Infection with HIV-1 is generally characterized by rapid viral mutation and evolution, 1–5 accompanied by hyperactivation of CD4+ and CD8+ T lymphocytes, 6–9 an imbalanced TH1 and 10–12 and progressive destruction of TH2 cytokine profile, CD4+ T cells, especially the HIV-1–specific subsets. 13 While cytokines and chemokines can mediate any of these virologic and immunologic features during HIV-1 infection, substantial interindividual differences in both early and later manifestations of HIV-1 disease 14 clearly imply the involvement of host genetic mediation of HIV-1 infection and pathogenesis. 15–17
The genes encoding various cytokines, chemokines, and their receptors often carry promoter and occasionally coding sequence variations that are functionally important. 18,19 Associations of genetic polymorphisms with varying clinical outcomes during HIV-1 infection have been repeatedly studied for the chemokine receptor and ligand genes CCR2, CCR5, CCL2 (MCP1), CCL3 (MIP1A), CCL5 (RANTES), and CXCL12 (SDF1). 16,17,20 Other immunogenetic studies of HIV/AIDS cohorts have examined single nucleotide polymorphisms (SNPs) in various cytokine genes like IFNG, 21,22IL4, 23–26IL6, 27IL10, 26,28IL12B, 29 and TNF. 29,30 Consistent findings have often been lacking in cross-cohort comparisons, most likely because the allelic distribution and haplotypic relationships of SNP variants can differ starkly between ethnic groups. 31–33 Such ethnic/racial specificity has been documented repeatedly in earlier studies. 20,34–40 Our genotyping of cytokine and chemokine gene variants in ethnically diverse North Americans now further supports such phenomena.
MATERIALS AND METHODS
Subjects and DNA Samples
The 579 patients, mostly African-American and Hispanic (Latino), were derived from the Reaching for Excellence in Adolescent Care and Health (REACH) project and the HIV-1 Epidemiology Research on Outcome (HERO) study, which targeted adolescents and adults, respectively. 41–43 For REACH participants, analyses were performed for all 183 HIV-1–seronegative (HIV−) participants and for the 227 HIV-1–seropositive (HIV+) participants with repeated virologic and immunologic outcome measures at various visit intervals when antiretroviral therapy (ART) was not taken. 44,45 The remaining 173 (Hispanics of Puerto Rican descent) were HERO participants living in Bronx, New York City. HIV-1− participants from both cohorts were considered at high risk of infection due to self-reported sexual behavior, injection drug use, or both. 41–43 Genomic DNA for each individual was extracted from 2 × 10 6 peripheral blood mononuclear cells using the QIAamp blood kit (QIAGEN, Chatsworth, CA). All DNA samples were diluted to 200 ng/μL and stored at 4°C in TE buffer (10 mM of Tris-HCl [pH 8.0] and 2 mM of EDTA) before use.
HIV-1–Related Outcome Measures
HIV-1 seropositivity could be assessed in both the REACH and HERO cohorts despite their differences in selection criteria, age, ethnic distributions, risk behaviors, treatment protocols, and follow-up strategy. For HIV+ REACH subjects with quarterly follow-up visits, CD4+ T lymphocytes were quantified by flow cytometry in National Institute of Allergy and Infectious Disease (NIAID)–certified laboratories at each clinical site. 46 Plasma HIV-1 RNA concentration (viral load) was measured in a centralized laboratory using either nucleic acid sequence–based amplification (NASBA) or NucliSens assays (Organon Teknika, Durham, NC) as previously described. 47 The lower limits of detection for the NASBA and NucliSens assays were 400 and 80 copies/mL, respectively. Multiple HIV-1 RNA measurements (transformed to log10) and absolute CD4+ cell counts were retained for 207 AIDS-free REACH adolescents for a 1-year period (up to 4 visits) when ART was not taken. 44 Analyses of immunologic outcome based on CD4+ T-cell counts were omitted in the HERO cohort because estimated duration of HIV-1 infection at enrollment was unreliable.
Polymerase Chain Reaction–Based Single Nucleotide Polymorphism Genotyping
Common SNPs for 13 genes (IFNG, IL1A, IL1B, IL1R1, IL1RN, IL2, IL4, IL4R, IL6, IL10, IL12B, TGFB1, and TNF;Table 1) in the cytokine system were typed by polymerase chain reaction (PCR) with sequence-specific primers (SSPs; Department of Transplantation Immunology, University of Heidelberg, Heidelberg, Germany) following procedures recommended by the 13th International Histocompatibility Workshop (IWHG) Cytokine Polymorphisms Component (CPC). 33 Reliability of genotyping results was verified in an initial analysis of 50 reference DNA samples distributed by the 13th IWHG. PCR SSP kits were also purchased from another supplier (Pel-Freez Clinical Systems, Brown Deer, WI) to achieve identical resolution (specificity). SNP typing for 3 chemokine genes (CCL2, CCL5, and CXCL12; see Table 1) relied on PCR SSP procedures developed in our own laboratory (J. Tang et al, unpublished data). The strategies were similar to those adopted for the typing of CCR2 and CCR5 SNPs and haplotypes as described elsewhere for the REACH and HERO cohorts. 37,44 In brief, representative genotypes (homozygous and heterozygous) were identified by sequencing and/or restriction fragment length polymorphisms. PCR SSP protocols were optimized using these reference DNA samples before being applied to test samples. Two SNPs (T-3575A and C-2763A) in the distal IL10 promoter were classified by the PCR SSP technique used earlier in a white (European American) cohort. 48
General Population Genetic Analyses
For each study population defined by ethnicity, the allele and carriage (population) frequencies of individual SNP variants were established by direct counting using SAS (Statistical Analysis Software, version 8.5; SAS Institute, Cary, NC), with the numbers of chromosomes (2N) and individuals (n) serving as the denominators, respectively. Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD) were assessed using the PopGene v1.32 statistical package. 49 Deviation (P < 0.05) from HWE was deemed indicative of sample selection bias or evolutionary advantage for particular SNP genotypes in a given population. Adjacent SNP alleles in strong LD form the basis for assigning local haplotypes; LD analyses were unnecessary for haplotypic combinations directly revealed by PCR SSP.
Genetic Association Analyses
Genetic associations with HIV-related outcome measures were evaluated in 4 steps. First, the overall allelic and/or genotypic heterogeneity between HIV+ and HIV− groups was measured by exact tests based on metropolis algorithms 50 and row by column (R×C) contingency tables. A statistical P value of <0.05 was taken to suggest overall genetic effect. 51 Second, carriage of individual alleles and haplotypes was compared between HIV+ and HIV− groups using χ2 or Fisher exact tests. Third, relationships between genetic variation and log10 HIV-1 virus load and CD4+ T-cell counts in HIV+ patients were defined by generalized linear regression model (GLM) statistics in SAS. Mixed models were also applied to the analyses of repeated measures at multiple (2–4) follow-up intervals when ART was not taken. Fourth, SNP variants showing marginal (P < 0.10) associations with HIV-1 infection and virologic/immunologic outcomes were further tested in multivariable regression and mixed model analyses, with statistical adjustments for race, gender, prior exposure to ART, visit interval (duration of follow-up), and the effects of known genetic factors (HLA, CCR2, and CCR5) in each cohort. 37,45
Polymerase chain reaction with SSPs successfully defined 24 common SNPs for 11 cytokine and chemokine genes (IFNG, IL2, IL4, IL4R, IL6, IL10, IL12B, TNF, CCL2, CCL5, and CXCL12) for most subjects. Only one DNA sample from an HIV− REACH subject had to be eliminated from analyses, and success in typing the 176 HERO Hispanics ranged from 96% to 100% for all loci except IFNG (56%;Table 2). Additional SNP typing for the IL1A, IL1B, IL1R1, IL1RN, and TGFB1 loci was available for about 50% of REACH and HERO samples due to frequent problems with several PCR reactions. Allele assignments for DNA from 6 chimpanzees (Pan troglodytes) were obtained for all but 3 SNPs (IL4R- 551 and the codon 10 and codon 25 SNPs in TGFB1). Homozygous sequences were detected in all chimpanzee samples (see Table 1); those defined at CCL2 and CCL5 loci confirmed several earlier reports. 52,53
Ethnic Differences, Hardy-Weinberg Equilibrium, and Haplotypes
For all but 3 (IL4–1098, IL10–3575, and IL12B 3′ untranslated region [UTR]) SNPs, the distribution of variant alleles differed widely by ethnicity, as did the contrasts in genotypic frequencies (P < 0.01 or P < 0.001) between African-Americans and Hispanics (see Table 2). For those 3 possible genotypes (2 homozygous and 1 heterozygous) defined for the IL10–1082 SNP, the expected and observed frequencies deviated from HWE in all racial groups, as documented by 21 laboratories participating in the 13th IWHG CPC. 33 Other more sporadic deviations from HWE varied from each ethnic group to another, without apparent correlation with sample size or HIV-1 serostatus (see Table 2).
The presence of multiple SNP haplotypes in IL4, IL6, IL10, TNF, CCL2 (MCP1), and CCL5 (RANTES) genes (Table 3) closely matched those reported elsewhere. 26,33,39,52–54 The exclusive LD between IL10–819T and IL10–592A was also confirmed. For SNPs at IL6 and IL10 loci not linked through PCR SSP, uniformly strong LD (Δ = 0.04–0.09; P < 0.001) justified their analysis as distinct haplotypes (see Table 3).
Cytokine and Chemokine Gene Variants in Relation to HIV-1 Serostatus
When the distribution of SNP variants (alleles as well as haplotypes) was compared between HIV+ and HIV− individuals within each ethnic group (see Tables 2, 3); differences with a nominal P < 0.05 (by univariate analyses) were found mostly in a race-specific manner. For example, the increased presence of IL4–33C allele (P = 0.03) in HIV+ versus HIV− African-Americans from REACH derived entirely from the association of −33C/C homozygosity (relative odds [RO] = 2.13, 95% confidence interval (CI): 1.20–3.70, unadjusted P = 0.01), but data from the other REACH and HERO Hispanic subsets did not display these relationships. Likewise, a disproportional decrease of CXCL12 801A (also known as SDF1–3′A) in HIV+ compared with HIV− HERO Hispanics (P = 0.02) could not be replicated in REACH patients.
In multivariable analyses of individual cohorts (Table 4), IL4R 551A/A was associated with the absence of HIV-1 infection in REACH participants (RO = 0.53; P = 0.009), whereas IL10–1082A/A, CCL5 ACT, and CXCL12 801A were independently associated with HIV-1 serostatus in HERO participants (RO = 0.13–0.32; P = 0.001–0.04). Carriage of 2 haplotypes, IL4 GTC and IL10 TCGCC, differed between HIV+ and HIV− groups regardless of ethnicity or cohort (see Table 3), but only the latter remained an unfavorable contributing factor (see Table 4; RO = 2.25; P < 0.001) after adjustment for gender, ethnicity, cohort, and CCR5–Δ32 (a 32–base pair [bp] deletion mutation defined earlier in both cohorts). 37,44
Cytokine and Chemokine Gene Variants in Relation to Early Virologic and Immunologic Outcomes in HIV-1–Infected Adolescents
In analyses restricted to 207 HIV-infected and AIDS-free REACH adolescents, the only SNP genotypes associated with contrasting immunologic outcomes (CD4+ T-cell count) were those defined by the −590 SNP in IL4 promoter (Fig. 1). More specifically, participants with the IL4–590T/T genotype consistently had higher (+87–131 cells/μL) CD4+ T-cell counts during 4 ART-free visit intervals when compared with others without IL4–590T/T (adjusted P = 0.004; see Fig. 1). Despite a clear linear correlation between CD4+ T-cell counts and virologic outcomes (HIV-1 viral load) in subjects who were IL4–590T/T–positive (Pearson r = −0.552; P < 0.0001) and IL4–590T/T–negative (Pearson r = −0.523; P < 0.0001), differences in viral load between the IL4–590T/T–positive and IL4–590T/T–negative subjects were quite modest (<0.10 log10 copies/mL; P > 0.50 by mixed model analyses) during the first 3 visit intervals; the difference at the fourth visit approached 0.25 log10 copies/mL (P = 0.19). Additional analyses of SNP haplotypes at IL4 and at other loci revealed no other appreciable effect on HIV-1 viral load or CD4+ T-cell counts in univariate or multivariable models (data available on request).
Analyses of Partial Data for Additional Loci
Polymerase chain reaction with SSP-based SNP typing for the IL1A, IL1B, IL1R1, IL1RN, and TNFB1 loci was not as informative due to technical difficulties. Use of alternative genotyping techniques, including DNA sequencing, appeared necessary. Nonetheless, partial data from these loci showed no trend for association with either HIV-1 infection (P > 0.25) or immunologic/virologic outcomes (P > 0.50). Meanwhile, there are no known associations of IL1A, IL1B, IL1R1, IL1RN, or TNFB1 variants with HIV-1–related outcomes.
Racial/ethnic specificity (population heterogeneity) poses a major obstacle to both epidemiologic and experimental analyses of HIV-1 infection and pathogenesis. Our demonstration that cytokine and chemokine gene variants differ markedly in their allelic and haplotypic frequencies between ethnic groups is consistent with earlier documentation of population-specific genetic associations with HIV-1–related outcomes. 24,25,28,36,53 More specifically, similarities between positive findings observed in the REACH and HERO cohorts and others reported elsewhere varied from one locus to another. CXCL12 (SDF1) 801A has been seen less frequently in HIV+ than in HIV− Thai sex workers exposed to HIV-1 clade E viruses, 55 and the association between IL4–590T/T with delayed immunodeficiency in REACH cohort mirrors the reportedly slower progression to AIDS in French HIV-1+ subjects carrying the IL4–590T (also known as −589T or −549T). 24,26 On the other hand, our findings on the ACT haplotype of CCL5 (RANTES) contradicted the earlier suggestion that the AC haplotype (at −403 and −28 relative to the transcription start site or −471 and −96 relative to the translation start site) was more common in HIV+ than HIV− European Americans. 53 In 2 other reports, variants found on the CCL5 ACT haplotype have demonstrated unfavorable effects on HIV-1 seroconversion and disease progression (relative hazards of AIDS = 1.57–1.89; P = 0.002–0.08) in US white (European American) populations. 39,56 The mechanisms underlying these seemingly conflicting genetic relationships remain to be defined.
In a separate analysis of individual T-lymphocyte responses to HIV-1–specific 20mer peptides, the IL4 promoter −590T/T genotype in a subset of REACH participants was further associated with increased magnitude of interferon-γ (INFγ)–secreting T cells, as enumerated by ELISpot assay (J. Tang and P.A. Goepfert et al, unpublished data). Thus, delayed immunodeficiency associated with IL4–590T/T in the REACH cohort (see Fig. 1) might reflect sustained HIV-specific immunity as a possible mechanism. A somewhat puzzling aspect was the lack of association between IL4–590T/T with cell-free HIV-1 viral load, which has been widely considered as a reliable predictor of HIV-1 pathogenesis and clinical course of disease progression. 57,58 A modest inverse correlation between viral load and CD4+ T-cell count (Pearson r = −0.52 to −0.55) was confirmed in the REACH subjects, suggesting that 27% to 30% (ie, r2) of the variability in CD4+ T-cell counts could be captured by measuring differences in viral load. The origins of this dissociation between 2 related outcome measures could be 2-fold. First, viral load was universally lower in REACH adolescents (mostly female) than usually observed in adult male populations such that the power to reveal a statistically significant association across a narrower range of values was reduced in the REACH adolescent cohort. Second, there is preliminary evidence that IL4 genotypes might confer greater influence on HIV-1 coreceptor usage than on virus-host equilibration, as seen in HIV-infected Japanese. 23
Our study of male and female adolescents and adults from distinct ethnic groups was intended to yield more generalizable findings. Major risk factors for HIV-1 infection also differed between these cohorts, with sexual exposure accounting for most of the risk in the REACH project and injection drug use in the HERO study. 41–43 Relatively consistent associations of 2 haplotypes at the IL4 and IL10 loci with HIV-1 infection supported the belief that immunogenetic relationships can be independent of age, gender, ethnicity, and risk behavior. Similarly, robust observations have been reported for chemokine receptor gene (CCR2 and CCR5) variants on chromosome 3, including CCR5- Δ32 on the HHG*2 haplotype. 40,59–61 Nevertheless, CCR5–Δ32 was too rare in the HERO study and REACH project 37,44 to confound the observed relationships of cytokine gene polymorphisms (see Table 4).
On average, an SNP occurs at least once in every 500- to 1000-bp region of genomic sequences. 31,62 The few SNPs being studied here are expected to tag some but not all common clusters of genetic variants at the candidate loci. For example, 2 other IL4 SNPs (G–1136A and G+45A) described more recently contribute to several haplotypes. 63 Several intronic SNPs have been found in a French population. 26 Likewise, new IL10 SNPs have been documented in populations of African, European, or mixed ancestries. 54,64 Continuing discovery of potential IL4 and IL10 effects encourages more systematic evaluation of polymorphisms at these loci. Data from other ongoing HIV/AIDS cohorts differing in study design, research foci, experimental methodologies, and cohort characteristics should also become valuable in testing the validity of other less consistent immunogenetic findings.
The authors thank investigators and staff [listed in J Adolesc Health. 2001;29(Suppl):5–6] of the Adolescent Medicine HIV/AIDS Research Network (1994–2001) as well as others participating in the HERO study for their valuable contributions. We are further indebted to patients in the respective cohorts for their cooperation, to C.A. Rivers and A. Myracle for technical assistance, to A. Moore and D. Buono for data management, and to P.N. Fultz, PhD, for use of chimpanzee DNA samples.
1. Wei X, Ghosh SK, Taylor ME, et al. Viral dynamics in human immunodeficiency virus type 1 infection
2. Ho DD, Neumann AU, Perelson AS, et al. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection
3. Wolinsky SM, Korber BT, Neumann AU, et al. Adaptive evolution of human immunodeficiency virus-type 1 during the natural course of infection
4. Walker BD, Goulder PJ. AIDS. Escape from the immune system. Nature
5. Moore CB, John M, James IR, et al. Evidence of HIV-1
adaptation to HLA-restricted immune responses at a population level. Science
6. Giorgi JV, Detels R. T-cell subset alterations in HIV-infected homosexual men: NIAID Multicenter AIDS cohort study. Clin Immunol Immunopathol
7. Prince HE, Kleinman S, Czaplicki C, et al. Interrelationships between serologic markers of immune activation and T lymphocyte subsets in HIV infection
. J Acquir Immune Defic Syndr Hum Retrovirol
8. Kestens L, Vanham G, Gigase P, et al. Expression of activation antigens, HLA-DR and CD38, on CD8 lymphocytes during HIV-1 infection
9. Ramzaoui S, Jouen-Beades F, Gilbert D, et al. During HIV infection
, CD4+ CD38+ T-cells are the predominant circulating CD4+ subset whose HLA-DR positivity increases with disease progression and whose V beta repertoire is similar to that of CD4+ CD38- T-cells. Clin Immunol Immunopathol
10. Clerici M, Shearer GM. A Th1→Th2 switch is a critical step in the etiology of HIV infection
. Immunol Today
11. Clerici M, Shearer GM. The Th1-Th2 hypothesis of HIV infection
: new insights. Immunol Today
12. Altfeld M, Addo MM, Kreuzer KA, et al. T(H
)1 to T(H
)2 shift of cytokines in peripheral blood of HIV-infected patients is detectable by reverse transcriptase polymerase chain reaction but not by enzyme-linked immunosorbent assay under nonstimulated conditions. J Acquir Immune Defic Syndr
13. Douek D, Brenchley J, Betts M, et al. HIV preferentially infects HIV-specific CD4+ T cells. Nature
14. Collaborative Group on AIDS Incubation and HIV Survival Including the CASCADE EU Concerted Action. Time from HIV-1
seroconversion to AIDS and death before widespread use of highly-active antiretroviral therapy: a collaborative re-analysis. Concerted Action on SeroConversion to AIDS and Death in Europe. Lancet
15. Fauci AS, Pantaleo G, Stanley S, et al. Immunopathogenic mechanisms of HIV infection
. Ann Intern Med
16. O’Brien SJ, Nelson GW, Winkler CA, et al. Polygenic and multifactorial disease gene association in man: lessons from AIDS. Annu Rev Genet
17. Kaslow RA, Tang J, Dorak MT. The role of host genetic variation in HIV infection
and its manifestations. In: Wormser GP, ed. AIDS and Other Manifestations of HIV Infection
. 4th ed. New York: Elsevier Science. 2004; in press.
18. Bidwell J, Keen L, Gallagher G, et al. Cytokine
gene polymorphism in human disease: on-line databases. Genes Immun.
19. Haukim N, Bidwell JL, Smith AJ, et al. Cytokine
gene polymorphism in human disease: on-line databases, Supplement 2. Genes Immun.
20. Modi WS, Goedert JJ, Strathdee S, et al. MCP-1-MCP-3-Eotaxin gene cluster influences HIV-1
21. Bream JH, Carrington M, O’Toole S, et al. Polymorphisms of the human IFNG gene noncoding regions. Immunogenetics
22. An P, Vlahov D, Margolick JB, et al. A tumor necrosis factor-inducible promoter variant of interferon-γ accelerates CD4+ T cell depletion in human immunodeficiency virus-α-infected individuals. J Infect Dis
23. Nakayama EE, Hoshino Y, Xin X, et al. Polymorphism in the interleukin-4 promoter affects acquisition of human immunodeficiency virus type 1 syncytium-inducing phenotype. J Virol
24. Nakayama EE, Meyer L, Iwamoto A, et al. Protective effect of interleukin-4 -589T polymorphism on human immunodeficiency virus type 1 disease progression: relationship with virus load. J Infect Dis
25. Kwa D, Van Rij RP, Boeser-Nunnink B, et al. Association between an interleukin-4 promoter polymorphism and the acquisition of CXCR4 using HIV-1
26. Vasilescu A, Heath SC, Ivanova R, et al. Genomic analysis of Th1-Th2 cytokine
genes in an AIDS cohort: identification of IL4
haplotypes associated with the disease progression. Genes Immun.
27. Foster CB, Lehrnbecher T, Samuels S, et al. An IL6
promoter polymorphism is associated with a lifetime risk of development of Kaposi sarcoma in men infected with human immunodeficiency virus. Blood
28. Shin HD, Winkler C, Stevens JC, et al. Genetic restriction of HIV-1
pathogenesis to AIDS by promoter alleles of IL10. Proc Natl Acad Sci USA
29. Price P, Morahan G, Huang D, et al. Polymorphisms in cytokine
genes define subpopulations of HIV-1
patients who experienced immune restoration diseases. AIDS
30. Brinkman BMN, Keet IPM, Miedema F, et al. Polymorphisms within the human tumor necrosis factor-alpha region in human immunodeficiency virus type 1-seropositive persons. J Infect Dis
31. Stephens JC, Schneider JA, Tanguay DA, et al. Haplotype variation and linkage disequilibrium in 313 human genes. Science
32. Judson R, Salisbury B, Schneider J, et al. How many SNPs does a genome-wide haplotype map require?Pharmacogenomics
33. Louie LG, Wallenstein E, Schmelzer K, et al. Report of the anthropology group from the Cytokine
Polymorphism Component. In: Hansen J, Dupont B, eds. HLA 2002: Proceedings of 13th International Histocompatibility Workshop and Congress,
Vol. II. Seattle: IHWG Press. 2004; in press.
34. Mummidi S, Ahuja SS, Gonzalez E, et al. Genealogy of the CCR5 locus and chemokine
system gene variants associated with altered rates of HIV-1
disease progression. Nat Med
35. Mummidi S, Ahuja SS, McDaniel BL, et al. The human CC chemokine
receptor 5 (CCR5) gene. Multiple transcripts with 5′-end heterogeneity, dual promoter usage, and evidence for polymorphisms within the regulatory regions and noncoding exons. J Biol Chem
36. Gonzalez E, Bamshad M, Sato N, et al. Race-specific HIV-1
disease modifying effects associated with CCR5
haplotypes. Proc Natl Acad Sci USA
37. Tang J, Rivers C, Karita E, et al. Allelic variants of human beta-chemokine
receptor 5 (CCR5
) promoter: evolutionary relationships and predictable association with HIV-1
disease progression. Genes Immun.
38. Mummidi S, Bamshad M, Ahuja SS, et al. Evolution of human and non-human primate CC chemokine
receptor 5 gene and mRNA. Potential roles for haplotype and mRNA diversity, differential haplotype-specific transcriptional activity, and altered transcription factor binding to polymorphic nucleotides in the pathogenesis of HIV-1
and simian immunodeficiency virus. J Biol Chem
39. An P, Nelson GW, Wang L, et al. Modulating influence on HIV/AIDS by interacting RANTES gene variants. Proc Natl Acad Sci USA
40. Tang J, Shelton B, Makhatadze NJ, et al. Distribution of chemokine
genotypes and their relative contribution to human immunodeficiency virus type 1 (HIV-1
) seroconversion, early HIV-1
RNA concentration in plasma, and later disease progression. J Virol
41. Schoenbaum EE, Hartel D, Selwyn PA, et al. Risk factors for human immunodeficiency virus infection
in intravenous drug users. N Engl J Med
42. Rogers AS, Futterman DK, Mosciki AB, et al. The REACH project of the adolescent medicine HIV/AIDS research network: design, methods, and selected characteristics of participants. J Adolesc Health
43. Wilson CM, Houser J, Partlow C, et al. The REACH (Reaching for Excellence in Adolescent Care and Health) project: study design, methods, and population profile. J Adolesc Health
44. Tang J, Wilson CM, Schaen M, et al. CCR2
genotypes in HIV type 1-infected adolescents: limited contributions to variability in plasma HIV type 1 RNA concentration in the absence of antiretroviral therapy. AIDS Res Hum Retroviruses.
45. Tang J, Wilson CM, Meleth S, et al. Host genetic profiles predict virological and immunological control of HIV-1 infection
in adolescents. AIDS
46. Douglas SD, Rudy B, Muenz L, et al. Peripheral blood mononuclear cell markers in antiretroviral therapy-naive HIV-infected and high risk sero-negative adolescents. Adolescent Medicine HIV/AIDS Research Network. AIDS
47. Holland CA, Ellenberg JH, Wilson CM, et al. Relationship of CD4+ T cell counts and HIV type 1 viral loads in untreated, infected adolescents. Adolescent Medicine HIV/AIDS Research Network. AIDS Res Hum Retroviruses
48. Yee LJ, Tang J, Gibson AW, et al. Interleukin 10 polymorphisms as predictors of sustained response in antiviral therapy for chronic hepatitis C infection
49. Yeh F, Yang R, Boyle T, et al. POPGENE, the user-friendly shareware for population genetic analysis. Molecular Biology and Biotechnology Centre, University of Alberta, Canada. Available online since 1997 at: http://www.ualberta.ca/~fyeh/index.htm
50. Raymond ML, Rousset F. An exact test for population differentiation. Evolution.
51. Klitz W, Bugawan TL, Panelo A, et al. Association of CTLA-4 variation with type I diabetes in Filipinos. Immunogenetics
52. Gonzalez E, Rovin BH, Sen L, et al. HIV-1 infection
and AIDS dementia are influenced by a mutant MCP-1 allele
linked to increased monocyte infiltration of tissues and MCP-1 levels. Proc Natl Acad Sci USA
53. Gonzalez E, Dhanda R, Bamshad M, et al. Global survey of genetic variation in CCR5, RANTES, and MIP-1α: impact on the epidemiology of the HIV-1
pandemic. Proc Natl Acad Sci USA
54. Gibson AW, Edberg JC, Wu J, et al. Novel single nucleotide polymorphisms in the distal IL-10 promoter affect IL-10 production and enhance the risk of systemic lupus erythematosus. J Immunol
55. Sriwanthana B, Hodge T, Mastro TD, et al. HIV-specific cytotoxic T lymphocytes, HLA-A11, and chemokine
-related factors may act synergistically to determine HIV resistance in CCR5 delta32-negative female sex workers in Chiang Rai, northern Thailand. AIDS Res Hum Retroviruses
56. McDermott DH, Beecroft MJ, Kleeberger CA, et al. Chemokine
RANTES promoter polymorphism affects risk of both HIV infection
and disease progression in the Multicenter AIDS Cohort Study. AIDS
57. Mellors JW, Rinaldo CR, Jr, Gupta P, et al. Prognosis in HIV-1 infection
predicted by the quantity of virus in plasma. Science
58. Saag MS, Holodniy M, Kuritzkes DR, et al. HIV viral load markers in clinical practice. Nat Med
59. Liu R, Paxton WA, Choe S, et al. Homozygous defect in HIV-1
coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection
60. Mangano A, Gonzalez E, Dhanda R, et al. Concordance between the CC chemokine
receptor 5 genetic determinants that alter risks of transmission and disease progression in children exposed perinatally to human immunodeficiency virus. J Infect Dis
61. Kawamura T, Gulden FO, Sugaya M, et al. R5 HIV productively infects Langerhans cells, and infection
levels are regulated by compound CCR5
polymorphisms. Proc Natl Acad Sci USA
62. Cargill M, Altshuler D, Ireland J, et al. Characterization of single-nucleotide polymorphisms in coding regions of human genes. Nat Genet
63. Modi WS, O’Brien TR, Vlahov D, et al. Haplotype diversity in the interleukin-4 gene is not associated with HIV-1
transmission and AIDS progression. Immunogenetics
64. Moraes MO, Santos AR, Schonkeren JJ, et al. Interleukin-10 promoter haplotypes are differently distributed in the Brazilian versus the Dutch population. Immunogenetics
65. Liu HL, Chao D, Nakayama EE, et al. Polymorphism in RANTES chemokine
promoter affects HIV-1
disease progression. Proc Natl Acad Sci USA
66. Winkler C, Modi W, Smith MW, et al. Genetic restriction of AIDS pathogenesis by an SDF-1 chemokine
gene variant. ALIVE Study, Hemophilia Growth and Development Study (HGDS), Multicenter AIDS Cohort Study (MACS), Multicenter Hemophilia Cohort Study (MHCS), San Francisco City Cohort (SFCC). Science