Publications

BACKGROUND

Interferon-α (IFN-α) treatment suppresses HIV-1 viremia and reduces the size of the HIV-1 latent reservoir. Therefore, investigation of the molecular and immunologic effects of IFN-α may provide insights that contribute to the development of novel prophylactic, therapeutic and curative strategies for HIV-1 infection. In this study, we hypothesized that microRNAs (miRNAs) contribute to the IFN-α-mediated suppression of HIV-1. To inform the development of novel miRNA-based antiretroviral strategies, we investigated the effects of exogenous IFN-α treatment on global miRNA expression profile, HIV-1 viremia, and potential regulatory networks between miRNAs and cell-intrinsic anti-HIV-1 host factors in vivo.

METHODS

Global miRNA expression was examined in longitudinal PBMC samples obtained from seven HIV/HCV-coinfected, antiretroviral therapy-naïve individuals before, during, and after pegylated interferon-α/ribavirin therapy (IFN-α/RBV). We implemented novel hybrid computational-empirical approaches to characterize regulatory networks between miRNAs and anti-HIV-1 host restriction factors.

RESULTS

miR-422a was the only miRNA significantly modulated by IFN-α/RBV in vivo (p<0.0001, paired t test; FDR<0.037). Our interactome mapping revealed extensive regulatory involvement of miR-422a in p53-dependent apoptotic and pyroptotic pathways. Based on sequence homology and inverse expression relationships, 29 unique miRNAs may regulate anti-HIV-1 restriction factor expression in vivo.

CONCLUSIONS

The specific reduction of miR-422a is associated with exogenous IFN-α treatment, and likely contributes to the IFN-α suppression of HIV-1 through the enhancement of anti-HIV-1 restriction factor expression and regulation of genes involved in programmed cell death. Moreover, our regulatory network analysis presents additional candidate miRNAs that may be targeted to enhance anti-HIV-1 restriction factor expression in vivo.

Endometriosis, the most common cause of chronic pelvic pain, is an estrogen-dependent disease in which classic estrogen receptors (ERα, ERβ) play an important role. Although recent evidence suggests that the novel G protein-coupled estrogen receptor (GPR30) also plays a key role in the progression of endometriosis, whether it is also involved in endometriosis pain is still unknown. Here we tested the hypothesis that GPR30 expressed by nociceptors contributes to endometriosis pain. Intramuscular injection of the GPR30 agonists raloxifene or 17β-estradiol produced a fast-onset, persistent, mechanical hyperalgesia at the site of the injection. Intrathecal antisense (AS) oligodeoxynucleotides (ODN), but not mismatch (MM) ODN, targeting mRNA for GPR30 markedly inhibited its protein expression in nociceptors and attenuated the mechanical hyperalgesia induced by local raloxifene or 17β-estradiol. Pretreatment with the GPR30 antagonist G-36 also inhibited the hyperalgesia induced by raloxifene or 17β-estradiol in naive control rats. Surgical implant of autologous uterine tissue onto the gastrocnemius muscle, which induces endometriosis-like lesions, produced local mechanical hyperalgesia. Intrathecal AS, but not MM, ODN targeting GPR30 mRNA reversibly inhibited the mechanical hyperalgesia at the site of endometriotic lesions. Finally, intralesional injection of the GPR30 antagonist G-36 also inhibited the mechanical hyperalgesia at the site of ectopic uterine tissue. We conclude that local GPR30 agonists produce persistent mechanical hyperalgesia in naive female rats, whereas local GPR30 antagonists inhibit mechanical hyperalgesia in a model of endometriosis pain. Thus, GPR30 expressed by nociceptors innervating ectopic uterine lesions might play a major role in endometriosis pain.

Many orally administered, small-molecule, targeted anticancer drugs, such as dasatinib, exhibit pH-dependent solubility and reduced drug exposure when given with acid-reducing agents. We previously demonstrated that betaine hydrochloride (BHCl) can transiently re-acidify gastric pH in healthy volunteers with drug-induced hypochlorhydria. In this randomized, single-dose, three-way crossover study, healthy volunteers received dasatinib (100 mg) alone, after pretreatment with rabeprazole, and with 1500 mg BHCl after rabeprazole pretreatment, to determine if BHCl can enhance dasatinib absorption in hypochlorhydric conditions. Rabeprazole (20 mg b.i.d.) significantly reduced dasatinib Cmax and AUC0-∞ by 92 and 78%, respectively. However, coadministration of BHCl significantly increased dasatinib Cmax and AUC0-∞ by 15- and 6.7-fold, restoring them to 105 and 121%, respectively, of the control (dasatinib alone). Therefore, BHCl reversed the impact of hypochlorhydria on dasatinib drug exposure and may be an effective strategy to mitigate potential drug-drug interactions for drugs that exhibit pH-dependent solubility and are administered orally under hypochlorhydric conditions.

Inter-individual variation in gene regulatory elements is hypothesized to play a causative role in adverse drug reactions and reduced drug activity. However, relatively little is known about the location and function of drug-dependent elements. To uncover drug-associated elements in a genome-wide manner, we performed RNA-seq and ChIP-seq using antibodies against the pregnane X receptor (PXR) and three active regulatory marks (p300, H3K4me1, H3K27ac) on primary human hepatocytes treated with rifampin or vehicle control. Rifampin and PXR were chosen since they are part of the CYP3A4 pathway, which is known to account for the metabolism of more than 50% of all prescribed drugs. We selected 227 proximal promoters for genes with rifampin-dependent expression or nearby PXR/p300 occupancy sites and assayed their ability to induce luciferase in rifampin-treated HepG2 cells, finding only 10 (4.4%) that exhibited drug-dependent activity. As this result suggested a role for distal enhancer modules, we searched more broadly to identify 1,297 genomic regions bearing a conditional PXR occupancy as well as all three active regulatory marks. These regions are enriched near genes that function in the metabolism of xenobiotics, specifically members of the cytochrome P450 family. We performed enhancer assays in rifampin-treated HepG2 cells for 42 of these sequences as well as 7 sequences that overlap linkage-disequilibrium blocks defined by lead SNPs from pharmacogenomic GWAS studies, revealing 15/42 and 4/7 to be functional enhancers, respectively. A common African haplotype in one of these enhancers in the GSTA locus was found to exhibit potential rifampin hypersensitivity. Combined, our results further suggest that enhancers are the predominant targets of rifampin-induced PXR activation, provide a genome-wide catalog of PXR targets and serve as a model for the identification of drug-responsive regulatory elements.