Distinct gene-expression profiles associated with the susceptibility of pathogen-specific CD4 T cells to HIV-1 infection.

In HIV infection, CD4 responses to opportunistic pathogens such as Candida albicans are lost early, but CMV-specific CD4 response persists. Little is currently known about HIV infection of CD4 T cells of different pathogen/antigen specificities. CFSE-labeled PBMCs were stimulated with CMV, tetanus toxoid (TT), and C albicans antigens and subsequently exposed to HIV. HIV infection was monitored by intracellular p24 in CFSE(low) population. We found that although TT- and C albicans-specific CD4 T cells were permissive, CMV-specific CD4 T cells were highly resistant to both R5 and X4 HIV. Quantification of HIV DNA in CFSE(low) cells showed a reduction of strong-stop and full-length DNA in CMV-specific cells compared with TT- and C albicans-specific cells. ß-Chemokine neutralization enhanced HIV infection in TT- and C albicans-specific cells, whereas HIV infection in CMV-specific cells remained low despite increased entry by ß-chemokine neutralization, suggesting postentry HIV restriction by CMV-specific cells. Microarray analysis (Gene Expression Omnibus accession number: GSE42853) revealed distinct transcriptional profiles that involved selective up-regulation of comprehensive innate antiviral genes in CMV-specific cells, whereas TT- and C albicans-specific cells mainly up-regulated Th17 inflammatory response. Our data suggest a mechanism for the persistence of CMV-specific CD4 response and earlier loss of mucosal Th17-associated TT- and C albicans-specific CD4 response in AIDS.

Expression of genes associated with immunoproteasome processing of major histocompatibility complex peptides is indicative of protection with adjuvanted RTS,S malaria vaccine.

BACKGROUND: Patterns of expressed genes in the peripheral blood mononuclear cells of persons who were receiving RTS,S/AS01 or RTS,S/AS02 malaria vaccine and were undergoing experimental challenge with mosquito-borne falciparum malaria were examined to identify markers associated with protection. METHODS: Thirty-nine vaccine recipients were assessed at study entry; on the day of the third vaccination; at 24 h, 72 h, and 2 weeks after vaccination; and on day 5 after challenge. Of 39 vaccine recipients, 13 were protected and 26 were not. Eleven vaccine recipients exhibited delayed onset of parasitemia. All infectivity control subjects developed parasitemia. Prediction analysis of microarrays identified genes corresponding with protection. Gene set enrichment analysis identified sets of genes associated with protection after the third vaccination and before challenge. RESULTS: After the third vaccination and before challenge, differential expression of genes in the immunoproteasome pathway distinguished protected and nonprotected persons. At 5 days after challenge, differential expression of genes associated with programmed cell death distinguished between subjects protected and not protected from malaria blood-stage infection. CONCLUSIONS: The up-regulation of genes associated with the efficient processing of major histocompatibility complex peptides suggests a potential role of the vaccine in conferring major histocompatibility complex class 1-mediated protection and may represent a useful surrogate marker of vaccine efficacy without the need for challenge.

Activated PD-1+ CD4+ T cells represent a short-lived part of the viral reservoir and predict poor immunologic recovery upon initiation of ART.

OBJECTIVE: Activated (CD38HLA-DR) PD-1 CD4 T cells are strongly associated with virus replication and disease progression in untreated HIV-1 infection, and viral persistence in individuals on ART. Few studies have examined cell-associated viral load (CAVL) in different activated CD4 T-cell populations to measure relative contributions to viral reservoirs. DESIGN: Longitudinal assessment of HIV-1 chronically infected Ugandans initiating ART, to investigate activated CD4 T-cell populations and their contribution to viral reservoirs. METHODS: We followed 32 HIV-1 chronically infected individuals from Kampala, Uganda, and determined their CD4 T-cell counts and viral load at baseline, 6, and 12 months after the initiation of ART. T-cell populations were sorted based on activation profiles and gag DNA was measured to determine CAVL within these populations. Soluble factors associated with inflammation were measured in plasma using a multiplexed platform. RESULTS: Concomitant with viral load decline and CD4 T-cell count rebound, the activated PD-1 CD4 T-cell population contracted upon initiation of ART. Baseline levels of activated PD-1 CD4 T cells correlated with plasma levels of IP-10 and TNFRII. Interestingly, a higher baseline level of activated PD-1 CD4 T cells was associated with poorer CD4 T-cell recovery after 12 months of ART. This population contributed significantly to the cell-associated HIV DNA load at baseline, whereas their contribution declined on ART, indicating high turnover. CONCLUSION: Activated PD-1 CD4 T cells are predictors of poor immunologic recovery on ART and may represent a short-lived component of HIV-1 reservoirs.

Silencing of Lactotransferrin expression by methylation in prostate cancer progression.

BACKGROUND: Cancer cells gain selection advantages by the coordinated silencing of protective and by the activation of cell proliferation/cell survival genes. Evaluations of epithelial cell transcriptome of benign and malignant prostate glands by laser capture microdissection (LCM) identified Lactotransferrin (LTF) as the most significantly downregulated gene in prostate cancer (CaP) cells (p < 10(-6)). Frequent downregulation, significant association of LTF with PSA recurrence-free survival in CaP patients and the established anti-tumorigenic effects of LTF in experimental cancer models have provided impetus to evaluate LTF expression features and mechanisms in CaP specimens. METHODS: LTF mRNA expression analysis was performed in LCM derived benign and malignant prostate epithelial cells by using Affymetrix GeneChip and QRT-PCR. LTF protein expression was assessed in tissue specimens by immunohistochemistry and in serum samples from CaP patients compared to healthy male control by using ELISA. Mechanism of LTF downregulation was analyzed in 5-azadeoxycytidine treated LNCaP and LAPC4 cells using MALDI-TOF MS. Proliferation and cell cycle analysis of CaP cells by FACS flow cytrometry was assessed in LNCaP cell cultures. RESULTS: Quantitative analysis of LTF mRNA expression in tumor cells revealed marked downregulation of LTF with significant associations to decreased PSA recurrence-free survival of CaP patients (n = 100, p < or = 0.0322). Moreover, low levels of LTF protein expression was observed in tumor tissues as well as in sera from CaP patients (p < or = 0.0001). LTF promoter downstream CpG island methylation was found in LNCaP and LAPC4 cells. Furthermore, replenishing of LTF by supplementing growth media with LTF protein resulted in reduced cell growth. Cell cycle analysis revealed robust increases in apoptosis in response to LTF treatment. CONCLUSION: This study highlights the potential for LTF in chemoprevention and to become a biologically relevant prognostic marker of CaP, suggesting that silencing of the LTF gene may be causally linked to CaP progression.

Frequent overexpression of ETS-related gene-1 (ERG1) in prostate cancer transcriptome.

Transcription factors encoded by the ETS family of genes are central in integrating signals that regulate cell growth and differentiation, stress responses, and tumorigenesis. This study, analysing laser microdissected paired benign and malignant prostate epithelial cells from prostate cancer (CaP) patients (n=114; 228 specimen) by GeneChip and quantitative real-time RT-PCR, identifies ETS-related gene (ERG), a member of the ETS transcription factor family, as the most frequently overexpressed proto-oncogene in the transcriptome of malignant prostate epithelial cells. Combined quantitative expression analysis of ERG with two other genes commonly overexpressed in CaP, AMACR and DD3, revealed overexpression of at least one of these three genes in virtually all CaP specimen (54 of 55). Comprehensive evaluation of quantitative ERG1 expression with clinicopathological features also suggested that ERG1 expression level in prostate tumor cells relative to benign epithelial cells is indicator of disease-free survival after radical prostatectomy.

HIV DNA Set Point is Rapidly Established in Acute HIV Infection and Dramatically Reduced by Early ART.

HIV DNA is a marker of HIV persistence that predicts HIV progression and remission, but its kinetics in early acute HIV infection (AHI) is poorly understood. We longitudinally measured the frequency of peripheral blood mononuclear cells harboring total and integrated HIV DNA in 19 untreated and 71 treated AHI participants, for whom 50 were in the earliest Fiebig I/II (HIV IgM-) stage, that is ≤2weeks from infection. Without antiretroviral therapy (ART), HIV DNA peaked at 2weeks after enrollment, reaching a set-point 2weeks later with little change thereafter. There was a marked divergence of HIV DNA values between the untreated and treated groups that occurred within the first 2weeks of ART and increased with time. ART reduced total HIV DNA levels by 20-fold after 2weeks and 316-fold after 3years. Therefore, very early ART offers the opportunity to significantly reduce the frequency of cells harboring HIV DNA.

Androgen-induced expression of endoplasmic reticulum (ER) stress response genes in prostate cancer cells.

Evaluations of androgen regulated gene (ARG) repertoire provide new insights into the androgen receptor (AR) mediated signaling at the transcriptional level. Definition of ARGs having critical functions in the biology of normal and malignant prostate should aid in identifying new bio-markers and therapeutic targets for prostate cancer (CaP). Using Affymetrix HuGene FL oligonucleotide arrays, temporal expression profiles of ARGs in widely used hormone responsive LNCaP cells, were analysed by hierarchical clustering methods and functional classification. ARGs in response to different androgen concentrations showed temporal co-regulation of genes involved in specific biochemical pathways. This study focuses on our new observations of the coordinated androgen induction of genes (NDRG1, PDIR, HERPUD1, ORP150) involved in the endoplasmic reticulum (ER) stress response pathway. Expression analysis of the two selected ER stress responsive genes, NDRG1 and HERPUD1 in primary CaPs revealed a significantly reduced tumor associated expression. Intriguing linkage of the androgen signaling to ER stress responsive genes, a protective response to protein unfolding or protein damage resulting from cellular stress signals, suggests that androgens may induce such stress signals in CaP cells. Decreased CaP associated expression of two ER stress responsive genes also suggests that possible abrogation of this pathway in prostate tumorigenesis.

Identification and utilization of inter-species conserved (ISC) probesets on Affymetrix human GeneChip platforms for the optimization of the assessment of expression patterns in non human primate (NHP) samples.

BACKGROUND: While researchers have utilized versions of the Affymetrix human GeneChip for the assessment of expression patterns in non human primate (NHP) samples, there has been no comprehensive sequence analysis study undertaken to demonstrate that the probe sequences designed to detect human transcripts are reliably hybridizing with their orthologs in NHP. By aligning probe sequences with expressed sequence tags (ESTs) in NHP, inter-species conserved (ISC) probesets, which have two or more probes complementary to ESTs in NHP, were identified on human GeneChip platforms. The utility of human GeneChips for the assessment of NHP expression patterns can be effectively evaluated by analyzing the hybridization behaviour of ISC probesets. Appropriate normalization methods were identified that further improve the reliability of human GeneChips for interspecies (human vs NHP) comparisons. RESULTS: ISC probesets in each of the seven Affymetrix GeneChip platforms (U133Plus2.0, U133A, U133B, U95Av2, U95B, Focus and HuGeneFL) were identified for both monkey and chimpanzee. Expression data was generated from peripheral blood mononuclear cells (PBMCs) of 12 human and 8 monkey (Indian origin Rhesus macaque) samples using the Focus GeneChip. Analysis of both qualitative detection calls and quantitative signal intensities showed that intra-species reproducibility (human vs. human or monkey vs. monkey) was much higher than interspecies reproducibility (human vs. monkey). ISC probesets exhibited higher interspecies reproducibility than the overall expressed probesets. Importantly, appropriate normalization methods could be leveraged to greatly improve interspecies correlations. The correlation coefficients between human (average of 12 samples) and monkey (average of 8 Rhesus macaque samples) are 0.725, 0.821 and 0.893 for MAS5.0 (Microarray Suite version 5.0), dChip and RMA (Robust Multi-chip Average) normalization method, respectively. CONCLUSION: It is feasible to use Affymetrix human GeneChip platforms to assess the expression profiles of NHP for intra-species studies. Caution must be taken for interspecies studies since unsuitable probesets will result in spurious differentially regulated genes between human and NHP. RMA normalization method and ISC probesets are recommended for interspecies studies.

Patterns of gene expression in peripheral blood mononuclear cells of rhesus macaques infected with SIVmac251 and exhibiting differential rates of disease progression.

Using the Affymetrix HuGeneFL GeneChip, the global expression patterns of genes in the peripheral blood mononuclear cells (PBMCs) of rhesus macaques, infected with SIVmac251 and exhibiting rapid, typical, or slow rates of disease progression, were examined. Assessments of the change in gene expression (fold change), the temporal coordination of gene expression (self-organizing map analysis), and the similarities and significant differences in gene expression across the groups were performed on samples taken before infection and 3 and 7 weeks postinfection. An upregulation of the p27 interferon-inducible gene and of genes associated with cellular activation and immune response was observed in all three groups. Rapidly progressing animals exhibited a modest number of genes with a change in expression of 3-fold or greater, typically progressing animals exhibited the greatest number, and slowly progressing animals exhibited the fewest. Self-organizing map cluster analysis indicated that rapidly progressing animals exhibited the least coordinated gene expression over the three study time points, typically progressing animals exhibited a moderate degree, and animals with slow progression exhibited the most coordinated gene expression. Mann-Whitney U analysis indicated that differences in gene expression were most pronounced between the rapidly and slowly progressing groups and least pronounced between the rapidly and typically progressing animals. These observations elucidate distinct features of gene expression in animals with different rates of disease progression.

Impact of viral infection on the gene expression profiles of proliferating normal human peripheral blood mononuclear cells infected with HIV type 1 RF.

Exploiting the power of high-density gene arrays, the simultaneous expression analysis of 5600 cellular genes was executed on proliferating peripheral blood mononuclear cells (PBMCs) from three normal human donors that were infected in vitro with the T cell tropic laboratory strain of HIV-1, RF. Profiles of expressed genes were assessed at 1, 12, 24, 48, and 72 hr postinfection and compared with those of matched uninfected PBMCs. Viral infection resulted in an overall increase in the number of genes expressed with peaks of expression at 1, 12, and 48 hr postinfection. Functional clustering of genes whose expression level in infected PBMCs varied by 2-fold or greater from levels in the controls indicated that cellular activation markers, proteins associated with immune cell function and with transcription and translation, exhibited increased expression subsequent to viral infection. Gene families exhibiting a decline in gene expression were confined to the 72 hr time point and included genes associated with catabolism and a subset of genes involved with cell signaling and synthetic pathways. Self-organizing map (SOM) cluster analysis identified temporal patterns of coordinated gene expression in infected PBMCs including genes associated with the immune response, the cytoskeleton, and ribosomal subunit structural proteins required for protein synthesis.

Osteoblast-specific factor 2 expression in prostate cancer-associated stroma: identification through microarray technology.

OBJECTIVES: To better understand the gene expression patterns in tumor-associated stroma, laser-capture-microdissections from clinical specimens were analyzed by genome-wide-expression microarray technology. The epithelial-stromal interaction plays a critical role in prostate development, reactive changes, and tumorigenesis. Diverse microarray technologies have been used to characterize the molecular changes in prostate cancer. Even though these gene expression studies are compromised by the heterogeneity of the tumor, as well as by the difficulty associated with collecting appropriate counterparts to represent normal prostate cells, the gene array data from tumors have shown promising results. Currently, little is known about the tumor-associated stromal gene expression profile in prostate cancer. METHODS: Matching benign and malignant epithelial cell-related stroma cells were subjected to microarray platforms. RESULTS: The prostatatic stroma expressed several osteogenic molecules. In particular, one of the genes, OSF2, was upregulated in tumor-associated stroma compared with benign epithelial cell associated stroma, which was further validated by immunohistochemical examination. CONCLUSIONS: These data show that the combination of laser capture dissection with computational enhancement of epithelial and stromal microarray data is a useful tool to assess gene expression changes in prostate cancer stroma.

CD4+ T-cell decline after the interruption of antiretroviral therapy in ACTG A5170 is predicted by differential expression of genes in the ras signaling pathway.

Patterns of expressed genes examined in cryopreserved peripheral blood mononuclear cells (PBMCs) of seropositive persons electing to stop antiretroviral therapy in the AIDS Clinical Trials Group Study A5170 were scrutinized to identify markers capable of predicting the likelihood of CD4+ T-cell depletion after cessation of antiretroviral therapy (ART). A5170 was a multicenter, 96-week, prospective study of HIV-infected patients with immunological preservation on ART who elected to interrupt therapy. Study entry required that the CD4 count was greater than 350 cells/mm(3) within 6 months of ART initiation. Median nadir CD4 count of enrollees was 436 cells/mm(3). Two cohorts, matched for clinical characteristics, were selected from A5170. Twenty-four patients with an absolute CD4 cell decline of less that 20% at week 24 (good outcome group) and 24 with a CD4 cell decline of >20% (poor outcome group) were studied. The good outcome group had a decline in CD4+ Tcell count that was 50% less than the poor outcome group. Significance analysis of microarrays identified differential gene expression (DE) in the two groups in data obtained from Affymetrix Human FOCUS GeneChips. DE was significantly higher in the poor outcome group than in the good outcome group. Prediction analysis of microarrays (PAM-R) identified genes that classified persons as to progression with greater than 80% accuracy at therapy interruption (TI) as well as at 24 weeks after TI. Gene set enrichment analysis (GSEA) identified a set of genes in the Ras signaling pathway, associated with the downregulation of apoptosis, as significantly upregulated in the good outcome group at cessation of ART. These observations identify specific host cell processes associated with differential outcome in this cohort after TI.

Impact of antiretroviral treatment on gene expression in peripheral blood mononuclear cells from SIVmac251-infected macaques.

BACKGROUND: A survey of gene expression in peripheral blood mononuclear cells from cynomolgus macaques infected with SIVmac251 was conducted to ascertain the impact of viral infection and successful antiretroviral (ARV) intervention on gene transcription at peak seroconversion, viral set point, and after treatment with 9-R 2 phosphonomethoxypropyl adenine and beta -2'3' dideoxy-3'-thia-5 fluorocytidine. METHODS: Robust multichip average-normalized data sets generated on Affymetrix GeneChips were analyzed using Significance Analysis of Microarrays (SAM), to determine differential gene expression. Unsupervised learning algorithms and gene-ontology tools were used to elucidate hierarchical relationships and to define the function of significantly enriched biological categories of differentially regulated genes. Gene networks associated with immune response and inflammation impacted by ARV treatment were derived by use of Pathway Architect software. RESULTS: Viral infection results in down-regulation of gene expression, which is greatest by the viral set point. Of the 3647 genes down-regulated at the viral set point, 1033 were up-regulated as the result of successful ARV treatment. There is significant overlap in the identity of these genes. CONCLUSIONS: Intervention with successful ARV treatment in macaques infected with SIVmac251 results in the partial reversal of the down-regulated gene expression characteristic of early viral infection.

Relationship between chloroquine toxicity and iron acquisition in Saccharomyces cerevisiae.

Chloroquine is one of the most effective antimalarials, but resistance to it is becoming widespread. However, we do not fully understand either the drug's mode of action or the mechanism of resistance. In an effort to expand our understanding of the mechanism of action and resistance associated with chloroquine, we used Saccharomyces cerevisiae as a model eukaryotic system. To aid in the discovery of potential drug targets we applied the transcriptional profiling method to identify genes transcriptionally responsive to chloroquine treatment in S. cerevisiae. Among the genes that were differentially expressed with chloroquine treatment were a number of metal transporters involved in iron acquisition (SIT1, ARN2, ARN4, and SMF2). These genes exhibit similar expression patterns, and several are known to be regulated by AFT1, a DNA binding protein, which responds to iron levels in the cell. We investigated the role of chloroquine in iron metabolism by using a variety of approaches, including pharmacological, genetic, and biochemical techniques. For these experiments, we utilized yeast lacking the major iron uptake pathways (FET3 and FET4) and yeast deficient in SIT1, encoding the major up-regulated iron siderophore transporter. Our experiments show that yeast genetically or environmentally limited in iron availability has increased sensitivity to chloroquine in pharmacological assays and that the addition of iron rescues these cells from chloroquine killing. 55FeCl3 accumulation was inhibited in the presence of chloroquine, and kinetic analysis demonstrated that inhibition was competitive. These results are consistent with deprivation of iron as a mechanism of chloroquine killing in yeast.