Neurodevelopmental outcome of preterm infants enrolled in myo-inositol randomized controlled trial.

OBJECTIVE: This study evaluates the 24-month follow-up for the NICHD Neonatal Research Network (NRN) Inositol for Retinopathy Trial. STUDY DESIGN: Bayley Scales of Infants Development-III and a standardized neurosensory examination were performed in infants enrolled in the main trial. Moderate/severe NDI was defined as BSID-III Cognitive or Motor composite score <85, moderate or severe cerebral palsy, blindness, or hearing loss that prevents communication despite amplification were assessed. RESULTS: Primary outcome was determined for 605/638 (95%). The mean gestational age was 25.8 ± 1.3 weeks and mean birthweight was 805 ± 192 g. Treatment group did not affect the risk for the composite outcome of death or survival with moderate/severe NDI (60% vs 56%, p = 0.40). CONCLUSIONS: Treatment group did not affect the risk of death or survival with moderate/severe NDI. Despite early termination, this study represents the largest RCT of extremely preterm infants treated with myo-inositol with neurodevelopmental outcome data.

Effects of Myo-inositol on Type 1 Retinopathy of Prematurity Among Preterm Infants <28 Weeks' Gestational Age: A Randomized Clinical Trial.

Importance: Previous studies of myo-inositol in preterm infants with respiratory distress found reduced severity of retinopathy of prematurity (ROP) and less frequent ROP, death, and intraventricular hemorrhage. However, no large trials have tested its efficacy or safety. Objective: To test the adverse events and efficacy of myo-inositol to reduce type 1 ROP among infants younger than 28 weeks' gestational age. Design, Setting, and Participants: Randomized clinical trial included 638 infants younger than 28 weeks' gestational age enrolled from 18 neonatal intensive care centers throughout the United States from April 17, 2014, to September 4, 2015; final date of follow-up was February 12, 2016. The planned enrollment of 1760 participants would permit detection of an absolute reduction in death or type 1 ROP of 7% with 90% power. The trial was terminated early due to a statistically significantly higher mortality rate in the myo-inositol group. Interventions: A 40-mg/kg dose of myo-inositol was given every 12 hours (initially intravenously, then enterally when feeding; n = 317) or placebo (n = 321) for up to 10 weeks. Main Outcomes and Measures: Type 1 ROP or death before determination of ROP outcome was designated as unfavorable. The designated favorable outcome was survival without type 1 ROP. Results: Among 638 infants (mean, 26 weeks' gestational age; 50% male), 632 (99%) received the trial drug or placebo and 589 (92%) had a study outcome. Death or type 1 ROP occurred more often in the myo-inositol group vs the placebo group (29% vs 21%, respectively; adjusted risk difference, 7% [95% CI, 0%-13%]; adjusted relative risk, 1.41 [95% CI, 1.08-1.83], P = .01). All-cause death before 55 weeks' postmenstrual age occurred in 18% of the myo-inositol group and in 11% of the placebo group (adjusted risk difference, 6% [95% CI, 0%-11%]; adjusted relative risk, 1.66 [95% CI, 1.14-2.43], P = .007). The most common serious adverse events up to 7 days of receiving the ending dose were necrotizing enterocolitis (6% for myo-inositol vs 4% for placebo), poor perfusion or hypotension (7% vs 4%, respectively), intraventricular hemorrhage (10% vs 9%), systemic infection (16% vs 11%), and respiratory distress (15% vs 13%). Conclusions and Relevance: Among premature infants younger than 28 weeks' gestational age, treatment with myo-inositol for up to 10 weeks did not reduce the risk of type 1 ROP or death vs placebo. These findings do not support the use of myo-inositol among premature infants; however, the early termination of the trial limits definitive conclusions.

Quaking promotes monocyte differentiation into pro-atherogenic macrophages by controlling pre-mRNA splicing and gene expression.

A hallmark of inflammatory diseases is the excessive recruitment and influx of monocytes to sites of tissue damage and their ensuing differentiation into macrophages. Numerous stimuli are known to induce transcriptional changes associated with macrophage phenotype, but posttranscriptional control of human macrophage differentiation is less well understood. Here we show that expression levels of the RNA-binding protein Quaking (QKI) are low in monocytes and early human atherosclerotic lesions, but are abundant in macrophages of advanced plaques. Depletion of QKI protein impairs monocyte adhesion, migration, differentiation into macrophages and foam cell formation in vitro and in vivo. RNA-seq and microarray analysis of human monocyte and macrophage transcriptomes, including those of a unique QKI haploinsufficient patient, reveal striking changes in QKI-dependent messenger RNA levels and splicing of RNA transcripts. The biological importance of these transcripts and requirement for QKI during differentiation illustrates a central role for QKI in posttranscriptionally guiding macrophage identity and function.

Genetic analysis of the localization of APOBEC3F to human immunodeficiency virus type 1 virion cores.

UNLABELLED: Members of the APOBEC3 family of cytidine deaminases vary in their proportions of a virion-incorporated enzyme that is localized to mature retrovirus cores. We reported previously that APOBEC3F (A3F) was highly localized into mature human immunodeficiency virus type 1 (HIV-1) cores and identified that L306 in the C-terminal cytidine deaminase (CD) domain contributed to its core localization (C. Song, L. Sutton, M. Johnson, R. D'Aquila, J. Donahue, J Biol Chem 287:16965-16974, 2012, http://dx.doi.org/10.1074/jbc.M111.310839). We have now determined an additional genetic determinant(s) for A3F localization to HIV-1 cores. We found that one pair of leucines in each of A3F's C-terminal and N-terminal CD domains jointly determined the degree of localization of A3F into HIV-1 virion cores. These are A3F L306/L368 (C-terminal domain) and A3F L122/L184 (N-terminal domain). Alterations to one of these specific leucine residues in either of the two A3F CD domains (A3F L368A, L122A, and L184A) decreased core localization and diminished HIV restriction without changing virion packaging. Furthermore, double mutants in these leucine residues in each of A3F's two CD domains (A3F L368A plus L184A or A3F L368A plus L122A) still were packaged into virions but completely lost core localization and anti-HIV activity. HIV virion core localization of A3F is genetically separable from its virion packaging, and anti-HIV activity requires some core localization. IMPORTANCE: Specific leucine-leucine interactions are identified as necessary for A3F's core localization and anti-HIV activity but not for its packaging into virions. Understanding these signals may lead to novel strategies to enhance core localization that may augment effects of A3F against HIV and perhaps of other A3s against retroviruses, parvoviruses, and hepatitis B virus.

Signals in APOBEC3F N-terminal and C-terminal deaminase domains each contribute to encapsidation in HIV-1 virions and are both required for HIV-1 restriction.

Human cytidine deaminases APOBEC3F (A3F) and APOBEC3G (A3G) inhibit human immunodeficiency virus type-1 (HIV-1) replication. In the absence of HIV-1 Vif, A3F and/or A3G are incorporated into assembling virions and exert antiviral functions in subsequently infected target cells. Encapsidation of A3F or A3G within the protease-matured virion core following their incorporation into virions is hypothesized to be important for the antiviral function of these proteins. In this report, we demonstrated that A3F was quantitatively encapsidated in the mature virion core. In distinct contrast, A3G was distributed both within and outside of the virion core. Analysis of a series of A3F-A3G chimeras comprised of exchanged N- and C-terminal deaminase domains identified a 14 amino acid segment in the A3F C-terminal deaminase domain that contributed to preferential encapsidation and anti-HIV activity. Amino acid residue L306 in this C-terminal segment was determined to be necessary, but not sufficient, for these effects. Amino acid residue W126 in the N-terminal deaminase domain was determined also to contribute to preferential encapsidation and antiviral activity of A3F. Analysis of the A3F (W126A L306A) double mutant revealed that both residues are required for full anti-HIV function. The results reported here advance our understanding of the mechanisms of A3F virion encapsidation and antiviral function and may lead to innovative strategies to inhibit HIV-1 replication.

Oral cyclosporin A inhibits CD4 T cell P-glycoprotein activity in HIV-infected adults initiating treatment with nucleoside reverse transcriptase inhibitors.

PURPOSE: P-glycoprotein limits the tissue penetration of many antiretroviral drugs. The aim of our study was to characterize the effects of the P-glycoprotein substrate cyclosporin A on T cell P-glycoprotein activity in human immunodeficiency virus-infected participants in the AIDS Clinical Trials Group study A5138. METHODS: We studied P-glycoprotein activity on CD4 and CD8 T cells in 16 participants randomized to receive oral cyclosporin A (n=9) or not (n=7) during initiation antiretroviral therapy (ART) that did not include protease or non-nucleoside reverse transcriptase inhibitors. RESULTS: CD4 T cell P-glycoprotein activity decreased by a median of 8 percentage points with cyclosporin A/ART (difference between cyclosporin A/ART vs. ART only, P= 0.001). Plasma trough cyclosporin A concentrations correlated with the change in P-glycoprotein activity in several T cell subsets. CONCLUSIONS: Oral cyclosporin A can inhibit peripheral blood CD4 T cell P-glycoprotein activity. Targeted P-glycoprotein inhibition may enhance the delivery of ART to T cells.

Tariquidar, a selective P-glycoprotein inhibitor, does not potentiate loperamide's opioid brain effects in humans despite full inhibition of lymphocyte P-glycoprotein.

BACKGROUND: Loperamide, a potent opioid, has been used as an in vivo probe to assess P-glycoprotein activity at the blood-brain barrier, because P-glycoprotein inhibition allows loperamide to cross the blood-brain barrier and exert its central opioid effects. In humans, studies with nonselective and moderately potent inhibitors resulted in mild opioid effects but were confounded by the concurrent inhibition of loperamide's metabolism. The authors studied the effect of the highly selective, potent P-glycoprotein inhibitor tariquidar on loperamide's central opioid effects. METHODS: In a randomized, double-blind, crossover study, nine healthy subjects received on 2 study days oral loperamide (32 mg) followed by an intravenous infusion of either tariquidar (150 mg) or placebo. Central opioid effects (pupil diameter, sedation) were measured for 12 h, and blood samples were drawn up to 48 h after drug administration to determine plasma loperamide concentrations and ex vivo P-glycoprotein activity in T lymphocytes. Values for pupil diameter and loperamide concentrations were plotted over time, and the areas under the curves on the tariquidar and placebo study day were compared within each subject. RESULTS: Tariquidar did not significantly affect loperamide's central effects (median reduction in pupil diameter area under the curve, 6.9% [interquartile range, -1.4 to 12.1%]; P = 0.11) or plasma loperamide concentrations (P = 0.12) but profoundly inhibited P-glycoprotein in lymphocytes by 93.7% (95% confidence interval, 92.0-95.3%). CONCLUSION: These results suggest that despite full inhibition of lymphocyte P-glycoprotein, the selective P-glycoprotein inhibitor tariquidar does not potentiate loperamide's opioid brain effects in humans.

The HIV-1 Vif PPLP motif is necessary for human APOBEC3G binding and degradation.

The HIV-1 virion infectivity factor (Vif) is required during viral replication to inactivate the host cell anti-viral factor, APOBEC3G (A3G). Vif binds A3G and a Cullin5-ElonginBC E3 ubiquitin ligase complex which results in the proteasomal degradation of A3G. The Vif PPLP motif (amino acids 161-164) is essential for normal Vif function because mutations in this motif reduce the infectivity of virions produced in T-cells. In this report, we demonstrate that mutation of the Vif PPLP motif reduces Vif binding to A3G without affecting its interaction with ElonginC and Cullin5. We demonstrate that the failure of the Vif mutant to bind A3G resulted in A3G incorporation into assembling virions with loss of viral infectivity.

Profile of the retina by optical coherence tomography in the pediatric age group.

PURPOSE: To establish normative values of the retina in the pediatric population using optical coherence tomography (OCT). DESIGN: Prospective observational case control study. METHODS: Prospective study examining macular thickness and nerve fiber layer thickness in children with no ocular disease. After clinical examination, patients meeting the inclusion and exclusion criteria underwent OCT scanning. RESULTS: Thirty-two eyes were examined for macular thickness and 25 eyes for nerve fiber layer thickness. Normative values are found in the Table. The average foveal thickness for children is 221 microns vs 182 microns in adults. CONCLUSION: This study demonstrates normative values of retinal thickness and retinal nerve fiber layer (RNFL) thickness in the pediatric age group. Children have slightly thicker maculas than adults; the RNFL thickness is comparable to adults.

Multilocus genetic interactions and response to efavirenz-containing regimens: an adult AIDS clinical trials group study.

OBJECTIVE: For the HIV-1 reverse transcriptase inhibitor efavirenz, variant drug transporter gene ABCB1 may predict virologic response but not plasma efavirenz exposure. Conversely, variant drug metabolizing enzyme gene CYP2B6 predicts greater plasma efavirenz exposure but not virologic response. We examined whether long-term responses to efavirenz, and/or plasma efavirenz exposure, are better predicted by multilocus genetic interactions than by individual polymorphisms. MATERIALS AND METHODS: We studied antiretroviral-naïve study participants randomized to receive efavirenz (with or without nelfinavir) plus two nucleoside analogues in study ACTG 384, and who had DNA available for analysis. Participants were followed up for up to 3 years. Nine single nucleotide polymorphisms in ABCB1, CYP2B6, CYP3A4, CYP3A5 and CYP2C19 were identified. Gene-gene interactions were identified using multifactor dimensionality reduction. RESULTS: Among 340 efavirenz recipients, higher efavirenz AUC24 h values were associated with a single locus model involving CYP2B6 516G>T (73% accuracy; P<0.001). This was also the best model among blacks (69% accuracy; P<0.001), whereas among whites the best model involved a gene-gene interaction between CYP2B6 516G>T and ABCB1 2677G>T (82% accuracy, P<0.001). Among 155 participants who received efavirenz without nelfinavir, virologic failure was associated with a two-locus interaction between ABCB1 2677G>T and CYP2B6 516G>T (65% accuracy, P<0.001). Toxicity failure was best predicted by an interaction between ABCB1 2677G>T and ABCB1 3435C>T (71% accuracy, P<0.001). CONCLUSIONS: Multilocus genetic interactions between variant drug metabolism and transporter genes may predict efavirenz pharmacokinetics and treatment responses. This finding may have implications for better individualizing antiretroviral therapy.

Drug transporter and metabolizing enzyme gene variants and nonnucleoside reverse-transcriptase inhibitor hepatotoxicity.

This nested case-control study examined relationships between MDR1, CYP2B6, and CYP3A4 variants and hepatotoxicity during antiretroviral therapy with either efavirenz- or nevirapine-containing regimens. Decreased risk of hepatotoxicity was associated with MDR1 3435C-->T (odds ratio, 0.254; P=.021). An interaction between MDR1 and hepatitis B surface antigen status predicted risk with 82% accuracy (P<.001).

Pharmacogenetics of long-term responses to antiretroviral regimens containing Efavirenz and/or Nelfinavir: an Adult Aids Clinical Trials Group Study.

BACKGROUND: Efavirenz and nelfinavir are metabolized by cytochrome P-450 (CYP) 2B6 and CYP2C19, respectively, with some involvement by CYP3A. Nelfinavir is a substrate for P-glycoprotein, which is encoded by MDR1. The present study examined associations between genetic variants and long-term responses to treatment. METHODS: Adult AIDS Clinical Trials Group study 384 randomized antiretroviral-naive subjects to receive efavirenz and/or nelfinavir plus 2 nucleoside analogues, with follow-up lasting up to 3 years. Population pharmacokinetics were estimated from a nonlinear mixed-effects model. Polymorphisms in CYP2B6, CYP2C19, CYP3A4, CYP3A5, and MDR1 were characterized. RESULTS: The 504 participants in the genetic study included 340 efavirenz recipients and 348 nelfinavir recipients (184 of the 504 participants received both efavirenz and nelfinavir). Of the participants, 49% were white, 31% were black, and 19% were Hispanic. Plasma exposure to efavirenz and nelfinavir in each population was significantly associated with the polymorphisms CYP2B6 516G-->T and CYP2C19 681G-->A, respectively. Among efavirenz recipients, the MDR1 position 3435 TT genotype was associated with decreased likelihood of virologic failure and decreased emergence of efavirenz-resistant virus but not with plasma efavirenz exposure. Among nelfinavir recipients, a trend toward decreased virologic failure was associated with the polymorphism CYP2C19 681G-->A. CONCLUSIONS: Genetic variants predict plasma exposure to efavirenz and nelfinavir, and they may predict virologic failure and/or emergence of drug-resistant virus. These associations with treatment responses must be validated in other studies.

The diagnostic concordance of actinic keratosis and squamous cell carcinoma.

Diagnostic concordance of intraepithelial malignancy is generally only fair. Because the diagnosis of actinic keratosis (AK) and squamous cell carcinoma (SCC) is not uniform and because such terms are not consonant with the nomenclature of other human epithelial malignancies, nomenclature revisions have been attempted. One hundred dermatopathologists were solicited to review 15 tissue sections representing a spectrum of varying thickness epidermal malignancy and to choose either AK or SCC as the diagnosis. Among the 77 participating dermatopathologists, intraclass correlation was high for what was perceived as AK, SCC, and their differentiation. Development of a two-tiered diagnostic system that retains our present diagnostic capabilities, but better fits the pathobiology of superficial epidermal malignancy is suggested. Davis DA, Donahue JP, Bost JE, Horn TD. The diagnostic concordance of actinic keratosis and squamous cell carcinoma.

Implications of T-cell P-glycoprotein activity during HIV-1 infection and its therapy.

OBJECTIVES: P-glycoprotein (P-gp) may reduce antiretroviral efficacy by decreasing disposition of HIV-1 protease inhibitors into tissues and cells. In contrast, P-gp overexpression in vitro can inhibit HIV-1 replication, and some drugs induce P-gp expression. To explore which of these mechanisms predominate in vivo, this study characterized relationships between T-cell P-gp activity and clinical parameters in HIV-infected adults. METHODS: P-gp activity was quantified in total and naive CD4+ and CD8+ T cells of HIV-infected adults by flow cytometry using the substrate dye DiOC2(3). Demographic, virologic, immunologic, and treatment factors were obtained from medical records. Factors associated with P-gp activity were identified using multivariate linear regression. RESULTS: A total of 185 subjects (22% female; 34% African American) were studied, of whom 131 (71%) were receiving antiretroviral treatment. There was marked interindividual variability in P-gp activity. By multivariate analysis, higher CD4+ T-cell P-gp activity was associated with lower log10 HIV-1 RNA (P = 0.005), but not treatment or demographic factors. P-gp activity was correlated across T-cell subsets. CONCLUSIONS: The inverse relationship between P-gp activity and plasma HIV-1 RNA is most consistent with an inhibitory effect on viral replication rather than drug disposition. Antiretroviral drug class did not independently predict P-gp activity.

Effects of nelfinavir and its M8 metabolite on lymphocyte P-glycoprotein activity during antiretroviral therapy.

The efflux pump P-glycoprotein decreases drug penetration into cells and tissues. To determine whether nelfinavir or its metabolites inhibit P-glycoprotein in lymphocytes from a healthy volunteer, whole blood cells from human immunodeficiency virus-negative donors were incubated either in human plasma to which nelfinavir or its M8 metabolite were added ex vivo or in plasma from human immunodeficiency virus-positive patients receiving nelfinavir. The 50% P-glycoprotein inhibitory concentrations of purified nelfinavir and M8 were 10.9 micromol/L and 29.5 micromol/L, respectively, for CD4(+) T cells and 19.3 micromol/L and >48 micromol/L, respectively, for CD8(+) T cells. Significant inhibitory activity was present in plasma from 27 of 46 patients (59%) receiving nelfinavir. Plasma nelfinavir concentrations correlated with percent inhibition on CD4(+) (rho = 0.85, P <.0001) and CD8(+) (rho = 0.83, P <.0001) T cells. The M8 concentrations correlated weakly with both inhibition and nelfinavir concentrations. On the basis of our findings in lymphocytes from a healthy volunteer exposed to plasma from human immunodeficiency virus-positive patients, we believe it is likely that CD4(+) and CD8(+) lymphocytes in patients receiving nelfinavir as therapy for human immunodeficiency virus may have P-glycoprotein inhibited by plasma concentrations of nelfinavir.

Characterization of expression of a functionally conserved Helicobacter pylori methyltransferase-encoding gene within inflamed mucosa and during in vitro growth.

Methylation of bacterial DNA can regulate microbial growth and virulence. Expression of hpyIM, a conserved methyltransferase of the gastric pathogen Helicobacter pylori, was quantitated in gastric biopsy specimens from 41 H. pylori-infected patients and during growth in vitro, by quantitative reverse transcriptase-polymerase chain reaction and/or RNA slot-blot analysis, to determine whether levels of transcription were associated with pathologic outcome, as based on both severity of gastritis and inflammatory cytokine levels, or were regulated by bacterial growth phase. The effects that hpyIM inactivation has on bacterial morphology were determined by electron microscopy. Expression of hpyIM varied dramatically within colonized gastric tissue, and levels were not related to either colonization density, severity of inflammation, mucosal IL-8 concentrations, or clinical disease. In vitro, hpyIM expression was higher during log-phase growth and was required for normal bacterial morphology, suggesting that hpyIM expression may be growth-phase regulated within the gastric niche.

Inactivation of a Helicobacter pylori DNA methyltransferase alters dnaK operon expression following host-cell adherence.

The Helicobacter pylori hpyIM gene encodes a type II DNA methyltransferase that is highly conserved among strains. To investigate the potential role of M.HpyI methyltransferase activity in controlling gene expression in H. pylori, we analyzed gene transcription profiles in wild-type strain J166 and an isogenic hpyIM mutant strain using gene arrays. This analysis showed that the expression of a majority of genes was unaffected by hpyIM mutation, especially in exponential phase cultures. However, in stationary phase cultures and in cells adherent to AGS gastric epithelial cells in vitro, loss of hpyIM function altered the expression of the stress-responsive dnaK operon. Complementation of the hpyIM mutation using a shuttle plasmid encoding a wild-type copy of the gene re-established the wild-type pattern of dnaK operon expression. These data suggested that hpyIM, encoding a DNA methyltransferase, may have a role in H. pylori physiology that supersedes its original function in a type II restriction-modification system.