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1.  Validation of the Gen-Probe Aptima Qualitative HIV-1 RNA Assay for Diagnosis of Human Immunodeficiency Virus Infection in Infants 
Journal of Clinical Microbiology  2013;51(12):4137-4140.
The qualitative Roche HIV-1 DNA Amplicor assay has been used for the past 20 years to diagnose HIV infection in infants and young children but is being phased out; hence, alternative assays must be found. The Gen-Probe Aptima qualitative HIV-1 RNA assay is currently the only FDA-cleared HIV-1 nucleic acid assay approved for diagnosis, but data on the use of this assay with infant plasma are limited. We assessed Aptima's performance using control material for reproducibility and limit of detection and 394 plasma samples (0.2 to 0.5 ml) from HIV-exposed infected and uninfected infants and children for analytical sensitivity and specificity. Assays to assess within-run repeatability and between-run reproducibility indicated that the controls with 10,000 (5 of 5), 200 (5 of 5), 100 (16 of 16), 50 (12 of 12), and 25 (20 of 20) HIV-1 RNA copies/ml (cp/ml) were always positive, and negatives were always negative (20 of 20). The limit of detection was 14 cp/ml, as determined by probit analysis. The analytic sensitivity of the assay was 99.5% (189/190 samples; 95% confidence interval [CI], 97.1 to 99.9%) and specificity was 99.5% (199/200 samples; 95% CI, 97.2 to 99.9%). These results suggest that the assay is suitable for early infant diagnosis of HIV-1.
doi:10.1128/JCM.01525-13
PMCID: PMC3838099  PMID: 24088864
2.  Venous thromboembolism in adults with sickle cell disease: A serious and under-recognized complication 
The American journal of medicine  2013;126(5):443-449.
BACKGROUND
Sickle cell disease is recognized as a hypercoagulable state; however the frequency and characteristics of venous thromboembolism in sickle cell patients have not been well-defined.
PURPOSE
To establish the prevalence and risk factors for venous thromboembolism in a large cohort of patients with sickle cell disease and determine the relationship between venous thromboembolism and mortality.
METHODS
We performed a cross-sectional study of 404 sickle cell disease patients cared for at the Sickle Cell Center for Adults at Johns Hopkins. Demographic, sickle cell disease-specific comorbidity, and venous thromboembolism data were collected on all patients.
RESULTS
101 patients (25%) had a history of venous thromboembolism with a median age at diagnosis of 29.9 years. A history of non-catheter-related venous thromboembolism was found in 18.8% of patients. Sickle variant genotypes conferred a higher risk of non-catheter-related venous thromboembolism compared to sickle cell anemia genotypes (SS/Sβ0) (relative risk (RR) 1.77, 95% confidence interval (CI) 1.18–2.66). Tricuspid regurgitant jet velocity ≥ 2.5 m/s was also associated with non-catheter-related venous thromboembolism (RR 1.65, CI 1.12–2.45). Thirty patients (7.4%) died during the study period. Adjusting for all variables, non-catheter-related venous thromboembolism was independently correlated with death (RR 3.63, CI 1.66–7.92).
CONCLUSION
Venous thromboembolism is common in adults with sickle cell disease. Sickle variant genotypes and tricuspid regurgitant jet velocity ≥ 2.5m/s are associated with non-catheter-related venous thromboembolism. In addition, non-catheter-related venous thromboembolism appears to be an independent risk factor for death in our cohort. These results suggest that disease-specific prophylaxis and treatment strategies for venous thromboembolism should be investigated in sickle cell disease patients.
doi:10.1016/j.amjmed.2012.12.016
PMCID: PMC3627211  PMID: 23582935
3.  Expression of p16INK4a as a biomarker of T-cell aging in HIV-infected patients prior to and during antiretroviral therapy 
Aging cell  2012;11(5):916-918.
Summary
The p16INK4a tumor suppressor gene is a mediator of cellular senescence and has been suggested to be a biomarker of ‘molecular’ age in several tissues including T-cells. To determine the association of both active and suppressed HIV infection with T-cell aging, T-cell p16INK4a expression was compared between 60 HIV+ suppressed subjects, 23 HIV+ untreated subjects, and 18 contemporaneously collected HIV-negative controls, as well as 148 HIV-negative historical samples. Expression did not correlate with chronologic age in untreated HIV+ patients, consistent with an effect of active HIV replication on p16INK4a expression. In patients on cART with suppressed viral loads, however, p16INK4a levels were similar to uninfected controls and correlated with chronologic age, with a trend toward an inverse correlation with CD4 count. These data show that p16INK4a is a reliable biomarker of T cell aging in HIV+ patients with suppressed viral loads and suggest that poor CD4 cell recovery on cART may be associated with increased T-cell expression of p16INK4a, a marker of cellular senescence.
doi:10.1111/j.1474-9726.2012.00856.x
PMCID: PMC3697001  PMID: 22738669
4.  COMPARISON OF DIFFERENT TRUNK ENDURANCE TESTING METHODS IN COLLEGE‐AGED INDIVIDUALS 
Objective:
Determine the reliability of two different modified (MOD1 and MOD2) testing methods compared to a standard method (ST) for testing trunk flexion and extension endurance.
Participants:
Twenty‐eight healthy individuals (age 26.4 ± 3.2 years, height 1.75 ± m, weight 71.8 ± 10.3 kg, body mass index 23.6 ± 3.4 m/kg2).
Method:
Trunk endurance time was measured in seconds for flexion and extension under the three different stabilization conditions. The MOD1 testing procedure utilized a female clinician (70.3 kg) and MOD2 utilized a male clinician (90.7 kg) to provide stabilization as opposed to the ST method of belt stabilization.
Results:
No significant differences occurred between flexion and extension times. Intraclass correlations (ICCs3,1) for the different testing conditions ranged from .79 to .95 (p <.000) and are found in Table 3. Concurrent validity using the ST flexion times as the gold standard coefficients were .95 for MOD1 and .90 for MOD2. For ST extension, coefficients were .91 and .80, for MOD1 and MOD2 respectively (p <.01).
Conclusions:
These methods proved to be a reliable substitute for previously accepted ST testing methods in normal college‐aged individuals. These modified testing procedures can be implemented in athletic training rooms and weight rooms lacking appropriate tables for the ST testing.
Level of Evidence:
3
PMCID: PMC3474305  PMID: 23091786
Core; stabilization; trunk endurance
5.  Potent Antioxidant Dendrimers Lacking Pro-oxidant Activity 
Free radical biology & medicine  2010;50(8):918-925.
It is well known that antioxidants have protective effects against oxidative stress. Unfortunately, in the presence of transition metals, antioxidants including polyphenols with potent antioxidant activities may also exhibit pro-oxidant effects, which may irreversibly damage DNA. Therefore, antioxidants with strong free radical scavenging abilities and devoid of pro-oxidant effects would be of immense biological importance. We report two antioxidant dendrimers with a surface rich in multiple phenolic hydroxyl groups, benzylic hydrogens and electron donating ring substituents that contribute to their potent free radical quenching property. In order to minimize their pro-oxidant effects, the dendrimers were designed with a metal chelating tris(2-aminoethyl)amine (TREN) core. The dendritic antioxidants were prepared by attachment of six syringaldehyde or vanillin molecules to TREN by reductive amination. They exhibited potent radical scavenging properties: 5 times stronger than quercetin and 15 times more potent than Trolox according to the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The antioxidant dendrimers also protected low-density lipoprotein, lysozyme and DNA against 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced free radical damage. More importantly, unlike quercetin and Trolox, the two TREN antioxidant dendrimers did not damage DNA via their pro-oxidant effects when incubated with physiological amounts of copper ions. The dendrimers also showed no cytotoxicity towards Chinese hamster ovary cells.
doi:10.1016/j.freeradbiomed.2010.10.699
PMCID: PMC3047602  PMID: 20977937
Polyphenolic dendrimer; antioxidant; pro-oxidant; lipoprotein; lysozyme; DNA
6.  Addition of 7 Days of Zidovudine plus Lamivudine to Peripartum Single-Dose Nevirapine Effectively Reduces Nevirapine Resistance Postpartum in HIV-Infected Mothers in Malawi 
Background
We assessed whether 7 days of zidovudine+lamivudine postpartum with single-dose nevirapine at labor decreases nevirapine resistance in HIV-infected women in Malawi.
Methods
HIV-infected pregnant women receiving intrapartum single-dose nevirapine and 7 days of zidovudine+lamivudine (n=132), and women receiving intrapartum single-dose nevirapine alone (n=66) were followed from an antenatal visit through 6 weeks postpartum. Plasma specimens at 2 and 6 weeks postpartum were tested for genotypic resistance to nevirapine by population sequencing and sensitive real-time PCR. Poisson regression was used to determine predictors of postpartum nevirapine resistance.
Results
Median HIV RNA was similar at entry (4.27 log vs. 4.35 log, p=0.87), differed at 2 weeks postpartum (2.67 log vs. 3.58 log, p<0.0001), but not at 6 weeks postpartum (4.49 log vs. 4.40 log, p=0.79), between single-dose nevirapine/zidovudine+lamivudine and single-dose nevirapine groups, respectively. Nevirapine resistance, measured by population sequencing and sensitive real-time PCR, was significantly less common in those receiving single-dose nevirapine/zidovudine+lamivudine compared to single-dose nevirapine, respectively, at 2 weeks (10% (4/40) vs. 74% (31/42), p<0.0001) and 6 weeks postpartum [10% (11/115) vs. 64% (41/64), p<0.0001; adjusted relative risk=0.18, 95% confidence interval (0.10–0.34)].
Conclusions
The significant decrease in nevirapine resistance conferred by one week of zidovudine+lamivudine should help policymakers optimize peripartum HIV prophylaxis recommendations.
PMCID: PMC2913302  PMID: 20672451
7.  Oligodendrocyte progenitor cells proliferate and survive in an immature state following treatment with an axolemma-enriched fraction 
ASN NEURO  2011;3(1):e00053.
The ability of an AEF (axolemma-enriched fraction) to influence the proliferation, survival and differentiation of OPC (oligodendrocyte progenitor cells) was evaluated. Following addition of AEF to cultured OPC, the AEF associated with the outer surface of OPC so that subsequent metabolic events were likely mediated by direct AEF-OPC contact. Addition of AEF to the cultured OPC resulted in a dose- and time-dependent increase in proliferation that was partially dependent on Akt (protein kinase B) and MAPK (mitogen-activated protein kinase) activation. The major mitogen in an AEF-SE (soluble 2.0 M NaCl extract of the AEF) was identified as aFGF (acidic fibroblast growth factor) and accounted for 50% of the mitogenicity. The remaining 50% of the mitogenicity had properties consistent with bFGF (basic fibroblast growth factor) but was not unequivocally identified. Under conditions that limit the survival of OPC in culture, AEF treatment prolonged the survival of the OPC. Antigenic and morphological examination of the AEF-treated OPC indicated that the AEF treatment helped the OPC survive in a more immature state. The potential downstream metabolic pathways potentially activated in OPC by AEF and the consequences of these activated pathways are discussed. The results of these studies are consistent with the view that direct contact of axons with OPC stimulates their proliferation and survival while preventing their differentiation.
doi:10.1042/AN20100035
PMCID: PMC3063503  PMID: 21345173
acidic fibroblast growth factor (aFGF); axolemma-enriched fraction (AEF); axonal–oligodendrocyte signalling; CNS trophic factors; multiple sclerosis; oligodendrocyte differentiation; oligodendrocyte progenitor cell; AEF, axolemma-enriched fraction; AEF-SE, soluble 2.0 M NaCl extract of the AEF; aFGF, acidic fibroblast growth factor; Akt, protein kinase B; BCA, bicinchoninic acid; BDNF, brain-derived neurotrophic factor; bFGF, basic fibroblast growth factor; CNPase, 2′,3′-cyclic nucleotide 3′-phosphodiesterase; CNS, central nervous system; CREB, cAMP-response-element-binding protein; DAPI, 4′,6-diamidino-2-phenylindole; F-12, Ham's F12 nutrient medium; DMEM–F12, Dulbecco's modified Eagle medium nutrient mixture F-12; DSHB, Developmental Studies Hybridoma Bank; ERK, extracellular-signal-regulated kinase; FCS, fetal calf serum; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; GalC, galactosylcerebroside; GFAP, glial fibrillary acidic protein; LIF, leukaemia inhibitory factor; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; NRG, neuregulin; OPC, oligodendrocyte progenitor cell; RIPA buffer, radio immunoprecipitation assay buffer
8.  Synthesis and antioxidant properties of dendritic polyphenols 
doi:10.1016/j.bmcl.2009.09.088
PMCID: PMC2988400  PMID: 19822425
9.  HIV-1 Inhibits Autophagy in Bystander Macrophage/Monocytic Cells through Src-Akt and STAT3 
PLoS ONE  2010;5(7):e11733.
Autophagy is a homeostatic mechanism of lysosomal degradation. Defective autophagy has been linked to various disorders such as impaired control of pathogens and neurodegeneration. Autophagy is regulated by a complex array of signaling pathways that act upstream of autophagy proteins. Little is known about the role of altered regulatory signaling in disorders associated with defective autophagy. In particular, it is not known if pathogens inhibit autophagy by modulation of upstream regulatory pathways. Cells infected with HIV-1 blocked rapamycin-induced autophagy and CD40-induced autophagic killing of Toxoplasma gondii in bystander (non-HIV-1 infected) macrophage/monocytic cells. Blockade of autophagy was dependent on Src-Akt and STAT3 triggered by HIV-1 Tat and IL-10. Neutralization of the upstream receptors VEGFR, β-integrin or CXCR4, as well as of HIV-1 Tat or IL-10 restored autophagy in macrophage/monocytic cells exposed to HIV-1-infected cells. Defective autophagic killing of T. gondii was detected in monocyte-derived macrophages from a subset of HIV-1+ patients. This defect was also reverted by neutralization of Tat or IL-10. These studies revealed that a pathogen can impair autophagy in non-infected cells by activating counter-regulatory pathways. The fact that pharmacologic manipulation of cell signaling restored autophagy in cells exposed to HIV-1-infected cells raises the possibility of therapeutic manipulation of cell signaling to restore autophagy in HIV-1 infection.
doi:10.1371/journal.pone.0011733
PMCID: PMC2908694  PMID: 20661303
10.  The public health approach to identify antiretroviral therapy failure: high-level nucleoside reverse transcriptase inhibitor resistance among Malawians failing first-line antiretroviral therapy 
AIDS (London, England)  2009;23(9):1127-1134.
Background
Over 150 000 Malawians have started antiretroviral therapy (ART), in which first-line therapy is stavudine/lamivudine/nevirapine. We evaluated drug resistance patterns among patients failing first-line ART on the basis of clinical or immunological criteria in Lilongwe and Blantyre, Malawi.
Methods
Patients meeting the definition of ART failure (new or progressive stage 4 condition, CD4 cell count decline more than 30%, CD4 cell count less than that before treatment) from January 2006 to July 2007 were evaluated. Among those with HIV RNA of more than 1000 copies/ml, genotyping was performed. For complex genotype patterns, phenotyping was performed.
Results
Ninety-six confirmed ART failure patients were identified. Median (interquartile range) CD4 cell count, log10 HIV-1 RNA, and duration on ART were 68 cells/μl (23–174), 4.72 copies/ml (4.26–5.16), and 36.5 months (26.6–49.8), respectively. Ninety-three percent of samples had nonnucleoside reverse transcriptase inhibitor mutations, and 81% had the M184V mutation. The most frequent pattern included M184V and nonnucleoside reverse transcriptase inhibitor mutations along with at least one thymidine analog mutation (56%). Twenty-three percent of patients acquired the K70E or K65R mutations associated with tenofovir resistance; 17% of the patients had pan-nucleoside resistance that corresponded to K65R or K70E and additional resistance mutations, most commonly the 151 complex. Emergence of the K65R and K70E mutations was associated with CD4 cell count of less than 100 cells/μl (odds ratio 6.1) and inversely with the use of zidovudine (odds ratio 0.18). Phenotypic susceptibility data indicated that the nucleoside reverse transcriptase inhibitor backbone with the highest activity for subsequent therapy was zidovudine/lamivudine/tenofovir, followed by lamivudine/tenofovir, and then abacavir/didanosine.
Conclusion
When clinical and CD4 cell count criteria are used to monitor first-line ART failure, extensive nucleoside reverse transcriptase inhibitor and nonnucleoside reverse transcriptase inhibitor resistance emerges, with most patients having resistance profiles that markedly compromise the activity of second-line ART.
doi:10.1097/QAD.0b013e32832ac34e
PMCID: PMC2896488  PMID: 19417582
Africa; antiretroviral failure; public health approach; resistance; resource-limited setting
11.  Transmitted Antiretroviral Drug Resistance among Acute and Recent HIV Infections in North Carolina, 1998 to 2007 
Antiviral therapy  2009;14(5):673-678.
Background
Transmitted drug resistance (TDR) limits antiretroviral options, complicating management of HIV-positive patients. HIV disproportionately affects the Southern United States (US), but available national estimates of TDR prevalence principally reflect large metropolitan centers outside this region.
Methods
The Duke/UNC Acute HIV Program has collected data on acute or recent HIV infections (ARHI) in North Carolina (NC) since 1998. Acute infections represent antibody-negative, RNA-positive subjects; recent infection was determined by history of HIV testing, or concordance between detuned ELISA and antibody avidity assays. Genotypic sequence data from the earliest collected pre-treatment plasma sample were analyzed with the Stanford HIV Database and screened for Surveillance Drug Resistance Mutations (SDRMs).
Results
253 individuals with ARHI between 1998 and May 2007 had complete genotypic sequence data for analysis; 39.5% were acute infections, 78.7% were male, 64.8% were non-white, and 53.8% were men who have sex with men. The overall prevalence of TDR was 17.8%, with SDRMs for non-nucleoside reverse transcriptase inhibitors (NNRTIs) in 9.5% of the cohort. Mutations for nucleos(t)ide RT inhibitors (NRTIs) were detected in 7.5%, and for protease inhibitors (PIs) in 3.2%. K103N was the most common mutation (7.5%). Thymidine analogue mutations were found in 4.7% of samples; the most common PI SDRM was L90M (2.4%). Dual-or triple-class antiretroviral resistance was rare, encountered in only six samples (2.4%).
Conclusions
The prevalence of TDR in NC is similar to estimates from US metropolitan areas. These findings have implications for initial regimen selection and secondary prevention efforts outside of large, metropolitan HIV epicenters.
PMCID: PMC2860724  PMID: 19704170
HIV Infections/epidemiology; HIV Infections/transmission; North Carolina/epidemiology; Drug resistance, viral; Antiretroviral therapy, highly active
12.  Nevirapine Resistance in Human Immunodeficiency Virus Type 1-Positive Infants Determined Using Dried Blood Spots Stored for Up to Six Years at Room Temperature▿  
Journal of Clinical Microbiology  2009;47(4):1209-1211.
Dried blood spots that had been stored ambiently for 3 to 6 years lost approximately 1 log10 of human immunodeficiency virus type 1 (HIV-1) RNA, but the majority could still be genotyped for resistance. Nevirapine resistance was found in 7/16 (43.5%) HIV-1-positive HIVNET 024 infants at 4 to 6 weeks, but no resistance was found at other time points.
doi:10.1128/JCM.00254-09
PMCID: PMC2668311  PMID: 19225101
13.  Qualitative Human Immunodeficiency Virus RNA Analysis of Dried Blood Spots for Diagnosis of Infections in Infants▿  
Journal of Clinical Microbiology  2008;47(1):220-222.
The Gen-Probe Aptima human immunodeficiency virus type 1 (HIV-1) RNA assay was adapted for the diagnosis of HIV infection in infants by using dried blood spots. The assay was 99% sensitive (128/129) and 100% specific (162/162). This may prove useful in resource-limited settings, since it precludes the need for a phlebotomist and maintenance of a cold chain.
doi:10.1128/JCM.01521-08
PMCID: PMC2620881  PMID: 19005148
14.  Comparison of Two Rapid Human Immunodeficiency Virus (HIV) Assays, Determine HIV-1/2 and OraQuick Advance Rapid HIV-1/2, for Detection of Recent HIV Seroconversion▿  
Journal of Clinical Microbiology  2008;46(10):3482-3483.
Modified protocols of two rapid tests were compared with a less sensitive (LS) (detuned) enzyme immunoassay (EIA) for their abilities to distinguish recent human immunodeficiency virus (HIV) seroconversion from long-term infections. The results for samples from 100 HIV-positive patient that had previously been tested by the Vironostika LS EIA had a 97% concordance with the results of the Determine HIV 1/2 assay and 93% concordance with those of the OraQuick HIV 1/2 assay.
doi:10.1128/JCM.00665-08
PMCID: PMC2566129  PMID: 18685013
15.  Independent Evolution of Human Immunodeficiency Virus Type 1 env V1/V2 and V4/V5 Hypervariable Regions during Chronic Infection▿  
Journal of Virology  2007;81(10):5413-5417.
Using DNA heteroduplex tracking assays, we characterized human immunodeficiency virus type 1 env V4/V5 genetic populations in multiple blood plasma samples collected over an average of 7 months from 24 chronically infected human subjects. We observed complex and dynamic V4/V5 genetic populations in most subjects. Comparisons of V4/V5 and V1/V2 population changes over the course of the study showed that major shifts in genetic populations frequently occurred in one region but not the other, and these observations were independently confirmed in one subject by single-genome sequencing. These results suggest that the V1/V2 and V4/V5 regions of env often evolve independently during chronic infection.
doi:10.1128/JVI.02554-06
PMCID: PMC1900199  PMID: 17329337
16.  Sequential Turnover of Human Immunodeficiency Virus Type 1 env throughout the Course of Infection▿ †  
Journal of Virology  2006;80(21):10591-10599.
We examined the rates of variant population turnover of the V1-V2 and V4-V5 hypervariable domains of the human immunodeficiency virus type 1 (HIV-1) gp120 molecule in longitudinal plasma samples from 14 men with chronic HIV-1 infection using heteroduplex tracking assays (HTA). Six men had high rates of CD4+ T-cell loss, and eight men had low rates of CD4+ T-cell loss over 2.5 to 8 years of infection. We found that V1-V2 and V4-V5 env populations changed dramatically over time in all 14 subjects; the changes in these regions were significantly correlated with each another over time. The subjects with rapid CD4 loss had significantly less change in their env populations than the subjects with slow CD4 loss. The two subjects with rapid CD4 loss and sustained low CD4 counts (<150/μl for at least 2 years) showed stabilization of their V1-V2 and V4-V5 populations as reflected by low levels of total change in HTA pattern and low HTA indices (a novel measure of the emergence of new bands and band distribution); this stabilization was not observed in other subjects. The stabilization of env variant populations at low CD4 counts following periods of rapid viral evolution suggests that selective pressure on env, likely from new immune responses, is minimal when CD4 counts drop dramatically and remain low for extended periods of time.
doi:10.1128/JVI.00644-06
PMCID: PMC1641766  PMID: 16956948
17.  Effect of a Protease Inhibitor-Induced Genetic Bottleneck on Human Immunodeficiency Virus Type 1 env Gene Populations 
Journal of Virology  2005;79(16):10627-10637.
The initiation of drug therapy or the addition of a new drug to preexisting therapy can have a significant impact on human immunodeficiency virus type 1 (HIV-1) populations within a person. Drug therapy directed at reverse transcriptase and protease can result in dramatic decreases in virus load, causing a contraction in the virus population that represents a potential genetic bottleneck as a subset of virus with genomes carrying resistance mutations repopulate the host. While this bottleneck exerts an effect directly on the region that is being targeted by the drugs, it also affects other regions of the viral genome. We have applied heteroduplex tracking assays (HTA) specific to variable regions 1 and 2 (V1/V2) and variable region 3 (V3) of the HIV-1 env gene to analyze the effect of a genetic bottleneck created by the selection of resistance to ritonavir, a protease inhibitor. Subjects were classified into groups on the basis of the extent of the initial drop in virus load and the duration of virus load reduction. Subjects with a strong initial drop in virus load exhibited a loss of heterogeneity in the env region at virus load rebound; in contrast, subjects with a weak initial drop in virus load exhibited little to no loss of heterogeneity at virus load rebound in either region of env examined. The duration of virus load reduction also affected env populations. Subjects that had prolonged reductions exhibited slower population diversification and the appearance of new V1/V2 species after rebound. The longer reduction of virus load in these subjects may have allowed for improved immune system function, which in turn could have selected for new escape mutants. Subjects with rapid rebound quickly reequilibrated the entry env variants back into the resistant population. When the pro gene developed further resistance mutations subsequent to virus load rebound, no changes were observed in V1/V2 or V3 populations, suggesting that the high virus loads allowed the env populations to reequilibrate rapidly. The rapid equilibration of env variants during pro gene sequence transitions at high virus load suggests that recombination is active in defining the HIV-1 sequence population. Conversely, part of the success of suppressive antiviral therapy may be to limit the potential for evolution through recombination, which requires dually infected cells.
doi:10.1128/JVI.79.16.10627-10637.2005
PMCID: PMC1182671  PMID: 16051855
18.  CXCR4-Dependent Infection of CD8+, but Not CD4+, Lymphocytes by a Primary Human Immunodeficiency Virus Type 1 Isolate 
Journal of Virology  2004;78(22):12288-12296.
We recently isolated from an infant an X4-syncytium-inducing (SI) human immunodeficiency virus type 1 (HIV-1) variant (92US143-T8) that was able to infect CD8+ lymphocytes independently of CD4. Although it was CD4 independent, the 92US143-T8 isolate also maintained the ability to infect CD4+ cells. In the present study, we investigated the role of CXCR4 in the infection of CD4+ and CD8+ cells by this primary isolate. The expression of CXCR4 was down modulated in CD8+ lymphocytes after infection with the 93US143-T8 isolate. Infection of CD8+ lymphocytes by the 93US143-T8 isolate was prevented by treatment with AMD3100, a specific antagonist for CXCR4, indicating CXCR4-dependent infection. Interestingly, AMD3100 treatment had no inhibitory role in the infection of purified CD4+ lymphocytes by the same isolate. Furthermore, AMD3100 treatment failed to prevent infection of known CD4+ CXCR4+ T-cell lines (MT-2 and CEM) by the 93US143-T8 isolate. In fact, virus replication in the CD4+ cells was often enhanced in the presence of AMD3100. Viruses produced from the infected CD4+ cells in the presence of AMD3100 maintained an unchanged envelope genotype and an SI phenotype. For the first time, these results provide evidence of CXCR4-dependent infection of CD8+ lymphocytes by a primary HIV-1 isolate. This study also shows a different mode of infection for the CD4+ and CD8+ lymphocytes by the same HIV-1 variant. Finally, our findings suggest that a more careful evaluation is necessary before the random use of AMD3100 as a new entry inhibitor in patients harboring SI HIV-1 strains.
doi:10.1128/JVI.78.22.12288-12296.2004
PMCID: PMC525083  PMID: 15507616
19.  Multiple V1/V2 env Variants Are Frequently Present during Primary Infection with Human Immunodeficiency Virus Type 1 
Journal of Virology  2004;78(20):11208-11218.
Human immunodeficiency virus type 1 (HIV-1) exists as a complex population of multiple genotypic variants in persons with chronic infection. However, acute HIV-1 infection via sexual transmission is a low-probability event in which there is thought to be low genetic complexity in the initial inoculum. In order to assess the viral complexity present during primary HIV-1 infection, the V1/V2 and V3 variable regions of the env gene were examined by using a heteroduplex tracking assay (HTA) capable of resolving these genotypic variants. Blood plasma samples from 26 primary HIV-1-infected subjects were analyzed for their level of diversity. Half of the subjects had more than one V1/V2 viral variant during primary infection, indicating the frequent transmission of multiple variants. This observation is inconsistent with the idea of infrequent transmission based on a small transmitting inoculum of cell-free virus. In chronically infected subjects, the complexity of the viral populations was even greater in both the V1/V2 and the V3 regions than in acutely infected subjects, indicating that in spite of the presence of multiple variants in acute infection, the virus does pass through a genetic bottleneck during transmission. We also examined how well the infecting virus penetrated different anatomical compartments by using the HTA. Viral variants detected in blood plasma were compared to those detected in seminal plasma and/or cerebral spinal fluid of six individuals. The virus in each of these compartments was to a large extent identical to virus in blood plasma, a finding consistent with rapid penetration of the infecting variant(s). The low-probability transmission of multiple variants could be the result of transient periods of hyperinfectiousness or hypersusceptibility. Alternatively, the inefficient transfer of a multiply infected cell could account for both the low probability of transmission and the transfer of multiple variants.
doi:10.1128/JVI.78.20.11208-11218.2004
PMCID: PMC521858  PMID: 15452240
21.  Isolation of CD4-Independent Primary Human Immunodeficiency Virus Type 1 Isolates That Are Syncytium Inducing and Acutely Cytopathic for CD8+ Lymphocytes 
Journal of Virology  2004;78(3):1243-1255.
Previous studies have established the existence of CD4-independent simian immunodeficiency virus, human immunodeficiency virus type 2 (HIV-2), and laboratory strains of HIV-1. However, whether CD4-independent viruses may also exist in HIV-1-infected patients has remained unclear. We have recently reported the isolation of viruses from an AIDS patient that were able to infect CD8+ cells independent of CD4, using CD8 as a receptor. Using a similar approach, here we examined viruses from 12 randomly selected patients (obtained from the AIDS Research and Reference Program, National Institutes of Health) for the presence of CD4-independent HIV-1. CD4-independent variants were isolated from infected CD8+ cells from the viral quasispecies of 7 of 12 patients. The CD4-independent isolates were able to infect primary CD8+ cells as well as a CD4− CD8+ T-cell line. Soluble CD4 and blocking anti-CD4 or -CD8 antibody had no effect on infection of CD8+ cells. Remarkably, two of the seven CD4-independent isolates, but not their parental bulk viruses, induced syncytia and caused acute death of infected CD8+ cells. Some of the CD4-independent variants were also able to infect U87 cells that were negative for CD4, CD8, and common HIV coreceptors, suggesting a novel entry mechanism for these isolates. The CD4-independent isolates were derived from adults and children infected with subtypes A, B, and D. Although no common motif for CD4 independence was found, novel sequence changes were observed in critical areas of the envelopes of the CD4-independent viruses. These results demonstrate that HIV-1-infected patients can frequently harbor viruses that are able to mediate CD4-independent infection of CD8+ cells. In addition, this study also provides evidence of primary HIV-1 variants that are syncytium inducing and acutely cytopathic for CD8+ lymphocytes.
doi:10.1128/JVI.78.3.1243-1255.2004
PMCID: PMC321385  PMID: 14722279
22.  Turnover of env Variable Region 1 and 2 Genotypes in Subjects with Late-Stage Human Immunodeficiency Virus Type 1 Infection 
Journal of Virology  2003;77(12):6811-6822.
The env gene of human immunodeficiency virus type 1 (HIV-1) includes some of the most genetically diverse regions of the viral genome, which are called variable regions 1 through 5 (V1 through V5). We have developed a heteroduplex tracking assay to detect changes in variable regions 1 and 2 of env (V1/V2-HTA). Using sequences from two molecular clones as probes, we have studied the nature of longitudinal virus population changes in a cohort of HIV-1-infected subjects. Viral sequences present in 21 subjects with late-stage HIV-1 infection were initially screened for stability of the virus population by V1/V2-HTA. The virus populations at entry comprised an average of five coexisting V1/V2 genotypic variants (as identified by HTA). Eight of the 21 subjects were examined in detail because of the dynamic behavior of their env variants over an approximately 9-month period. In each of these cases we detected a single discrete transition of V1/V2 genotypes based on monthly sampling. The major V1/V2 genotypes (those present at >10% abundance) from the eight subjects were cloned and sequenced to define the nature of V1/V2 variability associated with a discrete transition. Based on a comparison of V1/V2 genotypic variants present at entry with the newly emerged variants we categorized the newly emerged variants into two groups: variants without length differences and variants with length differences. Variants without length differences had fewer nucleotide substitutions, with the changes biased to either V1 or V2, suggestive of recent evolutionary events. Variants with length differences included ones with larger numbers of changes that were distributed, suggestive of recall of older genotypes. Most length differences were located in domains where the codon motif AVT (V = A, G, C) had become enriched and fixed. Finally, recombination events were detected in two subjects, one of which resulted in the reassortment of V1 and V2 regions. We suggest that turnover in V1/V2 populations was largely driven by selection on either V1 or V2 and that escape was accomplished either through changes focused in the region under selection or by the appearance of a highly divergent variant.
doi:10.1128/JVI.77.12.6811-6822.2003
PMCID: PMC156159  PMID: 12768001
23.  Characterization of Human Immunodeficiency Virus Type 1 in Saliva and Blood Plasma by V3-Specific Heteroduplex Tracking Assay and Genotype Analyses 
Journal of Virology  2001;75(10):4936-4940.
The gp120 V3-encoding region of human immunodeficiency virus type 1 (HIV-1) RNA derived from the saliva and blood plasma of 11 individuals was characterized by heteroduplex tracking assay and sequence analyses. R5-like viral variants were identified in both fluids of all subjects. X4-like variants were detected in the plasma and/or saliva of three subjects, indicating that X4-like variants are not excluded from the saliva compartment. Viral subpopulations were similar in both fluids of most subjects, suggesting that HIV-1 in oral fluids and blood may stem from a common source. These findings raise the possibility of using saliva as a noninvasive fluid for evaluating and monitoring viral evolution in infected persons.
doi:10.1128/JVI.75.10.4936-4940.2001
PMCID: PMC114251  PMID: 11312368
24.  Effects of Genital Tract Inflammation on Human Immunodeficiency Virus Type 1 V3 Populations in Blood and Semen 
Journal of Virology  2000;74(19):8946-8952.
We have examined cell-free viral populations in the blood plasma and seminal plasma compartments of men infected with subtype C human immunodeficiency virus type 1 (HIV-1) using the V3-specific heteroduplex tracking assay (V3-HTA). We studied two cohorts of subjects who had visited either a sexually transmitted disease (STD) clinic for genital tract inflammation in the form of urethritis (n = 43) or a dermatology clinic (controls, n = 14) in Malawi. We have previously shown that the presence of urethritis is associated with an eightfold increase in virus load in the seminal plasma compartment (M. S. Cohen et al., Lancet 349:1868–1873, 1997). The purpose of this study was to determine whether genital tract inflammation and its treatment caused genetic instability in cell-free HIV-1 populations. In a cross-sectional analysis at study entry, three-fourths of the STD and control subjects had multiple V3 populations in their blood while 60% of the STD subjects and 79% of the control subjects had multiple V3 populations in their semen. Overall, one-fourth of all of the subjects showed discordance between results with blood and semen specimens when samples were compared for the presence and absence of subpopulations. When differences in the relative levels of abundance of bands were also taken into account, two-fifths of all of the subjects showed discordance between the compartments. Among the subset of subjects in whom multiple virus populations could be detected, half showed discordance between the compartments. There were no differences between STD and control cohorts for these comparisons of the compartments in this cross-sectional analysis at study entry. Longitudinal analysis of the viral populations from two separate clinic visits over 1 to 4 weeks showed that the complexity of each V3 population as measured by Shannon entropy was different in blood and semen at the two time points, indicating that the blood and semen constitute different compartments for HIV-1. The seminal plasma compartment was more dynamic than the blood plasma compartment for the STD subjects who were treated for urethritis, with changes being noted in the presence or absence of V3-HTA bands in the semen of 29% of these subjects but in the blood of only 9% of these subjects. However, the changes were generally small. Overall, our results suggest that 40% of male subjects show discordance between seminal and blood viral populations and that the complexity of each V3 population was different between the two compartments. Both of these results point to the partial independence of the seminal compartment as a viral niche within the body.
PMCID: PMC102090  PMID: 10982338
25.  Patterns of Changes in Human Immunodeficiency Virus Type 1 V3 Sequence Populations Late in Infection 
Journal of Virology  2000;74(18):8494-8501.
We have used a V3-specific heteroduplex tracking assay (V3-HTA) with probes from two different human immunodeficiency virus type 1 (HIV-1) subtypes to examine the extent and pace of HIV-1 evolution late in infection. Twenty-four subjects with advanced HIV-1 infection (CD4+ T-cell count, <100/μl) and stable viral loads were studied using blood plasma samples collected over a study period of approximately 9 months, during which time most of the subjects were treated with reverse transcriptase inhibitors. The V3-HTA patterns from the first and last time points were evaluated initially to determine the amounts of change in V3 sequence populations, which were primarily changes in abundance in preexisting sequence populations. Three of the 24 subjects had major changes (greater than 50% total change in the relative abundance of the sequence populations), 11 subjects had intermediate changes (10 to 50% total change), and 10 subjects had minimal changes (less than 10% total change). The average total amount of change was between two- and threefold greater in subjects with X4-like variants, although there was no correlation between average viral load and the presence of X4-like variants. V3-HTA patterns in monthly samples from 11 of the subjects were also compared. In two subjects, the amount of change exceeded 40% in a 1-month period. Overall, the pace of change in V3 populations varied between subjects and was not constant within a subject over time. Sequence analysis of the V3 variants showed that R5-like variants (not containing any X4-associated substitutions) continued to be maintained in three subjects in the presence of X4-like variants, indicating that X4 variants do not always outgrow R5 variants. The coreceptor usage of the V3 sequences from two subjects was determined using a cell fusion assay. One subject had an X4 variant that was maintained at a low level for at least 9 months, during which time the predominant variants were R5X4 (dualtropic), while in the second subject the reverse situation was observed. One of the dualtropic variants had a novel sequence motif in V3, suggesting another evolutionary pathway to altered tropism. These studies begin to probe the complexities and pace of V3 evolution in vivo, revealing dynamic patterns of change among multiple V3 sequence variants in a subset of subjects.
PMCID: PMC116361  PMID: 10954550

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