Microarrays-Enabled Hypothesis Generation: The Suspect Role of FNBP-1 in Neuropsychiatric Pathogenesis Associated with HIV and/or HCV Infection.

OBJECTIVE: The spectrum of neuropsychiatric illness (NI) associated with the Human Immunodeficiency Virus (HIV) and/or the Hepatitis C Virus (HCV) is far reaching and significantly impacts the clinical presentation and outcome of infected persons; however, the etiological and pathophysiological background remains partially understood. The present work was aimed to investigate the potential significance of formin binding protein 1 (FNBP-1)-dependent pathways in NI-pathogenesis by elaborating on previous microarray-based research in HIV and/or HCV-infected patients receiving interferon-α (IFN-α) immunotherapy via a rigorous data mining procedure. METHODS: Using microarray data of peripheral whole blood (PB) samples obtained from HCV mono-infected persons (n=25, Affymetrix® HG-U133A_2) 12 h before and after the 1st dose of pegylated IFN-α (PegIFN-α), we re-applied the same analytical algorithm that we had developed and published in an earlier study with HIV/HCV co-infected subjects (N=28, Affymetrix® HG-U133A), in order to evaluate reproducibility of potential NI-related molecular findings in an independent cohort. RESULTS: Among 28 gene expression profiles (HIV/HCV: N=9 vs. HCV: N=19) selected by applying different thresholds (a Mean Fold Difference value (MFD) in gene expression of ≥ 0.38 (log2) and/or P value from <0.05 to ≤ 0.1) FNBP-1 was identified as the only overlapping marker, which also exhibited a consistent upregulation in association with the development of NI in both cohorts. Previous functional annotation analysis had classified FNBP-1 as molecule with significant enrichment in various brain tissues (P<0.01). CONCLUSION: Our current findings are strongly arguing for intensifying research into the FNBP-1-related mechanisms that may be conferring risk for or resistance to HIV- and/or HCV-related NI.

Distinct and overlapping genomic profiles and antiviral effects of Interferon-λ and -α on HCV-infected and noninfected hepatoma cells.

Recently, several SNPs in the region of the IL28B (IFN-λ) gene have been associated with spontaneous clearance of hepatitis C virus (HCV) and enhanced cure rates for IFN-alfa-based therapies, suggesting a potential correlation between IFN-λ and the ability to clear HCV. To understand the mechanism of IFN-λ's as compared to IFN-α's antiviral activity, we performed a comprehensive analysis of their anti-HCV effects, whole genome transcriptome profiling with validation, and signalling of IFN-α and IFN-λ using J6/JFH-1 and Huh7.5 cells in vitro. IFN-λ and IFN-α exhibited comparable anti-HCV activity and gene expression profiles in Huh7.5 cells. While the majority of genes induced by IFN-α and IFN-λ were similar, IFN-λ exhibits profound, but delayed kinetics of IFN-stimulated genes (ISG) induction, while IFN-α induced more rapid induction of ISGs. Furthermore, the increased induction of ISG expression by IFN-λ correlated with up-regulation of IFN-λ receptor (IL-28RA) expression and more prolonged activation of the Jak-STAT signalling pathway. The findings from our comparative analysis of IFN-α and IFN-λ in HCV-infected and noninfected cells support the clinical use of IFN-λ as a potential alternative to IFN-α in the treatment of chronic hepatitis C.

CCL5 mRNA is a marker for early fibrosis in chronic hepatitis C and is regulated by interferon-α therapy and toll-like receptor 3 signalling.

Mechanisms causing liver fibrosis during chronic hepatitis C virus infection (cHCV) are not sufficiently understood. This study was aimed to identify biomarkers for early fibrosis (EF) and to investigate their potential role in cHCV-related fibrogenesis. To this end, peripheral whole blood (PB) samples from 36 patients with cHCV recruited from two independent cohorts were subjected to microarray analysis 12 h before initiation of peginterferon-alpha (Peg-IFN-α) and ribavirin therapy. Liver biopsies were evaluated using the Batts-Ludwig staging (BL-S) classification system for fibrosis. We showed that gene expression profiles (N = 8) distinguished between EF (BL-S: 0,1) and late fibrosis (LF; BL-S: 2,3,4) with 88.9% accuracy. Fibrosis-related functional annotations for chemokine-'C-C-motif'' ligand 5 (CCL5) provided foundation for focused investigation, and qRT-PCR confirmed that CCL5 mRNA levels (PB) reliably discriminate EF from LF (accuracy: 86.7%). Positive correlations (P < 0.05) with CCL5 mRNA levels and EF discovered gene expression profiles (PB) reflecting stable expression of IFN-α receptor 1, negative regulation of the MyD88-dependent toll-like receptor (TLR) pathway and decreased expression of TLR3 in vivo. Remarkably, Peg-IFN-α suppressed CCL5 mRNA levels (PB) in EF in vivo. These findings along with results from parallel in vitro investigation into the effect of IFN-α or poly I:C (TLR3-agonist) on CCL5 gene expression in hepatic stellate cells (HSC) attest to the multi-site involvement of these pathways in regulating fibrogenesis. In conclusion, we identified novel, reliable biomarkers for EF and exposed functional properties of the molecular network regulating CCL5 biosynthesis in peripheral or hepatic cell types with key roles in cHCV-related liver and/or immune pathogenesis.

Defective plasmacytoid dendritic cell-NK cell cross-talk in HIV infection.

HIV viremia is associated with a wide range of immune dysfunctions that contribute to the immunocompromised state. HIV viremia has been shown to have a broad effect on several immune cell types and/or their interactions that are vital for mounting an effective immune response. In this study, we investigated the integrity of plasmacytoid dendritic cell (pDC)-NK cell interactions among HIV viremic, aviremic, and seronegative individuals. We describe a critical defect in the ability of pDCs from HIV-infected individuals to secrete IFN-alpha and TNF and subsequently activate NK cells. We also describe an inherent defect on NK cells from HIV-infected individuals to respond to pDC-secreted cytokines. Furthermore, we were able to demonstrate a direct effect of HIV trimeric gp120 on NK cells in vitro similar to that described ex vivo. Finally, we were able to establish that the HIV gp120-mediated suppressive effect on NK cells was a result of its binding to the integrin alpha(4)beta(7) expressed on NK cells. These findings suggest a novel mechanism by which HIV is capable of suppressing an innate immune function in infected individuals.

Transcriptomic response to differentiation induction.

BACKGROUND: Microarrays used for gene expression studies yield large amounts of data. The processing of such data typically leads to lists of differentially-regulated genes. A common terminal data analysis step is to map pathways of potentially interrelated genes. METHODS: We applied a transcriptomics analysis tool to elucidate the underlying pathways of leukocyte maturation at the genomic level in an established cellular model of leukemia by examining time-course data in two subclones of U-937 cells. Leukemias such as Acute Promyelocytic Leukemia (APL) are characterized by a block in the hematopoietic stem cell maturation program at a point when expansion of clones which should be destined to mature into terminally-differentiated effector cells get locked into endless proliferation with few cells reaching maturation. Treatment with retinoic acid, depending on the precise genomic abnormality, often releases the responsible promyelocytes from this blockade but clinically can yield adverse sequellae in terms of potentially lethal side effects, referred to as retinoic acid syndrome. RESULTS: Briefly, the list of genes for temporal patterns of expression was pasted into the ABCC GRID Promoter TFSite Comparison Page website tool and the outputs for each pattern were examined for possible coordinated regulation by shared regelems (regulatory elements). We found it informative to use this novel web tool for identifying, on a genomic scale, genes regulated by drug treatment. CONCLUSION: Improvement is needed in understanding the nature of the mutations responsible for controlling the maturation process and how these genes regulate downstream effects if there is to be better targeting of chemical interventions. Expanded implementation of the techniques and results reported here may better direct future efforts to improve treatment for diseases not restricted to APL.

Gene expression profiles in hepatitis C virus (HCV) and HIV coinfection: class prediction analyses before treatment predict the outcome of anti-HCV therapy among HIV-coinfected persons.

Therapy for hepatitis C virus (HCV) infection in human immunodeficiency virus (HIV)-infected patients results in modest cure rates. Gene expression patterns in peripheral blood mononuclear cells from 29 patients coinfected with HIV and HCV were used to predict virological response to therapy for HCV infection. Prediction analysis using pretherapy samples identified 79 genes that correctly classified all 10 patients who did not respond to therapy, 8 of 10 patients with a response at the end of treatment, and 7 of 9 patients with sustained virological response (86% overall). Analysis of 17 posttreatment samples identified 105 genes that correctly classified all 9 patients with response at the end of treatment and 7 of 8 patients with sustained virological response (94% overall). Failure of anti-HCV therapy was associated with elevated expression of interferon-stimulated genes. Gene expression patterns may provide a tool to predict anti-HCV therapeutic response.

A comparison of microLC/electrospray ionization-MS and GC/MS for the measurement of stable isotope enrichment from a [2H2]-glucose metabolic probe in T-cell genomic DNA.

Measurement of the proliferation of lymphocytes and other high-turnover cell populations in vivo can be accomplished through the incorporation of an isotopically labeled DNA precursor into actively dividing cells and the subsequent determination of the isotope enrichment in the isolated genomic DNA from selected cell populations. Two published gas chromatography/mass spectrometry (GC/MS) methods were successfully modified by our laboratory whereby a postinjection methylation reaction, rather than silylation or acetylation, was used to form a volatile derivative of deoxyadenosine (dA). We also developed a second robust microcapillary liquid chromatography-electrospray ionization (microLC-ESI)/MS method that is faster and more sensitive than the GC/MS method and does not require sample derivatization. Following administration of [6,6-(2)H(2)]-glucose to human immunodeficiency virus-infected patients, peripheral blood was drawn; cells were obtained by lymphapheresis and fractionated. DNA was isolated from the desired cell subtypes and enzymatically hydrolyzed to the free deoxyribonucleosides. The digest was analyzed using both capillary GC/MS and microLC/ESI-MS to measure the levels of the dA and [(2)H(2)]-dA or their reaction products. Sample enrichments were calculated by comparison to standard curves prepared from dA and [(2)H(2)]-dA. The microLC/ESI-MS method required fewer cells, less sample preparation, shorter analysis times, and a single calibration curve. Overall, the microLC/ESI-MS method is superior to the GC/MS method in terms of precision and accuracy, while providing a 4-fold increase in sensitivity (from 20 pmol at 0.2% [(2)H(2)]-dA enrichment to 5 pmol at 0.1% [(2)H(2)]-dA enrichment).

Identification of dynamically distinct subpopulations of T lymphocytes that are differentially affected by HIV.

We examined the effects of human immunodeficiency virus infection on the turnover of CD4 and CD8 T lymphocytes in 17 HIV-infected patients by 30 min in vivo pulse labeling with bromodeoxyuridine (BrdU). The percentage of labeled CD4 and CD8 T lymphocytes was initially higher in lymph nodes than in blood. Labeled cells equilibrated between the two compartments within 24 h. Based on mathematical modeling of the dynamics of BrdU-labeled cells in the blood, we identified rapidly and slowly proliferating subpopulations of CD4 and CD8 T lymphocytes. The percentage, but not the decay rate, of labeled CD4 or CD8 cells in the rapidly proliferating pool correlated significantly with plasma HIV RNA levels for both CD4 (r = 0.77, P < 0.001) and CD8 (r = 0.81, P < 0.001) T cells. In six patients there was a geometric mean decrease of greater than 2 logs in HIV levels within 2 to 6 mo after the initiation of highly active antiretroviral therapy; this was associated with a significant decrease in the percentage (but not the decay rate) of labeled cells in the rapidly proliferating pool for both CD4 (P = 0.03) and CD8 (P < 0.001) T lymphocytes. Neither plasma viral levels nor therapy had an effect on the decay rate constants or the percentage of labeled cells in the slowly proliferating pool. Monocyte production was inversely related to viral load (r = -0.56, P = 0.003) and increased with therapy (P = 0.01). These findings demonstrate that HIV does not impair CD4 T cell production but does increase CD4 and CD8 lymphocyte proliferation and death by inducing entry into a rapidly proliferating subpopulation of cells.

Evidence for increased T cell turnover and decreased thymic output in HIV infection.

The effects of HIV infection upon the thymus and peripheral T cell turnover have been implicated in the pathogenesis of AIDS. In this study, we investigated whether decreased thymic output, increased T cell proliferation, or both can occur in HIV infection. We measured peripheral blood levels of TCR rearrangement excision circles (TREC) and parameters of cell proliferation, including Ki67 expression and ex vivo bromodeoxyuridine incorporation in 22 individuals with early untreated HIV disease and in 15 HIV-infected individuals undergoing temporary interruption of therapy. We found an inverse association between increased T cell proliferation with rapid viral recrudescence and a decrease in TREC levels. However, during early HIV infection, we found that CD45RO-CD27high (naive) CD4+ T cell proliferation did not increase, despite a loss of TREC within naive CD4+ T cells. A possible explanation for this is that decreased thymic output occurs in HIV-infected humans. This suggests that the loss of TREC during HIV infection can arise from a combination of increased T cell proliferation and decreased thymic output, and that both mechanisms can contribute to the perturbations in T cell homeostasis that underlie the pathogenesis of AIDS.

Interleukin-2 induced immune effects in human immunodeficiency virus-infected patients receiving intermittent interleukin-2 immunotherapy.

To characterize the immunological effects of intermittent IL-2 therapy, which leads to selective increases in CD4+ T lymphocytes in HIV-infected patients, 11 patients underwent extensive immunological evaluation. While IL-2 induced changes in both CD4+ and CD8+ cell number acutely, only CD4+ cells showed sustained increases following discontinuation of IL-2. Transient increases in expression of the activation markers CD38 and HLA-DR were seen on both CD4+ and CD8+ cells, but CD25 (a chain of the IL-2 receptor) increased exclusively on CD4+ cells. This increase in CD25 expression was sustained for months following discontinuation of IL-2, and was seen in naive as well as memory cells. IL-2 induced cell proliferation, but tachyphylaxis to these proliferative effects developed after 1 week despite continued IL-2 administration. It thus appears that sustained CD25 expression selectively on CD4+ cells is a critical component of the immunological response to IL-2, and that intermittent administration of IL-2 is necessary to overcome the tachyphylaxis to IL-2-induced proliferation.

Impact of HIV-1 infection and highly active antiretroviral therapy on the kinetics of CD4+ and CD8+ T cell turnover in HIV-infected patients.

To evaluate the effects of HIV infection on T cell turnover, we examined levels of DNA synthesis in lymph node and peripheral blood mononuclear cell subsets by using ex vivo labeling with BrdUrd. Compared with healthy controls (n = 67), HIV-infected patients (n = 57) had significant increases in the number and fraction of dividing CD4(+) and CD8(+) T cells. Higher percentages of dividing CD4(+) and CD8(+) T cells were noted in patients with the higher viral burdens. No direct correlation was noted between rates of T cell turnover and CD4(+) T cell counts. Marked reductions in CD4(+) and CD8(+) T cell proliferation were seen in 11/11 patients 1-12 weeks after initiation of highly active antiretroviral therapy (HAART). These reductions persisted for the length of the study (16-72 weeks). Decreases in naive T cell proliferation correlated with increases in the levels of T cell receptor rearrangement excision circles. Division of CD4(+) and CD8(+) T cells increased dramatically in association with rapid increases in HIV-1 viral loads in 9/9 patients 5 weeks after termination of HAART and declined to pre-HAART-termination levels 8 weeks after reinitiation of therapy. These data are consistent with the hypothesis that HIV-1 infection induces a viral burden-related, global activation of the immune system, leading to increases in lymphocyte proliferation.

HIV-1 and T cell dynamics after interruption of highly active antiretroviral therapy (HAART) in patients with a history of sustained viral suppression.

Identifying the immunologic and virologic consequences of discontinuing antiretroviral therapy in HIV-infected patients is of major importance in developing long-term treatment strategies for patients with HIV-1 infection. We designed a trial to characterize these parameters after interruption of highly active antiretroviral therapy (HAART) in patients who had maintained prolonged viral suppression on antiretroviral drugs. Eighteen patients with CD4(+) T cell counts >/= 350 cells/microliter and viral load below the limits of detection for >/=1 year while on HAART were enrolled prospectively in a trial in which HAART was discontinued. Twelve of these patients had received prior IL-2 therapy and had low frequencies of resting, latently infected CD4 cells. Viral load relapse to >50 copies/ml occurred in all 18 patients independent of prior IL-2 treatment, beginning most commonly during weeks 2-3 after cessation of HAART. The mean relapse rate constant was 0.45 (0.20 log(10) copies) day(-1), which was very similar to the mean viral clearance rate constant after drug resumption of 0.35 (0.15 log(10) copies) day(-1) (P = 0.28). One patient experienced a relapse delay to week 7. All patients except one experienced a relapse burden to >5,000 RNA copies/ml. Ex vivo labeling with BrdUrd showed that CD4 and CD8 cell turnover increased after withdrawal of HAART and correlated with viral load whereas lymphocyte turnover decreased after reinitiation of drug treatment. Virologic relapse occurs rapidly in patients who discontinue suppressive drug therapy, even in patients with a markedly diminished pool of resting, latently infected CD4(+) T cells.

Effects of cold exposure on estrous behavior and neural estrogen receptor in Syrian hamsters.

Two experiments examined the effects of 72-h exposure to reduced environmental temperature (5 degrees C) on steroid-induced estrous behavior and neural estrogen-receptor immunoreactivity (ERIR) in ovariectomized Syrian hamsters. Cold exposure significantly inhibited sexual receptivity induced by sequential injections of estradiol benzoate (2.5 microg) and progesterone (500 microg), but only if the animals were not permitted to overeat (limited to 110% of ad lib intake at 22 degrees C). The suppression of sexual receptivity was accompanied by decreases in the number of detectable ERIR cells in the ventromedial hypothalamus (VMH) and by increases in the number of ERIR cells in the medial preoptic area (mPOA). The cold-induced decreases in estrous behavior and in VMH ERIR cells were prevented by lesions of the area postrema (AP), but AP lesions did not prevent the increases in mPOA ERIR cells. Thus, cold exposure mimics the effects of treatment with metabolic inhibitors, experimental diabetes, food deprivation, and insulin-induced fattening on these endpoints. These findings are consistent with the hypothesis that dwelling in the cold affects reproduction indirectly via its actions on metabolic fuel availability, rather than by acting directly on neuroendocrine processes.

Leptin facilitates and inhibits sexual behavior in female hamsters.

Food deprivation decreases fertility in female mammals in part by inhibiting sexual behaviors. Genetically obese ob/ob mice, like food-deprived wild-type animals, are also infertile; treatment of ob/ob mice with leptin, the adipocyte-derived protein that they lack, corrects some of their reproductive deficiencies. We tested the hypothesis that leptin treatment would prevent the suppression of sexual receptivity that is caused by food deprivation in female Syrian hamsters. Instead, we found that treatment with murine leptin facilitated female sexual behavior in ad libitum-fed hamsters, but not in food-deprived animals. In food-deprived hamsters, leptin treatment actually intensified the inhibition of lordosis. Food deprivation decreased detectable estrogen receptor immunoreactivity (ERIR) in the ventromedial hypothalamus (VMH), but the leptin-induced changes in female sexual behavior were not accompanied by parallel changes in VMH ERIR. Thus leptin facilitates estrous behavior in hamsters, but it does not overcome the lordosis-inhibiting metabolic cues produced by acute food deprivation. Because circulating leptin levels are directly related to body fat content, an implication of these findings is that elevated levels of adipose tissue could have a positive influence on sexual responsiveness.

HIV envelope-directed signaling aberrancies and cell death of CD4+ T cells in the absence of TCR co-stimulation.

HIV-1 infection in CD4(+) T cells initiates a viral cytopathic effect (CPE) that is dependent on the activation of intracellular protein tyrosine kinases (PTK). PTK in T cells are also activated during the course of TCR or CD4 receptor engagement and the manner of receptor engagement may generate signals leading either to cell proliferation, tolerance induction (anergy) or programmed cell death (PCD). We have identified PTK triggered during the interaction of cells stably expressing surface HIV envelope (gp 120/gp41; HIVenv) and CD4(+)T cells, which leads to extensive and rapid individual cell death. We have found that killing is accompanied by tyrosine phosphorylation and activation of the CD4-associated p56(ICK) kinase, and by activation of a second member of the scr family of PTK, p59(fyn) kinase, normally associated with T cell stimulation through the TCR. Interestingly, in contrast with normal T cell signaling, the zeta subunit of the TCR fails to become tyrosine-phosphorylated during signaling accompanying HIV-directed cell killing. Downstream activation of the ZAP-70 PTK also does not occur. Unlike T cell apoptosis triggered by soluble HIVenv glycoproteins, which requires co-stimulation of CD4 and the antigen-specific TCR, T cell killing by membrane-associated HIVenv does not require TCR co-stimulation, because aberrant signaling and cell death are triggered by CD4(+) but TCR- cell lines. These results are the first report where dual activation of the Lck and Fyn PTK does not result in normal downstream signaling through the ZAP PTK, We suggest by analogy to SCID resulting from ZAP-70 mutations, that the dissociation of upstream PTK activation from ZAP-70 signaling contributes to T cell depletion by HIV and to the development of AIDS.

Saccharomyces cerevisiae U14 small nuclear RNA has little secondary structure and appears to be produced by post-transcriptional processing.

Yeast U14 small nuclear (sn) RNA is required for normal processing of rRNA. The sequence and folding properties of U14 were analyzed in the present study, with the aim of defining the structures of natural U14 subspecies and characterizing the folding properties of free U14 RNA. Natural U14 was determined to consist of four subspecies of 125-128 nucleotides, none containing a 5'-cap structure. Length heterogeneity occurs at both ends and is presumed to reflect post-transcriptional processing of U14 precursors. Results from nuclease and chemical probing revealed that U14 has surprisingly little secondary structure overall. Three essential sequence elements conserved among all U14 RNAs occur in regions that are largely single-stranded, i.e. box C, box D, and a 13-nucleotide segment complementary to 18 S rRNA; a non-essential 14-nucleotide sequence complementary to 18 S rRNA is also unpaired. Two non-conserved segments required for activity are part of a stably folded 32-base domain that is unique to yeast U14. Finally, a 5'-, 3'-stem shown earlier to be required for U14 accumulation appears to exist only in precursors to U14 and not in protein-free mature RNA. The implications of these results are discussed in terms of U14 synthesis and function.

Escherichia coli 4.5S RNA gene function can be complemented by heterologous bacterial RNA genes.

The essential 4.5S RNA gene of Escherichia coli can be complemented by 4.5S RNA-like genes from three other eubacteria, including both gram-positive and gram-negative organisms. Two of the genes encode RNAs similar in size to the E. coli species; the third, from Bacillus subtilis, specifies an RNA more than twice as large. The heterologous genes are expressed efficiently in E. coli, and the product RNAs resemble those produced by cognate cells. We conclude that the heterologous RNAs can replace E. coli 4.5S RNA and that the essential function of 4.5S RNA is evolutionarily conserved. A consensus structure is presented for the functionally related 4.5S RNA homologs.