<%BANNER%>

Differential Impact of HIV-1 Protease Inhibitors on Subsets of CD4+ T-Lymphocytes

Permanent Link: http://ufdc.ufl.edu/UFE0021809/00001

Material Information

Title: Differential Impact of HIV-1 Protease Inhibitors on Subsets of CD4+ T-Lymphocytes
Physical Description: 1 online resource (134 p.)
Language: english
Creator: Gavegnano, Christina
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: Medicine -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS), affects nearly 40 million people. HIV-1 targets cells expressing CD4 and one of two major chemokine coreceptors, CXCR4 (X4) or CCR5 (R5). HIV-1 enters into cells via sequential interaction with CD4 receptor and either X4 or R5 coreceptors. Protease Inhibitors (PI) are a class of antiretroviral agents used to treat HIV-1 infected individuals. PIs are substrates for six known efflux transporters, most notably pglycoprotein (p-gp). P-gp is a member of the ATP Binding Cassette (ABC) efflux transporter family, which displays highly variable expression among lymphocytes. Lymphocytes are a primary target of infection and represent a heterogeneous population of cells that differ in receptor and coreceptor expression, activation state, surface expression of activation markers, and effector or memory function. Previous studies in our laboratory determined that significantly more PI is required to inhibit viral replication for otherwise identical viruses targeted to R5 versus X4 expressing T lymphocytes. The hypothesis is that differences in efflux kinetics and peak intracellular PI levels in subsets of HIV-1 target cells within the lymphocyte population are responsible for requirement of significantly higher IC50 in the R5 versus X4 expressing lymphocytes. Three questions were posed relative to subsets of CD4+ T lymphocytes to test the hypothesis: Optimal conditions at which to observe peak intracellular PI levels and efflux kinetics, receptor expression profile, and Rh-123 assay to observe p-gp activity. Results are as follows: Using the Rh-123 assay to observe p-gp activity, it was determined that a subset of lymphocytes that are R5+/CD4+/CD45RA-/CD45RO+ displayed p-gp activity comparable to the highest known p-gp activity in any human cell, NK cells. Cells expressing CD4 and R5 are targets for HIV-1 infection by an R5 using virus. The population of cells displaying high p-gp activity represents one of the populations for R5 infection within the lymphocyte population, demonstrating a link between p-gp activity in the R5+/CD4+/CD45RA/CD45RO+ cells and the requirement for more drug in viruses targeted to R5 expressing T lymphocytes. Although PI are substrates for other efflux transporters, and p-gp is not the sole determining factor for efflux and bioavailability, the conclusion is that p-gp activity is higher in a subset of the R5 target cells relative to cells representing the X4 HIV-1 targets. As p-gp activity is linked directly to intracellular bioavailability of PI, we can also conclude that these data demonstrate a link between p-gp activity and differences in IC50 for HIV-1 targeted to R5 versus X4 expressing T lymphocytes. These data may have implications for in vivo replication dynamics, therapy outcome, persistence of R5 infection, and viral reservoirs for drug resistant viruses emerging under the selective pressure of suboptimal levels of drug. Suboptimal levels of ART is an ongoing problem in treating HIV-1 infection and presents obstacles including emergence of resistant mutations and clearance of virus from infected cells with low bioavailability Understanding dynamics of virus/host/drug interaction and identifying mechanisms for decreased bioavailability of ART can lead to design of novel therapeutics to increase intracellular concentrations of drug.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Christina Gavegnano.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Goodenow, Maureen M.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021809:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021809/00001

Material Information

Title: Differential Impact of HIV-1 Protease Inhibitors on Subsets of CD4+ T-Lymphocytes
Physical Description: 1 online resource (134 p.)
Language: english
Creator: Gavegnano, Christina
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: Medicine -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS), affects nearly 40 million people. HIV-1 targets cells expressing CD4 and one of two major chemokine coreceptors, CXCR4 (X4) or CCR5 (R5). HIV-1 enters into cells via sequential interaction with CD4 receptor and either X4 or R5 coreceptors. Protease Inhibitors (PI) are a class of antiretroviral agents used to treat HIV-1 infected individuals. PIs are substrates for six known efflux transporters, most notably pglycoprotein (p-gp). P-gp is a member of the ATP Binding Cassette (ABC) efflux transporter family, which displays highly variable expression among lymphocytes. Lymphocytes are a primary target of infection and represent a heterogeneous population of cells that differ in receptor and coreceptor expression, activation state, surface expression of activation markers, and effector or memory function. Previous studies in our laboratory determined that significantly more PI is required to inhibit viral replication for otherwise identical viruses targeted to R5 versus X4 expressing T lymphocytes. The hypothesis is that differences in efflux kinetics and peak intracellular PI levels in subsets of HIV-1 target cells within the lymphocyte population are responsible for requirement of significantly higher IC50 in the R5 versus X4 expressing lymphocytes. Three questions were posed relative to subsets of CD4+ T lymphocytes to test the hypothesis: Optimal conditions at which to observe peak intracellular PI levels and efflux kinetics, receptor expression profile, and Rh-123 assay to observe p-gp activity. Results are as follows: Using the Rh-123 assay to observe p-gp activity, it was determined that a subset of lymphocytes that are R5+/CD4+/CD45RA-/CD45RO+ displayed p-gp activity comparable to the highest known p-gp activity in any human cell, NK cells. Cells expressing CD4 and R5 are targets for HIV-1 infection by an R5 using virus. The population of cells displaying high p-gp activity represents one of the populations for R5 infection within the lymphocyte population, demonstrating a link between p-gp activity in the R5+/CD4+/CD45RA/CD45RO+ cells and the requirement for more drug in viruses targeted to R5 expressing T lymphocytes. Although PI are substrates for other efflux transporters, and p-gp is not the sole determining factor for efflux and bioavailability, the conclusion is that p-gp activity is higher in a subset of the R5 target cells relative to cells representing the X4 HIV-1 targets. As p-gp activity is linked directly to intracellular bioavailability of PI, we can also conclude that these data demonstrate a link between p-gp activity and differences in IC50 for HIV-1 targeted to R5 versus X4 expressing T lymphocytes. These data may have implications for in vivo replication dynamics, therapy outcome, persistence of R5 infection, and viral reservoirs for drug resistant viruses emerging under the selective pressure of suboptimal levels of drug. Suboptimal levels of ART is an ongoing problem in treating HIV-1 infection and presents obstacles including emergence of resistant mutations and clearance of virus from infected cells with low bioavailability Understanding dynamics of virus/host/drug interaction and identifying mechanisms for decreased bioavailability of ART can lead to design of novel therapeutics to increase intracellular concentrations of drug.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Christina Gavegnano.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Goodenow, Maureen M.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021809:00001


This item has the following downloads:


Full Text

PAGE 1

1 DIFFERENTIAL IMPACT OF HIV-1 PROTEA SE INHIBITORS ON SUBSETS OF CD4+ T-LYMPHOCYTES By CHRISTINA GAVEGNANO A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORI DA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007

PAGE 2

2 2007 Christina Gavegnano

PAGE 3

3 This thesis is not dedicated to any particular party.

PAGE 4

4 ACKNOWLEDGMENTS I acknowled ge funding source disability supp lement-5R01HD032259 and all parties who offered support both professionally and personally.

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES.........................................................................................................................9 ABSTRACT...................................................................................................................................12 CHAP TER 1 INTRODUCTION AND BACKGROUND........................................................................... 15 Human Immunodeficiency Type 1.........................................................................................15 HIV-1 Genomic Organization................................................................................................ 16 HIV-1 Life Cycle....................................................................................................................16 Targets of HIV-1 Infection.....................................................................................................17 Antiretroviral Therapy (ART)................................................................................................ 17 Entry Inhibitors and Mechanism of Action..................................................................... 18 T-20.................................................................................................................................18 Selzentry..........................................................................................................................19 Reverse Transcriptase Inhibito rs and Mechanism of Action.......................................... 19 Protease Inhibitors and Mechanism of Action................................................................ 19 Resistance to Antiretroviral Therapy............................................................................... 20 Interaction Between Drug, Cells, and Virus........................................................................... 20 Bioavailability of ART....................................................................................................21 Efflux transporters for which PI are substrates........................................................ 21 Substrate specificity fo r efflux transporters ............................................................. 22 Interaction between substrate and efflux transporter...............................................22 Activity state of efflux transporters.......................................................................... 22 Activity and expression of efflux transporters when substrate is present ................ 23 Presence of multiple substrates................................................................................ 23 Route of orally administered HIV-1 Protease Inhibitors ......................................... 24 Impact of MDR1 genetic polymorphisms on intracellular bioavailability of PI ...... 24 Mechanisms to Study Intracellula r Bioavailability of ART ............................................25 HPLC and mass spectrometry.................................................................................. 25 Radio labeled PI.......................................................................................................25 Rhodmaine-123........................................................................................................ 26 Staining for efflux tran sporter expression ................................................................ 27 Western blot for efflux transporters......................................................................... 27 Use of efflux transporter +/cell lines..................................................................... 28 RT-PCR for Efflux Transporters.............................................................................. 28 Studies Observing Relationship Between PI, Efflux Transporters, and Subsets of HIV-1 Target Cells ......................................................................................................28

PAGE 6

6 Studies assessing p-gp mediated efflux of PI........................................................... 29 Studies using Rh-123 to assess p-gp activity in lym phocyte subsets ....................... 29 Impact of ART Independent of Antiviral Effect............................................................. 30 Therapy Response Groups...............................................................................................32 Hypothesis..............................................................................................................................33 Specific Aim 1a......................................................................................................................33 Specific Aim 1b......................................................................................................................34 Specific Aim 1c......................................................................................................................34 Significance................................................................................................................... ..35 2 MATERIALS AND METHODS........................................................................................... 49 Determination of Peak In tracellular PI Levels ....................................................................... 49 Efflux Studies: Intracellular Pr otease Inhibitor E xperiments................................................. 50 Receptor Expression Profile Study in CD4+ T lymphocytes.................................................52 Determining p-gp activity and Inhibition of Rhodam ine-1, 2,3 (Rh-123) Efflux by PIs....... 53 3 RESULTS...............................................................................................................................59 Review of Hypothesi s and Experim ents................................................................................. 59 Question 1: Confirmation of Ability to Observ e Peak Intracellular PI Lev els in PBMC...... 60 Question 2: Assessing Intra a nd Inter-assay Variability......................................................... 62 Question 3: Determining Optimal Conditions to Observe Bas eline Intracellular PI Levels Above Limit of Detection in PBMC....................................................................... 63 Question 4: Optimal Conditions to Observe Efflux Kinetics of Protease Inhibitors in PBMC ..................................................................................................................................64 Question 5: Receptor Expression Profile For Subsets of T-lymphocytes in Optimal Conditions to Observe Intracellular Protease Inhibitor Levels in PBMC ........................... 66 Question 6: Are There Enough CCR5 Cells to Perform Efflux St udy in Unstimulated PBMC?................................................................................................................................67 Question 7: Alternative A pproach and Rh-123 Assay ..........................................................69 P-gp Activity in Lymphocyte Subsets.............................................................................70 Protease Inhibitors are S ubstrates for p-gp......................................................................70 4 DISCUSSION.........................................................................................................................84 Questions Posed to Test Hypothesis of Thesis ....................................................................... 84 Relating Peak Intracellular PI Data to Toxicity and Published Data ...................................... 85 Intracellular PI Studies: Para m eters That Impact Intracel lular PI Levels and Efflux............ 86 Total Number of R5 Target Cells: Options and Alternative Methods.................................... 86 Detained Analysis and Interpretation of Rh-123 Studies ....................................................... 87 Lipophilicity of Drug: Parameters for Anal ysis and Interpreta tion of Rh-123 Data .......87 Affinity of PI for p-gp: Analysis and Interpretation of Rh-123 Data .............................88 Presence of Intracellular Pr oteins : Parameters for Anal ysis and Interpretation of Rh-123 Data.................................................................................................................89 Modulation of p-gp by PI: Parameters for Analysis and Interpretation of Rh-123 Data ..............................................................................................................................90

PAGE 7

7 Activity State of p-gp: Parameters for An alysis and Interpreta tion of Rh-123 Data ......91 Pitfalls of Rh-123 Study.........................................................................................................92 Alternative Methods to Assess p-gp Activity and Expression ............................................... 93 Follow Up to Rh-123: Direct Assessme nt of Efflux Transporter Expression ........................ 93 Relationship Between p-gp Activity and IC50........................................................................95 Relationship Between Fitness, IC50, and Target Cells............................................................ 95 Relationship of Results to Thera py Outcom e and Disease Progression................................. 97 Impact of PI Independent of Antiviral Effect: Relationship to Therapy Outcom e................. 97 Relationship Between Efflux Kinetics, Peak Intracellular PI Levels, and Cell Cycle Studies .................................................................................................................................98 Significance and Implications for Diffe rentia l p-gp Activity in CD4+/CD45RA/CD45RO+/CCR5+ T lymphocytes....................................................................................99 APPENDIX A IMPACT OF HIV-1 PROTEASE INHIBITORS ON CELL CYCLE PROGRESSION IN PBMC ..............................................................................................................................100 Introduction................................................................................................................... ........100 Methods for PHA or Anti-CD3mAb Stimulation................................................................. 101 Methods for Assessment of PI Effect on Cell Cycle Progression........................................ 102 Results...................................................................................................................................103 Discussion.............................................................................................................................103 B RAW DATA FOR RECEPTOR EXPR ESSION AND INT RACELLULAR PI STUDIES..............................................................................................................................112 LIST OF REFERENCES.............................................................................................................122 BIOGRAPHICAL SKETCH.......................................................................................................134

PAGE 8

8 LIST OF TABLES Table page 1-1. Summary of HIV-1 coreceptor use and tropism................................................................ 39 1-2. Efflux transporters for which PI are subs trates and their expression in HIV-1 target cells. ................................................................................................................................44 1-3. Methods to assess efflux transporte r expression o r activity and intracellular bioavailability of drug. .....................................................................................................45 3-1. Receptor expression profile fo r CD4 enrich ed lymphocytes:............................................ 79 B-1. Intracellular PI leve ls reported in ng/mL and M for PBMC treated with 1, 10, or 100 M IDV or RTV for 3, 18, or 48 hours. ...................................................................114 B-2. Intracellular concentration of RTV in U937 cells treated with 1.0 M RTV for 18 hours, reported in ng/m L and mM. ................................................................................ 115 B-3. Baseline intracellular RTV levels for unstim ulated or PHA stimulated PBMC treated with 1 M RTV for 18 hours at 37oC in 2 % or 10% serum........................................... 116 B-4. Intracellular RTV levels in stimulat ed PBMC in 10 % serum treated with 1.0 M or 10.0 M RTV for 18 hours prior to obs ervation of RTV efflux at 4oC or 37oC for 0 (baseline; immediately after 18 hour drug load), 10, 20, 40, or 60 minutes.................... 117 B-5. Efflux of RTV in stimulated or unstimulated PBMC in 2 % serum treated with 1.0 M RTV for 18 hours...................................................................................................... 119 B-6. Efflux of RTV in stimulated or unstimulated PBMC in 10 % serum treated with 1.0 M RTV for 18 hours. ..................................................................................................... 120

PAGE 9

9 LIST OF FIGURES Figure page 1-1. Global distribution of HIV-1 by groups, subtypes, and recom binant forms, organized by geographical location.................................................................................................... 36 1-2. Structure of HIV-1 virion. ................................................................................................37 1-3. Genomic organization of HIV-1........................................................................................ 37 1-4. HIV-1 Life Cycle. ............................................................................................................38 1-5. Mechanism of action for HIV-1 fusion inhibitor T20.......................................................40 1-6. Mechanism of action for HIV-1 Re verse Transcriptase Inhibitors. ................................. 41 1-7. Structure of HIV-1 Protease with Protease Inhibitor bound. ............................................. 42 1-8. Route of orally administered HIV-1 Protease Inhibitors. ................................................43 1-9. Effect of PI on mech anism of Rhodam ine-123. ............................................................... 46 1-10 Viral and immune responses to HIV-1 antiretroviral therapy. ........................................ 47 1-11. Significantly more drug is required to i nhibit HIV-1 replication for viruses targeted to otherwise identical viruses targeted to R5 versus X4 expressing T-lym phocytes. I..... 48 2-1. Experimental design for pilot peak intracellular P roteas e Inhibitor study. ..................... 55 2-2. Experimental design for Pr otease Inhibitor efflux study. .................................................. 56 2-3. Sample calculation for conversion of ng/mL to M f or intracellular Protease Inhibitor experiments. ...................................................................................................... 57 2-4. CD4+ T cell enrichment procedure. ................................................................................ 58 3-1. Peak intracellular PI levels in PBMC treated with 1, 10, 100 M IDV or RTV. .............. 72 3-2. Intra-assay and inter-assay variability in PI efflux study. ................................................ 73 3-3. Baseline intracellular RTV levels. ................................................................................... 74 3-4. Efflux of RTV in stimulated PBMC. ............................................................................... 75 3-5. Baseline intracellular RTV levels fro m Protease Inhibitor efflux study............................ 76 3-6. Efflux of RTV in stimulated or unstimulated PBMC in 2 % serum. ............................... 77

PAGE 10

10 3-7. Receptor expression profile fo r CD4 enrich ed lymphocytes............................................. 78 3-8. Establishment of gate and Rh -123 efflux in total PBMC. .............................................. 80 3-9.1. Rh-123 activity in NK cells, CD4 and CD8 T lymphocytes. ...........................................81 3-9.2. Rh-123 activity in subsets of HIV-1 target cells. ............................................................. 82 3-10. Efflux of Rh-123 in the presence of IDV, RTV, or R-Verapam il. .................................. 83 A-1. Effect of PHA on cell cycl e progression in PBMC. ...................................................... 107 A-2. Experimental design for ce ll cycle P I study in PBMC.................................................... 108 A-3. Effect of PI on cell cycl e progression in PBMC. ........................................................... 109 A-4. Effect of IDV, RTV, and NFV on c ell cycle progression in PBMC. ............................. 110 A-5. Pitfalls for Propidium Iodide or 7AAD in concert with receptor expression stain. .........111 B-1. Receptor expression profile for CD4 enriched lymphocytes, donor B............................ 112 B-2. Receptor expression profile for CD4 enriched lymphocytes, donor C............................ 113

PAGE 11

11 LIST OF ABBREVIATIONS ABC ATP-Binding Cassette ART Antiretroviral Therapy BCRP Breast Cancer Resistance Protein BLQ Below Limit of Quantitation HIV-1 Human Immunodeficiency Virus Type 1 IN Integrase MRP Multidrug Resistance Transporter Protein NNRTI Non Nucleoside Reverse Transcriptase Inhibitor NRTI Nucleoside Reverse Transcriptase Inhibitor p-gp p-glycoprotein PI Protease Inhibitor PIres Protease Inhibitor Resistant PIsen Protease Inhibitor Sensitive PR Protease Rh-123 Rhodamine 123 RT Reverse Transcriptase R5 CCR5 X4 CXCR4

PAGE 12

12 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science DIFFERENTIAL IMPACT OF HIV-1 PROTEA SE INHIBITORS ON SUBSETS OF CD4+ T-LYMPHOCYTES By Christina Gavegnano December 2007 Chair: Maureen Goodenow Major: Medical Sciences Human immunodeficiency virus type 1 (H IV-1), the causative agent of acquired immunodeficiency syndrome (AIDS), affects ne arly 40 million people. HIV-1 targets cells expressing CD4 and one of two major chemokine coreceptors, CXCR4 (X4) or CCR5 (R5). HIV-1 enters into cells via sequential interact ion with CD4 receptor and either X4 or R5 coreceptors. Protease Inhibitors (PI) are a cla ss of antiretroviral agents used to treat HIV-1 infected individuals. PIs are substrates fo r six known efflux transp orters, most notably pglycoprotein (p-gp). P-gp is a member of the ATP Binding Cassette (ABC) efflux transporter family, which displays highly variable expression among lymphocytes. Lymphocytes are a primary target of infec tion and represent a heterogeneous population of cells that differ in receptor and coreceptor expression, activation state, surface expression of activation markers, and effector or memory function. Previous studi es in our laboratory determined that significantly more PI is required to inhibit viral replication for otherwise identical viruses targeted to R5 versus X4 expressing T lymphocytes. The hypothesis is that differences in efflux kinetics and peak intracellular PI levels in subsets of HIV-1 target cells within the lymphocyte population ar e responsible for requirement of significantly higher IC50 in the R5 versus X4 expressing lymphocytes. Three questions were

PAGE 13

13 posed relative to subsets of CD4+ T lymphocytes to test the hypothesis: Optimal conditions at which to observe peak intracellular PI levels an d efflux kinetics, receptor expression profile, and Rh-123 assay to observe p-gp activity. Results are as follows: Using the Rh-123 assa y to observe p-gp activity, it was determined that a subset of lymphocytes that are R5+/CD4+/CD45RA-/C D45RO+ displayed p-gp activity comparable to the highest known p-gp activity in any human cell, NK cells. Cells expressing CD4 and R5 are targets for HIV1 infection by an R5 using viru s. The population of cells displaying high p-gp activity represents one of the populations for R5 infection within the lymphocyte population, demonstrating a link between p-gp activity in the R5+/CD4+/CD45RA/CD45RO+ cells and the requirement for more drug in viruses targeted to R5 expressing T lymphocytes. Although PI are substrates for other efflux transporters, and p-gp is not the sole determining factor for efflux and bi oavailability, the conclusion is th at p-gp activity is higher in a subset of the R5 target cells relative to cells re presenting the X4 HIV-1 targets. As p-gp activity is linked directly to intracellular bioavailability of PI, we can also conclude that these data demonstrate a link between p-gp ac tivity and differences in IC50 for HIV-1 targeted to R5 versus X4 expressing T lymphocytes. These data may have implications for in vivo replication dynamics, therapy outcome, persistence of R5 inf ection, and viral reservoirs for drug resistant viruses emerging under the selective pressure of suboptimal levels of drug. Suboptimal levels of ART is an ongoing problem in treating HIV-1 infection and presents obstacles including emergence of resistant mutations and clearance of virus from infected cells with low bioavailability Understanding dynamics of virus/host/drug inte raction and identifying

PAGE 14

14 mechanisms for decreased bioavailability of AR T can lead to design of novel therapeutics to increase intracellular co ncentrations of drug.

PAGE 15

15 CHAPTER 1 INTRODUCTION AND BACKGROUND Human Immunodeficiency Type 1 HIV-1 is a retrovirus belonging to the genus Lentivirus. Infections by lentiviruses are characterized by rem arkably complex interactions w ith the host and a chronic course of disease. Common features of disease include long and va riable incubation period s, persistent viral replication, neurologic manifest ations, and destruction of speci fic hematologic or immunologic cells 29, 45,100. All lentiviruses exhibit a common mor phogenesis and morphology, a tropism for macrophages, extensive genetic and antigenic variability, and the presence of additional regulatory genes not found in other groups of retroviru ses. Lentiviruses have been isolated from several animal species including sheep, goats, horses, cattle, cats, monkeys, and humans. HIV-1 and HIV-2 are the only known human lentiviruses 29. Since its identification in the early 1980's, Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Syndrome (AIDS), has escalated to a global pandemic. An estimated 39.5 million people are living with HIV-1 and 2.9 million people died of AIDS-related illnesses in 2006. There were 4.3 million new infections in 2006 with 2.8 million (65%) of these occurring in sub-Sahara n Africa and important increases in Eastern Europe and Central Asia, where there are some indications that infection rates have risen by more than 50% since 2004 (WHO, 2006). The dis ease is characterized by susceptibility to opportunistic infection and a seve re and steady decline in CD4+ T cells 29, 45,48,100. Groups and subtypes according to geographica l location classify HIV-1. Group M (major) contains subtypes A-K, and is most prevalen t worldwide. The most common subtypes in group

PAGE 16

16 M are subtype B, which predominates in North America, Europe, parts of South America and India, subtype C which predominates in sub-Sa haran Africa, and subtype E which predominates in Southeastern Asia. Two new groups, N (new) and O (outliner), have been identified in Africa and Eastern Europe (Figure 1-1) 29,92. HIV-1 Genomic Organization HIV-1 is a s ense strand RNA virus which packages 2 copies of a positive-sense RNA strand genome of approximately 10,000 nucleotides (10 kb) into each virion. The genome is flanked by two identical long te rminal repeats (LTR) and cont ains the three major genes gag, pol, and env as well as accessory proteins Vif, Vpr, Vpu, Tat, Rev, and Nef. The structural proteins matrix (p17MA), capsid (p24CA), nucleocapsid (p7NC), and p6, are encoded in gag Enzymes protease (PR), reverse transcriptase (RT), and integrase (IN) are encoded in pol The env gene encodes the envelope glycoproteins gp120 and gp41, wh ich are required for viral attachment and entry (Figure 1-2, 1-3). 29, 45,92,100 HIV-1 Life Cycle HIV-1 gp120 on the surf ace of the virion binds to CD4 receptor on the host cell. This causes conformational changes in gp120, which then expose coreceptor-binding sites, and cellular chemokine coreceptors CCR5 or CXCR4 are engaged. After attachment, the viral envelope (trimeric complexes of glycoproteins gp120-gp41) fuse with the lipid membrane of the target cell, releasing the viral core into cy toplasm of the host cell. Viral RNA is reverse transcribed into double-stranded DNA via viral enzyme RT. The DNA translocates to the nucleus and integrates into the host genome via viral enzyme IN. Transcription of the RNA and translation of gene products occur next, follo wed by assembly at the plasma membrane, budding of the newly formed virion, and protease processing resulting in a mature, infectious virion (Figure 1-4). 27, 29, 45,92,100

PAGE 17

17 Targets of HIV-1 Infection HIV-1 entry into cells is mediated by targ et cell expression of CD4 and appropriate chemokine coreceptor, primarily CXCR4 or CCR5. CD4/CCR5 cells include macrophages (t 1/2 weeks) and a small subset of ac tivated memory T lymphocytes (t 1/2 upon primary exposure to antigen = years, t 1/2 upon secondary exposure to antigen = days) while CXCR4 is expressed by most CD4 T lymphocytes, transformed lymphocytic or monocytic lineages, and macrophages. R5 viruses are consistently macrophage tropic (M-tropic) and displa y M-R5 phenotype, while X4 viruses display heterogeneous phenotypes. The majority of primary X4 viruses replicate in both macrophages and T cell lines, use CXCR4 eith er alone or in combination with CCR5 and are dual (D)-tropic, whereas X4 viruses which do not exhibit tropism for macrophages, but maintain ability to replicate in T cell lines, are T-X4 phenotypic. Viruses that can productively replicate in R5 expressing lymphocytes, but no t macrophages, are termed L-R5 (Table 1-1). 8, 14, 17,29,32,45, 48, 49, 80 R5 viruses predominate early in infection, whereas in ~ 50 % of patients, X4 viruses emerge and are associated with rapid progression to AIDS. Ly mphocytes represent the primary target of HIV-1 infection, are a sentinel part of the adaptive immune response, and function to circulate between the peripheral blood and secondary lymphoid organs until antigen is encountered. Macrophages are part of the innate immune response, are phagocytic, and represent a small fraction of infected cells (Table 1-1). 8,29, 48,100-101 Antiretroviral Therapy (ART) Four class es of ART are used to control HI V-1 infection: entry inhibitors, nucleoside reverse transcriptase inhibitors (NRTI), non-nucleoside reverse tran scriptase inhibitors (NNRTI), and protease inhibitors (PI). Ot her classes of drugs currently in clinical trials to treat HIV-1 infection include 1. Immune based therapies focused on CD4 T-cell reconstitution via

PAGE 18

18 administration of T-cell prolifera tive cytokines, notably IL-2, a nd 2. Integrase inhibitors, which function to inhibit HIV-1 integration into the host cell genome. 11,29,100,111,116 Entry Inhibitors and Mechanism of Action There are tw o FDA approved entry inhibitors: T-20, a fusion inhibitor, and Selzentry, a CCR5 inhibitor. Other possibilities currently bei ng studied for entry inhi bitors include soluble CD4, siRNA directed silencing of CD4 mRNA e xpression, inhibition of chemokine coreceptor engagement by HIV-1 (targeting other binding sites on CCR5 than Selzentry), and molecules that target the envelope-CD4 interaction (Figure 1-5). 11, 29, 100,116 T-20 Fusion betw een the virus and the host cell is a cr itical and necessary step in the viral life cycle. Without this event, rel ease of the viral core into the ho st cell cytoplasm cannot occur. T20 was approved by the U.S. Food and Drug Ad ministration (FDA) in 2003 and is a 36kDa protein which functions as a dominant negative molecule, inhibiting form ation of the hairpin structure necessary for fusion to occur (Figure 1-6). Disadvantages of T-20 treatment for HIV-1 infection include cost of synthesis, the large amount of T-20 re quired for efficacy, and the size of the molecule, which prohibits oral route, and must be injected. Clinic al trials and studies conducted after FDA approval have demonstrat ed that T-20 is most efficacious when administered in combination with multiple drugs for which the virus is sensitive, but displayed limited ability to reduce viral loads when administ ered alone or in combination with drugs that the virus was resistant to. This correlates the use of T20 with administration of drugs that the virus is sensitive to, potentially limiting the circ umstances that T20 can be successfully used (Figure 1-5). 11, 29,111,116

PAGE 19

19 Selzentry Selzentry is a CCR5 inhibitor that was approved by the FDA in August 2007. The molecule inhibits viral entry via binding to CCR5 coreceptor, inhibiti ng the receptor/coreceptor vira l envelope interactio n required for HIV-1 entry into the host cell. Selzentry provides an advantage over other drugs, presenting an option for treatment of HIV-1 infected individuals with multiple drug resistance to other ART, and is approved for these individuals. Little is known about the long-term effect of Selzentry treatment, and thus is not approved for HIVpositive patients who have drug sensitive strains at this time. 111,116 Reverse Transcriptase Inhibitors and Mechanism of Action The two clas ses of Reverse Transcriptase Inhi bitors are NRTI and NNRTI. NRTI were the first anti-HIV drugs used clini cally, and require intracellula r phosphorylation for activation. NRTI compete with endogenous nucleotides fo r incorporation into the growing viral DNA strand, however they lack a 3 hydroxyl terminus thus incorporation of the NRTI into the growing DNA strand results in termination of the DNA strand, as the next phosphodiester bond is not formed (Figure 1-6). 29,100,115 NNRTI do not require in tracellular phosphorylation for ac tivation, have a limited effect on other cellular enzymes, and bind directly to RT inhibiting its enzymatic activity (Figure 1-6). 29,100,116 Protease Inhibitors and Mechanism of Action PI target HIV-1 PR, an aspartic protease com prised of two 99 amino acid monomers, which are only active in homodimeric form. PR cleaves Gag and Gag-pol, resulting in a mature infectious virion. PIs are competitive inhibitors, whic h compete for binding in the active site with the natural substrate. Once bound, PI cannot be cl eaved, resulting in inactivation of the enzyme (Figure 1-7.) 25-27, 29,100,116

PAGE 20

20 There are currently ten PI approved by the F DA for treatment of HIV infection, with the most recent FDA approval occurring in June 2006, for TMZ-114. All PI belong to the competitive small-molecule inhibitor family of drugs. 29,100,40,116 Two experimental PI which have not yet been evaluated by the FDA are in clin ical trials, and other PI in various stages of development exist. 116 They include: Agents which irreversibly modify the activ e site aspartates, resulting in diminished requirement for high concentration of inhi bitor and possibly reduc ing development of resistance mutations Agents targeting dimer interface of the en zyme, preventing formation of the homodimer required for activity Defective PR monomers which function as dominant negative macrom olecular inhibitors, forming heterodimeric comple xes with wt PR monomers Resistance to Antiretroviral Therapy Resistan ce is conferred by multiple mechanisms. 1,6,9-10,12,20,24,25-27,29,40,49-50,61,63,100,117 Viral RT error rate (mutation rate is approximately 1 in 104 errors per base incorporated; one new mutation is introduced into each ne w copy of the genome), allowing for rapid accumulation of mutations in virus-encoded drug targets (RT, PR, Env) Mutations which reduce sensitivity to PI are found in patients nave to PI therapy Accumulation of protease inhibitor resistant (PIres) mutations (due to suboptimal drug levels) Cross Resistance Interaction Between Drug, Cells, and Virus The goal in treating HIV-1 infection is to delay progression to AIDS by controlling viral replication and preventing or reve rsing immune deficiency. The end result of administration of a PI containing regimen in vivo is far more complicated than er adication of virus via antiviral therapy. Many parameters impact therapy out come and disease progression, and involve a complex interplay between drug, cells, and virus.

PAGE 21

21 Bioavailability of ART Bioavailability of ART is dependent of multiple factors including: 1,2,4,5,6,9,12,1822,35,37,40,42,44,51,56,57,60,63-66,68,70-73,76,77,81,93,94,98,100,104,106,107,109,112 Route of administration: oral (Figure 1-8) versus injection Percent plasma protein binding of drug (which impacts amount of free drug available to enter cells, and efflux kinetics of drug) Percent of drug bound to intracellular proteins (w hich impacts their ability to inhibit viral replication) Size of drug Lipophilicity of drug Affinity of drug for efflux transporters Expression of efflux transporte rs in HIV-1 infected cells Variable efflux transporter expre ssion between patients (7-fold) Ability of drug to modulate efflux transporter expression Drug metabolism in the intestinal lumen (applicable for orally administered drugs) Co-administration of other ART, which may boos t the bioavailability or compete for binding to efflux transporters Polymorphisms of the MDR1 gene Affinity of the drug for the binding pocket of the efflux transporter Efflux transporters for which PI are substrates There are six known effl ux transporters for which PI are substrates, which display differential expression, and activity across cell types (Table 1-2). 1,2,6,12,19,22,33,37,51,5660,65,71,75,77,81,93,106 P-glycoprotein (p-gp/MDR1), multidrug resistance transporter protein 1 (MRP1), MRP2, MRP5 and BCRP (Breast Cancer Resistance Protein) are expressed in lymphocytes. P-gp, MRP1, MRP4, MRP5 are e xpressed in macrophages (Table 1-2).

PAGE 22

22 Each of these efflux transpor ters belongs to the ATP bindi ng cassette family of efflux transporters (ABC). There are forty nine currently known member s of the ABC family, most of which are approximately 1500 AA in size and compri sed of two equal or unequal halves, contain one or two binding pockets, possess a membrane spanning domain and a transmembrane domain, and efflux substrate via an ATP de pendent mechanism (Figure 1-9). 1,5,18,19,33,43,70 Substrate specificity for efflux transporters A wide variety of structurally unrelated potentially cytotoxic co m pounds are substrates for the ABC family of efflux transporters including antibiotics, immunosuppressive agents, chemotherapeutic agents, steroids, -blockers, and HIV-1 protease inhibitors. 1,2,5,19,20,22,43,56,57,60,65,68,70,70,77,81,106 Interaction between substrate and efflux transporter There is no shuttle mechanism for substrat e delivery to binding pocket(s) of efflux transporters. Most ABC efflux transporters possess two binding pockets for substrate, which increases the diversity of the transporter for su bstrate. Although two binding pockets exist, the conformation dictates that only one binding pocket may be occupied by substrate at one time. Thus, although two binding pockets exist, the efflux transporter may only efflux one substrate at a time. Substrate that is in close physical pr oximity to the binding pocket will be effluxed, whereas substrate that is physically distant fr om the transporter will remain intracellular.1,5,18,19,70 Activity state of ef flux transporters ABC e fflux transporters remain in a confor mationally inactive state until substrate binds to the substrate-binding pocket. This event c onfers a conformational change, which allows for activation of the efflux transporter, resulting in efflux of substrate fr om the cell. Activity state of the transporter is not dictated by any factor other than presence of substrate in binding pocket of

PAGE 23

23 efflux transporter. Upon efflux of the substrat e, the efflux transporte r will again adopt the conformationally inactive stat e until another substrate bi nds to the binding pocket.1,5,18,19,70 Activity and expression of efflux tr ansporters when substrate is present In all cases, presence of PI activates the e fflux transporter, allowing for efflux of PI via the ATP dependent mechanism, but does not increa se expression levels of the transporter (with the exception of NFV). Of the ten PI, NFV is the only known PI that can not only activate the efflux transporter, but increas e efflux transporter expression. Depending on the substrate, increase in efflux transporter expr ession may occur, as is the case with some NRTI. This finding has been demonstrated in multiple HIV-1 ta rget cells including primary lymphocytes, macrophages, and T cell lines. 1,18,19,21,42,44,65,70,71,77,81, 93 Presence of multiple substrates Multiple agents are substrates for ABC efflux transporters. In HIV-1 infection, combination ART dictates the concomitant administration of multiple drugs. In an HIV-1 infected individual, multiple drugs may compet e for binding to efflux transporters, and the presence of drugs with higher affinity for th e efflux transporter-binding pocket can affect intracellular drug levels, where affinity is de fined as ability of Drug A to bind to efflux transporter, conferring preferen tial efflux, when both Drug A and Drug B are present. When a drug with higher affinity is present with a drug of lower affinity, the higher affinity drug competitively inhibits the lower affinity drug. The higher affinity drug will be preferentially effluxed from the cell, and the lower affinity drug will remain largely intracellular. In this way, presence of multiple drugs in a system directly affects intracellular drug levels and ability of drug to be effluxed. 1,5,18,19,70

PAGE 24

24 Route of orally administered HIV-1 Protease Inhibitors Protease Inhibitors are administered orally, and are highly metabolized by cytochromes, most notably CYP-450a and CYP450b in the intestinal lumen 1,4,37,106. The drug that is not metabolized traverses the intestinal lumen and ci rculates in the periphery, where the PIs are highly bound to plasma proteins (lowest perc ent bound = 60 % for IDV, highest percent bound = 98 % for Nelfinavir [NFV]). Of the PI that is free (non-plasma protein bound) to traverse the lipid bilayer and enter cells, lipoph ilicity dictates whether passiv e diffusion occurs, or whether entry must occur via active transport 1,4,37,106. Upon entry into cells, PI s may bind to intracellular proteins, or, depending on effl ux transporter expression (PIs are substrates for ATP-BindingCassette Family of efflux transporters), become ra pidly effluxed from cells (Figure 1-8). PIs that are effluxed may again enter cells either by passive diffusion or active transport. 1,5,18,19,70 Metabolism, plasma protein bi nding, and efflux of PI presen t obstacles in treating HIV-1 infection. 1,2,4,5,6,9,18,19,35,43-44,57,60,70,71,81,84,85,94,106,116,117 Identifying mechanisms for decreased bioavailability of ART, and PIs in particular, can lead to design of novel therapeutic s to increase intracellular concentrations of drug. Impact of MDR1 genetic polymorphisms on intracellular bioavailability of PI Many studies have been conducted to elucidat e the relationship between efflux transporter expression, bioavailability of PI, and ability to control HIV-1 replication in infected patients. 6,9,12,20,39-41,44,57,58,61,71,72,77 Conflicting results have been report ed in the literature, often as a function of differences in cohorts or cell type studied. Some studi es report that in HIV-infected patients, IC50 of PI is inversely correlated with MDR1 gene overexpression in lymphocytes and that undetectable viral loads are associated with the use of low-dose RTV (as a boost) 1,20,40. Other studies report the c ontrary and state that there is a mi nimal effect of MDR1 and CYP3A5 genetic polymorphisms on the pharmacokinetics of IDV in HIV-infected patients, but do not

PAGE 25

25 address impact of other PI either alone or in concert with RTV 104. The data to date are unable to consistently draw a direct link between MDR1 genetic pol ymorphisms and intracellular bioavailability of drug, although few studies overlap in methods applied, reducing ability to directly compare results between studies. Mechanisms to Study Intracellular Bioavailability of ART Multip le mechanisms are routinely employed to study the intracellular bioavailability of ART, in particular HIV-1 PI, either directly or indirectly (Table 1-3). 1,19-22,30,4144,56,57,59,60,63,65,66,68,71,73,77,83,93,97,98,106,107,112 HPLC and mass spectrometry High perform ance liquid chromatography c oupled with mass spectrometry provides a direct assessment of intracellular drug availa ble. This method is applied after methanol extraction of intracellular fraction of drug. Lim itations for this method include total cell number required to observe intracellular PI levels above the limit of detection for the assay, and inability to observe intracellular fracti on of drug within subsets of cells without prior sorting of subpopulations (Table 1-3). Radio labeled PI Use of radio labeled PI is em ployed to discover intracellular PI levels within cells. Radio labeled forms of PI are available for most PI, and intracellular PI can be observed within the population of cells treated with the radio labeled drug by detecti ng radioactivity of the sample. Limitations of this method include inability to concomitantly observe PI levels in cells while assessing receptor expression prof iles, thus precluding this method from assessing intracellular PI levels within subsets of cells without prior sorting of subpopulations (Table 1-3).

PAGE 26

26 Rhodmaine-123 Rhoda mine-123 (Rh-123) is a fluorescent dye that can be detected on FL1 via flow cytometry. Hofmann, et al 68, first reported this method in 1992 The group first established a concert between Rh-123 intracellular accumulation and p-gp expression using cell lines that were positive and null for p-gp. The group assessed Rh-123 accumulation in cells isolated from patients suffering from acute myeloid leukemia (A ML), a cell type known to have significantly elevated levels of p-gp, both in the presence and absence of a specific p-gp inhibitor. For cells without inhibitor, Rh-123 efflux was rapid, wh ereas in the presence of inhibitor, Rh-123 accumulated intracellularly and did not efflux (Figure 1-9). To confirm the hypothesis that Rh-123 accumulation relates to p-gp activity and expression, single color flow cytometry was perf ormed using a specific flurochrome conjugated monoclonal antibody against p-gp, MRK-16, in the AML cell lines (which rapidly effluxed Rh123) and cell lines null for p-gp (which did not efflux Rh-123) Analysis confirmed that p-gp levels were high in the cells that rapidly efflux Rh-123, but were abse nt in alternate cell lines null for p-gp 68. Shortcomings of this method are that this method provides indirect assessment of p-gp expression, instead observing p-gp activity. P-gp activity correlates with p-gp expression in most cases. In a system where p-gp expression le vels may be altered by presence of substrate (for example, NFV), this method would not be ideal, and would require follow up with specific anti-p-gp monoclonal antibodies, we stern blot analysis, RT-PCR, or use of p-gp+/cell lines to directly assess expression and modulation of p-gp by PI. 1,19-22,30,41 44,56,57,59,60,63,65,66,68,71,73,77,84,93,97,98,106,107,112 Comprehensive discussion regarding shortcomings, pitfalls, and alternative approach es to Rh-123 assay appears in the discussion section of this thesis, as it relates directly to the data reported in the thesis.

PAGE 27

27 Many studies have been performed to study effl ux transporter expression within subsets of cells. Various combinations of the assays described above (Rh-123, p-gp+/cell lines, RT-PCR, western blot, flow cytometry) have been em ployed. A summary of these data and methods employed is as follows: 1,19-22,30,41-44,56,57,59,60,63,65,66,68,71,73,77,84,93,97,98,106,107,112 Staining for efflux transporter expression Another commonly used method for indirect as sessment of bioavailability of ART is the indirect measure of efflux transporter expr ession via flurochrome-conjugated monoclonal antibodies. There are antibodies for all known efflux transpor ters for which PI are substrates, presenting a diverse method to assess six efflux tr ansporters simultaneously within a target cell population via flow cytometry. This method can also be employed with receptor expression staining, allowing for concomitant determination of efflux transporter expression within subsets of cells (Table 1-3). Shortcomings of this approach include the f act that although this is a direct measure of efflux transporter expression, it is an indirect method for determin ing intracellular drug levels. This method must be performed in tandem with other experiments, which can validate and support the results from this assay. Until recently, many of the antibodies commercially available were limited, thus truncating the ability to perform complex experiments to observe many parameters simultaneously. Western blot for efflux transporters Western blot analysis can be perform ed with antibodies specific for efflux transporters on cellular lysates obtained from PI or drug treated cells. In this way, direct assessment of efflux transporter expression as a function of time and exposure to PI can be observed (Table 1-3).

PAGE 28

28 Use of efflux transporter +/cell lines Cell lines that are high p-gp expr es sers as determined from fl ow cytometry direct stain, Rh-123 efflux, casein efflux, wester n blot, RT-PCR or assessment of intracellular concentration of drugs that are substrates for p-gp but not othe r efflux transporters provi de an excellent model to study effect of PI on p-gp activity and expression. Tumorogenic cell lines frequently over express efflux transporters as a mechanism of re sistance to chemotherapeutic agents, which are substrates for p-gp or other e fflux transporters. CEM-MDR cells, which express 30-fold more pgp than CEM cells, and CEM-MRP cells, which expr ess fivefold more MRP1 protein than CEM cells present a model to specifically study p-gp and MRP1. Another mechanism of obtaining a cell line expressing high levels of efflux transporters is transfection of a cell line with MDR1 gene. L-MDR1 cells are a human epithelial kidney cell line transfected with expression vector expr essing MDR1 cDNA, and P388/dx cells are a monocytic leukemia cell line that express high le vels of p-gp. Both cell lines are commonly employed to assess direct modulation of efflux transporter activity and expression by PI and other drugs (Table 1-3). RT-PCR for Efflux Transporters Reverse Transcriptase-PCR provides a direct m echanism to observe modulation of mRNA transcripts by presence of substr ate. Transcript levels before, during, and after addition of PI can be observed in a system comprised of cells ex pressing p-gp, allowing for direct assessment of efflux transporter expres sion (Table 1-3). Studies Observing Relationship Betw een PI, Efflux Transporters, and Subsets of HIV-1 Target Cells Many studies have been performed to study one or more aspects of efflux transporter expression and activity and its relationship to PI and subs ets of HIV-1 target cells. The impact of

PAGE 29

29 these data relative the findings of this thesis are addressed in the di scussion. A summary of studies encompassing these data are as follows: 1,19-22,30,4144,56,57,59,60,63,65,66,68,71,73,77,84,93,97,98,106,107,112 Studies assessing p-gp me diated efflux of PI Many studies have been perform ed to st udy the interaction between PI and efflux transporter activity and expression. Re sults are diverse and appear below. 1,19-22,30,4144,56,57,59,60,63,65,66,68,71,73,77,84,93,97,98,106,107,112 PI inhibit Rh-123 efflux PI are substrates for p-gp (using Rh-123 assay) There is differential p-gp activ ity within total PBMC population There is differential p-gp expression within subsets of lymphocytes (assessed with direct stain for efflux transporters in concert with receptor expression stain) NFV but not other PI in creases p-gp expression in vitro but not in vivo Increased expression and activity of p-gp is not related to intrace llular HIV-1 RNA and DNA levels Hierarchy for lipophilicity: NFV > S QV > RTV > IDV (in PBMC assessed in vitro ) Hierarchy for intracellular accumulation of PI: NFV > SQV > RTV > IDV (in PBMC assessed in vitro) PI interfere with p-gp function in targets of HIV-1 inf ection within the lymphocyte population ABC efflux transporters p-gp and MRP1 limit the intracellular bioavailability of IDV in macrophages (in vitro) Studies using Rh-123 to assess p-gp activity in lymphocyte subsets Upon establishm ent by Hofmann, et al that Rh-123 is a substrate for p-gp 68, Rh-123 has been employed by many groups to assess p-gp activity. The impact of these data relative the findings of this thesis are a ddressed in the discussion.

PAGE 30

30 Schuitemaker et al sought to determine if p-gp expressi on or activity in HIV-1 target cells within the lymphocyte populat ion correlates with intr acellular viral RNA or DNA 89. To answer this question, they studied Rh-123 levels in CD4+ T lymphocytes cells isolated from HIV-1 infected patients that were subsequently gated on CD45RO. The CD45RO cells were gated to observe two distinct populati ons: RO+/Rh-123 dim (high p-gp activity) and RO-/Rh-123 bright (low p-gp activity). The group determined that intracellular HIV-1 RNA and DNA was significantly lower in cells with high-p-gp activity, thus conclu ding that the potential efflux function of p-gp on PIs may be clinically less re levant than the effect of p-gp on intracellular HIV-1 replication. Gastl, et al 73 assessed Rh-123 activity within ly mphocyte subsets by gating on CD4, CD8, CD45RA and CD45RO, or on CD16/CD56 (NK cells). The group determined that CD4+/CD45RA+ cells displa y higher p-gp activity than CD4+/CD45RO+ cells. Benedetti et a l 112 assessed p-gp activity with Rh-123 a ssay and direct p-gp expression in subsets of PBMC gated on CD3, CD4, CD8, CD16 and CD56. The group determined that p-gp activity is highest in NK cells and that p-gp ac tivity is significantly higher in CD3+/CD8+ T cells relative to CD3+CD4+ T cells, but that pgp activity is not always a good correlate for p-gp expression. Gupta et al 7 studied Rh-123 activity in CD4 and CD8 T cells isolated from HIV-1 infected patients. The group determined that p-gp activity is low in chronically infected HIV-1 infected individuals with viral loads that predict disease progression. The group also stated that the study population would need to be increas ed to confirm these results. Impact of ART Independent of Antiviral Effect Little is known about the eff ect on the cellular level of entr y inhibitors independent of their antiviral effect, and no potentially benefici al effects on the imm une system have been

PAGE 31

31 clearly mapped to T-20 or Selzentr y treatment. Both NRTI and NNRTI have been implicated in modulation of host cell functions independent of their antiviral effect, most notably modulation efflux transporter function, activity and expression, and modulation of proteasome function 20,88,89. Extensive literature exists to demonstrate that PIs have an effect on cells independent of their antiviral effect.2,4,12,15,21,23,38,44,52,62,78,79,87-91,102,102,113-115 Effects include the following: Inhibition of cell cycle progression via G1 block in primary PBMC and cell lines Restoration of levels of circul ating levels of cytokines in HI V-1 infected patients to levels observed in healthy patients (Type 2 to Type 1 switch) Inhibition of apoptosis both in vitro and ex vivo for both infected and bystander cells Modulation of antigen presentation Inhibition of proliferation in lymphocytes Modulation of lipoprotein metabolism Modulation of dendr itic cell maturation In a significant percen tage of children and adolescents receiving a PI containing regimen, immune reconstitution is achieved but not concom itant with decrease in viral burden (viral failure, immune success; VF/IS) (Figure 1-10), impli cating a role for PI in immune reconstitution independent of its antiviral effect. Immune reconstitution is highly favorable fo r patient prognosis and leads to delayed progression to AIDS. 29,34-36 This discordant response is not determined solely by host factors alone or simple genetic ch anges in the viral genome. 13, 23-27, 39, 43, 46, 61, 83 ART induced discordant responses are multifactorial and i nvolve a complex interplay between phenotypic properties of the virus, ART eff ect and interaction with host cells and the ability of the immune system to control viral replic ation in the presence of ART.13, 23-27, 39, 43, 46, 60, 83 The effect of PI on

PAGE 32

32 the immune response independent of its antiviral effect, and the imp lications of differential effect of PI within subsets of HIV-1 target cells and in other cells, constitutes a complex set of questions relating to therapy out come and progression to AIDS. Therapy Response Groups Plasm a virus levels and CD4 T cell counts are us ed to classify patient response to ART. Patients who exhibit sustained s uppression of viral rep lication (viral success) and significant improvement in CD4 T cell counts (immune succe ss) are classified as Viral Success Immune Success (VS/IS). This occurs in 25-30 % of children. Patients who fail to maintain suppression of viral replication (viral failu re) and do not exhibit improvement in CD4 T cell counts (immune failure) are classified as Vira l Failure Immune Failure (VFIF) This occurs in 15-20 % of pediatric patients. Patients who exhibit sustai ned reconstitution of T cell counts (immune success), despite transient viral suppression and s ubsequent rebound to levels that predict disease progression are classified as Viral Failure Immune Success (V F/IS). Discordant response (VF/IS) occurs in 25-40 % of childre n ad adolescents, and is maintain ed in adults in a variety of cohorts (Figure 1-10). 39, 100, 116,118 The goal when treating HIV-infected patie nts is to delay pr ogression to AIDS by controlling viral replicat ion and preventing or reversing immu ne deficiency, and viral loads and T cell counts directly correlate with clinical disease progression29,34,36. For example, children who display discordant response ex hibit lower clinical disease pr ogression rates than those of control groups with similar viral levels w ho do not display immune reconstitution (implicating critical role of immune reconstitu tion in favorable clinical prognosis) 46,100,108. In addition, chronic HIV infection and high viral loads global ly impair immunity via multiple mechanisms such as CD4 T cell attrition by direct infection and apoptosis, HI V-induced uncontrolled activation of adaptive and innate immune response, impairment of thymic output, and evasion of

PAGE 33

33 HIV-specific immunity. 23,34,36,29,53,54,103 Therefore, controlling viral replication while maintaining or restoring functional immunity remains sentinel in treating HIV-infected individuals. Hypothesis Previous studies in our laboratory determ ined that significantly more drug (188 fold for IDV and 141 fold for RTV) is required to inhibit viral replication for otherwise identical viruses targeted to R5 versus X4 expressi ng T-lymphocytes (Figure 1-.11). The hypothesis of this thesis is that di fferential bioavailability, and specifically differences in efflux kinetics and peak intracellu lar PI levels in subsets of lymphocytes are responsible for requirement of significantly higher IC50 in the R5 subset of HIV-1 target cells. Specific Aim 1a Previous studies in our laboratory demonstrated that significantly m ore drug is required to inhibit viral replication for othe rwise identical viruses targeted to R5 versus X4 T lymphocytes (Figure 1-11). To test the hypothe sis that differences in efflux ki netics and peak intracellular PI levels in subsets of HIV-1 target cells within the lymphocyte population are responsible for these data, the optimal conditions at which to observe peak intracellular PI levels and efflux kinetics in subsets of CD4+ T lymphocytes were determin ed, using U937 promonocytic cell line, and total PBMC. Using U937 and PBMC provided a model to observe inter and intraassay variability, as well as optimized conditions. This specific aim wa s addressed with the following questions: Published data demonstrates that peak intrace llular PI levels in PB MC are conferred by 18 hours extracellular PI treatment at 37oC. Can we recapitulate similar values in our laboratory? What is the inter-assay and intra-assay variability for this study? What are the optimal conditions to observe ba seline intracellular PI levels above limit of detection in PBMC? (Variables include seru m concentration in extracellular media and stimulation state of cells)

PAGE 34

34 What are the optimal conditions to observe e fflux kinetics of PI in PBMC? (Variables include serum concentration in extracellular media, temperature, time, and stimulation state of cells) Specific Aim 1b Upon optim ization of conditions at which to obser ve peak intracellular PI levels and efflux kinetics, the receptor expression profile for th ese PBMC was observed. This experiment was conducted to determine if the total number of R5 positive HIV-1 target cells within the lymphocyte population was high enough to sort, a nd employ peak intracellular PI and efflux kinetics studies as optimized above. To address this specific aim, the following questions were posed: What is the receptor expression profile for subs ets of T-lymphocytes for conditions that are optimal to observe intracellular PI levels in PBMC? The limiting factor is the R5 subset of HIV-1 target cells within the lymphocyte population. Will there be enough of this ly mphocyte subpopulation under the optimized conditions? Specific Aim 1c It was determ ined that the conditions at wh ich peak intracellular PI levels and efflux kinetics can be observed results in a total number of R5 positive HIV-1 target cells within the lymphocyte population which may be too low to so rt and subsequently employ efflux and peak intracellular PI studies. Alte rnative approach was employed using a Rhodamine-123 assay (Rh123) to observe p-gp activity within subsets of CD4+ T lymphocytes. A specific question was posed as a means to determine an alternative approach, ultimately culminating in the use of Rh123 collaborative study, as follows: If the cell number is too low to obtain enough of the R5 subset, what alternative approaches can be employe d to test the hypothesis?

PAGE 35

35 Significance Suboptim al levels of ART is an ongoing problem in treating HIV-1 infection and presents obstacles including emergence of resistant mutations and clearance of virus from infected cells with low bioavailability. 1,6,9,10,21,40,43,76,77,81 Understanding dynamics of virus/host/drug interaction and identifying mechanisms for decrea sed bioavailability of ART can lead to design of novel therapeutics to increase intr acellular concentr ations of drug.

PAGE 36

36 Figure 1-1. Global distribution of HIV-1 by groups, subtypes, and recombinant forms, organized by geographical location. Group M (major) c ontains subtypes A-K. Two new groups, N (new) and O (outliner), have been id entified in Africa and Eastern Europe 29,92.

PAGE 37

37 Figure 1-2. Structure of HIV-1 virion. HIV-1 is comprised of a nucleocapsid core, which contains two copies of the viral genome, core proteins, and reverse transcriptase (RT). The viral envelope, surrounds the core and consists of transmembrane gp41), and the surface subunit (gp120) 29, 45,92,100. Figure 1-3. Genomic organization of HIV-1. Two identical long te rminal repeats (LTR; purple) flank the viral genome, which includes the main genes gag, pol and env (blue), and accessory genes vif vpr, vpu tat rev and nef (red) 29, 45,92,100. This figure was obtained from Goodenow Lab Shar e drive and is unpublished.

PAGE 38

38 Figure 1-4. HIV-1 Life Cycle. Attachment of HIV-1 to host cell via CD4 and coreceptor (1); fusion; release of viral RNA into the cytoplasm of the host cell (2); Reverse Transcription (3); Integra tion (4); translation of ge ne products (5); budding and assembly at plasma membrane and pr ocessing by HIV-1 PR producing a mature virion (6) 27, 29, 45,92,100. This figure was obtained from Goodenow Lab Share drive and is unpublished.

PAGE 39

39 Table 1-1. Summary of HIV-1 coreceptor use and tropism. Tropism and coreceptor phenotype designation of virus appears in left column. Target cell coreceptor use appears in middle column. Target cell tropis m appears in the far right column8, 14, 17,29,32,45, 48, 49, 80. This table was created by Christin a Gavegnano in powerpoint and is not published.

PAGE 40

40 Figure 1-5. Mechanism of action for HIV-1 fusion inhibitor T20. Without fusion inhibitor, virus engages CD4 receptor followed by coreceptor and virus/host cell fusion is subsequently mediated (left panel). T20 pr events formation of intermediate hairpin structure required for fusion, thus preventi ng fusion of virus with host cell membrane (right panel) 11, 29,111,116. This figure was created fr om scratch in powerpoint by Christina Gavegnano and is not published.

PAGE 41

41 Figure 1-6. Mechanism of action for HIV-1 Reve rse Transcriptase Inhibitors. NRTI compete with endogenous nucleotides for incorpor ation into growing viral DNA strand, but lack a 3hydroxyl terminus. (B) Incor poration of NRTI into growing DNA strand results in termination of DNA strand (the next phosphodiester bond is not formed). (C) NNRTI bind directly to RT, inhibiting its enzymatic activity. 29,100,116 This figure was created from scratch in powerpoint by Christina Gavegnano and is not published.

PAGE 42

42 Figure 1-7. Structure of HIV-1 Protease with Proteas e Inhibitor bound. PI target HIV-1 PR, an aspartic protease comprised of two id entical 99 amino acid monomers. PI are competitive inhibitors, which compete for binding in the active site with the natural substrate. Once bound, PI cannot be cleaved, resulting in inactivation of the enzyme. Backbone (green ribbon), active sites (red), and non active s ites (blue). PI is bound to the active site (pink) 25-27, 29,100,116. This figure was obtained from the Goodenow lab share drive and is unpublished.

PAGE 43

43 Figure 1-8. Route of orally admi nistered HIV-1 Protease Inhibitors HIV-1 PI are administered orally and are highly metabolized in the in testinal lumen by cytochromes. Upon entry into the circulating peripher y, PI are highly bound to plasma proteins, which inhibits their ability to enter cells. Depending on the PI, 2-32 % of initial dose of PI is available to enter cells. 1,2,4,5,6,9,12,18-22,35,37,40,42,44,51,56,57,60,63-66,68,7073,76,77,81,93,94,98,100,104,106,107,109,112 This figure was created from scratch by Christina Gavegnano using powerpoint and is unpublished.

PAGE 44

44 Table 1-2. Efflux transporters fo r which PI are substrates and their expression in HIV-1 target cells. P-glycoprotein (p-gp/MDR1), MRP1, MRP2, MRP5 and BCRP are expressed in lymphocytes. P-gp, MRP1, MRP4, MRP5 are expressed in macrophages. 1,2,4,5,6,9,12,18-22,35,37,40,42,44,51,56,57,60,63-66,68,70-73,76,77,81,93,94,98,100,104,106,107,109,112 This table was created from scratch in powerpoint by Christina Gavegnano and is unpublished.

PAGE 45

45 Table 1-3. Methods to assess efflux transporter expression or activity and intracellular bioavailability of drug. Assays are listed in left column. Whether the assay is a direct or indirect measure of effl ux transporter expression or in tracellular drug levels is listed in middle and right columns, respectiv ely. This figure was created from scratch by Christina Gavegnano in powerpoint and is not published. 1,19-22,30,4144,56,57,59,60,63,65,66,68,71,73,77,84,93,97,98,106,107,112

PAGE 46

46 Figure 1-9. Effect of PI on m echanism of Rhodamine-123. (A) Rh-123 passively diffuses into cells and can be effluxed by p-gp. (B) PI bi nd with greater affinity to p-gp than Rh123, therefore PI are preferentially effl uxed by p-gp, and Rh-123 accumulates inside the cells in a ratio that is proportional to the amount of PI that has been effluxed by pgp. 68 This figure was created from scra tch by Christina Gavegnano in powerpoint and is not published.

PAGE 47

47 Figure 1-10. Viral and immune responses to HIV-1 antiretroviral th erapy. Median log10 viral RNA copies/mL (Y axis) versus weeks (X axis) (A). Median CD4 T-cell counts per microliter (Y axis) versus weeks (X axis )(B). Upon ART treatment, VFIF viral loads do not decrease and CD4 T-cell counts do not increase; VSIS viral loads decrease and CD4-T cell counts rebound; VFIS viral loads transiently decrease but rebound to levels similar to pretherapy and CD4 Tcell counts rebound. This figure was created by Sarah K. Ho and is unpublished. 39,100, 116,118

PAGE 48

48 Figure 1-11. Significantly more drug is required to inhibit HIV-1 replication for viruses targeted to otherwise identical viruses targeted to R5 versus X4 expressing T-lymphocytes. IC50 curve taken from day 8 of 10 day infec tion for LAI (TX4 or MR5 envelope as indicated) pretherapy or postth erapy variants in the presen ce of RTV (panel A, B) or IDV (panel C, D) in CXCR4 or CCR5 e xpressing PBMC. More drug is required to inhibit X4 posttherapy relative to X4 pretherapy variant (A, filled circles, open circles, respectively), and mo re drug is required to i nhibit posttherapy R5 variant relative to R5 pretherapy vari ant (B, filled squares, open squares, respectively). More drug is required to inhibit X4 posttherapy relative to X4 pretherapy variant (C, filled circles, open circles, respectively), and mo re drug is required to inhibit posttherapy R5 variant relative to R5 pretherapy va riant (D, filled squares, open squares, respectively). Data represent mean and SEM of three independent donors. This figure was created by Sarah K. Ho and is unpublished.

PAGE 49

49 CHAPTER 2 MATERIALS AND METHODS Determination of Peak In tracellula r PI Levels This experiment was performed to answer part of specific aim 1a, question 1. PBMC for this study were prepared by Ficoll (Histopaque 1077; Sigma Diagnostics, St. Louis, Mo.) gradient density centrifugation from leukopack obtained from LifeSouth/Civitan Blood (Gainesville, FL). PBMC were re suspended at a concentration of 106 cells/mL and stimulated for 72 hr at 37oC, 5 % CO2, with 10 mg/mL phyto-hemagglutinin (PHA) (Sigma Diagnostics) in RPMI 1640 medium (Gibco/Invitrogen, Grand Is land, N.Y.) supplemented with 30 U/mL human interleukin-2 (Boehringer Mannheim), 2 mM Lglutamine and 100 U of penicillin per mL, 100 g of streptomycin (Gibco/Invitrogen) per mL, and 10 % heatinactivated fetal calf serum (Gibco/Invitrogen). Three days post-stimulation, 15 x 106 cells were incubated with 1, 10, and 100 M of the protease inhibitors IDV and RTV (obtained from NIH AIDS Reagent and Reference Program, Bethesda, MD) in duplicate in T-25 flasks (S tarstedt, St. Louis, MO) (at 37C, 5 % CO2). At 3, 18, and 48 hours post-treatment, 5 x 10 cells were removed from each flask for drug extraction. The cells were aliquoted into 15 mL conica l tubes (Starstedt) washed 3 times at 4oC in 5 mL of ice cold 1 X Dulbeccos Phosphate buffered saline (PBS, Gibco/Invitrogen), and centrifuged (Beckman Coulter, Fullerton, CA) at 700 x g for 6 minutes at 4C. Following the third wash, the cells were counted and extracted in 70% CH3OH overnight at 4C rotating, with a final concentration of 1x106 c/mL.

PAGE 50

50 The following day, the samples were spun at 700 x g for 6 minutes at 4C, and the supernatant fraction containing th e intracellular drug, was transf erred to a 1.8 mL cryovial and stored at -20C until shipped to collaborators for mass spectroscopy analysis. To minimize drug loss, all steps were performed within an hour at 4C. Liquid Chromatography (LC) followed by Mass Spectrometric analysis (for confirmation of LC readout) by Lane Bushman from collaborator Courtney Fletchers laboratory (University of Colorado) was performed on CH3OH extracts to determine intracel lular [PI] in each sample. For LC, a methods internal standard (RTV or IDV analog) was added to each sample. Sample was then dried, resuspended in mobile phase (in a blank matrix of cellular lysate in CH3OH), and injected into the system (Figure 2-1). The labeli ng/designation for all duplicate samples that were NOT forwarded to Colorado, both for these studies and all other intracellular PI studies is as follows: Date-concentration of extracellular drug -timepoint for efflux-temperature of efflux-cell type. For example, a sample obtained on January 1, 2005 treated with 1 M RTV, allowed to efflux for 10 minutes at 4oC in PBMC would read as follows: 20050101-1 M RTV-4oC-10 minutes-PBMC. All samples were stored in 81 place boxes in 1.5 mL cryovials at -80oC at the completion of each experiment. Efflux Studies: Intracellular Pr otease Inhibitor Experiments This experim ent was performed to answer part of specific aim 1a, question 2-4. PBMC were isolated from leukopack obtained from Life South/Civitan (Gainesville, FL) as described previously, and either PHA stimulated (10 g/mL PHA for 72 hours at 37oC) or immediately exposed to 1.0 M or 10.0 M RTV containing RPMI 1640 medium with 2.0 % or 10.0 % fetal calf serum supplemented with 2 mM L-glutam ine and 100 U of penicillin per mL, 100 g of streptomycin per mL. Cells were resu spended at a concentration of 1.0 x 106 cells/mL for 18

PAGE 51

51 hours at 37oC, 5 % CO2. U937 promonocytic cel l line (American Type Culture Collection, Manassas, VA) were treated analogously with th e exception that U937 were not treated with PHA before exposure to RTV containing medi a. After 18 hr. exposure to drug, 5.0 x 106 cells were aliquoted into 15 mL coni cal tubes, centrifuged at 4oC at 700 x g for 6 minutes to remove PI containing media, and washed th ree times in ice cold 1X PBS (4oC, 700 x g) to remove residual drug. Intracellular le vels of PI were determined at baseline (immediately after washings), 10, 20, 40, 60, and 120 minutes post washing at 4oC, 20oC, or 37oC. At each time point, cells were imme diately centrifuged (4oC, 700 x g) to remove residual drug. Efflux was then determined over the time course of base line (immediately after washings), 10, 20, 40, 60, and 120 minutes post washing at 4oC, 20oC, and 37oC. At each time point, cells were immediately centrifuged (4oC, 700 x g) for 6 minutes and resuspended in ice-cold 70% CH3OH for 4 hours at 4oC. Collaborator Lane Bushman of Cour tney Fletchers laboratory performed liquid Chromatography (LC) followed by Mass Spectro metric analysis (for confirmation of LC readout) (University of Colorado) to determine intracellular [PI] in each sample (Figure 2-2). A positive control sample was prepared by spiking 1 mL of 70 % CH3OH with RTV stock to a final concentration of 10 uM to confirm concentration of RTV stock used for the experiments. For LC, a methods internal standard (RTV analog) was added to each sample. Sample was then dried, resuspended in mobile phase (in a blank matrix of cellular lysate in CH3OH), and injected into the system (Figure 2-2). Concentration of ng/mL was converted to M by first converting ng/mL into g/mL and dividing this value by the MW of the PI to obtain mol. To determine the intracellular concentration of drug per cell, the mol value was divided by the volume of 1.0 x 106 cells, which is the total number of cells in 1mL. This cell number to volume ratio (1.0 x 106 cells/mL)

PAGE 52

52 corresponds to the concentration of cells in the sample where the intracellular PI was quantified. Therefore, using this value to determine M readout yields an intracellular PI value that correlates to the concentration of drug in each cell (Figure 2-3). For U937 and PBMC, standard error of the mean (SEM) and stan dard deviations were calculat ed to determine intraassay and interassay variability, respect ively (Sigma Stat 3.0). Receptor Expression Profile Study in CD4+ T lymphocytes This experim ent was performed to answer part of specific aim 1b, questions 5 and 6. CD4+ lymphocytes were isolated from leukopack obtained from Lifesouth/ Civitan (Gainesville, FL) using human CD4+ T cell enrichment cocktail (Stemcell Technology, Seattle, WA), which depleted cells expressing CD8, CD16, CD 14 (monocytes), CD36, CD56, and CD66b), and enriched for CD4+ T lymphocytes (Figur e 2-4). Cells were cultured at 1x106 cells/mL in RPMI 1640 and resuspended in either 2 % or 10 % heat inactivated fetal calf serum containing media, with or without PHA stimulation, and supplem ented with 2 mM L-glutamine and 100 U of penicillin per mL 100 g of streptomycin (Gibco/Invitrogen). At timepoints of baseline (immediately after isolation), 1, 3, and 5 days, cells were aliquoted into 50 mL conical t ubes (Starstedt), and centrifuged for 10 minutes at 1000 rpm, and residual media was removed. Cells were suspended in FACS wash [phos phate-buffered saline (PBS) containing 10% fetal calf serum, 2% normal human AB seru m (Sigma), and 0.02% sodium azide (Fisher Scientific)], and flurochrome conjugated monoc lonal antibodies specific for CD4, CD14, CCR5, or CXCR4, or isotype contro ls for 30 minutes at 4oC. Fluorescence-tagged monoclonal antibodies specific for CCR5 (CCR5-PE), CXCR 4 (CXCR4-APC), CD14 (CD14-FITC), and CD4 (CD4-PerCP) were obtained from BD Phar mingen (San Diego, CA), as were isotype

PAGE 53

53 matched control monoclonal antibodies. After in cubation, cells were subjected to centrifugation for 8 minutes at 1000 rpm at room temperature with cold FACS wash, and fixed in 1% ice-cold paraformaldehyde (Sigma). Coreceptor expression by subsets of CD4 T cells was observed via initial gate drawn around CD4+ population (R1). R1 was then assess ed for CD14 expression on a plot of CD14 versus forward scatter. R1 was also assesse d for either CXCR4 expr ession or CCR5 expression on a plot of coreceptor expressi on (either CXCR4 or CCR5) for the R1 population versus percent of cells. Gates for CD4+/CCR5+ and CD4+/CXC R4+ lymphocytes were established based on plot of isotype matched controls for either CXCR4 or CCR5. Data acquisition was performed using an FACS Vantage flow cytometer (Bec ton Dickinson) and Fl oJo Software, Version 7.0,Treestar, MD). Experiments were conducted in three independent donors, with technical duplicates per donor. Standard error of the mean (SEM) and standard deviation were determined using Sigma Stat 3.0. Determining p-gp activity and Inhibition of Rh odamine-1, 2,3 (Rh-123) Efflux by PIs Collaborator Matthew Morrow at University of South Florida, Sleasman laboratory, performed Rh-123 studies. This experiment was conducted to answer specific aim 1c, question 7. PBMC from fresh whole blood we re isolated using Histopaque density gradient centrifugation as described previously. PBMC were cultured at 1x106 cells/mL in complete RPMI-1640 media (10% fetal calf serum, 2 mM L-gl utamine, 100U/mL penicillin, and 100 g/mL streptomycin) were treated for 30 minutes at 37oC with 0.5 g/mL Rh-123 (Molecular Probes Invitrogen) either alone or in the presence of 10 M R-Verapamil (Sigma), 10 M (RTV), 10 M (IDV), or 10 M (NFV). After 30 minutes, the cells were transferred to fresh complete RPMI-1640 lacking Rh-123 and PI. The cells were cultured at 37oC in 5% CO2 and samples were collected at 1 and 2.5 hours time points for analysis of intra cellular levels of Rh-123 using flow cytometry.

PAGE 54

54 Cells were washed with 1X PBS at 4oC, and then stained for surface CD3 (CD3-), CD4 (CD4-), CD8 (CD8-), CCR5 (CCR5-), CD45RA (CD45RA-), and CD45RO (CD45RO-) using cold buffers to inhibit further Rh-123 efflux. All anti bodies were obtained from BD Biosciences (San Jose, CA). Data acquisition was performed usi ng an LSR-II flow cytometer (BD Biosciences), and data analysis was performed using FlowJo software, version 7 (Treestar, Portland, OR).

PAGE 55

55 Figure 2-1. Experimental design for pilot peak intracellular Protease Inhibitor study. PBMC were isolated from healthy donor and imme diately subjected to PHA stimulation for 72 hours. After 72 hours, 1, 10, or 100 M IDV, RTV, or NFV was added to extracellular media, and cells were harveste d at 4, 18, or 48 hours post addition of PI.

PAGE 56

56 Figure 2-2. Experimental desi gn for Protease Inhibitor efflux study. PBMC or U937 were treated with 1 or 10 M RTV containing media for 24 hours prior to removal of RTV containing media. Methanol extraction was subsequently performed on cells to obtain intracellular fr action of drug.

PAGE 57

57 Figure 2-12. Sample Conversion calculation from ng/mL to M PI Figure 2-3. Sample calculation for conversion of ng/mL to M for intracellular Protease Inhibitor experiments. Gram value wa s converted to mol, and mol value was converted to molarity using the equation mol/L, where L volume is determined by the volume of cells in the sample.

PAGE 58

58 Figure 2-4. CD4+ T cell enrichment procedure. Leukopack is subjected to antibody cocktail containing antibodies specific for monocytes (CD14), and depletion of all cells that do not express CD4. The antibodies bind to the unwanted cells, and subsequently cross-link with the erythrocytes. Upon cen trifugation, the unwanted cells are pulled to the bottom of the tube, whereas the enri ched population of desi red cells appears in a monolayer.

PAGE 59

59 CHAPTER 3 RESULTS Review of Hypothesis and Experiments To test the hypothesis that re quirem ent for significantly more drug for otherwise identical viruses targeted to R5 versus X4 expressing Tlymphocytes is related to bioavailability of intracellular PI, three specific aims (1a, 1b, 1c) were conducted, which were delineated into seven distinct questions. Specifi c aim 1a sought to determine the optimal conditions at which to observe peak intracellular PI levels and efflux ki netics in CD4+ T lymphocytes. This specific aim was addressed with the following questions: Published data demonstrates that peak intrace llular PI levels in PB MC are conferred by 18 hours extracellular PI treatment at 37oC. Can we recapitulate similar values in our laboratory? What is the inter-assay and intra-assay variability for this study? What are the optimal conditions to observe ba seline intracellular PI levels above limit of detection in PBMC? (Variables include seru m concentration in extracellular media and stimulation state of cells) What are the optimal conditions to observe e fflux kinetics of PI in PBMC? (Variables include serum concentration in extracellular media, temperature, time, and stimulation state of cells) Specific aim 1b sought to determine if there ar e enough of the R5 HIV-1 target cells within PBMC, and to determine the recepto r expression profile of these ce lls. To address this specific aim, the following questions were posed: What is the receptor expression profile for subs ets of T-lymphocytes for conditions that are optimal to observe intracellular PI levels in PBMC? The limiting factor is the R5 subset of HIV-1 target cells within the lymphocyte population. Will there be enough of this ly mphocyte subpopulation under the optimized conditions?

PAGE 60

60 Specific aim 1c was employed as an alternative approach to sorting cells, direct stain for efflux transporter expression, or other potential experiments discu ssed in the introduction of this thesis. A specific question was posed as a m eans to determine an alternative approach, ultimately culminating in the use of Rh-123 collaborative study, as follows: If the cell number is too low to obtain enough of the R5 subset, what alternative approaches can be employe d to test the hypothesis? Question 1: Confirmation of Ability to Observe Peak Intracellular PI Levels in PBMC Intracellular bioavailability of drug is fr equently determined by High Performance Liquid Chromatography (HPLC) with tandem mass spectrome tric analysis to confirm the LC readout 1, 30. Previous literature has established that peak intracellular PI levels are conferred in total PBMC upon treatment with PI co ntaining media for 18 hours at 37oC. 1, 20,40,41,43 Before further efflux studies were performed via these methods, recapitulation of analogous experiments in the literature was performed to confir m ability to observe similar results, and to confirm that 18 hours of PI treatment yields p eak intracellular PI levels. Total PBMC were treated with 1, 10, or 100 M IDV or RTV after 72 hr. PHA stimulation in the absence of drug, and sample s were taken at 4, 18, and 48 hours post addition of drug. For PBMC treated with 1 M IDV, intracellular IDV levels were ~3.0 M at 4 hours and 18 hours, and decreased to ~ 2.0 M at the end of the timecour se. For PBMC treated with 10 M IDV, intracellular IDV levels were 13 M at 4 hours and remained constant for the remainder of the timecourse. For PBMC treated with 100 M IDV, intracellular IDV levels were 106 M at 4 hours, increased to 126 M at 18 hours, and remained constant through the remainder of the timecourse (Figure 3-1). For PBMC treated with 1 M RTV, intracellular RTV levels were 2.3 M at 4 hours, increased to 5 M at 18 hours, and decreased to 1.1 M at the end of the timecourse. For PBMC

PAGE 61

61 treated with 10 M RTV, intracellular RTV levels were ~ 20 M at 4 hours and 18 hours, and decreased to 12 M by the end of the timecourse For PBMC treated with 100 M RTV, intracellular RTV levels were 839 M at 4 hours, increased to 2.1x10 M at 18 hours, and decreased to ~ 900 M by the end of the timecourse (Figur e 3-1). It should be noted that technical replicates could not be performed, as the size of the study dictated use of an entire leukopack, and that this pilot study was conducte d in one donor. Kinetics for IDV at all concentrations, and for RTV at 1 and 10 M follow that of reported findings, however the kinetics for PBMC did not (Figure 3-1). Five out of six parameters tested were analogous to published findings relative to timeframe at which peak intracellu lar PI levels conferred (~18 hours), and the actual intracellular M values observed. It is hypothesized that the ki netics reported in this pilot experiment for 100 M RTV treatment is not correct and may have en countered error. Exam ples of error include pipetting error, misreading of LC readout by the operator of the machiner y, loading the incorrect sample into the LC for readout, or insufficien t resuspension of sample before readout. The pilot experiment conducted to answer question 1 posed in this thesis, in tandem with published literature regarding toxicity of PI in PBMC determined that 18 hours of extracellular PI treatment at 1, 10, or 100 M PI confers peak intracellular PI levels. Specifically, intracellular PI levels are lower at timepoints observed before and after the 18-hour ti mepoint. Timepoint of 18 hours to confer peak intracellu lar PI levels in PBMC is also in concert with published literature 1, 20,40,41,43. Specific points regarding toxicity and relationship between data obtained in this thesis to published findings and toxicity are reported in the di scussion section of this thesis. Relative to optimal conditions to be used for subsequent efflux studies, parameters are defined by the following parameters:

PAGE 62

62 Ability to observe intracellular PI levels above the limit of detection (which for efflux studies is ~ 1 M) Time of extracellular PI treatment that confers peak intracellular PI levels Concentration of extracellular PI that is not toxic to the cells This pilot study confirmed that intracellular PI levels for IDV and RTV in PBMC can be observed above the limit of detection for 1, 10, and 100 M IDV or RTV treatment, and that the peak intracellular PI levels are conferred upon 18 hours extracellular PI treatment. Toxicity studies in the literature demonstrate th at this range of concentrations (1-100 M IDV or RTV) is not toxic to the cells and does not induce apoptosis. The conclusion from question 1 is that all future studies with PI and PBMC will involve ex tracellular PI treatment w ithin the concentration ranges 1-100 M. This experiment should be repeated in multiple independent donors to confirm statistical significance and ability to recapitulate published findings. Question 2: Assessing Intra and Inter-assay Variability Upon confirmation that the employed techni que yields similar re sults to published findings for intracellular PI levels over time, a nd that 18 hours of PI treatment yields peak intracellular PI levels, the next step was to perform a study to determine intra and inter-assay variability. This was achieved us ing U937 promonocytic cell line. U937 cells were treated with 1 or M RTV for 18 hours at 37oC, and intracellular PI levels were quantified using the methods described above. Techni cal duplicates or triplicates were performed within each expe riment, and the experiment was conducted in duplicate. The baseline intracellular PI levels ( M values immediately after 18 hours of extracellular 1 M RTV treatment at 37oC) are reported as follows: Mean M value within one experiment (intra-assay variability) was 3.2 M (SEM 0.67), and the mean across experiments (inter-assay variability)

PAGE 63

63 was 4.0 M (Standard Deviation 1.5). The conclusion from question 2 is that both intra and inter-assay variability are low (Figure 3-2). Question 3: Determining Optimal Conditions to Observe Baseline Int racellular PI Levels Above Limit of Detection in PBMC Question 1 was designed to test ability to recapitulate published findings, but also determined that with 1 M extracellular PI trea tment for 18 hours at 37oC in 10 % serum with stimulated PBMC, intracellular PI levels are above the limit of detection. As question 1 represents a pilot experiment, and was conducted in a single donor, quest ion 3 tested two donors and assessed two different crite ria that can impact intracellu lar PI levels. Conducting the experiment across multiple donors and with different variables that may affect intracellular PI levels assures that optimal parameters will be determined, and that they will allow for intracellular levels above limit of det ection, independent of donor tested. PBMC were cultured with either 2 % or 10 % se rum containing media, and with or without 72 hour PHA stimulation. 1 M RTV was applied to cultures for 18 hours at 37oC either immediately after PBMC isola tion (for non-PHA stimulated PBMC), or after 72-hour PHA stimulation (for PHA stimulated PBMC). Baseline intracellular PI levels (immediately after 18 hour load of cells with PI) for stimulated PBMC in 2 % serum and 10 % serum were 4.7 M (SEM 0.2) and 7.6 M (SEM 1.6), respectively (Figure 3-3 B). Baseline intracellular PI levels (immediately after 18 hour load of cells with PI) for nonPHA-stimulated PBMC in 2 % serum and 10 % serum were 6.6 M (SEM 1.9) and 4.9 M (SEM 0.2), respectively (Figure 3-3 A). The conclusion from question 3 is that stimulation state of cells and concentration of serum in ex tracellular media has little effect on baseline intracellular PI levels. Experiment should be repeated in a minimum of three independent

PAGE 64

64 donors, with replicates per donor, to confirm statistical significance and ability to observe results across donors. Question 4: Optimal Conditions to Observe Effl ux Kinetics of Protease Inhibitors in PBMC The next step was to determ ine the optimal conditions at which to observe efflux of drug. Multiple parameters can affect efflux of drug from cells including temperature, stimulation state of cells, and time. 1,5,6,18,19,20,30,42-44 Upon establishment that peak intracellular PI levels can be observed for stimulated and unstimulated PBMC, with 2 % or 10 % serum in extracellular media treated with RTV, the next experiment was designed to determine how serum concentration, PHA stimulation, and temperature of efflux affect effl ux kinetics of PI in total PBMC Temperature affects efflux, as ATP-dependent efflux machinery is active at 37oC, and inactive at 4oC. Stimulation of cells has been linked to increased efflux transporter expr ession, resulting in incr eased ability to efflux intracellular drug. Time affects efflux, as exposure of cells to inhibitory efflux conditions (4oC) will initially result in inhibition of efflux (via inhibition of ATP-dependent efflux machinery). Cells loaded with drug will, as a function of time, begin to passively diffuse drug over time, independent of temperature. 1,5,6,18,19,20,29,42-44 Optimizing these conditions would allow for determination of which combination of serum, stimulation, and efflux temperature would be st facilitate observing differences in baseline as well as efflux kinetics in subsets of PBMC Stimulated PBMC were treated with media containing 1 or 10 uM RTV and 10 % serum for 18 hrs. After 18 hr. RTV treatment, efflux was observed at 4oC or 37oC at baseline (immediately af ter 18 hour RTV load at 37oC), 10, 20, 40, and 60 minutes. Data for 10 M RTV is n=1, and 1 M RTV is n=2 with technical duplicates, however only one sample was sent to Denver, CO., for analysis. Independent of extracellular

PAGE 65

65 concentration of RTV applied, th e majority of drug was retained intracellularly throughout the timecourse at 4oC (Figure 3-4, blue circles, red circles), while nearly all PI effluxed to levels below limit of quantitation (Below Limit of Quantitation [BLQ]; ~ 1 uM) within the first ten minutes at 37oC (Figure 3-4, blue triangles red triangles). These findings demonstrated that RTV was able to enter cells, as baseline values were 7.6 M (+/-1.6 M) and 12.2 M (+/-3.4 M) for 1 or 10 uM RTV treatment respectively (Fi gure 3-5), but that effl ux occurs very rapidly as a function of 37oC temperature (Figure 3-4, blue triangles, red triangles). Treatment of stimulated PBMC with 1 uM RT V in 10 % serum did not yield conditions at which efflux can be observed above limit of det ection, therefore parameters were modified. A study was then performed with a reduced serum c oncentration (2 %) and performed the efflux at an intermediate temperature (20oC) as well as 4oC and 37oC, using stimulated and unstimulated PBMC treated with 1 uM RTV for 18 hrs. As a control, condition of 10 % serum was tested again. Data for 2 % serum is n=1. All data including baseline intracellular levels were BLQ. Hypothesized reasons for error and discussion regarding these possible errors have been removed from this thesis. For experiment with 2 % serum, as expected, nearly all PI was maintained intracellularly throughout the timecourse independent of stimula tion criteria for cells effluxed at 4oC (Figure 3-6 A, B, short, green lines) with the exception of the 120 minute timepoint for stimulated PBMC. The 120-minute timepoint intracellular level proved to be irrelevant, as nearly all RTV was effluxed from the cells within 10 minutes at 37o C for stimulated PBMC (Figure 3-6 A, blue lines, filled circles) and unstimulated PBMC (Fi gure 3-6 B, blue lines, filled tr iangles). For cells effluxed at 20oC, ~ 50 % of RTV was effluxed within the first ten minutes and RTV had effluxed BLQ by 120 minutes for unstimulated PBMC (Figure 3-6 B, red line, filled triangle). For stimulated

PAGE 66

66 PBMC effluxed at 20oC, ~ 15 % of RTV had effluxed by the 10 minute timepoint, whereas nearly all RTV had effluxed by the 20 minute time point and remained BLQ for the remainder of the timecourse (Figure 3-6 A, red line, filled triangles). BLQ is ~ 1 M. Data are n=1 with technical duplicates/donor, but only one replicate was sent to Denver, CO., for analysis. No follow up experiments were performed to confir m efflux pattern of RTV in PBMC resuspended in 2 % serum in other independent donors. The conclusion drawn from question 4 is that optimal conditions to observe baseline intracellular levels and efflux kinetics for PBMC include use of unstimulated PBMC maintained in 2 % serum and effluxed at 20oC, but that experiment should be repeated in a minimum of three independent donors, with replicates per donor to confirm statistical significance and ability to observe results across donors. Question 5: Receptor Expression Profile For S ubsets of T-lymphocytes in Optimal Conditions to Observe Intracellular Protease Inhibitor Levels in PBMC Question 4 determined that the optimal paramete rs to observe intracellu lar PI levels are in unstimulated PBMC in 2 % serum. A study was performed in three i ndependent donors with technical duplicates/donor, howev er data from only one rep licate was saved and analyzed (summary of data for all donors in Table 3-1) to determine the receptor expression profile of CD4, CXCR4 and CCR5, within a population of unstimulated lymphocytes enriched for CD4 expression and depleted of CD 14 expressing cells (monocytes, which also express CD4). In the CD4 enriched population CD4 enriched T lymphocytes were subjected to gating on CD4, demonstrating that ~ 80 % of enriched cells were CD4 positive (Figure 3-7 A). A gate was drawn around these CD4 positive cells, and that population of cells was subjected to CD14 gate, demonstrating that zero percent of CD4 positive cells were CD14 positive (Figure 3-

PAGE 67

67 7 B). This demonstrates that none of the CD4 positive cells are monocytes, which express CD14. Next, the CD4 positive population was subjected to gate to determine percentage of CXCR4+, CCR5+, and CXCR4+/CCR5+ cells. Gate was established based on isotype matched controls. It was determined that CXCR4+/CD 4+ lymphocytes represent ~ 80 % of the CD4+ lymphocyte population (Figure 3-7 C). CCR5+/CD4+ lymphocytes represent ~ 20 % of CD4+ lymphocyte population (Figure 3-7 D), and that the CCR5+ cells are dim, and do not display significant delineation between CCR5+ and CCR5subpopulations (Figure 3-7 D). ~ 15 % of the CD4+ lymphocytes were CD4+/CXCR4+/CCR5+ (Figure 3-7 E). Data are representative from one of three independent donors tested. All three donors displayed va lues that are within two standard deviations of the mean. All values obtained were +/7 % of each other. Summary of data from the three donors te sted appear in Table 3-1. Pilo t study to increas e intensity of CCR5 signal with FASER kit (Miltenyi Biotec, Auburn, CA), which provides an excellent foundation to determine methods to obtain di stinct CCR5+ population, was not performed. Further studies to modify stimulation and media conditions of cells to increase CCR5 expression, and decrease double positive X4+/R5+ subpopulations were not performed. Question 6: Are There Enough CCR5 Cells to Perform Efflux Study in Unstimulated PBMC? As CCR5 ex pression is low in unstimulated PBMC it was mathematically calculated to be possible, but decided to be unfeasible, to obt ain the necessary number of cells from one leukopack of freshly isolated, non PHA-stimulated PBMC and examine efflux differences between R5 and X4 cells. In addition, CCR5 positive lymphocytes display dim receptor expression, which presents significant difficu lty in sorting this population of cells, and differentiating this population of cells from isotype or from CXCR4+ cells, impeding ability to

PAGE 68

68 set gate around population of CCR5+ cells with confidence. Pilot study to increase intensity of CCR5 signal with FASER kit (Miltenyi Biotec, Auburn, CA), which provides an excellent foundation to determine methods to obtain di stinct CCR5+ population, was not performed. Further studies to modify stimulation and media conditions of cells to increase CCR5 expression, and decrease double positive X4+/R5+ subpopul ations were not performed. One leukopack can yield ~ 7.0x108 PBMC, of which ~ 50 %, or ~ 5.6x108 are CD4+ lymphocytes, and of which ~7.0x107 cells are CD4+/CCR5+/CXCR4-. For one efflux study, ~5.0x106 cells/condition is a minimum requirement. Twelve conditions are required for efflux study (baseline, 10, 20, 40, 60, and 120 minute time points) as well as counter cells for 4oC and 37oC (tubes which are treated analog ously to all other cells, but whose sole purpose is to obtain an accurate cell count at the c onclusion of the study). Thus, 14 parameters are necessary, totaling ~ 7.0 x 107 CD4+/CCR5+/CXCR4lymphocytes. In or der to obtain these target cells, a live gate cell sort must be conducted. These calculations represent a c onservative calculation, and experiments to study smaller total number of cells for efflux study, and to determine if 7.0x107 could be feasible for efflux studies we re not conducted. Antibodies for CD4, CCR5, CXCR4, and CD14 must be used. To stain the ~ 5.6 x 108 CD4+ lymphocytes isolated with each of these markers would total ~ $2,000 ($260/an tibody X 6.5 total bottles of Ab (1.3 bottles of anti-CD4+ 1.3 bottles of anti-CXCR4 + 1.3 bo ttles anti-CCR5 + 1.3 bottles of anti-CD14 + 1.3 bottles-isotype matched controls) + ($50/hr flow cytometer X 3.5 hours at flow rate demonstrated to not induce apoptosis in lymphocytes ). Viability of sorted cells was not tested, and the expectation is that not 100 % if cells w ould be viable after undergoing sort. The number of calculated R5 target cells not accounting for loss of unviable cells af ter the sort is ~20-40 million cells more than the threshold requireme nt for the efflux study. Although the method of

PAGE 69

69 sorting coupled with subsequent efflux study co uld have mathematically provided enough cells to perform the efflux study, this idea was termin ated in favor of alternative method (Rh-123 assay) described below. Further studies to obtain a flurochrome whic h does not significantly overlap with isotype, presenting brighter CCR5 receptor expression prof ile, coupled with additi onal studies including viability assessment over time of sorted cells and repetition of study in multiple independent donors were not conducted. Studies including modification of stimul ation criteria for cells and media conditions that may decrease double positi ve X4+/R5+ lymphocytes were not performed, but present an excellent foundation from which to launch further studie s to obtain distinct populations of X4 and R5 HIV-1 target cells w ithin the lymphocyte population. Due to these limitations/stringencies, an alternative approach was employed to observe p-glycoprotein activity within populations of cells repres enting the X4 and R5 HIV-1 targ et cells within the lymphocyte population via flow cytometry. Question 7: Alternative Approach and Rh-123 Assay Many alternative methods and approaches co uld have been employed as an alternative method, as discussed in the introduction, and addr essed in the discussi on. The Rh-123 study was chosen as an alternative appro ach to sorting subpopulations of HIV-1 target cells within the lymphocyte population. As part of a collaborative study, the experiments in this section were performed by Matt Morrow in th e laboratory of Dr. John Sleasma n, University of South Florida. Rh-123 can be fluorescently detected via FL1 and is a substrate for p-gp68. Therefore, cells that are p-gp negative will retain Rh-123 (Rh-123 bright ) whereas cells that express p-gp will efflux Rh-123 (Rh-123 dim). Rh-123 provides a mechanism to observe p-gp activity and was used to assess p-gp activity in subsets of HIV-1 target cells within the lymphocyte population. Rh12-3

PAGE 70

70 in tandem with PI treatment to cells was then em ployed to confirm that PI are substrates for p-gp, and that this could be observed in the eight-color flow analysis that was used in this study. P-gp Activity in Lymphocyte Subsets To explore p -gp activity as a potential mechanism for differences in PI levels required to inhibit replication in R5 versus X4 lymphocytes, Rh-123 efflux wa s assessed in several subsets of lymphocytes. Initial gates were established to determine Rh-123 fluorescence in total PBMC and demonstrated heterogeneous p-gp activity wi thin the lymphocyte popul ation (Figure 3-8). Further gating on subpopulations of lymphocytes wa s then established. CD4+ lymphocytes, NK cells (CD16+/CD56+), and CD8+ T cells. Tota l PBMC, CD4+ Tlymphocytes, and CD8+ T lymphocytes contain cells with heterogeneous p-gp expression, and NK cells display high p-gp expression (highest of any known human cell) (Figure 3-9.1 A, B, C). Separate gates were established on total PBMC to obtain CD3+/CCR5+ and CD3+/CCR5cell populations (Figure 39.2 D). Further gating was established to obtain CD45RA+/and CD 45RO+/lymphocytes within the CD3/CCR5 positive and negative populations (Figure 3-9.2 E, F). Each of these subsets represents either th e X4 or R5 HIV-1 target cells within the lymphocyte population. A subset of cells representing the R5 HIV-1 target cells demonstrated the lowest Rh-123 fluorescence relative to all other subsets tested (Figure 3-9.2 F, circled population). As Rh-123 fluorescen ce is inversely proportional to p-gp activity, this result indicated high p-gp expression analogous to NK ce ll p-gp expression in this population of cells relative to all other subsets of lymphocytes tested. This expe riment was conducted in one donor without technical replicates. Protease Inhibitors are Substrates for p-gp The next goal was to de monstrate that IDV and RTV are substrates for p-gp within the confines of our experimental conditions, thus establishing that p-gp levels correlate with

PAGE 71

71 intracellular PI levels. Although published literature defines this fact, no study has addressed this question with eight-color analysis. Therefore, confirmation of the ability to recapitulate this observation with these additional parameters was performed. Rh-123 activity was assessed in the presence of IDV, RTV, and known inhibitor of p-gp, R-Verapamil. Molecules with higher affinity for p-gp than Rh-123 will displace Rh-123 and thus preferentially efflux from cells. Therefore, p-gp positive cells in the presence of Rh-123 and substrate of higher affinity than Rh-123 will be Rh-123 bright. CD3+/CD4+/CCR5+ lymphocytes subsequently gated on CD45RA and CD45RO expression were loaded with Rh-123 for 30 minutes (Figure 3-10 A, shaded), allowed to efflux for 1 hour at 37oC after Rh-123 load (Figure 3-10 A, red), allowed to efflux for 1 hour in the presence of 10 M R-Verapamil, IDV, or RTV (Figure 3-10 A, blue). Treatment with R-Verapamil, IDV or RTV was sufficient to inhibit Rh123 efflux in all populations of cells (F igure 3-10 B). These findings demonstrated that IDV and RTV are substrates for p-gp and that this interacti on can be defined with a complex panel of fluorochromes. Together, these findings further validate the hypothesis that differential p-gp activity directly correlates wi th variable levels of PI found w ithin lymphocyte subsets. This experiment was conducted in one do nor without technical replicates This experiment must be repeated in multiple independent donors to confir m statistical significance and ability to observe results across donors.

PAGE 72

72 Figure 3-1. Peak intracellular PI le vels in PBMC treated with 1, 10, 100 M IDV or RTV. Peak intracellular PI levels were conferred upon ~ 18 hours extracellular PI treatment for both IDV and RTV, independent of extrace llular concentration a pplied (A, B, C). This experiment represents n=1, wit hout technical replicates. Blue = 1 M PI tx, red = 10 M PI tx, green = 100 M PI tx.

PAGE 73

73 Figure 3-2. Intra-assay and inter-a ssay variability in PI efflux study. U937 were treated with 1 M RTV, and baseline intracellular PI levels were observed after 18 hours extracellular PI treatment. This figur e contains data from two independent experiments with technical duplicates or trip licates per experiment. Error bars for the data on the left bar indicate SEM and erro r bars for the data on the right indicate standard deviation. Mean M value within one experiment (intra-assay variability; left bar) was 3.2 M (SEM 0.67), and the mean across experiments (inter-assay variability; ri ght bar) was 4.0 M (Standard Deviation 1.5), and all values obtained fell within two standard deviations of the m ean, demonstrating that both variability is low.

PAGE 74

74 Figure 3-3. Baseline intracellula r RTV levels. (A) For unstimulated, (B) for PHA stimulated PBMC treated with 1 M RTV treatment for 18 hours at 37oC in 2 % or 10% serum. Baseline intracellular PI levels (immediately after 18 hour load of cells with PI) for non-PHA-stimulated PBMC in 2 % serum and 10 % serum were 6.6 M (SEM 1.9) and 4.9 M (SEM 0.2), respectively (A). Baseline intracellular PI levels (immediately after 18 hour load of cells with PI) for stimulated PB MC in 2 % serum and 10 % serum were 4.7 M (SEM 0.2) and 7.6 M (SEM 1.6), respectively (B). Data from 10 % serum is from two donors, with technical duplicates/donor. Data from 2 % serum is from one donor, with technical duplicates /donor. Error for data indicates SEM.

PAGE 75

75 Figure 3-4. Efflux of RTV in stimulated PB MC. Nearly all RTV remained intracellular throughout the timecourse for cells maintained at 4oC (filled circles), whereas nearly all RTV was effluxed from the cells within 10 minutes at 37oC (filled triangles), independent of extracellular concentration of RTV app lied. Red lines indicate 10 M extracellular RTV treatment whereas blue lines indicate M RTV extracellular treatment. Data for 1.0 M RTV is representative from one donor of two donors tested. Data from the se cond donor treated with 1.0 M were all BLQ. Data for 10.0 M RTV is n=1. Technical duplicates/donor were conducted, but only one replicate was sent to Denver, CO., for analysis. BLQ is ~ 1 M.

PAGE 76

76 Figure 3-5. Baseline intracellular RTV levels from Protease Inhibitor efflux study. Although nearly all RTV had effluxed from cells within 10 minutes at 37oC, baseline intracellular RTV levels were significantly above the limit of detection. For 1 M RTV treatment, intracellular RTV levels were nearly 8 M, and for 10 M RTV treatment, intracellular levels were ~ 12 M (left bar, right bar, respectively). Data are from two independent donors, with tec hnical duplicates/donor. Error represents SEM.

PAGE 77

77 Figure 3-6. Efflux of RTV in stimulated or unst imulated PBMC in 2 % serum. Nearly all RTV remained intracellular throughout the tim ecourse for cells maintained at 4oC for stimulated PBMC (A, green lines, filled circles) and unstimulated PBMC (B, green lines, filled triangles) with the exception of the 120 minute timepoint for stimulated PBMC which was BLQ. Nearly all RTV was effluxed from the cells within 10 minutes at 37o C for stimulated PBMC (A, blue lin es, filled circles) and unstimulated PBMC (B, blue lines, filled triangles). For efflux at 20oC, ~ 50 % of RTV was effluxed within the first ten minutes a nd RTV had effluxed BLQ by 120 minutes for unstimulated PBMC (B, red line, filled tria ngle). For stimulated PBMC effluxed at 20oC, ~ 15 % of RTV had effluxed by the 10 minute timepoint. Nearly all RTV had effluxed by the 20 minute timepoint and remained BLQ for remainder of the timecourse (A, red line, filled triangles). BLQ is ~ 1 M. Data are n=1 with technical duplicates/donor, but only one replicate was sent to Denver, CO., for analysis.

PAGE 78

78 Figure 3-7: Receptor expression profile for CD4 enriched lymphocytes. CD4 enriched population was gated on CD4 expression (A). ~ 80 % of enriched cells were CD4 positive. CD4 positive lymphocytes were then subjected to gate on CD14 (B). None of the CD4 enriched cells expressed CD14 (monocytes) (B). CD4 positive lym phocytes were subjected to gate on CXCR4 (C), CCR5 (D), or dual plot for CXCR4/CCR5 (E). Percent of CD4/CXCR4 cells is ~ 75 % of enriched population, and percent of CD4/CCR5 cells is ~ 20 % of enriched population. Percent cells positive for both CXCR4 and CCR5 is ~15 % (E). All gates were established for isotype matched controls. Data are representative from one of three independent donors tested. All three donors displayed values that are within two st andard deviations of the mean, indicating statistically insignificant difference in values obtained. Summary of data from the three donors tested appear in Table 3-1.

PAGE 79

79 Table 3-1: Receptor expression profile for CD 4 enriched lymphocytes: Summary from three independent donors. Percent of CD4 enri ched cells that are CD4+, CD4+/CXCR4+, CD4+/CCR5+, and CD4+/CCR5+/CXCR4+. A ll three donors displa yed values that are within two standard devi ations of the mean, indicati ng statistically insignificant differences in values obtained.

PAGE 80

80 Figure 3-8. Establishment of gate and Rh-123 efflux in total PBMC. Gate was drawn around lymphocyte population (A). Rh-123 loaded cells displayed high Rh-123 fluorescence (B, blue), whereas lymphocytes displaye d heterogeneous Rh-123 fluorescence (B, red). Data represent one donor tested without technical replicates. Rh-123 Fluorescence

PAGE 81

81 Figure 3-9.1. Rh-123 activity in NK cells, CD4 and CD8 T lymphocytes. Gates were established to determine Rh-123 fluorescen ce in NK cells, CD8, CD4, cells. NK cells display low Rh-123 fluorescence. CD8 cells display heterogeneous Rh-123 fluorescence, and exhibit two distinct popul ations; one with high Rh-123 fluorescence one with lower Rh-123 fluorescence. CD 4 cells display heterogeneous Rh-123 fluorescence and appear to contain a mixture of cells; some with high Rh-123 fluorescence (overlap with Rh-123 loaded cells), some with lower Rh-123 fluorescence (overlap with NK cells or CD8 cells). Data represent one donor tested without technical replicates. Rh-123 Fluorescence Rh-123 Fluorescence

PAGE 82

82 Figure 3-9.2. Rh-123 activity in subsets of HIV-1 target cells. Separate gates were established on total PBMC to obtain CD3+/CCR5+ and CD3+/CCR5cell populations (D). Further gating was establis hed to obtain CD45RA+/a nd CD45RO+/lymphocytes within the CD3/CCR5 positive and negative pop ulations (E, F). A subset of cells representing the R5 HIV-1 target cells de monstrated the lowest Rh-123 fluorescence relative to all other subset s tested (F, circled populati on. Data represent one donor tested without techni cal replicates.

PAGE 83

83 Figure 3-10. Efflux of Rh-123 in the pres ence of IDV, RTV, or R-Verapamil. CD3+/CD4+/CCR5+ lymphocytes subse quently gated on CD45RA and CD45RO expression were loaded with Rh-123 for 30 mi nutes (A, shaded), allowed to efflux for 1 hour at 37oC after Rh-123 load (A, red), allowed to efflux for 1 hour in the presence of 10 M R-Verapamil, IDV, or RTV (A, blue). Efflux was observed at one hour (B, red), or two hours (B, blue). Treatment w ith R-Verapamil or IDV inhibited ~80 % of Rh-123 efflux. RTV inhibited ~ 30 % of Rh-123 efflux (B). Data represent one donor tested without tech nical replicates.

PAGE 84

84 CHAPTER 4 DISCUSSION Questions Posed to Test Hypothesis of Thesis The hypothesis of this thesis is that decreased intrac ellular bioavailability of PI within the R5 relative to X4 lymphocyte subsets is a possible mechanism responsible for requirement of significantly higher IC50 in the R5 subset. PI are substrates for multiple efflux transporters, most notably p-glycoprotein (p-gp), a member of th e ABC Efflux transporter family. ABC efflux transporters are highly variable th roughout tissue and be tween cell types. 1,2,4-6,11,18-20,22,33,37,4244,60,64,66,68,71,73 A series of specific aims were designed, with specific questions posed to test the hypothesis as follows: Specific Aim 1a was designed to determine optimal conditions to observe peak intracellular PI levels and efflux kinetics in CD 4+ T lymphocytes, and was answered with the following questions: Published data demonstrates that peak intrace llular PI levels in PB MC are conferred by 18 hours extracellular PI treatment at 37oC. Can we recapitulate similar values in our laboratory? What is the inter-assay and intraassay variability for this study? What are the optimal conditions to observe ba seline intracellular PI levels above limit of detection in PBMC? (Variables include seru m concentration in extracellular media and stimulation state of cells) What are the optimal conditions to observe e fflux kinetics of PI in PBMC? (Variables include serum concentration in extracellular me dia, temperature, time, and stimulation state of cells) Specific aim 1b was designed to determine if th ere are enough R5 HIV-1 target cells in the lymphocyte population to perform sorting. To answer this question, the following questions were posed: What is the receptor expression profile for subs ets of T-lymphocytes for conditions that are optimal to observe intracellular PI levels in PBMC?

PAGE 85

85 The limiting factor is the R5 subset of HIV-1 target cells within the lymphocyte population. Will there be enough of this lymphocyte subpopulation under the optimized conditions? Specific aim 1c was designed as an alternative approach, as other follow up experiments and methods were not performed. This aim sought to determine the p-gp activity in subsets of lymphocytes representing X4 or R5 HIV-1 targets. This aim employed a Rh-123 flow cytometric based assay, which focused upon the following question: If the cell number is too low to obtain enough of the R5 subset, what alternative approaches can be employed to test the hypothesis? Relating Peak Intracellular PI Data to Toxicity and Published Data Relativ e to the extracellular concentration of PI applied in this pilot study, previous studies in the literature have dem onstrated that up to ~ 400 M extracellular treatment of PBMC with PI does not result in apoptosis, as determined by multiple methods to observe both mitochondrial and downstream indicators of apoptosis (DNA fragmentation). 87, 88,91,102,113 Studies conducted in the Goodenow laboratory focusing solely on mech anism related observed with the CellTiterBlue Assay (Promega, Madison, WI) which is base d on the ability of livi ng cells to convert a redox dye (resazurin) into a fluoresce nt end product (resorufin). Viab le cells retain the ability to reduce resazurin into resorufin. Nonviable cells rapidly lose me tabolic capacity, do not reduce the indicator dye, and thus do not generate a fluorescent signal 3,47. This marker for apoptosis is downstream of early indicators of apoptosis such as A nnexin V, but upstream of markers for late stage apoptosis including DNA fragmentation. The study performed in the Goodenow laboratory (Steven Pomeroy) demonstrated that up to 1000 M IDV is not toxic to PBMC. Although this concentration is higher th an that reported in the literature87, 87,91,102,113, it is important to note that the st udy in the Goodenow lab did not observe in tandem any other upstream or downstream markers of apoptosis, and that other indicators of apoptosis may have

PAGE 86

86 been positive but undetected in the assay performed in the G oodenow laboratory. Taking these data into consideration, we can c onclude from this pilot study ab ility to recapitu late published data in concert with the fact th at 18 hour extracellular PI treatment confers peak intracellular PI levels. Intracellular PI Studies: Parameters That Im pact Intracellular PI Levels and Efflux Multip le parameters can affect intracellular PI concentrations, including temperature, stimulation state of the cell, time, and concentr ation of serum in the extracellular media. Relative to baseline intra cellular PI levels (immediately after 18 hour load of cells with PI at 37oC), stimulation state and concentration of se rum in the extracellular media can affect intracellular PI levels.1,5,6,12,18,19,35,37,40, 43,56, 57, 64,106,109 Keeping these factors in mind, we were able to design a study to assess the impact of temperature, time, and concentration of extracellul ar serum on intracellular PI levels. Our data demonstrate that efflux above limit of detec tion can occur when e fflux is observed at 20oC in unstimulated PBMC maintained in 2 % serum cont aining media. Keeping this fact in mind, the next step in the discussion of these data was to determine the best possible mechanism to test the hypothesis of this thesis. Total Number of R5 Target Cells: Options and Alternative Methods Although it is m athematically possible to obt ain enough of the cells representing the R5 target cells within th e lymphocyte population, other options exist that could present a more feasible mechanism to test the hypothesis of this thesis. Methods include use of efflux transporter +/cell lines, RT-PCR, western blot an alysis, and direct stain for efflux transporter expression. These experiments were not perf ormed, although they, in concert with the preliminary data obtained in th is thesis, provide an excelle nt foundation from which further

PAGE 87

87 studies can be launched. Disc ussion regarding each method, its pitfalls, potential method of execution and analysis appear below. Detained Analysis and Int erpretation of Rh-123 Studies Alternative m ethod of Rh-123 assay was employed to assess p-gp activity within subsets of T lymphocytes. When analyzing Rh-123 data multiple parameters must be considered. A summary of parameters, followed by discussion is as including: 1,2,4,5,6,9,12,1822,35,37,40,42,44,51,56,57,60,63-66,68,70-73,76,77,81,93,94,98,100,104,106,107,109,112 Lipophilicity of drug Affinity of PI for p-gp Presence of intracellular proteins that may bind PI (and prevent efflux of PI) Ability of PI to modulate p-gp expression levels Activity state of p-gp during timefram e that PI efflux is being studied Combined effect of parameters 1-3 on Rh-123 efflux Presence of other efflux transporters a nd their potential eff ect on the system Lipophilicity of Drug: Parameters for Anal ysis and Interpretation o f Rh-123 Data Of the ten FDA approved PI, NFV displays the greatest intracellular accumulation in vitro as assessed in cell lines and primary lymphocytes. The rank order of intracellular accumulation is NFV > RTV > IDV. These data were demonstrated in homogeneous p-gp expressing cell lines and in total PBMC 1,2, 19,21,56,57, 65. Relative to the data reported in this thesis, consideration for lipophilicity of drugs tested (IDV and RTV) must occur. The data reported in this thesis demonstrate that R-Verapamil, a known substrate for p-gp, and inhibitor of Rh-123 efflux, inhibits nearly 80 % of Rh-123 efflux, whereas RTV and IDV inhibit ~ 30 % and ~ 80 %, respectively. Taken alone, thes e data would indicate that R-Ve rapamil and IDV display similar affinity for p-gp whereas RTV di splays significantly less affin ity for p-gp (and thus cannot out compete Rh-123 for p-gp mediated efflux).

PAGE 88

88 Rank order of intracellular a ccumulation mirrors rank orde r of lipophilicity of PI, correlating higher lipophilicity with greater ability to enter cells and higher intracellular PI levels. No shuttle mechanism exists to trans port substrate to p-gp for efflux. Therefore, the greater the intracellular concen tration of drug, the higher the lik elihood that the drug will come in contact with the efflux transporter and become effluxed. This principle holds true relative to ability to inhibit Rh-123 efflux. If more RTV is present inside the cell, then inhibition of Rh-123 will occur more readily than if little RTV was present intracellularl y, as more drug is available to out compete Rh-123 for binding to p-gp. Data reported in this thesis demonstrate that RTV inhibits significantly less Rh-123 relative to IDV (~30 % versus ~80 %). These data in the context of the correlation between intracellular accumulation and ability to inhibit Rh -123 efflux would indicate that IDV displays higher intracellula r accumulation than RTV. As the Rh -123 assay cannot directly determine intracellular levels of drug, a direct correlation be tween intracellular PI levels and ability to inhibit Rh-123 efflux cannot be drawn to te st the hypothesis for observed results. It is important to understand that mechanisms responsible for data are multifactorial, and likely include factors that cannot be directly measured with the Rh-123 assay. A carefully designed set of experiments to elucidate mechanis ms responsible for these data is necessary to test how hierarchical intracellular accumulation of PI affects the data reported in this thesis. Comprehensive discussion occurs in the alternativ e methods section of th is thesis relative to approaches to elucidate mechanisms responsible for data, and their relationship to the factors bulleted above. Affinity of PI for p-gp: Analysis and Interpretation of Rh -123 Data Drugs bind with differential affinity to efflux transporters. Multiple studies have demonstrated that Rh-123 is a substrate for p-gp 68, however differential affinity of PI within

PAGE 89

89 lymphocytes, and in subsets of lymphocytes, is largely undefined. Although RTV displays greater intracellular accumulation than IDV 44, RTV may display lower affinity for p-gp than IDV, rendering RTV less effective in competitiv e inhibition of Rh-123 efflux. In addition, affinity of RTV and IDV for other efflux transporters relative to their affinities for p-gp, and relative to each other, is not defined. If RTV displays higher affinity for MRP1, for example, relative to p-gp, then RTV could be primarily and preferentially effluxed from the cell via a p-gp independent mechanism. Rh-123 assay can indir ectly detect efflux of PI via p-gp dependent mechanisms, but not other efflux transporters. The contribution of other efflux transporter expression, activity, and affinity of PI for thes e transporters cannot be detected within the confines of the experiment conducted. Therefore, in the context of the data reported in this thesis, it is difficult to explain one specific mechanism responsible for the results. A carefully designed se t of experiments to elucidate mechanisms responsible for these data is necessary to test how affinity of PI affects the data reported in this thesis. Comprehensive di scussion occurs in the alternative methods section of this thesis relativ e to approaches to elucidate mechanisms responsible for data. Presence of Intracellular Prot eins: Parameters for Analysis and Interpretation of Rh-123 Data To understand the m echanism responsible fo r our data, other parameters must be considered. PI are designed for therapeutic f unction of inhibiting HIV-1 Protease, which is an aspartic protease. PI are have been shown to bind and interact with ce llular proteases, most notably caspases (cysteine proteases). 77,79,88,102,103 PI have also been implicated as inhibitors of cellular proteasome function.89,90 It is hypothesized, but currently undefined, that PI binding/interaction with intracellular proteins is not limited to caspases or proteasomes.

PAGE 90

90 Upon binding, PI are subsequently unavailable to bind to efflux transporters, and are often physically immobilized, preventing their ability to bind to efflux transporters 1,18,19,40,70. Whether RTV and IDV bind to different proteins, bind to pr oteins with different affinities, or whether these proteins display differentia l expression levels w ithin subsets of cells, is unknown. Our data observed ability of RTV, IDV, or R-Verapamil ability to inhibit Rh-123 efflux in the subpopulation of lymphocytes representing the hi gh p-gp expressing R5 HIV-1 target cells. These data may or may not hold true when observed in a total lymphocyte population, within other subsets of lymphocytes, or across mu ltiple donors. The conclusion is multi-fold: RTV may bind to proteins that IDV does not, t hus impeding its ability to inhibit Rh-123 efflux Protein expression levels that RTV or IDV may bind to could be different in the population of cells observed relative to other cell populations. RTV and IDV may display different percent inhibition of Rh-123 in other subsets of cells or in a total population of PBMC. These experiments must be repeated in multiple donors to confirm results, and coupled with tandem experiments to elucidate mechanisms res ponsible for results (dis cussed in alternative methods section of this thesis). Further analysis and/or experiments must be pe rformed to answer these questions. Assessing percent inhibition of Rh-123 efflux by RTV and IDV in other subsets of ly mphocytes and in total PBMC would determine if RTV inhibits a greate r percentage of Rh-123 efflux within different cell populations. Testing ability of IDV and RTV to inhibit Rh -123 efflux in homogeneous cell lines (and/or total PBMC) presents a follow up expe riment to test ability recapitulate published data, ruling out technical error as mechanism for results. Modulation of p-gp by PI: Parameters for Analysis and Interpretation of Rh-123 Data NFV, but not other PI or RVerapam il (p-gp inhibitor) in crease p-gp and MRP1 expression in vitro 20,43. As p-gp expression indirectly correlates with p-gp activity 68, it is important to understand the relationship between drug, efflux transporter expression, and Rh-123 efflux.

PAGE 91

91 Increased p-gp expression in a cell will result in more rapid efflux of Rh-123 relative to cells with lower p-gp expression. IDV and RT V do not increase efflux p-gp expression 20,43, therefore presence of these drugs cannot account for mo re rapid efflux of Rh-123 as a function of increased p-gp expression. IDV and RTV do not increase MRP1 transporter expression, however effect of these drugs on MRP2, MRP4 MRP5, or BCRP is unknown. If PI increase expression of these efflux transporters, then the PI will consequently be effluxed more rapidly. This effect would be undetectable within the co nfines of the Rh-123 assay, as it observes only pgp mediated efflux and does not consid er efflux by other mechanisms. As no data is currently available to support or deny the idea that PI may modulate MRP2, MRP4, MRP5, or BCRP, pilot experiments could be performed to address and fill current gaps in knowledge. Western blot, RT-PCR, and direct staining for effl ux transporters with specific flurochrome conjugated mAb for cel ls treated with or without PI over time, with appropriate controls presents options for execution of these pilot studies. Activity State of p-gp: Parameters for An alysis and Interpretation of Rh-123 Data In all cases, presence of PI w ithin the binding pocket of the efflux transporter activates the efflux transporter, allowing for efflux of PI via the ATP de pendent m echanism. Efflux transporters remain in a basal, non-active stat e until substrate-binding pocket is engaged by substrate. As no shuttle mechanism exists to transport substrate to efflux transporter (subsequently conferring activation of the transporter), activation occurs when substrate is in close physical proximity to substrate binding pocket.5,18,19,70 Points above addressed binding of PI to intracellular proteins and inability of PI to bind to substrate binding pocket once bound to intracellular proteins. As limited data exist regarding the profile of intracellular proteins to which PI bind (other than caspa ses and proteasomes), and with what affinity, it is difficult to determine what, if any, role this protein/PI interaction may have on ability of PI to interact with

PAGE 92

92 p-gp and subsequently confer ac tivation. Independent of thes e gaps in knowledge, we have demonstrate that IDV, RTV, and R-Verapamil i nhibit Rh-123 efflux, something that could not occur if drugs were completely bound to intracellular proteins. The stat istical significance of these data is unknown, as n=1 and no technical replicates were perf ormed within the donor tested. These experiments must be repeat ed, and followed up as described below to confirm/validate results. Pitfalls of Rh-123 Study Although Rh-123 provides an excellent m echanism to observe p-gp activity within subsets of cells, the assay displa ys shortcomings and pitfalls. Thes e shortcomings can be addressed by performing a comprehensive and carefully planne d set of follow up experiments to elucidate specific mechanisms responsible for Rh-123 efflux da ta. A list of shortcomings and pitfalls are summarized below followed by discussion for potent ial execution of experiments to rectify each shortcoming/pitfall. Assay is indirect measure of pgp expression (observes p-gp activity) P-gp activity correlates with, but is not always directly prop ortional with, p-gp expression Requires further follow-up experiments with direct assessments of p-gp expression to confirm hypotheses about p-gp expression obt ained from Rh-123 p-gp activity studies Assay cannot discern mechanism responsible for Rh-123 efflux: (are results a function of pgp activity alone or potential modulation of p-gp expression levels (or other efflux transporter levels) that may occur as a functi on of presence of substr ate [as occurs with NFV]) ? Rh-123 can only indirectly assess intracellular bioa vailability of PI. It is twice removed from intracellular PI levels (p-gp activ ity is indirectly related to p-gp expression, which correlates with, but is not a direct measure of, intracellular PI levels) Rh-123 assay is specific for p-gp and cannot be used to determine activity of other efflux transporters for which PI are substrates or their impact on efflux of PI by p-gp

PAGE 93

93 Alternative Methods to Assess pgp Activity an d Expression Many assays are commonly used to dire ctly assess not only p-gp expression, but intracellular bioavailability of PI within cells.1, 19-22,30,41-44,56,57,59,60,63,65,66,68,71,73,77,84,93,97,98,106,107,112 When performed in tandem, these experiment s can establish relati onships between efflux transporter expression and intr acellular levels of drug. In the study performed in this thesis, follow up experiments can be performed to confirm and/or test the hypothesis that differential p-gp activity is an indicator of differential p-gp expression, which directly correlat es with intracellular PI levels. This study sought to determine the mechanism(s) responsible for significantly higher IC50 in R5 versus X4 using viruses in T lymphocytes. Relative to the in itial question posed, additional fo llow up experiments to directly assess efflux transporter expression in these subsets as well as intracellular PI levels within these subsets could be performed and present an excellent starting point from which to launch further studies. Follow Up to Rh-123: Direct Assessment of Efflux Transporter Expression Flow cytometry presents the m ost feasible method for directly asse ssing p-gp expression. Flurochrome conjugated mAb specific for each of the six efflux transporters for which PI are substrates (p-gp, MRP1, MRP2, MRP4, MRP5, BCRP) are commercially available and have been demonstrated in numerous studies to pr ovide accurate assessment of efflux transporter expression as confirmed with comparative analysis of analogous cells subjected to western blot and RT-PCR analysis. 42,66,68,71,72 Performing p-gp expression studies will allow for confirmation of p-gp expression that is hypothesized from p-gp activity prof ile obtained with Rh-123 data. The mechanisms to directly assess intracellular PI levels exist: 29,42,62

PAGE 94

94 HPLC and tandem mass spectrometry conducted on cellular lysates of cells previously treated with PI Radio-labeled PI In the case of this study, pre sorting of cells must occur for HPLC and mass spectrometry, or radio-labeled PI to be employed within subsets of lymphocytes, and pres ents potential problems with total cell number and constraints. In the context of the data obtained in the thesis, an excellent follow up to confirm hypothesis of diffe rential p-gp expression in lymphocyte subsets could be flow cytometry with mAb specific for p-gp with gating on specific lymphocyte subsets. In addition, the data set could be expanded to en compass all efflux transporters for which PI are substrates, allowing for a comprehensive analysis of relationship between efflux transporters for which PI are substrates, and expression within X4 and R5 HIV-1 target cells within the lymphocyte population. Pilot experiments to assess appropriate combin ation of fluorochromes and combinations, as well as reproducibility across donors must first be conducted to achieve the end goal. In addition, use of cell line with known levels of e fflux transporter expression could be employed as a preliminary study to test inter and intra assay variability, an d ability to observe efflux transporters within the parameters of the newly designed experiment. Direct assessment of efflux tr ansporter expression presents an excellent foundation to assess levels of all know efflux transpor ters for which PI are substrates within the X4 and R5 HIV-1 target cells. It is important to note that performing stain for efflux transporter expression would not fully elucidate the mechanisms responsible for higher IC50 in R5 versus X4 T lymphocytes. Instead, these data would provide an excellent foundation from whic h to launch further studies to elucidate the complex relationship between dr ug, efflux transporter act ivity, expression, and intracellular accumulation of drug.

PAGE 95

95 Relationship Between p-gp Activity and IC50 The questions posed above culminated in the fi nding that a subset of lymphocytes that are R5+/CD4+/CD45RA-/CD45RO+ displayed p-gp leve ls comparable to the highest known p-gp levels in any human cell, NK cells67. Cells expressing CD4 and R5 are targets for HIV-1 infection by an R5 using virus. The population of cells displaying high pgp activity represents one of the populations for R5 infection within the lymphocyte population, demonstrating a link between p-gp activity in the R5+/CD4+/CD45RA/CD45RO+ cells and the requirement for more drug in viruses targeted to R5 expressing T lymphocytes. Although PI are substrates for other efflux transporters, and p-gp is not the sole determining factor for efflux and bioavailability,1, 2,4,5,6,9,12,18-22,35,37,40,42,44,51,56,57,60,63-66,68,7073,76,77,81,93,94,98,100,104,106,107,109,112 these data demonstrate that p-gp activity is higher in a subset of the R5 target cells relative to cells representing the X4 HIV-1 targets. As p-gp activity is linked directly to intracellular bioavailability of PI, these data demonstrate a conclusive link between pgp activity and differences in IC50 for otherwise identical viruse s targeted to R5 versus X4 expressing T lymphocytes. Follow up experime nts must be performed to confirm this hypothesis, as described in the alternative met hods section of this thesis, in concert with repetition of the reported data in multiple independent donors. Relationship Between Fitness, IC50, and Target Cells The primary goal of the thesis centers around significant differences in IC50 for otherwise identical viruses targeted to R5 versus X4 expre ssing T lymphocytes. It is important to relate the fitness of these viruses to IC50, as this relationship further implic ates difference in target cell as a modulator of not only IC50 values but also replicative capacity. Previous studies in our laboratory demonstr ated that X4 using recombinant viruses containing posttherapy PIres gag-pol fragment display a significant reduction in p24 production

PAGE 96

96 relative to otherwise identical reco mbinant viruses with pretherapy PIsen gag-pol fragment whereas pre and posttherapy R5 recombinant viruse s replicate to similar levels. These studies were conducted with otherwise identical viruses ta rgeted to X4 or R5 expressing T lymphocytes, implicating differences in target cell as a modulat or or differential replic ative capacity. Although published findings report that R5 viruses display a fitness advantag e relative to X 4, these reports did not use recombinant viruses, nor was there a direct comparison of LAIX4 envelope with JRFL V1-V5 envelope, which were used in our studies. 8,17, 96 Fitness studies in our lab were expanded to address differences in target cell relative to IC50. It was determined that significantly more drug (188 fold for IDV, 141 fold for RTV) is required to inhibit viral replica tion for otherwise identical viruse s targeted to R5 versus X4 T lymphocytes. As the only difference for these viru ses are the target cell, this finding suggested that differences in the cells, not virus, are responsible for the outcome. Relating data from our laboratory to published findings from other groups is important in determining the full impact of data obtained from this thesis. Schuitemaker97 et al reported that intracellular HIV-1 RNA and DNA was significantly lower in cel ls with high-p-gp activity relative to low p-gp activity (as assessed by Rh-123 assay), thus concluding that the potential efflux function of p-gp on PIs may be clinically less relevant than the effect of p-gp on intracellular HIV-1 replication. To correlate these data to findings from this thesis, consideration for target cell population must occur. The gr oup used Rh-123 assay in concert with CD45RO stain to delineate two groups: RO+/high-p-gp activity, and RO-/lo w-p-gp activity cells. Gating solely on CD45RO is not a surrogate marker for X4 versus R5 HIV-1 target cells within the lymphocyte population. As no gating or stain for CD45RA, CCR5, or CXCR4 was performed, the analysis conducted by Schuitemaker is focu sing upon two populations of cells which may co-

PAGE 97

97 express varying levels and combinations of CXCR4 and or CCR5. In addition, our data demonstrate that ~30 % of CD4+ lymphocytes co-express CD45RA and CD45RO, demonstrating that gate on CD45RO does not eliminate inclusion of CD45RA. Although Schuitemakers data presents an interesting correlation between intracellular HIV-1 RNA and DNA and p-gp activity, it is important to note that this correlation is dr awn based on a population of cells that is different from the cells tested in this thesis. Despite different target cell analysis, one cannot discount eith er the findings from this thesis or Schuitemakers data. Instead, the data from this thesis, in concert with Schuitemakers findings, present an excellent foundation from which to launch fu rther studies to confirm the hypothesis of this thesis, and conduct in vivo studies to observe p-gp activity and viral RNA or DNA within the R5 and X4 HIV-1 lymphocyte targets. Relationship of Results to Therapy Outcome and Disease Progression Findings fro m this thesis impact multiple in teractions between drug, host, and virus. Although differences in bioavailab ility of PI within lymphocyte subsets explains significantly higher IC50 for otherwise identical viruses targeted to R5 versus X4 T-lymphocytes, these data impact other determinants in HIV-1 infection. This study draws a link between differential p-gp activity in R5 versus X4 T lymphocytes, and may indicate that differences between these target cells is not limited to p-gp activity. Furthermor e, findings from this th esis also provided a foundation from which to launch further studies to determine how PI impact HIV-1 target cells, and to determine how these events relate to therapy outcome and disease progression. Impact of PI Independent of Antiviral Effect: Relationship to Therapy Outcome When treating HIV infected patients, the goal is to delay progression to AIDS by controlling viral replicati on and preventing or revers ing immune deficiency 29,34-36. However, in a significant percentage of children and adoles cents, immune reconstitution is achieved but not

PAGE 98

98 concomitant with decrease in viral burden, imp licating a role for ART, and specifically PI, in immune reconstitution independent of its antiviral effect 13, 23-27, 39, 43, 46, 61, 83. Sentinel events that favor immune reconstitution and decreased susceptibility to HIV1 infection have been observed upon addition of PI either in vivo or in vitro Such extravirologic properties of PI include modulation of antigen presentati on, cell cycle arrest, cell prolif eration, ABC efflux transporter activity, lipoprotein metabolism, dendritic cell maturation, and inhibition of spontaneous and activation induced apoptosis of both HIV-1 infected and uninfected T lymphocytes 2,4,12,15,21,23,38,44,52,60,62,78,79,87-91,102,103,113-115 Immune reconstitution is highly favorable fo r patient prognosis and leads to delayed progression to AIDS 29,34-36. ART induced discordant response s are multifactorial and involve a complex interplay between phenotypic properties of the virus, ART effect and interaction with host cells, and the ability of the immune system to control viral replication in the presence of ART. The effect of PI on the immune respons e independent of its an tiviral effect, and the implications of differential effect of PI within subsets of HIV-1 target cells and in other cells, constitutes a complex set of questions relating to therapy outcome and progression to AIDS. Relationship Between Efflux Kinetics, Peak In tracellular PI Levels, and Cell Cycle Studies Mechanisms correlating failure of therapy w ith R5 emergence are not fully understood. This foundation of this study sought determine if e fflux kinetics and peak intracellular PI levels in subsets of HIV-1 target cells could be respons ible for requirement of significantly more drug required to inhibit viral replica tion for otherwise identical viruse s targeted to R5 versus X4 Tlymphocytes. This correlation re lates not only to the mechanism responsible for requirement of significantly more drug for the R5 subset, but also relates to ther apy outcome for HIV-1 infected individuals, and may explain emergence and pers istence of R5 infecti on. In addition, these findings in concert with published data demonstra ting effect of PI on cells independent of their

PAGE 99

99 antiviral effect, provide a base from which to launch further studies to determine additional impact of PI on cells independent of their antiviral effect. The appendix of this thesis addr esses a pilot study designed to determine the effect of IDV, RTV, and NFV on cell cycle progress ion in total PBMC. It was de termined that IDV, RTV, and NFV induce a G1 block in a dose dependent manner. This study was conducted with the idea that further studies could be e xplored to determine the mechanisms responsible for G1 block, to relate these data to potential differences in subsets of HIV-1 target cells, and to draw a correlation between these data and therapy outcome for patients receiving a PI containing regimen. Further studies were not conducted on this project, however findings present an excellent foundation for further experiments. Significance and Implications for Diffe rential p-gp Activity in CD4+/CD45RA/CD45RO+/CCR5+ T lymphocytes These data m ay have implications for in vivo replication dynamics, therapy outcome, persistence of R5 infection, a nd viral reservoirs for drug resi stant viruses emerging under the selective pressure of suboptimal levels of drug. Suboptimal levels of ART is an ongoing problem in treating HIV-1 infection and presen ts obstacles including em ergence of resistant mutations and clearance of virus from in fected cells with low bioavailability 1,6,9,10,21,40,43,76,77,81. Understanding dynamics of virus/host/drug in teraction and identif ying mechanisms for decreased bioavailabil ity of ART can lead to design of novel therapeutics to increase intracellular concentrations of drug.

PAGE 100

100 APPENDIX A IMPACT OF HIV-1 PROTEASE INHIBITORS ON CELL CYCLE PROGRESSION IN PBMC Introduction Previous studies have establis hed that PI have an effect on cells independent of their antiv iral activity against HIV-1. Sentinel even ts that favor immune reconstitution and decreased susceptibility to HIV-1 infection have been obs erved. Such extravirol ogic properties of PI include modulation of antigen presentation, cel l cycle arrest, cell proliferation, ABC efflux transporter activity, lipoprote in metabolism, dendritic cell maturation, and inhibition of spontaneous and activation induced apoptosis of both HIV-1 infect ed and uninfected T lymphocytes. 2,4,12,15,21,23,38,44,520,62,78,79,87-91,102,103,113-115 The objective of the experiments in this appendix was to discover a role for PI in m odulating cells that may influence virus/host cell interactions and explain a role for PI in impacting therapy outc ome independent of its antiviral effect. A unique paradigm is observed in 25-40 % of children and adolescents who, following initiation of a PI containing regimen, exhibi t a VF/IS discordant response (sustained reconstitution of T cell immunity despite transient viral supp ression and subsequent rebound to levels which predict disease progression). This discordant response is not determined solely by host factors alone or simple genetic changes in the viral genome. AR T induced discordant responses are multifactorial and involve a comple x interplay between phenotypic properties of the virus, ART effect and interaction with host cells, and the ability of the immune system to control viral replication in the presence of ART. 39, 100, 116,118 The experiments in this appendix were based on one specific facet of the VF/IS discordant response: effect of PI on cells independent of their an tiviral effect. The experiments were based on the hypothesis that PI modulation of cell cycle progression in lymphocytes may

PAGE 101

101 be a determinant in therapy outcome. Cells th at are actively progressi ng through the cell cycle are significantly more susceptible to HIV-1 inf ection relative to quiescent, non-cycling cells. If PI can induce cell cycle arrest in HIV-1 target cells, this would render th ese cells significantly less susceptible to HIV-1 infection. This hypothe sis could represent a model where PI favorably impact cells independent of their antiviral effect. Pahwa, 2001 et al reported that IDV inhibits cell cycle progression via a G1 block in CD4+ T-lymphocytes in a dose dependent manner 23. The study in this appendix sought to determine if other PI (RTV and NFV) modulat e cell cycle progression in lymphocytes, and to relate these data to therapy outcome reported for patients rece iving a RTV or NFV containing regimen. Significance When treating HIV infected patients, the goal is to delay progression to AIDS by controlling viral replicat ion and preventing or reversing immu ne deficiency. However, in a significant percentage of childre n and adolescents, immune reconstitution is achieved but not concomitant with decrease in viral burden, imp licating a role for ART, and specifically PI, in immune reconstitution independent of its antivi ral effect. Immune reconstitution is highly favorable for patient prognosis and l eads to delayed progression to AIDS. 39, 100, 116,118 Determining mechanisms responsible for favorab le effect of PI on immune reconstitution independent of its antiviral effect may lead to design of novel therapeutics, which exploit host immune response. Methods for PHA or Anti-CD3mAb Stimulation The studies in this appendix were conducted with the intent of determining effect of PI on cell cycle progression in PBMC and corre lating these data with fitness and IC50 data previously obtained in the Goodenow laboratory. All fitness and IC50 studies were conducted on PHA

PAGE 102

102 stimulated PBMC, thus PHA as a means of stimul ation to induce cell cycle progression was first studied. Published data from Pahwa, et al determined that IDV induced a G1 block in cell cycle progression but did so using anti-CD3mAb stimul ated lymphocytes. This group subjected the lymphocytes to PI for 18 hours prior to 48-hour stimulation with anti-CD3mAb in the absence of PI. Therefore, in the context of the original intent or these studies and the published data, both PHA and anti-CD3mAb was tested for its ability to induce cell cycle progression in PBMC. Cryopreserved PBMC isolated as described previously from one donor were cultured at a concentration of 1.0x106 c/mL for 18 hours at 37oC in RPMI 1640 supplemented with 30 U/mL human interleukin-2, 2 mM L-glutamine and 100 U of penicillin per mL, 100 g of streptomycin per mL, and 2% heatinactivated fetal calf seru m. After 18 hours, PBMC were treated with 0.5 g/mL anti-CD3mAb (Pharmingen) for 48 hours, or 0, 10, 25, 50, or 100 g/mL PHA for 48 or 72 hours at 37oC. Cells were then centrifuged in 15 mL conical tubes and centrifuged at 1000 rpm for 10 minutes. Residual media was remove d, and cell pellets were subjected to three washings in ice cold 1X PBS at 1000 rpm for 10 mi nutes. After washings, cell pellets were fixed in 70% ethanol for 1 hour and incubated w ith Propidium Iodide (Invitrogen) (50 g/mL) and RNAse (Invitrogen) (100 g/mL). Samples were kept in th e dark at 4C for 30 minutes and immediately analyzed for cell-cycle profile using FACS Vantage Flow Cytometer (Beckman Dickinson), Mod-Fit Software (Verify Software, Inc.). Methods for Assessment of PI Effect on Cell Cycle Progression Cryopreserved PBMC isolated as described previously from one donor were cultured at a concentration of 1.0x106 c/mL for 18 hours at 37oC with 0, 10, 25, 50, or 100 M IDV, RTV, or NFV in RPMI 1640 supplemented with 30 U/mL human interleukin-2, 2 mM L-glutamine and 100 U of penicillin per mL, 100 g of streptomycin per mL, and 2% heatinactivated fetal calf

PAGE 103

103 serum. After 18 hours, PB MC were treated with 0.5 g/mL anti-CD3mAb (Pharmingen) in drug free media (figure A-2)_. Technical triplic ates were prepared for each sample. After 48-hour anti-CD3mAb treatment, cells were then centrifuged in 15 mL conical tubes and centrifuged at 1000 rpm for 10 minutes. Residual media was removed, and cell pellets were subjected to three washings in ice cold 1X PBS at 1000 rpm for 10 minutes. After washings, cell pellets were fixed in 70% ethanol for 1 hour and incubated with Propidium Iodide (Invitrogen) (50 g/mL) and RNAse (Invitrogen) (100 g/mL). Samples were kept in the dark at 4C for 30 minutes and immediately analyzed for cell-cycl e profile using FACS Vantage Flow Cytometer (Beckman Dickinson), Mod-Fit Software (Verif y Software, Inc.), and Sigma Stat 3.0 (SEM based on technical triplica tes) (Figure A-1). Results For quiescent, non-Anti-CD3m Ab, non-PHA stim ulated PBMC, ~ 85 % of PBMC were in G1, ~ 15 % of PBMC were in S, and no PB MC were in G2/M phase. For Anti-CD3mAb stimulated PBMC, ~ 50 % of cells were in G1 wher eas the other half of cells were in S or G2/M (Figure A-3, A-4). For PBMC treated with PHA, percent of cells in Go/G1 ranged from 25-50 % independent of time exposed to PHA or concen tration of PHA applied (Figure A1). IDV, RTV, and NFV inhibited cell cycle progression in a dose-dependent manner,. Nearly 80 % of PBMC arrested in G1 upon exposure to 100 M IDV, whereas nearly 90 % of cells were arrested in G1 upon exposure to 50 M RTV or NFV, with the remaining cells in S phase (Figure A-3, A-4). Discussion This study dem onstrated that IDV, RTV, and NFV inhibit cell cycle progression in PBMC via a G1 block in a dose de pendent manner. Specifically, 50 M RTV and NFV induced

PAGE 104

104 a G1 block for a significan t percentage of cells (> 80 %) and IDV induced a G1 arrest upon 100 M treatment. In vivo the peak plasma level of extracellular PI that are non-plasma protein bound and available to traverse the lip id bilayer and enter cells is ~ 8.0 M for IDV, 11.0 M for RTV, and ~ 3.0 M for NFV. The concentration required to induce a G1 block in total PBMC in vitro is significantly higher (~ 50 M). This study was conducte d on a heterogeneous population of cells including monocytes, NK cells, and multiple subsets of T-lymphocytes. These cells display significantly different levels of efflux transporter expression and activity. These experiments answered a critical question relative to PI effect on cell cycle progression in PBMC, however analysis of physiologically relevant c oncentrations of drug in specific subsets of lymphocytes, or HIV-1 target cells within the lymphocyte population cannot be determined from this pilot experiment, and must be cons idered in further experiments. We can determine that a con centration of PI that is not physiologically relevant to in vivo pharmacokinetics results in a G1 block when observing the total PBMC population. Published literature and findings from the objectives in th e parent thesis demons trate differential efflux transporter activity and e xpression, coupled with signi ficant differences in IC50 for otherwise identical viruses targeted to R5 versus X4 T-lymphocytes. The hypothesis is that physiologically relevant concentrations of PI (3-11 M PI) may induce a G1 block in particular subsets of lymphocytes which display lower efflux transporte r activity and expression but not in subsets which display high efflux transporter activity and e xpression (where high is defined as similar to NK cells, which display highest p-gp activity of any known human cell) 68. Relative to the IC50 studies and the Rh-123 studies, we can hypothesize that subsets of lymphocytes that may arrest in G1 upon exposure to physiologically releva nt concentrations of PI include the X4 HIV-1 target cells, and that the R5 HIV-1 target cells (in the lymphocyte

PAGE 105

105 population) represent cells that would likely undergo a G1 arrest. No follow up experiments to confirm these hypotheses were performed, however these ideas present an excellent foundation for future studies to answer these questions. Ability to test the hypothesis relies on abil ity to observe cell cycle progression within subsets of T-lymphocytes, and presents technical lim itations on two fronts: 1) ability to separate enough of the R5 HIV-1 target cells, and the financ ial constraints associated with this task, and 2) inability to observe receptor expression profiles in cells permeablized to allow for DNA stain (either Propidium Iodide or 7-Amino-actinomyc in D (7AAD) to enter cells (Figure A-5). Propidium Iodide is a fluorescent vital dye that stains DNA. It does not cross the PM of cells that are viable or in the early stages of apoptosis because they main tain PM integrity. Vital cells must be permeablized via exposure to 70 % etha nol, allowing for the dye to enter cells and subsequently bind to DNA. 7AAD, an alternativ e to Propidium Iodide, does not readily enter vital cells, and manufacturer prot ocol recommends permeablization of cells with ethanol, which precludes concomitant recep tor expression profile st udies (Figure A-4). Administration of a PI containing regimen in vivo is often associated with CD4 T cell reconstitution 46,100,108. These results may not be solely dete rmined by the antiviral activity of PI, as many reports of favorable effects of PI on immunity have been re ported independent of antiviral activity.2,4,12,15,21,23,38,44,5260,62,78,79,87-91,102,103,113-115 Here, a pilot study was conducted, where findi ngs may indicate that IDV, RTV, and NFV may prolong cell survival either dire ctly or indirectly vi a cell cycle arre st. In patients receiving a PI containing regimen, PI modulation of cellu lar functions may favorab ly impact CD4 T cell immunity and immunologic outcome independe nt of its antiviral effect. Although no experiments were conducted to answer these que stions, the pilot experi ments in this appendix

PAGE 106

106 present an excellent foundation from which to launc h further studies to determine effect of PI on cells independent of their antiviral effect. Understanding specific mechanisms responsible for these effects could lead to design of novel therap eutics, which directly af fect these events.

PAGE 107

107 Figure A-1. Effect of PHA on cell cycle pr ogression in PBMC. (A) Representative chromatograph of cell cycle progression for PHA treated PBMC. (B) PBMC treated with 0, 10, 25, 50, or 100 mg/mL PHA for 48 ho urs, or (C) 72 hours prior to 24 hour quiescence Non PHA treated PBMC are 100 % in Go/G1 phase whereas PHA treated PBMC are 25-50 % in Go/G1 phase (B C). Data are representative of one donor tested without replicates.

PAGE 108

108 Figure A-2. Experimental design for cell cycl e PI study in PBMC. After PBMC isolation, PBMC were resuspended in media cont aining 2 % fetal bovine serum and 0, 10, 25, 50, or 100 M IDV, RTV, or NFV for 24 hours prior to stimulation with 0.5 g/mL anti-CD3 mAb in drug free media for 48 hours. At 72 hours post PI treatment, Propidium Iodide cell cycle analysis was performed. Cells were treated with 0, 10, 25, 50, or 100 M IDV, RTV, or NFV.

PAGE 109

109 Figure A-3. Effect of PI on cell cycle progression in PBMC. Gate was drawn around lymphocyte population (A). Unstimulated PBMC were 85 % in G1 and 15 % in S phase and anti-CD3mAb stimulated PBMC we re ~50 % in G1, 40 % in S, and 10 % in G2/M (B). PBMC treated with 100 M IDV were ~ 80 % in G1, 19 % in S, and 2 % in G2/M. PBMC treated with 50 M NFV were 95 % G1, 5 % S, and0 % in G2/M. PBMC treated with 50 M RTV were 94 % in G1, 4 % in S, and 2 % in G2/M. Data are representative of n=1 with technical triplicates.

PAGE 110

110 Figure A-4. Effect of IDV, RT V, and NFV on cell cycle progre ssion in PBMC. IDV, RTV, and NFV inhibited cell cycle progression in a dose dependent manner via a G1 block. IDV inhibited arrested nearly 80 % of cells in G1 upon 100 M treatment (yellow), and RTV and NFV arrested ~ 95 % of cells in G1 upon 50 M treatment (red and blue, respectively). Red dotte d line indicates percentage of quiescent cells in G0/G1 and black dotted line indicates percentage of anti-CD3 mAb stimulated cells in G0/G1. Data are n=1 with technical tripli cates. Error bars represent SEM.

PAGE 111

111 Figure A-5. Pitfalls for Propidium Iodide or 7AAD in concert with receptor expression stain. Vital cells possess an intact lipid bilayer, which cannot be penetrated by Propidium Iodide or 7AAD (A) and intact receptors (not shown). Cells permeablized by exposure to ethanol results in a non-intact lipid bilayer, which can be penetrated by Propidium Iodide or 7AAD (B), and disrupt ed receptor expressi on (not shown).

PAGE 112

112 APPENDIX B RAW DATA FOR RECEPTOR EXPRESSI ON AND INT RACELLULAR PI STUDIES Figure B-1. Receptor expression profile for CD4 enriched lymphocytes, donor B. CD4 enriched population was gated on CD4 expression (A). ~ 80 % of enriched cells were CD4 positive (A). CD4 positive lymphocytes were then subjected to gate on CD14 (A). None of the CD4 enriched cells expr essed CD14 (monocytes) (B). CD4 positive lymphocytes were subjected to gate on CXCR4 (C) or CCR5 (D), or gate on CXCR4+/CCR5+ (E). Percent of CD4/ CXCR4 cells is ~ 80 % of enriched population, and percent of CD 4/CCR5 cells is ~ 20 % of enriched population. Percent of CD4+/CXCR4+/CCR5+ T ly mphocytes is ~ 20 %.

PAGE 113

113 Figure B-2. Receptor expression profile for CD4 enriched lymphocytes, donor C. CD4 enriched population was gated on CD4 expression (A). ~ 80 % of enriched cells were CD4 positive (A). CD4 positive lymphocytes were then subjected to gate on CD14 (B). None of the CD4 enriched cells expr essed CD14 (monocytes) (B). CD4 positive lymphocytes were subjected to gate on CXCR4 (C) or CCR5 (D), or gate on CXCR4+/CCR5+ (E). Percent of CD4/ CXCR4 cells is ~ 72 % of enriched population, and percent of CD 4/CCR5 cells is ~ 25 % of enriched population. Percent of CD4+/CXCR4+/CCR5+ T ly mphocytes is ~ 15 %.

PAGE 114

114 Table B-1. Intracellular PI leve ls reported in ng/mL and mM for PBMC treated with 1, 10, or 100 mM IDV or RTV for 3, 18, or 48 hours. Each value was converted from ng/mL as cited in figure 2-3. Ng/mL were converted to grams, then converted to mol using the equation g/MW=mol. Mol were conve rted to molarity using the equation M=mol/L, where the volume was calculated using the volume of 1 million cells (the number of cells in the sample tested). Drug Extracelluar [C] Applied to Cells ( M) Time Cells Exposed to Drug (Hours) Intracellular PI Level (ng/mL) Intracellular PI Level ( M) IDV 1 3 0.92 3.3 IDV 1 18 0.88 3.1 IDV 1 48 0.61 2.1 IDV 10 3 3.72 13.1 IDV 10 18 3.12 11.1 IDV 10 48 3.27 11.5 IDV 100 3 30.3 106 IDV 100 18 37.0 130 IDV 100 48 36.4 128 RTV 1 3 0.66 2.3 RTV 1 18 1.39 4.9 RTV 1 48 0.32 1.1 RTV 10 3 5.76 20 RTV 10 18 5.36 18.6 RTV 10 48 3.38 11.7 RTV 100 3 242 839.1 RTV 100 18 3088 2059 RTV 100 48 594 1000

PAGE 115

115 Table B-2. Intracellular concen tration of RTV in U937 cells treated with 1.0 mM RTV for 18 hours, reported in ng/mL and mM. Each va lue was converted from ng/mL as cited in figure 2-3. Ng/mL were converted to gr ams, then converted to mol using the equation g/MW=mol. Mol were converted to molarity using the equation M=mol/L, where the volume was calculated using the volume of 1 million cells (the number of cells in the sample tested). Sample I.D. (experiment and replicate #) Intracellular RTV Level (ng/mL) Intracellular RTV Level ( M) Expt. #1, Replicate # 1 6.4 6.9 Expt. #1 Replicate # 2 3.2 4.5 Expt. #2, Replicate #1 1.7 2.9 Expt. #2, Replicate #2 2.0 3.6 Expt. #2, Replicate #3 1.6 2.9

PAGE 116

116 Table B-3. Baseline intracellular RTV levels for unstimulated or PHA stimulated PBMC treated with 1 M RTV for 18 hours at 37oC in 2 % or 10% serum. Each value was converted from ng/mL as cited in figure 23. Ng/mL were converted to grams, then converted to mol using the equation g/MW=m ol. Mol were converted to molarity using the equation M=mol/L, where the volum e was calculated using the volume of 1 million cells (the number of cells in the sample tested). Donor and Replicate PHA Stimulated? [Serum] in Media (%) Intracellular RTV Level (ng/mL) Intracellular RTV Level ( M) Donor #1, Replicate #1 YES 2 0.29 4.6 Donor #1, Replicate #2 YES 2 0.32 5 Donor #1, Replicate #3 YES 2 0.28 4.4 Donor #1, Replicate #1 NO 2 0.34 5.4 Donor #1, Replicate #2 NO 2 0.65 10.2 Donor #1, Replicate #3 NO 2 0.26 4.1 Donor #2, Replicate #1 YES 10 0.66 10.4 Donor #2, Replicate #2 YES 10 0.50 7.6 Donor #1, Replicate #1 NO 10 BLQ BLQ Donor #1, Replicate #2 NO 10 BLQ BLQ Donor #2, Replicate #1 YES 10 BLQ BLQ Donor #2, Replicate #2 YES 10 BLQ BLQ

PAGE 117

117 Table B-4. Intracellular RTV levels in stimulat ed PBMC in 10 % serum treated with 1.0 mM or 10.0 mM RTV for 18 hours prior to observati on of RTV efflux at 4oC or 37oC for 0 (baseline; immediately after 18 hour drug lo ad), 10, 20, 40, or 60 minutes. Each value was converted from ng/mL as cited in figur e 2-3. Ng/mL were converted to grams, then converted to mol using the equation g/MW=mol. Mol were converted to molarity using the equation M=mol/L, where the volume was calculated using the volume of 1 million cells (the number of cells in the sample tested). Donor # [RTV] Applied to PBMC Time Cells Allowed to Efflux(Minutes) Temperature Of Efflux (C) Intracellular RTV (ng/mL) Intracellular RTV M) 1 1.0 0 4 BLQ BLQ 1 1.0 10 4 BLQ BLQ 1 1.0 20 4 BLQ BLQ 1 1.0 40 4 BLQ BLQ 1 1.0 60 4 BLQ BLQ 1 1.0 0 37 BLQ BLQ 1 1.0 10 37 BLQ BLQ 1 1.0 20 37 BLQ BLQ 1 1.0 40 37 BLQ BLQ 1 1.0 60 37 BLQ BLQ 1 10.0 0 4 0.98 11.2 1 10.0 10 4 0.98 11.2 1 10.0 20 4 0.94 10.8 1 10.0 40 4 1.5 17.6 1 10.0 60 4 1.96 17.8 1 10.0 0 37 0.34 6.8 1 10.0 10 37 BLQ BLQ 1 10.0 20 37 BLQ BLQ 1 10.0 40 37 BLQ BLQ 1 10.0 60 37 BLQ BLQ 2 1.0 0 4 0.50 7.6 2 1.0 10 4 0.39 5.9 2 1.0 20 4 0.45 6.6 2 1.0 40 4 0.39 5.9 2 1.0 60 4 0.82 12.4

PAGE 118

118 2 1.0 0 37 0..67 10.4 2 1.0 10 37 BLQ BLQ 2 1.0 20 37 BLQ BLQ 2 1.0 40 37 BLQ BLQ 2 1.0 60 37 BLQ BLQ 2 10.0 10 37 BLQ BLQ 2 10.0 20 37 BLQ BLQ 2 10.0 40 37 BLQ BLQ 2 10.0 60 37 BLQ BLQ

PAGE 119

119 Table B-5. Efflux of RTV in stimulated or uns timulated PBMC in 2 % serum treated with 1.0 mM RTV for 18 hours. Efflux of RTV was observed at 40C, 20oC, or 37oC for 0, 10, 20, 40, 60, or 120 minutes post 18 hour RTV treatment. Each value was converted from ng/mL as cited in figure 2-3. PHA Stimulated? Time Cells Allowed to Efflux(Minutes) Temperature Of Efflux (C) Intracellular RTV (ng/mL) Intracellular RTV M) YES 0 4 0.29 4.6 YES 10 4 0.32 5 YES 20 4 0.29 4.6 YES 40 4 BLQ BLQ YES 60 4 0.29 4.6 YES 120 4 BLQ BLQ YES 0 20 0.32 5 YES 10 20 0.27 4.3 YES 20 20 BLQ BLQ YES 40 20 BLQ BLQ YES 60 20 BLQ BLQ YES 120 20 BLQ BLQ YES 0 37 0.28 4.4 YES 10 37 BLQ BLQ YES 20 37 BLQ BLQ YES 40 37 BLQ BLQ YES 60 37 BLQ BLQ YES 120 37 BLQ BLQ NO 0 4 0.52 8.3 NO 10 4 0.52 8.3 NO 20 4 0.5 7.8 NO 40 4 0.53 8.6 NO 60 4 0.57 9.1 NO 120 4 0.52 8.3 NO 0 20 0.65 10.2 NO 10 20 0.31 5.3 NO 20 20 BLQ BLQ NO 40 20 0.40 6.3 NO 60 20 0.44 6.9 NO 120 20 BLQ BLQ NO 0 37 0.26 4.1 NO 10 37 BLQ BLQ NO 20 37 BLQ BLQ NO 40 37 BLQ BLQ NO 60 37 BLQ BLQ NO 120 37 BLQ BLQ

PAGE 120

120 Table B-6. Efflux of RTV in stimulated or uns timulated PBMC in 10 % serum treated with 1.0 mM RTV for 18 hours. Efflux of RTV wa s observed at 40C, 20oC, or 37oC for 0, 10, 20, 40, 60, or 120 minutes post 18 hour RTV treatment. Each value was converted from ng/mL as cited in figure 2-3. PHA Stimulated? Time Cells Allowed to Efflux(Minutes) Temperature Of Efflux (C) Intracellular RTV (ng/mL) Intracellular RTV M) YES 0 4 BLQ BLQ YES 10 4 BLQ BLQ YES 20 4 BLQ BLQ YES 40 4 BLQ BLQ YES 60 4 BLQ BLQ YES 120 4 BLQ BLQ YES 0 20 BLQ BLQ YES 10 20 BLQ BLQ YES 20 20 BLQ BLQ YES 40 20 BLQ BLQ YES 60 20 BLQ BLQ YES 120 20 BLQ BLQ YES 0 37 BLQ BLQ YES 10 37 BLQ BLQ YES 20 37 BLQ BLQ YES 40 37 BLQ BLQ YES 60 37 BLQ BLQ YES 120 37 BLQ BLQ NO 0 4 BLQ BLQ NO 10 4 BLQ BLQ NO 20 4 BLQ BLQ NO 40 4 BLQ BLQ NO 60 4 BLQ BLQ NO 120 4 BLQ BLQ NO 0 20 BLQ BLQ NO 10 20 BLQ BLQ NO 20 20 BLQ BLQ NO 40 20 BLQ BLQ NO 60 20 BLQ BLQ NO 120 20 BLQ BLQ NO 0 37 BLQ BLQ NO 10 37 BLQ BLQ NO 20 37 BLQ BLQ NO 40 37 BLQ BLQ NO 60 37 BLQ BLQ NO 120 37 BLQ BLQ

PAGE 121

121

PAGE 122

122 LIST OF REFERENCES 1. Acosta, E. P., T. N. K akuda, R. C. Brundage, P. L. Anderson, and C. V. Fletcher .2000. Pharmacodynamics of human immunode ficiency virus type 1 protease inhibitors. Clin. Infect. Dis. 30 Suppl 2:S151-S159. 2. Agarwal, S, Pal, D, Mitra, AK. 2007 Both P-gp and MRP2 mediate transport of Lopinavir, a protease inhib itor.. Int. J. Pharm. 339 (1-2):139-47. 3. Ahmed, S.A., Gogal, R.M. and Walsh, J.E. 1994. A new rapid and simple nonradioactive assay to monitor and determine the proliferation of lymphocytes: An alternative to [3H]thymidine incorp oration assays. J. Immunol. Meth. 170 211. 4. Albermann, A., Hubertus, F., Winnerthal, S., Zgraggen, K., Volk, C., Hoffmann, MM, Haefeli, WE, Weiss, J. 2005. MRP1/ABCC1, MRP2/ABCC2, BCRP/ABCG2, and PXR in peripheral blood m ononuclear cells and their rela tionship with the expression in intestine and liver. Biochemical Pharmacology. 70:949. 5. Ambudkar, SV., Kim, I., Sauna, Z. 2006. The power of the pump: Mechanisms of action of P-glycoprotein (ABCB1). European Journal of Pharm. Sci. 27:392. 6. Anderson, PL., Fletvher, CV. 2004. Updated clinical pharm acologic considerations for HIV-1 protease inhibitors. 1(1):33-9. 7. Andreana, A, Aggarwal, S, Gollapudi, S, Wien, D, Tsuruo, T, Gupta, S. 1996. Abnormal expression of a 170-kilodalton P-glycoprotein encoded by MDR1 gene, a metabolically active efflux pump, in CD4+ a nd CD8+ T cells from patients with human immunodeficiency virus type 1 infect ion. AIDS Res Hum Retroviruses. 10;12(15):14571462. 8. Arie, K., Gali, Y., El-Abdellati, A., Heyndrickx, L., Janssens, W., Vanham, G 2006. Replicative fitness of CCR5-using and CX CR4-using human immunodeficiency virus type 1 biological clones. Virology 347 ; 65 74 9. Aquaro S, Svicher V, Schols D, Pollicita M, Antinori A, Balzarini J, Perno CF. 2006. Mechanism s underlying activity of antir etroviral drugs in HIV-1-infected macrophages: new therapeutic st rategies. J. Leukoc. Biol. 80 (5):1103-10. 10. Aquaro, S., C. F. Perno, E. Balestra, J. Balzarini, A. Cenci, M. Francesconi, S. Panti, F. Serra, N. Villani, and R. Calio 1997. Inhibition of replication of HIV in

PAGE 123

123 primary monocyte/macrophages by different anti viral drugs and comparative efficacy in lymphocytes. J. Leukoc. Biol. 62:138-143. 11. Baldwin, CE., Sanders, RW., Berkout, B. 2003. Inhibiting HIV-1 Entry with Fusion Inhibitors. Current Medicinal Chemistry. 10;1633-1642 1633 12. Barbara, L., Rutella, S., Leone, G., Vella, S., Cauda, R 2001. HIV-Protease Inhibitors Contribute to P-Glycoprotein Efflux Function Defect in Peripheral Blood Lymphocytes From HIV-Positive Patients Receiving HAART. Journal of Acquired Immune Def. Synd. 27(4), 1, 321-330. 13. Barrie, K. A., E. E. Perez, S. L. Lamers, W. G. Farmerie, B. M. Dunn, J. W. Sleasman, and M. M. Goodenow 1996. Natural variation in HIV-1 protease, Gag p7 and p6, and protease cleavage sites within ga g/pol polyproteins: amino acid substitutions in the absence of protease inhibitors in mothers and children infected by human immunodeficiency virus type 1. Virology 219:407-416. 14. Bleul, CC., Wu, L., Hoxie, JA., Springer, TA., Mackay, CR. 1997. PNAS. The HIV coreceptors CXCR4 and CCR5 are differentia lly expressed and regulated on human T lymphocytes. 94:1925-1930. 15. Bode, H., L. Lenzner, O. H. Kraemer, A. J. Kroesen, K. Bendfeldt, J. D. Schulzke, M. Fromm, G. Stoltenburg-Didinger, M. Zeitz, and R. Ullrich. 2005. The HIV protease inhibitors saquinavir ritonavir, and nelfinavir induce apoptosis and decrease barrier function in human intestinal epithelial cells. Antivir. Ther. 10:645-655. 16. Boulassel, M. R., G. H. Smith, N. Gilmore, M. Klein, T. Murphy, J. MacLeod, R. LeBlanc, J. Allan, P. Rene, R. G. Lalonde, and J. P. Routy 2003. Interleukin-7 levels may predict virological response in advanced HIV-1-infected patients receiving lopinavir/ritonavir-based therapy. HIV. Med. 4:315-320. 17. Brockman, M., Tanzi, G ., Walker, B., Allen, T. 2006. Use of a novel GFP reporter cell line to examine replicati on capacity of CXCR4and CCR5-tropic HIV-1 by flow cytometry. Journal of Virological Methods 131;134. 18. Callaghan, R., Ford, RC., Kerr, DI 2005. The translocation mechanism of Pglycoprotein. FEBS Letters. 580:1056. 19. Cascorbi, I 2006. Role of pharmacogenetics of ATP-binding cassette transporters in the pharmacokinetics of drugs. Pharmacology & Therapeutics 112 (2006) 457 473. 20. Chaillou, S., Durant, J, Garrafo, R. Georgenthum, E. Roptin, C., Clevenbergh, P., Dunais, B, Mondain, V., Roger, PM., Dellamonica, P. 2002. Intracellular Concentration of Protease Inhi bitors in HIV-1Infected Patients: Correlation with MDR1 Gene Expression and Low Dose of Ritonavir. HIV-1 Clin. Trials. 3(6):493.

PAGE 124

124 21. Chandler, B., Almond, L., Ford, J., Ow en, P., Hoggard, P., Khoo, S., Back, D 2003. The Effects of Protease Inhibitors and Nonnucleoside Reverse Transcriptase Inhibitors on P-Glycoprotein Expression in Pe ripheral Blood Mononuclear Cells In Vitro. JAIDS. 33:551. 22. Chang C, Bahadduri PM, Polli JE, Swaan, PW, Ekins S. 2006. Rapid identification of P-glycoprotein substrates and inhibitors. Drug Metab Dispos. 34(12):197684 23. Chavan, S., S. Kodoth, R. Pahwa, and S. Pahwa 2001. The HIV protease inhibitor Indinavir inhibits cell-cycle progression in vitro in lym phocytes of HIV-infected and uninfected individuals. Blood 98:383-389. 24. Chun, T. W., D. Engel, S. B. Mizell, L. A. Ehler, and A. S. Fauci 1998. Induction of HIV-1 replication in latently infected CD4+ T cells using a combination of cytokines. J. Exp. Med. 188:83-91. 25. Clemente, J. C., R. M. Coman, M. M. Thiaville, L. K. Janka, J. A. Jeung, S. Nukoolkarn, L. Govindasamy, M. gbandj e-McKenna, R. McKenna, W. Leelamanit, M. M. Goodenow, and B. M. Dunn 2006. Analysis of HIV1 CRF_01 A/E Protease Inhibitor Resistance: Structural Determinants for Maintaining Sensitivity and Developing Resistance to Atazanavir. Biochemistry 45:5468-5477. 26. Clemente, J. C., R. Hemrajani, L. E. Blum, M. M. Goodenow, and B. M. Dunn 2003. Secondary mutations M36I and A71V in the human immunodeficiency virus type 1 protease can provide an advantage for the emergence of the primary mutation D30N. Biochemistry 42:15029-15035. 27. Clemente, J. C., R. E. Moose, R. Hemraj ani, L. R. Whitford, L. Govindasamy, R. Reutzel, R. McKenna, M. gbandje-McKe nna, M. M. Goodenow, and B. M. Dunn 2004. Comparing the accumulation of activean d nonactive-site mutations in the HIV-1 protease. Biochemistry 43:12141-12151. 28. Coberley, C. R., J. J. Kohler, J. N. Brown, J. T. Oshier, H. V. Baker, M. P. Popp, J. W. Sleasman, and M. M. Goodenow 2004. Impact on genetic networks in human macrophages by a CCR5 strain of human immunodeficiency virus type 1. J. Virol. 78:11477-11486. 29. Coffin, JM., Hughes, SH., Varmus, HE. 1997. Retroviruses. 30. Compain S Durand-Gasselin L Grassi J, Benech H 2007. Im proved method to quantify intracellular zidovudine monoand triphosphate in peripheral blood mononuclear cells by liquid chromatogra phy-tandem mass spectrometry. J. Mass Spectrom. 42(3):389-404.

PAGE 125

125 31. Cruikshank, W. W., K. Lim, A. C. Theodore, J. Cook, G. Fine, P. F. Weller, and D. M. Center. 1996. IL-16 inhibition of CD3-depe ndent lymphocyte activation and proliferation. J. Immunol. 157:5240-5248. 32. David, S. A., M. S. Smith, G. J. Lopez, I. Adany, S. Mukherjee, S. Buch, M. M. Goodenow, and O. Narayan 2001. Selective transmission of R5-tropic HIV type 1 from dendritic cells to resting CD4+ T cells. AIDS Res. Hum. Retroviruses. 17 :59-68. 33. Deeley, RG., Cole, SPC. 2005. Substrate recognition and transport by multidrug resistance protein 1 (ABCC1). FE BS Letters. 580 (2006) 1103. 34. Detels, R., A. Munoz, G. McFarlane, L. A. Kingsley, J. B. Margolick, J. Giorgi, L. K. Schrager, and J. P. Phair 1998. Effectiveness of potent antiretroviral therapy on time to AIDS and death in men with known HIV infection duration. Multi center AIDS Cohort Study Investigators. JAMA 280:1497-1503. 35. Dou H, Morehead J, Destache CJ, Kingsley JD, Shlyakhtenko L, Zhou Y, Chaubal M, Werling J, Kipp J, Rabinow BE, Gendelman HE. 2007. Laboratory investigations for the m orphologic, pharmacokinetic, and an ti-retroviral properties of indinavir nanoparticles in human monocytederived macrophages. Virology. 358(1):148-58. 36. Dybul, M., A. S. Fauci, J. G. Bartlett, J. E. Kaplan, and A. K. Pau 2002. Guidelines for using antiretroviral agents among HIV-inf ected adults and adolescents. Ann. Intern. Med. 137:381-433. 37. Eagling, VA., Profit, L., Back, DJ. 1999. Inhibition of the CYP3A4-mediated metabolism and P-glycoprotein-mediated tran sport of the HIV-1 protease inhibitor saquinavir by grapefruit juice co mponents. J. Clin. Pharmacol. 48, 543. 38. Ergun-Longmire B Lin-Su K Dunn AM Chan L Ham K, Sison C, Stavola J, Vogiatzi MG 2006. Effects of protease inhibito rs on glucose tolerance, lipid m etabolism, and body composition in children and adolescents infected with human immunodeficiency virus. Endocr. Prac. 12(5):514-21. 39. Essajee, S. M., M. Kim, C. Gonzalez, M. Rigaud, A. Kaul, S. Chandwani, W. Hoover, R. Lawrence, H. Spiegel, H. Po llack, K. Krasinski, and W. Borkowsky 1999. Immunologic and virologic responses to HAART in severely immunocompromised HIV-1-infected children. AIDS 13:2523-2532. 40. Fletcher, CV 2007. Translating efficacy into effectiv eness in antiretroviral therapy: beyond the pill count. Drugs. 67(14):1969-79. 41. Ford, J., M. Boffito, A. Wildfire, A. Hill, D. Back, S. Khoo, M. Nelson, G. Moyle, B. Gazzard, and A. Pozniak 2004. Intracellula r and plasma pharmacokinetics of

PAGE 126

126 saquinavir-ritonavir, administered at 1,600/100 milligrams once daily in human immunodeficiency virus-infected pati ents. Antimicrob. Agents Chemother. 48:23882393. 42. Ford, J., P. G. Hoggard, A. Owen, S. H. Khoo, and D. J. Back 2003. A simplified approach to determining P-glycoprotein e xpression in peripheral blood mononuclear cell subsets. J. Immunol. Methods 274:129-137. 43. Ford, J., S. H. Khoo, and D. J. Back 2004. The intracellular pharmacology of antiretroviral protease inhibito rs. J. Antimicrob. Chemother. 54:982-990. 44. Ford, J., E. R. Meaden, P. G. Hoggard, M. Dalton, P. Newton, I. Williams, S. H. Khoo, and D. J. Back 2003. Effect of protease inhi bitor-containing regimens on lymphocyte multidrug resistance transporte r expression. J. Antimicrob. Chemother. 52:354-358. 45. Gayle HD, Hill GL 2001. Global impact of human i mmunodeficiency virus and AIDS. Clin Microbiol Rev. 14:327-35. 46. Ghaffari, G., D. J. Passalacqua, J. L. Caicedo, M. M. Goodenow, and J. W. Sleasman 2004. Two-year clinical and immune outcomes in human immunodeficiency virus-infected children who reconstitute CD4 T cells without control of viral replication after combination antiretr oviral therapy. Pediatrics 114:e604-e611. 47. Gloeckner, H., Jonuleit, T. and Lemke, H.D. 2001. Monitoring of cell viability and cell growth in a hollow-fiber bioreactor by use of the dye Alamar Blue. J. Immunol. Meth. 252 :131. 48. Goodenow, MM., Collman, RG. 2006. HIV-1 coreceptor preference is distinct from target cell tropism: a dual-parameter no menclature to define viral phenotypes J. Leukoc. Biol. 80(5):965-72. 49. Goodenow, M. M., S. L. Rose, D. L. Tuttle, and J. W. Sleasman 2003. HIV-1 fitness and macrophages. J. Leukoc. Biol. 74 :657-666. 50. Gonzalez, LMF., Brindeiro, RM., Aguiar, RS., Pereira, HS., Abreu, CM., Soares, MA., Tanuri, A 2004. Impact of Nelfinavir Re sistance Mutations on In Vitro Phenotype, Fitness, and Replication Capacity of Human Immunodefici ency Virus Type 1 with Subtype B and C Proteases. Antimicrob. Agents and Chem. 48(9):3552. 51. Granfors MT Wang JS Kajosaari LI Laitila J Neuvonen PJ Backman JT .Differential inhibition of cytochrom e P450 3A4, 3A5 and 3A7 by five human immunodeficiency virus (HIV) protease inhibito rs in vitro. 2006. Basic Clin. Pharmacol. Toxicol. 98(1):79-85. 52. Gruber A Wheat JC Kuhen KL Looney DJ Wong-Staal F. 2001. Differential effects of HI V-1 protease inhi bitors on dendritic cell imm unophenotype and function. J. Biol. Chem. 276(51):47840-3.

PAGE 127

127 53. Herbeuval, J. P., A. Boasso, J. C. Grivel, A. W. Hardy, S. A. Anderson, M. J. Dolan, C. Chougnet, J. D. Lifson, and G. M. Shearer 2005. TNF-related apoptosis-inducing ligand (TRAIL) in HIV-1-infected patient s and its in vitro production by antigenpresenting cells. Blood 105:2458-2464. 54. Herbeuval, J. P., J. C. Grivel, A. Boasso, A. W. Hardy, C. Chougnet, M. J. Dolan, H. Yagita, J. D. Lifson, and G. M. Shearer 2005. CD4+ T-cell death induced by infectious and noninfectious HIV-1: role of type 1 in terferon-dependent, TRAIL/DR5mediated apoptosis. Blood 106:3524-3531. 55. Herbeuval, J. P., J. Nilsson, A. Boasso, A. W. Hardy, M. J. Kruhlak, S. A. Anderson, M. J. Dolan, M. Dy, J. Andersson, and G. M. Shearer 2006. Differential expression of IFN-{alpha} and TRAIL/DR5 in lymphoid tis sue of progressor ve rsus nonprogressor HIV-1-infected patients. Proc Natl. Acad. Sci. U. S. A 103:7000-7005. 56. Hoetelmans, RM., Meenhorst, PL., Mulder, JW., Burger, DM., Koks, CH., Beijnen, JH. 1997. Clinical pharmacology of HIV protea se inhibitors: focus on saquinavir, indinavir, and ritonavir. PHar. World Sci. 19(4):159-75. 57. Huisman, MT., Smit, JW., Wiltshire, HR., Hoetelmans, RM., Meijnen, JH., Schinkel, AH 2001. P-glycoprotein limits oral avai lability, brain, and fetal penetration of Saquinavir even with high doses of Ritonavir. Mol. Pharmacol. 59(4):806-13. 58. Isgro, A., A. Aiuti, I. Mezzaroma, L. Ruc o, M. Pinti, A. Cossarizza, and F. Aiuti 2005. HIV type 1 protease inhibitors enhance bone marrow progenitor cell activity in normal subjects and in HIV t ype 1-infected patients. AI DS Res. Hum. Retroviruses 21:51-57. 59. Johnes, K., Horrard, PG., Khoo, S., Maher, B., Back, DJ 2000. Effect of alpha 1 acid glycoprotein on the intracellular accumu lation of the HIV protease inhibitors Saquinavir, ritonavir and Indinavir in vitro J. Clin. Pharmacol. 51:91-102. 60. Jorajuria S, Dereuddre-Bosquet N, Becher F, Martin S, Porcheray F, Garrigues A, S, Dormont D, Clayette P 2004. ATP binding cassette multidrug transporters limit the anti-HIV activity of zidovudi ne and indinavir in infected human macrophages Antivir Ther. 9(4):519-28. 61. Kaplan SS, Ferrari G, Wrin T Hellmann NS, Tomaras GD, Gryszowka VE Fiscus SA Weinhold KJ Hicks CB 2005. Longitudinal assessm ent of immune response and viral characteristics in HIVinfected patients with pr olonged CD4(+)/viral load discordance AIDS Res. Hum. Retroviruses. 21 (1):13-6.

PAGE 128

128 62. Kedzierska, K., S. M. Crowe, S. Turville, and A. L. Cunningham 2003. The influence of cytokines, chemokines and their receptors on HIV-1 replication in monocytes and macrophages. Rev. Med. Virol. 13 :39-56. 63. Khoo, S. H., P. G. Hoggard, I. Williams, E. R. Meaden, P. Newton, E. G. Wilkins, A. Smith, J. F. Tjia, J. Lloyd, K. Jones, N. Beeching, P. Carey, B. Peters, and D. J. Back 2002. Intracellular accumulation of human immunodeficiency virus protease inhibitors. Antimicrob. Agents Chemother. 46 :3228-3235. 64. Khoo S HIV and pharm acogenomics in 2007. 2007. Pharmacogenomics. 8(1):25-7. 65. Kim, A., Dintaman, S., Waddell, D., Silverman, J 1998. Saquinavir, an HIV Protease Inhibitor, Is Transported by P-Glycoprotein. Journal of Pharm acology and Experimental Therapeutics. 286: 1439 66. Kobayashi, Y., Yamashiro, T., Nagatake, H ., Yamamoto, T., Watanabe, N., Tanaka, H., Shigenobu, K., Tsuruo, T 1994. Expression and function of multidrug resistance p-glycoprotein in a cultured natural killer cell-rich population revealed by MRK16 monoclonal antibody and AHC-52. Biochem. Pharmacol. 48(8):1641-6. 67. Ledergerber, B., M. Egger, M. Opravil, A. Telenti, B. Hirschel, M. Battegay, P. Vernazza, P. Sudre, M. Flepp, H. Furrer, P. Francioli, and R. Weber 1999. Clinical progression and virological failu re on highly active antire troviral therapy in HIV-1 patients: a prospective cohort st udy. Swiss HIV Cohort Study. Lancet 353:863-868. 68. Ludescher, C., Thaler, J., Drach, D., Drach, J., Spitaler, M., Gatt ringer, C., Huber, H., Hofmann, J. 1992. Detection of activity of P-gl ycoprotein in human tumour samples using rhodamine 123. Br. J. Haematol. (1):161-8. 69. Lee, C., B. Tomkowicz, B. D. Freedman, and R. G. Collman 2005. HIV-1 gp120induced TNF-{alpha} production by primar y human macrophages is mediated by phosphatidylinositol-3 (PI-3) kinase and m itogen-activated protein (MAP) kinase pathways. J. Leukoc. Biol. 78:1016-1023. 70. Loo, TW., Clarke, DM 2005. Do drug substrates enter the common drug-bindning pocket of p-glycoprotein through gates? Biochemical and Biophysical Research Communications. 329:419. 71. Lucia, MB, Savarino, A, Straface, E, Golotta, C, Rastrelli, E, Matarrese, P, Rutella, S, Malorni, W, Cauda, R. 2005. Role of lymphocyte multidrug resistance protein 1 in HIV infection: expression, function, and conseq uences of inhibition. J. Acquir. Immune Defic. Syndr. 40(3):257-66. 72. Lucia, B., Rutella, S., Giuseppe, S., Vella, S., Cauda, R 2001. HIV-Protease Inhibitors Contribute to P-Glycoprotein Efflux Function Defect in Peripheral Blood Lymphocytes From HIV-Positive Patients Receiving HAART. AIDS Journal of Acquired Immune Deficiency Syndromes. 27(4), 1 321-330

PAGE 129

129 73. Ludescher, C., Pall, G., Irschick, E., Gastl, G 1998. Differentia l activity of pglycoprotein in normal blood lymphocyte subsets. British Journal of Haematology. 101:722-727. 74. Lum, J. J., D. J. Schnepple, and A. D. Badley 2005. Acquired T-cell sensitivity to TRAIL mediated killing during HIV infection is regulated by CXCR4-gp120 interactions. AIDS 19:1125-1133. 75. Mammano, F., Petit, C., Clavel, F. 1998. Resistance-Associated Loss of Viral Fitness in Human Immunodeficiency Virus Type 1: Phenotypic Analysis of Protease and gag Coevolution in Protease I nhibitor-Treated Patients. 72(9): 7632-7637. 76. McDonald, CK., Kuritzkes, DR. 1997. Human immunodeficiency virus type 1 protease inhibitors. 157 (9):951-9. 77. Meaden, E. R., P. G. Hoggard, P. Newton, J. F. Tjia, D. Aldam, D. Cornforth, J. Lloyd, I. Williams, D. J. Back, and S. H. Khoo 2002. P-glycoprotein and MRP1 expression and reduced ritonavir and sa quinavir accumulation in HIV-infected individuals. J. Antimicrob. Chemother. 50:583-588. 78. Melvin AJ Lennon S Mohan KM, Purnell JQ 2001. Metabo lic abnormalities in HIV type 1-infected children tr eated and not treated with prot ease inhibitors. Aids Res. Hum. Retro. 17(12):1117-23. 79. Morita, A., N. Suzuki, Y. Matsumoto, K. Hirano, A. Enomoto, J. Zhu, and K. Sakai 2000. p41 as a possible marker for cell death is generated by caspase cleavage of p42/SETbeta in irradiated MOLT-4 ce lls. Biochem. Biophys. Res. Commun. 278:627632. 80. Nicholson, J., Browning, SW., Hengel, RL., Lew, E., Gallagher, LE., RimLand, D. McDougal, SJ. 2001. CCR5 and CXCR4 Expression on Memory and Naive T Cells in HIV-1 Infection and Response to Highly Activ e Antiretroviral Thera py. AIDS Journal of Acquired Immune Deficiency Syndromes. 27(2):105-115. 81. Owen, A., Chandler, B., Back, DJ 2005. The implications of P-glycoprotein in HIV: friend or foe? Fundamental & Clinical Pharmacology 19:283. 82. Palella, F.J., Jr. et al. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection 1998. HIV Outpatient Study Investigators N Engl J Med. 338: 53-60. 83. Perez, E. E., S. L. Rose, B. Peyser, S. L. Lamers, B. Burkhardt, B. M. Dunn, A. D. Hutson, J. W. Sleasman, and M. M. Goodenow 2001. Human immunode ficiency virus type 1 protease genotype pred icts immune and viral respon ses to combination therapy with protease inhibitors (PIs) in PI-naive patients. J. Infect. Dis. 183:579-588.

PAGE 130

130 84. Perno, C. F., F. M. Newcomb, D. A. Davi s, S. Aquaro, R. W. Humphrey, R. Calio, and R. Yarchoan 1998. Relative potency of protease inhibitors in monocytes/macrophages acutely and chronically infected with human immunodeficiency virus. J. Infect. Dis. 178:413-422. 85. Perno, C., Svicher, V., Schols, D., Pollicita, M., Balzarini, J., Aquaro, S 2006. Therapeutic strategies toward s HIV-1 infection in macropha ges. Antiviral Res. 71(23):293-300 86. Peruzzi, M., C. Azzari, L. Ga lli, A. Vierucci, and M. M. De 2002. Highly active antiretroviral therapy restor es in vitro mitogen and an tigen-specific T-lymphocyte responses in HIV-1 perinatally infected children despite viro logical failure. Clin. Exp. Immunol. 128:365-371 87. Phenix, B. N., J. B. Angel, F. Mandy, S. Kravcik, K. Parato, K. A. Chambers, K. Gallicano, N. Hawley-Foss, S. Cassol, D. W. Cameron, and A. D. Badley 2000. Decreased HIV-associated T cell apoptosis by HIV protease inhibitors. AIDS Res. Hum. Retroviruses 16:559-567. 88. Phenix, B. N., C. Cooper, C. Owen, and A. D. Badley 2002. Modulation of apoptosis by HIV protease inhibitors. Apoptosis. 7:295-312. 89. Piccinini M Rinaudo MT Anselmino A Buccinn B Ramondetti C Dematteis A Ricotti E Palmisano L Mostert M Tovo PA 2005. The HIV protease inhibitors nelfinavir and saquinavir, but not a variety of HIV reverse tran scriptase inhibitors, adversely affect hum an proteas ome function. Antivir. Ther. 10(2):215-23. 90. Piccinini M Rinaudo MT Chiapello N Ricotti E Baldovino S Mostert M Tovo PA 2002. The hum an 26S proteasome is a targ et of antiretroviral agents. AIDS. 16(5):693-700. 91. Pilon, A., Lum, Jl, Dardon, J., Phenix, B., Douglas, R., Badley, A. 2002. Induction of Apoptosis by a Nonnucleoside Human Im munodeficiency Virus Type 1 Reverse Transcriptase Inhibitor. Antimic robial Agents and Chemotherapy. 46(8):2687. 92. Piot P, Bartos M, Ghys PD, Walker N, Schwartlander B 2001. The global impact of HIV/AIDS. Nature. 2001. 410:968-73. 93. Profit, L., Eagline, V., Back, D. 1999. Modulation of Pglycoprotein function in human lymphocytes and Caco-2 cell monolay ers by HIV-1 protease inhibitors AIDS. 13:1623. 94. Rodrguez-Nvoa S, Barreiro P Jimnez-Ncher I Soriano V 2006. Overview of the pharm acogenetics of HIV therapy Pharmacogenomics J. 6(4):234-45.

PAGE 131

131 95. Ronaldson PT, Bendayan R. 2006. HIV-1 viral envelope glycoprotein gp120 triggers an inflamm atory response in cultured ra t astrocytes and regu lates the functional expression of P-glycoprotei n. Mol. Pharmacol. 70(3):1087-98. 96. Roy, A., Schweighardt, B., Eckstein, L., Goldsmith, M., McCune, J. 2005. Enhanced Replication of R5 HIV-1 Over X4 HIV-1 in CD4+CCR5+CXCR4+ T Cells. J. Acquired Immune Def. Synd. 40:267. 97. Sankatsing SU, Cornelissen M, Kloost erboer N, Crommentuyn KM, Bosch TM, Mul FP, Jurriaans S, Huitema AD, Beijne n JH, Lange JM, Prins JM, Schuitemaker H. 2007. Antiviral Activity of HIV Type 1 Proteas e Inhibitors Nelfinavir and Indinavir in vivo is not Influenced by P-gl ycoprotein Activity on CD4+ T cells. AIDS Res. Hum. Retroviruses. 23(1):19-27. 98. Sankatsing, S., Beijnen, J., Schinkel, A., Joep, M ., Lane, A., Prinsl, JM. 2004. PGlycoprotein in Human Immunodeficiency Virus Type 1 Infection and Therapy. Antimicrobial Agents and Chemotherapy. 48(4):1073. 99. Schweighardt, B., Roy, A., Meiklejohn, D., Grace, E., Moretto, W., Heymann, J., Nixon, D. 2004. R5 Human Immunodefi ciency Virus Type 1 (HIV-1) Replicates More Efficiently in Primary CD4 T-Cell Cultures Than X4 HIV-1. Journal of Virology. 17(78); 9164. 100. Sleasman, J. W. and M. M. Goodenow 2003. 13. HIV-1 infecti on. J. Allergy Clin. Immunol. 111:S582-S592. 101. Sleasman, J. W., R. P. Nelson, M. M. Gooden ow, D. Wilfret, A. Hutson, M. Baseler, J. Zuckerman, P. A. Pizzo, and B. U. Mueller 1999. Immunoreconstitution after ritonavir therapy in childre n with human immunodeficien cy virus infection involves multiple lymphocyte li neages. J. Pediatr. 134:597-606. 102. Sloand, E. M., P. N. Kumar, S. Kim, A. Chaudhuri, F. F. Weichold, and N. S. Young 1999. Human immunodeficiency virus t ype 1 protease inhibitor modulates activation of peripheral blood CD4(+) T cells and decreases their susceptibility to apoptosis in vitro and in vivo. Blood 94 :1021-1027. 103. Smith, D. J., M. J. McGuire, M. J. Tocci, and D. L. Thiele. 1997. IL-1 beta convertase (ICE) does not play a requis ite role in apoptos is induced in T lymphoblasts by Fasdependent or Fas-independent CTL effector m echanisms. J. Immunol. 158:163-170. 104. Solas C, Simon N, Drogoul MP, Quaranta S, Frixon-Marin V, Bourgarel-Rey V, Brunet C, Gastaut JA, Durand A, Lacarelle B, Poizot-Martin I 2007. Minimal effect of MDR1 and CYP3A5 genetic polymorphism s on the pharmacokinetics of indinavir in HIV-infected patients. Br J Clin Pharmacol. 64(3):353-62.

PAGE 132

132 105. Song, W., C. M. Wilson, S. Allen, C. Wang, Y. Li, R. A. Kaslow, and J. Tang. 2006. Interleukin 18 and human immunodeficiency vi rus type I infection in adolescents and adults. Clin. Exp. Immunol. 144:117-124. 106. Storch CH, Theile D, Lindenm aier H, Haefeli WE, Weiss J 2007. Comparison of the Inhibitory Activity of AntiHIV Drugs on P-glycoprotein. Biochemical Pharmacology. 15;73(10):1573-81 107. Strmer E, von Moltke LL, Perloff MD, Greenblatt DJ 2002. Differential modulation of P-glycoprotein expression and activity by non-nucleoside HIV-1 reverse transcriptase inhibitors in cell culture. Pharm Res. 19(7):1038-45. 108. Sufka SA, Ferrari G, Gryszowka VE, Wrin T Fiscus SA Tomaras GD, Staats HF Patel DD Sempowski GD Hellmann NS Weinhold KJ Hicks CB 2003. Prolonged CD4+ cell/v irus load discordance during treatment with protease inhibitor-based highly active antiretroviral th erapy: immune respons e and viral control. J Infect Dis. 187(7):1027-37. 109. Turriz iani, O., Antonelli, G. 2004. Host factors and e fficacy of antiretroviral treatment. New Microbio. 27(2 Suppl 1):63-9. 110. Tuttle, D. L., C. R. Coberley, X. Xie, Z. C. Kou, J. W. Sleasman, and M. M. Goodenow 2004. Effects of human immunodeficien cy virus type 1 infection on CCR5 and CXCR4 coreceptor expression on CD4 T lymphocyte subsets in infants and adolescents. AIDS Re s. Hum. Retroviruses 20 :305-313. 111. Vasquez, E. 2007. Maraviroc--new HIV drug. Emerging options need to be used wisely. Posit. Aware. 2007. 18(4):18-9 112. Vasquez EM, Petrenko Y Jacobssen V, Sifontis NM Testa G Sankary H Benedetti E 2005. An assessm ent of P-glycoprotein expr ession and activity in peripheral blood lymphocytes of transplant candidates. Transplant Proc. 37(1):175-7. 113. Weichold, F. F., J. L. Bryant, S. Pati, O. Barabitskaya, R. C. Gallo, and M. S. Reitz, Jr. 1999. HIV-1 protease inhibitor ritonavir modulates susceptibility to apoptosis of uninfected T cells. J. Hum. Virol. 2:261-269. 114. Whelan KT Lin CL, Cella M McMichael AJ Austyn JM Rowland-Jones SL. 2003. The HIV protease inhibitor indinavir red uces immature dendritic cell transendothelial migratio n. Eur. J. Immunol. 33 (9):2520-30.

PAGE 133

133 115. Wolf, T., S. Findhammer, B. Nolte, E. B. Helm, and H. R. Brodt 2003. Inhibition of TNF-alpha mediated cell death by HIV-1 specifi c protease inhibitors. Eur. J. Med. Res. 8:17-24. 116. Yeni, P. 2006. Update on HAART in HIV-1. Journal of Hepatology 44 (2006) S100 S103 117. Zaccarellil, M., Tozzi, V., Perno, CF., Antiori, A. 2004. The challenge of antiretroviral-drug-resistant HI V: is there any possible clin ical advantage? Curr. HIV Res. 2:283-292. 118. Zennou, R., Mammano, F., Paulous, S., Mathez, D., Clavel, F. 1998. Loss of Viral Fitness Associated with Multiple Gag and Gag-Pol Processing Defects in Human Immunodeficiency Virus Type 1 Variants Select ed for Resistance to Protease Inhibitors In Vivo J. Virology. 72 (4): 3300-3306. 119. Zhang, M., J. Drenkow, C. S. Lankford, D. M. Frucht, R. L. Rabin, T. R. Gingeras, C. Venkateshan, F. Schwartzkopff, K. A. Clouse, and A. I. Dayton 2006. HIV regulation of the IL-7R: a viral mechanism for enhancing HIV-1 replication in human macrophages in vitro. J. Leukoc. Biol. 79(6):1328-38.

PAGE 134

134 BIOGRAPHICAL SKETCH Christina Gavegnano earned a high school diplom a from Nort h Marion High School, Ocala, Florida, and a Bachelors of Science De gree in Microbiology and Cell Science from the University of Florida, Gainesville, Florida.