Dual anti-HIV mechanism of clofarabine.

BACKGROUND: HIV-1 replication kinetics inherently depends on the availability of cellular dNTPs for viral DNA synthesis. In activated CD4(+) T cells and other rapidly dividing cells, the concentrations of dNTPs are high and HIV-1 reverse transcription occurs in an efficient manner. In contrast, nondividing cells such as macrophages have lower dNTP pools, which restricts efficient reverse transcription. Clofarabine is an FDA approved ribonucleotide reductase inhibitor, which has shown potent antiretroviral activity in transformed cell lines. Here, we explore the potency, toxicity and mechanism of action of clofarabine in the human primary HIV-1 target cells: activated CD4(+) T cells and macrophages. RESULTS: Clofarabine is a potent HIV-1 inhibitor in both activated CD4(+) T cells and macrophages. Due to its minimal toxicity in macrophages, clofarabine displays a selectivity index over 300 in this nondividing cell type. The anti-HIV-1 activity of clofarabine correlated with a significant decrease in both cellular dNTP levels and viral DNA synthesis. Additionally, we observed that clofarabine triphosphate was directly incorporated into DNA by HIV-1 reverse transcriptase and blocked processive DNA synthesis, particularly at the low dNTP levels found in macrophages. CONCLUSIONS: Taken together, these data provide strong mechanistic evidence that clofarabine is a dual action inhibitor of HIV-1 replication that both limits dNTP substrates for viral DNA synthesis and directly inhibits the DNA polymerase activity of HIV-1 reverse transcriptase.

Synergistic reduction of HIV-1 infectivity by 5-azacytidine and inhibitors of ribonucleotide reductase.

Although many compounds have been approved for the treatment of human immunodeficiency type-1 (HIV-1) infection, additional anti-HIV-1 drugs (particularly those belonging to new drug classes) are still needed due to issues such as long-term drug-associated toxicities, transmission of drug-resistant variants, and development of multi-class resistance. Lethal mutagenesis represents an antiviral strategy that has not yet been clinically translated for HIV-1 and is based on the use of small molecules to induce excessive levels of deleterious mutations within the viral genome. Here, we show that 5-azacytidine (5-aza-C), a ribonucleoside analog that induces the lethal mutagenesis of HIV-1, and multiple inhibitors of the enzyme ribonucleotide reductase (RNR) interact in a synergistic fashion to more effectively reduce the infectivity of HIV-1. In these drug combinations, RNR inhibitors failed to significantly inhibit the conversion of 5-aza-C to 5-aza-2'-deoxycytidine, suggesting that 5-aza-C acts primarily as a deoxyribonucleoside even in the presence of RNR inhibitors. The mechanism of antiviral synergy was further investigated for the combination of 5-aza-C and one specific RNR inhibitor, resveratrol, as this combination improved the selectivity index of 5-aza-C to the greatest extent. Antiviral synergy was found to be primarily due to the reduced accumulation of reverse transcription products rather than the enhancement of viral mutagenesis. To our knowledge, these observations represent the first demonstration of antiretroviral synergy between a ribonucleoside analog and RNR inhibitors, and encourage the development of additional ribonucleoside analogs and RNR inhibitors with improved antiretroviral activity.

CCR7/CCL19 controls expression of EDG-1 in T cells.

T lymphocytes circulate between the blood, tissues, and lymph. These T cells carry out immune functions, using the C-C chemokine receptor 7 (CCR7) and its cognate ligands, CCL19 and CCL21, to enter and travel through the lymph nodes. Distinct roles for each ligand in regulating T lymphocyte trafficking have remained elusive. We report that in the human T cell line HuT78 and in primary murine T lymphocytes, signaling from CCR7/CCL19 leads to increased expression and phosphorylation of extracellular signal-regulated kinase 5 (ERK5) within eight hours of stimulation. Within 48-72 h we observed peak levels of endothelial differentiation gene 1 (EDG-1), which mediates the egress of T lymphocytes from lymph nodes. The increased expression of EDG-1 was preceded by up-regulation of its transcription factor, Krüppel-like factor 2 (KLF-2). To determine the cellular effect of disrupting ERK5 signaling from CCR7, we examined the migration of ERK5(flox/flox)/Lck-Cre murine T cells to EDG-1 ligands. While CCL19-stimulated ERK5(flox/flox) naïve T cells showed increased migration to EDG-1 ligands at 48 h, the migration of ERK5(flox/flox)/Lck-Cre T cells remained at a basal level. Accordingly, we define a novel signaling pathway that controls EDG-1 up-regulation following stimulation of T cells by CCR7/CCL19. This is the first report to link the two signaling events that control migration through the lymph nodes: CCR7 mediates entry into the lymph nodes and EDG-1 signaling controls their subsequent exit.

Single-Strand Consensus Sequencing Reveals that HIV Type but not Subtype Significantly Impacts Viral Mutation Frequencies and Spectra.

A long-standing question of human immunodeficiency virus (HIV) genetic variation and evolution has been whether differences exist in mutation rate and/or mutation spectra among HIV types (i.e., HIV-1 versus HIV-2) and among HIV groups (i.e., HIV-1 groups M-P and HIV-2 groups A-H) and HIV-1 Group M subtypes (i.e., subtypes A-D, F-H, and J-K). To address this, we developed a new single-strand consensus sequencing assay for the determination of HIV mutation frequencies and spectra using the Illumina sequencing platform. This assay enables parallel and standardized comparison of HIV mutagenesis among various viral vectors with lower background error than traditional methods of Illumina library preparation. We found significant differences in viral mutagenesis between HIV types but intriguingly no significant differences among HIV-1 Group M subtypes. More specifically, HIV-1 exhibited higher transition frequencies than HIV-2, due mostly to single G-to-A mutations and (to a lesser extent) G-to-A hypermutation. These data suggest that HIV-2 RT exhibits higher fidelity during viral replication, and taken together, these findings demonstrate that HIV type but not subtype significantly affects viral mutation frequencies and spectra. These differences may inform antiviral and vaccine strategies.

Sex and estrogen influence drug abuse.

Evidence is accumulating that the etiology, epidemiology, consequences and mechanisms that underlie drug abuse are different in males and females. In this review, we present examples of sex differences in all phases of drug abuse, including acquisition, steady-state maintenance, escalation, dysregulation, withdrawal, relapse and treatment. Most reported findings are based on laboratory research in animals, but there are corroborating reports from human clinical and epidemiological studies. In all phases of drug abuse, females seem to be more sensitive to the rewarding effects of drugs than males, and estrogen is a major factor that underlies these sex differences.

Effects of gender and menstrual cycle phase on food-maintained responding under a progressive-ratio schedule in cynomolgus monkeys.

Clinical and preclinical data suggest that fluctuations in ovarian steroid hormones across the menstrual/estrous cycle influence spontaneous feeding behavior in females. The effects of gender, menstrual cycle phase, and ovarian hormone fluctuations on food-maintained responding under a progressive-ratio schedule were investigated in four female and three male cynomolgus monkeys. Females were studied across 21 menstrual cycles, and ovulatory cycles were defined by analysis of ovarian steroid hormone levels. Data were analyzed for the early and mid-follicular phase and the mid- and late luteal phase of the menstrual cycle. Progressive-ratio break points for food were significantly higher in males than in females (p < 0.01). However, progressive-ratio break points did not vary consistently as a function of menstrual cycle phase during ovulatory cycles. There were no systematic patterns of progressive-ratio break points in anovulatory menstrual cycles. Only one female monkey reached significantly higher break points during the mid- and late luteal phases in comparison to the mid-follicular phase of the menstrual cycle (p < 0.05). There was also a significant positive correlation between progressive-ratio break points and progesterone levels and a significant negative correlation with estradiol in that monkey. Although fluctuations in ovarian steroid hormones may influence food consumption under some conditions, consistent patterns of food-maintained responding were not detected during ovulatory menstrual cycles in cynomolgus monkeys.

Effect of short- vs. long-term estrogen on reinstatement of cocaine-seeking behavior in female rats.

Estrogen effects on cocaine-induced reinstatement of lever responding were examined in sham-operated, vehicle-treated (SH+VEH), ovariectomized (OVX+VEH), and OVX female Wistar rats with estrogen replacement (OVX+EB). The effect of long- (64+/-1.56 days) and short-term (9 days) EB treatment on reinstatement of cocaine-seeking behavior was compared in Experiment 1 and 2, respectively, in order to compare the effect of EB when it was present during the development vs. expression of reinstatement of cocaine-seeking behavior. Rats were trained to self-administer 0.4 mg/kg/inf cocaine. After the acquisition criteria were met, rats continued to respond for cocaine for 2 h/day for a 14-day maintenance period. Cocaine was then replaced with saline and the 21-day extinction period commenced. Subsequently, rats were tested for reinstatement of lever responding on the previously drug-paired lever after alternating daily injections of saline or cocaine. In both experiments, there were no differences between groups in self-administration behavior during training, maintenance, or extinction. In Experiment 1, SH+VEH and chronically treated OVX+EB rats had greater cocaine-induced reinstatement than OVX+VEH rats. In Experiment 2, short-term treated OVX+EB rats also showed enhanced cocaine-induced reinstatement compared to OVX+VEH rats. The results indicate that EB-mediated enhancement of cocaine-induced reinstatement is dependent on EB presence during the expression of reinstatement but not during the formation of stimulus-reward associations during the development of cocaine-reinforced behavior.

Sex differences in the vulnerability to drug abuse: a review of preclinical studies.

Clinical and preclinical findings indicate that males and females differ on several aspects of drug reinforcement. Females are more vulnerable than males during transition periods of drug use that are characteristic of drug addiction and relapse. Females are also more sensitive than males to the reinforcing effects of stimulants. It has been suggested that ovarian hormones contribute to the mechanisms of action underlying these sex differences. This review examines the preclinical literature on sex differences and ovarian hormonal influences on drug self-administration in animals. It summarizes the findings on the effects of these variables during different phases of drug addiction. Possible differences in the mechanisms of action of drugs of abuse due to interactions with sex differences or ovarian hormonal factors are considered. The animal literature on sex differences in drug abuse treatment effectiveness is also discussed.

The effects of cocaine on gonadal steroid hormones and LH in male and female rhesus monkeys.

Cocaine stimulates significant increases in estradiol, testosterone (T), and luteinizing hormone (LH) in rhesus monkeys, but the temporal interactions between the gonadal steroid hormones and LH have not been determined. The effects of i.v. cocaine (0.8 mg/kg) or saline placebo administration on estradiol, T, and LH were compared in follicular phase female and male rhesus monkeys. Samples for hormone analysis were collected at 2-min intervals for 20 min, then at 10-min intervals for 50 min. Peak plasma cocaine levels were detected at 4 min and pharmacokinetic analyses showed no significant gender differences. Baseline hormone levels were equivalent before saline and cocaine administration, and saline did not alter LH or estradiol levels. In females, when baseline estradiol levels were low (< 100 pg/ml), LH increased significantly within 8 min after cocaine administration (P < 0.05), but when baseline estradiol levels were high (> 100 pg/ml), LH levels did not change significantly after cocaine administration. Estradiol and T increased significantly after LH, within 16 min after cocaine administration (P < 0.01-0.001). In males, significant LH increases were detected at 16 min after cocaine administration (P < 0.05-0.001), but estradiol and T did not change significantly. Thus, cocaine may stimulate significant increases in estradiol and T in females but not in males. These rapid hormonal changes may contribute to cocaine's abuse-related effects, as well as to disruptions of the menstrual cycle during chronic cocaine administration.

Sex differences in the acquisition of IV methamphetamine self-administration and subsequent maintenance under a progressive ratio schedule in rats.

RATIONALE: Previous work indicates that female rats initiate cocaine use sooner than male rats and reach significantly higher break points (BPs) for a single injection of cocaine under a progressive ratio (PR) schedule compared to male rats. OBJECTIVES: The present study extends previous work examining sex differences to the acquisition of methamphetamine (METH) (0.02 mg/kg) and maintenance of METH-maintained responding under a PR schedule. METHODS: An automated priming procedure that has previously been shown to be sensitive to sex differences was used for the acquisition of drug self-administration. A PR schedule that has been shown to be sensitive in detecting sex differences in maintenance levels of cocaine-reinforced responding was used for the maintenance phase of the experiment. RESULTS: A greater percentage of female rats met the acquisition criterion for METH (0.02 mg/kg) self-administration compared to male rats (55.6% versus 11.1%, respectively), and they did so at a significantly faster rate. Under stable fixed-ratio 1 (FR1) conditions (after acquisition and 5 days before the PR schedule) female rats responded for significantly more METH (0.02 mg/kg) infusions compared to males. Dose-response curves obtained under the PR schedule during maintenance indicated that female rats self-administered significantly more METH infusions compared to male rats. CONCLUSIONS: These data suggest that female rats are more vulnerable to the acquisition of METH self-administration, and they are more motivated to self-administer METH compared to male rats under a PR schedule during the maintenance phase.

Biological basis of sex differences in drug abuse: preclinical and clinical studies.

The recent focus on drug abuse in women has brought attention to numerous differences between women and men. In this review, we discuss both preclinical and clinical findings of sex differences in drug abuse as well as mechanisms that may underlie these differences. Recent evidence suggests that the progression to dependence and abuse may differ between women and men; thus, different prevention and treatment strategies may be required. Similar sex differences in drug sensitivity and self-administration have been reported in laboratory animal studies. Females appear to be more vulnerable than males to the reinforcing effects of psychostimulants, opiates, and nicotine during many phases of the addiction process (e.g. acquisition, maintenance, dysregulation-escalation, relapse). Male and female animals differ in their behavioral, neurological, and pharmacological responses to drugs. Although the role of sex in the mechanisms of drug action remains unclear, preclinical and clinical studies indicate that ovarian hormones, particularly estrogen, play a role in producing sex differences in drug abuse. Future research is necessary to provide information on how to design more effective drug abuse treatment programs and resources that are sex specific.

Caffeine dependence in teenagers.

This study identifies and characterizes symptoms of caffeine dependence in adolescents. Thirty-six adolescents who consumed caffeine daily and had some features of caffeine dependence on telephone screen were scheduled for outpatient evaluation. Evaluation included the Diagnostic Interview Schedule for Children-IV-Youth Version (DISC-IV) and modified DISC-IV questions that assessed caffeine dependence based on DSM-IV substance dependence criteria. Of 36 subjects, 41.7% (n=15) reported tolerance to caffeine, 77.8% (n=28) described withdrawal symptoms after cessation or reduction of caffeine intake, 38.9% (n=14) reported desire or unsuccessful attempts to control use, and 16.7% (n=6) endorsed use despite knowledge of physical or psychological problems associated with caffeine. There was no significant difference in the amount of caffeine consumed daily by caffeine dependent versus non-dependent teenagers. These findings are important due to the vast number of adolescents who drink caffeinated beverages.

Sex differences in the escalation of intravenous cocaine intake following long- or short-access to cocaine self-administration.

Preclinical data have indicated that extended access to cocaine self-administration (e.g., 6-12 h/day) facilitates an escalation in daily cocaine intake that is not seen when rats are given shorter (e.g., 1-2 h/day) access to cocaine for self-administration. Data from studies with rats have shown that females self-administer more cocaine than males during all phases of drug abuse (e.g., acquisition, maintenance, and reinstatement). The purpose of this study was to examine potential differences between males and females in the escalation of intravenous cocaine intake following a differential access (e.g., 1 vs. 6 h) period of cocaine self-administration. Four groups of rats were compared: (1) long-access (LgA; 6 h) females; (2) LgA males; (3) short-access (ShA; 1 h) females; and (4) ShA males. Animals were given LgA or ShA to intravenous cocaine (0.5 mg/kg/infusion) self-administration under an Fr 1 schedule for 21 days. Subsequently, access conditions were made equal (3 h) across groups, and dose-response curves for cocaine were compared. Results revealed that the LgA groups' dose-response curves were significantly elevated above those of ShA groups. Additionally, the dose-response curve of LgA female rats was significantly elevated above that of LgA male rats. These results suggest that female rats are more sensitive than male rats to factors that contribute to the escalation of cocaine intake (e.g., extended access conditions).

Influence of estrogen in the acquisition of intravenously self-administered heroin in female rats.

Previous research indicates that female rats acquire cocaine and heroin self-administration at a faster rate than male rats, and female rats with endogenous estrogen, or ovariectomized (OVX) rats with estrogen replacement acquire cocaine self-administration more rapidly than female rats with estrogen either surgically or chemically blocked. The purpose of this investigation was to extend the above findings to the acquisition of heroin (0.0075 mg/kg) self-administration in female rats. An automated autoshaping procedure was used to train rats to self-administer heroin. Three groups of female rats were compared: (1) OVX+estradiol benzoate (OVX+EB), (2) OVX+vehicle (OVX+VEH), and (3) sham-operated+vehicle (SH+VEH). Results revealed that OVX+EB rats acquired heroin self-administration in significantly fewer days compared to OVX+VEH rats. Additionally, OVX+EB rats that met the acquisition criteria self-administered a significantly greater number of heroin infusions during the last 5 days of the acquisition period compared to OVX+VEH rats. These results indicate that OVX+EB rats initiate heroin use sooner than OVX+VEH rats and consume greater amounts of heroin during the last 5 days of acquisition compared to OVX+VEH female rats.

Rapid genetic modification of mouse embryonic stem cells by Inducible Cassette Exchange recombination.

Embryonic stem cell (ESC) differentiation is a useful means by which to produce large quantities of cells in vitro representing early stages of embryonic development. A conditional gene expression system allows interrogation of factors at specific time points in the differentiation of ES cells to defined cell types. We have developed a method for rapidly generating conditional inducible murine ES cells by targeting genes into an Inducible Cassette Exchange (ICE) locus. The ICE locus encodes a doxycycline-inducible floxed Cre, which replaces itself with an incoming floxed gene of interest. The derivative cell lines, selected in G418, thus bear doxycycline-inducible transgenes. We provide detailed methods for performing ICE recombination and generating derivative doxycycline-inducible cell lines.