Sequential waves of gene expression in patients with clinically defined dengue illnesses reveal subtle disease phases and predict disease severity.

BACKGROUND: Dengue virus (DENV) infection can range in severity from mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Changes in host gene expression, temporally through the progression of DENV infection, especially during the early days, remains poorly characterized. Early diagnostic markers for DHF are also lacking. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated host gene expression in a cohort of DENV-infected subjects clinically diagnosed as DF (n = 51) and DHF (n = 13) from Maracay, Venezuela. Blood specimens were collected daily from these subjects from enrollment to early defervescence and at one convalescent time-point. Using convalescent expression levels as baseline, two distinct groups of genes were identified: the "early" group, which included genes associated with innate immunity, type I interferon, cytokine-mediated signaling, chemotaxis, and complement activity peaked at day 0-1 and declined on day 3-4; the second "late" group, comprised of genes associated with cell cycle, emerged from day 4 and peaked at day 5-6. The up-regulation of innate immune response genes coincided with the down-regulation of genes associated with viral replication during day 0-3. Furthermore, DHF patients had lower expression of genes associated with antigen processing and presentation, MHC class II receptor, NK and T cell activities, compared to that of DF patients. These results suggested that the innate and adaptive immunity during the early days of the disease are vital in suppressing DENV replication and in affecting outcome of disease severity. Gene signatures of DHF were identified as early as day 1. CONCLUSIONS/SIGNIFICANCE: Our study reveals a broad and dynamic picture of host responses in DENV infected subjects. Host response to DENV infection can now be understood as two distinct phases with unique transcriptional markers. The DHF signatures identified during day 1-3 may have applications in developing early molecular diagnostics for DHF.

Plasmodium inui infection reduces the efficacy of a simian immunodeficiency virus DNA vaccine in a rhesus macaque model through alteration of the vaccine-induced immune response.

Human immunodeficiency virus type 1 and malaria are co-endemic in many areas. We evaluated the effects of Plasmodium inui infection on the performance of a simian immunodeficiency virus (SIV) DNA vaccine. Rhesus macaques were infected with P. inui by transfusion of whole blood from a persistently infected animal. Animals with and animals without P. inui infection were then vaccinated 4 times with an SIV DNA vaccine encoding SIVgag, SIVpol, and SIVenv. Animals were subsequently challenged with thirty 50% rhesus monkey infectious doses of SIVmac251 6 weeks after the last vaccination. P. inui-infected immunized animals showed a significantly higher viral load than animals without P. inui infection (P = .010, by the Wilcoxon rank sum test). The higher viral loads in the P. inui-infected animals were durable and were observed at all sampling time points across the study (P = .00245, by the Wilcoxon rank test). The P. inui-infected animals also had correspondingly lower CD4(+) cell counts. There were fewer vaccine-specific CD4(+) and CD8(+) cells in the P. inui-infected animals, compared with uninfected animals. Of importance, P. inui infection seemed to decrease the number of CD8(+) cells that could proliferate or secrete interferon γ, although the number of CD8(+) cells capable of secreting tumor necrosis factor α following in vitro stimulation was increased. This study demonstrated that P. inui infection had an influence on the immune response to an SIV DNA vaccine and decreased the vaccine's efficacy.

A double-blind randomized phase I clinical trial targeting ALVAC-HIV vaccine to human dendritic cells.

BACKGROUND: We conducted a novel pilot study comparing different delivery routes of ALVAC-HIV (vCP205), a canarypox vaccine containing HIV gene inserts: env, gag and pol. We explored the concept that direct ex vivo targeting of human dendritic cells (DC) would enhance the immune response compared to either conventional intramuscular or intradermal injections of the vaccine alone. METHODOLOGY/PRINCIPAL FINDINGS: Healthy HIV-1 uninfected volunteers were administered ALVAC-HIV or placebo by intramuscular injection (i.m.), intradermal injection (i.d.) or subcutaneous injection (s.q.) of autologous ex vivo transfected DC at months 0, 1, 3 and 6. All vaccine delivery routes were well tolerated. Binding antibodies were observed to both the ALVAC vector and HIV-1 gp160 proteins. Modest cellular responses were observed in 2/7 individuals in the DC arm and 1/8 in the i.m. arm as determined by IFN-γ ELISPOT. Proliferative responses were most frequent in the DC arm where 4/7 individuals had measurable responses to multiple HIV-1 antigens. Loading DC after maturation resulted in lower gene expression, but overall better responses to both HIV-1 and control antigens, and were associated with better IL-2, TNF-α and IFN-γ production. CONCLUSIONS/SIGNIFICANCE: ALVAC-HIV delivered i.m., i.d. or s.q. with autologous ex vivo transfected DC proved to be safe. The DC arm was most immunogenic. Proliferative immune responses were readily detected with only modest cytotoxic CD8 T cell responses. Loading mature DC with the live viral vaccine induced stronger immune responses than loading immature DC, despite increased transgene expression with the latter approach. Volunteers who received the autologous vaccine loaded mature DC developed a broader and durable immune response compared to those vaccinated by conventional routes. TRIAL REGISTRATION: ClinicalTrials.gov NCT00013572.

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.

The accuracy of physicians' perceptions of patients' suffering: findings from two teaching hospitals.

PURPOSE: How accurately physicians perceive patient suffering remains unclear. The authors sought to quantitatively compare physicians' estimates of their patients' suffering with the patients' ratings of their own suffering, using a paired survey. METHOD: Six major domains of suffering (DOSs) were derived from narrative descriptions of suffering by physicians and patients in a preliminary multicenter pilot study. From September 2005 through July 2006 at two teaching hospitals in Washington, DC, and Bethesda, Maryland, before a clinical encounter between a patient and physician, patients rated the impact of each of these DOSs on their overall suffering. After the same encounter, physicians rated the same DOSs according to their perception of suffering experienced by that patient. Patient responses were compared with physician responses using the Wilcoxon signed ranks and Spearman correlation tests. RESULTS: Two hundred twenty-seven adult patients and their treating physicians completed the survey. Cooperation rates among patients and physicians were 94% and 97%, respectively. For two of the six DOSs (pain and physically nonpainful symptoms), there was no significant difference between the physicians' estimates of suffering and the patients' ratings of the DOS. For the remaining four DOSs (communication, emotional factors, loss, and systems factors), there was significant disagreement between the physicians' estimates and the actual suffering of the patients (P < .01). When all six DOSs were combined to ascertain how well perceptions of overall suffering correlated, significant discordance was also observed between physicians' perceptions and patients' descriptions (P < .001). CONCLUSIONS: This study suggests that the physicians who participated might need more training in the recognition of patient suffering. Because these physicians were trained in medical schools across the country, the deficiencies noted here may not be limited to only the physicians in this study. More studies of physicians' ability to detect and manage suffering are needed, especially at nonteaching hospitals.

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.

Functional genomic relationships in HIV-1 disease revealed by gene-expression profiling of primary human peripheral blood mononuclear cells.

BACKGROUND: An assessment of biomarkers from an analysis of human peripheral blood mononuclear cell gene-expression profiles was made, to acquire an understanding of transcriptional changes associated with human immunodeficiency virus type 1 (HIV-1) infection in vivo. METHODS: Supervised learning algorithms were used to create signature gene sets that could be used to distinguish seropositive from seronegative samples and delineate changes in disease status during the early stages of infection. Bioinformatic tools were used to classify persons and to functionally characterize groups of differentially expressed genes, to elucidate the impact of viral infection on host cell gene-expression patterns. RESULTS: A 10-gene signature set that could be used to accurately determine the HIV-1 serostatus was identified. A 6-gene signature set was used to distinguish seropositive persons exhibiting differential changes in CD4(+) T cell counts, with 93% accuracy. Functional classification of differentially expressed genes in HIV-1 indicated a preponderance of down-regulated genes with functions related to the immune response and apoptosis. Hierarchical cluster analysis in persons whose CD4(+) T cell counts increased, compared with that in persons whose CD4(+) T cell counts decreased, was characterized by the down-regulation of genes associated with apoptosis, mitochondrial function, protein biosynthesis, and RNA binding. CONCLUSIONS: Gene-expression profile analysis of a complex infectious virus, such as HIV-1, may be useful to elucidate the functional genomic relationships associated with viral infection.

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.

Microarray analysis of acute and delayed gene expression profile in rats after focal ischemic brain injury and reperfusion.

Temporal changes in gene expression were measured using DNA microarrays after 30-min or 2-hr transient middle cerebral artery occlusion (MCAo) in rats. Total RNA was extracted from the injured hemisphere at 30 min, 4 hr, 8 hr, 24 hr, 3 days, and 7 days after MCAo for GeneChip analysis using Affymetrix U34 Rat Neurobiology arrays (1,322 functional genes). In total, 267 genes were expressed differentially: 166 genes were upregulated, 94 genes were downregulated, and 7 genes were biphasically up- and downregulated. Among all differentially expressed genes, 88 were newly identified as associated with ischemic brain injury. Most affected genes were distributed among 12 functional categories. Immediate early genes, transcription factors, and heat shock proteins were upregulated as early as 30 min after MCAo, followed by the upregulation of inflammation, apoptosis, cytoskeletal, and metabolism genes, which peaked within 4-24 hr of injury. Neurotrophic growth factors exhibited a sustained upregulation beginning 24 hr after MCAo and persisting through 7 days post-injury. Three classes of genes were downregulated with distinct temporal patterns: ion channel genes and neurotransmitter receptor genes were downregulated between 8-24 hr after injury, whereas synaptic proteins genes were downregulated between 3-7 days after MCAo. Downregulation of synaptic protein gene expression after ischemic injury is of particular interest because of its conspicuously delayed pattern as a functional group, which has not been reported previously and may play a role in post-injury recovery.

Long-term efficacy of routine access to antiretroviral-resistance testing in HIV type 1-infected patients: results of the clinical efficacy of resistance testing trial.

The long-term efficacy of making resistance testing routinely available to clinicians has not been established. We conducted a clinical trial at 6 US military hospitals in which volunteers infected with human immunodeficiency virus type-1 were randomized to have routine access to phenotype resistance testing (PT arm), access to genotype resistance testing (GT arm), or no access to either test (VB arm). The primary outcome measure was time to persistent treatment failure despite change(s) in antiretroviral therapy (ART) regimen. Overall, routine access to resistance testing did not significantly increase the time to end point. Time to end point was significantly prolonged in the PT arm for subjects with a history of treatment with > or =4 different ART regimens or a history of treatment with nonnucleoside reverse-transcriptase inhibitors before the study, compared with that in the VB arm. These results suggest that routine access to resistance testing can improve long-term virologic outcomes in HIV-infected patients who are treatment experienced but may not impact outcome in patients who are naive to or have had limited experience with ART.

Biological threat detection via host gene expression profiling.

With the increased threat posed by biological weapons, detection techniques for biothreat pathogens are critically needed to monitor and assess the severity of the illness once exposure has occurred. Current approaches for detecting biological threats are either time-consuming or highly specific but provide little information regarding pathogenicity. Genotyping of pathogens by PCR provides a fast and definitive means for identifying pathogens, but reliance on pathogen genotypic endpoints has several limitations. Current progress in DNA microarrays technology provides an alternative way to address the issues faced by traditional detection systems through host gene expression profiles of peripheral blood cells. We discuss the advantages and critical issues facing the use of host gene expression profiling for biological threat detection.

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.