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,1
* Department of Laboratory Medicine, University of Washington School of Medicine, Seattle, Washington, USA; and
Program in Infectious Disease, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
1 Correspondence: Department of Laboratory Medicine, 960 Republican St., Seattle, WA 98109. E-mail: tzhu{at}u.washington.edu
ABSTRACT
The role of blood monocytes in HIV-1 infection is a relatively new field of interest. What happens to HIV-1 in monocytes and their relationship to CD4+ T cells before, during, and after suppressive antiretroviral therapy (ART) is largely unstudied. Here, considering that diversity is a good indicator of continued replication over time, we evaluated the effect of ART on HIV-1 in blood monocytes and CD4+ T cells by examining the diversity of HIV-1 from 4 infected patients who underwent and stopped therapy. We determined diversity and compartmentalization of HIV-1 between blood monocytes and CD4+ T cells in each patient in relationship to their ART regimens. Our data indicate that the rate of HIV-1 diversity increase in monocytes during therapy was significantly higher than in CD4+ T cells (P<0.05), suggesting that HIV-1 present in monocytes diversify more during therapy than in CD4+ T cells. Increased rates of HIV-1 compartmentalization between monocytes and CD4+ T cells while on therapy were also observed. These results suggest that ART inhibits HIV-1 replication in CD4+ T cells more than in blood monocytes and that better treatments to combat HIV-1 in monocytes/macrophages may be needed for a more complete suppression of HIV replication.
Key Words: treatment • leukocyte • diversity
INTRODUCTION
Since the advent of highly active antiretroviral therapy (HAART), people infected with HIV-1 have been able to live longer and healthier lives with the disease. However, recent discoveries about viral reservoirs [1 2 3 4 5 6 7 8 9 ] and drug sanctuaries [10 ] (review in [11 ]) in the body, which are relatively unaffected by drugs, have helped explain the difficulties in eradicating HIV-1 with HAART. Not only can these reservoirs maintain a silent virus population during therapy, they can continually produce virus and replenish viral pools in patients even during effective therapy [1 2 3 4 5 ].
Blood monocytes are released into the blood from bone marrow and circulate in the peripheral blood from 1 to 3 days before primarily differentiating into immature DC’s and different tissue macrophages. Thus, the identification of persistent HIV-1 in blood monocytes itself suggests ongoing renewal of infected monocytes by virus replication and/or recent infection of monocytes or their precursor cells [12 ]. Findings of HIV-1 replication in CD14+ monocytes support the theory that the HIV-1 pool in monocytes can be renewed as a result of viral replication [12 ]. Monocytes could also be infected by viruses produced from tissues as infected monocytes carry provirus and differentiate into tissue macrophages where HIV can productively replicate [12 13 14 15 16 ]. It is possible that although infected monocytes produce relatively low amounts of virus while circulating in peripheral blood, they are a major carrier of virus into tissue sites where a greater amount of HIV-1 can be subsequently produced [12 13 14 15 16 ].
Recent studies on patients with and without HAART demonstrated that virus isolated from blood monocytes can evolve separately from virus in CD4+ T cells, suggesting an independent evolutionary pathway within monocyte-macrophage cell lines [12 , 13 ]. Later studies showed similar results of distinct HIV-1 populations in blood monocytes and CD4+ T cells in patients with prolonged, suppressive HAART [14 , 15 ]. Taken together, these studies suggest that HIV-1 in blood monocytes could represent replicating HIV-1 in the presence of antiretroviral therapy (ART), and thus should be the target for new ART drugs to achieve a more complete suppression of HIV-1 replication.
To further determine the influence of ART on HIV-1 genetic evolution and replication in monocytes compared with CD4+ T cells, we examined sequences of the second constant region to the fifth variable region (C2 to V5) of HIV-1 gp120 from CD14+ monocytes, CD4+ T cells, and plasma samples from various time points before, during, and after ART from 4 patients. Compartmentalization calculations and diversity analyses were performed in order to determine what impact therapy had on the evolution of the virus between the two cell types. We found that ART has a consistent effect on the rate that HIV-1 evolves in these two cell types, allowing diversity in monocytes to increase throughout therapy while adversely impacting the rate of diversity increase in CD4+ T cells. Compartmentalization was also seen to occur either during therapy or soon after.
MATERIALS AND METHODS
Study patients
Four patients with varying conditions of discontinued therapy were chosen from the Seattle primary infection cohort and were recruited by the University of Washington Primary Infection Clinic [16
, 17
]. They were chosen based on availability of samples and variety of therapy situations. All were homosexual males; 3 were Caucasians (1180, 1175, and 1396) and 1 was Hispanic (1184). As shown in Fig. 1A
, 1180 was late-treated; receiving a combination of ddl, Efavirenz, and 3TC 1247 days post-seroconversion. This patient stayed on therapy and was effectively treated (maintaining a viral load at or near undetectable) for 439 days and was followed for an additional period of 924 days after therapy was stopped. Patient 1175 (Fig. 2A
) began therapy with AZT, 3TC, and Indinavir 264 days post-seroconversion. His only deviation from this drug regimen during his 777 days on therapy was a 74-day-long drug study with an unknown drug or placebo. The latest time point studied fell 1575 days after the end of therapy. Interestingly, with this therapy, the patient was unable to maintain plasma viral load at or below detectable levels after 464 days. The viral load continued to increase even when the medication was taken for the last 406 days of therapy and reached up to 1710 copies/ml. Patient 1396 (Fig. 3A
) was early treated and began a regimen of 3TC, d4T, and Indinavir 30 days after seroconversion. This regimen was continued for 611 days until Indinavir was replaced with Nevirapine. This new regimen was followed for another 420 days for a total of 1031 days of therapy. The latest time point studied from this patient was 985 days post-therapy. These drugs were effective for the entire duration of therapy. Patient 1184 (Fig. 4A
) was very early treated, and began a combination of AZT, 3TC, and Indinavir just 1 day after seroconversion. He was effectively treated for 353 days and followed for 1928 days after stopping therapy.
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Isolation of HIV-1 DNA and RNA
Genomic DNA containing HIV-1 viral DNA was isolated from CD4+ T cells and CD14+ monocytes using the QiAmp DNA mini kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol as described previously [12
, 13
]. HIV-1 viral RNA was isolated and purified from plasma using the QiAmp Viral RNA Mini Kit (Qiagen) according to the manufacturer’s protocol. This RNA was then reverse transcribed into cDNA as described previously [12
, 13
].
PCR, cloning, sequencing, and sequence analysis
Cellular DNA and reverse transcribed cDNA was used for two rounds of nested PCR. The C2-V5 region of the env gene was amplified with outer primers P5-3 (residues 6848 to 6877 of the HIV-1 HXB2 sequence in GenBank; 5‘-ATCCTTTGAGCCAATTCCCATACATTATTG-3‘) and P2 [12
] and inner primers P5 [12
] and P120 (residues 7978 to 8001of the HIV-1 HXB2 sequence in GenBank; 5‘-AAGAACCCAAGGAACATAGCTCC-3‘). The PCR conditions used have been described previously [12
, 13
]. All PCR products were gel-purified and cloned using the pcR2.1 TOPO TA Cloning Kit, (Invitrogen, Carlsbad, CA) with restriction digest mapping to identify positive clones. To avoid template re-sampling, a special procedure of limiting dilution PCR, cloning, and sequencing with the Big Dye Terminator kit (Big Dye v3.1 Cycle, Foster City, CA) was used [13
]. Sequences were edited with Sequencher 4.1 to eliminate possible computer error within the sequences, then aligned with Clustal X [18
] and edited again using MacClade [19
] software for misaligned insertions and deletions. Phylogenetic trees were created from the MacClade files using PAUP *4.0b10 software (Sinauer Associates, Inc., Sunderland, MA). These neighbor-joining, maximum likelihood trees used the HKY85 model of substitution with estimates on the transition/transversion ratio and 
-shape parameters for gamma distribution. Distance branches were also calculated with this software and used to calculate genetic distances between sequences (diversity) using the HKY85+G+I model of evolution. P values for diversity were obtained using an unpaired t test with Welch’s Correction (GraphPad Software, Inc., San Diego, CA). Compartmentalization P values were calculated using Slatkin and Maddison’s method for measuring the restriction of gene flow among populations [20
], which was changed slightly for use in HIV-1 populations [21
]. A maximum likelihood tree was generated, and each compartment was designated a number (Monocytes=1, T Cells=2), and the minimum number of steps between the compartments was measured. A total of 1000 random trees were then generated, and the distribution of steps was compared with the number of steps in the original tree. If the number of steps in the original tree falls well outside the random distribution of steps (inside 5%), compartmentalization has occurred (P<0.05).
Nucleotide sequence numbers in Genbank: DQ852990–DQ853378.
RESULTS
HIV-1 sequence diversity in blood monocytes and CD4+ T cells during the course of discontinued ART
1180
At 14 days post-seroconversion, HIV-1 C2-V5 sequences were homogeneous in both blood monocytes and CD4+ T cells, which is consistent with previous findings [13
]. After 1194 days without ART, HIV-1 within CD4+ T cells had a significantly higher diversity than in the monocytes (P=0.001, Fig. 1C
and 1D
). Interestingly, from 50 days before therapy (1197 days post-seroconversion in Fig. 1A
and 1C
) to 98 days after the end of therapy (1734 days post -seroconversion), the rate of increase of HIV-1 diversity within monocytes was significantly higher than in CD4+ T cells (P=0.0097). During this period the rate of increase of diversity within monocytes was significant, while not significant in CD4+ T cells (CD4+ T cells, P=0.15; Monocytes, P=0.001). However, 924 days after stopping ART (2560 days post-seroconversion, Fig. 1A
and 1C
), HIV-1 sequences in the CD4+ T cells had become more diverse, whereas HIV-1 diversity in monocytes remained stable.
1175
Patient 1175, similar to 1180, had a higher rate of HIV-1 diversity in monocytes compared with CD4+ T cells during therapy, although this was not quite significant (P=0.07). Diversity during this time for both CD4+ T cells and monocytes was significant (P<0.001), indicating replication during therapy. At 464 days after the initiation of ART, it could be seen that the diversity of HIV-1 in the monocytes is significantly higher than in CD4+ T cells (P=<0.001 Fig. 2C
and 2D
); however, between 313 days before therapy to 1031 days after therapy the monocyte derived HIV-1 diversifies at a slightly higher rate than the CD4+ T cells (Fig. 2A)
. At the latest time point, 1575 days after the end of therapy, the CD4+ T cell virus had become significantly more diverse than the monocyte viruses (P=<0.001).
1396
Patient 1396 received early treatment, 30 days after seroconversion, and maintained low levels of plasma viral RNA until ceasing ART 1052 days post-seroconversion. As previously reported in patients with suppressive HAART [12
], the virus in the two cell types remained nearly homogenous, although HIV-1 in monocytes increased in diversity at a slightly higher rate than CD4+ T cells in the latter part of therapy (not significant) (Fig. 3C
and 3D)
. However, after stopping therapy, the viral load rebounded (Fig. 3A)
and the CD4+ T cell virus became significantly more diverse than the monocyte virus at day 1377 (P=<0.001, 325 days after stopping therapy) and 2021 days (P=<0.001, 985 days after stopping therapy) post-seroconversion (Fig. 3C
and 3D)
.
1184
Patient 1184 was very early treated, just 1 day after seroconversion. No difference was found in diversity at seroconversion; however, by day 353 post-therapy (707 days post-seroconversion, Fig. 4C
and 4D
), HIV-1 diversity was higher in the monocytes than in CD4+ T cells. During therapy, the rate of diversification was greater in the monocytes than in the CD4+ T cells, although this was not quite significant (P=0.056). After 2091 days post-seroconversion, the CD4+ T cell viruses were more diversified than the monocyte viruses (P=0.007, Fig. 3C
and 3D
).
Phylogenetic compartmentalization between CD4+ T Cells and monocytes during the course of discontinued ART
1180
During the first 1197 days in which patient 1180 was untreated, no evidence of compartmentalization was found (P=0.115, Fig. 1D
). However, after just 389 days of therapy (1454 days post-seroconversion), the virus showed significant compartmentalization (P=0.014) between the CD4+ T cells and monocytes. This compartmentalization of HIV-1 was maintained throughout the rest of the follow-up period. Plasma samples were taken 98 and 924 days after the cessation of therapy. The first time point indicated that plasma sequences are similar to both CD4+ T cells and monocytes, while the latter time point exhibited compartmentalization between the plasma and each cell type (See Fig. 1B
for phylogenetic tree of sequences).
1175
At seroconversion no compartmentalization was detectable; however, it was observed 464 days after the initiation of therapy (P=0.008, Fig. 2D
). At 582 and 1031 days after the cessation of therapy, the compartmentalization disappeared and did not return until 1575 days after therapy was stopped (2616 post-seroconversion, P=<0.001, Fig. 2D
; see Fig. 2B
for phylogenetic tree of sequences).
1396
This early treated patient displayed compartmentalization 801 days after initiating therapy (P=0.016, Fig. 3D
). He continued therapy for another 221 days, and the viral populations remained compartmentalized throughout the follow-up period of 985 days after the cessation of therapy (see Fig. 3B
for phylogenetic tree of sequences).
1184
Patient 1184 was the earliest treated, 1 day post-seroconversion, and showed compartmentalization 353 days after the end of therapy (P=0.011, Fig. 4D
), which is 707 days after seroconversion, and maintained this compartmentalization throughout the follow up period, which ended 1928 days after the cessation of therapy (see Fig. 4B
for phylogenetic tree of sequences).
Phylogenetic relationship of HIV-1 in plasma to monocytes and CD4+ T cells during the course of discontinued ART
1180
Plasma samples were taken at 1734 and 2560 days post-seroconversion. At 1734 days post-seroconversion (207 days after the initiation of therapy), several clusters of HIV-1 in monocytes and CD4 + T cells can be seen (Fig. 1B)
. Plasma viruses in this time point were similar to HIV-1 in both CD4+ T cells and blood monocytes (Fig. 1B)
. Plasma sequences are associated with the various groupings of T cells and monocytes, indicating that rebounding virus might be associated with monocytes as well as CD4+ T cells. The latest time point (924 days post-therapy) more clearly shows compartmentalization. Most of the CD4+ T cell viral sequences at this time point are sequestered at the bottom of the tree, and most of the monocyte and plasma viruses are located near the middle. Calculations show that the plasma, monocytes, and CD4+ T cells are all compartmentalized at this point (Fig. 1D)
; however, several plasma sequences that are similar to both CD4+ T cells and monocytes could be seen in the tree.
1175
The phylogenetic tree for this patient is interesting, as it could be seen that viruses have persisted from the beginning of infection throughout the study period. Similar sequences from every time point are located in the top portion of the tree, which indicates a conserved sequence throughout infection. A plasma sample was taken 464 days after the initiation of therapy. At this time, one monocyte sequence can be seen at the top of the tree (Fig. 2B)
along with almost the entire corresponding CD4+ T cell virus population. The rest of the monocytes for this time point are located in the center of the tree, with a couple at the bottom. Interestingly, this is also where the plasma sequences are located. Thus, although the calculations indicate compartmentalization of all three compartments (Fig. 2D)
, it can be seen from the tree (Fig. 2B)
that the closest sequences, by distance, to the plasma viruses are the monocyte-derived sequences, indicating a possible association of monocytes and replicating virus during therapy. A plasma sample was also taken at the latest time point, 1575 days after ending therapy. Nearly all monocyte sequences at this time are located in the founding viral population portion of the tree at the top while the CD4+ T cell-derived HIV-1 population is located outside this area with the plasma samples, visibly representing compartmentalization. Again, the calculations indicate compartmentalization of the plasma virus from the other two cell types. However, the tree also displays a number of plasma sequences in close proximity to the grouping of CD4+ T cells, indicating a connection between CD4+ T cells and plasma at the latest time point, which is expected after 1575 days without therapy when CD4+ T cells again become the major site of virus production.
1396
HIV-1 in the first plasma sample for this patient, taken 52 days after the cessation of therapy is slightly separated from the CD4+ T cells and monocytes. However, the plasma compartment is a similar distance from both, indicating a mutual contribution to the plasma virus from monocytes as well as CD4+ T cells near the end of therapy. The next time point, which was taken 325 days after therapy, also clearly shows compartmentalization between CD4+ T cells and monocytes. At this time point, according to the tree, the closest related sequences are the CD4+ T cell viruses, again suggesting that the major virus production occurs in CD4+ T cells in the absence of ART.
1184
The first plasma sample for patient 1184 was taken 353 days after the end of therapy. Groups of monocyte-derived HIV-1 sequences at this time are located at the top of the tree and in two other places near the center. CD4+ T cell sequences are generally located in the top portion of the tree. The plasma sequences were calculated to be associated with both CD4+ T cells and monocytes at this time point and can be seen in the tree as well. The latest time point, taken 1737 days post-therapy for the CD4+ T cells and monocytes and at 1928 days after stop therapy for the plasma, indicates compartmentalization of all three compartments. The separation of CD4+ T cells and monocytes can be seen in the tree, as most of the monocyte viruses are located at the lower portion of the tree while the majority of the CD4+ T cell viruses are located above them. The HIV-1 sequences in the plasma are separated from the HIV-1 sequences of both CD4+ T cells and monocytes; however, the tree indicates that the closest compartment is the monocytes, as most of the plasma sequences are located in the bottom portion of the tree where the majority of the monocyte derived HIV-1 sequences are.
DISCUSSION
It has been shown that HIV-1 within monocytes can replicate at low levels during ART [12 ] and that monocytes may serve as an important compartment for maintaining the monocyte/macrophage reservoir [12 , 13 , 22 ]. It has also been shown that ART drugs may not penetrate monocytes/macrophages as well as CD4+ T cells [23 24 25 ] (reviewed in [26 ]). Given low levels of replication in blood monocytes and reduced permeability of monocytes to drugs, new HIV-1 infections of cells during therapy would be more likely to occur in monocytes and that this, along with suppression of the virus in CD4+ T cells, could cause an increase in viral diversity in monocytes as compared with CD4+ T cells and an increased rate of compartmentalization during therapy [12 13 14 15 ]. The present longitudinal study of 4 individuals with discontinued ART presents a unique opportunity to examine the correlation between HIV-1 diversity and ART within a given individual during the course of discontinued therapy. Our study demonstrates in vivo evidence of a reduced effect of ART on the diversity of HIV-1 circulating in blood monocytes as compared with HIV-1 in CD4+ T cells.
One way to test the effects of ART on HIV-1 in CD4+ T cells and blood monocytes is to measure the diversity change over the course of therapy. Diversity measures the genetic distance between all sequences in a group of sequences and has been shown to be a good indicator of continued replication over time. This distance usually increases faster in CD4+ T cells due to the fact that roughly 99% of HIV-1 viral particles are produced by CD4+ T cells in treatment naïve patients. Our results demonstrate that, during therapy, an increase in diversity of the HIV-1 in monocytes compared with CD4+ T cells frequently occurs. In the case of patient 1180, after 1197 days of nontreatment, HIV-1 diversity in the CD4+ T cells was significantly higher (2.5%) than in the monocytes (1.5%, P=0.001). However, the increase of HIV-1 diversity within monocytes was significantly higher compared with CD4+ T cells over the course of therapy (P=0.0097), which may indicate a stronger suppression of therapy on HIV-1 in CD4+ T cells compared with the monocytes compartment. We observed a similar pattern of higher rates of diversity increase in HIV-1 derived from blood monocytes compared with CD4+ T cells during therapy, as well as an opposite trend in the absence of therapy in all patients. This trend is especially evident in patients 1180, 1175, and 1184; however, 1396 showed a slight increase in the rate of HIV-1 diversity within the CD4+ T cells during the early portion of therapy before displaying a slightly higher increase of diversity of HIV-1 in monocytes during the latter part of therapy.
Using Slatkin and Maddison’s test to estimate gene-flow restriction between compartments [20 ], we have shown that HIV-1 evolved differently and independently between monocytes and CD4+ T cells in our patient cohort. Similar observations were seen in previous studies on therapy-naïve HIV-1 individuals [13 ] and patients with ART [13 14 15 ]. Interestingly, the compartmentalization pace between monocytes and CD4+ T cells was higher during therapy than in the off-therapy period. This was especially evident in patient 1180, which showed no compartmentalization for the first 1197 days in which he was untreated. However, just 207 days after the initiation of therapy, compartmentalization occurred. Even more interesting is patient 1175, who displayed evidence of compartmentalization just 2 years post-HIV-1 seroconversion. The loss of compartmentalization just after stopping therapy is also interesting; this was the only patient who showed this phenomenon. The interesting aspect of this patient with respect to this study is that it took nearly 5 years for the virus to compartmentalize again without drugs. This finding again demonstrates that, without ART, compartmentalization occurs at a slower rate than in the presence of therapy [13 ]. One explanation for this phenomenon could relate to the fact that drug treatment may inhibit HIV-1 replication to a lesser extent in monocytes/macrophages than in CD4+ T cells as reported [23 24 25 ] (reviewed in [26 ]) . This unequal drug effect may augment the difference in HIV-1 replication capacity in monocytes/macrophages and CD4+ T cells. As the virus continues to replicate in the monocyte/macrophage compartment compared with the more drug-permissive CD4+ T cell compartment, an enhancement of compartmentalization during treatment becomes more possible. In 3 cases compartmentalization occurred during therapy, and 1 case exhibited compartmentalization within 1 year after stopping therapy, which suggests that ART plays a role in this process.
It has been reported extensively that the amount of viral genetic diversity seen in a given individual coincides, at least in part, with selective pressures for structural changes imposed by the immune system [27 28 29 30 31 32 ], antiretroviral drug pressure [33 , 34 ], or preferential tropism and replication for target cells [35 36 37 38 39 ]. Therefore, the observed HIV-1 compartmentalization between monocytes and CD4+ T cells could be a result of host factors (immune response and preferential tropism) alone (in untreated patients) [13 ] or a combination of host factors and ART [12 ]. An important consideration when evaluating the biological significance of both diversity and compartmentalization is the possibility that an increasing viral load, combined with diminishing CD4 and CD8 T cells, will force the virus to be shuttled to other cell types, such as macrophages, which could be a physical and biological explanation for the compartmentalization and diversity data presented here. However, for all patients the CD4 count never reaches below 438 and the CD8 count never reaches below 336, with the exception of the latest time point for 1184, which is expected to be low considering the viral load was over 1 million copies/ml and over 1700 days after the end of therapy. As seen in Figs. 1D , 2D , 3D and 4D , the rest of the time points have relatively high CD4 and CD8 counts. This finding indicates that a declining T cell population is not responsible for inducing the virus to infect monocytes more prevalently during therapy in our study subjects.
The phylogenetic analysis for each patient suggests that monocytes may play a role in the rebounding viral population after the cessation of therapy, as it was shown to provide replication-competent virus to the plasma during and after therapy. This was seen in both patients 1180 and 1184, where the plasma virus was shown to cluster closely to both CD4+ T cells and monocytes directly following therapy, as well as in patient 1396 where plasma virus sampled just after stopping therapy contained some sequences with homology to both CD4+ T cells and monocytes.
HIV-1 infected CD14+ monocytes, after circulating in the peripheral blood for 1 to 3 days, may differentiate into macrophages and disseminate into various tissues. HIV-1 within these tissue macrophages then replicate, and some preferentially infect blood monocytes again [12 13 14 15 16 ]. This circular pathway involving monocyte-macrophage-selected HIV-1 strains may allow for HIV-1 in monocytes/macrophages to become a separate compartment during therapy [12 13 14 15 16 ]. However, no results directly support this hypothesis. Furthermore, in vitro studies have demonstrated that fresh monocytes are highly resistant to HIV-1 infection [40 41 42 43 44 ]. Although recent studies indicate that HIV-1 replication occurs in blood monocytes in vivo, it remains unclear as to what extent HIV-1 can infect and replicate in blood monocytes in vivo [12 13 14 15 16 ]. The data presented here indicate a possible role for monocytes/macrophages as a separate drug sanctuary in the blood, which in turn may affect the evolution of drug-resistant mutations and the overall persistence of HIV-1 in the body during therapy. Limitations of the present study include a lack of a large patient sample size, shortage of sequences for analysis due to technical difficulties for some time points, and only a few plasma time points studied. While further studies with a larger cohort and more intensive sampling should be conducted in order to confirm the findings of the present study, our findings indicate that the creation of better drug regimens may be necessary to combat HIV-1 infection.
ACKNOWLEDGEMENTS
This work was supported by Public Health Service grants AI 49109, AI45402, AI55336, and AI57005. We thank David Nickle and James Mullins (University of Washington) for help with data analysis; Blythe McLoughlin, Amanda Woodward, and Leah Hampson (University of Washington) for technique assistance; Claire Stevens, Janine Maenza, and Ann Collier (University of Washington) for the recruitment of study subjects; John McNevin and Julie McElrath (Fred Hutchinson Cancer Research Center) for providing samples, and the study participants for their time and effort.
Received March 4, 2006; revised June 30, 2006; accepted July 3, 2006.
REFERENCES
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