Maryanne Vahey

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.

Antiviral effect and ex vivo CD4+ T cell proliferation in HIV-positive patients as a result of CD28 costimulation.

Because stimulation of CD4+ lymphocytes leads to activation of human immunodeficiency virus-type 1 (HIV-1) replication, viral spread, and cell death, adoptive CD4+ T cell therapy has not been possible. When antigen and CD28 receptors on cultured T cells were stimulated by monoclonal antibodies (mAbs) to CD3 and CD28 that had been immobilized, there was an increase in the number of polyclonal CD4+ T cells from HIV-infected donors. Activated cells predominantly secreted cytokines associated with T helper cell type 1 function. The HIV-1 viral load declined in the absence of antiretroviral agents. Moreover, CD28 stimulation of CD4+ T cells from uninfected donors rendered these cells highly resistant to HIV-1 infection. Immobilization of CD28 mAb was crucial to the development of HIV resistance, as cells stimulated with soluble CD28 mAb were highly susceptible to HIV infection. The CD28-mediated antiviral effect occurred early in the viral life cycle, before HIV-1 DNA integration. These data may facilitate immune reconstitution and gene therapy approaches in persons with HIV infection.

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.

Drug Resistance Patterns, Genetic Subtypes, Clinical Features, and Risk Factors in Military Personnel with HIV-1 Seroconversion

Genetic variability is a central feature of HIV-1. The high frequency of mutations during HIV-1 replication leads to the development of viral quasi-species in vivo and contributes to genetic heterogeneity among HIV-1 isolates (1, 2). There is a growing appreciation that HIV-1 genetic diversity, including the existence of distinct genetic subtypes and the evolution of drug-resistant genotypes, can greatly affect the diagnosis and treatment of HIV-1 infection (1, 2). On the basis of DNA sequence analysis, HIV-1 has been classified into genetic subtypes, with subtypes A through I making up the major HIV-1 group (group M). The more genetically diverse groups, O and N, have also recently been described (1). For reasons that are not clear, HIV-1 subtypes are variably dispersed throughout the world. Some regions, such as central Africa and eastern Europe, have multiple circulating subtypes, whereas the distribution in other regions is more restricted (1, 3). Differences among HIV-1 subtypes can affect the sensitivity of some diagnostic assays of antibody (4) and plasma HIV-1 RNA (5, 6). Although non-subtype B infection has been reported in the United States, the prevalence in the U.S. population is unknown (7, 8). Widespread use of antiretroviral drugs has led to transmission of drug-resistant HIV-1, but the prevalence of resistant mutations in treatment-naive persons has not been thoroughly studied. Because all U.S. Navy and U.S. Marine Corps personnel are screened for HIV-1 infection at 1- to 3-year intervals, HIV-1 infection is often detected early and the seroconversion period can be estimated (9). This permits an investigation of the epidemiologic correlates and risk behaviors associated with the acquisition of non-subtype B and drug-resistant infections. Thus, we determined the prevalence of non-subtype B infection and genotypes associated with antiretroviral drug resistance in a well-characterized cohort of military personnel with recently acquired HIV-1 infection. Methods Study Design United States Navy or Marine Corps personnel with HIV-1 seroconversion who are assigned to military bases west of the Mississippi River in the United States or in the Pacific region overseas are referred to the Navy Medical Center San Diego for initial and follow-up HIV-1 evaluations. Between February 1997 and February 1998, 99 of 141 personnel referred were eligible on the basis of documented seroconversion within the past 3 years. Ninety-five of 99 patients enrolled and signed a consent form approved by the institutional review board of the Navy Medical Center San Diego. Thirty-two patients were being referred for their first HIV evaluation, and 63 were enrolled during a follow-up visit. Blood was obtained to determine genetic subtype and the presence of antiretroviral drug resistance. All patients completed a self-administered risk factor questionnaire labeled with a unique code number. Surveys were sealed in an envelope and placed in a locked drop box that was emptied weekly by off-site data entry personnel. Clinical research staff extracted clinical and laboratory information from the medical record and sent this information, identified by code number, to the data entry site. Laboratory Analysis Seroconversion was documented by a previous negative result on whole-virus HIV-1 enzyme immunoassay followed by a positive result on enzyme immunoassay and a confirmatory positive result on Western blot assay. Lymphocyte subset analysis was performed by flow cytometry, and plasma HIV-1 RNA (available after July 1996) was measured by quantitative reverse transcription polymerase chain reaction assay (Amplicor HIV-1 Monitor assay, Roche Molecular Systems, Branchburg, New Jersey). Genetic subtyping of HIV-1 was performed by using a two-step algorithm. Sera were screened by using a competitive binding enzyme immunoassay with peptides derived from the third variable loop of the HIV-1 envelope glycoprotein. For samples that were serologically reactive to non-subtype B peptides, DNA was extracted from corresponding peripheral blood mononuclear cells, and sequence analysis was performed over a 640-base pair segment of the HIV-1 envelope gene (10). Testing for viral drug resistance was successfully performed in 31 of the 32 therapy-naive patients. Plasma-derived viral RNA was reverse transcribed into complementary DNA, amplified by polymerase chain reaction, and directly sequenced by using an automated ABI Sequencer (Applied Biosystems, Foster City, California). Consensus DNA sequences for the protease and reverse transcriptase genes from each participant were examined for mutations associated with HIV-1 antiretroviral resistance (11). Statistical Analysis Descriptive analysis of demographics, risk behaviors, and laboratory results were performed. Duration of HIV infection was calculated starting from the time of onset, which we estimated as the midpoint between the last negative and first positive result on HIV enzyme immunoassay. Bivariate analyses using odds ratios and 95% CIs determined by the Fisher exact method were done to compare the treatment-naive patients with drug-resistant infection and those with drug-sensitive infection. Statistical analyses were done by using Epi-Info, version 6.02 (Centers for Disease Control and Prevention, Atlanta, Georgia). Results Patients Characteristics and risk exposures of the cohort are summarized in Table 1. Duration of infection and laboratory results are presented for the entire cohort on initial evaluation and for the treatment-naive cohort at the time of enrollment and antiretroviral testing. Risk behaviors known to be associated with acquisition of HIV-1 were reported during the period of seroconversion in all but two patients. Table 1. Characteristics and Risk Exposures of Military Personnel with HIV-1 Seroconversion Laboratory Analysis The CD4 cell counts, plasma HIV RNA levels, and results of syphilis and hepatitis serologic testing in study patients are summarized in Table 1. Combined serologic and genetic analysis revealed that 7 of 95 patients were infected with HIV-1 genetic subtype E (7.4%); the remaining patients were infected with subtype B. Eight of 31 treatment-naive patients (26% [95% CI, 12% to 46%]) had one or more primary mutations that have been associated with phenotypic drug resistance (11) (Table 2). All reverse transcriptase mutations were associated with resistance to zidovudine, lamivudine, or nevirapine-delavirdine. Of the 4 patients with reverse transcriptase mutations, 2 (patients 1 and 2) had mutations that could confer resistance to both nucleoside and non-nucleoside reverse transcriptase inhibitors. Table 2. Drug-Resistant Mutations in Eight Treatment-Naive Patients Characteristics of Patients with Subtype E Infection and Drug-Resistant Mutations Infection with HIV-1 subtype E was documented in 6 men and 1 woman; all 6 men reported sexual contact during short deployments in Thailand. The female patient reported having sex with a man on active duty in the United States. Compared with subtype B-infected patients, subtype E-infected patients were more likely to be heterosexual (100% and 38%), to have had overseas exposure (86% and 27%), and to report sex with commercial sex workers (86% and 15%). Comparisons between patients with wild-type (n=23) and treatment-resistant (n=8) genotypes are listed in Table 1. Discussion We found a high prevalence of non-B genetic subtypes and antiretroviral drug-resistant mutations among treatment-naive military personnel with recently acquired HIV-1 infection. Seven of 95 patients (7.4%) were infected with HIV-1 subtype E. Eight of 31 treatment-naive patients (26%) had primary drug-resistant mutations; of these 8 patients, 3 had mutations in the reverse transcriptase gene only, 4 had mutations in the protease gene only, and 1 had mutations in both the reverse transcriptase and protease genes. Although our study is limited to a single clinical referral center and the sample is small, the results are generally similar to those of other studies that focused on selected drug-resistant mutations. Among several recent studies, 6% to 13% of treatment-naive patients had mutations for zidovudine resistance (12-14). Although data on the prevalence of mutations in the protease gene have not yet been published, transmission of HIV-1 that is resistant to multiple reverse transcriptase and protease inhibitors has been reported (15). Our data suggest that patients infected with drug-resistant virus were more likely to have acquired HIV in the United States and to report sexual contact with a person who is known to be infected with HIV; they were also less likely to be heterosexual. Prospective studies of the treatment responses of drug-naive patients with resistant genotypes have not been performed, but some data suggest that baseline nucleoside analogue mutations can diminish the potency and duration of viral suppression by commonly used nucleoside analogue combinations (16). In addition, several reports suggest an inherent decrease in susceptibility to reverse transcriptase or protease inhibitors in some HIV-1 genetic subtypes (17). Thus, both the transmission of acquired mutations and the genetic subtype of HIV-1 may have important implications for treatment of HIV-1 disease. Although we (7) and others (8) have previously described the introduction of non-subtype B infections into the United States, the current study is the first use of a large cohort of recently infected persons to describe the prevalence and associated risk factors for non-subtype B infection. Six of the seven patients infected with HIV-1 subtype E reported sexual contact during short deployments to Thailand; in contrast, most cases of subtype B infection were acquired in the United States. The acquisition of sexually transmitted diseases and HIV during overseas travel is not unique to the military; 5% to 20% of travelers report having sex with a new partner while abroad (18).

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.

Common and divergent immune response signaling pathways discovered in peripheral blood mononuclear cell gene expression patterns in presymptomatic and clinically apparent malaria

ABSTRACT Using genome-wide expression profiles from persons either experimentally challenged with malaria-infected mosquitoes or naturally infected with Plasmodium falciparum malaria, we present details of the transcriptional changes that occur with infection and that either are commonly shared between subjects with presymptomatic and clinically apparent malaria or distinguish these two groups. Toll-like receptor signaling through NF-κB pathways was significantly upregulated in both groups, as were downstream genes that function in phagocytosis and inflammation, including the cytokines tumor necrosis factor alpha, gamma interferon (IFN-γ), and interleukin-1β (IL-1β). The molecular program derived from these signatures illuminates the closely orchestrated interactions that regulate gene expression by transcription factors such as IRF-1 in the IFN-γ signal transduction pathway. Modulation of transcripts in heat shock and glycolytic enzyme genes paralleled the intensity of infection. Major histocompatibility complex class I molecules and genes involved in class II antigen presentation are significantly induced in 90% of malaria-infected persons regardless of group. Differences between early presymptomatic infection and natural infection involved genes that regulate the induction of apoptosis through mitogen-activated protein (MAP) kinases and signaling pathways through the endogenous pyrogen IL-1β, a major inducer of fever. The induction of apoptosis in peripheral blood mononuclear cells from patients with naturally acquired infection impacted the mitochondrial control of apoptosis and the activation of MAP kinase pathways centered around MAPK14 (p38α and p38β). Our findings confirm and extend findings regarding aspects of the earliest responses to malaria infection at the molecular level, which may be informative in elucidating how innate and adaptive immune responses may be modulated in different stages of infection.

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.

Serum Levels of Virus Burden in Early-Stage Human Immunodeficiency Virus Type 1 Disease in Women

The fundamental clinical, viral, and immunologic features of early-stage human immunodeficiency virus type 1 (HIV-1) disease were examined in a seroprevalent cohort of 28 men and 14 women assessed longitudinally at three equally dispersed time points over a mean of 43 months. There were no gender differences in the relative risk of developing AIDS-defining end points or death. The median serum RNA levels assessed at the three study time points were 3.3-, 4.9-, and 1.5-fold lower, respectively, in women than in men. This suggests that while serum virus load may be as powerful a correlate of disease status in women as it is in men, the absolute values of the virus levels may be different in the 2 populations. These observations may have implications for the interpretation of levels of virus burden in women for the assessment of disease progression, transmission, and treatment.

The acute neurotoxicity of mefloquine may be mediated through a disruption of calcium homeostasis and ER function in vitro

BackgroundThere is no established biochemical basis for the neurotoxicity of mefloquine. We investigated the possibility that the acute in vitro neurotoxicity of mefloquine might be mediated through a disruptive effect of the drug on endoplasmic reticulum (ER) calcium homeostasis.MethodsLaser scanning confocal microscopy was employed to monitor real-time changes in basal intracellular calcium concentrations in embryonic rat neurons in response to mefloquine and thapsigargin (a known inhibitor of the ER calcium pump) in the presence and absence of external calcium. Changes in the transcriptional regulation of known ER stress response genes in neurons by mefloquine were investigated using Affymetrix arrays. The MTT assay was employed to measure the acute neurotoxicity of mefloquine and its antagonisation by thapsigargin.ResultsAt physiologically relevant concentrations mefloquine was found to mobilize neuronal ER calcium stores and antagonize the pharmacological action of thapsigargin, a specific inhibitor of the ER calcium pump. Mefloquine also induced a sustained influx of extra-neuronal calcium via an unknown mechanism. The transcription of key ER proteins including GADD153, PERK, GRP78, PDI, GRP94 and calreticulin were up-regulated by mefloquine, suggesting that the drug induced an ER stress response. These effects appear to be related, in terms of dose effect and kinetics of action, to the acute neurotoxicity of the drug in vitro.ConclusionsMefloquine was found to disrupt neuronal calcium homeostasis and induce an ER stress response at physiologically relevant concentrations, effects that may contribute, at least in part, to the neurotoxicity of the drug in vitro.

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.