The role of RNAi and microRNAs in animal virus replication and antiviral immunity

The closely related microRNA (miRNA) and RNAi pathways have emerged as important regulators of virus–host cell interactions. Although both pathways are relatively well conserved all the way from plants to invertebrates to mammals, there are important differences between these systems. A more complete understanding of these differences will be required to fully appreciate the relationship between these diverse host organisms and the various viruses that infect them. Insights derived from this research will facilitate a better understanding of viral pathogenesis and the host innate immune response to viral infection.     

Survival of human bone marrow cells after in vitro treatment with 12 anticancer drugs and implications for tumor drug sensitivity assays

Summary We investigated the responsiveness of human normal granulocyte-macrophage colony-forming units in culture (GM-CFUC) continuously exposed in vitro to 1 of 12 anticancer drugs. All drugs except bleomycin showed a simple negative exponential dosesurvival curve. The in vitro toxicity of drugs in GM-CFUC did not always correlate with the relative myelosuppressive potency observed in vivo. In addition, tumor specimens from 38 patients mainly with ovarian cancer were cultured in a human tumor colony-forming assay and continuously exposed to drugs at low, intermediate, and high concentrations capable of killing 40%, 78%, and 99% of GM-CFUC, respectively. The most active drugs were cis-platinum, velban, 5-fluorouracil, and 5-fluoro-ara-AMP. Dosesurvival curves of bone marrow progenitor cells may serve as an in vitro reference system for selecting appropriate drug concentrations of myelosuppressive drugs in drug-sensitivity assays of human tumors.This work was supported by NIH grants CA 28153, CA 14528, CA 23272, the Max Kade Foundation, New York, and the Leukemia Society of America     

A sibling-augmented case-only approach for assessing multiplicative gene-environment interactions

Family-based designs protect analyses of genetic effects from bias that is due to population stratification. Investigators have assumed that this robustness extends to assessments of gene-environment interaction. Unfortunately, this assumption fails for the common scenario in which the genotyped variant is related to risk through linkage with a causative allele. Bias also plagues other methods of assessment of gene-environment interaction. When testing against multiplicative joint effects, the case-only design offers excellent power, but it is invalid if genotype and exposure are correlated in the population. The authors describe 4 mechanisms that produce genotype-exposure dependence: exposure-related genetic population stratification, effects of family history on behavior, genotype effects on exposure, and selective attrition. They propose a sibling-augmented case-only (SACO) design that protects against the former 2 mechanisms and is therefore valid for studying young-onset disease in which genotype does not influence exposure. A SACO design allows the ascertainment of genotype and exposure for cases and exposure for 1 or more unaffected siblings selected randomly. Conditional logistic regression permits assessment of exposure effects and gene-environment interactions. Via simulations, the authors compare the likelihood-based inference on interactions using the SACO design with that based on other designs. They also show that robust analyses of interactions using tetrads or disease-discordant sibling pairs are equivalent to analyses using the SACO design.     

Using Case-parent Triads to Estimate Relative Risks Associated with a Candidate Haplotype

Estimating haplotype relative risks in a family-based study is complicated by phase ambiguity and the many parameters needed to quantify relative risks for all possible diplotypes. This problem becomes manageable if a particular haplotype has been implicated previously as relevant to risk. We fit log-linear models to estimate the risks associated with a candidate haplotype relative to the aggregate of other haplotypes. Our approach uses existing haplotype-reconstruction algorithms but requires assumptions about the distribution of haplotypes among triads in the source population. We consider three levels of stringency for those assumptions: Hardy-Weinberg Equilibrium (HWE), random mating, and no assumptions at all. We assessed our method's performance through simulations encompassing a range of risk haplotype frequencies, missing data patterns, and relative risks for either offspring or maternal genetic effects. The unconstrained model provides robustness to bias from population structure but requires excessively large sample sizes unless there are few haplotypes. Assuming HWE accommodates many more haplotypes but sacrifices robustness. The model assuming random mating is intermediate, both in the number of haplotypes it can handle and in robustness. To illustrate, we reanalyze data from a study of orofacial clefts to investigate a 9-SNP candidate haplotype of the IRF6 gene.     

Isoflavones in urine, saliva, and blood of infants: data from a pilot study on the estrogenic activity of soy formula

In the United States, about 25% of infant formula sold is based on soy protein, which is an important source of estrogenic isoflavones in the human food supply. Nevertheless, few studies report isoflavone levels in infants. We did a partly cross-sectional and partly longitudinal pilot study to examine children's exposure to isoflavones from different feeding methods. A total of 166 full-term infants between birth and 1 year of age were recruited into soy formula, cow milk formula, or breast milk regimens according to their feeding histories. A total of 381 urine, 361 saliva, and 88 blood samples were collected at 382 visits. We used automated online solid-phase extraction coupled to high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for measuring three isoflavones (daidzein, genistein, and equol) in urine, and used similar LC/MS/MS techniques for saliva and blood spots. Concentrations of daidzein and genistein were undetectable in most blood or saliva samples from children fed breast milk or cow milk formula. The proportion of non-detectable values was somewhat lower in urine than in the other matrices. Concentrations of equol were detectable only in a few urine samples. For both daidzein and genistein, urine contained the highest median concentrations, followed by blood and then saliva. Urinary concentrations of genistein and daidzein were about 500 times higher in the soy formula-fed infants than in the cow milk formula-fed infants. The correlations between matrices for either analyte were strikingly lower than the correlation between the two analytes in any single matrix. We did not find significant correlations between isoflavone concentrations and the levels of certain hormones in children fed soy formula. Our results, based on much larger numbers of infants, strongly confirm previous reports, but whether phytoestrogens in soy formula are biologically active in infants is still an open question. We plan further longitudinal studies focusing on physical and developmental findings reflecting the effects of estrogen exposure.     

Pooled exposure assessment for matched case-control studies

Exposure assessment using biologic specimens is important for epidemiology but may become impracticable if assays are expensive, specimen volumes are marginally adequate, or analyte levels fall below the limit of detection. Pooled exposure assessment can provide an effective remedy for these problems in unmatched case-control studies. We extend pooled exposure strategies to handle specimens collected in a matched case-control study. We show that if a logistic model applies to individuals, then a logistic model also applies to an analysis using pooled exposures. Consequently, the individual-level odds ratio can be estimated while conserving both cost and specimen. We discuss appropriate pooling strategies for a single exposure, with adjustment for multiple, possibly continuous, covariates (confounders) and assessment of effect modification by a categorical variable. We assess the performance of the approach via simulations and conclude that pooled strategies can markedly improve efficiency for matched as well as unmatched case-control studies.     

Suicide and pesticide use among pesticide applicators and their spouses in the agricultural health study

Background: An association may exist between pesticide exposure and suicide.Objective: We sought to evaluate the existence of an association between pesticide use and suicide using data from the Agricultural Health Study (AHS), a prospective cohort study of licensed pesticide applicators and their spouses in Iowa and North Carolina.Methods: Via linkage to state mortality files and the National Death Index, we identified 110 suicides occurring between enrollment in the AHS (from 1993 to 1997) and 31 May 2009, among 81,998 cohort members contributing 1,092,943 person-years of follow-up. The average length of follow-up was 13.3 years. AHS participants provided data on pesticide use and potential confounders via self-administered questionnaires at enrollment. We evaluated several measures of pesticide use: use of any pesticide, ever use of 50 specific pesticides, cumulative lifetime days of use and intensity-adjusted cumulative lifetime days of use of 22 specific pesticides, and ever use of 10 functional and chemical classes of pesticides. We used Cox proportional hazards regression models to estimate adjusted hazard ratios and 95% confidence intervals.Results: After adjusting for age at enrollment, sex, number of children in family, frequency of alcohol consumption during the past 12 months, and smoking status, we found no association between prior pesticide use and suicide in applicators and their spouses. Results were the same for applicators and spouses together or for applicators alone and were consistent across several measures of pesticide use.Conclusions: Our findings do not support an association between moderate pesticide use and suicide.     

Optimal sampling of rat liver tissue for toxicogenomic studies

Different degrees of a toxic response between and within the various lobes of the liver have been observed in rodents following treatment with acetaminophen. This study was designed to compare 2 sampling methods of the rat liver for gene-expression analysis. Ten male Fischer 344/N rats, 12-14 weeks of age, were treated with vehicle (0.5% aqueous ethyl cellulose) or acetaminophen (APAP, 1500 mg/kg) and sacrificed 24 hours following dose administration. Two representative sections were collected from the left liver lobe, stained with hematoxylin and eosin (H&E), and evaluated independently by 2 pathologists. The central core of the left lobe was cubed and frozen. Five random cubes were conserved, while the remaining left lobe core was pulverized. From each of the 10 animals, 2 random cubes and 2 samples from the homogeneous, pulverized samples were prepared for microarray analysis. Histopathologic evaluation revealed a variable response of centrilobular necrosis within the left lobe. Multiple methods used to analyze the microarray data indicated that sampling technique was not a major contributor to the variability observed in the gene expression data; however, only the powdered samples clustered for all animals, even those with disparate histopathologic results. Additionally, a powdered sample provided the advantages of a homogenous sample pool and the ability to use sample aliquots for other analyses to include proteomics, metabonomics, and other molecular techniques.