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1.  Amniotic Fluid Exhibits an Innate Inhibitory Activity Against HIV Type 1 Replication in Vitro 
Indirect evidence suggests that amniotic fluid (AF) may play a role in the pathogenesis of in utero HIV-1 transmission. The purpose of this study was to evaluate the potential innate inhibitory role of AF on HIV replication, which may contribute to protection of the fetus against intrauterine transmission. AF was collected from term HIV-1-negative women undergoing scheduled cesarean section. The inhibitory effect of AF against HIV-1BA-L replication was tested in vitro with or without the addition of protease inhibitor cocktail (PIC) in PHA-stimulated PBMC cultures. Quantitative measurement of human neutrophil peptides 1-3 (HNP1-3) was performed on all AF samples, using an ELISA assay. AF exhibited a dose-dependent inhibitory activity against HIV-1BA-L replication, with all samples (n=12) reaching significant inhibitory effect using 50% AF. In vitro, this activity decreased over time, but was able to be sustained with the addition of PIC. The HNP1-3 concentration in AF samples (n=12) ranged from undetectable (<41 pg/ml, n=3) to >250,000 pg/ml with a median of 5,146 pg/ml. AF exhibited a significant and dose-dependent innate inhibitory activity against HIV-1 replication, which was present in all AF samples tested. This effect was prolonged in the presence of PIC, suggesting that the inhibitory factor was in the cell-free protein fraction. The HNP1-3 concentration in AF was in the subinhibitory range for HIV with no correlation between its concentration and the HIV-1 inhibitory activity. These data show the presence of a significant innate inhibitory activity against HIV in AF.
PMCID: PMC3537303  PMID: 22998428
2.  Short Communication: Enhanced CD8+ T Cell Apoptosis in HIV-Infected Adolescents with Virologic Failure on Protease Inhibitor-Based Therapy 
In this study, we investigated the possibility of differential effects of protease inhibitor (PI)-containing (PI+) and PI-sparing (PI−) antiretroviral therapies (ART) on CD8+ T cell apoptosis. We retrospectively analyzed both PD-1 expression and CD8+ T cell apoptosis in a cross-sectional study of HIV-positive adolescents and young adults (mean age = 17.4 years), with perinatally or behaviorally acquired HIV infection. Fifty-one specimens of cryopreserved peripheral blood mononuclear cells (PBMCs) were analyzed using 7-color flow cytometry: 20 from patients receiving PI+ ART, 14 from PI− ART, and 17 from the untreated. The results showed that percentages of PD-1+ CD8+ T cells were strongly correlated with plasma viral loads regardless of treatment (p = 0.0001). The percentage of PD-1+ CD8+ T cells was also positively associated with percentages of Annexin V+ CD8+ T cells (p = 0.04) in the PI+-treated group. The fraction of apoptotic (Annexin V+) CD8+ T cells was associated with viral load in the patients receiving ART that contained one or more protease inhibitors (p = 0.029), but not in the PI− or untreated groups. In summary, we found a direct correlation between PD-1 expression on CD8+ T cells and HIV levels that was not affected by types of medications used in the ART of those adolescents, suggesting that virological success is necessary for PD-1 downregulation. CD8+ T cell apoptosis was linked to high levels of PD-1 expression and HIV viremia. However, there was a higher degree of apoptosis among viremic patients receiving PI therapy, suggesting an immunologically adverse effect of continuing PI+ therapy after virological failure.
PMCID: PMC2932552  PMID: 20507209
3.  Interleukin-15 Enhances Cytotoxicity, Receptor Expression, and Expansion of Neonatal Natural Killer Cells in Long-Term Culture 
Newborn infants have a higher susceptibility to various pathogens due to developmental defects in their host defense system, including deficient natural killer (NK) cell function. In this study, the effects of interleukin-15 (IL-15) on neonatal NK cells was examined for up to 12 weeks in culture. The cytotoxicity of fresh neonatal mononuclear cells (MNC) as assayed by K562 cell killing is initially much less than that of adult MNC but increases more than eightfold after 2 weeks of culture with IL-15 to a level equivalent to that of adult cells. This high level of cytotoxicity was maintained for up to 12 weeks. In antibody-dependent cellular cytotoxicity (ADCC) assays using CEM cells coated with human immunodeficiency virus gp120 antigen, IL-15 greatly increased ADCC lysis by MNC from cord blood. IL-15 increased expression of the CD16+ CD56+ NK markers of cord MNC fivefold after 5 weeks of incubation. Cultures of neonatal MNC with IL-15 for up to 10 weeks resulted in a unique population of CD3− CD8+ CD56+ cells (more than 60%), which are not present in fresh cord MNC. These results show that IL-15 can stimulate neonatal NK cells and sustain their function for several weeks, which has implications for the clinical use of IL-15.
PMCID: PMC515280  PMID: 15358647
4.  Performance of the Applied Biosystems ViroSeq Human Immunodeficiency Virus Type 1 (HIV-1) Genotyping System for Sequence-Based Analysis of HIV-1 in Pediatric Plasma Samples 
Journal of Clinical Microbiology  2001;39(4):1254-1257.
The ViroSeq HIV-1 Genotyping System is a commercially available, integrated sequence-based system for analysis of human immunodeficiency virus type 1 (HIV-1) drug resistance. We evaluated the performance of this system by analyzing HIV-1 in pediatric plasma samples. Plasma samples from children 4 months to 17 years of age were obtained from a clinical trial protocol (PACTG 377). Children in PACTG 377 were randomized to four treatment arms, including different combinations of antiretroviral drugs. HIV-1 genotyping was performed using samples collected prior to antiretroviral therapy (baseline) and at the time of virologic failure. Performance of the genotyping system was compared in three university laboratories. A total of 196 samples were analyzed, including 135 baseline and 61 failure samples. Plasma volumes ranged from 0.05 to 0.5 ml, and viral loads ranged from 1,084 to 3,484,991 copies/ml. PCR products suitable for sequencing were obtained for 192 of the 196 samples. Complete sequences for protease and reverse transcriptase were obtained for all of these 192 samples. For 180 samples, data were obtained from both DNA strands for the entire region analyzed. There was no evidence of sample cross-contamination based on phylogenetic analysis of HIV-1 sequences. Performance of the genotyping system was similar in three laboratories. This genotyping system performs well for analysis of HIV-1 in pediatric plasma samples, including those with low volume and low viral load. The availability of this system should facilitate studies of HIV-1 drug resistance.
PMCID: PMC87920  PMID: 11283037
5.  Enhancement of Antibody-Dependent Cellular Cytotoxicity of Neonatal Cells by Interleukin-2 (IL-2) and IL-12 
Newborn infants are more susceptible to infections due in part to deficiencies in the cytotoxic functions of their lymphocytes. We investigated the ability of interleukin-2 (IL-2) and IL-12 to enhance the cytotoxicity of neonatal (cord blood) and adult mononuclear cells (MNCs) in both natural killer (NK) cell and antibody-dependent cellular cytotoxicity (ADCC) assays. The cytotoxic activity of cord blood MNCs was less than 50% that of adult MNCs in most assays prior to exposure to cytokines. Incubation with IL-2 (100 U/ml) or IL-12 (1 ng/ml) for 18 h increased the NK cell activity (using K562 target cells) of both cord blood and adult MNCs, and the combination of IL-2 and IL-12 increased cord blood cytotoxicity threefold, making the cytotoxicity of cord blood cells equivalent to that of adult cells treated with the same cytokines. In ADCC assays with chicken erythrocyte targets, the combination of IL-2 and IL-12 increased the cytotoxicities of both cord blood and adult MNCs, with greater enhancement again seen with cord blood cells. In assays with NK cell-resistant CEM cells coated with human immunodeficiency virus (HIV) gp120 antigen in the presence of hyperimmune anti-HIV immunoglobulin, ADCC of cord blood MNCs was about 50% that of adult MNCs; ADCC of cord blood MNCs increased two- to threefold with the addition of IL-2 and IL-12, whereas ADCC of adult MNCs did not increase. Incubation of cord blood cells, but not adult cells, with IL-2 or IL-12 for 1 week increased the percentage of CD16+/CD56+ cells two- to fivefold and enhanced ADCC activity. Thus, IL-2 and IL-12 greatly enhance both the NK cell and ADCC activities of neonatal MNCs and increase the number of NK cells in longer-term culture.
PMCID: PMC121400  PMID: 9455889

Results 1-5 (5)