Addition of the CCR5 inhibitor Maraviroc (MVC) to ongoing antiretroviral therapy increases CD4+ T cell counts in some virologically suppressed patients with suboptimal CD4+ T cell recovery. To understand the mechanisms by which MVC elicits increases in CD4+ T cell counts, the present study was undertaken to identify host factors (i.e. genes) that are modulated and are correlated with CD4+ T cell recovery during the 24 weeks of MVC intensification in 32 subjects. Median changes of CD4+ T cell counts over 24 weeks of MVC compared to baseline were 38 cells/mm3 (p < 0.001). The median slope of CD4+ T cell recovery was 39 cells/mm3 per year before initiation of MVC and 76 cells/mm3 per year during MVC intensification, however, this increase was not statistically significant (p = 0.33). Microarray analysis (N = 31,426 genes) identified a single differentially expressed gene, tumor necrosis factor alpha (TNF), which was modestly (1.44-fold, p < 0.001) downregulated by MVC at week 24 compared to baseline. TNF differential expression was evaluated using an independent method of droplet digital PCR, but the difference was not significant (p = 0.6). Changes in gene expression did not correlate with CD4+ T cell recovery or any changes in the CD4+ T cell maturation, proliferation and activation phenotypes. In summary, our data suggest that modest improvements of CD4+ T cell counts during MVC intensification cannot be explained by changes in gene expression elicited by MVC. However, the modest changes in T cell composition, including reduction of the percentages of Tregs, proliferating CD4+ T cells and senescent CD8+ T cells, suggest immunologically favorable effects of MVC.

HIV is able to outpace the innate immune response, including that mediated by interferon (IFN), to establish a productive infection. Primary macrophages, however, may be protected from HIV infection by treatment with type I IFN before virus exposure. The ability of HIV to modulate the type I IFN-mediated innate immune response when it encounters a cell that has already been exposed to IFN remains poorly defined. The optimal pretreatment time (12 h) and the most potent HIV-inhibitors (e.g., IFN-α2 and -ω) were identified to investigate the ability of HIV to modulate an established type I IFN response. Gene expression at the level of the entire transcriptome was then compared between primary macrophages treated with type I IFNs, as opposed to treated with IFNs and then infected with HIV. Although HIV was not able to establish a robust infection, the virus was able to downregulate a number of IFN-stimulated genes (ISGs) with a fold change greater than 1.5 (i.e., AXL, IFI27, IFI44, IFI44L, ISG15, OAS1, OAS3, and XAF1). The downregulation of OAS1 by the presence of HIV was confirmed by real-time quantitative polymerase chain reaction. In conclusion, even though HIV replication is significantly inhibited by IFN pretreatment, the virus is able to downregulate the transcription of known antiviral ISGs (e.g., IFI44, ISG15, and OAS1).

Deoxyribonucleic acid (DNA) of the human immunodeficiency virus (HIV) provides the most sensitive measurement of residual infection in patients on effective combination antiretroviral therapy (cART). Droplet digital PCR (ddPCR) has recently been shown to provide highly accurate quantification of DNA copy number, but its application to quantification of HIV DNA, or other equally rare targets, has not been reported. This paper demonstrates and analyzes the application of ddPCR to measure the frequency of total HIV DNA (pol copies per million cells), and episomal 2-LTR (long terminal repeat) circles in cells isolated from infected patients. Analysis of over 300 clinical samples, including over 150 clinical samples assayed in triplicate by ddPCR and by real-time PCR (qPCR), demonstrates a significant increase in precision, with an average 5-fold decrease in the coefficient of variation of pol copy numbers and a >20-fold accuracy improvement for 2-LTR circles. Additional benefits of the ddPCR assay over qPCR include absolute quantification without reliance on an external standard and relative insensitivity to mismatches in primer and probe sequences. These features make digital PCR an attractive alternative for measurement of HIV DNA in clinical specimens. The improved sensitivity and precision of measurement of these rare events should facilitate measurements to characterize the latent HIV reservoir and interventions to eradicate it.

ABSTRACT To determine the influence of asymptomatic genital viral infections on the cellular components of semen and blood, we evaluated the associations between the numbers and activation statuses of CD4 + and CD8 + T lymphocytes in both compartments and the seminal levels of cytomegalovirus (CMV), herpes simplex virus (HSV), and human immunodeficiency virus 1 (HIV). Paired blood and semen samples were collected from 36 HIV-infected antiretroviral-naïve individuals and from 40 HIV-uninfected participants. We performed multiparameter flow cytometry analysis (CD45, CD45RA, CD3, CD4, CD8, and CD38) of seminal and blood cellular components and measured HIV RNA and CMV and HSV DNA levels in seminal and blood plasma by real-time PCR. Compared to HIV-uninfected participants, in the seminal compartment HIV-infected participants had higher levels of CMV ( P < 0.05), higher numbers of total CD3 + ( P < 0.01) and CD8 + subset ( P < 0.01) T lymphocytes, and higher CD4 + and CD8 + T lymphocyte activation (RA-CD38 + ) ( P < 0.01). Seminal CMV levels positively correlated with absolute numbers of CD4 + and CD8 + T cells in semen ( P < 0.05) and with the activation status of CD4 + T cells in semen and in blood ( P < 0.01). HIV levels in semen ( P < 0.05) and blood ( P < 0.01) were positively associated with T-cell activation in blood. Activation of CD8 + T cells in blood remained an independent predictor of HIV levels in semen in multivariate analysis. The virologic milieu in the male genital tract strongly influences the recruitment and activation of immune cells in semen and may also modulate T-cell immune activation in blood. These factors likely influence replication dynamics, sexual transmission risk, and disease outcomes for all three viruses.

Objective: To identify a pre-HAART gene expression signature in peripheral blood mononuclear cells (PBMCs) predictive of CD4(+) T-cell recovery during HAART in HIV-infected individuals.Design: This retrospective study evaluated PBMC gene expression in 24 recently HIV-infected individuals before the initiation of HAART to identify genes whose expression is predictive of CD4(+) T-cell recovery after 48 weeks of HAART.Methods: The change in CD4(+) T-cell count (Delta CD4) over the 48-week study period was calculated for each of the 24 participants. Twelve participants were assigned to the 'good' (Delta CD4 >= 200 cells/mu l) and 12 to the 'poor' (Delta CD4 < 200 cells/mu l) CD4(+) T-cell recovery group. Gene expression profiling of the entire transcriptome using Illumina BeadChips was performed with PBMC samples obtained before HAART. Gene expression classifiers capable of predicting CD4(+) T-cell recovery group (good vs. poor), as well as the specific Delta CD4 value, at week 48 were constructed using methods of Class Prediction.Results: The expression of 40 genes in PBMC samples taken before HAART predicted CD4(+) T-cell recovery group (good vs. poor) at week 48 with 100% accuracy. The expression of 22 genes predicted a specific Delta CD4 value for each HIV-infected individual that correlated well with actual values (R=0.82). Predicted Delta CD4 values were also used to assign individuals to good vs. poor CD4(+) T-cell recovery groups with 79% accuracy.Conclusion: Gene expression in PBMCs can be used as biomarkers to successfully predict disease outcomes among HIV-infected individuals treated with HAART. (C) 2010 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins

Purpose: The purpose of this study was to determine the cytokine-related pathogenesis of human immunodeficiency virus retinopathy in human autopsy eyes.Methods: Fresh autopsy eyes were procured from clinically diagnosed patients with acquired immunodeficiency syndrome who had died as a result of disease-related complications; eyes were immediately immersed in RNAlater. Clean 2-mm trephines were used to punch individual pathologic retina in areas of cotton-wool spots and control punches. Total RNA was extracted using the TRIzol extraction protocol, and the optimal density of the RNA was measured at an optical density of 260 nm. [Delta]Ct (cytokine) values were calculated using the comparative cytokine analysis method. The results are expressed as a mean fold modulation and as a statistical comparison of Ct values controlling for retinal areas without a lesion in the same eye.Results: The fold modulations and the statistical comparisons of the cytokines studied in tissues from cotton-wool spots and control retina, respectively, regulated on activation normal T cell expressed and secreted (RANTES), macrophage inflammatory protein 1 beta, macrophage inflammatory protein 1 alpha (5.32x, P = 0.04), and Bcl-2-associated X protein (1.24x, P = 0.05) had a marked elevation of fold modulation and were statistically significant compared with control tissue. Interleukin-8 (1.09x, P = 0.18), interleukin-4, and interleukin-10 (2.7x, P = 0.30) were not significantly expressed in cotton-wool spots.Conclusion: Certain inflammatory human immunodeficiency virus-associated and apoptotic cytokines are expressed in cottonwool spots in eyes with human immunodeficiency virus retinopathy. RETINA 30: 952-957, 2010

Nearly all cervical cancers are associated with the high-risk subtypes of human papillomavirus (HPV) expressing the E6 and E7 oncoproteins. The E6 and E7 oncoproteins reduce cellular levels of the p53 and the retinoblastoma (pRb) tumor suppressors, respectively, and represent an important component of the malignant phenotype. Several groups have shown that treatment with cidofovir suppresses levels of E6 and E7, restoring cellular p53 and pRb levels, in turn slowing cell replication and increasing the susceptibility of the cancer cells to radiation and apoptosis. Recently, our group synthesized alkoxyalkyl esters of cidofovir, which were found to be >100 times more active than unmodified cidofovir in vitro against various double-stranded DNA viruses, including cytomegalovirus, herpes simplex virus, adenoviruses, cowpox, vaccinia, and variola viruses. We compared the activity of octadecyloxyethyl-cidofovir (ODE-CDV) and oleyloxyethyl-cidofovir (OLE-CDV) with that of unmodified cidofovir against both HPV-negative and HPV-positive cervical cancer cells. We compared the antiproliferation activity in CaSki, HeLa, and Me-180 cells, prototypical HPV-positive cell lines bearing the HPV-16, HPV-18, and HPV-68 high-risk subtypes, with the activity in C33A cells, a cervical cancer cell line lacking HPV, and in nonmalignant primary human foreskin fibroblast cells. OLE-CDV and ODE-CDV were several logs more potent than cidofovir in CaSki, Me-180, HeLa, and C33A cervical cancer cells as determined by 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt proliferation assay. Cell cycle analysis indicates that the cidofovir analogues interfere with passage of dividing cells through the S phase. ODE-CDV and OLE-CDV were 500 to 17,000 times more active than cidofovir in inhibiting the growth of cervical cancer cells. ODE-CDV and OLE-CDV showed selectivity for cervical cancer cells versus nonmalignant human foreskin fibroblast cells and warrant further investigation as potential therapies for cervical cancer.

Human immunodeficiency virus type 1 (HIV-1) alters gene expression in infected cells, leading to cellular dysfunction. We uncovered a number of host cell genes that are modulated in both CD4(+) T cell lines and primary CD4(+) T lymphocytes infected with HIV-1, using high-density oligonucleotide probe microarray technology. We focused on one gene in particular, nuclear factor I-B2 (NFI-B2), because of its high level of expression. NFI-B2 is a member of the nuclear factor I family of nuclear proteins, which are known to be involved in viral and cellular transcription. To better understand the role of NFI-B2 during HIV-1 infection, we generated a Jurkat T cell line that constitutively expressed NFI-B2. After infection with HIV-1, these cells produced fewer viruses because of a downregulation of surface CD4 expression. The surface expression of the coreceptor, CXCR4, remained unchanged. Furthermore, levels of CD4 mRNA were reduced in NFI-B2-producing cells, suggesting that expression of NFI-B2 impairs CD4 transcription. Modulation of NFI-B2 by HIV-1 may represent yet another mechanism by which HIV infection reduces cell surface expression of CD4.

We measured apoptosis of subsets of T lymphocytes by single-cell analysis of caspase activation, to confirm high turnover of chemokine receptor CCR5(+) T cells in subjects with acute, primary human immunodeficiency virus type 1 (HIV-1) infection (PHI). High levels of spontaneous apoptosis, consisting mainly of CD8(+) T lymphocytes, were closely associated with increases in the activation markers Ki-67, CD38, and the HIV coreceptor CCR5 and with decreases in Bcl-2 and the interleukin (IL)-7 receptor at the single-cell level. Increased expression of Ki-67 and CCR5 ex vivo, as well as increased apoptosis, was seen in all T cell receptor beta-chain variable region (TCRBV) subfamilies studied. The addition of IL-2 or IL-15, but not IL-7, significantly inhibited caspase activation, increased Bcl-2 expression, and rapidly initiated proliferation in vitro of CD8(+) T cells expressing CCR5 and multiple TCRBV subfamilies. Furthermore, IL-15 receptor alpha-chain messenger RNA levels were increased in peripheral blood mononuclear cells during PHI. These results suggest that CCR5(+)Ki-67(+)Bcl-2(dim) activated T cells generated during PHI traffic via blood to tissue sites, where the cells may survive and/or further proliferate under the local influence of IL-2 or IL-15. Understanding cytokine effects on CCR5(+) T cells will be important in understanding chronic HIV-1 replication and pathogenesis.

CD4 + T-cell depletion is a characteristic of human immunodeficiency virus type 1 (HIV-1) infection. In this study, modulation of mRNA expression of 6800 genes was monitored simultaneously at eight time points in a CD4 + T-cell line (CEM-GFP) during HIV infection. The responses to infection included: (1) >30% decrease at 72 h after infection in overall host-cell production of monitored mRNA synthesis, with the replacement of host-cell mRNA by viral mRNA, (2) suppression of the expression of selected mitochondrial and DNA repair gene transcripts, (3) increased expression of the proapoptotic gene and its gene p53-induced product Bax, and (4) activation of caspases 2, 3, and 9. The intense HIV-1 transcription resulted in the repression of much cellular RNA expression and was associated with the induction of apoptosis of infected cells but not bystander cells. This choreographed host gene response indicated that the subversion of the cell transcriptional machinery for the purpose of HIV-1 replication is akin to genotoxic stress and represents a major factor leading to HIV-induced apoptosis.

HIV-1 induces apoptosis and leads to CD4+ T-lymphocyte depletion in humans. It is still unclear whether HIV-1 kills infected cells directly or indirectly. To elucidate the mechanisms of HIV-1-induced apoptosis, we infected human CD4+ T cells with HIV-1. Enzymatic analysis with fluorometric substrates showed that caspase 2, 3, and 9 were activated in CD4+ T cells with peak levels 48 h after infection. Immunoblotting analysis confirmed the cleavage of pro-caspase 3 and 9, and of specific caspase substrates. Release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria was observed in HIV-infected cells. The cytochrome c and AIF release preceded the reduction of the mitochondrial transmembrane potential and nuclear chromatin condensation. H IV infection led to phosphorylation of p53 at the Ser15 residue, detectable as early as 24 h after infection. The p53 phosphorylation was followed by increased mRNA and protein expression of p21, Bax, HDM2, and p53. Up-regulation of surface FasL expression, accompanied by a down-regulation of Fas-associated proteins (FADD, DAXX, and RIP), was observed 72 h after infection. Our results suggest that HIV activates the p53 pathway, leading to cytochrome c and AIF release with ensuing caspase activation.

TRAIL is a member of the Tumour Necrosis Factor (TNF) Family and a potent inducer of apoptosis in many breast carcinoma cell lines but not in normal human mammary epithelial cells. In vivo administration of soluble TRAIL causes regression of breast cancer xenografts without causing measurable toxicity. Combining it with other traditional anti-cancer therapies enhances the efficacy of TRAIL treatment. The basis for the resistance of normal breast epithelial cells to TRAILinduced apoptosis will be investigated using filter arrays and high-density microarrays. Using a similar approach, the synergy between TRAIL and other cancer therapies will be studied.