Does endoscopic balloon dilatation improve intranasal drug delivery to the olfactory cleft? A pre-clinical cadaver feasibility study.

BACKGROUND: Although systemic steroids have been shown to improve olfactory function, topical steroids have not demonstrated the same efficacy. This could a result of limited drug delivery to the narrow, superiorly placed olfactory cleft. This study aimed to examine the penetration of intranasal drugs to the olfactory cleft following endonasal balloon dilatation. METHODS: Balloon dilatation was performed in 12 thawed, fresh-frozen cadaver specimens. In the Mygind position, nasal drops mixed with blue food dye were administered into the nostril before and after the dilatation procedure. Endoscopic videos were recorded to assess dye staining of the olfactory cleft and osteo-meatal complex using a 4-point Likert scale. RESULTS: Prior to balloon dilatation, the mean penetration of nasal drops into the olfactory cleft was 1.34, which improved significantly (p < 0.05) to 2.66 following the procedure. There was no change in dye penetration into the osteo-meatal complex after balloon dilatation. CONCLUSION: The results of this exploratory study suggest that balloon dilatation may improve the delivery of nasal drops to the olfactory cleft area. The clinical applicability and impact on olfactory function will require further assessment.

Spectroscopic and Structural Characterization of Water-Shared Ion-Pairs in Aqueous Sodium and Lithium Hydroxide.

The structures of the ion-pairs formed in aqueous NaOH and LiOH solutions are elucidated by combining Raman multivariate curve resolution (Raman-MCR) experiments and ab initio molecular dynamics (AIMD) simulations. The results extend prior findings to reveal that the initially formed ion-pairs are predominantly water-shared, with the hydroxide ion retaining its full first hydration-shell, while direct contact ion-pairing only becomes significant at higher concentrations. Our results confirm previous experiments and simulations indicating greater ion-pairing in aqueous LiOH than NaOH as well as at high temperatures. Our results further imply that NaOH and LiOH ion-pairing free energies have an approximately linear (rather than square-root) dependence on ion concentration (in the molar range), with positive enthalpies and entropies that increase with concentration, thus implying that water-mediated interactions enthalpically disfavor and entropically favor ion-pair formation.

Genotype-phenotype correlation in a large English cohort of patients with autosomal recessive ichthyosis.

BACKGROUND: Recessive forms of congenital ichthyosis encompass a group of rare inherited disorders of keratinization leading to dry, scaly skin. So far, 13 genes have been implicated, but there is a paucity of data on genotype-phenotype correlation in some populations. OBJECTIVES: We compiled an English cohort of 146 individuals with recessive ichthyosis and assessed genotype-phenotype correlation. METHODS: Deep phenotyping was undertaken by history-taking and clinical examination. DNA was screened for mutations using a next-generation sequencing ichthyosis gene panel and Sanger sequencing. RESULTS: Cases were recruited from 13 National Health Service sites in England, with 65% of patients aged < 16 years at enrolment. Pathogenic biallelic mutations were found in 83% of cases, with the candidate gene spread as follows: TGM1 29%, NIPAL4 12%, ABCA12 12%, ALOX12B 9%, ALOXE3 7%, SLC27A4 5%, CERS3 3%, CYP4F22 3%, PNPLA1 2%, SDR9C7 1%. Clinically, a new sign, an anteriorly overfolded ear at birth, was noted in 43% of patients with ALOX12B mutations. The need for intensive care stay (P = 0·004), and hand deformities (P < 0·001), were associated with ABCA12 mutations. Self-improving collodion ichthyosis occurred in 8% of the cases (mostly TGM1 and ALOX12B mutations) but could not be predicted precisely from neonatal phenotype or genotype. CONCLUSIONS: These data refine genotype-phenotype correlation for recessive forms of ichthyosis in England, demonstrating the spectrum of disease features and comorbidities, as well as the gene pathologies therein. Collectively, the data from these patients provide a valuable resource for further clinical assessment, improving clinical care and the possibility of future stratified management. What's already known about this topic? Recessive forms of ichthyosis are rare but often difficult to diagnose. Mutations in 13 genes are known to cause recessive forms of ichthyosis: ABCA12, ALOX12B, ALOXE3, CERS3, CYP4F22, LIPN, NIPAL4, PNPLA1, SDR9C7, SLC27A4, SULT2B1, ST14 and TGM1. Some phenotypic features may associate with certain gene mutations, but paradigms for genotype-phenotype correlation need refining. What does this study add? The genotypic spectrum of recessive ichthyosis in England (based on 146 cases) comprises TGM1 (29%), NIPAL4 (12%), ABCA12 (12%), ALOX12B (9%), ALOXE3 (7%), SLC27A4 (5%), CERS3 (3%), CYP4F22 (3%), PNPLA1 (2%) and SDR9C7 (1%). New or particular phenotypic clues were defined for mutations in ALOX12B, ABCA12, CYP4F22, NIPAL4, SDR9C7 and TGM1, either in neonates or in later life, which allow for greater diagnostic precision. In around 17% of cases, the molecular basis of recessive ichthyosis remains unknown.

Dynamics and Vibrational Spectroscopy of Alcohols in Ionic Liquids: Methanol and Ethanol.

The structure, dynamics, and vibrational spectroscopy of dilute HOD, methanol, and ethanol in the 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, [emim][NTf2], ionic liquid (IL) are investigated with molecular dynamics (MD) simulations. The structure of the ILs around the solutes is qualitatively similar, where the OD bond of the deuterated alcohols donates an interaction to an [NTf2] anion and the [emim] cations interact with the oxygen atom of the OD group. The slowest time scale for the reorientational dynamics of the OD bond varied considerably for HOD, methanol, and ethanol (27, 71, and 87 ps, respectively). In contrast, the slowest time scales for spectral diffusion of the OD vibrational frequency were 11 ps for each of the three solutes, which indicates that the dynamics of the IL is relatively unchanged by the presence of the alcohols at dilute concentration. The theoretical results for the reorientational and spectral diffusion dynamics compare favorably with prior two-dimensional infrared (2D IR) spectroscopic measurements.

Slow Acceptance of Universal Antiretroviral Therapy (ART) Among Mothers Enrolled in IMPAACT PROMISE Studies Across the Globe.

The PROMISE trial enrolled asymptomatic HIV-infected pregnant and postpartum women not eligible for antiretroviral treatment (ART) per local guidelines and randomly assigned proven antiretroviral strategies to assess relative efficacy for perinatal prevention plus maternal/infant safety and maternal health. The START study subsequently demonstrated clear benefit in initiating ART regardless of CD4 count. Active PROMISE participants were informed of results and women not receiving ART were strongly recommended to immediately initiate treatment to optimize their own health. We recorded their decision and the primary reason given for accepting or rejecting the universal ART offer after receiving the START information. One-third of participants did not initiate ART after the initial session, wanting more time to consider. Six sessions were required to attain 95% uptake. The slow uptake of universal ART highlights the need to prepare individuals and sensitize communities regarding the personal and population benefits of the "Treat All" strategy.

Counter Cations Affect Transport in Aqueous Hydroxide Solutions with Ion Specificity.

The anomalously high mobility of hydroxide and hydronium ions in aqueous solutions is related to proton transfer and structural diffusion. The role of counterions in these solutions, however, is often considered to be negligible. Herein, we explore the impact of alkali metal counter cations on hydroxide solvation and mobility. Impedance measurements demonstrate that hydroxide mobility is attenuated by lithium relative to sodium and potassium. These results are explained by ab initio molecular dynamics simulations and experimental vibrational hydration shell spectroscopy, which reveal substantially stronger ion pairing between OH- and Li+ than with other cations. Hydration shell spectra and theoretical vibrational frequency calculations together imply that lithium and sodium cations have different effects on the delocalization of water protons donating a hydrogen bond to hydroxide. Specifically, lithium leads to enhanced proton delocalization compared with sodium. However, proton delocalization and the overall diffusion process are not necessarily correlated.

Circulating 25-hydroxyvitamin D, nasopharyngeal airway metabolome, and bronchiolitis severity.

Low circulating 25-hydroxyvitamin D (25OHD) levels are a risk factor for acute respiratory infection (eg, bronchiolitis) in children. However, little is known about the relation of circulating 25OHD with the many downstream functional molecules in target organs-such as the airway-and with clinical outcomes. In this prospective multicenter study of infants (age <1 year) hospitalized with bronchiolitis, we measured serum 25OHD levels and profiled the metabolome of 144 nasopharyngeal airway samples. Among 254 metabolites identified, we defined a set of 20 metabolites that are related to lower serum 25OHD and higher vitamin D-binding protein levels. Of these metabolites, 9 metabolites were associated with a significantly higher risk of positive pressure ventilation use. These metabolites were glycerophosphocholines esterified with proinflammatory fatty acids (palmitate, arachidonate, linoleate, and stearate), sphingomyelins, alpha-hydroxyisovalerate, 2-hydroxybutyrate, and 3-(4-hydroxyphenyl)lactate (all FDR<0.05). Based on the multicenter data, vitamin D-related airway metabolites were associated with risks of bronchiolitis severity.

Molecular Autopsy for Sudden Death in the Young: Is Data Aggregation the Key?

The Scripps molecular autopsy study seeks to incorporate genetic testing into the postmortem examination of cases of sudden death in the young (<45 years old). Here, we describe the results from the first 2 years of the study, which consisted of whole exome sequencing (WES) of a cohort of 50 cases predominantly from San Diego County. Apart from the individual description of cases, we analyzed the data at the cohort-level, which brought new perspectives on the genetic causes of sudden death. We investigated the advantages and disadvantages of using WES compared to a gene panel for cardiac disease (usually the first genetic test used by medical examiners). In an attempt to connect complex clinical phenotypes with genotypes, we classified samples by their genetic fingerprint. Finally, we studied the benefits of analyzing the mitochondrial DNA genome. In this regard, we found that half of the cases clinically diagnosed as sudden infant death syndrome had an increased ratio of heteroplasmic variants, and that the variants were also present in the mothers. We believe that community-based data aggregation and sharing will eventually lead to an improved classification of variants. Allele frequencies for the all cases can be accessed via our genomics browser at https://genomics.scripps.edu/browser.

Corrigendum: Molecular Autopsy for Sudden Death in the Young: Is Data Aggregation the Key?

[This corrects the article on p. 72 in vol. 4, PMID: 29181379.].

Direct visualization of shear waves in viscoelastic fluid using microspheres.

Wormlike micellar fluids, being viscoelastic, support shear waves. Shear waves in 500 mM CTAB-NaSal micellar fluid were visualized by seeding the fluid with 212-250 µm diameter polyethylene microspheres. This method was compared to visualization through birefringence induced by shear stress in the fluid. Measured shear wave speeds were 733 and 722 mm/s, respectively, for each technique. Particle displacement was a sinusoidal function of time and displacement amplitude decreased quadratically with distance from the source. This supports the possibility of using particle amplitude measurements as a measure of attenuation even at low fluid concentration where birefringence visualization techniques fail.

Markers of renal disease and function are associated with systemic inflammation in HIV infection.

OBJECTIVES: Both renal disease and systemic inflammation predict non-AIDS-defining events and overall mortality in HIV-infected patients. Here, we sought to determine the relationships between renal disease and circulating inflammation markers. METHODS: We performed a secondary analysis of AIDS Clinical Trials Group Study A5224s to determine if markers of renal disease [urine protein:creatinine ratio (uPCR), urine albumin:creatinine ratio (uACR), and estimated glomerular filtration rate (eGFR), using Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) creatinine and cystatin C-creatinine] were associated with markers of systemic inflammation [high-sensitivity C-reactive protein, interleukin-6, tumour necrosis factor (TNF)-α, soluble TNF-α receptor I (sTNFRI), sTNFRII, and soluble vascular cellular and intercellular adhesion molecules]. We correlated these renal and inflammatory markers prior to antiretroviral initiation and after 96 weeks of therapy. RESULTS: We found that eGFR (estimated using CKD-EPI cystatin C-creatinine), uPCR, and uACR were significantly correlated with most assessed markers of systemic inflammation prior to antiretroviral initiation. uPCR and eGFR (using CKD-EPI cystatin C-creatinine), but not uACR, remained significantly correlated with most of the assessed inflammatory markers after 96 weeks of antiretroviral therapy (ART). Most of these correlations, although statistically significant, were < 0.50. eGFR using CKD-EPI creatinine was much less frequently associated with inflammation markers and only significantly correlated with sTNFR1 at week 0 and with sTNFRI and II at week 96. CONCLUSIONS: Renal disease and function were associated with systemic inflammation in HIV infection, both before and after ART. Systemic inflammation may partially explain the relationships between proteinuria, albuminuria, and reduced renal function and future adverse outcomes.

Cytomegalovirus (CMV) and human immunodeficiency virus (HIV) burden, CMV end-organ disease, and survival in subjects with advanced HIV infection (AIDS Clinical Trials Group Protocol 360).

We undertook a prospective study to analyze cytomegalovirus (CMV) end-organ disease (EOD) in subjects with advanced human immunodeficiency virus (HIV) infection. Of 403 individuals without prior CMV EOD who were followed up for a median of 151 weeks, 56 died and 21 developed CMV EOD. Twenty of the subjects with CMV EOD had CD4 cell counts of < or =50 cells/mm3 and HIV RNA level of >10,000 copies/mL of plasma at baseline; in these 20 subjects, an increase of CMV DNA level to greater than the quantification limits was associated with CMV EOD. A CD4 cell count of < or =100 cells/mm3 and an HIV RNA level of >10,000 copies/mL of plasma at baseline, a CMV DNA level of >200 copies/mL of blood during follow-up, or development of CMV EOD were all associated with decreased survival. HIV-infected subjects with CD4 cell counts of < or =50 cells/mm3 and HIV RNA levels of >10,000 copies/mL of plasma should have blood fractions screened for CMV DNA; if CMV DNA is detected, CMV prophylaxis might be considered.

Associations of CCR5, CCR2, and stromal cell-derived factor 1 genotypes with human immunodeficiency virus disease progression in patients receiving nucleoside therapy.

Genotype data for CCR5, CCR2, and stromal cell-derived factor 1 (SDF-1) were obtained from 354 human immunodeficiency virus type 1 (HIV-1)-positive subjects who were being treated with nucleosides. Associations with HIV-1 load, HIV syncytium-inducing (SI) phenotype, CD4 cell count, and disease progression were analyzed. No differences in HIV-1 load or CD4 cell count were observed between wild type (+) and variant genotypes. Changes from non-SI to SI viral phenotype were more frequent in heterozygotes with a 32-bp deletion (Delta32) in the CCR5 gene than in + homozygotes (40% vs. 7%; P=.01). In a multivariate analysis, heterozygous CCR5 Delta32 was associated with reduced hazard of progression (hazard ratio, 0.32; P=.02). Subjects homozygous for the SDF-1 3'A variant had more-rapid disease progression (P=.008). The SDF-1 homozygous 3'A variant was related to more-rapid disease progression, and CCR5 Delta32 was associated with reduced rates of hazard for disease progression in nucleoside-treated subjects.

Considerations in choosing a primary endpoint that measures durability of virological suppression in an antiretroviral trial.

OBJECTIVES: At present, many clinical trials of anti-HIV-1 therapies compare treatments by a primary endpoint that measures the durability of suppression of HIV-1 replication. Several durability endpoints are compared. DESIGN: Endpoints are compared by their implicit assumptions regarding surrogacy for clinical outcomes, sample size requirements, and accommodations for inter-patient differences in baseline plasma HIV-1-RNA levels and in initial treatment response. METHODS: Virological failure is defined by the non-suppression of virus levels at a prespecified follow-up time T(early virological failure), or by relapse. A binary virological failure endpoint is compared with three time-to-virological failure endpoints: time from (i) randomization that assigns early failures a failure time of T weeks; (ii) randomization that extends the early failure time T for slowly responding subjects; and (iii) virological response that assigns non-responders a failure time of 0 weeks. Endpoint differences are illustrated with Agouron's trial 511. RESULTS: In comparing high with low-dose nelfinavir (NFV) regimens in Agouron 511, the difference in Kaplan-Meier estimates of the proportion not failing by 24 weeks is 16.7% (P = 0.048), 6.5% (P = 0.29) and 22.9% (P = 0.0030) for endpoints (i), (ii) and (iii), respectively. The results differ because NFV suppresses virus more quickly at the higher dose, and the endpoints weigh this treatment difference differently. This illustrates that careful consideration needs to be given to choosing a primary endpoint that will detect treatment differences of interest. CONCLUSION: A time from randomization endpoint is usually recommended because of its advantages in flexibility and sample size, especially at interim analyses, and for its interpretation for patient management.

In vivo antagonism with zidovudine plus stavudine combination therapy.

Human immunodeficiency virus (HIV)-infected subjects receiving zidovudine were randomized either to add stavudine (d4T) or didanosine (ddI) to their current regimen or to switch to ddI or d4T monotherapy. After 16 weeks of therapy, the mean reduction in HIV RNA from baseline was 0.14 log(10) copies/mL in patients receiving d4T or zidovudine plus d4T. In subjects receiving ddI or ddI plus zidovudine, reductions were 0.39 and 0.56 log(10), respectively. CD4 cell counts remained stable or showed modest increases in all arms except the zidovudine plus d4T arm. Patients receiving zidovudine plus d4T showed progressive declines in CD4 cell counts with a median of 22 cells/mm(3) below baseline by 16 weeks. Examination of intracellular levels of d4T-triphosphate in 6 subjects was consistent with previous in vitro studies demonstrating pharmacologic antagonism between zidovudine and d4T. Analysis of these data suggests that zidovudine and d4T should not be prescribed in combination and that ddI provides greater antiviral activity than d4T in zidovudine-treated patients.

A randomized, controlled, double-blind study comparing the survival benefit of four different reverse transcriptase inhibitor therapies (three-drug, two-drug, and alternating drug) for the treatment of advanced AIDS. AIDS Clinical Trial Group 193A Study Team.

OBJECTIVE: The primary objective was to compare the effects of dual or triple combinations of HIV-1 reverse transcriptase inhibitors with respect to survival. The time to new HIV disease progression or death, toxicities, the change in CD4 cells, and plasma HIV-1 RNA concentrations in a subset of study subjects were evaluated. DESIGN: This was a multicenter randomized, double-blind, placebo-controlled study. SETTING: The study was conducted among 42 adult AIDS Clinical Trials Group sites and 7 National Hemophilia Foundation centers. PATIENTS: 1313 HIV-infected patients with CD4 counts < or = 50 cells/mm3 participated in this study, which was conducted from June 1993 to June 1996. INTERVENTION: Patients were randomized to one of four daily regimens containing 600 mg of zidovudine: zidovudine alternating monthly with 400 mg didanosine; zidovudine plus 2.25 mg of zalcitabine; zidovudine plus 400 mg of didanosine; or zidovudine plus 400 mg of didanosine plus 400 mg of nevirapine (triple therapy). MAIN OUTCOME MEASURES: The main outcome was survival (i.e., time to death). RESULTS: A significant difference in survival time was found between the four treatment groups, favoring those assigned to triple therapy (p = .02). A significant difference was also found in the delay of disease progression or death among the four treatment arms favoring the group assigned to triple therapy (p = .002). Baseline CD4 cell counts and plasma HIV-1 RNA concentrations as well as changes of CD4 counts at week 8 predicted survival for subjects in the virology substudy. CONCLUSIONS: In the pre-protease inhibitor era, a combination of triple reverse transcriptase inhibitors prolonged life and delayed disease progression in AIDS patients with advanced immune suppression.

Likelihood-based inference for the genetic relative risk based on affected-sibling-pair marker data.

Using genetic marker data from affected sibling pairs, we study likelihood-based linkage analysis under quasi-recessive, quasi-dominant, and general single-locus models. We use an epidemiologic parameterization under a model where the marker locus is closely linked to the putative disease susceptibility gene. This model and parameterization allow inferences about the relative risk associated with the susceptible genotype. We base inferences on approximate likelihoods that focus on the affected siblings in the sibship and, using these likelihoods, we derive closed-form maximum likelihood estimators for model parameters and closed-form likelihood ratio statistics for tests that the relative risk associated with the susceptible genotype is one. Under the general single-locus model, our likelihood ratio test is the same as the iteratively computed triangle test proposed by Holmans (1993, American Journal of Human Genetics 52, 362-374) for the case where marker identity-by-descent is known; our derivation gives a closed form for the test statistic. We present quartiles of the distribution of parameter estimates and critical values for the exact null distribution of our likelihood ratio test statistics; we also give large-sample approximations to their null distributions. We show that the powers of our likelihood ratio tests exceed the powers of more commonly used nonparametric affected-sibling-pair tests when the data meet the inheritance model assumptions used to derive the test; we also show that our tests' powers are robust to violation of model assumptions. We conclude that our model-based inferences provide a practical alternative to more common affected-sibling-pair tests when investigators have some knowledge about the mode of inheritance of a disease and that our methods may sometimes be useful for comparing the genetic relative risk with environmental relative risks.

An examination of methods for sample size recalculation during an experiment.

In designing experiments, investigators frequently can specify an important effect that they wish to detect with high power, without the ability to provide an equally certain assessment of the variance of the response. If the experiment is designed based on a guess of the variance, an under-powered study may result. To remedy this problem, there have been several procedures proposed that obtain estimates of the variance from the data as they accrue and then recalculate the sample size accordingly. One class of procedures is fully sequential in that it assesses after each response whether the current sample size yields the desired power based on the current estimate of the variance. This approach is efficient, but it is not practical or advisable in many situations. Another class of procedures involves only two or three stages of sampling and recalculates the sample size based on the observed variance at designated times, perhaps coinciding with interim efficacy analyses. The two-stage approach can result in substantial oversampling, but it is feasible in many situations, whereas the three-stage approach corrects the problem of oversampling, but is less feasible. We propose a procedure that aims to combine the advantages of both the fully sequential and the two-stage approaches. This quasi-sequential procedure involves only two stages of sampling and it applies to the stopping rule from the fully sequential procedure to data beyond the initial sample which we obtain via multiple imputation. We show through simulations that when the initial sample size is substantially less than the correct sample size, the mean squared error of the final sample size calculated from the quasi-sequential procedure can be considerably less than that from the two-stage procedure. We compare the distributions of these recalculated sample sizes and discuss our findings for alternative procedures, as well.

A single morphogenetic field gives rise to two retina primordia under the influence of the prechordal plate.

Two bilaterally symmetric eyes arise from the anterior neural plate in vertebrate embryos. An interesting question is whether both eyes share a common developmental origin or they originate separately. We report here that the expression pattern of a new gene ET reveals that there is a single retina field which resolves into two separate primordia, a suggestion supported by the expression pattern of the Xenopus Pax-6 gene. Lineage tracing experiments demonstrate that retina field resolution is not due to migration of cells in the median region to the lateral parts of the field. Removal of the prechordal mesoderm led to formation of a single retina both in chick embryos and in Xenopus explants. Transplantation experiments in chick embryos indicate that the prechordal plate is able to suppress Pax-6 expression. Our results provide direct evidence for the existence of a single retina field, indicate that the retina field is resolved by suppression of retina formation in the median region of the field, and demonstrate that the prechordal plate plays a primary signaling role in retina field resolution.

Transcriptional repression by human adenovirus E1A N terminus/conserved domain 1 polypeptides in vivo and in vitro in the absence of protein synthesis.

The human adenovirus E1A 243R protein (243 residues) transcriptionally represses a set of cellular genes that regulate cellular growth and differentiation. We describe two lines of evidence that E1A repression does not require cellular protein synthesis but instead involves direct interaction with a cellular protein(s). First, E1A 243R protein represses an E1A-repressible promoter in the presence of inhibitors of protein synthesis, as shown by cell microinjection-in situ hybridization. Second, E1A 243R protein strongly represses transcription in vitro from promoters of the E1A-repressible genes, human collagenase, and rat insulin type II. Repression in vitro is promoter-specific, and an E1A polypeptide containing only the N-terminal 80 residues is sufficient for strong repression both in vivo and in vitro. By use of a series of E1A 1-80 deletion proteins, the E1A repression function was found to require two E1A sequence elements, one within the nonconserved E1A N terminus, and the second within a portion of conserved region 1 (40-80). These domains have been reported to possess binding sites for several cellular transcription regulators, including p300, Dr1, YY1, and the TBP subunit of TFIID. The in vitro transcription-repression system described here provides a powerful tool for the further analysis of molecular mechanism and the possible role of these cellular factors.