Volunteer Bias in Recruitment,Retention, and Blood Sample Donation in a Randomised Controlled Trial Involving Mothers and Their Children at Six Months and Two Years: A Longitudinal Analysis

Background

The vulnerability of clinical trials to volunteer bias is under-reported. Volunteer bias is systematic error due to differences between those who choose to participate in studies and those who do not.

Methods and Results

This paper extends the applications of the concept of volunteer bias by using data from a trial of probiotic supplementation for childhood atopy in healthy dyads to explore 1) differences between a) trial participants and aggregated data from publicly available databases b) participants and non-participants as the trial progressed 2) impact on trial findings of weighting data according to deprivation (Townsend) fifths in the sample and target populations. 1) a) Recruits (n = 454) were less deprived than the target population, matched for area of residence and delivery dates (n = 6,893) (mean [SD] deprivation scores 0.09[4.21] and 0.79[4.08], t = 3.44, df = 511, p<0.001). b) i)As the trial progressed, representation of the most deprived decreased. These participants and smokers were less likely to be retained at 6 months (n = 430[95%]) (OR 0.29,0.13–0.67 and 0.20,0.09–0.46), and 2 years (n = 380[84%]) (aOR 0.68,0.50–0.93 and 0.55,0.28–1.09), and consent to infant blood sample donation (n = 220[48%]) (aOR 0.72,0.57–0.92 and 0.43,0.22–0.83). ii)Mothers interested in probiotics or research or reporting infants’ adverse events or rashes were more likely to attend research clinics and consent to skin-prick testing. Mothers participating to help children were more likely to consent to infant blood sample donation. 2) In one trial outcome, atopic eczema, the intervention had a positive effect only in the over-represented, least deprived group. Here, data weighting attenuated risk reduction from 6.9%(0.9–13.1%) to 4.6%(−1.4–+10.5%), and OR from 0.40(0.18–0.91) to 0.56(0.26–1.21). Other findings were unchanged.

Conclusions

Potential for volunteer bias intensified during the trial, due to non-participation of the most deprived and smokers. However, these were not the only predictors of non-participation. Data weighting quantified volunteer bias and modified one important trial outcome.

Trial Registration

This randomised, double blind, parallel group, placebo controlled trial is registered with the International Standard Randomised Controlled Trials Register, Number (ISRCTN) 26287422. Registered title: Probiotics in the prevention of atopy in infants and children.     

A Genome-Wide Scan for Breast Cancer Risk Haplotypes among African American Women

Genome-wide association studies (GWAS) simultaneously investigating hundreds of thousands of single nucleotide polymorphisms (SNP) have become a powerful tool in the investigation of new disease susceptibility loci. Haplotypes are sometimes thought to be superior to SNPs and are promising in genetic association analyses. The application of genome-wide haplotype analysis, however, is hindered by the complexity of haplotypes themselves and sophistication in computation. We systematically analyzed the haplotype effects for breast cancer risk among 5,761 African American women (3,016 cases and 2,745 controls) using a sliding window approach on the genome-wide scale. Three regions on chromosomes 1, 4 and 18 exhibited moderate haplotype effects. Furthermore, among 21 breast cancer susceptibility loci previously established in European populations, 10p15 and 14q24 are likely to harbor novel haplotype effects. We also proposed a heuristic of determining the significance level and the effective number of independent tests by the permutation analysis on chromosome 22 data. It suggests that the effective number was approximately half of the total (7,794 out of 15,645), thus the half number could serve as a quick reference to evaluating genome-wide significance if a similar sliding window approach of haplotype analysis is adopted in similar populations using similar genotype density.     

Hydrophylita (Lutzimicron) emporos Shih & Polaszek (Hymenoptera: Trichogrammatidae) from Taiwan,Parasitising Eggs,and Phoretic on Adults,of the Damselfly Psolodesmus mandarinus mandarinus (Zygoptera: Calopterygidae)

Hydrophylita emporos n. sp. reared from eggs of Psolodesmus mandarinus mandarinus McLachlan (Zygoptera: Calopterygidae) in Taiwan is described. This is the first species of Hydrophylita to be described from the Old World, and the first record of phoresy in the genus. Adult females were observed aggregating at the base of the female damselfly’s abdomen. When the damselfly begins ovipositing, females move to the tip of the abdomen, enter the water and quickly locate eggs for parasitising. The article contains links to video footage of this process.     

Attenuated PLD1 association and signalling at the H452Y polymorphic form of the 5-HT2A receptor

The 5-HT_2A receptor (5-HT_2AR) is implicated in psychotropic changes within the central nervous system (CNS). A number of polymorphisms have been reported in the 5-HT_2AR gene; one of these results in a non-synonymous change, H452Y, in the carboxy-terminal tail of the receptor protein. The minor allele (9% occurrence) has been statistically associated with CNS dysfunction such as impaired memory processing and resistance to neuroleptic treatment in schizophrenic patients. We investigated the impact of H452Y mutation of the 5-HT_2AR expressed in COS7 cells on distinctly coupled intracellular signalling pathways from the receptor, focusing on the heterotrimeric G protein-independent phospholipase D (PLD) pathway, compared to the conventional Gq/11-linked phospholipase C (PLC) pathway. The H452Y mutation selectively attenuated PLD signalling, which as in the wild-type receptor, was mediated by a molecular complex involving PLD1 docked to the receptor's carboxy-terminal tail domain. Co-immunoprecipitation and GST-fusion protein experiments revealed that the H452Y mutation selectively reduced PLD1 binding to the receptor. Experiments with blocking peptides to mimic short sections of the 5-HT_2AR tail sequence revealed that the peptide spanning residue 452 strongly reduced PLD but not PLC responses of the receptor. Similar observations were made when assessing both PLD responses and PLD-dependent cellular proliferation elicited by activation of 5-HT_2ARs natively expressed in MCF-7 cells. Overall these findings indicate that the H452Y polymorphic variant of the 5-HT_2AR displays selective disruption of its PLD signalling pathway. This may potentially play a role in the CNS dysfunction associated with the H452Y allele of the 5-HT_2AR.     

Characterization of the Structure and Melting Behavior of the Loop I fragment of ColE1 RNA I

Abstract

We have synthesized two RNA fragments: a 42-mer corresponding to the full loop I sequence of the loop I region of ColE1 antisense RNA (RNA I), plus three additional Gs at the 5′-end, and a 31-mer which has 11 5′-end nucleotides (G(-2)-U9) deleted. The secondary structure of the 42-mer, deduced from one- and two-dimensional NMR spectra, consists of a stem of 11 base-pairs which contains a U-U base-pair and a bulged C base, a 7 nucleotide loop, and a single-stranded 5′ end of 12 nucleotides. The UV-melting study of the 42-mer further revealed a multi-step melting behavior with transition temperatures 32°C and 71°C clearly discernible. In conjunction with NMR melting study the major transition at 71°C is assigned to the overall melting of the stem region and the 32°C transition is assigned to the opening of the loop region. The deduced secondary structure agrees with that proposed for the intact RNA I and provides structural bases for understanding the specificity of RNase E.     

Activation of the Endoplasmic Reticulum Calcium Sensor STIM1 and Store-Operated Calcium Entry by Rotavirus Requires NSP4 Viroporin Activity

Rotavirus nonstructural protein 4 (NSP4) induces dramatic changes in cellular calcium homeostasis. These include increased endoplasmic reticulum (ER) permeability, resulting in decreased ER calcium stores and activation of plasma membrane (PM) calcium influx channels, ultimately causing a 2- to 4-fold elevation in cytoplasmic calcium. Elevated cytoplasmic calcium is absolutely required for virus replication, but the underlying mechanisms responsible for calcium influx remain poorly understood. NSP4 is an ER-localized viroporin, whose activity depletes ER calcium, which ultimately leads to calcium influx. We hypothesized that NSP4-mediated depletion of ER calcium activates store-operated calcium entry (SOCE) through activation of the ER calcium sensor stromal interaction molecule 1 (STIM1). We established and used a stable yellow fluorescent protein-expressing STIM1 cell line (YFP-STIM1) as a biosensor to assess STIM1 activation (puncta formation) by rotavirus infection and NSP4 expression. We found that STIM1 is constitutively active in rotavirus-infected cells and that STIM1 puncta colocalize with the PM-localized Orai1 SOCE calcium channel. Expression of wild-type NSP4 activated STIM1, resulting in PM calcium influx, but an NSP4 viroporin mutant failed to induce STIM1 activation and did not activate the PM calcium entry pathway. Finally, knockdown of STIM1 significantly reduced rotavirus yield, indicating STIM1 plays a critical role in virus replication. These data demonstrate that while rotavirus may ultimately activate multiple calcium channels in the PM, calcium influx is predicated on NSP4 viroporin-mediated activation of STIM1 in the ER. This is the first report of viroporin-mediated activation of SOCE, reinforcing NSP4 as a robust model to understand dysregulation of calcium homeostasis during virus infections.     

Neuraminidase-Inhibiting Antibody Is a Correlate of Cross-Protection against Lethal H5N1 Influenza Virus in Ferrets Immunized with Seasonal Influenza Vaccine

In preparing for the threat of a pandemic of avian H5N1 influenza virus, we need to consider the significant delay (4 to 6 months) necessary to produce a strain-matched vaccine. As some degree of cross-reactivity between seasonal influenza vaccines and H5N1 virus has been reported, this was further explored in the ferret model to determine the targets of protective immunity. Ferrets were vaccinated with two intramuscular inoculations of trivalent inactivated split influenza vaccine or subcomponent vaccines, with and without adjuvant, and later challenged with a lethal dose of A/Vietnam/1203/2004 (H5N1) influenza virus. We confirmed that vaccination with seasonal influenza vaccine afforded partial protection against lethal H5N1 challenge and showed that use of either AlPO₄ or Iscomatrix adjuvant with the vaccine resulted in complete protection against disease and death. The protection was due exclusively to the H1N1 vaccine component, and although the hemagglutinin contributed to protection, the dominant protective response was targeted toward the neuraminidase (NA) and correlated with sialic acid cleavage-inhibiting antibody titers. Purified heterologous NA formulated with Iscomatrix adjuvant was also protective. These results suggest that adjuvanted seasonal trivalent vaccine could be used as an interim measure to decrease morbidity and mortality from H5N1 prior to the availability of a specific vaccine. The data also highlight that an inducer of cross-protective immunity is the NA, a protein whose levels are not normally monitored in vaccines and whose capacity to induce immunity in recipients is not normally assessed.     

Assessing breeding success in common woodland birds using a novel method

Capsule Repeated counts of fledged broods can provide a useful estimate of breeding success for most common woodland birds.

Aims To assess the efficacy of comparing fledged-brood survey data with territory mapping using simple mark–recapture analysis techniques to provide an estimate of breeding success for common woodland birds that does not involve finding nests.

Methods Three observers undertook territory mapping surveys of adults, followed by counts of fledged broods four times a week during May–July 2007 in two 15 ha woods each, both in southern England. Using known fledging to maturity periods, these counts were used to calculate daily detection probabilities for broods of ubiquitous species. These enabled fledged brood territory occupancy probabilities (i.e. brood to territory ratios) to be estimated that take account of the possibility that broods were present but missed by surveys.

Results Of the 19 species found in all six woods, mean daily detection probability estimates for fledged broods of 17 species ranged from 0.17 to 0.50 with significant variation between woods for 12 species, but within region/observer for four species. The mean probability of detecting a brood at least once was over 75% using four visits per week and over 50% using two visits. Only for Great Spotted Woodpeckers Dendrocopos major and Garden Warblers Sylvia borin was the fledging period too short and the daily detection probability too low to provide a reasonable estimate of the territory occupancy probability.

Conclusion Daily detection probabilities for fledged broods of most common woodland birds were sufficiently high to enable useable estimates of fledged-brood territory occupancy probabilities to be made based on a survey programme involving two or three visits per week between late May and the end June. The method used may have application as a means of providing a relatively easily derived productivity index for woodland bird monitoring programmes or for research studies.