can both activate Raf protein serine/threonine kinases which might be involved in cellular radioresistance (198). Current knowledge about the effects of UVR on HIV-1 replication, as gained from previous studies, includes : 1. Exposure of chronically HIV-infected cells in vitro to U V B and U V C radiation activated HIV replication (199). 2. Exposure of cells to U V C before HIV infection in vitro also increased viral replication (200). 3. U V C and U V B at cytotoxic fluences would activate HIV-1 replication. However, U V A at such fluences was ineffective in this regard (201-203). 4. Combined treatment with U V A and a photosensitizer resulted in strong activation of HIV-1 replication in vitro (204). 5. Very low fluences were needed to induce expression of HIV-1 LTR in fibroblasts of patients with xeroderma pigmentosum, as compared to normal cells, suggesting a relationship between the effect of UVR and the capability of cellular DNA repair (205). Overall, the mechanisms by which UVR activates HIV-1 replication are complex, and may include direct immune suppression, UV-induced release of extracellular factor(s) (i.e. IL-1, TNF-a, activation of N F - K B , bulky DNA damage, loosening of chromatin structure, modification of protein kinases, activation of proto-oncogenes, and mutagenesis (206-218). Both positive and negative results are reported in the recent published literature regarding the effects of ionizing radiation on HIV-1 replication in vitro. A few papers report that x-rays activate HIV-1 LTR expression, thereby enhancing viral replication. It was also demonstrated that ionizing radiation was able to activate NF-kB, but could not increase HIV-1 gene expression in stable transfected HIVcat/Hela cells (219-222). To our knowledge, no data other than those generated in this present study address the issue of differences in HIV-1 replication based on the timing of radiation (before or after infection 111 in acute infection models). The mechanisms by which ionizing radiation modulates viral replication are still unknown. Using acute and chronic infection models to study low dose ionizing radiation effects on HIV-1 replication may help to provide some meaningful insight into the interaction. It will be necessary to do the specific experiments to evaluate the interaction, as the effect of ionizing radiation on viral replication is probably different from that of UVR, and this needs to be clarified. Determining the effect of ionizing radiation on the activation of lymphocytes is the first step to address this question, as we know that the exogenous factors and cellular activation are important in establishing productive viral replication. We could then go on to determine any effect of the timing of ionizing radiation on alterations in viral replication kinetics. We have specifically measured the effect of low doses of ionizing radiation on IL-2R expression in mitogen-stimulated cells. Secondly, HIV-1 replication was examined in PBMC that were subjected to ionizing radiation before acute viral infection. To extend and confirm these results, the change in HIV-1 replication was examined in CD4 cells and recombined CD4 and CD8 cells, each of which had been exposed to ionizing radiation before acute viral infection. This (along with "mixing" experiments of irradiated and non-irradiated cells) would help establish whether direct radiation of the target cell is required for there to be any effect of radiation, or whether a soluble factor (as could be secreted by a CD8 cell exposed to ionizing radiation) could affect the kinetics of infection of other cells in the same culture medium. Experiments were also carried out to determine if exposure to ionizing radiation after acute infection of target cells exerted any effects similar to those observed in our previous models. The effects of ionizing radiation were then examined in PBMC isolated from HIV-1-infected patients, as well as in appropriate continuous cell lines carrying the HIV-1 genome (8E5 and H9RF). Finally, to explore some of the mechanisms by which low dose radiation activates viral replication, low concentrations of H2O2 were used as an oxidative stress to mimic the stimulatory effect of ionizing radiation. The results of our work in this thesis demonstrate that HIV-1 infection or low 112 dose radiation can increase IL-2Ra expression on the cell surface of PBMC stimulated with a mitogen. The effect is enhanced if the cells are subjected to both acute HIV-1 infection and low dose radiation. Interestingly, the surface expression of IL-2Ra in non-irradiated cells was stimulated by culture supernatants removed from irradiated cells. Low dose radiation exposure increased viral replication in PBMC if the radiation was administrated before viral infection. The stimulatory effect on viral replication was further confirmed and extended in isolated CD4 and recombined CD4 and CD8 cells when the radiation was performed before viral infection. Mixing experiments implicates a role for a soluble factor (not yet defined) in our observations. The dominant effect of low-dose radiation on HIV-1 replication in PBMC was suppressive if the radiation was administered immediately following or 18 hours after acute viral infection. Although no effect of ionizing radiation on HIV-1 replication in chronically infected cell lines was observed, there was some enhancement of HIV-1 infection in PBMC taken from chronically infected patients, especially those with pre-existing evidence of high-level replication in the circulation to start with (positive plasma viremia). 7.2 Radiation, Free Radicals, Cell Activation, and IL-2Ra Expression The importance of cell activation in HIV-1 infection is well known (223). Activation of CD4 cells is essential for effective HIV-1 replication, although quiescent CD4 cells can be infected and produce low level viral replication. The kinetics of viral infection is dependent on the interplay of host transcriptional activators (NF-kB, TNF-a, IL-1 and IL-6) and viral regulatory proteins (tat, rev and nef). HIV-1 gene expression is regulated, via the 5' LTR, by the same mitogenic signals that induce T cell activation. For instance, it is known that PHA can activate N F - K B (223). Over-expression of protein-tyrosine kinase p59 f y n stimulates the HIV-1 promoter in transfected Jurkat cells when they are treated with ConA and PMA to mimic antigen stimulation via N F - K B (217). The addition of TNF-a to many cell types activates N F - K B that subsequently promotes HIV-1 gene expression. The level of circulating TNF-a is elevated in HIV-1-infected patients and correlates with opportunistic infections, high plasma viremia, decreased CD4 counts 113 and disease progression (188). CD4 is down-regulated following HIV-1 infection and this may render infected cells resistant to super-infection, facilitate release of virus, and limit the normal response of cells to antigen. The mechanisms by which CD4 is down-regulated relates to nef-induced changes in CD4 endocytosis, and retention (and degradation) of newly synthesized CD4 in the ER (225). In the present study, as would have been predicted, CD4 down-regulation occurred after acute HIV-1 infection of PHA-stimulated PBMC. Both the plasma membrane and the nucleus are known targets of radiation, effects that may be mediated by two different signaling systems (226). Reactive oxygen species (ROS) and ceramide (activation of sphingomyelinase produces ceramide) are considered to be second messenger molecules, activating RAS, RAF, and MAP kinases (227). The energy of y-radiation is high enough to cause molecular ionization and the generation of free radicals including superoxide anion (02~), hydroxyl (HO), peroxyl (ROO) and alkoxyl (RO) radicals, as well as hydrogen peroxide (H2O2). The indirect radiation effect is dependent on the presence of reactive oxygen species and therefore it is fundamentally similar to the effect of oxidative stress (228), with active and/or suppressive effects on lymphocytes (229, 230). Under physiological conditions, T cell activation is the response to a foreign peptide antigen, presented in the context of self-MHC. The recognition of antigen by the T cell antigen-receptor complex results in ligation of the TCR complex resulting in activation of several protein tyrosine kinases (PTKs) such as p56 l ck, p59 f y n, ZAP-70 and Syk. These trigger early membrane/intracellular signal transduction events including tyrosine phosphorylation of membrane and cytoplasmic proteins, hydrolysis of plasma membrane inositol phospholipids, elevation of cytoplasmic C a + + concentrations, activation of Ras, activation of several serine/threonine protein kinases. PKC-mediated phosphorylation of I K B leads to the generation of free N F - K B , which is then translocated to the nucleus. Following these events a variety of genes such as cellular proto-oncogenes (ex: c-myc, c-fos), cytokine genes (ex: IL-2, INF-y) and cytokine receptor genes (ex: I L -114 2Roc) are transcriptionally activated. New cell surface molecules such as IL-2Ra are expressed and effector cytokines like IL-2, IFN-y are released. Eventually, autocrine IL-2 production and subsequent receptor-mediated T lymphocyte proliferation is initiated. Activated T cells secrete IL-2 which further amplifies IL-2Ra expression and IL-2 production. MAP kinases stimulate transcription factors in response to extra-cellular signals such as mitogens, cytokines, growth factors, U V radiation and stress-inducing agent, similar to what occurs with polyclonal mitogen stimulation with ConA and PHA (231-234). It is postulated that exposure to ionizing radiation may mimic this entire process, by a similar mechanism. IL-2Ra was chosen in this study as a marker of lymphocyte activation in response to HIV-1 infection and low dose y-radiation (235-237). In vitro, acute HIV-1 infection of human PBMC could result in elevated IL-2 secretion via CD3 and CD28 receptors. The HIV-1 protein tat can enhance IL-2 expression (238), an effect that is mediated by the CD28-responsive element in the IL-2 promoter. In the presence of the reverse-transcriptase inhibitor azidothymidine (AZT), no enhancement of IL-2 secretion is observed (239). The production of hydrogen peroxide by human monocytes in response to IL-2 may have an amplifying effect on this process (240). The action of IL-2 on immune cells is mediated by binding to IL-2 receptor proteins. The IL-2 receptor consists of a, P and y chains. Resting lymphocytes do not express the IL-2Ra chain, but they are induced to do so quickly and transiently following stimulation with antigen or mitogen. The IL-2Ra chain contributes to the formation of high-affinity IL-2R, and the cytoplasmic domains of P and y chains are then required for signal transduction (241). It was reported, that IL-2 and IFN-y regulate expression of the subunit of IL-2 receptor differentially, the former at the transcriptional and the latter at the post-transcriptional level (242). In the early stages of HIV-1 infection, it was reported that decreased IL-2Ra expression was partially responsible for the reduction of the T cell proliferative response to antigens, mitogens and IL-2 stimulation (243). IL-2 production 115 was restored in murine retrovirus infection that had progressed to AIDS when it was treated with vitamin E supplementation (244). The significance of IL-2 for HIV-1 replication in PBMC relates to its effects on activation, proliferation and cytokine production (245). In normal cells, IL-2a gene transcription is initiated within 3 hours of activation. It was known that chronic T cell stimulation could lead to shedding of IL-2Ra. In the present studies, our results show that acute HIV-1 infection could induce IL-2Ra expression on PBMC when they were stimulated with PHA, even in the presence of IL-2-containing culture medium. The percentage of IL-2Ra expression on the surfaces of irradiated lymphocytes was also elevated when they were stimulated with ConA, as compared to non-irradiated cells. However, no significant difference in IL-2Ra could be observed in the samples exposed to ionizing radiation and then infected with HIV-1, as compared to samples infected with HIV-1 without radiation exposure. This indicates that both HIV-1 infection and low dose of radiation could induce IL-2Ra expression on cell surface. However, the mechanism and/or intensity of the two effects overlapped, indicating that the same signal transduction pathway may mediate both effects. This common pathway may be oxidative stress, which is known to be quite active in many aspects of HIV-1 disease pathogenesis, including enhanced viral replication (246-249). Based on the results showing that 0.25 mGy of y-radiation could increase IL-2Roc expression on PBMC when they were stimulated with ConA, signal activation by ionizing radiation might not require DNA damage, as such a radiation dose is well below that required for such an effect to occur. These data indicating that low-dose ionizing radiation may modulate HIV-1 replication (at least in vitro) led to the design of further experiments to evaluate this phenomenon in more depth. 7.3 Acute Infection Models, PBMC of HIV-1-infected individuals and Stable HIV-1-infected CD4+ cell lines We used acute HIV-1-infection models and PBMC of HIV-1-infected patients as well as stable HIV-1-infected CD4+ cell lines to study the effects of ionizing radiation on 116 viral replication. In the acute infection model, radiation was administered before or after acute infection of PBMC isolated from normal donors. In the clinical infection model, PBMC were isolated from HIV-1-infected patients, and then exposed to radiation in vitro. The advantage of using PBMC (rather than infected or transfected cell lines) was that the result would more closely represent in vivo conditions. The disadvantage of using PBMC is that they are a heterogeneous cell population, and may lead to the generation of results that are more difficult to interpret. The situation is further complicated by the great variation between donors, which may make it more difficult to draw a clear conclusion. This being said, we were able to reproducibly demonstrate a distinct stimulatory effect of low dose ionizing radiation on HIV-1 replication in a model where ionizing radiation was administered prior to acute infection by standard laboratory protocols. This effect was especially noticeable at a dose of 50 cGy, particularly in cells where lower levels of endogenous viral replication were observed. To expand on this observation, the magnitude of any observed effects in isolated CD4 cells was examined. The results show that HIV-1 replication in purified CD4 cells was also enhanced by prior exposure to ionizing radiation. Similar effects were observed in purified CD4 cells treated with low concentrations of H2O2, which may be the intermediate in the observed ionizing radiation effect. Differential radiation effects in CD4 cells, recombined CD4 and CD8 cells and bulk PBMC indicate a multifactorial effect of ionizing radiation, which may also be mediated by soluble factors as well as increased synthesis of free radicals (and other intermediates) in the infected cell. Different effects on HIV-1 replication based on the timing of radiation (before or after acute infection) were also examined. When radiation was administered to PBMC immediately after or 18 hours following acute HIV-1 infection, significant reductions in viral replication were observed. In PBMC isolated from HIV-1-infected patients, (which more closely mimics the in vivo situation,) we hypothesized that exposure to gamma radiation at doses of 5-50 cGy might increase viral replication in culture. However, the results were not convincing, and no significant differences were observed between irradiated and non-irradiated samples. In 8 patients with low levels of viral replication in 117 the circulation, there was no evidence of viral replication in cultures established from both control and irradiated samples. The culture initiated from the PBMC of patients with more evidence of ongoing replication (positive plasma p24 antigen levels) showed evidence of productive HIV-1 in culture under control conditions. Exposure to 50 cGy increased viral replication as compared to the control culture at days 14, 17 and 21 in culture. Typically, less than 1% of PBMC of HIV-1 infected patients within this pool is infected. In addition to CD4 cells, CD8+ T cells, N K cells, B cells and monocytes are also can be infected with HIV. Radiation may have differential effects on patients with variable viral loads as low dose of radiation enhanced HIV-1 replication in PBMC isolated from clinically HIV-1-infected-patients with positive p24, but not with undetectable p24 viremia. HIV-1-infected CD4+ cell lines (8E5, 8E5L and H9RF) were used to determine the generalizability of the observation. Unfortunately, it could not be confirmed. It could be hypothesized that radiation may also stimulate viral replication by T helper cell type switching (250, 251). With this in mind, the level of IL-10 in culture supernatants was measured in samples generated from experiments demonstrating the greatest stimulatory effect of radiation on HIV-1 replication. It is known that IL-10 is produced by type 2 helper T cells, which act to suppress cytokine production by type 1 helper T cells, as well as exert immunosuppressive effects on macrophage function and immunostimulatory effects on B cells (252). Previous studies have demonstrated that IL-10 can inhibit y-IFN production in IL-2- or PHA-activated PBMC. Production of IL-10 increased in the PBMC of HIV-1-infected patients and may be correlated with a worsening of their immune status (250). In our experiments, no increase in IL10 production was measured in two experiments where radiation- induced viral stimulatory effect was evident. To understand whether this radiation stimulatory effect is related to T cell type switching, it is necessary to measure other Thl/2 type associated cytokines following radiation and viral infection. We are thus left with the likelihood of free radical- induced signal transduction as 118 the most plausible mechanism for any observed effect. The results show that HIV-1 can infect purified CD4 cells but does not replicate well in CD4 cells, as productive HIV-1 infection is only induced in cells exposed to gamma radiation before viral infection. Oxidative stress, such as exposure to low concentrations of H2O2 could mimic but did not exactly parallel the observed radiation effects. Although a 50 cGy dose caused a noticeable radiation-induced viral stimulatory effect, there was no clear dose-response effect over the range of 1 -200 cGy. Genetic variability among different donors, the non-specific interaction of y- rays and DNA, and complex regulation of HIV-1 replication may all contribute to this lack of clarity. The reason why a dose of 50 cGy produced the most significant viral stimulatory effect is not known, but it is consistent with the results of Prasad et al. (253) who observed that 50 cGy gamma radiation best induced expression and binding activity of NF-KB. It may be argued that, in spite of a great variation in HIV-1 infection among different donor cells, it is significant that a consistent radiation stimulatory effect on HIV-1 replication could still be observed. Recently, it has been shown that radiosensitivity at low doses is dose-dependent. Dual phenomena have been proposed that there is low dose hypersensitivity (HRS) when the radiation dose is below a threashold and increased radiosensitivity (IRR) when the dose is above a threshold. A typical threshold is about 5-40 cGy. The dual low-dose dependent radiosensitivity phenomenon may reflect changes in the amount, rate or type of DNA synthesis or repair (261). 50 cGy is the dose just above the threshold. In our studies 50 cGy gamma radiation has demonstrated the most significant HIV stimulatory effect. This trigger type low dose radiation-induced enhanced HIV-1 replication may be related to the changes of some gene expressions, the changes of DNA structure and conformation, the modulation of DNA repair and the shifting of the balance of immune responses. 119 Low dose ionizing radiation did not alter the level of HIV-1 replication in 8E5, 8E5L and H9RF cells. This supports a critical effect of the timing of radiation in determining the viral stimulatory or suppressive effect. In the cell lines, most of the individual cells are infected, and this may more closely mimic the "post-infection" exposure to radiation. Conversely, in the PBMC taken from patients, the majority of the cells may not, in fact, be carrying HIV-1. Therefore, this may be more similar to the "pre-infection" exposure model. 7.4 Differential Radiation Effects and Cell-Cell Communication The HIV-1 replication cycle involves many steps, the first being attachment and fusion with the target cell. It was reported in binding studies using lectins that 0.25-5 Gy doses of ionizing radiation induced plasma membrane alterations. The extent of perturbation of the plasma membrane depends on the doses, the cell type as well as the physiological condition of the cells. It has already been demonstrated that ionizing radiation can alter virus receptors, i.e. the attachment and penetration of the virus could be enhanced or retarded at different timings of radiation and infection (248-251). The importance of viral lipid composition in the phenomenon of HIV-1 infectivity was confirmed in experiments demonstrating that rising temperature, lipophilic drugs, and liposomes can reduce HIV-1 infection, while pre-incubation of HIV-1 with a synthetic lipid enhances infection of A3.01 and H9 cells (252). Although the present experiments did not address this issue, ionizing radiation-induced cell membrane changes could explain a number of our observations. Theoretically, radiation can affect the natural course of viral replication in two ways: one is by exerting its effect directly on the HIV-1 genome by altering DNA that results in gene up-regulation; and the other is by affecting HIV-1 replication indirectly by activating/suppressing cell-associated processes. It was known that low doses of radiation could induce immune suppression, but could also stimulate immune responses under certain circumstances. Therefore, the effect of ionizing radiation may be through 120 activating cellular genes that are associated with HIV-1 replication, or by a change in the appropriate immune effector systems controlling viral replication. It seems that after HIV-1 entry into cells, the stimulatory effect of ionizing radiation disappears or is reduced, with a suppressive effect being observed in certain cases. This is further supported by the results that show radiation enhances HIV-1 replication in HIV-1-infected PBMC isolated from HIV-1 positive patients in the setting of higher baseline replication rates. It suggests that radiation-stimulated viral replication might happen in the case where there are viral particles present that can infect other cells, and/or where a proportion of the cells are not infected. This observation was further supported by the fact that radiation failed to activate latent infection in 8E5L cells and there was no radiation modulation of HIV-1 replication in 8E5 and H9RF cells (all of which are infected). Like UVR, ionizing radiation can induce soluble mediator(s) that have an effect on cell-to-cell communication and HIV-1 promoter activation (259). Most recently, a "bystander effect" of radiation has been observed in many studies (260-267). The present study shows that when irradiated cells are mixed in culture with non-irradiated cells, the same magnitude of stimulation of IL-2Ra is observed on the surface of these mixed cells as if the experiment is carried out with all the cells being irradiated. This implicates a role for radiation-induced soluble factor(s). The stimulatory effect does not extend to non-irradiated cells when mixed with irradiated cells after 24 hours of radiation using non-irradiated medium, which further indicates that irradiated cells release soluble factor(s) that would be present in the medium. When irradiated cells were mixed in culture with non-irradiated cells, and then stimulated with PHA and infected with HIV-1, the stimulatory effect of radiation was moderate, and clearly aborted by the addition of normal non-irradiated cells. When the radiation stimulatory effect was strong, such as in the sample receiving 50 cGy, the stimulatory effects could be extended from irradiated to non-irradiated cells. With the administration of radiation after infection, the results from mixed cell cultures demonstrated that the inhibitory effects could be extended from irradiated to non-121 irradiated cells, but could not be transferred by culture medium, arguing against a role for any soluble mediator in these observations. Radiation-induced intracellular ROS increase may be one of the mechanisms underline this bystander effect on HIV replication. 7.5. Suggestions for Future Experiments Results from the present study showed that low dose radiation has a significant influence on HIV-1 replication. The doses evaluated in this thesis are the lowest ones which still show a significant biological response and are well below those leading to DNA damage. In theory, these low doses of radiation may activate lymphocytes (particularly CD4 cells) by inducing IL-2Rot expression that may enhance HIV-1 replication, or may suppress HIV-1 replication by activating appropriate antiviral immune responses. Other investigators have already recognized UVR-enhanced HIV-1 promoter expression in vitro, but whether UV exposure is safe for HIV-1- infected patients is a complex question and has not been clearly resolved. Although X-ray-enhanced HIV-1 replication and LTR transcription have been reported (266), the present study is the first time the issue has been addressed based on the timing of the radiation, and the first model in which both viral stimulatory and suppressive effects were observed. Clearly, further studies are needed to monitor viral replication in HIV-1 infected patients receiving ionizing radiation for therapeutic purposes. It has been reported that low dose total body radiation can act as a potent anti-retroviral agent in vivo. Our results based on radiation of acute infected cells supports this previous finding. Conversely, radiation exposure prior to infection significantly enhanced HIV-1 infection when cells were stimulated with a mitogen. This was especially noticeable at 50 cGy. The highest stimulatory effects were found when acute infection following host cell radiation was delayed for 24 hours, corresponding to approximately one cell generation. The stimulatory effects were more obvious in the early stages of the viral life cycle (perhaps before the integration of viral DNA). The present study is the first to show that similar results can be obtained by mixing irradiated 122 (50 cGy) and non-irradiated cells, indicating that cell-to-cell communication plays an important role in modulating viral replication. This may well be linked to our demonstration of a trigger-like enhancement of IL-2Ra on PBMC, a response that required mitogen stimulation in addition to radiation exposure. The nature of a possible "trigger" effect at 50 cGy merits further study. Finally and most importantly, the mechanism of expression of the differential effects we have observed has not yet been fully elucidated. The stimulatory effect of low doses of radiation is due to host cells becoming more susceptible to viral infection rather than to latent viral DNA becoming transcriptionally active. However, the interaction of HIV-1, cells and radiation is quite complex, and further work will be required to fully understand all of the interactions at a molecular level. Without this knowledge, the application of radiation as a therapeutic agent for HIV-1-related conditions (or for HIV-1 infection itself) cannot be done with complete insight. However, epidemiological studies on HIV-1-infected patients who were exposed to radiation would be important and helpful to address the issue of clinical relevance. Extra precautions such as administration of antiretroviral drugs should be considered when HIV-1-infected patients are exposed to therapeutic radiation. As viral replication is continuous throughout the entire course of HIV-1 infection, it will be important to explore the beneficial or deleterious effects of low dose radiation to then make specific recommendations that may be applicable in clinical practice. 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